Medical Laboratory Science Examination Review

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Linda J. Graeter Associate Professor Medical Laboratory Science Program University of Cincinnati Cincinnati, Ohio Elizabeth G. Hertenstein Assistant Professor Medical Laboratory Science Program University of Cincinnati Cincinnati, Ohio Charity E. Accurso Assistant Professor Medical Laboratory Science Program University of Cincinnati Cincinnati, Ohio Gideon H. Labiner Associate Professor Medical Laboratory Science Program University of Cincinnati Cincinnati, Ohio 3251 Riverport Lane St. Louis, Missouri 63043

Elsevier’s Medical Laboratory Science Examination ISBN: 978-1-4557-0889-5

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Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. With respect to any drug or pharmaceutical products identified, readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

Library of Congress Cataloging-in-Publication Data Elsevier’s medical laboratory science examination review / [edited by] Linda J. Graeter, Elizabeth G. Hertenstein, Charity E. Accurso, Gideon H. Labiner. – First edition. p.; cm. Medical laboratory science examination review Includes bibliographical references and index. ISBN 978-1-4557-0889-5 (pbk.: alk. paper) I. Graeter, Linda J., editor. II. Hertenstein, Elizabeth G., editor. III. Accurso, Charity E., editor. IV. Labiner, Gideon H., editor. V. Title: Medical laboratory science examination review. [DNLM: 1. Clinical Laboratory Techniques–Examination Questions. QY 18.2] RB37 616.07056–dc23 2014016504

Executive Content Strategist: Kellie White Content Development Manager: Billie Sharp Content Development Specialist: Betsy McCormac Publishing Services Manager: Catherine Jackson Senior Project Manager: Rachel E. McMullen Design Direction: Maggie Reid

Printed in the United States of America CONTRIBUTORS

Brenda C. Barnes, PhD, MT(ASCP)SBBCM Mark W. Ireton, MA, BS, MLS(ASCP)CM Director, Medical Laboratory Science Program, Blood Bank Technologist II Associate Professor Hoxworth Blood Center Allen College University of Cincinnati Waterloo, Iowa Cincinnati, Ohio

Janelle M. Chiasera, PhD, MT(ASCP) Paul R. Labbe, MS, MCLT Chair,The Department of Clinical and Diagnostic Sciences, Vice President Information Resources Professor CompuNet Clinical Laboratories The University of Alabama, Birmingham Dayton, Ohio Birmingham, Alabama Joel E. Mortensen, PhD, HCLD, FAAM Sandy Cook, MS, MT(ASCP) Department of Pathology and Laboratory Medicine Assistant Professor Cincinnati Children’s Hospital Medical Center Clinical Laboratory Services Cincinnati, Ohio Ferris State University Big Rapids, Michigan Susan King Strasinger, DA, MLS(ASCP) Faculty Associate Melanie J. Giusti, BS, MLS(ASCP)CM The University of West Florida Program Manager Pensacola, Florida Medical Laboratory Science Program College of Allied Health Sciences University of Cincinnati Cincinnati, Ohio

v This page intentionally left blank ACKNOWLEDGMENTS

We are grateful to all contributing authors and reviewers who dedicated time and effort during the development of this book. A special word of recognition and appre- ciation for their dedication to Medical Laboratory education is offered to Melanie Giusti, MLS(ASCP)CM; Lara Kolar, MT(ASCP); John Landis, MS, MT(ASCP); Jennifer Macht, BS MT(ASCP), CHT (ABHI); Ryan McGough, MS, MT(ASCP); Erin Rumpke, MS, MT(ASCP); and Beth Warning, MS, MLS (ASCP)CM. Last but not least, our sincere thanks are extended to our Elsevier colleagues: Ellen Wurm-Cutter, Content Manager; Amy Whittier, Content Development Specialist; and all others at Elsevier who were involved in this project. Their assistance, thought- ful advice, and continued support were invaluable as we navigated through the various steps in completing the book.

vii This page intentionally left blank PREFACE

The Medical Laboratory Science Review is intended to certification or licensure examinations. The Evolve web- serve as a review tool for candidates who are preparing site was created with that in mind and includes printable to sit for certification or licensure examinations in Med- study worksheets and additional review materials. Stu- ical Laboratory Science. However, the integral nature of dents are able to generate individualized study files this review book provides a review for individuals seeking from these materials. The website also includes 1000 to strengthen knowledge in the topics related to the clin- additional multiple-choice questions that are different ical laboratory. Therefore this text can provide a multi- from those in the book. From these materials, students purpose review. The text is ideal for those preparing can create additional practice examinations focusing for Medical Laboratory Science (MLS) or categorical on the content area of their choice. Technologist/Scientist (Blood Banking, Chemistry, Hematology, Microbiology, and Molecular Biology) certification examinations sponsored by the American Soci- EXAMINATION PREPARATION ety of Clinical Pathology (ASCP) Board of Certification or for the American Medical Technologists (AMT)- Preparation for certification and licensure examinations supported certification examination. Additionally, this is sometimes a daunting and intimidating process. text will be helpful for those preparing for the Medical We encourage students to recognize the time and effort Laboratory Technology (MLT) certification examina- placed in successfully completing their respective education, although the scope of some of the advanced topics tional programs and the knowledge gained while doing are outside the required competencies for the MLT level. so. Preparing for the examination then becomes a struc- The outline format enhances learning and comprehension tured plan that provides for a review of the knowledge for each professional career entry track. Others who gained. would benefit from using this review text to support their Shortly before program completion, thoroughly review studies are those seeking advanced degrees, ASCP Spe- the ASCP Board of Certification website. Be sure to review cialist certification, Physician Assistant students, and the requirements that must be met to sit for the examina- Pathology Residents. Additional uses include serving as tion, along with the recommended dates to submit the a reference book for students and educators, providing application. We encourage students to sit for the examina- continuing education review and to refresh knowledge. tion within 6 months of program completion. Students The book’s preface is followed by review materials who wait longer tend to have a more difficult time review- encompassing all major areas of the laboratory, divided ing and preparing for the examination. On the website, into 11 chapters. Each chapter includes a comprehensive you will also find content outlines and a distribution of bulleted summary of didactic information. The chapter the content areas that will aid in your planning. Details summary outlines provide a thorough but efficient review are included about the cost, length, and structure of the of key content information. Each chapter is followed by examination. Review the examination preparation guide. 30 to 100 multiple-choice questions. The questions • Complete a practice examination or set of review include representation of the three question types (I, II, questions. Record your answers on a separate piece III) to enhance recall, interpretation, and problem-solving of paper so that you can continue to practice with skills. Each question includes an explanation of the cor- the same questions. rect answer. The book’s final section is a comprehensive ○ After reviewing missed questions, make a list of the practice examination designed using the ASCP Board of specific content areas that were missed (e.g., ane- Certification guidelines. mias, streptococci, liver enzymes) and then design The chapters and examination questions were written a study schedule using a calendar that allows more by Medical Laboratory educators and clinical experts, all time to review the more challenging areas. of whom are recognized for expertise in their respective ○ Plan 1 to 2 weeks for a thorough review of each area of practice. All chapters and multiple-choice ques- content area. tions underwent a peer-review process as the content • Create mini study guides for your more challenging was developed. or weaker areas. The study guides should include The companion website—Evolve to accompany Med- the following: ical Laboratory Science Review—was developed to ○ Graphical representations of a disease or process enhance each candidate’s preparation process by provid- ○ Concise charts or tables ing additional review materials. Students often benefit ○ A brief paragraph explaining the topic or ques- from a variety of study approaches when preparing for tion or a short outline

ix x Preface

Preparing the guides is an active learning exercise that Practice, practice, practice! It is always helpful to ad- will help in reviewing the specific content and in main- dress questions you previously reviewed. Use the questions taining focus on weaker topics. It is human nature to in this text and those from the online companion site. gravitate toward favorite topics, but it is also necessary The day of the examination ...breathe deeply! If you to focus on the areas that are more challenging. Compile are not sure of the location of the testing center, take a the study guides in a binder organized by content area. test drive to the center a week before the examination. The guides will be great tools to review the week before Have a scheduled plan for the day. Be well rested and the actual examination be sure to eat a good meal before arriving at the testing When studying the review questions or old examina- center. If you have prepared, your efforts will be evident tions: in your success! • Provide a rationale as to why you can rule out incorrect answers and rule in correct ones. NOTE: Although it is this book’s intent to properly • Pay attention to small details that will help rule out prepare readers for their certification examination, use a wrong answer. of this book alone does not guarantee passage of certifi- • Writing your rationales out in sentence form is cation or licensure examinations. another great review tool. • Create your own question rationales. CONTENTS

Color insert follows p. 20

1 Microbiology, 1 Joel E. Mortensen and Linda J. Graeter 2 Mycology, Virology, and Parasitology, 50 Linda J. Graeter and Joel E. Mortensen 3 Hematology, 90 Sandy Cook 4 Hemostasis, 139 Charity E. Accurso 5 Clinical Fluid Analysis, 159 Sue King Strasinger 6 Immunology and Serology, 180 Elizabeth G. Hertenstein 7 Immunohematology and Blood Transfusion Medicine, 200 Brenda C. Barnes and Elizabeth G. Hertenstein 8 Clinical Chemistry, 228 Janelle M. Chiasera 9 Molecular Diagnostics, 262 Gideon H. Labiner 10 Laboratory Operations, 283 Paul R. Labbe and Linda J. Graeter 11 Laboratory Calculations, 298 Melanie J. Giusti and Mark W. Ireton

APPENDIX A Answers and Rationales to Certification Preparation Questions, 312 B Mock Examination, 357 C Examination Preparation Worksheet, 367 D Color Insert Figure Credit Lines, 370

xi This page intentionally left blank CHAPTER 1 Microbiology

Joel E. Mortensen and Linda J. Graeter

STAINED SMEARS • Proper staining technique ○ Underdecolorization • Gram stain ○ Overdecolorization • Gram stain history • Special considerations: Sputum specimens ○ Developed by Hans Christian Gram in 1884 ○ >25 epithelial cells/lpf¼saliva ○ Became the major bacterial staining method ○ Few epithelial cells, many PMN cells:—Specimen • Most bacteria are stained by this method more likely to yield a pathogen ○ Exceptions include Legionella, Mycoplasma, ○ Examine properly stained area Chlamydia, and others ○ Recognize normal oral flora • Gram stain procedure ○ Report or reject ○ Crystal violet ○ Sputum only, may not apply to aspirates or ○ Gram’s iodine children ○ Decolorizer • Special considerations: Urine ○ Acetone and alcohol either alone or together ○ Urine specimens ○ Safranin § 1 cell per oil immersion field¼approximately • Gram stain mechanism 1 105 CFU/mL ○ Differences in the microbial cell wall are visualized § Not commonly performed § Cell walls of gram-negative cells have higher • Quantitation lipid content than gram-positive cells ○ No organisms seen ○ Crystal violet penetrates both types ○ Few per slide¼Rare ○ Iodine is added, forming the crystal violetiodine ○ 0 to 2 per field¼Few (CV-I) complex (mordant) ○ 2 to 10 per field¼Moderate ○ Decolorizer dissolves the lipid layer from the ○ More than 10 per field¼Many gram-negative cells allowing the CV-I complex to wash out ○ Counterstain is applied to dye the decolorized GROWING BACTERIA IN THE gram-negative cells LABORATORY—MEDIA • Clinical utility ○ A true STAT test in microbiology • Types of media ○ Judge adequacy of a specimen • Bacteriology ○ Recognition specific morphologies ○ Routine ○ Indicate need for additional tests ○ Fastidious ○ Expand clinical diagnostic picture ○ Anaerobes • Limitations ○ Mycoplasma, Ureaplasma ○ Only partial bacterial identification • Mycobacteriology ○ Some organisms do not stain • Mycology ○ “No organisms seen” does not rule out infection • Virology ○ Normal flora can mask pathogens ○ Viruses ○ Human error ○ Chlamydia ○ Organisms do not stain as expected • Constituents of media • Diagnostic considerations • Agar ○ Cell identification ○ Gelatinous seaweed extract § Epithelial cells ○ 1% to 2% agar in plates § Polymorphonuclear (PMN) cells • Nutrients § Bacteria ○ Hydrolyzed proteins

1 2 CHAPTER 1 Microbiology

§ Animal • Chocolate agar § Plant ○ Casein peptones ○ Carbohydrates, sugars ○ Meat peptones • Enrichments ○ Corn starch ○ Yeast extracts, blood ○ Hemoglobin (V factor) ○ • Buffers IsoVitaleX Enrichment (X factor) § ○ Stable pH for growth Used for specimens from which fastidious • pH indicators organisms may be isolated § ○ Neutral red: Red to colorless Haemophilus spp., Neisseria spp., Brucella or ○ Phenol red: Yellow to red Capnocytophaga spp. • ○ Thymol blue: Yellow to green/blue MacConkey agar ○ ○ Others Peptone base with lactose, crystal violet, and bile • Inhibitors salts § Lactose to provide fermentable sugar (Lactose ○ Dyes positive vs negative) § Crystal violet, eosin, and methylene blue § Crystal violet—inhibit gram-positive bacteria ○ Bile salts § Bile salts—inhibit gram-positive bacteria § Sodium deoxycholate ○ Neutral red pH indicator ○ Sodium chloride ○ Selective for gram-negative organisms ○ Sodium citrate ○ Differential for lactose fermentation ○ Antibiotics § Lactose positive • Selective § Escherichia coli: Dry, flat, dark pigment • Contains inhibitory agents to all organisms except § Klebsiella/Enterobacter: Mucoid one being sought § Citrobacter: Late fermenter • Selects for certain organisms to the disadvantage of § Serratia: Late, red pigment (some) others § Lactose negative • Example § Proteus: Swarming ○ Colistin nalidixic acid agar (CNA) § Morganella, Providencia, Edwardsiella, ○ MacConkey agar Hafnia • Differential § Pseudomonas • Allows organism tobe morphologically distinguished § Salmonella from other organisms with different characteristics § Shigella • Examples ○ Used for various patient and environmental ○ Sheep blood agar (SBA) samples ○ MacConkey agar • XLD • Key media for routine aerobic cultures ○ Yeast extract • Supportive ○ Sodium deoxycholate ○ Blood agar § Inhibits gram-positive organisms ○ Chocolate agar ○ Phenol red: pH indicator • Selective/differential for gram-negative bacilli ○ Lactose and sucrose in excess, xylose in lower ○ MacConkey agar amounts ○ Xylose lysine desoxycholate (XLD) or Hektoen § Lactose and sucrose to provide fermentable (HE) for stool sugar • Selective/differential for gram-positive organisms ○ Lysine: Lysine decarboxylase ○ CNA § Salmonella decarboxylate the lysine shift the ○ Phenylethyl alcohol agar (PEA) pH indicator to red • Examples ○ Sodium thiosulfate, ferric ammonium citrate ○ Blood agar § H2S production § Casein peptones: Group of proteins from milk ○ Yellow: Ferments the excess carbohydrates (or § Soybean peptones xylose only), causes large pH drop, yellow § 5% sheep blood (E. coli) § Approximately 1% agar-agar ○ Colorless or red: No fermentation, no H2S § Used for general growth of gram-positive and (Shigella and Providencia) gram-negative aerobes ○ Red with black center § The most common supportive media because § Ferments xylose, produces low pH, then most organisms grow on it, but it is also differ- decarboxylates lysine, produces high pH ential because of hemolytic pattern § H2S production (Salmonella) CHAPTER 1 Microbiology 3

• HE • Chopped meat–glucose and thioglycolate broth are ○ Meat peptones and yeast extract most common ○ Bile salts • Use should be limited to fluids and tissue § Inhibit gram-positive organisms ○ Not swabs ○ Lactose, sucrose, salacin § Lactose and sucrose to provide fermentable sugar Growth Requirements—Other Important ○ Indicators: Bromophenol blue and acid fuchsin Components § pH indicators • Atmosphere requirements ○ Ferric ammonium citrate for H2S production • 3% to 5% CO ○ 2 Differential for Salmonella and Shigella • Room air ○ Yellow-orange colonies: Lactose fermenter • Increased CO2 and N, decreased O2 (E. coli) ○ Microaerophilic: 5% O2, 10% CO2, 85% N ○ Colorless/green colonies with unchanged medium: ○ Anaerobic: 85% N2, 10% H2,5%CO2 Non–lactose fermenter (Shigella, Providencia) • Temperature requirements ○ Black colonies: H2S production (Salmonella) • 35 C Room temperature (25 Cto30 C) • CNA • 42 C ○ Casein peptones • 4 C ○ Digest of animal tissue • pH and moisture requirements ○ Yeast and beef extract • pH ○ Corn starch ○ Most are 6.5 to 7.5 ○ 5% sheep blood ○ Buffers maintain pH of media ○ CNA: inhibits most gram-negative organisms • Moisture ○ Used for samples with mixed flora ○ 70% humidity is optimal for good growth • PEA ○ Prevents drying of media ○ Casein peptones ○ Soybean peptones ○ 5% sheep blood STRUCTURE AND METABOLISM ○ Phenylethyl alcohol to inhibit gram-negative organisms • Bacteria are prokaryotes: Single-cell organisms lacking ○ Some laboratories use CNA rather than PEA membrane-bound nuclei • because of gram-negative breakthrough Eukaryotes are organisms with a defined nucleus • • Key media for routine anaerobic Mammalian and plant cells are eukaryotic • • Basic: Blood agar Reproduction of prokaryotic cells is by binary fission • ○ Agar base supplemented with 5% sheep blood, Simple division of one cell into two cells • hemin, and vitamin DNA replication and formation of a separating ○ Agar base usually trypticase soy or brain heart membrane and cell wall infusion • Can be approximately 20 minutes ○ Good nonselective medium for initial isolation of anaerobes Bacteria • Selective for anaerobes: Supplemented PEA ○ Vitamin K and hemin are added to PEA agar • Cells must acquire nutrients, produce energy, and syn- ○ Suppresses growth of facultative gram-negative thesize macromolecules bacilli • It is important to study these areas so that bacteria can ○ All anaerobes grow well on this medium be isolated and identified in the laboratory • Selective for gram-negative bacilli: Kanamycin- • Bacterial growth requirements vancomycin–laked blood agar (K-V agar) • Nutrients acquired by active transport across the ○ Aminoglycoside helps separate aerobes and cell membrane from the environment anaerobes in mixed cultures • Requirements for all bacteria ○ Limit the swarming of Proteus spp. ○ A carbon source (for cellular constituents) ○ Kanamycin and vancomycin permit growth of ○ A nitrogen source (for proteins) only gram-negative anaerobes ○ Energy source: Adenosine triphosphate (ATP) (to ○ Laked blood stimulates the growth of some perform cellular tasks) anaerobes ○ Trace elements • Back-up broth § Iron • Broth medium serves as a check for agar plates § Calcium • Useful when the primary shows no growth § Zinc 4 CHAPTER 1 Microbiology

§ Copper • Most bacteria are placed into two groups (deter- § Manganese mined by differences in the cell wall) § Cobalt ○ Gram-positive § Phosphorus ○ Gram-negative § Sulfur • Chromosome § Potassium • Single, long, supercoiled, circular DNA molecule § Magnesium • Prokaryotic cell contains no nucleus • Oxygen growth requirements • Attached to the cell membrane • Obligate aerobe: Live and grow in air, cannot grow • Bacterial chromosomes contain genetic information anaerobically to code for between 850 to 6500 products • Facultative anaerobe: Can grow aerobically and ○ Enzymes, proteins, and RNA molecules anaerobically ○ Human chromosome contains approximately • Aerotolerant anaerobe: Grows better anaerobically, 30,000 genes but can tolerate low levels of air • Plasmids • Obligate anaerobe: Grows only anaerobically, poi- • Small, circular double-stranded DNA soned by air • Not part of the chromosome • Microaerophilic: Increased CO2 or other enriched • May contain several to several hundred genes environment • One plasmid, multiple copies of same plasmid, or • Bacterial metabolism more than one type • Production of ATP • Antibiotic resistance genes common ○ Drives other metabolic processes • Can be exchanged between a donor and a recipient • Substrates ! glucose ! metabolic pathway ! energy in conjugation • Fermentation versus respiration • Ribosomes ○ Fermentation • Sites of protein synthesis § Glucose is converted into pyruvate • A 70S prokaryotic ribosome comprises a 30S subu- § Embdem-Meyerhof pathway or glycolysis nit and a 50S subunit § Fermentation is metabolism in the absence • Estimated approximately 15,000 ribosomes in the of O2 cytoplasm of an E. coli cell § Anaerobic • Cell membrane § Net gain: 2ATP, NADH2 • Bacterial membrane similar in structure and func- § Pyruvate can then enter several other cycles tion to eukaryotic cell membrane § End-products vary depending on cycle entered • Membrane consists of proteins and phospholipids ○ Aerobic respiration • Bilayer, with charged or polar groups facing out- § Glucose usage under aerobic conditions ward and the noncharged portions in between § Pyruvate enters Krebs cycle (tricarboxylic acid • Selectively permeable [TCA] cycle) • Many enzymes are attached to membrane § Nicotinamide adenine dinucleotide (NADH) ○ Metabolic reactions take place at membrane and flavin adenine dinucleotide (FADH) enter • Bacterial cell wall the electron transport chain • Cell wall defines the shape of bacterial cells § Net gain: 38 ATP (including ATP from • Main constituent is complex polymer— fermentation) peptidoglycan § End-products: CO2 and H2O ○ Many sugar molecules (polysaccharide) linked ○ Application to the clinical laboratory by small peptide (short protein) chains § Systems to detect fermentation or respiration ○ Peptidoglycan is found only in bacteria § Acid detection (pH indicators) • Thickness of the cell wall and its exact composition § Gas detection vary with the species of bacteria § Alcohol detection • Gram-positive cell walls have thick layer of • Different carbon sources peptidoglycan • Not all bacteria undergo respiration • Gram-negative walls have much thinner layer of ○ Some lack the enzymes needed peptidoglycan and an outer membrane ○ Some cannot survive in O2 • Within the cell wall of gram-negative bacteria is • Potential biochemical tests for organism lipopolysaccharide (LPS) identification ○ Part of LPS protrudes from the cell surface—O ○ Tests based on presence or absence of these spe- antigen cific enzymes • Cellular morphology and arrangement • Cellular structure • Glycocalyx • Bacteria are prokaryotic • Some bacteria have layer of material located outside • Bacteria are small (0.2-2 mm diameter,1-6 mmlength) cell wall CHAPTER 1 Microbiology 5

• Glycocalyx is a slimy, gelatinous material produced ○ Capsules near the cell membrane and secreted outside of the ○ Pili cell wall ○ Other extracellular proteins • Two types of glycocalyx • Bacterial taxonomy: Shared morphologic, physiologic, ○ Slime layer: Not highly organized, not firmly and genetic traits attached • Species § Pseudomonas and Staphylococcus ○ Basic taxonomic group ○ Capsule: Highly organized and firmly attached ○ Composed of related individuals with shared to the cell wall characteristics that resemble one another § Usually polysaccharides, may be combined ○ Complete definition difficult with lipids and proteins • Genus (genera) § Protects from engulfment by white blood • Family cells (WBCs) • Order ○ Both may protect the bacterium from antibiotics • Class division • Flagella • Kingdom • Enable bacteria to move in liquid environment • Bacterial names are binomial ○ Water, intestinal tract, blood, or urine ○ Genus and species • Consist of three or more protein appendages ○ Treated as Latin and written in italic twisted together ○ Genus can be abbreviated in written material • Number and arrangement of flagella are character- after first used istic of species § Staphylococcus aureus • Single flagellum at one end to multiple flagella cov- § S. aureus ering the entire cell surface • Name changes are designated with ( ) • Pili ○ Stenotrophomas (Xanthomonas) maltophilia • Pili or fimbriae are short, hairlike structures ○ Usually on external surface of gram-negative FAMILY STAPHYLOCOCCACEAE organisms • Much thinner than flagella, rigid structure, not • General characteristics associated with motility • Gram-positive cocci in clusters or tetrads • Originate in cytoplasm and extend through the • Catalase positive plasma membrane, cell wall, and capsule • Aerobic to facultative anaerobic • Two types—sex pilus and attachment pilus • Nonmotile ○ Adherence pili anchor to surfaces • Staphylococcus: Greek staphyle means “bunch of § Tissues in animal’s body grapes” and coccos means “granule” § Usually quite numerous • Genus Staphylococcus: General characteristics ○ Sex pilus transfers genetic material • All ferment glucose § Cell possessing a sex pilus is donor cell • Differentiated by coagulase test § Attach to another cell (usually of the same • Coagulase-positive are considered S. aureus species) • Coagulase-negative species § Genetic material, usually a plasmid, trans- ○ Staphylococcus epidermidis ferred through the hollow sex pilus ○ Staphylococcus saprophyticus • Spores • Approximately 30 other species • Some bacteria form thick-walled structures ○ Bacillus and Clostridium Staphylococcus aureus ○ Means of survival when moisture or nutrients low • Most clinically important • Formed during sporulation • Causes numerous infections ○ Copy of the chromosome • Important hospital pathogen ○ Some cytoplasm enclosed in thick protein coats • Antibiotic resistance has become a major issue (again) • Resistant to heat, cold, drying, most chemicals, • Epidemiology boiling • Humans are natural reservoir for S. aureus • Survive for many years in soil or dust • Asymptomatic colonization is far more common • Virulence factors than infection • Bacteria that cause disease are termed pathogenic • Colonization of nasopharynx and perineum skin because of various factors occurs shortly after birth and recurs • Examples • Transmission occurs by direct contact with a colo- ○ Exotoxins nized carrier ○ Endotoxin ○ Carriage rates from 25% to 50% 6 CHAPTER 1 Microbiology

○ Higher in injection drug users; patients with dia- • Abscess betes, dermatologic conditions, or long-term • Fibrin wall around a core of organisms and indwelling intravascular catheters; and health leukocytes care workers • Pathogenesis • Young children have higher rates • Ability to elaborate proteolytic enzymes may facil- • Colonization may be transient or persistent itate the process • Clinical disease • Nondisseminated: Local disease (e.g., boils) • Causes suppurative (pus-forming) infections and • Dissemination results in pneumonia, bone and joint toxin diseases infection • Infections can be superficial or invasive • Toxin disease: Either toxin alone or in combination ○ Superficial skin lesions: Boils, sties, furuncles, with invasion impetigo • Toxins ○ Invasive: Pneumonia, mastitis, arthritis, menin- • Enterotoxins gitis, osteomyelitis, and endocarditis ○ Heat-stable exotoxins that cause diarrhea and • Toxin diseases: Food poisoning, scalded skin syn- vomiting drome, toxic shock disease ○ Enterotoxins A and D are resistant to gastric and ○ Scalded skin syndrome digestive acids § Extensive exfoliative dermatitis (skin ○ Toxins are preformed in foods sloughing) ○ Symptoms (i.e., nausea, vomiting, abdominal § In adults, occurs in chronic renal failure and pain, and cramping) appear 2 to 8 hours after immunocompromised individuals ingestion and resolve within 8 hours § Mortality in adults can be as high as 50% • Enterotoxin F (TSST-1) § Localized¼Bullous impetigo (large pustule) • Epidermolytic toxin § Generalized¼Profuse peeling of the epider- ○ Sloughing of the skin mal layer of skin ○ Widespread systemic immune responses ○ Toxic shock syndrome • Exfoliative toxin § Toxic shock syndrome toxin-1 (TSST-1) ○ Similar to TSST-1 but a different site in skin associated ○ Cytolytic toxins: Extracellular factors that affect § Superantigen red blood cells (RBCs) and WBCs § Characteristic rash • Hemolysins § Multisystem disease: High fever, hypotension, ○ Alpha a-Hemolysin: Destroys RBCs, platelets, and shock tissue § Identified in both sexes ○ Beta b-Hemolysin: Destroys RBCs § Higher prevalence with tampon use ○ Gamma d-Hemolysin: Causes injury, less lethal § Most patients recover: 2% to 5% mortality • Leukocidin • Oxacillin-resistant S. aureus ○ Panton-Valentine leukocidin: Exotoxin lethal • Oxacillin-resistant S. aureus (ORSA) and to PMNs methicillin-resistant S. aureus (MRSA) are resistant ○ May suppress phagocytosis to antibiotics • Enzymes ○ Methicillin, oxacillin, nafcillin, penicillin, and • Coagulase: Causes coagulation of surroundings amoxicillin • Hyaluronidase: Hydrolyzes hyaluronic acid in con- ○ Frequently other agents nective tissue • Hospital acquired versus community acquired • Lipase: Aids colonization by acting on sebaceous • Historically, among persons in hospitals and health glands care facilities who have weakened immune systems • Fatty acid–modifying enzyme: Breaks down antista- • Infections acquired by persons who have not been phylococcal lipids made by the host recently hospitalized are known as community- • Protein A acquired (CA-ORSA) infections ○ In S. aureus cell wall • Infections in the community are usually skin ○ Binds Fc portion of immunoglobulin (avoid infections and occur in otherwise healthy people phagocytosis) • May be more pathogenic than hospital-acquired (HA-ORSA) infection • Pathogenesis and virulence factors Coagulase-Negative Staphylococci • Carriage of the organism • Disseminated via hand to body sites and breaks in • Clinically important the skin • Staphylococcus epidermidis • Eczema or minor dermatitis • Staphylococcus saprophyticus CHAPTER 1 Microbiology 7

• Normal flora of skin and mucous membranes ○ Beta (b): Lysis of the sheep RBCs in the blood • Approximately 30 other species agar plates Staphylococcus epidermidis ○ Gamma (g): Nonhemolytic • Infections • Classification • Predominantly hospital acquired • 85 species at present • Predisposing factors • Hemolytic pattern on 5% sheep blood agar ○ Catheterization, prosthetic heart valves, immu- • Serologic group (Lancefield) of the b-hemolytic nosuppressive therapy group: A, B, C, D, F, G . . . T • Bacteremia • Taxonomic/genetic related now used • Endocarditis • b-Hemolytic streptococci • Most common cause of hospital-acquired urinary • Streptococcus pyogenes: Group A tract infection (UTI) • Streptococcus agalactiae: Group B Staphylococcus saprophyticus • Streptococcus group C, F, G • Normal flora of the mucous membranes of the • Others urogenital tract • Many found predominately in animals • Causes UTI • Young, sexually active women Streptococcus Species • Considered significant in urine cultures even if it is found in small numbers Streptococcus pyogenes • Group A Streptococcus Laboratory Diagnosis • Not the exact same as S. pyogenes, approximately 5% Streptococcus anginosus • Media • Spread from person to person • Mannitol salt • Carriers do exist • CNA • Clinical disease • PEA • Causes relatively common, significant diseases • CHROMagar—media containing patented chro- • Pharyngitis: “Strep throat” mogenic substrates that can be formulated to pro- • Skin infections vide specific colors to develop in colonies of a • Otitis particular genus or species • Sinusitis • CHROMagar MRSA • Sepsis • S. aureus • Scarlet fever • Colonies are medium to large, ivory to yellow, and • Some less common diseases beta-hemolyic ○ Pneumonia • Catalase positive and coagulase positive ○ Meningitis • Mannitol salt positive ○ Fasciitis: Flesh-eating bacteria • S. epidermidis • Complications • Colonies are small to medium, nonhemolytic, white ○ Rheumatic heart disease • Coagulase negative ○ Glomerulonephritis • Biochemicals required to identify • Rheumatic fever • Often not speciated ○ Complication of pharyngitis • S. saprophyticus ○ Can cause chronic, progressive damage to heart • Colonies are large, with approximately 50% pro- ○ Pathogenesis poorly understood ducing a yellow pigment • Acute glomerulonephritis • Coagulase negative ○ Complication of pharyngitis or cutaneous • Novobiocin resistant ○ Circulating immune complexes deposit in glomeruli FAMILY STREPTOCOCCACEAE ○ Inflammatory response causes damage • Pathogenesis and virulence factors • Genus Streptococcus • Enters through the respiratory tract or skin contact • Gram-positive cocci in chains or pairs • Either local disease or spread • Catalase negative ○ Local disease alone or with S. aureus • Small, grayish colonies on sheep blood agar ○ Systemic spread leads to disease and • Categorized by Lancefield groups complications ○ Alpha (a): conversion of hemoglobin to methe- • M protein and lipoteichoic acid for attachment moglobin resulting in a green zone in the blood • Hyaluronic acid capsule: Inhibits phagocytosis agar around a colony • Extracellular products 8 CHAPTER 1 Microbiology

○ Pyrogenic (erythrogenic) toxin, which causes the • Group D laboratory diagnosis rash of scarlet fever • Small, white colonies with hemolysis on blood ○ Streptokinase agar ○ Streptodornase (DNase B) • Enterococcus and group D bile esculin positive ○ Streptolysins • Enterococcus grows in higher NaCl concentration • Laboratory diagnosis and is PYR positive • GPC in chains • a-Hemolytic streptococci • Small, transparent, smooth, b-hemolytic colonies • Streptococcus pneumoniae • Bacitracin susceptible • Viridans group streptococci • L-pyrrolidonyl arylamidase (PYR) positive • Sometimes enterococcal species • Latex or other grouping tests are usually used in • Nutritionally variant streptococci clinical laboratories Streptococcus pneumoniae • Susceptibility testing and treatment • No Lancefield grouping • All isolates are penicillin susceptible • Infects humans exclusively, no reservoir is found in • Susceptibility testing not appropriate in almost all nature settings • Carrier rate of S. pneumoniae in the normal human ○ Exceptions are penicillin-allergic patients nasopharynx is 20% to 40% ○ Manual method usually used • Clinical disease Streptococcus agalactiae • Pneumococcal pneumonia is most common in • Group B Streptococcus elderly, debilitated, or immunosuppressed • Normal flora in female genital tract and gastrointesti- ○ Often after viral infection damages the respira- nal (GI) tract tory ciliated epithelium • Clinical significance ○ Incidence peaks in the winter • Neonatal sepsis and meningitis ○ Community-acquired pneumonia • UTI • Otitis media • Pneumonia in elderly • Meningitis (most common cause in adults) • Pathogenesis and virulence factors • Septicemia • In early-onset neonatal disease, organism is trans- • Laboratory diagnosis mitted vertically from the mother • GPC in pairs, lancet • In late-onset (from 7 days to 3 months age) menin- • Colonies are round and usually wet, glistening, gitis is acquired horizontally, in some instances as a mucoid nosocomial infection • a-Hemolysis • Virulence • Optochin susceptible ○ Capsule • Bile soluble ○ Hemolysin • Susceptibility and treatment ○ Hyaluronidase • Susceptibility testing appropriate for isolates from ○ Proteases normally sterile body sites • Laboratory diagnosis • Most automated methods do not work well • Grayish-white, slightly mucoid colonies • Penicillin resistance an issue • Small zone of b-hemolysis: Larger colony than Other a-Hemolytic Streptococci group A • Viridans streptococci • Hippurate and CAMP test positive • Normal flora • Latex or other grouping tests are usually used in • Upper respiratory tract clinical laboratories • Urogenital tract • Susceptibility testing and treatment • Clinical significance: Subacute bacterial endo- • All isolates are penicillin susceptible carditis • Other agents are not always active • Identification ○ Susceptibility testing not appropriate in almost • a-Hemolytic all settings • Optochin resistance ○ Exceptions are penicillin-allergic patients • Examples: Streptococcus mutans, Streptococcus Group D Streptococcus salivarius, Streptococcus anginosus group, Strepto- • Normal flora coccus gallolyticus (bovis) • GI tract • Susceptibility testing and treatment • Urogenital tract • Some penicillin resistance has been reported • Group D antigen, although not always b-hemolytic • Susceptibility testing not usually appropriate • Streptococcus gallolyticus because the isolates are rarely clinically significant • Streptococcus equinus • If the cause of true disease, susceptibility testing • Clinical significance: Bacteremia and endocarditis may be appropriate CHAPTER 1 Microbiology 9

FAMILY ENTEROCOCCACEAE • Leuconostoc • Micrococcus • Enterococcus Species Pediococcus • General characteristics • 38 species • Uncommonly isolated • Most common isolates from humans • Vitamin B6/pyridoxyl is required for lab growth of • Enterococcus faecalis Abiotrophia and Granulicatella • Enterococcus faecium • May be mistaken for Staphylococcus or • Enterococcus avium Streptococcus spp. • Enterococcus casseliflavus • Limited pathogenic potential but possible in the • Enterococcus gallinarum young, the old or the immunocompromised • General characteristics • May be difficult to identify, rule out Staphylococcus • Gram-positive cocci typically in pairs and short or Streptococcus may be all that is possible chains • Facultative anaerobe AEROBIC GRAM-POSITIVE BACILLI • Catalase negative • Epidemiology • Aerobic gram-positive bacilli represent a tax- • Normal flora of the GI and urogenital tract of onomically and genetically diverse group of organ- humans and animals isms • One of the top three nosocomial pathogens • As human pathogens, notable diseases include liste- • Clinical significance riosis, anthrax, erysipelas, and diphtheria, although • UTI incidence in North America is very low • Wounds • General characteristics • Abdominal and pelvic infections • Similarities • Nosocomial infections ○ Capable of growth in the presence of O2 • Endocarditis and bacteremia ○ Retention of crystal violet after alcohol decolor- • Virulence factors ization step of Gram stain thus appearing purple • Fimbriae: Attachment to epithelial cells in color (gram-positive) • Adhesins: Attachment to intestinal tract ○ Rod shaped • Bacteriocins: Inhibits growth of other intestinal ○ Generally grow easily after 24 hours on non- bacteria selective agars such as SBA • Gelatinase: Hydrolizes collagen and hemoglobin • General characteristics • Laboratory diagnosis • Phenotypic diversity includes • Small, white colonies ○ Cell size • a-Hemolytic or nonhemolytic, may be b-hemolytic ○ Approximately 0.2 to 1.5 mm wide, 0.5 to 10 mm • Both Enterococcus and group D are bile esculin long positive ○ Production of spores • Enterococcus grows in higher NaCl concentration ○ Microscopic appearance may vary and PYR positive ○ Regular “rod” shape • Susceptibility testing and treatment § Rounded, square, or slightly pointed ends • Inherently resistant to cephalosporins ○ Coryneform with club-shaped cells arranged • Less susceptible than streptococci to penicillin and such that they resemble the letters V and L ampicillin ○ Filamentous: Cells form long chains • Vancomycin (vancomycin-resistant enterococcus ○ Filamentous with rudimentary or true branching [VRE]) • Initial grouping of aerobic gram-positive bacilli • Resistant to trimethoprim and sulfamethoxazole • Spore forming • Most isolates should be tested but limited ○ Bacillus spp. reporting • Non–spore forming • Treatment often limited to vancomycin, aminogly- ○ Listeria, Erysipelothrix, Corynebacterium, and cosides, and maybe fluoroquinolones others • Some newer agents may be useful: Linezolid, daptomycin Bacillus Species MISCELLANEOUS GRAM-POSITIVE COCCI • General characteristics • More than 50 species, all are found in soil • Abiotrophia • All Bacillus sp. form endospores • Aerococcus • All are catalase positive • Granulicatella • Spore formation 10 CHAPTER 1 Microbiology

• Endospores are unique to Bacillus spp. among aerobes • Rhinorrhea (runny nose) rare • Transition from vegetative cells to spores under • Incubation period: 1 to 7 days (possibly ranging up harsh and desiccated environments preserves cell to 42 days) viability for long periods • Case fatality • Not always evident in Gram smears, but visualiza- ○ Without antibiotic treatment: 97% tion of spores confirms Bacillus genus ○ With antibiotic treatment: 75% • Special stains are used to better visualize spores • Anthrax: GI • Spores can be “weaponized” for use as infectious • Abdominal distress, usually accompanied by aerosols in biological attack bloody vomiting or diarrhea, followed by fever ○ Highly infectious and signs of septicemia Bacillus cereus • GI illness sometimes seen as oropharyngeal ulcera- • Most common disease is food poisoning tions with cervical adenopathy and fever • Less common opportunistic infection • Develops after ingestion of contaminated, poorly • Epidemiology cooked meat • Common agent in food poisoning • Incubation period: 1 to 7 days • Two forms of food poisoning • Case-fatality: 25% to 60% (role of early antibiotic ○ Diarrheal: Meat, 24 hours, self-limiting treatment is undefined) ○ Emetic: Fried rice, 10 hours, self-limiting • Anthrax: Complications ○ Above symptoms caused by two distinct toxins • 5% develop meningitis • • Opportunistic disease Coma and death occur 1 to 6 days after exposure ○ Serious ocular infection • Recovery confers immunity ○ Wound infection • Vaccines available to high-risk groups • Pathogenesis and virulence • Antibiotics used after exposure • Food poisoning is toxin mediated ○ Ciprofloxacin, tetracycline (60-day treatment) • Laboratory diagnosis • Pathogenesis and virulence factors • Colonies are large, spreading, beta-hemolytic • Cutaneous infection remains localized • Catalase positive • Inhalation and GI cases often proceed to bacterial Bacillus anthracis sepsis with high morbidity and mortality • Most virulent and significant human disease • Laboratory diagnosis • Epidemiology • Colonies are medium to large irregular, gray. • Zoonotic disease in herbivores (e.g., sheep, goats, Medusa head projections, non-hemolytic cattle) follows ingestion of spores in soil • Catalase positive • Human infection typically acquired through con- • If suspected, stop working with the isolate and contact with anthrax-infected animals or animal prod- tact State Health Laboratory ucts (no person-to-person spread) • Less typical is infection through intentional expo- Listeria Species sure (bioterrorism) • Clinical presentation: Anthrax • General characteristics ○ Cutaneous: Direct contact with infected material • Appear on Gram stain as gram-positive short rods ○ Inhalation: Aspiration of spore aerosol (wool or coccobacilli sorters disease) • Grow aerobically ○ Gastrointestinal: Eating of contaminated meat • No spores • Anthrax: Cutaneous • Not acid fast • Form most commonly encountered in naturally • Able to grow at 4 C, unlike many other bacteria occurring cases • Taxonomy and history • Incubation period: 1 to 12 days • Six species; type species is Listeria monocytogenes, ○ Begins as a papule, progresses to a vesicular stage, the most important to human disease then to a depressed black necrotic ulcer (eschar) • Found widespread in nature; habitat is soil and ○ Edema, redness, or necrosis without ulceration decaying vegetation, but carried by numerous may occur humans and animals • Case-fatality • Epidemiology ○ Without antibiotic treatment: 20% • Easy access to food processing and represents major ○ With antibiotic treatment: 1% threat to food chain • Anthrax: Inhalation • Most virulent of the Listeria and common human • Begins as a “viral-like” illness, characterized by pathogen is L. monocytogenes myalgia, fatigue, fever, with or without respiratory • Disease: Listeriosis symptoms, followed by hypoxia ○ Predominantly a food-borne illness (ingestion) CHAPTER 1 Microbiology 11

• Cutaneous: Occupational exposure • Lesions painful, with edema, inflammation, and ○ Meat processors and veterinarians possible local arthritis • High mortality rate • Systemic disease possible in immunocompromised • Clinical disease individuals • Affects high-risk populations • Laboratory diagnosis ○ Elderly, immunocompromised hosts • Specimen of choice ○ Pregnant women and their babies ○ Skin biopsy • Complications of pregnancy • Gram stain of suspect lesion ○ Placentitis and/or amnionitis ○ Pleomorphic gram-positive Bacillus ○ Infection passed to fetus (congenital) • Cultivation § Premature birth, abortion, or stillborn birth ○ SBA with 5% to 10% CO2 § Neonatal meningitis § H2SpositiveonTripleSugarIronAgar(TSI)slant • Meningitis • Sepsis Corynebacterium Species Listeria monocytogenes • Laboratory diagnosis • A diverse group of gram-positive bacilli commonly • Gram smear of normally sterile body fluids with referred to as coryneforms characteristic morphology • Differentiated by chemotaxonomic means such as • Culture recovery from appropriate specimens analysis of cell wall components ○ Blood, cerebrospinal fluid (CSF), amniotic • Exhibits a clublike morphology that reflects its name fluid, placenta, genital tract, stool, respiratory (coryne means “club” in Greek) secretion • General characteristics • Recognition of aerobic gram-positive bacilli • Small gram-positive bacilli ○ b-Hemolytic (clear zone) • May resemble Chinese letters ○ Biochemical profile • Characteristic colonial morphology: dry ○ Motility characteristics: umbrella pattern Corynebacterium diphtheriae ○ Growth at 4 C • Epidemiology Erysipelothrix • Significant human pathogen is C. diphtheriae, • Non–spore forming gram-positive rod which manifests as a respiratory disease or less com- • Related to Listeria spp. monly as a cutaneous infection • E. rhusiopathiae is the species of clinical interest • Acquired by person-to-person contact and spread • Agent of swine erysipelas, widespread disease through close contact with carriers who harbor in pigs organisms • Cause of erysipeloid, a cutaneous infection in • Although a global pathogen, is rare in the United humans occasionally acquired by contact with States because of childhood vaccination programs infected animals • Clinical disease • General characteristics • Normally encountered as a respiratory ailment • Small gram-positive bacilli (0.2-0.5 mm wide 0.8- • Common symptoms include sore throat with low- 2.5 mm long) grade fever • Occur singly, in short chains or filaments • Adherent membrane of the tonsils, pharynx, or nose • Aerobic or facultative anaerobic O2 requirements is a hallmark of disease • Nonmotile • Swelling of neck is usually present in severe disease • Catalase negative • Cutaneous diphtheria can occur and manifests as • Wide temperature range growth on complex nutri- infected skin lesions but without a characteristic ent media such as SBA appearance • Epidemiology and clinical disease • Complications • Widespread in nature ○ Myocarditis • Colonizes variety of animals, fish, and birds, but ○ Kidney and liver inflammation particularly pigs ○ Peripheral neuropathy • Resists cold and alkaline environment ○ Airway obstruction • In animals causes erysipelas, ranging from cutane- ○ Death (occurs in 5% to 10% of respiratory ous to systemic disease cases) • In humans, erysipeloid is a cutaneous infection con- • Pathogenesis and virulence factors sisting of localized cellulitis after acquisition • Invasion through skin abrasion, injury, or bite ○ Bacteria colonize and proliferate in local tissues ○ Occupational hazard among animal handlers, of the throat, creating pseudomembrane farming personnel, veterinarians • Toxigenesis 12 CHAPTER 1 Microbiology

○ Bacteria produce an exotoxin that causes the FAMILY MYCOBACTERIACEAE death of eukaryotic cells and tissues by inhibition of cell protein synthesis • General characteristics ○ Causes heart and nerve damage and is responsi- • Slim, rod-shaped organisms that are 1 to ble for the lethal symptoms of the disease 10 mm long • Vaccination consists of a toxoid, which maintains the • Nonmotile and non–spore forming, obligate antigenicity of the toxin without the toxicity and aerobes prompts production of toxin-neutralizing antibody • Slow growing (3 to 40 days in culture) • Vaccination programs solely are responsible for the • Contain mycolic acids, complex, long-chain control of diphtheria in a population fatty acids • Laboratory diagnosis • Possess a cell envelope with a high lipid content • Specimens • Although mycobacteria are discussed in the section ○ Throat and nasopharyngeal on gram-positive bacilli, it is important to note that ○ Material under pseudomembrane useful with the exception of species classified as “rapid • Media growers,” mycobacteria do not stain with Gram ○ General media: 5% SBA reagents ○ Selective medium: Cystine-tellurite medium • All mycobacteria will stain acid fast with Ziehl- (CTBA), which selects against normal throat flora Neelsen or auromine O stains and are commonly § C. diphtheria colonies are black to gray referred to as an acid-fast bacillus (AFB) ○ Differential medium: Tinsdale, which allows • Acid-fast staining (Ziehl-Neelsen) recognition of suspected C. diphtheriae • Step 1 exposes the smear to carbol fuchsin, a § C. diphtheria colonies are black with red dye brown halos • Step 2 involves decolorization with hydrochloric • Gram stain of sample acid ○ Gram-positive “Chinese letters” appearance • Step 3 counterstains with methylene blue • Biochemicals • An acid-fast organism resists decolorization and ○ Urease negative remains red, whereas non–acid fast organisms ○ Nitrate positive stain blue ○ Catalase positive • Cell wall structure • Toxin testing (tests for toxin phage) • Mycolic acids are components of a variety of ○ In vivo: Culture and antitoxin in rabbit lipids found only in mycobacteria, Nocardia, and ○ In vitro: Immunodiffusion corynebacteria Corynebacteria Other Than Corynebacterium • The chain length of these mycolic acids is longest in diphtheriae Mycobacterium, intermediate in Nocardia, and • Corynebacteria are widespread as normal flora on the shortest in Corynebacterium spp. skin and mucosal membranes of humans and animals • This explains why mycobacteria are generally acid • Such infections range from local cutaneous to deep- fast, nocardia less acid fast, and corynebacteria are seated systemic infections non–acid fast • Corynebacterium jeikeium • Conversely, Mycobacteria will not stain with the • Important in nosocomial or immunocompromised Gram procedure and Nocardia and Corynebacte- • Line-related infections rium will stain as gram-positive • Antimicrobial resistance • Epidemiology and clinical disease Other Corynebacteria • The pulmonary disease tuberculosis, caused by • Because of the common presence of Coryneba- Mycobacterium tuberculosis complex, is an enor- cterium on human surfaces, significance should mous global problem be attached to their recovery only in the following ○ Estimated 1.7 billion people, one third of the cases world’s population, are infected • Isolated from otherwise sterile body sites • 8.4 million new cases per year and 2 to 3 million • If clearly predominant in mixed flora culture results deaths grown from well-collected sample • Infection risk proportional to intensity of exposure • If recovered in high count (>104 cfu/mL) from urine • Infection does not usually lead to disease as single organism • M. tuberculosis is exclusively a human pathogen • Significance is enhanced in the following cases • Mycobacterium bovis is a closely related animal ○ Multiple specimens positive for same pathogen that causes a disease in people that is organism indistinguishable from tuberculosis (TB). It is ○ Coryneform bacteria seen on direct Gram stain acquired from the ingestion of infected meat with strong leukocytic reaction or milk CHAPTER 1 Microbiology 13

Mycobacterium Species (Box 1-1) • Cell-mediated immunity develops 2 to 6 weeks after active infection (result of T cell, not B cell, Mycobacterium tuberculosis proliferation) • Natural history of M. tuberculosis infection • At that point, delayed-type hypersensitivity • M. tuberculosis is acquired through the inhalation response is demonstrable through a skin test; the of droplet nuclei antigen reagent is termed purified protein deriva- • The primary spread of the organism is via aerosol tive, so the test is known as the PPD test droplets from coughing • Patients who cannot raise a sufficient delayed-type • Bacteria not initially killed multiply in the phago- hypersensitivity response will develop miliary some of a macrophage, destroying the macrophage (disseminated) TB • Released organisms are ingested by other • Epidemiology and clinical disease macrophages • Increased risk for clinical disease after primary • Cytokines and chemokines produced attract other infection is found in young children, Native Amer- phagocytic cells, including monocytes, other alveo- icans, Native Africans, patients infected with the lar macrophages, and neutrophils human immune deficiency virus (HIV), intravenous • These cells eventually form a nodular structure drug abusers termed the tubercle or granuloma ○ Infants and the very young have a high mortality • The immunologic response to this infection is cellu- rate from primary infections lar (rather than humoral), and a delayed type hyper- • Highest risk for incidence and reactivation sensitivity reaction develops against tuberculin ○ Former for current prison inmates protein that manifests a positive skin test reaction ○ The homeless, the elderly • Dissemination of the organism to the lymph nodes ○ Foreign-born persons from TB-endemic areas and bloodstream occurs with deposition in liver, • Prophylaxis spleen, kidney, bone, brain, meninges, and other • Bacillus Calmette Gue´rin (BCG) vaccine is 70% parts of the lung with further granuloma formation. effective in preventing infection in a specific This is termed disseminated or miliary TB and on population radiography shows multiple “millet seed”–like • BCG is not used in countries with a low incidence of lesions TB because the ability to detect infection with M. • Natural history of TB tuberculosis with the PPD is lost • Reactivation of TB results when persisting bacteria • After a positive tuberculin test, isoniazid (INH) and in a host suddenly proliferate, because the granu- other drugs are taken for 6 to 9 months or rifampin loma formation does not eradicate the organisms for 4 to 9 months to prevent disease • 15% to 20% of primary disease reactivates at a • Drug-resistant strains later point, leading to caseous granuloma and cavity • Increased resistance to INH and other anti- formation, during which the disease is highly tuberculosis drugs noted in recent years; in contagious M. tuberculosis resistance is due to mutation, • Greatest risk for reactivation not plasmid transfer ○ Within 5 years of primary infection • Multiple drug–resistant strains pose severe prob- ○ Those at the extremes of age lems and are seen in populations with HIV ○ Pregnant women, immunosuppressed patients infection ○ Malnourished population, alcoholics, and • Most nontuberculous Mycobacterium spp. are patients with diabetes generally more resistant to drugs than M. • Most common presentation of TB is as a chronic pul- tuberculosis monary disease • Laboratory diagnosis • Chronic cough • Dual motivation in laboratory detection of M. • Chest pain tuberculosis • Shortness of breath ○ Diagnose and treat patient • Low-grade fever ○ Infection control • Fatigue § Stop contagion • Night sweats § Protect others • Loss of appetite Nontuberculous Mycobacteria • Weight loss Atypical Mycobacteria and Mycobacteria Other • Sputum production Than Tuberculosis (MOTT) • Immunity to TB • Environmentally acquired, not transmitted person to • Infection is contained in 80% to 85% of people person by respiratory means who recover without ever having symptoms of • Generally infects those with weakened immune sys- disease tems because of age, disease, or other factors 14 CHAPTER 1 Microbiology

BOX 1-1 Isolation and Identification of Mycobacterium

• Mycobacterium characteristics • AFB liquid detection systems • Slim rod-shaped organisms that are 1-10 mm long. • Specimen Processing • Nonmotile and nonspore forming, obligate aerobes • AFB Staining Techniques • Slow growing (3 to 40 days in culture) • Carbol fuchsin based • All Mycobacteria will stain “Acid-fast” with Ziehl-Neelson or • Heat fix suspension to slides auromine/rhodamine stains and are commonly referred to as Acid • 15 min @ 80 C or 2 hr @ 65 C Fast Bacilli or AFB • Ziehl-Nielson “requires HEAT step during staining” (tedious) • With the exception of the rapidly growing AFB, mycobacteria will • Kinyoun “COLD” modification of ZN preferred method not stain with the Gram’s procedure • Read on oil immersion (1000) • Lab safety is a major concern due to the potential of infectious • Auramine Rhodamine fluorescent stain aerosols • Organisms appear yellowish green against a black back- • Containment is crucial ground • Biosafety level 2 hoods • Can scan slide at low power (250) and confirm at 400 • Gowns, gloves, and masks • More sensitive for detection and faster scan of slide than ZN • Sealed centrifuge cups • Requires microscope with fluorescent optics • Negative pressure room and BSL 3 containment desirable • Incubation

• Mycobacterium Sample Preparation • Most species require 35º C-37º C, 5%-10% CO2 • Common samples: • Dark, high humidity, loose caps • NF likely to be present in sputum, bronchial wash or lavage, • Optimally at least 2 media skin lesions, urine, stool • Examine weekly for growth • Normally sterile body sites do not contain normal flora—pleural • Hold cultures for 6-8 weeks fluid, blood, spinal fluid, deep tissue biopsies • Lower incubation temperature if infection cutaneous due to • The complex lipid AFB cell wall allows procedures that decontam- possible mycobacteria other than TB (MOTT) inate normal bacterial flora but allow AFB to remain viable • Identification of Mycobacteria • Decontamination • Growth Rate—time for visible colonies to appear: • Bacterial flora eliminated or reduced • Rapid7 days • Mycobacteria release from mucus • Slow>7 days • Concentration aids in detection of low numbers • AFB Colony morphology • AFB are largely protected from decontamination • M. tuberculosis • Use of mildest procedure that controls decontamination rate • The color is typically buff, regardless of light advocated • Texture of the colony is rough • Expect contamination rate of 3%-5% • M. avium complex • No contamination means procedure is too harsh • Color is tan to buff regardless of light • AFB smear and culture follows decontamination • Texture of the colony is smooth Specimen Processing for AFB • M. kansasii • Culture Media—Slanted media in tubes preferred for safety reasons. • Color is yellow or reddish yellow • Texture of the colony is smooth • Egg based (whole or yolks)—Lowenstein-Jensen (LJ) • Preferred basic nonselective media • AFB Colony morphology • Gruft modification—Penicillin/Nalidixic acid—inhibitory to • Pigment Production among MOTT contaminants • Scotochromogens: grow with a deep yellow pigment regard- • Malachite green—inhibitory to routine bacteria less of light • • Agar based, conventional petri dish M. scofulaceum, M. gordonae • Middlebrook 7H10, 7H11 most popular • Photochromogens: develop yellow pigment only after expo- • Salts, vitamins, cofactors, tween/glycerol sure to light • “Thin” plates for early detection of colonies—10-12 days, • M. kanasii, M. marinum microscopically • Nonchromogens: no pigment beyond tan to buff colonies • Less commonly used than LJ slants for reasons of containment regardless of light • • Liquid media M. avium-intracellulare • 7H9 media most common • Note: M. tb complex produces buff colonies • Reduces turnaround time to average of 10 days • Pigment production among MOTT • Used in BACTEC system (14C labeled palmitic acid-detection of • Key Biochemical Tests 14 • Niacin test free CO2), and other continuous monitoring automated systems • M. tuberculosis accumulates considerable niacin in media. + • 0.5 mL PANTA (polymixin B, amphotericin B, nalidixic acid, Test early sign that isolate may be tuberculosis trimeth/sulfa, azlocillin)—added to processed specimens to •Niacinextractedandtestedwithstripforcolor prevent specimens development CHAPTER 1 Microbiology 15

BOX 1-1 Isolation and Identification of Mycobacterium—cont’d

• Semi Quantitative Catalase • Additional identification methods for mycobacteria • M. kansasii positive • GLC or HPLC • M. scrofulaceum positive • Analysis of long-chain fatty acids • M. tb and avium negative • Most health departments and CDC prefer method • Hydrogen peroxide added • Genetic probes • Positive test¼bubbles rising above 45 mm from baseline • DNA probes specific for hybridization for rRNA sequences— • Arylsulfatase use chemiluminometer • Rapid growers are only mycobacterium positive in 3 day test • Only single colony of organism needed • Helps to differentiate from similar looking Nocardia species • Rapid and specific detection of organism • Nitrate reduction • Available for M. tuberculosis, avium, intacellulare, kansasii • M. tb and M. kansasii are positive, M. avium is negative and gordonae • Red color on strip or reagent test is positive

Species Niacin Semi Quant Catalase Nitrate reduct. Aryl sulfatase Tween hydrol M. tuberculosis +- + - - M. kansasii -+ + - + M.scrofulaceum -+ - - - M. avium complex -- - - - M. gordonae -+ - V + M. fortuitum -+ + + V

• Can cause TB-like, cutaneous, or leprosy-like diseases General Collection Guidelines • Are not susceptible to certain common antituberculo- and Diagnosis sis antibiotics • • M. avium complex (MAC) is the most commonly Sterile body site areas can be set up directly onto cul- associated HIV-related systemic bacterial infection ture media • and manifests as a pulmonary pathogen, much CSF, pleural fluid, deep tissue biopsies • like TB Nonsterile sites require special processing: Deconta- • M. avium infection not associated with acquired mination and digestion • immunodeficiency syndrome is quite rare Sputum, bronchial washings, bronchoalveolar • Treatment of M. avium involves a long-term regimen lavage, skin biopsy • of multiple drug combinations, because this organism Likely to be overgrown with routine bacteria • does not always respond to the drug regimens used to AFB resists decontamination and digestion because treat M. tuberculosis of cell wall lipid content, whereas routine bacteria • Mycobacterium kansasii: Pulmonary disease in com- are destroyed • promised hosts (individuals infected with HIV) AFB: Safety precautions • • Mycobacterium marinum: Cutaneous disease from Avoid direct contact with organism • contact with contaminated water Aerosols present greatest hazard • • Mycobacterium scrofulaceum: Cervical adenitis in Class 2 biosafety hood ○ children (contaminated raw milk, soil, daily Maintenance: Airflow checks ○ products) High-efficiency particulate absorption (HEPA) • Mycobacterium fortuitum, Mycobacterium chelonae, filters: Sterile air current • Mycobacterium abscessus: Primarily skin and soft tis- Sealed centrifuge buckets • sue disease in various hosts Gown, gloves, masks, foot covers • • Mycobacterium leprae: Agent of leprosy Keep doors closed when specimens are open—slight Other Nontuberculous Mycobacteria negative air pressure created by hood • • Mycobacterium gordonae Laboratory diagnosis of TB • • Mycobacterium spp. commonly found in tap water AFB visible by Ziehl-Neelsen or fluorochrome • Generally nonpathogenic smears of sputum or appropriate sample • • Can confuse the reading of AFB smears if care not Recovery by culture (the gold standard) either on taken in sample preparation conventional AFB media or that which uses radio- metric detection (BACTEC) 16 CHAPTER 1 Microbiology

• Prolonged incubation time required (up to 8 weeks) • Spread person to person through inhalation or con- • Biochemical characterization tact with infected skin • Nucleic acid assays (NAA) ○ Silent phase: Multiplication of bacilli ○ Represent rapid diagnosis of TB ○ Intermediate phase: Peripheral nerves, sensory ○ Sensitivity of the NAA is approximately 95% in impairment patients with a positive AFB smear, but only 50% • Organism can be grown in the footpads of mice and in smear negative cases (U.S. Food and Drug nine-banded armadillos, not on artificial media Administration data) § Diagnosis usually made by clinical findings • Nontuberculous mycobacteria (atypicals, MOTT) and observation of AFB on direct smear of • Classified into Runyon groups based on lesions ○ Presence or absence of pigmentation ○ Pigment production: Light dependent or not FAMILY ENTEROBACTERIACEAE ○ Growth rate: Slow or fast (BOXES 1-2 AND 1-3) • Chromatography techniques allow species-level identification of mycobacteria based on their cellu- • Largest, most heterogeneous group of clinically impor- lar fatty acid and/or mycolic acid profiles (reference tant bacteria laboratories) • General characteristics • Specific DNA molecular probes are available for • Most are normal flora of the GI tract species identification • Gram-negative bacilli • BCG used outside the United States as an attenuated • Facultative anaerobes vaccine • Colony morphology is similar for most • Nontuberculous mycobacteria (MOTT) ○ Large, gray, spreading colonies • All other species not in the M. tuberculosis complex ○ Only Klebsiella and Enterobacter are mucoid • Present everywhere, generally environmental • All Enterobacteriaceae • Usually not transmitted person to person ○ Ferment glucose • Pathogenicity varies and often depends on host ○ Reduce nitrates to nitrites (rare exceptions) immune status (opportunistic infection) ○ Oxidase negative • Runyon: Four groups based on growth rate, ○ Most are motile by peritrichous flagella pigment production, and reaction of pigment • Serologic classification to light • Cell-associated antigens • M. avium complex (MAC) ○ O: Somatic antigens (heat stable) • Complex includes M. avium and Mycoplasma § Polysaccharide of the LPS intracellulare § Associated with endotoxin release • Most commonly isolated AFB among the MOTT ○ K: Capsular antigens (heat labile) • Ubiquitous in nature § Capsular polysaccharide • Acquired by inhalation or ingestion § Strains with K are more pathogenic • Patients with HIV are particularly at risk ○ H: Flagellar antigens (heat labile) • Nonphotochromogens: No pigment § Flagellar protein antigens • Smooth, cream-colored colonies § Responsible for motility • Greater drug resistance than M. tuberculosis • Clinical disease • Rapid growers • Based on the clinical infections produced • M. fortuitum-chelonae complex ○ Opportunistic • Grow in 7 days or less on solid media § Normal flora • Can grow in routine media and stain as gram- § Cause infections outside of natural habitat positive cells with diphtheroid-like morphology ○ Primary intestinal • Acquired from environmental sources or nosocomi- § Salmonella ally during surgery from contaminated objects or § Shigella fluids § Plesiomonas • Enter by inoculation into the skin § Yersinia enterocolitica • Can also cause chronic pulmonary infections • Opportunistic genera • M. leprae ○ Citrobacter • Cause of leprosy, which is also termed Hansen’s ○ Edwardsiella disease ○ Enterobacter • Chronic disease of the skin, mucous membranes, ○ Escherichia tissue ○ Hafnia • Rare in the United States, but cases exist in Texas ○ Klebsiella and Louisiana ○ Morganella CHAPTER 1 Microbiology 17

BOX 1-2 Tests Used in the Identification of Enteric Gram-Negative Bacilli

Routine Enterobacteriaceae Light orange to red positive Memebers of the family Enterobacteriaceae are usually divided into Yellow/no change—negative lactose fermenters (+) and non-lactose fermenters (-) Positive: Proteus spp. Lactose + Negative: E. coli E. coli—dark Indole Klebsiella—mucoid Ability to metabolize tryptophan by testing for indole Enterobacter—mucoid Indole+aldehyde yields a red color Citrobacter—late Indole—spot test Serratia—late, red pigment Saturate filter paper with reagent Lactose – Rub portion of colony onto paper Proteus—swarming Rapid development of color is positive test Morganella Positive: E. coli Providencia Negative: E. cloacae Edwardsiella Methyl Red and Voges-Proskauer (MR/VP) Hafnia Glucose metabolism and metabolic products Lactose is degraded into glucose and galactose Important Biochemical Tests Glucose used through Embden–Meyerhof–Parnas (EMP pathway) o-Nitrophenyl-p-D-galactopyranoside (ONPG) to produce pyruvic acid Some lactose fermenters lack permease and so are “slow” or Pyruvic acid use produces many mixed acids “late” Enterics take two separate pathways Non-lactose fermenters lack both Mixed acid pathway and butylene glycol pathway ONPG tests for b-galactosidase Two tests for end-products of these pathways The substrate is complexed to galactose Methyl Red test and Voges-Proskauer Cleavage causes a color change Escherichia coli is MR+and VP Positve: E. coli Enterobacter aerogenes and Klebsiella pneumoniae are MR and Negative: Salmonella enteritica VP+ Decarboxylase tests Pseudomonas aeruginosa is MR and VP Detects decarboxylation of specific amino acids Gelatinase Alkaline end products result Used to determine the production of proteolytic enzyme that Ornithine decarboxylase (ODC) digests gelatin Ornithine ! Putrescine Positive: Proteus vulgaris Lysine decarboxylase (LDC) Negative: Enterobacter aerogenes Lysine ! Cadaverine Carbohydrate fermentation Arginine dihydrolase (ADH) Various media used to determine the ability of bacteria to ferment Arginine ! Citrulline specific carbohydrates Decarboxylase tests The fermentation pattern can then be used as part of an identifi- Medium starts purple cation scheme Fermentation shifts medium acid—indictor to yellow Motility Test Decarboxylation shifts the pH alkaline—indicator to purple Single stab of the organism into the gelatin tube Mineral oil “traps” alkaline end products Incubate at 37 C for up to 7 days Control tube lacks amino acid—yellow Movement away from initial stab line is positive motility Simmons Citrate Positive: E. coli Ability of organism to use sodium citrate for metabolism and Negative: K. pneumoniae growth Identification of Enterobacteriaceae Indicator—bromophenol blue Identification is a process or flow Only streak slant 1. Growth on media Light inoculum 2. Gram stain Blue—positive (rise in pH) 3. Lactose fermentation Green—negative (no change) 4. Oxidase Positive: Klebsiella pneumoniae and Proteus mirabilis 5. Basic biochemical tests Negative: Escherichia coli and Shigella dysenteriae Identification of Enterobacteriaceae Urease Production API 20E

Ability of bacteria to hydrolyze urea to ammonia and CO2 The API-20E test kit is designed for the identification of enteric Ammonia release causes pH change bacteria Positive—bright pink Plastic strip holding twenty mini-test tubes is inoculated with saline Urease—method suspension of a pure culture Streak slant Rehydrates the dessicated medium in each tube Incubate 37 C Incubated 18-24 hours at 37 C

Continued 18 CHAPTER 1 Microbiology

BOX 1-2 Tests Used in the Identification of Enteric Gram-Negative Bacilli—cont’d

The reactions are converted to a seven-digit code No fermentation The code is fed into the manufacturer’s database Alkaline slant/Alkaline butt (K/K) or (K/NC) Identification usually as genus and species Non-enterics—able to degrade peptones Identification of gastrointestinal pathogens Only glucose fermentation Screen stool cultures Alkaline slant/Acid butt (K/A)—red/yellow Normal flora in lower bowel¼107/mL Too much acid in butt to revert to alkaline Anaerobes, diptheroids, enterococcus, streptococcus, enterics, yeasts Lactose (sucrose) fermentation Anaerobes/aerobes at 1000/1 Acid slant/Acid butt (A/A)—all yellow Determine which colonies deserve further attention H2S production—Black Initial Screening Steps Gas production—bubbles or split media Detectsuspiciouscoloniesamongstnumerousnormalfloraorganisms Lysine Iron Agar (LIA) Lactose fermenter vs. non-lactose fermenter Lysine, glucose, ferric ammonium citrate, sodium thiosulfate Growth on selective media Primarily used to detect lysine use

H2S producing colonies Good tool when used with TSI to screen stools for pathogens Non-sorbitol fermenters—E. coli O157 Suspected non-Enterobacteriaceae Beta hemolytic enteric organisms Vibrio Oxidase positive—suspected non-Enterobacteriaceae Aeromonas Suspected Enterobacteriaceae Campylobacter Conventional identification Tests for other enteric pathogens Automated identification Growth on specialized medium Screening tubes Oxidase and catalase TSI and LIA Gram stain Triple Sugar Iron (TSI) or KIA Suspected Campylobacter Screening ID of enteric pathogens Growth on specialized media at 42 C

Identical except TSI sucrose, KIA does not Atmosphere 5% O2, 10% CO2, 85% N2 Detects ability to produce gas and acid from fermentation and H2S Oxidase and catalase positive Both are used as slants: Curved gram-negative bacilli Slant—aerobic Sea gull shape Butt—anaerobic Stains very lightly 0.1% glucose, 1% sucrose, 1% lactose, phenol red, ferrous sulfate Hippurate hydrolysis positive Detects fermentation: Suspected Vibrio and Aeromonas If glucose is the only one fermented, the small amount of acid that Beta hemolytic on Blood agar is produced on the slant will be oxidized to a neutral product Comma-shaped gram-negative bacilli Slant remains alkaline (red) Non-lactose fermenter on MacConkey agar Butt—acid is not oxidized and turns yellow Thiosulfate citrate bile sucrose agar (TCBS) If lactose and sucrose are also fermented, there is so much acid that Yellow green colonies both the slant and butt become yellow Oxidase and catalase positive TSI—5 Reactions API or commercial systems usually identify

From Tille PM: Bailey & Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.

○ Proteus • Biochemical tests: Many ○ Providencia • Lactose fermentation and utilization of carbohy- ○ Serratia drates are key biochemical tests • Clinical disease ○ All ferment glucose, so lactose is used for initial • Most cause opportunistic and nosocomial infections differentiation ○ UTI • Rarely, laboratories use traditional tube tests ○ Pneumonia • More often use spot tests or limited workup on ○ Wound infections some specimen types ○ Catheter colonization • Commercially available in kits • Isolated from almost all body sites ○ API test strips • Laboratory diagnosis ○ Most large laboratories use automated identifi- • Oxidase negative cation systems • Colony morphology and Gram stain § Vitek (bioMerieux) • Culture: Use supportive and selective media to § MicroScan (Dade) recover pathogens § Phoenix (BD Bioscience) ○ MacConkey agar • Enteric pathogens ○ XLD • Food-borne ○ Hektoen ○ Salmonella CHAPTER 1 Microbiology 19

BOX 1-3 Biochemical Differentiation of Representative Enterobacteriacae

From Tille PM: Bailey & Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.

○ E. coli • Epidemiology ○ Campylobacter • Naturally occur in poultry products and reptiles ○ Yersinia • Food-borne infections account for 1.3 billion cases • Human to human of acute diarrhea with 3 million deaths worldwide ○ Shigella • Ingestion of contaminated food, water, or milk ○ Salmonella typhi • 40,000 cases annually in the United States ○ Helicobacter pylori • Centers for Disease Control and Prevention (CDC) • Water-borne and reports: In recent years a notable increase in ○ Vibrio cases related to a multidrug-resistant Salmonella ○ Aeromonas typhimurium ○ Plesiomonas ○ Case-fatality and hospitalization rates for this • Enterobacteriaceae pathogens strain are twice that of other Salmonella spp. • Salmonella • Clinical disease: Gastroenteritis • Shigella • Most common type of illness • E. coli • Diarrhea, low fever, nausea • Yersinia • Symptoms last 1 to 3 days • Plesiomonas • Positive stool cultures • Serotyping • No systemic involvement • Antigens: Heat stable • Clinical disease: Bacteremia/septicemia ○ A, B, C, etc. • Nontyphoidal bacteremia ○ 98% of human isolates are A through G • Salmonella choleraesuis • H antigens: Heat sensitive • High spiking fever ○ 1, 2, 3, etc. • Positive blood cultures • Vi (K) antigens: Virulence antigens • Few GI symptoms ○ Heat sensitive • Particularly invasive ○ May mask O antigens • Clinical disease: Enteric fever ○ Boil to remove Vi, retype • S. typhi: Typhoid fever 20 CHAPTER 1 Microbiology

○ Most serious ○ Dysentery characterized by a small volume of ○ Fever and GI involvement bloody, mucoid stools, and abdominal pain ○ Positive blood cultures during first week • Pathogenesis ○ Positive stool cultures during second week • Resist gastric acidity • Carrier state • Requires very few organisms to infect ○ Carry bacteria asymptomatically after infection ○ 10 to 200 organisms ○ Can shed organism unknowingly ○ Organism resistant to acid ○ Typhoid Mary • Cytotoxin (Shiga toxin) causes inflammation and • Pathogenesis ulcerative lesions • Ingested in food ○ Destroys epithelial cell • Survive passage through the gastric acid ○ Bloody, mucus-laden stools • Invade the mucosa of the small and large intestine • Laboratory diagnosis ○ Produce toxins • Lactose negative • Invasion of epithelial cells stimulates release of • TSI: Alkaline/acid, no gas, no H2S cytokines, which induce an inflammatory reaction • Urease negative • Inflammatory response causes diarrhea • Confirm with serotyping ○ May lead to ulceration and destruction of the • Report to health department mucosa • May disseminate from intestines to systemic disease • Laboratory diagnosis Escherichia coli • Lactose negative • May cause several types of diarrheal illnesses • H2S positive: Black colonies • Enterohemorrhagic E. coli is most important • Triple Sugar Iron Agar (TSI): Alkaline/acid, H2S, gas • E. coli O157:H7 ○ Butt is acid as a result of glucose fermentation • Hemorrhagic colitis: Pediatric • Methyl red, citrate, lysine decarboxylase, ornithine • Can lead to hemolytic uremic syndrome: Build-up decarboxylase, arginine dihydrolase positive of toxin in kidneys • Must confirm with serotyping • Bloody diarrhea with no PMNs • Reportable organism • Fever absent • Species Water-borne or food-borne: Often transmitted via Shigella ground beef • Serotyping (O antigen) • Laboratory diagnosis • A: Shigella dysenteriae (12 serotypes) • All E. coli ○ • B: Shigella flexneri (6 serotypes) Ferment glucose, lactose, and xylose ○ • C: Shigella boydii (23 serotypes) Indole and Methyl Red positive ○ • D: Shigella sonnei (1 serotype) No H2S or urease ○ • Groups A to C are physiologically similar; S. sonnei Citrate negative ○ can be differentiated biochemically Motile or nonmotile strains • Epidemiology • E. coli O157:H7 ○ • Human to human (fecal-oral route) MacConkey with sorbitol (SMAC) § ○ Very communicable Does not ferment sorbitol (i.e., clear colonies) ○ ○ Food—inoculated by humans Typical E. coli reactions § • S. sonnei: Most common in the United States Indole § • S. dysenteriae: Least recovered in the United Confirm identification with routine system ○ States Confirm with serotyping: O or H ○ ○ Most severe Reportable organism ○ Third World countries • Clinical disease Yersinia Species • Acute infection with onset of symptoms within 24 to 48 hours of ingestion • Clinically significant species • Average duration of symptoms in untreated adults ○ Yersinia pestis: Plague is 7 days ○ Yersinia enterocolitica ○ Organism may be cultivated from stools for • Epidemiology: Y. enterocolitica 30 days or longer ○ Food-borne and water-borne illness • Two basic clinical presentations ○ Blood transfusions ○ Watery diarrhea associated with vomiting and • Y. enterocolitica mild-to-moderate dehydration • Clinical disease CHAPTER 1 Microbiology 21

○ Gastroenteritis: May resemble appendicitis • Variable from 35 to 42 C ○ Seasonal and ethnic, chitterlings (small intestines • Generally aerobic of pig or other animals) Campylobacter jejuni • Laboratory diagnosis • Epidemiology • Grows better at 25 to 30 C • C. jejuni most common • Small, lactose-negative colonies on MacConkey • Food-borne gastroenteritis agar ○ Poultry and raw milk • Cefsulodin-irgasan-novobiocin (CIN) agar may be ○ Water used: produce pink colonies with red center • One of most common causes of human bacterial • TSI: Acid/acid, no gas gastroenteritis in numerous parts of the United • Routine identification system States • Clinical disease • Plesiomonas shigelloides Diarrhea, cramps, abdominal pain, fever within 2 to 5 days of exposure • Epidemiology • Bloody stool with high WBC count • Water-borne gastroenteritis: Freshwater • Lasts for 7 to 10 days • Mostly in tropics • Can be fatal • Clinical disease • Pathogenesis and virulence ○ Usually mild watery diarrhea • Susceptibility of host and strain virulence key ○ Human trial unsuccessful in setting up disease • Ingestion of contaminated food or water • Laboratory diagnosis • Penetrate the GI tract mucous lining • Routine culture conditions ○ Motility and shape • Gram-negative bacilli • Adhere to the gut enterocytes and release toxins • Growth on blood agar: No hemolysis ○ Enterotoxin and cytotoxins • Non–lactose fermenter on MacConkey agar • Laboratory diagnosis • Positive oxidase and indole most important initial • Special atmosphere and temperature screening tests ○ 42 C § Routine identification system ○ 5% O2, 10% CO2, 85% N2 • Campy blood agar ○ Brucella agar base with antibiotics MISCELLANEOUS ENTERIC PATHOGENS ○ Other selective media: Cefoperazone Vancomy- cin Amphotericin (CVA), Skirrow Medium • Campylobacter • Curved gram-negative bacilli • Vibrio ○ Sea gull shape • Aeromonas • Stains very lightly • Helicobacter pylori • Nonhemolytic, flat, gray, mucoid • Oxidase and catalase positive Family Campylobacteraceae • Darting motility on wet preparation • Hippurate hydrolysis positive • Campylobacter • Usually identified as Campylobacter spp. • At least 18 species and subspecies in Campylobacter • Arcobacter • Four species in Arcobacter FAMILY VIBRIONACEAE Campylobacter Species • Curved or S-shaped • 10 genera, including Vibrio • 0.5 to 5.0 mm long 0.5 to 1.0 mmwide • Gram-negative, non–spore forming rods Vibrio • Motile • Single, polar flagellum • 76 species currently recognized • Generally microaerophilic • Photobacterium damselae previously classified as Arcobacter Species Vibrio and shares ecology • Curved or S shaped • Oxidase-positive, facultative anaerobic, non–spore • 0.2 to 0.9 mm wide, 1 to 3 mm long forming, gram-negative bacilli • Gram-negative, non–spore forming bacilli • Comma-shaped cells • Motile • Typically found in saltwater • Single, polar flagellum • All members of genus are motile by single, polar • Grow at 15 to 30 C flagellum 22 CHAPTER 1 Microbiology

Vibrio cholerae • Pathogenesis and virulence factors • Epidemiology • Produces aerolysin • Water-borne illness ○ Cytotoxic enterotoxin • Found in plankton of fresh, brackish, and salt ○ Causes tissue damage in fish and amphibians water • Unclear pathogenesis in humans • Attached primarily to copepods in the zooplankton • Laboratory diagnosis • Coastal outbreaks usually follow zooplankton • Routine culture conditions blooms ○ Grows on MacConkey and blood agar • Pathogenesis and virulence ○ Usually b-hemolytic • Colonizes the GI tract • Gram-negative bacilli (straight) • Attaches to villi by pili • Lactose fermenter • Secretes a two-part toxin, cholera toxin • Positive oxidase and indole good screening tests • Toxin causes increased cyclic adenosine monophos- • API or routine identification system phate (cAMP) synthesis • Massive fluid efflux: Diarrhea • Clinical disease FAMILY HELICOBACTERACEAE • Cholera • Helicobacter, Sulfuricurvum, Sulfurimonas, Sulfuro- ○ Massive fluid loss: Death within 24 hours vum, Thiovulum, Wolinella ○ “Rice water stools”: WBCs and blood absent • Treatment ○ Replace fluids, antibiotics Helicobacter • Laboratory diagnosis • 29 species of Helicobacter recognized • Comma-shaped gram-negative bacilli • Exact mode of transmission is unknown • Non–lactose fermenter on MacConkey agar • Higher rate in undeveloped countries • Thiosulfate citrate bile sucrose agar (TCBS) • Clinical disease ○ Yellow-green colonies • One of the most transmitted human infections • Oxidase and catalase positive ○ Gastroenteritis • String test positive ○ Peptic ulcers Halophilic Vibrio Organisms ○ Associated with gastric cancer • Require 1% to 2% NaCl for growth • Pathogenesis and virulence factors • Cause gastroenteritis, wound infections, septicemia • Burrows into gastric mucosa • Vibrio parahaemolyticus: Gastroenteritis from seafood • Urease • Vibrio vulnificus: Septicemia from raw shellfish (lac- ○ Converts urea to ammonia and bicarbonate tose fermenter) ○ Ammonia neutralizes stomach acid • Vibrio alginolyticus: Wound and ear infections ○ Ammonia is toxic to the epithelial cells • Protease, catalase, and phospholipases • Damage to epithelial cells FAMILY AEROMONADACEAE § Elicits powerful immune response (ulcer) § Tissue biopsy is an important specimen in • Diseases mainly in fish and amphibians some settings • Frogs • Laboratory diagnosis ○ Red leg • Curved gram-negative bacilli ○ Fatal internal hemorrhaging • Growth on blood agar in 3 to 5 days • Fish • 35 C with high humidity ○ Develop ulcers, tail rot, fin rot, and hemorrhagic • Microaerophilic: 5% O , 10% CO , 85% N septicemia 2 2 2 • Positive: Oxidase, catalase, urease • Not usually grown Aeromonas hydrophila ○ Urease test on biopsy ○ Direct stain • Epidemiology and clinical disease ○ Stool antigen test • Organism ubiquitous in fresh and brackish water • Water-borne illnesses ○ Gastroenteritis FAMILY NEISSERIACEAE ○ Wound infections • Exposure to water, fish hook injuries • More than 20 genera ○ Bacteremia • General characteristics § Most often in young or old • Aerobic gram-negative diplococci CHAPTER 1 Microbiology 23

• Oxidase and catalase positive • Respiratory transmission ○ Neisseria elongate: Catalase negative, rod shaped • Mainly affects adolescents in overcrowded • Human reservoir environments ○ Respiratory and urogenital tract ○ College dormitories and the military ○ Sexual transmission (Neisseria gonorrhoeae) • Vaccine an important control measure • N. gonorrhoeae and Neisseria meningitidis are the • Clinical disease primary pathogens • First or second leading cause of community- acquired meningitis in the United States • Pathogenic Neisseria Species Also causes sepsis, conjunctivitis, disseminated organ infections, pneumonia without meningitis Neisseria gonorrhoeae • Many strains of N. meningitidis; clinically the most • Epidemiology important are A, B, C, Y, and W135 • Sexually transmitted by carrier • Laboratory diagnosis: Specimen collection and • Causes gonorrhea processing • Clinical disease • CSF • Males: Acute urethritis with dysuria and urethral ○ An amount greater than 1 mL of CSF is hand car- discharge ried to the laboratory ○ Not commonly asymptomatic ○ Specimen should not be refrigerated • Females: Colonizes endocervix causing ○ CSF specimen may be centrifuged ○ Vaginal discharge ○ Gram stain is prepared from the sediment ○ Dysuria ○ Sedimentisinoculated tochocolateand blood agar ○ Abdominal pain • Blood ○ Untreated may lead to pelvic inflammatory ○ Conventional blood culture systems disease (PID) • Skin scraping • Other sites of infection include ○ Petechiae may yield viable or stainable organisms ○ Eyes ○ Cut open lesion and collect fluid on swab ○ Throat • Laboratory diagnosis: Identification ○ Rectum • Gram-negative diplococci found intracellularly and • Can disseminate if untreated extracellularly ○ Less than 1% of infections • Grow well on sheep blood and chocolate agar ○ Purulent arthritis and septicemia • Grow on Thayer Martin selective agar • Laboratory diagnosis: Culture • Catalase and oxidase positive • Rapid transport critical for recovery • Cysteine trypticase agar (CTA) sugar oxidation: ○ Direct plating to selective media at the bedside glucose and maltose positive ○ Use of transport systems for Neisseria gonorrhoeae • Selective media to inhibit other bacteria and yeast Other (Nonpathogenic) Neisseria Species ○ Modified Thayer-Martin (MTM) or other selec- • Neisseria lactamica tive medium • Neisseria flavescens ○ Chocolate agar: Allows growth of other • Neisseria sicca saprophytes • Clinical significance • Incubation: • Normal flora of human upper respiratory tract ○ 3% to 7% CO incubator at 35 to 37 C 2 • Cause occasional infections ○ Humidity is important • Occasionally isolated from blood, genital tract, • Laboratory diagnosis: Identification and CSF • Gram-negative diplococci (some may appear as • Identification not appropriate unless isolated from tetrads) systemic site or pure culture • Small gray, translucent, raised colonies • Grows on chocolate, but usually not on blood agar • Catalase and oxidase positive FAMILY ALCALIGENACEAE § Cysteine trypticase agar (CTA) sugar oxidation: glucose positive • General characteristics Neisseria meningitidis • Small (0.2-0.7 mm) coccobacilli • Epidemiology • Fastidious, obligate aerobes that require • Human reservoir nicotinic acid ○ Upper respiratory tract of 3% to 30% of asymp- • Bordetella pertussis and Bordetella parapertussis tomatic individuals are nonmotile, Bordetella bronchiseptica is motile 24 CHAPTER 1 Microbiology

• Human respiratory tract is the only source of B. per- • Clinical disease tussis and B. parapertussis • Most human infections are wound infections/cellu- • Others infect birds and other mammals litis after cat bites ○ Pain, swelling, and serosanguinous drainage at the wound site Bordetella pertussis • Septic arthritis and osteomyelitis may occur after • Epidemiology deep puncture wounds • • Worldwide 60 million cases with 500,000 deaths Serious infections may occur in compromised • Endemic in most populations hosts • ○ Cycle often—3 to 4 years Respiratory tract infections • • Majority of cases in the United States occur in Bacteremia, endocarditis • August to November Central nervous system (CNS) infection • • No evidence of long-term carriage Eye infections after cat and dog scratches • ○ Immunity from vaccine or infection is not Laboratory diagnosis • lifelong Clinical history important • ○ Protection wanes after 3 to 5 years Specimens ○ ○ Immunity is undetectable by 12 years Pus, wound swab, tissue, sputum, blood • • Subclinical infections in adults may be common Growth requirements ○ • Adults usually the index cases in infants Growth on blood and chocolate agar ○ • Vaccine for adolescents recently approved No growth on MacConkey agar ○ • Clinical disease Incubate for 24 hours in CO2,at35 C • • Typical upper respiratory tract infection for 1 week Small, short, gram-negative bacilli • • Paroxysmal cough (out, out, out, whoop) Colonies ○ ○ Not always present Gray-green, convex, nonhemolytic, odor • • Long recovery Biochemicals ○ • Laboratory diagnosis Oxidase and catalase positive ○ • Direct fluorescent antibody (DFA) on nasal Indole positive ○ specimen Urease negative ○ • Culture of nasal specimen Commercial systems usually appropriate for • Polymerase chain reaction P. multocida • Serologic tests ○ Not generally available Other Species ○ Not helpful during acute phase Pasteurella ○ Difficult to interpret • Pasteurella canis • Pasteurella dagmatis • Found in mouths of canines FAMILY PASTEURELLACEAE • Pasteurella caballi • Infections associated with dog bites Pasteurella multocida • Upper respiratory tract of horses • Horse bite wounds • Reported in 1878 in fowl cholera–infected birds • 1880: Louis Pasteur • Most common species of Pasteurella isolated from Species humans Haemophilus • Grows on blood and chocolate • Genus includes • No growth on MacConkey agar • Haemophilus influenzae • Oxidase and catalase positive • Haemophilus aegyptius • Indole positive • Haemophilus haemolyticus • Epidemiology • Haemophilus parainfluenzae • Oral cavity of cats and dogs • Haemophilus ducreyi • Causative agents of several economically significant • New genus Aggregatibacter veterinary diseases • Haemophilus segnis, Haemophilus aphrophilus, ○ Cattle, buffaloes, sheep, goats, poultry, turkeys, and Actinobacillus actinomycetemcomitans rabbits, horses, and camels • Most members are nonpathogenic or opportunistic ○ Serious infectious diseases such as fowl cholera, pathogens bovine hemorrhagic septicemia, and porcine • Three major pathogenic species atrophic rhinitis • H. influenzae CHAPTER 1 Microbiology 25

• H. aegyptius Haemophilus ducreyi • H. ducreyi • Not normal flora: Sexually transmitted disease • Haemophilus: Derived from Greek for “blood (STD) lover” • Genital tract pathogen • Require growth factors present in blood • Genital chancres, ulcers • X Factor: Hemin, hematin • Epidemiology • V Factor: Nicotinamide adenine dinucleotide • Chancroid is rare in the United States (NADH) NADH ○ Localized endemic outbreaks occur in isolated • XV Factor strip test STD and prostitution populations • Both are found in chocolate agar • Annual global incidence approximately 6 million • Gram-negative pleomorphic coccobacilli or bacilli per year • Nonmotile • Chancroid more common in areas of low soc- • Aerobic or facultative anaerobic ioeconomic status such as Africa, Asia, and the • Oxidase and catalase positive Caribbean • Obligate parasite of mucous membranes of humans • More common in areas where the prevalence of and mammals HIV is high Haemophilus influenzae • Laboratory diagnosis • Often found as part of normal upper respiratory tract • 3% to 5% CO2 flora in humans • High humidity • Spread by droplets and close contact • Must be plated immediately • Clinical disease • Blood agar with X factor • Especially in children ○ Does not need V factor ○ Meningitis ○ Chocolate agar ○ Septicemia ○ Fastidious, will not satellite on blood agar ○ Epiglottitis • 2 to 10 days needed for growth ○ Pneumonia • Gram-negative coccobacillus: “School of fish” ○ Otitis pattern • Vaccine: Single biggest impact on pediatrics in last • Gray, yellow, or tan colonies 20 years ○ Nonmucoid • Pathogenesis and virulence • Catalase negative, oxidase positive • Polysaccharide capsule • Nucleic amplification is definitive test ○ Seven serogroups: a to f and e0 ○ Capsule type b (Hib) is the most clinically signif- Miscellaneous Haemophilus Species icant and virulent • Examples: H. parainfluenzae, H. haemolyticus ○ Immunoglobulin A (IgA) proteases • Normal flora in humans that occasionally cause upper • Outer membrane proteins and lower respiratory tract infections • Adherence factors • May lead to systemic infections resulting from inva- • Laboratory diagnosis sion of blood and tissue • Grows on chocolate agar ○ Requires factors X and V • Requires 3% to 5% CO2 Related Organisms • Satellites around S. aureus on SBA • Small translucent colonies Aggregatibacter aphrophilus • Gram-negative coccobacilli • Includes both species formally known as factor V– Haemophilus aegyptius independent (H. aphrophilus) and factor V–dependent • Conjunctivitis: “Pink eye” (H. paraphrophilus) strains • Brazilian purpuric fever Aggregatibacter segnis • Recurrent conjunctivitis • Formerly H. segnis • High fever Eikenella corrodens • Vomiting • General characteristics • Septicemia • All oxidase-positive, fastidious gram-negative • Shock bacilli • Mortality as high as 70% • Part of mouth flora in 40% to 70% of humans • Laboratory diagnosis • Clinical disease • Requires factors X and V • Frequently in infections from human bites • Grows on chocolate in 3% to 5% CO2 • May be mixed infections • Biochemical differentiation required • Laboratory diagnosis 26 CHAPTER 1 Microbiology

• Gram-negative coccobacilli § Can cause epidemics and isolated cases • Requires increased CO2 for growth (3%-10%) § Occurs sporadically (community-acquired) or • Oxidase positive as an epidemic • Catalase, urease, indole negative • Predisposing factors—both forms • Usually pit the agar during growth • Immunocompromised • Increased age Kingella Species • Heavy smoking • General characteristics • Exposure to high concentration of organisms • Four species in genus ○ Showers • Epidemiology ○ Air-conditioner cooling towers ○ Flora of the pharynx in young children • Pathogenesis and virulence factors ○ Transmitted from child to child • Intracellular pathogen • Clinical disease • Survive and multiply within macrophages • Osteomyelitis • Laboratory diagnosis • Bacteremia • Specimens • Endocarditis ○ Respiratory specimens are preferred • Joint infections ○ Urine for antigen testing • Laboratory diagnosis • Growth requirements • Fastidious short gram-negative coccobacillus ○ Cysteine and iron • Joint fluid into blood culture bottles ○ Buffered charcoal yeast extract (BCYE) media • Identification with Remel Rapid NH ○ 35 C, CO2 FAMILY LEGIONELLACEAE ○ Slow grower—hold for 2 weeks • Identification confirmation ○ Legionella pneumophila Serology testing ○ DNA probes • L. pneumophila is the primary pathogen ○ Reference laboratory • 14 serotypes • 01 most common FAMILY BRUCELLACEAE • General characteristics • Non–acid-fast, nonsporulating, and noncapsulated Brucella bacilli • Aerobic fastidious gram-negative bacilli; difficult to • Zoonotic disease with worldwide distribution stain • Acquired from animals or animal sources; most • Nonfermentative likely to contract are farmers, butchers, veterinar- • Oxidase and catalase positive ians, laboratory workers • Produces b-lactamase • Epidemiology • Epidemiology • Ingestion of contaminated dairy • Ubiquitous, natural sources • Clinical disease ○ Lakes, ponds, rivers • Undulant fever, Mediterranean fever, Malta fever • Human-made sources • Lymph nodes, blood, and reticuloendothelial sys- ○ Cooling towers, air-conditioning units, hot tubs/ tem affected spas, plumbing fixtures • Fever, chills, headache, hepatosplenomegaly • Illness acquired from breathing in organism; no • Laboratory diagnosis human-to-human transfer • Safety: BSL III cabinet • Clinical disease • Specimens • Described in 1976 ○ Blood, bone marrow, tissue • Epidemic of American Legion members ○ Transport blood and bone marrow in • Two diseases Isolator tube ○ Legionnaire’s disease ○ Inoculate solid media and blood culture bottles § Pneumonia: Fever, chills, cough, myalgia, ○ Tape plates headache, chest pain, sputum • Will grow on blood and chocolate agar § Can have a high mortality rate (30%) if not • MacConkey negative treated • Requires extended incubation for 7 to 10 days § Symptoms depend on person—asymptomatic • Automated blood culture systems will detect in to life-threatening 2 weeks ○ Pontiac fever • Small gram-negative coccobacilli § Milder form of disease, more like influenza, • Colonies no pneumonia, 0% mortality ○ Small, translucent, moist, nonhemolytic CHAPTER 1 Microbiology 27

• All are oxidase and catalase positive ○ Pseudomonas fluorescens • Urea and H2S results vary by species ○ Pseudomonas stutzeri • Do not put suspected Brucella in an automated sys- ○ Pseudomonas alcaligenes tem for identification • General characteristics • Additional tests • Oxidase positive ○ Serology • Aerobic gram-negative bacilli ○ Polymerase chain reaction (PCR) • Water and soil—ubiquitous in environment • Uses numerous substrates as energy FAMILY FRANCISELLACEAE • Identification and differentiating beyond P. aeruginosa and common species can be time con- Francisella tularensis suming • Epidemiology Pseudomonas aeruginosa • Transmission: Handling animals or carcasses • Clinical disease • Contaminated food or water • Wide range of diseases • Highly contagious and invasive • Septicemia • Clinical disease • UTI • Tularemia • Pneumonia • Multiple forms of the disease • Chronic lung infections • Laboratory diagnosis ○ Cystic fibrosis • Specimens • Skin and soft tissue infections ○ Safety: BSL III cabinet • Chronic lung infections: Cystic fibrosis ○ Ulcer swabs • Pulmonary infections a major cause of death ○ Lymph node biopsy • Repeat episodes of airway disease ○ Sputum • Chronic colonization/infections ○ Bone marrow • Exacerbations ○ Rarely isolated from blood • Chronic therapy • Growth requirements • Skin and soft tissue infections ○ Cysteine and iron • Intact skin is not a good medium for Pseudomonas ○ Grows on chocolate agar • Water is usually associated with infections ○ Increased CO ,35 C 2 • Immunocompromised patients are at risk for ○ Slow growth: 2 to 5 days infections • Faint staining, gram-negative coccobacilli • Pseudomonas folliculitis • Small, greenish, droplike colonies • Laboratory diagnosis • Biochemically inert • Presumptive identification ○ Catalase positive ○ Large colonies • Serologic tests ○ Grapelike odor • Molecular tests ○ Oxidase positive ○ Pyocyanin FAMILY PSEUDOMONADACEAE • For further confirmation ○ Growth at 42 C • More than 150 species listed ○ Glucose oxidation • Based on genetic analysis (16S rRNA sequence) Chry- ○ Pyoverdin (fluorescein) seomonas and Flavimonas are Pseudomonas spp. • Flavimonas oryzihabitans Other Pseudomonas Species • Chryseomonas luteola, Chryseomonas polytricha • Genus previously included most glucose nonferment- • P. fluorescens ing gram-negative bacilli • P. putida • Pseudomonas • P. stutzeri • Pseudo: Greek for “false” • Pseudomonas oryzihabitans • Monas: Greek for “a single unit” • Water and soil: Ubiquitous in environment • Used early in the history of microbiology to refer to all • Uses numerous substrates as energy germs • Identification and differentiating beyond P. aerugi- • Aeruginosa: Latin for oxidized copper nosa difficult • Compares to P. aeruginosa pigments in the laboratory ○ Molecular methods replacing traditional bio- • Most common isolates in the clinical laboratory chemical testing ○ P. aeruginosa • Inherently more resistant to antimicrobial agents ○ Pseudomonas putida than common gram-negative bacilli 28 CHAPTER 1 Microbiology

FAMILY MORAXELLACEAE • General characteristics • Aerobic nonfermentative gram-negative bacilli • Moraxella Species Most strains grow well on MacConkey agar • Oxidase negative • 19 species in the genus • Nonmotile • Only a few important in human medicine • Epidemiology • M. catarrhalis • Widely distributed in nature • M. bovis • Survive on various surfaces (both moist and dry) in • M. osloensis the hospital environment • Nonmotile, gram-negative coccobacilli • Strains have been isolated from food Moraxella catarrhalis • Isolated from healthy human skin • Commensals of the upper respiratory tract • Clinical disease • Isolated only from humans • Considered nonpathogenic to healthy individuals • Not associated with disease in healthy people • Most infections in immunocompromised • Clinical disease individuals • Causes mostly opportunistic infections • Frequently isolated in nosocomial infections • Can be isolated from patients with ○ Especially intensive care units ○ Ear infections • A. baumannii is a cause of nosocomial pneumonia ○ Bronchitis ○ Late-onset ventilator-associated pneumonia ○ Sinusitis • Other infections include ○ Pneumonia ○ Skin and wound infections ○ Predisposing pulmonary conditions (e.g., ○ Bacteremia chronic obstructive pulmonary disease) ○ Meningitis • Laboratory diagnosis • Laboratory diagnosis • Grow on simple nutrient agar • Coccobacilli ○ Blood agar at 35 C • Oxidase negative ○ Chocolate agar • Unable to reduce nitrate ○ No growth on Thayer-Martin • Difficult to identify some species § Differentiates it from N. gonorrhoeae because • Commercial systems appropriate for A. baumannii, of colistin but questionable for other species • Opaque, gray, smooth and dry colonies • Most strains grow well on MacConkey agar • Gram-negative coccobacilli ○ Except some A. lwoffii • Oxidase positive • Non–lactose fermenting • CTA sugar oxidation ○ May appear partially lactose fermenting on ○ Glucose: Negative MacConkey agar ○ Maltose: Negative ○ Lactose: Negative ○ Sucrose: Negative FAMILY BURKHOLDERIACEAE Other Moraxella Species • Normal flora and uncommon pathogens of human and Burkholderia Species other mammals’ upper respiratory tract • Speciation is beyond the scope of a routine Burkholderia cepacia laboratory • Epidemiology and clinical disease • Most susceptible to penicillins, cephalosporins, tetra- • Opportunistic and nosocomial pathogen cyclines, and aminoglycosides ○ Major pathogen of patients with cystic fibrosis • Psychrobacter phenylpyruvicus formerly Moraxella • Resistant to decontaminating agents phenylpyruvicus ○ Has been isolated from alcohol and iodine bottles • Laboratory diagnosis • Acinetobacter Species Aerobic gram-negative bacilli • Growth on blood, chocolate, and MacConkey agar • More than 10 species and multiple genomavars ○ Selective media usually used in patients with cys- • May be as many as 30 species tic fibrosis • Some are important in human medicine • Oxidase weakly positive • Acinetobacter baumannii ○ Routine laboratory test may be negative • Acinetobacter lwoffii • Commercial systems may identify complex • Acinetobacter haemolyticus • Highly resistant antibiotic susceptibility pattern CHAPTER 1 Microbiology 29

Burkholderia pseudomallei • Environmental organisms • Melioidosis • Water • Aggressive granulomatous pulmonary disease • Soil • Seen in Vietnam veterans • Opportunistic and nosocomial infections • Rarely seen in the United States • Cystic fibrosis • Automated systems may or may not identify correctly • Burns • Clinical history and communication with physician • Immunocompromised critical • Pathogenesis and virulence factors Burkholderia mallei • Opportunistic • Glanders • Multiple extracellular enzymes used for survival in • Primarily in horses, mules, and donkeys the environment • Usually by ingestion of contaminated food or water • Often resistant to multiple antimicrobial agents • Nodular lesions in the lungs and ulceration of the • Laboratory diagnosis mucous membranes • Aerobic gram-negative bacilli • Endemic in Africa, Asia, the Middle East, Central and • Grows on blood, chocolate, and MacConkey agar South America • Nonfermenters on MacConkey (lactose) • Not to be identified in a routine clinical laboratory • Oxidase positive • Motile

Stenotrophomonas maltophilia Alcaligenes Species • Aerobic • Nonfermentative • Alcaligenes faecalis • Gram-negative bacillus • Environmental organisms • They are motile by polar flagella ○ Water • Clinical disease ○ Soil • Opportunistic pathogen • Opportunistic and nosocomial infections • Serious infections in immunocompromised host ○ Cystic fibrosis • Nosocomial infections ○ Burns • Respiratory isolates are common ○ Immunocompromised • Other sites • Pathogenesis and virulence factors ○ Wounds • Opportunistic ○ Urine • Multiple extracellular enzymes used for survival in ○ Blood the environment • The major risk factor for infection in hospitalized • Often resistant to multiple antimicrobial agents patients is the implantation of medical devices • Laboratory diagnosis ○ Central venous catheters • Grow on blood, chocolate, and MacConkey agar ○ Urinary tract catheters • Gram-negative bacilli ○ Prosthetic heart valves • Nonfermenters on MacConkey (lactose) ○ Intraocular and contact lenses • Oxidase positive • Laboratory diagnosis • Motile • Growth on blood agar and chocolate agar: Green- lavender or yellow pigment • Non–lactose fermenter on MacConkey agar FAMILY CHLAMYDIACEAE • Oxidase negative • Catalase and esculin positive Chlamydia and Chlamydophila • Strongly oxidizes maltose • Three species in Chlamydia ○ Weak oxidation of glucose • Six species in Chlamydophila • Chlamydophila was recognized in 1999 FAMILY ALCALIGENACEAE • Chlamydia ○ Chlamydia muridarum ○ Achromobacter Species Chlamydia suis ○ Chlamydia trachomatis • Six species • Chlamydophila • Achromobacter xylosoxidans most common in clinical ○ Chlamydophila pneumoniae microbiology ○ Chlamydophila pecorum • Achromobacter denitrificans ○ Chlamydophila psittaci 30 CHAPTER 1 Microbiology

○ Chlamydophila abortus ○ Humans are the only natural host ○ Chlamydophila felis ○ Incidence is 300 to 500 cases per year in the ○ Chlamydophila caviae United States • Obligate intracellular pathogens ○ Male homosexuals are major reservoir of the • Three disease-causing species in humans disease • Chlamydia trachomatis • Clinical disease ○ STDs, eye infections • Urethritis, cervicitis, epididymitis • Chlamydophila pneumoniae • Pelvic inflammatory disease ○ Respiratory disease in humans, horses, koalas, • STD: Lymphogranuloma venereum and other animals • Trachoma • Chlamydophila psittaci ○ Not an STD, chronic conjunctivitis, blindness ○ Respiratory disease: Humans, birds • Inclusion conjunctivitis • Life cycle ○ Spread to infant from mother • Elementary bodies are the small (0.3-0.4 mm) infec- ○ No blindness tious form of the chlamydia • Pathogenesis ○ They possess a rigid outer membrane that is • Infects nonciliated columnar epithelial cells extensively cross-linked by disulfide bonds • Infiltration of PMNs ○ Because of their rigid outer membrane the ele- • Lymphoid follicle formation and fibrosis mentary bodies are resistant to harsh environ- • Clinical diseases from destruction of the cells and mental conditions encountered when the the host inflammatory response Chlamydia organisms are outside of their • Does not stimulate long-lasting immunity eukaryotic host cells ○ The elementary bodies bind to receptors on host Chlamydophila pneumoniae cells and initiate infection • Formerly classified as Chlamydia ○ Most Chlamydia organisms infect columnar • Humans only source of disease epithelial cells, but some can also infect • Atypical pneumonia macrophages • Epidemiology • Reticulate bodies are the noninfectious intracellular • Affects all age groups form of the Chlamydia organism ○ Most common among older age groups ○ They are the metabolically active replicating • Reinfection is common after a short period of form of the Chlamydia organism immunity ○ They possess a fragile membrane lacking the • Causes 10% of community-acquired pneumonias extensive disulfide bonds characteristic of the treated without hospitalization elementary bodies • Clinical disease Chlamydia trachomatis • Commonly causes mild-to-moderate respiratory • General characteristics illness • Only found in humans ○ Pneumonia, bronchitis, sinusitis, flulike illness • Most common bacterial STD pathogen • Pneumonia symptoms indistinguishable from other • Infections may be symptomatic or asymptomatic causes of pneumonia • Ocular infections: Biovar trachoma ○ Cough, fever, and difficulties breathing ○ 500 million people are infected worldwide • Incidence of asymptomatic appears high ○ 7 to 9 million people are blinded • May be severe in immunocompromised ○ Endemic in Africa, the Middle East, India, and • Laboratory diagnosis Southeast Asia • Tissue culture: historical gold standard ○ Infections occur mostly in children ○ Cultured on McCoy cells ○ Transmitted by droplets, hands, contaminated § Other cell lines will support the growth of clothing, flies, and by passage through an C. trachomatis infected birth canal ○ Stain 2 to 6 days after inoculation (iodine or fluo- • Genital tract infections: Biovar trachoma rescent antibody) ○ May be the most common bacterial STD in the • DFA United States ○ Detects outer membrane of elemental bodies ○ 50 million new cases occur yearly worldwide ○ Can be as sensitive as culture ○ In the United States, the highest infection rates • Molecular techniques occur in Native and African Americans ○ Nucleic acid amplification • Genital tract infections § PCR, ligase chain reaction, strand ○ STD that occurs sporadically in the United States displacement ○ Prevalent in Africa, Asia, and South America ○ DNA probes CHAPTER 1 Microbiology 31

Chlamydophila psittaci • Clinical disease: M. hominis • Epidemiology ○ Colonize male and female genital tract • Causative agent of psittacosis: Parrot fever ○ Role in genital tract diseases unclear • Natural reservoir can be any species of bird ○ Associated with adverse pregnancy outcomes • Present in tissues, feces, and feathers of symptom- • Clinical disease—U. urealyticum atic or asymptomatic birds • STD • Veterinarians, zoo keepers, pet shop employees at ○ Urethritis in males increased risk ○ Infertility, low birth weight, premature delivery • Clinical disease • Others • Incubation time of 7 to 15 days ○ Pneumonia, meningitis, bacteremia in newborns • Symptoms include fever, chills, headache, nonpro- ○ Bacteremia, abscesses, arthritis in immunocom- ductive cough, mild lung inflammation promised patients • Disease usually subsides in 5 to 6 weeks • Laboratory diagnosis • Asymptomatic infections are common ○ Mollicutes grow slowly by binary fission and • Seizure, coma, and death (5% mortality rate) produce “fried egg” colonies on agar plates can occur ○ Colonies of M. pneumoniae have a granular • Laboratory diagnosis and treatment and prevention appearance • Diagnosis is through serology ○ Because of slow growth, colonies may take up to ○ Fourfold rise in titer in paired sera 3 weeks to develop • Ureaplasma split urea FAMILY MYCOPLASMATACEAE FAMILY BARTONELLACEAE • Class Mollicutes: Meaning “soft skin” • Mycoplasmataceae • Bartonella • Grahmella Mollicutes of Humans • Rochalimaea • Grahamella and Rochalimaea may not have valid tax- • Approximately 120 species of the genus Mycoplasma onomic status • Seven species of the genus Ureaplasma • Lack cell walls Bartonella Species • Very small size • 0.2 to 0.8 mm in diameter • Approximately 19 described species • Very small genome • Three subspecies of Bartonella vinsonii • Require sterols for growth • Small (0.6 1.0 mm) gram-negative bacilli • Linked to respiratory infections by Roux and • Fastidious Nocard in 1898 Bartonella henselae • Isolated organism from bovine pleuropneumonia • Cat scratch disease • Probably evolved from gram-positive bacteria • Acquired after exposure to cats such as Lactobacillus, Bacillus, Streptococcus, ○ Scratches, bites Clostridium spp. • Disease usually benign • Not found growing freely as living organisms ○ Chronic regional lymphadenopathy • Depend on host for fatty acids, amino acids, nucleic • Laboratory diagnosis acid precursor, and cholesterol • Warthin-Starry silver staining • Epidemiology • DNA amplification • At least 16 species have been isolated from humans • Cultures not recommended • More than half are considered nonpathogenic ○ Blood or tissue transported in Isolator • Mostly Mycoplasma and Ureaplasma spp. ○ Chocolate agar • Associated with mucous membranes ○ Prolonged incubation with increased CO2 ○ Respiratory • Serology main testing ○ Urogenital • Cross-reactions with Coxiella burnetii, Chlamydia • Clinical disease spp., and other Bartonella spp. • Clinical disease: M. pneumoniae • IgG/IgM EIA ○ Common cause of mild pneumonia in people ○ Sensitivity 85% and specificity >98% younger than 40 years of age • Laboratory diagnosis, other than cat scratch disease § 15% to 50% of all pneumonia in adults and • Serology an even higher percentage of pneumonia in ○ Reference or research laboratory school-aged children ○ CDC 32 CHAPTER 1 Microbiology

FAMILY RICKETTSIACEAE • Borrelia ○ Borrelia burgdorferi: Lyme disease ○ Orientia Borrelia hermsii: Relapsing fever • Treponema pallidum: Syphilis • Orientia tsutsugamushi ○ Other treponemas: Yaws, pinta, bejel • Orientia were formerly classified as Rickettsia • General characteristics • Long, thin, spiral Rickettsia • Approximately 26 species Leptospira interrogans and Leptospira • General characteristics biflexa • Small intracellular bacteria • 14 species ○ 0.3 to 0.5 1to2mm • 6- to 20-mm coiled rods with hook • Gram-negative–like cell wall • Obligate aerobes ○ Contains LPS • Fastidious • Independent ATP and host ATP can be used • • No apparent genes for sugar metabolism, lipid syn- Epidemiology • Endemic worldwide thesis, and amino acid synthesis • Most common zoonotic infection worldwide • Divided into groups based on antigens • Most prevalent in the tropics and subtropics • Spotted fever group • • Contact: Animal urine Typhus group ○ • Directly Scrub typhus group ○ • Epidemiology: Rocky Mountain Spotted Fever Contaminated fresh water • (RMSF) Clinical disease • • Most common rickettsial disease in the United Leptospirosis • Severity of disease varies with serovar States • • 90% of patients mild disease Approximately 500 to 700 cases per year ○ • Originally described in Rocky Mountain area, but Low fever ○ Meningitis more common in South Central states ○ • Transmitted by the bite of an infected tick Rash • • Highest incidence from April through September 10%: Severe with hepatitis, general organ in- • Principal reservoir for R. rickettsii is hard tick volvement • ○ Transovarian passage occurs Laboratory diagnosis • • Clinical disease: RMSF Clinical presentation • • Culture Abrupt onset of fever, chills, headache, and myalgia ○ • Blood early 5 to 10 days after the tick bite ○ > • Rash common (90%) Urine late ( 2 weeks) • ○ 2 to 3 days after fever Molecular: PCR • ○ Begins on the hands and feet and spreads toward Serology: Enzyme immunoassay (EIA) the trunk ○ Palms and soles is common • Complications from vasculitis Borrelia ○ Respiratory failure, seizures, acute renal failure • Approximately 30 species of Borrelia • Mortality rate in untreated patients is 20% • Microaerophilic • Laboratory diagnosis • Temperature optima 28 to 30 C • Clinical • Generation time approximately 18 hr • Fluorescent or PCR for antigen in skin biopsies • Flagella determines helical shape ○ Reference laboratories • Flagella-negative mutants are straight bacilli • Serology is the major laboratory test • Lyme borreliosis ○ Weil-Felix test • Most common vector-borne disease in the United ○ Immunoflurorescent antibody (IFA) reagent is States available • Caused by B. burgdorferi • Transmitted by the hard ticks Ixodes spp. FAMILY LEPTOSPIRACEAE • Vector • Ixodes scapularis • Human disease ○ Deer tick or black-legged tick • Leptospira interrogans: Leptospirosis • Ixodes pacificus CHAPTER 1 Microbiology 33

• Clinical disease ○ Noninfectious stage • Stage 1 (3-30 days) ○ May last a lifetime or result in tertiary syphilis ○ 75% develop erythema chronicum migrans • Tertiary or late syphilis; noninfectious stage ○ May be other symptoms • Gummas • Stage 2 (1-7 months) ○ Granulomas ○ Cardiac and neurologic problems ○ No treponemes • Stage 3 (months to years) ○ Causes host response ○ Arthritis • Skin, subcutaneous tissue, deep tissue, bone • Laboratory diagnosis • Neurosyphilis • Diagnosis of Lyme borreliosis is clinical diagnosis • Congenital syphilis • Laboratory tests should not be used alone, can help • Caused by infection in utero with T. pallidum support a clinical diagnosis • A wide spectrum of severity exists • Laboratory tests • Laboratory diagnosis ○ Serology (enzyme immunoassay) • Non-treponemal tests include RPR and VDRL ○ Western blot • Treponemal tests include TP-PA and EIA § POS IgM¼2 of 3 protein bands § POS IgG¼5 of 10 protein bands ANAEROBIC BACTERIA MOST Treponema pallidum COMMONLY ISOLATED FROM INFECTION • Syphilis (BOX 1-4)

• STD (90%) • Bacteria: Relation to O2 ○ Caused by direct contact with lesions that con- • Spectrum of sensitivity to O2 tain T. pallidum ○ Strict anaerobes (<5% O2) • T. pallidum enters cracks in mucous membranes of ○ Aerotolerant anaerobes (>5% O2) genitals, anus, lips, and rectum during vaginal, oral, ○ Facultative anaerobes (aerobes) and anal sex ○ Strict aerobes • Four untreated stages: 1>2>latent>3 • Strict anaerobic bacteria • Congenital infection possible • Superoxide (SO) anion lethal to bacteria • Primary syphilis • Facultative and aerobic bacteria handle SO by • Single primary lesion on any cutaneous or mucous producing the enzyme superoxide dismutase membrane surface (SOD) • Base hard but painless: Hard chancre • SO converted to H2O2 and O2 • Appears in 3 weeks and disappears in 4 to 12 weeks • Catalase and peroxidase production assist in break- • Only diagnostic test is dark-field microscopy ing down H2O2 • Secondary syphilis • Strict anaerobes lack significant levels of SOD • 6 weeks to several months • Largely intolerant of O2 in environment • Cutaneous and mucous membrane lesions • Lack appropriate cytochrome system to use O2 as • Rough red skin rash terminal electron acceptor • Infectious, live treponemes • Energy solely by fermentation • Latent syphilis • Gram morphology no different from that in aerobic • Early latent period; 2 yrs or less organisms ○ Infectious lesions may reappear • Many anaerobic bacterial species found among the • Late latent period; over 2 yrs normal microbial flora of humans and animals

BOX 1-4 Identification of Anaerobic Bacteria

• Specimen selection: what not to culture • Appropriate specimens • Any specimen likely to be contaminated with normal or coloniz- • Blood ing flora (nasopharyngeal, gingival, bronchial washings, expec- • Other normally sterile sites (like pleural fluid) torated sputum, vaginal or cervical samples, voided urine, • Dental/sinus—aspiration of abscess or biopsy surface swabs) • Lung—aspirate or biopsy, thoracentesis • Minor wounds that are likely to respond to simple drainage • Abdomen—aspirate of abscess, peritoneal fluid, otherwise • Specimens from acute infections where anaerobes are unlikely sterile tissue to play a role (bacterial meningitis, routine urinary tract • Female genital tract—laparoscopy, surgical biopsy, aspirate of infection) abscess

Continued 34 CHAPTER 1 Microbiology

BOX 1-4 Identification of Anaerobic Bacteria—cont’d

• Bone—aspirate, biopsy • Incubate at 35 C-37 C • Soft tissue—surgical biopsy, aspiration of abscess • Use GasPak jars, bags, or anaerobic chambers • Transport of Specimens • Use methylene blue or resazurin indicator to validate anaerobic • Tissue specimens—anaerobic pouch or anaerobic transport conditions tube with medium • Do not expose plates to air for more than ½ hr • Fluid or aspirated pus • Routine media for the interpretation of aerobic growth should • Express into anaerobic transport vials OR be incubated in room air or low CO2 tension if available • Leave in syringe, discard needle & cover with sterile cap • Incubation time • Transport of Specimens • Inspect at 24 h for rapid growers, 48-72 h for others, 4-5 days • Swabs are strongly discouraged as aspirates or biopsy tissues total are far better specimens • Organisms grow more slowly than aerobes • If swab is unavoidable, it must be collected in a sterile surgical • Exceptions—incubate >7 days if Actinomyces is suspected field and transported in a special anaerobic swab device to lab as it grows very slowly • Primary Media • Dissection microscope is helpful to look at tiny colonies • Primary specimens should be plated on non-selective, selective, • Approach to Identification and differential media • Complete ID of anaerobes may be complicated, time consum- • All media should be supplemented with vitamin K and hemin ing, and expensive • Media should be fresh • General current approach includes: • Routinely used: • Focus on Bacteriodes fragilis recognition • Anaerobic SBA, LKV, BBE, CNA or PEA • Determination of mixed bacterial flora, both aerobes and • Routine aerobic SBA, MAC, CNA anaerobes

• Media has reducing agents to keep O2 levels low • Look for toxins of C. difficile • Laked kanamycin-vancomycin (LKV) blood agar • Tetanus, botulism, gas gangrene present as characteristic • Selects for gram anaerobic negative bacilli clinical pictures—seek Clostridium • Bacteroides bile esculin agar (BBE) • Use simple tests to presumptively group most commonly • Selective/differential for Bacteroides fragilis grp. encountered anaerobic organisms • CCFA Selective differential media for recognition of C. difficile • Let the Gram stain guide the work up • Cycloserine will inhibit gram-negative bacteria, while cefox- • Differential media and resistance to key antibiotics itin will inhibit both gram-positive and gram-negative organ- • Few key biochemical tests—Common anaerobic bacilli isms. C. difficile ferments the fructose in the medium • Bile—growth in the presence of 20% bile resulting in yellow colonies • Incorporated in BBE agar • Phenylethyl alcohol (PEA)—selects for GPC • Inhibits most anaerobes other than Bacteroides and • Selects for gram-positive bacilli and cocci Bilophilia • Egg Yolk Agar (EYA)—nonselective, differential medium • Kanamycin, vancomycin, colisitin susceptibility by disk • Egg yolk suspension allows detection of lecithinase and • Antibiotics disks can be placed on blood agar for all three lipase agents • Lecithin breakdown results in an opaque precipitate • Growth or inhibition in the presence of specific concentrations • Lipase enzyme hydrolyzes results in an iridescent sheen on of antibiotic can be used for presumptive identification the colony surface • Catalase • Thioglycolate broth • 15% hydrogen peroxide is used to test for the production of • Nonselective, reduced liquid media catase by anaerobes • Incubation of plates • Spot Indole • Anaerobes are most sensitive to oxygen during log phase of • Ability to metabolize tryptophan by testing for indole in either growth tubes or a spot test Bile Kanamycin Vancomycin Colistin Catalase Indole Prevotella SR R V - - Porphyromonas SR S R - + B. fragilis grp RR R R + V Fusobacterium VS R S - V Bilophila RS R S + - • Few key biochemical tests—anaerobic gram-negative bacilli • Lecithinase—see egg yolk agar Lecithinase Double zone of hemolysis CCFA yellow colonies Catalase Indole Clostridium perfringens ++ - -- Clostridium difficile -- + -- Clostridium septicum -- - -- Propionibacterium -- - ++ CHAPTER 1 Microbiology 35

Peptostreptococcus • Gram-positive bacillus that tends to form branches, sometimes with a beaded appearance • General characteristics • Common member of the mouth flora of humans • Gram-positive anaerobic cocci in chains • Aerotolerant anaerobe • Common member of the normal gut flora and • Very slow growing and difficult to recover in culture respiratory tract • Responds well to long-term therapy with penicillin • Almost always found, when clinically significant, in class of antibiotics coinfections with other anaerobes and facultative • Clinical syndromes anaerobes • Cervical-facial • Basically the only genus of the gram-positive anaer- ○ Most common, causes “lumpy jaw” obic cocci involved in disease ○ Slowly developing abscess, chronic infection Veillonella may erupt into sinus tracts on face or neck ○ Dental surgery or facial trauma predisposition • General characteristics • Pulmonary • Gram-negative anaerobic cocci (counterpart of the ○ Aspiration of mouth flora to lower respiratory aerobic Neisseria) tract • Common member of the mouth and intestinal flora ○ Primary pneumonia may result of humans ○ Radiography not specific to Actinomyces • Low incidence of pathogenicity ○ Chronic infection in which abscess is formed ○ If clinically significant, found in coinfections § Abscess, which can bore its way to surface and with other anaerobes and facultative anaerobes produce sinus tracts § Sulfur granules can be seen in the fluid Propionibacterium § Granules are actually aggregated microcolonies § • General characteristics Gram smear reveals the long filamentous • Gram-positive, non–spore forming, anaerobic Actinomyces bacillus • Abdominal ○ • Resembles Corynebacterium in morphology and Infection follows ingestion of organism ○ cell arrangement GI lesions can occur, not unlike those seen in pul- • Some strains are aerotolerant, yet yield better monary infection ○ growth under strictly anaerobic conditions Abscess can rupture through musculature and • Produces propionic acid skin to form tracts • Widely distributed as normal flora on skin and other • Genitourinary ○ body sites, including the respiratory tract, and may A. israelii may colonize the female genital tract as actasopportunisticpathogenwithothercopathogens normal flora ○ Associated with long-term use of intrauterine Propionibacterium acnes devices ○ Indistinguishable from pelvic inflammatory • General characteristics disease • Most common gram-positive, non–spore forming, § Vaginal discharge, abdominal pain, fever, uri- anaerobic rod encountered in clinical specimens nary discomfort, etc. • Slowly growing in culture ○ Treated with antibiotics and removal of the device • Common resident of the pilosebaceous glands of the human skin • Causative agent of acne vulgaris (pimples) GRAM-POSITIVE BACILLUS: • In addition to acne, P. acnes has been implicated in CLOSTRIDIUM other infectionss • General characteristics ○ Corneal ulcers • Gram-positive bacillus ○ Heart valves ○ Usually large bacilli ○ Prosthetic devices • Produce endospores ○ CNS shunts ○ May appear terminal or central • Opportunistic mixed infections with other flora ○ Excellent survival in environment • Highly susceptible to various b-lactam antimicro- • Are strictly anaerobic in metabolism bial agents such as penicillin G • Produce variety of potent toxins Actinomyces israelii • Clostridium spp. of clinical importance • Clostridium perfringens • General characteristics ○ Gas gangrene • Most common human pathogenic species ○ Food poisoning 36 CHAPTER 1 Microbiology

• Clostridium tetani • Food or wound botulism ○ Tetanus ○ Toxin: Botulism neurotoxin (BoTN) • Clostridium botulinum § Preformed in food source or made by organ- ○ Botulism ism contaminating a wound • Clostridium difficile § Blocks acetylcholine release at the neuromus- ○ Antibiotic associated diarrhea and colitis cular junction and causes an inhibition of muscle contraction ○ Symptoms Clostridium perfringens § Blurred vision, dizziness, muscle weakness, and flaccid paralysis • Nonmotile gram-positive anaerobic bacillus ○ Boiling food destroys the toxin • Part of human intestinal flora • Infant botulism • Minor opportunistic pathogen ○ Acquired by ingestion of food containing spores • Appears in mixed flora infection ○ Ranges from mild to fatal disease • Found universally in soil ○ Honey is the most common source of spores, • Potential to cause major myonecrosis called gas which then germinate in the child’s intestinal gangrene in wounds tract (contraindicated in children younger than • Gas gangrene 1 year of age) • Results from contaminated wounds ○ Toxin production causes symptoms of few days • Lesions progress from redness and swelling to duration and then often subside greenish blackish decoloration ○ Rare need for use of antitoxin • Toxin destroys muscle tissue ○ Symptoms • Gas bubbles present in blisters and under skin § Inability to suckle, constipation, flaccid paral- • Fatal within 48 hours without antibiotics ysis, muscle weakness • Treatment is surgical (debridement), antibiotics, and hyperbaric O2 chamber Clostridium difficile • Motile gram-positive anaerobic bacillus Clostridium tetani • Source ○ • Environmental bacteria: Soil Soil, air, water, human and animal feces • • Motile gram-positive anaerobic bacillus Use of broad-spectrum antibiotics lowers relative • Endospores contaminate puncture wounds, grow amount of other normal gut flora and allows C. diffi- anaerobically, and produce toxin to cause the disease cile to proliferate and infect large intestine • tetanus (lockjaw) Disease • • Tetanus C. difficile releases two enterotoxins (A and B) ○ • Painful and rapidly fatal syndrome Can cause diarrhea, but often causes pseudo- • Toxin binds to target nerve cells membranous colitis with destruction of intestinal ○ Inhibitory interneurons that regulate muscle con- lining (also called antibiotic associate colitis) • traction are blocked Symptoms ○ ○ Patients undergo single, constant muscle con- Watery diarrhea, abdominal cramps, fever, traction bloody stools, nausea, dehydration • ○ Infection can lead to respiratory failure Treatment: Oral metronidazole or vancomycin • • Treatment can include use of antitoxin Either or both of the following tests will confirm the • Protection is afforded by vaccination with toxoid to disorder ○ raise antibody against the toxin Immunoassay of stool extract for C. difficile toxins ○ Colonoscopy showing pathologic findings of Clostridium botulinum pseudomembranous colitis • Environmental bacteria: Soil • Motile gram-positive anaerobic bacillus ANAEROBIC GRAM-NEGATIVE BACILLI • Endospores may become airborne and contaminate food preparation followed by anaerobic storage (non- Bacteroides fragilis heated soups, canning of preserves) • Spores germinate to produce one of the most lethal • General characteristics toxins in the world and result in the disease • Gram-negative anaerobic bacillus botulism • Common member of the normal gut flora CHAPTER 1 Microbiology 37

• Cause of serious infections if the normal GI mucosal • Resistant barrier is breached ○ Strains are not inhibited by the usually achiev- • Can be carried to virtually any organ of the body via able systemic concentrations of the agent with bloodstream normal dosage schedules • Often found in coinfections with facultative ○ Clinical efficacy is unlikely anaerobes • Standard for all methods • The organism does not have a characteristic gram- • All testing is performed from a pure culture negative endotoxin • Bacterial suspensions are made in comparison to a turbidity standard ○ 5 105 CFU/mL broth dilution Other Gram-Negative Anaerobic Bacilli ○ 1 104 CFU/mL agar dilution ○ 1 108 CFU/mL diffusion Fusobacterium Species • Has spindle-shaped morphology • Found in respiratory and GI tracts ANTIMICROBIAL AGENTS • Found in mixed infections • Definitions Prevotella melaninogenica • Antibiotic: A chemical substance produced by a • Regular bacillus morphology microorganism that has the capacity to inhibit the • Found in respiratory and GI tracts growth of or kill other microorganisms • Cause of lung and dental infections • Antimicrobial: An agent that kills or suppresses • Grows a black-pigmented colony growth of microorganisms Porphyromonas Species • Antimicrobial effects • Regular bacillus morphology • Bacteriostatic agents prevent replication but do not • Purple-pigmented bacilli on agar kill their target • Mouth and genitourinary tract ○ Tetracyclines, macrolides, sulfonamides • Head, neck, and pleuropulmonary infections and peri- • Bacteriocidal agents result in cell death odontal disease ○ b-Lactams, vancomycin, fluoroquinolones • Mechanisms of antimicrobial action • Cell wall inhibitors ANTIMICROBIAL SUSCEPTIBILITY • Cell membrane inhibitors TESTING METHODS • Protein synthesis inhibitors ○ Ribosomes • Definitions • Other metabolic pathway inhibitors • Minimal inhibitory concentration (MIC) ○ Folate metabolism ○ Quantitative measure of the susceptibility of a • Cell wall synthesis inhibitors bacterial isolate to an antimicrobial agent • The b-lactams ○ The lowest concentration observed to inhibit ○ Penicillin growth of the isolate in vitro ○ Cephalosporins • Minimal bactericidal concentration (MBC) • Carbapenems and monobactams ○ Not commonly performed ○ Imipenem and meropenem ○ The lowest concentration of that antibiotic to kill ○ Aztreonam the bacterial isolate in vitro • Vancomycin • Definitions: breakpoints • Penicillins: Mechanism of action • Susceptible • Binds to penicillin-binding proteins ○ Organism can be inhibited by achievable serum • Stops transpeptidation (cell wall cross-linking) or tissue levels at the dosage of antimicrobial • Cell wall develops weak spots and bursts agent recommended for that type of infection • Bactericidal, synergistic with aminoglycosides ○ Favorable outcome • Works only on growing cells • Intermediate • Cephalosporins: Generation determined by the spec- ○ MICs approach usually attainable blood or tis- trum of activity sue levels • Carbapenems ○ Strains may be inhibited by certain antimicrobial • Imipenem/cilastatin, meropenem, doripenem agents in body sites where drugs may be • Broad spectrum, low MICs, but expensive concentrated • Problem with CNS toxicity in imipenem if overdosed § Macrolides and respiratory tract • Mechanism of action ○ Provides a buffer zone that prevents technical fac- ○ Same as other b-lactams tors from causing discrepancies in interpretations ○ Stable to many b-lactamases 38 CHAPTER 1 Microbiology

§ Except metallo–b-lactamases (e.g., S. malto- • Clindamycin philia and B. cepacia) • Quinupristin/dalfopristin • Vancomycin: Mechanism of action • Linezolid • Stops gram-positive cell wall peptidoglycan chain • Inhibitors of intermediate metabolism formation • Trimethoprim • Bactericidal for staphylococci and streptococci • Sulfa compounds • Bacteriostatic for enterococci • Trimethoprim/sulfamethoxazole • Mechanism of resistance • Mechanism of action ○ Gram-negative organisms are usually resistant ○ Stops folate synthesis ○ Enterococci modify the target site § VRE MICROBIOLOGY LABORATORY SAFETY: ○ Staphylococci have shown resistance STANDARD PRECAUTIONS • Cell membrane inhibitors • Polymyxin • Assume all patients are infectious for HIV, HBV, or • Colistin other blood-borne pathogens • Amphotericin • Limit access to the laboratory to trained • Polymyxin B, Colistin personnel only • Hydrophobic proteins that disrupt the gram- • Use barrier precautions at all times negative cell membrane • Gloves, masks, goggles, coats or gowns where • Active only against gram-negative bacteria indicated • Highly toxic: Renal, neurologic, nausea, vomiting, • Leave personal protective equipment in laboratory diarrhea and out of public areas • Rarely used • Thoroughly wash hands and other skin surfaces after • Useful for P. aeruginosa and other resistant gram- gloves are removed and immediately after any negative organisms contamination • DNA/RNA inhibitors • Use particular care with handling and disposal of • Fluoroquinolones sharps • Metronidazole • Rigorously follow needle stick policies • Nitrofurantoin • Refrain from • Rifampin • Eating, drinking, smoking, application of cosmetics • Fluoroquinolones • Insertion or removal of contact lenses • Ciprofloxacin, levofloxacin • Nail biting or pen or pencil chewing • Fluoroquinolones: Mechanism of action • Mouth-pipetting • Inhibit DNA gyrase and/or topoisomerase • General laboratory safety ○ Prevents DNA unwinding and blocks DNA • Threat: Chemicals; Defense: Active chemical synthesis hygiene plan, labeling and storage standards, train- • Bactericidal and concentration dependent ing, material data safety sheets (MSDS), disposal, • Not synergistic with other antibiotics use of fume hoods, spill kits and procedures, barrier • Metronidazole: Mechanism of action protection, eye wash stations • Damages DNA and other molecules directly • Threat: Fire—Defense: Training, extinguishers, • Resistance escape plan, elimination of open flames ○ Reduced uptake and metabolism, increasing in • Threat: Electrical—Defense: Active program of anaerobes checks and maintenance • Rifampin: Mechanism of action • Threat: Gas cylinders—Defense: Chaining into • Prevents RNA synthesis position in well-ventilated area, transport with ○ Inhibits the DNA-dependent RNA polymerase secure dollies • Bactericidal • Threat: Radiation—Defense: Radiation safety pro- • Intracellular activity grams, monitoring exposure among operators • Active gram-positive organisms ○ Mutation rate of this enzyme is high • Protein-synthesis inhibitors CERTIFICATION PREPARATION QUESTIONS • Macrolides ○ Erythromycin, clarithromycin, azithromycin, For answers and rationales, please see Appendix A. dirithromycin 1. Spores are found in select groups of bacteria. Which of • Aminoglycosides the following statements describes the major advan- ○ Gentamicin, tobramycin, amikacin, neomycin tage to the bacteria that possess these structures? • Tetracyclines a. Spores are resistant to heat, cold, drying, most ○ Tetracycline, doxycycline, minocycline chemicals, and boiling CHAPTER 1 Microbiology 39

b. Spores allow an organism to better control its local environment c. Spores allow bacteria to attach or adhere to host tissues d. Organisms with spores have a more efficient exchange of genetic material 2. Choose the binomial name that is correctly written. a. Staphylococcus Aureus b. Staphylococcus species aureus c. Staphylococcus aureus d. Staphylococcus aureus 3. Fermentation end-products are often used to aid in the identification of bacteria. Fermentation results in which of the following? a. Conversion of glucose to pyruvate FIGURE 1-1 (Courtesy Joel Mortensen, PhD. See also color plate 1.) b. Lactic acid, mixed acids, alcohols, CO2 production c. CO2 and water c. Many cells, many gram-positive cocci in pairs and d. Specific teichoic acids chains 4. The exchange of cellular DNA between two living d. More than 25 epithelial cells, probable oral con- bacterial cells that involves an intercellular bridge tamination, suggest recollect is which of the following processes? 9. 85% N2, 10% H2,5%CO2 is the environmental a. Transformation condition that best suits which type of organism? b. Transduction a. Aerobes c. Plasmidization b. Anaerobes d. Conjugation c. Capnophiles 5. Transduction is defined as which of the following? d. Microaerophiles a. The change of the bacterial genotypes through 10. Whichmediumcanbedescribedascontainingbilesalts the exchange of DNA from one cell to another and dyes (bromothymol blue and acid fuchsin) toselec- b. An internal change in the original nucleotide tively slow the growth of most nonpathogenic gram- sequence of a gene or genes within an organism’s negative bacilli found in the gastrointestinal tract genome and allow Salmonella spp. and Shigella spp. to grow? c. The process by which genetic elements such as a. Thayer-Martin plasmids and transposons excise from one geno- b. MacConkey mic location and insert into another c. PEA (phenylethyl alcohol) d. A mechanism that is mediated by viruses, by which d. Hektoen DNA from two bacteria may come together in one 11. Choose the group of bacteria that is described as cell, thus allowing for recombination catalase-positive, gram-positive cocci that grow fac- 6. A mordant that is applied after the primary stain to ultatively anaerobic and that form grapelike clusters. chemically bond the alkaline dye to the bacterial cell a. Neisseria spp. wall is which of the following? b. Rothia (Stomatococcus) spp. a. Safranin c. Staphylococcus spp. b. Crystal violet d. Micrococcus spp. c. Gram’s iodine 12. The slide coagulase test is a rapid screening test for d. Gram’s decolorizer the production of which of the following? 7. Which of the following bacteria should be considered a. Clumping factor important pathogens when reading gram-stained b. Free coagulase smears of soft tissue abscess? c. Extracellular coagulase a. Streptococcus pneumoniae d. Catalase b. Neisseria gonorrhoeae 13. The first identification test performed on a clinical c. Pseudomonas aeruginosa isolate of gram-positive, catalase-positive cocci d. Staphylococcus aureus should be which of the following? 8. The most appropriate interpretation of a gram- a. Penicillin test stained smear of a sputum specimen would be b. Gram stain which of the following? (gram-stained smear, c. Oxidase test 400.) d. Coagulase test a. Few epithelial cells, many PMNs 14. The Staphylococcus sp. that is more likely to cause b. Inadequate specimen, do not culture for anaerobes uncomplicated urinary tract infections in 40 CHAPTER 1 Microbiology

nonhospitalized hosts, especially sexually active b. They are a quick way to rule out streptococcal young women, is which of the following? pharyngitis and avoid giving antibiotics when a. Staphylococcus saprophyticus not needed b. Staphylococcus aureus c. They are always very sensitive and specific for c. Staphylococcus epidermidis streptococcal pharyngitis d. Staphylococcus intermedius d. They are a quick and accurate way to diagnose 15. The toxic shock syndrome toxin-1 is an important vir- bacterial and viral pharyngitis ulence factor in staphylococcal disease. This toxin is 22. The hemolysis of this Streptococcus spp. would best classified into which of the following groups of toxins? be described as which of the following? a. Cytolytic toxin b. Leukocidin c. Phospholipase d. Enterotoxin 16. Mannitol salt agar is selective and differential for which group of organisms? a. Staphylococcus spp. b. Enterococcus spp. c. Gram-positive cocci d. Streptococcus spp. 17. Within 5 hours of returning home from lunch at your most favorite fast food restaurant you feel very sick and are vomiting. Which of the following is the most likely causative organism? a. Staphylococcus aureus b. Vibrio parahaemolyticus c. Shigella sonnei d. Escherichia coli 18. The bacterial species that can be described as susceptible to bile and optochin, a-hemolytic, a major cause of bacterial meningitis, and often carrying an antiphagocytic capsule is which of the following? a. Enterococcus faecalis b. Streptococcus pneumoniae FIGURE 1-2 (Courtesy Joel Mortensen, PhD. See also color plate 2.) c. Streptococcus pyogenes d. Streptococcus agalactiae 19. The bacterial species that can be described as suscep- a. b-Hemolysis tible to penicillin and bacitracin, b-hemolytic, a major b. g-Hemolysis cause of bacterial pharyngitis, and often carrying an c. a-Hemolysis antiphagocytic M protein is which of the following? d. k-Hemolysis a. Enterococcus faecalis 23. Enterococcus spp. can be differentiated from most b. Streptococcus pyogenes Streptococcus spp. by which of the following tests? c. Streptococcus agalactiae a. Growth in presence of 6.5% salt d. Viridans streptococci b. Production of catalase 20. The bacterial species that can be described as able to c. Production of coagulase hydrolyze hippurate, b-hemolytic, a major cause of d. Growth on PEA medium neonatal meningitis and sepsis, and producer of the 24. A pure culture of a b-hemolytic Streptococcus sp. CAMP factor is which of the following? recovered from a leg ulcer gave the following a. Streptococcus pneumoniae reactions: b. Streptococcus pyogenes CAMP test¼Negative Hippurate hydrolysis¼Negative c. Streptococcus agalactiae Bile esculin slant¼No growth 6.5% Salt¼No growth d. Viridans streptococci PYR¼Negative Bacitracin¼Resistant 21. The rapid antigen detection methods for throat Optochin¼Resistant SXT¼Sensitive swabs used for screening patients for streptococcal pharyngitis can be best described by which of the fol- Which of the following is the most likely identifica- lowing statements? tion of this organism? a. They can be useful in quickly identifying most a. Streptococcus pyogenes cases of streptococcal pharyngitis b. Streptococcus agalactiae CHAPTER 1 Microbiology 41

c. Enterococcus faecalis 29. A skin lesion was opened and drained in surgery. The d. Streptococcus sp., not groups A, B, or D culture was positive for a gram-positive bacillus, 25. The ability to grow well at refrigerator temperatures is which gave the following growth characteristics a characteristic of which of the following organisms? and biochemical reactions: a. Mycobacterium gordonae MacConkey agar: No growth Catalase: Negative b. Listeria monocytogenes H2S on TSI: Positive Growth of blood agar, nonhemolytic c. Erysipelothrix Nonmotile No spores d. Bacillus cereus 26. A catalase-positive, gram-positive bacillus that is not These reactions are consistent with which of the acid-fast, does not branch, and does not form spores following organisms? could possibly belong to which group of bacteria? a. Listeria spp. a. Corynebacterium b. Group B b Streptococcus b. Bacillus c. Erysipelothrix spp. c. Nocardia d. Corynebacterium spp. d. Mycobacterium 30. Which of the following sets of tests provide the best 27. A throat culture was taken from a 6-year-old differentiation of Erysipelothrix from Listeria boy with a gray pseudomembrane covering his oro- monocytogenes? pharynx. A catalase-positive organism was isolated a. Gram-stained smear, oxidase, and optochin on cysteine-tellurite medium and subcultured to b. Gram-stained smear, catalase, and motility Tinsdale medium, where it grew as black colonies c. CAMP test, hydrogen sulfide production, esculin with brown halos. A Gram stain was performed on hydrolysis these colonies. Which of the following cellular mor- d. Reverse CAMP, gram-stained smear, b-hemolysis phologies was most likely seen? 31. Neonatal meningitis is an uncommon but sig- a. Gram-positive branching bacilli nificant disease. Two important causes of b. Gram-positive cocci in short chains this disease may be somewhat difficult to diff- c. Gram-positive bacilli in irregular clublike erentiate on preliminary observation. Which of shape the following sets of tests provide the best differen- d. Gram-positive cocci in grapelike clusters tiation of Streptococcus agalactiae from Listeria 28. A blood culture is positive for gram-positive bacilli monocytogenes? that gave the following growth characteristics and a. Gram-stained smear, oxidase, and optochin biochemical reactions: b. Gram-stained smear, catalase, and motility MacConkey agar: No growth Catalase: Positive c. CAMP test, hydrogen sulfide production,

H2S on TSI: negative Growth of blood agar, nonhemolytic b-hemolysis Nonmotile No spores d. Reverse CAMP, gram-stained smear, b-hemolysis 32. Which of the following tests is important as a part of These reactions are consistent with which of the the genus identification or as part of a preliminary following organisms? identification but is not used as a confirmatory iden- a. Listeria spp. tification of Bacillus anthracis? b. Group B b Streptococcus a. Demonstration of a capsule c. Erysipelothrix spp. b. Demonstration of spore formation d. Corynebacterium spp. c. Positive PCR test d. Lysis of the strain by specific bacteriophages 33. Bacillus anthracisandBacillus cereus can be differentiated in the laboratory by a variety of different test results. Which of the following sets of tests best differentiate these two species? a. Catalase and glucose fermentation b. Motility and lecithinase production c. Oxidase and b-hemolysis on 5% sheep blood agar d. Motility and b-hemolysis on 5% sheep blood agar 34. Which of the following specimens would be best for identifying Bacillus cereus as the cause of an outbreak of food poisoning? a. Blood b. Rectal swabs c. Stool samples FIGURE 1-3 (Courtesy Joel Mortensen, PhD. See also color plate 3.) d. Food 42 CHAPTER 1 Microbiology

35. A first morning sputum sample is received for acid- 40. The gram-negative bacillus that can be described as fast culture. The specimen is centrifuged, and the sed- oxidase-negative, nitrate-positive, indole-negative, iment is inoculated on two Lowenstein-Jensen slants citrate-positive, methyl red–positive, urease- that are incubated at 35 C with 5% to 10% CO2. negative, and H2S-positive is most likely which of After 1 week, the slants show abundant growth over the following? the entire surface. Stains reveal gram-negative bacilli. a. Klebsiella pneumoniae Which of the following should be done to avoid this b. Salmonella enteritidis problem? c. Escherichia coli a. Use a medium specifically designed for the d. Shigella sonnei growth of AFB 41. The swarming gram-negative bacillus that can be b. Dilute out the sediment before inoculation with described as oxidase-negative, nitrate-positive, saline indole-negative, and H2S-positive is mostly likely c. Decontaminate the specimen with NALC– which of the following? sodium hydroxide mixture a. Proteus aerogenes d. Incubate the tubes at room temperature to retard b. Proteus vulgaris bacterial growth c. Proteus mirabilis 36. A patient recently arrived in the United States from d. Escherichia coli Africa presents with a long-standing cutaneous 42. Profuse watery diarrhea (“rice water stools”), lead- lesion, which is cultured for bacteria, fungi, and ing to dramatic fluid loss, severe dehydration, and AFB. An AFB smear is made and is reported as pos- hypotension that frequently leads to death, is the itive for AFB. After 8 weeks of culture on both non- hallmark of which toxin activity? selective and selective AFB media, no colonies a. Cholera toxin appear. Which of the following organisms should b. Enteric endotoxin be suspected? c. Shiga toxin a. M. kansasii d. Toxin A b. M. tuberculosis 43. The selective medium thiosulfate citrate bile c. M. leprae saltssucrose(TCBS)agarisespeciallyformu- d. M. avium-intracellulare complex lated for isolating which pathogen from stool 37. The mycobacterial species that occur in humans and cultures? belong to the M. tuberculosis complex include which a. Vibrio spp. of the following? b. Salmonella spp. a. M. tuberculosis, nontuberculous Mycobacteria, c. Shigella spp. M. bovis, and M. africanum d. Plesiomonas spp. b. M. tuberculosis, M. gordonae, M. bovis BCG, 44. The majority of human infections with Cam- and M. africanum pylobacter spp. are caused by which of the c. M. tuberculosis, M. bovis, M. avium, and M. following? intracellulare a. Direct contact with carriers of the bacterium d. M. tuberculosis, M. bovis, M. bovis BCG, and M. b. Contamination of food, milk, or water with africanum animal feces 38. The Runyon system of classification is based on c. Multiplication of the organism in food products which of the following? d. Direct contact with persons infected with the a. Colony and microscopic morphology bacterium b. Biochemical characteristics 45. In the test for urease production, the presence of c. Growth rate and colonial pigmentation the enzyme hydrolyzes urea to which of the d. All of the above are correct following? 39. In identification of mycobacterial isolates, the Tween a. Ammonia and CO2 80 test involves which of the following? b. Putrescine a. An enzyme that is able to produce Tween c. Amines and CO2 80 from certain ingredients found in the d. Amines and water medium 46. The bacterial isolate on XLD agar shown in the b. Lipase that is able to hydrolyze polyoxyethylene image was isolated from a routine stool culture. sorbitan monooleate into oleic acid and polyox- Which of the following genera and species is the most yethylated sorbitol likely identification for this organism? c. The metabolism of niacin to nicotinic acid by a. Klebsiella pneumoniae enzymatic action b. Salmonella enteritidis d. Testing the isolate for susceptibility to c. Shigella sonnei Tween 80 d. Serratia marcescens CHAPTER 1 Microbiology 43

The organism is most likely which of the following? a. Klebsiella sp. b. Shigella sp. c. Salmonella sp. d. Escherichia coli 50. The best specimen for the isolation of Bordetella pertussis is which of the following? a. Throat swabs b. Sputum c. Nasopharyngeal aspirates d. Anterior nose swab 51. Organisms belonging to the genus Brucella are best described by which of the following statements? a. Gram-positive diplococci FIGURE 1-4 (Courtesy Joel Mortensen, PhD. See also color plate 4.) b. Gram-positive diphtheroid bacilli c. Gram-negative coccobacilli 47. The bacterial isolate shown below on CIN agar was d. Gram-negative bacilli isolated from a routine stool culture. Which of the 52. Serum samples collected on a patient with pneumo- following genera and species is the most likely iden- nia demonstrate a rising antibody titer to Legionella. tification for this organism? A bronchoalveolar lavage sample was collected and revealed a positive DFA test for Legionella, but no organisms were recovered from this specimen when it was cultured on the appropriate medium and incubated for 2 days at 35 CinCO2. Which of the following is the best explanation? a. Culture was not incubated long enough b. Antibody titer c. Specimen was incubated at the wrong temperature d. Positive DFA test result is a false positive 53. Of the following media, which provides the NAD necessary for the growth of Haemophilus spp.? a. 5% sheep blood agar b. Brain heart infusion agar c. Chocolate agar d. Nutrient agar 54. Performing the factor requirement test for Haemo- philus involves which of the following processes? FIGURE 1-5 (Courtesy Joel Mortensen, PhD. See also color plate 5.) a. Inoculation of unsupplemented media with a light suspension of the organism and placement of factors X and V disks on the agar surface a. Shigella flexneri b. Inoculation of liquid media, unsupplemented and b. Salmonella enteritidis supplemented with factors X and V c. Yersinia enterocolitica c. Detecting the presence of enzymes that convert a- d. Escherichia coli aminolevulinic acid (ALA) into porphyrins 48. Decarboxylation of the amino acids lysine, ornithine, d. Growth of the organism in the presence of bacte- and arginine results in the formation of which of the rial species that produce X and V factors as met- following products? abolic by-products a. Ammonia 55. Of the asaccharolytic, oxidase-positive bacilli that do b. Urea not grow on MacConkey agar, which one is among c. CO2 the HACEK group of bacteria known to cause sub- d. Amines acute bacterial endocarditis? 49. An organism was inoculated into a TSI tube and gave a. Eikenella corrodens the following reactions: b. Weeksella virosa Alkaline slant c. Pseudomonas maltophilia Acid butt d. Sphingomonas paucimobilis H2S: Not produced 56. Which of the following statements best completes the Gas: Not produced following thought: Presumptive identification of an 44 CHAPTER 1 Microbiology

oxidase-positive, gram-negative diplococcus on c. Fermenter Thayer-Martin medium from genital sites of a 6- d. Nonviable year-old female as Neisseria gonorrhoeae? 61. The oxidase test is a critical test when attempting to a. Provides the physician with quick and reliable identify nonfermenting gram-negative bacilli. This results at minimal cost test is designed to determine the presence of which b. May sometimes be incorrect, and a repeat culture of the following? should be collected a. b-Galactosidase c. May sometimes be incorrect and should not be b. Cytochrome oxidase reported until confirmed c. Glucose oxidizing enzymes d. Should be done only when venereal disease is d. Oxygen suspected 62. The blood culture of a patient with a central venous 57. The bacterial species that can be described as oxidase- catheter yielded a gram-negative bacillus growing on positive, glucose-positive, maltose-positive, sucrose- MacConkey agar with the following reactions: negative, lactose-negative, and a major cause of bac- Oxidase¼Negative Motility¼Positive terial meningitis is most likely which of the following? Glucose oxidative- Maltose oxidative-fermentative a. Neisseria meningitidis fermentative open¼Positive (strong) b. Neisseria gonorrhoeae open¼Positive (weak) c. Streptococcus pneumoniae Catalase¼Positive Esculin hydrolysis¼Positive d. Viridans group Streptococcus 58. The bacterial species that can be described as oxidase- Which of the following is the most likely identifica- positive, glucose-positive, maltose-negative, sucrose- tion of this organism? negative, lactose-negative, and a major cause of a. Burkholderia cepacia venereal disease is most likely which of the following? b. Pseudomonas aeruginosa a. Neisseria meningitidis c. Acinetobacter baumannii b. Neisseria gonorrhoeae d. Stenotrophomonas maltophilia c. Streptococcus pyogenes 63. Which organism is associated with the disease d. Viridans group Streptococcus Melioidosis? 59. Organisms belonging to the genus Neisseria are a. Burkholderia ralstonia described as which of the following? b. Burkholderia pseudomallei a. Gram-positive diplococci c. Burkholderia mallei b. Gram-negative diplococci d. Burkholderia cepacia c. Gram-negative coccobacilli 64. Differentiation of Stenotrophomonas maltophilia d. Gram-negative bacilli and Burkholderia cepacia is best accomplished by 60. The following were observed when the Hugh-Leifson which of the following tests? oxidative-fermentative test was performed on a bac- a. Oxidase test terial isolate. Which of the options below best b. Maltose and glucose medium describes the organism’s reaction? c. Tyrosine-enriched heart infusion agar a. Oxidizer d. Growth at 42 C b. Nonoxidizer 65. The respiratory culture of a patient with cystic fibrosis yielded a gram-negative bacillus with the following reactions: Oxidation Fermentation Oxidase¼Positive Motility¼Positive neg neg Glucose oxidative-fermentative Gelatin open¼Positive hydrolysis¼Positive Soluble green pigment on TSA slant Arginine dihydrolase¼Positive Growth at 42 C¼positive

Which of the following is the most likely identification of this organism? a. Burkholderia cepacia b. Pseudomonas aeruginosa c. Acinetobacter baumannii d. Stenotrophomonas xylosoxidans 66. Which test group best differentiates Acinetobacter FIGURE 1-6 (Photograph by Dr. WH Ewing, courtesy the Centers for Disease Control and Prevention, Public Health Image Library, baumannii from Pseudomonas aeruginosa? http://phil.cdc.gov/. See also color plate 6.) a. Oxidase, motility, nitrate reduction CHAPTER 1 Microbiology 45

b. Growth on MacConkey agar, catalase, nitrate has now spread to include his trunk. The medical team reduction has identified a list of possible organisms. Which of c. Growth on blood agar, oxidase, catalase the following is the most likely cause of this infection? d. TSI, urea, motility a. Q Fever 67. Which of the following sets of results represent the b. Ehrlichiosis most common reactions for Moraxella catarrhalis c. Rocky Mountain spotted fever when tested in CTA sugar tubes? d. Cat scratch disease a. Glucose: Negative; Maltose: Negative; Lactose: 74. Which of the following is a stage of venereal syphilis Negative; Sucrose: Negative that is characterized by the appearance of a chancre? b. Glucose: Positive; Maltose: Negative; Lactose: a. Primary syphilis Negative; Sucrose: Negative b. Secondary syphilis c. Glucose: Positive; Maltose: Positive; Lactose: c. Late syphilis Negative; Sucrose: Negative d. Tertiary syphilis d. Glucose: Positive; Maltose: Negative; Lactose: 75. Which of the following is a nontreponemal serologic Positive; Sucrose: Negative test in which soluble antigen particles are coalesced 68. A soluble, bright green pigment can be produced by to form larger particles that are visible as clumps Pseudomonas aeruginosa. This pigment is known as when they are aggregated by antibody? which of the following? a. Nontreponemal flocculation (NTF) a. Pyoverdin b. Fluorescent treponemal antibody absorption b. Pyocyanin (FTA-ABS) test c. Pyorubin c. Venereal Disease Research Laboratory d. Pyophena (VDRL) test 69. A small portion of a colony of a gram-negative bacilli d. T. pallidum particle agglutination (TP-PA) test was smeared onto a filter paper test system. One per- 76. A patient in a rural area of Massachusetts had a 5-cm cent tetramethyl-p-phenylenediamine dihydrochlor- red rash with an expanding margin on his back. The ide was added. At 10 seconds, a dark purple color lesion was obvious for approximately a month and developed where the colony was added to the paper. then resolved. Several weeks later, the patient experi- Which of the following statements best describes the enced episodes of partial facial paralysis and painful test results? joints. Which of the following is the most likely infec- a. Positive indole test tious agent in this case? b. Positive oxidase test a. Borrelia hermsii c. Positive urea test b. Borrelia burgdorferi d. Positive esculin test c. Leptospira interrogans 70. Characteristics of Mycoplasma and Ureaplasma d. Spirillum minor include which of the following? 77. A 16-year-old, sexually active patient comes to his a. They exhibit the presence of a thin gram-positive– physician’s office because of a circular, 1-cm lesion like cell wall with no cell membrane in the groin area which is ulcerated but not painful. b. They demonstrate rapid growth on MacConkey A rapid plasma reagin test is performed and is reac- agar, slow growth on basic nutrient agar tive with a titer of 1:16. Culture and gram-stain c. The have only a cell membrane with no cell wall smear results from an exudate of the lesion are neg- d. They exhibit rapid growth on MacConkey ative. Which of the following is the most likely cause medium and routine blood agar plates of this lesion? 71. Which of the following is a cause of nongonococcal a. Chlamydia trachomatis urethritis? b. Neisseria gonorrhoeae a. Mycoplasma hominis c. Treponema pallidum b. Mycoplasma pneumoniae d. Haemophilus ducreyi c. Ureaplasma urealyticum 78. The gram-stained smear shows an organism isolated d. Mycoplasma orale from a blood culture after bowel surgery. Under 72. Which of the following is the most sensitive method anaerobic incubation conditions, it grew as smooth, for the diagnosis of Chlamydia trachomatis? white, nonhemolytic colonies. The organism was not a. Cytology inhibited by colistin, kanamycin, or vancomycin and b. Culture hydrolyzed esculin. The most likely identification of c. Nucleic acid amplification this isolate is which of the following? d. Serologic testing a. Fusobacterium nucleatum 73. An 8-year-old boy from Oklahoma presents with a b. Fusobacterium varium 3-day history of fever, headache, and muscle aches. c. Bacteroides fragilis A rash first noted this morning on his ankles and wrists d. Prevotella melaninogenica 46 CHAPTER 1 Microbiology

c. Expression of results is by MIC for both d. The cost of the test is similar per drug 84. Which of the following clinical indications would most benefit from having quantitative (MIC) testing rather than qualitative (Sensitive, Intermediate, Resistant catagories) data from the laboratory? a. Urinary tract infection b. Bacterial meningitis c. Pneumonia caused by Mycoplasma d. Streptococcal pharyngitis 85. When performing antimicrobial susceptibility testing, the following definition of the minimum inhibitory concentration (MIC) is correct: a. The highest concentration of an antibiotic in a FIGURE 1-7 (Photograph by Dr. VR Dowell, Jr, courtesy the Centers dilution series that inhibits growth for Disease Control and Prevention, Public Health Image Library, b. The lowest concentration of an antibiotic in a http://phil.cdc.gov/. See also color plate 7.) dilution series that inhibits growth c. The lowest concentration of an antibiotic in a 79. Pseudomembranous colitis caused by Clostridium dilution series that kills the bacteria difficile is best confirmed by which of the following d. The lowest concentration of the antibiotic obtain- laboratory findings? able in the patient without toxicity a. Presence of the toxin in stool 86. In comparing quantitative MIC dilution testing to b. Isolation of C. difficile from stool qualitative agar disk diffusion testing, the higher c. Gas production in thioglycolate media the MIC of the drug for that organism: d. Gram stain of stool showing many gram-positive a. The smaller is the zone of inhibition bacilli b. The more susceptible the organism will appear on 80. Lecithinase production, double zone hemolysis on disk diffusion sheep blood agar, and gram-stained morphology c. The larger is the zone of inhibition are all useful criteria in the identification of which d. The more toxic is the drug to the patient of the following? 87. In a quality control (QC) procedure on a new a. Clostridium perfringens batch of Mueller-Hinton plates using a standard b. Streptococcus agalactiae QC stock strain of Staphylococcus aureus, the disk c. Escherichia coli inhibition zone sizes for three of the drugs tested d. Clostridium tetani were too small and fell below the expected QC range. 81. When activating a hydrogen and carbon dioxide gen- Which of the following is the most likely reason for erator system used for creating an anaerobic atmo- this observation? sphere, which of the following is an indication that a. These three antibiotic disks were outdated and the catalyst and generator envelope are functioning had lost potency properly? b. These three disks were faulty in that the antibiotic a. A decrease in temperature of the jar content was too high b. Bubble formation on the surface of the plates c. Bacterial suspension of Staphylococcus was prob- c. A change in color of the methylene blue indicator ably contaminated with another organism d. The formation of a visible cloud of gas d. The plates received insufficient incubation time 82. In a clinical specimen, the presence of sulfur granules 88. Which of the following definitions best fit the term strongly indicates the presence of which anaerobic urethritis? bacterium? a. Infection and or inflammation of the terminal a. Bacteroides fragilis portion of the lower urinary tract b. Actinomyces spp. b. The isolation of a specified quantitative count of c. Fusobacterium nucleatum bacteria in an appropriately collected urine spec- d. Clostridium tetani imen obtained from a person without symptoms 83. Which of the following are a common element or signs of urinary infection between using the E test and agar disk diffusion c. Dysuria, frequency, and urgency but yielding fewer (Kirby-Bauer) for antimicrobial susceptibility organisms than 105 colony-forming units of bacte- testing? ria per milliliter (CFU/mL) urine on culture a. Both establish an antibiotic gradient in agar d. Inflammation of the kidney parenchyma, calices b. Both create a circular zone of bacterial (cup-shaped division of the renal pelvis), and inhibition pelvis CHAPTER 1 Microbiology 47

89. The organism most commonly associated with otitis c. Macrophages media infections is associated with which of the fol- d. Squamous epithelial cells lowing positive test results? 95. Which of the following terms is used to describe an a. Coagulase increase of lymphocytes and other mononuclear cells b. VP (pleocytosis) in the cerebrospinal fluid and negative c. Optochin bacterial and fungal cultures? d. Bacitracin a. Meningoencephalitis 90. Which organism is most often responsible for b. Aseptic meningitis impetigo? c. Encephalitis a. Staphylococcus epidermidis d. Meningitis b. Streptococcus pyogenes 96. The culture of which sample routinely uses quantita- c. Enterococcus faecalis tion or the counting of bacterial cells present to assist d. Streptococcus agalactiae in the interpretation? 91. How do staphylococci spread so easily when infect- a. Blood ing the skin? b. Sputum a. They produce hyaluronidase, which hydrolyzes c. Urine hyaluronic acid present in the intracellular d. Abscess ground substance that makes up connective tissue 97. Gram staining and reading a glass slide with a mixed b. They produce lipase, which melts the fat under smear of Staphylococcus and Escherichia coli along the skin, making it easier to spread with each Gram staining run of specimens examined c. The hemolysins kill the white and red blood cells; within the microbiology laboratory that day is an then the protease liquefies the skin protein, allow- example of which of the following? ing easy penetration for the bacteria a. Quality assurance (QA) activity d. All of the above b. Quality control (QC) activity 92. Routine culture media for use with a specimen of c. National regulatory activity cerebrospinal fluid should include which of the fol- d. Office of Safety and Health Administration activity lowing sets of media? 98. Tracking the rate of skin organism contamination a. 5% sheep blood agar, Lowenstein Jensen agar, among a laboratory’s blood culture results on a 7H9 agar monthly basis and introducing specific training to b. 5% sheep blood agar, thioglycolate broth phlebotomists when rates exceed the norm would c. 5% sheep blood agar, MacConkey agar, be an example of which of the following? Sabourad dextrose agar a. Good laboratory practice d. 5% sheep blood agar, chocolate agar, thioglyco- b. Quality control late broth c. Universal standards 93. A college student is examined at the emergency d. Quality assurance department; he is disoriented with a fever, intense 99. Which of the following statements best defines headache, stiff neck, vomiting, and sensitivity “infectious substances”? to light. His friends say that he has been sick for a. Articles or substances capable of posing a risk to about 2 days and that his condition worsened over safety the last 3 hours. The physician does a complete b. Substances known or reasonably expected to blood count (CBC) and electrolytes. The electro- contain pathogens lytes are normal, but the patient’s white blood c. Patient samples containing bacteria count (WBC) is 12,000 cells/L. What test should d. Samples with class 3 pathogens the doctor order next? 100. Which of the following is an example of an inappro- a. Urine culture priate specimen or condition that would warrant b. Stool culture rejection for microbiology culture? c. Cerebrospinal fluid Gram stain and culture a. A nonsterile container for a stool culture d. Blood culture b. A swab of a skin and soft tissue infection 94. What cells are found in bacterial vaginosis? c. A tissue sample for anaerobic culture a. Clue cells d. A 24-hour urine sample for bacteriology b. Lymphocytes culture 48 CHAPTER 1 Microbiology

SELF-ASSESSMENT

Content Area: ______

Score on Practice Questions: ______

List the specific topics covered in the missed questions:

List the specific topics covered in the correct questions: CHAPTER 1 Microbiology 49

NOTES CHAPTER 2 Mycology, Virology, and Parasitology

Linda J. Graeter and Joel E. Mortensen

MYCOLOGY • Molds: Obligate hyphae • Yeasts: Unicellular, budding The Fungal Organism • Dimorphic: Two bodies or forms • Mycology terms • A group of nonmotile eukaryotic organisms that have • Perfect fungi definite cell walls, are devoid of chlorophyll, and ○ Sexual stage is known reproduce by means of spores (and conidia) • Fungi imperfecti • Heterotrophic ○ No known sexual stage • Hetero means “different,” and troph means • Conidia “nourishment” ○ Reproductive structures produced by an asexual • Eukaryotic (fungi) versus Prokaryotic (bacteria) mode • Capsule • Spore • Polysaccharide ○ Reproductive structures produced sexually, and ○ Much larger than bacterial capsule the asexual reproductive cells of the zygomycetes • Antiphagocytic, virulence • Conidiophore: Structure that supports conidia • Mostly in yeast ○ Annelloconidia: Produced by annellids ○ Cryptococcus neoformans: Encapsulated yeast ○ Phialoconidia: Produced by phialide • Fungal cell wall ○ Poroconidia: Produced from pores • Antigenic • Sporangium: Saclike structure where sporangio- • Multilayered spores are formed (Zygomycetes) ○ Polysaccharides (90%) • Asexual reproduction ○ Chitin • Arthroconidia: Directly from hyphae by modifica- ○ Proteins and glycoproteins (10%) tion of cell wall (barrels) • Provides shape and rigidity to cell • Blastoconidia: Budding of cell (mother and ○ Osmotic protection daughter) • Cell membrane • Chlamydoconidia: Directly from hyphae (swelling) • Bilayered phospholipids • Sexual reproduction • Sterols (ergosterol versus cholesterol) • Ascospore • Functions ○ Sexual spore formed in a saclike structure after ○ Protects cytoplasm meiosis ○ Regulates intake of nutrients • Zygospore ○ Facilitates capsule and cell wall synthesis ○ Round, thick-walled spore produced in a saclike • Cytoplasm structure by fusion of two hyphal tips • Nucleus, nucleolus, nuclear membrane, endoplas- • Basidiospore mic reticulum, mitochondria, vacuoles ○ Spore formed in a club-shaped reproductive • Mycology terms structure after meiosis • Hypha (plural: hyphae): Filamentous, tubular • Mycosis (mycoses) growth • Invasive treatments ○ True hyphae versus pseudohyphae • Immunosuppressive therapy • Septate (aseptate): Cross walls in hyphae • Immunocompromising infections • Mycelium (plural: Mycelia) ○ Human immune deficiency virus/acquired ○ Vegetative immunodeficiency virus (HIV/AIDS) ○ Aerial 50 CHAPTER 2 Mycology, Virology, and Parasitology 51

• Rise in common and uncommon mycoses • Systemic mycoses ○ Organisms and tissue infected ○ Any tissue • Five broad categories of fungal infections ○ Four organisms • Opportunistic fungi § “True” or “primary” pathogens ○ Immunocompromised patients ○ Endemic to specific geographic areas ○ Many different tissues § Must travel through the area to become infected ○ Ubiquitous: Environmental saprobes ○ Thermal dimorphs and yeasts ○ Monomorphs § “Two bodies” based on temperature § Same structural characteristics under all ○ Blastomyces, Coccidioides, Histoplasma, conditions Paracoccidioides ○ Aspergillus, Candida, Mucor, Rhizopus • Superficial mycoses BASIC CLINICAL MYCOLOGY ○ Infections of outer, “dead” layers ○ No host defense stimulation • Specimen collection ○ No pain or discomfort • Important factors in isolating and identifying a fun- ○ Usually treated because the infection is gal pathogen “unsightly” ○ Correct type of specimen ○ Exophiala, Malassezia, Piedraia, Trichosporon ○ Quality of specimen • Dermatophytic mycoses ○ Rapid transport ○ Skin, hair, nails ○ Use of appropriate culture media § Deeper than the superficial • Processed within 2 hours § Still no living skin penetration • Specimen transport ○ Produce secondary metabolites that irritate • Sterile, leak-proof container ○ Host defense causes itching ○ Dermatologic requires dry container ○ Sometimes cutaneous and superficial grouped ○ No transport media ○ Epidermophyton, Microsporum, Trichophyton • Processed within a few hours • Subcutaneous mycoses • Specimens can be refrigerated at 4 C ○ Muscle, bone, connective tissues ○ Only if processing is delayed ○ Traumatic inoculation ○ Blood and cerebrospinal fluid (CSF): 30 to 37 C § Thorns, scratch ○ Dermatologic: 15 to 30 C ○ Usually remain localized • Safety in the mycology laboratory ○ Cladosporium, Exophiala, Pseudallescheria, • Standard precautions Phialophora, Sporothrix ○ No smoking, eating, drinking, or applying cosmetics ○ Contact lenses (no removing or cleaning) TABLE 2-1 Major Medically Important Fungi ○ No mouth pipetting • Universal precautions Category Genus • Class 2 or 3 biosafety hoods Opportunistic fungi Aspergillus • Disinfectant – Phenol based Candida • Biohazard containers Mucor • Specimen processing: Methods Rhizopus • Direct inoculation Superficial mycoses Exophiala ○ Adding several drops of specimen to media Malassezia ○ For solid media, the specimen can be streaked Piedraia ○ Specimen types: Bronchial brush/wash, aspi- Trichosporon rates, CSF, swabs, body fluids, hairs, scrapings Dermatophytic mycoses Epidermophyton • Microsporum Concentration ○ Trichophyton Large volumes can be concentrated by Subcutaneous mycoses Cladosporium centrifugation Exophiala ○ Specimen types: Body fluids, CSF, urines Pseudallescheria • Minced (homogenized) Phialophora ○ Some solid specimens must be “destroyed” to Sporothrix expose a buried pathogen to the media Systemic mycoses Blastomyces ○ Specimen types: Nails, tissues, biopsies Coccidioides • Culture of fungi Histoplasma • Petri dishes or tubes Paracoccidioides ○ Oxygen requirements 52 CHAPTER 2 Mycology, Virology, and Parasitology

• Humidity • Dermatophyte test medium (DTM) • Subculture sometimes necessary ○ Dermatophytes from heavily contaminated spec- ○ Temperature range imens (pink-to-red color change) ○ Dimorphism ○ Commonly used in office practices ○ Sexual and asexual developmental structures • Media for subculture ○ Get rid of bacterial contamination ○ Potato dextrose agar (PDA) • Teasing needles ○ Potato flake agar (PFA) ○ Used more than bacteriologic loop ○ Incubation • Electric incinerator § Obligate filamentous: 25 or 37 C ○ Flame causes aerosols § Dimorphics: 25 and 37 C • Culture is very important § Yeast: 25 or 37 C ○ Can be main identification ○ Aerobic ○ No or few biochemicals ○ 3 to 4 weeks § Yeasts are the exception ○ Cornmeal agar for yeast morphology • Culture media § Recommended for promoting sporulation • Options • Pathogen versus contaminant ○ Test tubes for primary • The clinical picture § Less likely to become contaminated, less ○ Are patients’ symptoms consistent with fungal drying infection? ○ Petri dishes for subculture ○ Does this fungus normally cause these § Larger surface area for growth symptoms? • Use of inhibitory substances may be required • Laboratory findings ○ Chloramphenicol, gentamicin, cycloheximide ○ Fungal elements in tissue or other specimen ○ May encounter some fungal inhibition ○ Fungus grown in culture • Common media ○ More than one culture positive • Sabouraud dextrose agar (SDA) • Quality control ○ Most common, many fungi grow • Assessing quality of specimens ○ Emmon’s modification: Less glucose • Monitoring performance § Blastomyces dermatitidis ○ Tests, reagents, media, instruments • Mycosel and mycobiotic ○ Quality control culture collections ○ SDA+chloramphenicol+cycloheximide • Personnel ○ Selective recovery of dimorphs and ○ Performance evaluation dermatophytes ○ Proficiency testing • Brain heart infusion (BHI) agar • Laboratory identification ○ Enriched to enhance recovery C. neoformans of • Direct examination of clinical specimens and dimorphic transitions in Sporothrix and ○ Laboratory methods and tissue stains Paracoccidioides • Macroscopic/microscopic evaluation ○ Plates or tubes ○ Colony features and hyphae/conidia mor- ○ Broth+penicillin for Zygomycetes phology • BHI+gentamicin+chloramphenicol • Advanced methods ○ C. neoformans from contaminated specimen ○ Exoantigen, DNA probes, DNA sequencing • Sabouraud dextrose+BHI (SABHI) • Microscopic examination ○ Strengths of both ○ Direct examination can be used on several types ○ Enriched medium for Cryptococcus spp., ther- of specimens mally dimorphic fungi, etc. § Can identify yeast and filamentous forms • CHROMagar Candida § Culture is used regardless ○ Selective and differential for presumptive ○ Several preparations for direct examination identification of genus Candida from primary § Potassium hydroxide (KOH) preparation plates § Calcofluor white ○ Morphology and colors of the yeast colonies § India ink: Historical vary by species • KOH preparation ○ Candida albicans—light to medium green; ○ Examine hair, nails, skin scrapings, fluids, exu- Candida tropicalis—light blue to metallic-blue; dates, and biopsy specimens Candida krusei—light rose with a whitish ○ Can see important fungal elements border § Hyphae, yeast • Inhibitory mold agar (IMA) § Need reduced light or phase-contrast ○ Inorganic salts, chloramphenicol, gentamicin ○ 15% KOH added to specimen ○ Inhibits bacteria § Dissolves specimen quickly (fungi slowly) CHAPTER 2 Mycology, Virology, and Parasitology 53

§ Can be modified to include calcofluor white § Transferred to microscope slide § Binds to cell wall and fluoresces blue-white § Slide culture under ultraviolet light § Organism subcultured to a small piece • India ink of agar ○ Historically used with CSF specimens § Covered with a coverslip ○ Negative stain § Organism grows onto coverslip: Remove § Creates black background to visualize capsu- and examine lar material § Best method § C. neoformans • Examination of molds § More specific/sensitive tests are now available ○ Use one of the three methods listed previously, § Cryptococcal antigen test followed by addition of stain • Tissue examination: Stains § Lactophenol cotton blue (LPCB) ○ Giemsa, Wright-Giemsa • Dermatophyte identification § Histoplasmosis capsulatum (intracellular) ○ Hair perforation ○ Hematoxylin and eosin (H&E) § 5- to 10-mm sterile hair floated on sterile § Pink to pinkish-blue water and yeast extract ○ Meyer’s mucicarmine § Conidia or hyphae inoculated onto water § C. neoformans: Rose red surface ○ Gomori methenamine silver (GMS) § Remove hair shafts and observe in LPCB § Black weekly for 1 month ○ Papanicolaou stain § Trichophyton rubrum negative, Trichophyton § Pink to blue mentagrophytes positive ○ Periodic acid–Schiff (PAS) ○ Urease test § Red or purple § Tubes of urease agar are lightly inoculated • Macroscopic examination § 5 days at room temperature ○ Growth conditions § T. rubrum negative or weak, T. mentagro- § Yeasts: 2 to 3 days phytes positive § Molds ○ Trichophyton agars § Rapid: Less than 5 days § Originally numbers 1 to 4 § Intermediate: 6 to 10 days § Most laboratories use only 1 and 4 § Slow: More than 11 (sometimes 8 weeks) § Thiamine requirement § Dimorphism § Trichophyton agar 1 (without thiamine) ○ Pigment and Trichophyton agar 4 (with thiamine) § Front versus back of plate § 10 to 14 days, observe for growth ○ Texture ○ Rice grain growth § Dictated by presence and length of aerial hyphae § Sterile, nonfortified rice grain media § Glabrous: Leathery, waxy § 10 days, observe for growth § Velvety: Suede, plush § Microsporum canis versus Microsporum § Yeastlike: Looks like Staphylococcus audouinii § Cottony: Fluffy • Summary: Mold identification § Granular: Powdery • Specimen source or infection ○ Topography • Growth rate to reproductive structures § Rugose • Colony color front and back on plate § Radial grooves, “folded” • Microscopic morphology § Crateriform ○ Septate or aseptate hyphae § Central depression and raised edge ○ Conidiophore structure § Verrucous ○ Microconidia/macroconidia § Rough knobs ○ Other structures § Cerebriform • Advanced techniques § Brainlike ○ Exoantigen test • Examination of molds § Rapid information of immunoidentity ○ Three methods § Extract soluble antigen from unknown § Tease/cut preparation isolate § Organism removed directly from culture § Concentrate plate § React with antiserum specific to known § “Teased” apart with teasing needles fungi § Scotch tape preparation § Positive control necessary for definitive § Scotch tape pressed onto culture plate identification 54 CHAPTER 2 Mycology, Virology, and Parasitology

§ Test is read at 24 hours § Other agents can form germ tubes § Blastomyces, Coccidioides, Histoplasma § Not valid if read after 2 hours ○ DNA probe § “True” germ tube: C. albicans § Rapid kits that use nucleic acid hybridization § No constriction at base, where the tube to identify fungi in culture attaches to the mother cell § Highly specific to each fungus, because it is § A constricted base indicates C. tropicalis based on DNA sequence § Other species have germ tubes § Needs to be performed on cultured organisms § Candida stellatoidea (Sucrose assimilation § Not from specimens used to differentiate from C. albicans) § Developed for Coccidioides, Blastomyces, § Candida dubliniensis (no growth at 45 C) Histoplasma § Positive and negative controls are necessary § Specialized clinical laboratories are using ○ Fermentation/Assimilation DNA sequencing techniques to establish fun- § Fermentation gal identifications § Carbohydrate use in absence of oxygen • Laboratory identification of yeast § Assimilation • Macroscopic morphology § Which can be used as a sole carbon source? ○ Colony color and texture § Two systems (assimilation) ○ Color: White, tan, pink, salmon § API 20C (others): Strip test § Can have dematiaceous yeasts § Vitek: Automated ○ Texture: Mucoid, butterlike, velvety, wrinkled ○ Urea hydrolysis • Microscopic morphology: Wet preparation § Detected on simple urea agar ○ Hyphae § Rapid, easy ○ Pseudohyphae § Differentiates Cryptococcus from Rhodotorula ○ Blastoconidia § Positive: Pink • Cornmeal Tween 80 agar § Negative: Little to no change ○ Encourages development of chlamydospores ○ Temperature studies ○ Relationships among hyphae, pseudohyphae, § Cryptococcus spp. and others § Weak growth at 35 C and no growth at ○ Clear media: Can be observed under light 42 C microscope § Candida spp. ○ Specific organisms associated with specific § Several can grow well exceeding 45 C morphology • Order of events • Cornmeal agar morphology ○ Most yeasts ○ Used in conjunction with carbohydrate usage § Wet preparation ○ Four main morphology types § Germ tube § Hyphae § Germ tube negative and from sterile site § Pseudohyphae § Corn meal morphology § Arthroconidia § Physiologic/biochemical tests § Chlamydoconidia or blastoconidia § Temperature ○ Pseudohyphae and blastoconidia only § C. krusei THE OPPORTUNISTIC MOLDS § Candida parapsilosis § Candida kefyr • Most frequently isolated fungi § C. tropicalis • Opportunistic infections ○ Blastoconidia only • Infect those who are injured or debilitated § Candida glabrata • Common inhabitant of soil and organic debris § C. neoformans • Laboratory and environmental contaminant ○ Arthroconidia § Trichosporon beigelii Acremonium Species • Physiologic tests ○ Germ tube test • No known sexual stage: Fungi imperfecti § Filamentous outgrowth from blastoconidia • Filamentous fungus in plant debris and soil § Most basic and easiest to perform • Two more common species § Requires the use of serum or plasma • Acremonium falciforme § Some commercially made broths (will last • Acremonium kiliense longer) • Cause onychomycosis, keratitis, endocarditis, menin- § Overincubation and overinoculation are big- gitis, peritonitis, and osteomyelitis gest problems • Macroscopic CHAPTER 2 Mycology, Virology, and Parasitology 55

• Rapid grower • Most strains either do not grow at all or grow • White, cottony colonies weakly at 37 C • Microscopic • Microscopic • Hyaline, septate hyphae • Arthroconidia and coarse true hyphae are • Unbranched, solitary,erect phialides formed directly observed on the hyphal tips • Blastoconidia, conidiophores, and pseudohyphae • Conidia usually in clusters or fragile chains are absent • Therapy and susceptibility testing • Undifferentiated hyphae may be present • In vitro susceptibility • Arthroconidia observed • Limited data and minimum inhibitory concentra- ○ Either rectangular or rounded at the ends tion (MIC) breakpoints have not been defined ○ Do not alternate with normal cells • Newer azoles (voriconazole, posaconazole) exhibit good in vitro activity • Itraconazole MICs somewhat higher than MICs in Paecilomyces Species voriconazole • Sexual stage described: Teleomorph • MICs of caspofungin are relatively low • Soil, decaying plants, and food products • Several species • Fusarium Species Paecilomyces lilacinus and Paecilomyces variotii most common • No known sexual stage: Fungi imperfecti • Causes wide range of mycoses • Plants and soil • Emerging opportunistic pathogen • Normal mycoflora of commodities (rice) • Onychomycosis, sinusitis, otitis media, endocardi- • More than 20 species tis, osteomyelitis, peritonitis, and catheter-related • Fusarium solani, Fusarium oxysporum, Fusarium fungemia chlamydosporum • Macroscopic • Fusariosis • Rapid grower • Emerging cause of opportunistic mycoses • P. variotii is thermophilic • Disseminated infections have high mortality • Colonies are flat, powdery, or velvety • Trauma or inhaled conidia ○ Initially white and becomes yellow, yellow- • Macroscopic green, yellow-brown, olive-brown, pink, or vio- • Rapid grower, woolly to cottony, flat, spreading let, depending on the species colonies ○ Reverse is dirty white, buff, or brown • Front: White, cream, tan, salmon, cinnamon, yellow, • May resemble Penicillium spp. macroscopically and red, violet, pink, or purple microscopically ○ Reverse: Colorless, tan, red, dark purple, • Microscopic or brown • Septate, hyaline hyphae • Microscopic • Conidiophores are often branched • Macroconidia: Two or more cells, thick walled, • Phialides are swollen at the base and taper toward smooth, and cylindrical or sickle (canoe) shaped the apice • Usually grouped in pairs or brushlike clusters • Conidia are unicellular, hyaline to darkly colored, Geotrichum Species and form long chains • Lack known sexual stage • Found worldwide in soil, water, air, sewage, plants, Penicillium Species cereals, and dairy products • Found in normal human flora • Teleomorph described • Genus includes several species • Penicillium marneffei is thermal dimorph • More common: Geotrichum candidum, Geotrichum (Southeast Asia) clavatum, Geotrichum fici • Numerous species • May cause opportunistic infections in immunocom- • More common: Penicillium chrysogenum, Penicil- promised host lium citrinum • Infections acquired via ingestion or inhalation • Particularly virulent in patients with AIDS • Macroscopic • Keratitis, endophthalmitis, otomycosis, necrotizing • Produce rapid growing, white, dry, powdery-to- esophagitis, pneumonia, endocarditis, peritonitis, cottony colonies resembling ground glass and urinary tract infections (UTIs) • Colony may be yeastlike • P. marneffei often fatal • Optimal growth temperature is 25 C • Macroscopic 56 CHAPTER 2 Mycology, Virology, and Parasitology

• Rapid growing; velvety, woolly, or cottony • Rate of growth is usually rapid • Initially white and become blue-green, gray-green, ○ Usually matures in 3 days olive-gray, yellow, or pinkish ○ Some species are slower ○ Reverse is usually pale to yellowish • Temperature • Microscopic ○ A. fumigatus grows well at 45 C • Flask-shaped phialides • Macroscopic • Form brushlike clusters ○ First white then yellow, green, brown, or black • Conidia are round, unicellular, and form unbranch- • Microscopic ing chains at the tips of the phialides ○ Hyphae are septate ○ Unbranched conidiphore from a “foot cell” ○ Vesicles Scopulariopsis Species ○ Phialides cover the surface of the vesicle entirely (“radiate” head) or partially only at the upper • Soil, plant material, feathers, and insects surface (“columnar” head) • Unique in that it contains both hyaline and dematia- ○ Phialides are either uniseriate (attached to the ceous species vesicle directly) or biseriate (attached to the • Scopulariopsis brevicaulis (hyaline) vesicle via a supporting cell) metula • Scopulariopsis cinerea (dematiaceous) ○ Conidia form radial chains • Onychomycosis, especially of the toe nails • Disseminated infections: High mortality • Macroscopic • Grow moderately rapidly, granular to powdery ZYGOMYCETES • Front color is white initially and becomes light • Zygomycetes is the name of a class of fungi brown or buff • This class includes three orders: Mucorales, Mortierel- • Reverse color is usually tan with brownish center lales, and Entomophthorales • Microscopic • Most clinically significant are in Mucorales • Septate hyphae • Absidia, Cunninghamella, Mucor, Rhizomucor, • Conidiophores are hyphae-like and simple or Rhizopus branched • Zygomycosis • Sometimes incorrectly referred to as mucormycosis • Aspergillus Species Inhalation of sporangiospores, trauma (inoculation) • Can become invasive • More than 185 species • Sinus infections are common • Approximately 20 species have been described as • Typical presentation agents of infection in humans ○ Pulmonary, rhinocerebral, cutaneous, renal, or • Most common: Aspergillus fumigatus, Aspergillus meningeal involvement flavus, Aspergillus niger • Risks • Less common: Aspergillus clavatus, Aspergillus ○ Diabetes, leukopenia, immunosuppression, AIDS, glaucus group, Aspergillus nidulans burns, intravenous drug • Clinical disease • Rapid growth • Three clinical settings ○ More tissue damage, almost always fatal ○ Opportunistic infections ○ Rapidly fill plate in culture: “Lid lifters” ○ Allergic states • Differentiation ○ Toxin production ○ Presence (or absence) and location of rhizoids— • Opportunistic infections rootlike structures ○ Local infections ○ Branched or unbranched nature of ○ Local colonization in previously developed lung sporangiophore cavity ○ Size and shape of sporangium ○ Infection of every organ system has been ○ Macroscopic of similar described ○ Disseminated infections Mucor Species • Allergic reactions ○ Allergic bronchopulmonary aspergillosis • Several pathogenic species • Toxins • Many do not grow at 37 C ○ Aflatoxin • Rapid growth, fluffy (cotton candy), white initially ○ Veterinary diseases and becomes grayish-brown in time • Laboratory Identification • Reverse is white CHAPTER 2 Mycology, Virology, and Parasitology 57

• Aseptate or sparsely septate, broad hyphae, sporangio- • Tineas: Skin phores long and branched with terminal sporangia • Piedras: Hair • No rhizoids • Nonliving layer of the skin and extrafollicular hair • Lack of systemic immune response Species • Specimens are cultured onto SDA Rhizopus • Sometimes with antibiotics • Several important species • Diagnosis • 50% of all zygomycoses • Appearance of lesion • 90% of rhinocerebral infections • Skin scrapings • Grow very rapidly, cotton-candy white initially and • Hair shafts turns gray to yellowish-brown in time • Four main infections • Reverse side is white • Tinea versicolor • Pathogenic species of Rhizopus can grow well at 37 C • Tinea nigra • Broad, aseptate hyphae, long unbranched • White piedra sporagiophores • Black piedra • Rhizoids are produced • Tinea versicolor • Malassezia furfur ○ Superficial infection of the keratinized layers Rhizomucor Species of skin ○ • Rare cause of zygomycosis Normal flora of skin (90% asymptomatic) ○ • Normally fatal No known reason for predisposition ○ • Colony similar to that of Mucor Clinical picture § • Microscopic Patches of hypopigmented or hyperpigmented • Intermediate to Mucor and Rhizopus lesions § • Short rhizoids and branched sporangiophores Brown or fawn, scaling, redness § Chest, back, shoulders, arms, abdomen (itch, burn) Absidia Species ○ Specimen: Skin § Direct examination: KOH will show yeastlike • 21 species and hyphal forms • Absidia corymbifera is the only clinically significant ○ Culture: Lipophilic organism species § Add oil overlay and incubate at 37 C • Rapid growth, woolly to cottony, and olive-gray ○ Microscopic colonies § Thick-walled hyphae and “yeast,” some • Reverse side uncolored budding • Broad aseptate hyphae, sporangiophores branched § Spaghetti and meatballs and arise in groups of two to five • Tinea nigra • Sporangiospores are one-celled and round to oval • Hortaea werneckii • Exophilia and Cladosporium werneckii Cunninghamella Species obsolete names • Central and North America, Southeast Asia, Africa, • Seven species Europe • Cunninghamella bertholletiae is the only known • Most likely environmental human and animal pathogen • Clinical disease • Rapid growing, cottony, and white to tannish-gray ○ Synonyms: Pityriasis nigra, tinea nigra ○ Reverse is pale palmaris • Aseptate or sparsely septate broad hyphae, sporan- ○ Infection of keratinized skin layers of hand giophores long and branched ○ More common in those under 25 years of age and • Vesicles females • Sporangiophores are erect and form short lateral ○ Dark skin on one hand (usually only one) branches, each of which terminates in a swollen ○ Flat, brown lesion vesicle § Possibility of melanoma must be ruled out ○ Direct examination: KOH preparation SUPERFICIAL MYCOSES ○ Culture § Required to differentiate from melanoma • Among the most prevalent of human infectious diseases • H. werneckii • Mycotic infections of hair, skin, and nails ○ Macroscopic 58 CHAPTER 2 Mycology, Virology, and Parasitology

§ Colonies grow slowly and mature within ○ Not usually required 21 days ○ Slow grower (25 C) § Initially pale in color, moist, shiny, and • Does not penetrate hair shaft yeastlike • Piedraia hortae § Colonies become velvety, olive black, and cov- ○ Macroscopic ered with a thin layer of mycelium § Colonies are slow growing § The reverse side is black § Small, folded, dark brown to black § Does not grow at 37 C § May produce a reddish-brown diffusible ○ Microscopic features pigment § Septate hyphae, yeastlike conidia, and § Reverse side is black chlamydospores ○ Microscopic § Hyaline initially and become olive colored § Septate hyphae, asci, and ascospores § Annellides present § Asci are ellipsoid, solitary, or in clusters § Annelloconidia are intercalary and lateral and contain eight ascospores § Septate, thick-walled hyphae formed § Hyphae pigmented • White piedra • Caused by Trichosporon spp. SUBCUTANEOUS MYCOSES ○ Most commonly T. beigelii ○ T. beigelii may not have taxonomic status • Four major infections caused by several fungi • infection of hair of beard and mustache • Mycetoma • Environmental (soil and air) • Chromoblastomycosis ○ South and North America, Far East, Europe • Phaeohyphomycosis • Clinical disease • Sporotrichosis ○ Soft white to tan nodules • Common to all ○ Surround hair shaft, separated easily from hair • Lesion develops at site of inoculation (localized) ○ Hair breaks at nodule • Soil saprophytes that are moderately slow growers ○ Can become systemic in immunocompromised • Most commonly accepted • Direct examination: KOH preparation • Cladophialophora, Exophiala, Fonsecaea, Phialo- • Culture phora, Wangiella, Pseudallescheira/Scedosporium, ○ Not normally required Sporothrix schenckii ○ SDA: Yeastlike colonies • Most infections are due to traumatic inoculation • Microscopic • Common in tropics and subtropics ○ Nodule surrounding hair • Some of these fungi cause more than one type of sub- • Trichosporon spp. cutaneous infection ○ Macroscopic • Most are dematiaceous fungi § Colonies are rapid growing • Dematiaceous versus hyaline § Yeastlike, may be smooth, wrinkled, • Conidiation of dematiaceous fungi raised, folded • Cladosporium type § White to cream colored ○ Resembles a tree, in which conidiophore is the § Urease production characteristic trunk and branched chains of conidia form the • Microscopic branches ○ Many pseudohyphae and hyphae • Phialophora type ○ Blastoconidia are unicellular and variable in ○ Short conidiophores+phialide, vase shaped, con- shape idia extruded from phialide and then cluster ○ Arthroconidia produced • Rhinocladiella type • Black piedra ○ Stalked conidiophores that become knobby as • Piedraia hortae conidia are produced, conidia produced sequen- • Fungal infection on hair (scalp) tially until a Cladosporium type of conidiation is • Forms black, stony, hard nodules reached • Central and South America, Southern Asia, Africa • Laboratory identification of subcutaneous fungi • Swimming in rivers and stagnant waters • Specimens collected by aspiration • Clinical picture ○ Large amount of material, reduces chances that ○ Nodules firmly attached, can be microscopic to specimen will dry out visible by naked eye, hair feels rough • Granules observed and noted • Direct examination • SDA with and without antibiotics ○ KOH preparation ○ PDA for subculture • Culture • Biochemicals are available, but rarely done CHAPTER 2 Mycology, Virology, and Parasitology 59

Mycetoma • Cladisporium carrionii obsolete name • Very slow grower (up to 30 days) • General • Colonies are gray-green to black on surface and • Chronic granulomatous disease of feet (lower reverse, cottony extremities) • Pigmented, septate hyphae ○ Madura foot or maduromycosis • Cladosporium type of conidiation • Enlarged nodules, sinus drainage, bone destruction Fonsecaea pedrosoi • Exudate contains granules • Causes chromoblastomycosis and phaeohyphomycosis • No lymphatic system involvement (remain • Traumatic injury localized) • Gray-green to black, cottony colony within 21 days • Two types: Eumycotic and actinomycotic • Pigmented, septate hyphae • Laboratory procedures • All three types of conidiation • Direct examination: KOH preparation • Phialophora, Cladosporium, Rhinocladiella • Identification granules, colorless or pigmented sep- Fonsecaea compacta tate hyphae • Conidial heads of Cladosporium type of conidiation ○ Actinomycotic granules: Mycelium with hyphae are more compact 1 mm in diameter Phialophora verrucosa ○ Eumycotic granules: Wide hyphae (2-4 mm) • Causes chromoblastomycosis and phaeohyphomycosis terminating in chlamydoconidia • Autoinoculation and lymphatic system • Macroscopic Chromoblastomycosis • Olive-green to black, velvety • Microscopic • General • Pigmented, septate hyphae • Localized disease of skin and subcutaneous tissue • Only Phialophora type of conidiation • Verrucoid (wartlike) lesions on feet, legs, hands, Pseudallescheria boydii and buttocks • Scedosporium apiospermum: Name for alternate • Soil saprophytes that are introduced by trauma asexual stage (worldwide), dematiaceous • Major etiologic agent of mycetoma in the United States • Spreads through the body lymphatics or by and Europe autoinoculation • Different from other subcutaneous • Laboratory procedures • Grows rapidly, hyaline, has a sexual form • Direct examination: KOH exudate, crusts from • Macroscopic lesion • White to brownish-gray, fluffy colonies • Microscopic: Single-celled or clusters of single cells, • Microscopic dark pigment • Hyaline, septate hyphae • Culture: SDA at room temperature, hold for • Single anelloconidia produced on an anellophore 6 weeks (conidiophore) • Looking for three types of conidiation Exophiala jeanselmei ○ Cladosporium type • Cause of mycetoma and phaeohyphomycosis ○ Rhinocladiella type • Minor trauma and contaminated fomites ○ Phialophora type • Young cultures • Phaeohyphomycosis • Appear as black yeasts • Infection of subcutaneous tissue • Mature cultures ○ Classically: Infection with a dematiaceous • Velvety colonies fungus • Sticklike conidiophores with clustered conidia ○ The others have become distinct Wangiella dermatitidis § Mycetoma • Causes pheohyphomycosis § Chromoblastomycosis • Macroscopic § Sporotrichosis • Initially resemble black yeast ○ Miscellaneous dematiaceous fungi: Introduced • Longer 10 days, olive-gray to black velvety or gla- through trauma brous colony • Systemic infection is a disease of the • Wangiella spp. grow better at 40 to 42 C immunocompromised • Microscopic • KOH preparation shows pigmented hyphae • Pigmented, septate hyphae Cladophialophora carrionii • Conidiophores are indistinguishable from vegetative • Cause of chromoblastomycosis hyphae, except that conidia are clustered at ends • No shoes, trauma • Similar morphology to E. jeanselmei 60 CHAPTER 2 Mycology, Virology, and Parasitology

Acremonium Species • Microsporum • Etiologic agent of mycetomas, corneal infections, and • Macroconida numerous, thick-walled, rough nail infections • Microconida usually present • See Opportunistic Fungi section • Epidermophyton • Macroconida numerous, thin and thick Dermatophytes walled, smooth • Microconida not formed • Dermatophytosis: Infections of keratinized tissue (hair, Microsporum audouinii skin, nails) • Anthropophilic: Person to person • Most common: Ringworm • Children • Three major genera • Positive Wood’s lamp fluorescence • Trichophyton • Rare distorted macroconidia, rare microconidia • Microsporum • Light-tan front • Epidermophyton • Reverse salmon to colorless • Intermediate to slow growers • No growth on sterile rice media • Worldwide distribution Microsporum canis • Routes of infection • Zoophilic • Defined in three ways • Wood’s lamp fluorescence ○ Geophilic: Soil to man • Skin and hair ○ Zoophilic: Animal to human • Bright yellow colony reverse ○ Anthropophilic: Person to person • Especially on PDA • Approximately 43 accepted species • Large spindle-shaped macroconidia • Types of infections • 3 to 15 cells, tapering ends ○ Tinea barbae: Facial hair • Many microconidia ○ Tinea capitis: Scalp Microsporum gypseum ○ Tinea corporis: Arms, legs, and trunk • Geophilic ○ Tinea cruris: “Jock itch” affects the groin area • Rapid grower ○ Tinea faciei: Face • Not commonly infective ○ Tinea manuum: Hands • Skin and hair ○ Tinea pedis: “Athlete’s foot” • Powdery/granular buff-to-brown colony ○ Tinea unguinum: Fingernails and toenails • “Rowboat”-shaped macroconidia ○ Hair and hair follicles • Six or fewer septa § Favic: Hair follicle, crusty lesions Microsporum nanum § Ectothrix: Colonizes outside of shaft • Zoophilic § Endothrix: Hair follicle first, growth • Flat beige, brown, or white colony inside shaft • Small macroconidia ○ Nail and nail bed • One or two septa § Onychomycosis • Rare cause of tinea corporis in humans ○ Skin Epidermophyton floccosum • Laboratory diagnosis • Anthropophilic ○ Specimens: Hair, skin scraping, nail scraping or • Tinea cruris (+ pedis, + unguium) clipping • Does not infect hair ○ KOH preparation (+ calcofluor) • No microconidia § Hair infections • Smooth-walled, club-shaped, groupings of macroconi- § Endothrix dia (beaver’s tail) § Ectothrix • Colony: Khaki-yellow § Skin and nail infections Trichophyton mentagrophytes § Septate hyphae • Anthropophilic and zoophilic ○ Wood’s lamp • Infects all three keratinized tissues • Culture • Most common cause of athlete’s foot ○ SDA with and without inhibitory agents • Buff and powdery to white, cottony colony ○ 30 C for 4 weeks • Reverse side may be yellow, brown, colorless, ○ Colony morphology or red ○ Microconidia and macroconidia • Spiral hyphae • Trichophyton • Round clustering microconidia and cigar-shaped • Macroconida rare, thin-walled, smooth macroconidia • Microconida numerous • Urease positive, perforates hair CHAPTER 2 Mycology, Virology, and Parasitology 61

Trichophyton rubrum • Clusters of blastoconidia along pseudohyphae, ter- • Anthropophilic: Person to person minal chlamydoconidia • Ectothrix, if infect hair • C. albicans CMT morphology • Most commonly infects skin and nails • Germ tube positive, sucrose positive • White fluffy colony • C. stellatoidea (sucrose negative) • Reverse side red • Macroscopic morphology • Tear-drop shaped microconidia Candida tropicalis • Can produce macroconidia • Second most common Candida spp. • Urease negative, does not perforate hair • Vaginitis, intestinal disease, systemic infections, Trichophyton tonsurans meningitis • Anthropophilic • Infections are aggressive and very difficult to treat with • Endothrix traditional antifungals • Most common endothrix dermatophyte in the • Macroscopic United States • Creamy, glabrous with mycelial fringe • “Black dot” tinea capitis • Microscopic • Beige-to-olive granular colony with brown rust edge • Blastoconidia are single or small random clusters • Size and shape variation in microconidia along pseudohyphae • Requires thiamine • C. tropicalis CMT morphology Trichophyton schoenleinii Candida parapsilosis • Anthropophilic • Major cause of nosocomial infections • Slow growing • Indwelling catheter • Endothrix • Macroscopic • Colonies orange/brown and wrinkled when young, flat • Creamy, glabrous when mature • Microscopic • No macroconidia, rare microconidia • Relatively short, crooked or curved pseudohyphae • Antler-shaped hyphae • C. parapsilosis CMT morphology Candida kreusi THE CLINICALLY SIGNIFICANT YEASTS • Rarely isolated as a cause of endocarditis and vaginitis • Macroscopic • Significant part of the normal flora • Creamy, flat colonies • Skin and mucous membranes • Microscopic • Infections are often endogenous • Pseudohyphae and elongated blastoconidia, • Opportunists branch like trees • Greater immune suppression results in a greater vari- • C. krusei CMT morphology ety of yeast infections Torulopsis glabrata • Yeasts are most frequently isolated fungi • Also referred to as Candida glabrata • Most commonly found as fungemia • Candida Species Endocarditis, meningitis, UTI • Macroscopic • Common normal flora of skin, mucosa, and • Creamy, smooth, moist digestive tract • Microscopic • Can cause many infections • Blastoconidia only (on CMT), no pseudohyphae • Vulvovaginitis, thrush, pulmonary infections, eye Saccharomyces cerevisiae infections, meningitis, endocarditis, and dissemi- • The “working yeast” nated infections • Bread, beer, wine • Opportunist • Can occasionally be normal flora • Causative agent of thrush • Increasingly isolated from immunocompromised • Indicator of immunosuppression • Macroscopic • HIV, prolonged antimicrobial therapy, and chemo- • Creamy, smooth, moist therapy: Can be serious and become disseminated • Microscopic Candida albicans • Yeast cells and short pseudohyphae • Most common cause of yeast infection Cryptococcus Species • Can cause disease in any site when host defense is • Causative agent of meningitis and pulmonary altered disease • Macroscopic • C. neoformans • Creamy • Major cause of opportunistic infection in patients • Microscopic with AIDS 62 CHAPTER 2 Mycology, Virology, and Parasitology

• Found in soil contaminated with pigeon excreta Pneumocystis jiroveci • Meningitis: Predilection for central nervous system • Group is contested • All species are surrounded by a capsule • Yeast-protozoa-fungus • Gives the mucoid colony appearance • Opportunistic • India ink detects capsule: Negative stain • AIDS • Being replaced by latex agglutination for cryptococ- • Cellular immunity cal antigen • Pneumocystis pneumonia ○ India ink has low detection rate • Fever, nonproductive cough, shortness of breath • Do not produce true hyphae or pseudohyphae on corn- • Destroys alveolar cells meal agar, blastoconidia only • Laboratory diagnosis • All species are urease positive • Must demonstrate the organism in tissue, lavage, or • Nitrate variable sputum • Phenol oxidase • Cannot culture except in animal • C. neoformans • GMS commonly used stain • Causes melanin production on caffeic acid agar or ○ “Deflated ball” bird seed agar • Fluorescent antibody available • Dark colony color • Sugar assimilation also varies DIMORPHIC FUNGI Rhodotorula Species • Bright, salmon-colored colonies • Mycoses that involve major body systems or more • Closely related to Cryptococcus than one kind of tissue • Capsule production • Some include the opportunists in this category • Urease positive • These do not need “situational” help • Some are nitrate positive • Thermal dimorphism • Not common agents of disease • Blastomyces dermatitidis • Do cause some opportunistic infections • Coccidioides immitis Geotrichum candidum • Histoplasma capsulatum • Normal flora in intestinal tract • Paracoccidioides brasiliensis • Causes rare infections in immunocompromised • Sporothrix schenckii • Macroscopic • Penicillium marneffei • White, moist, yeast-like • Clinical disease • Microscopic • Cause multiple kinds of infections, varying • True hyphae, segment into arthroconidia, no severities blastoconidia • Primary infection is pulmonary Trichosporon beigelii ○ Incidence of benign infection is far greater than • Cause of white piedra fatal disseminated disease • Personal hygiene disease ○ Immunocompetent: Asymptomatic and resolves • Emerging agent of disseminated infection spontaneously • Mostly in cancer patients ○ Common cold or flu symptoms • Produces arthroconidia and blastoconidia on • Can progress to acute or chronic disease cornmeal agar • “Granulomatous” lesions in lungs Malassezia furfur ○ Granuloma: Collection of macrophages, giant • Normal skin flora in 90% of humans cells, and proteinaceous material (wall off • Tinea versicolor infection) • Catheter-related infections in patients on long-term • Yeast forms can enter lymph through macrophage intravenous lipids and disseminate to other organ systems • Macroscopic • Disseminated infections are fast moving and • Cream/brown wrinkled normally fatal • Microscopic • Specimen processing • Yeastlike cells • Tissue specimens: Minced Sporobolomyces Species • Pleural fluid and CSF: Concentrated • Most often recovered from environmental samples • Mucus or pus: Mucolytic agent • Rarecauseofinfectioninimmunocompromisedpatients • When dimorph is suspected • Macroscopic ○ Quick transport • Salmon-colored smooth colonies ○ Do not hold at room temperature (bacteria may • Microscopic overgrow) • Oval, elongate yeast cells, projectile spores ○ Do not refrigerate CHAPTER 2 Mycology, Virology, and Parasitology 63

• Microscopic • Laboratory identification • KOH preparation: Easiest, quickest • Specimen sources ○ Calcofluor ○ Sputum • H&E, PAS, and GMS for in situ tissue staining ○ Aspirated pus from lymph node and subcutane- • Culture media ous tissue ○ Primary isolation ○ Skin scrapings and biopsies § SDA or SABHI with and without ○ Blood, urine, CSF (systemic) antimicrobials • Collection and handling ○ Incubation at 30 C ○ Aseptic, plated promptly ○ BHI is sometimes recommended for better recov- • Macroscopic morphology ery on primary culture ○ 25 C: Slow growth of mold on SDA, white-to- § PDA for subculture beige waxy colony • Laboratory ○ 37 C: Yeast appears after 10 to 15 days, on • Risk factors enriched media ○ CAN CAUSE LABORATORY-ACQUIRED • Microscopic morphology INFECTIONS ○ Mold: Fine, septate, hyaline hyphae ○ Extreme caution must be taken when these § Conidia directly on hyphae or on lateral organisms are handled conidiophores ○ Transmitted by respiratory route ○ Yeast: Hyaline, large cells, budding § All cultures handled in biosafety hood § Slide cultures should not be performed Coccidioides immitis • Diagnosis: Immunologic methods ○ Most are not strong antigens • Endemic in hot, semiarid climates § Cellular not humoral responses • Southwestern United States and northern Mexico ○ Antibody detection • Valley fever § Positive: Exposure (not necessarily infection) • Saprobe in mold form (desert soil) § Those in endemic area are positive • Small threat to immunocompetent § Most patients with AIDS patients will be • Occupational hazard negative (no Ab) • Most virulent of all agents of human mycoses • Laboratory identification • Causes mild infection in everyone who inhales it ○ Exoantigen test • Clinical disease § Detects cell free extracts of the fungus • Primary pulmonary coccidiodomycosis § Material extracted from mold phase is reacted ○ Asymptomatic and self-limiting with known antisera • Disseminated rate in immunocompromised much § More definitive identification than colony higher than that of other fungal agents morphology alone • Specimen sources • Molecular methods ○ Sputum ○ Skin scraping ○ Blastomyces dermatitidis Blood, urine • Laboratory identification • Endemic to North America • Macroscopic morphology • Mississippi River valley ○ 3 to 5 days on SDA/SABHI • Gilchrist’s disease ○ Arthroconidia in 7 to 10 days • Most likely a soil saprobe ○ Colonies are white and cottony (cobwebs) • Found in wood, tree bark, rotting vegetation, ○ Yeast form not found in laboratory river banks § Spherules can be experimentally formed • Biggest threat to immunocompromised • Microscopic morphology • 12 cases of laboratory-acquired disease ○ Septate hyaline hyphae • Blastomycosis § Wide arthroconidia: Barrel shaped • Chronic granulomatous disease affecting lungs, § Disjuncture cells skin, and mucous membranes ○ Cultures are extremely hazardous because of • Chronic cutaneous blastomycosis many arthroconidia ○ Ulcerated lesions ○ Exposed or mucocutaneous tissues Histoplasma capsulatum • Systemic blastomycosis ○ Involves any organ: Bone lesions and osteomye- • Worldwide litis are often encountered • Endemic to Mississippi River and Ohio River valleys 64 CHAPTER 2 Mycology, Virology, and Parasitology

• Soil saprobe with high nitrogen content (chicken, bird, • Laboratory identification and bat guano) • Specimen sources • Spelunker’s disease ○ Exudates and pus from lesions • Mostly occupational hazard ○ Tissue biopsy • Clinical disease • Macroscopic • Histoplasmosis ○ Mold in 3 to 5 days at 25 C ○ Chronic granulomatous lung disease ○ Mature colonies are dark and flat ○ 5% progress to an acute fulminating, rapidly ○ Yeast at 37 C (white or tan) fatal disease (mostly in children) • Microscopic ○ Organism found in macrophages ○ Mold: Delicate thin hyphae, septate, frequently • Patients with AIDS are at high risk found as ropes, conidiophores produce multiple • First found in histiocytes (histo) conidia in flowerets arrangements ○ No actual capsule (capsulatum) ○ Two types of conidia • Laboratory identification § Small oval, unicellular conidia • Macroscopic § Large, dark walled spheres ○ Slow growing mold § Yeast: Cigar shaped at 37 C ○ Tan, fluffy colonies ○ Yeast form in 10 to 15 days on enriched media FUNGUS-LIKE BACTERIA • Microscopic ○ Fine septate hyphae, microconidia and • Actinomycetes macroconidia • Three major genera ○ Macroconidia become tuberculate with age • Actinomyces, Nocardia, and Streptomyces ○ Yeast cells bud at narrow neck • Others: Rhodococcus, Actinomadura, and Nocardiopsis • All higher bacteria: Paracoccidioides brasiliensis • Thought to be fungi for years • • Endemic to northwest, central, and southern South Some species form aerial mycelia in culture • America, Central America, and southern Mexico Clinical manifestations are similar to those of systemic • Soil saprobe of acid soil fungal infection • Causes paracoccidioidomycosis • Actinomyces are anaerobic, Nocardia and Streptomy- • Asymptomatic and self-limiting ces are aerobic • • Can disseminate to other tissues Nocardia stain partially acid-fast, Actinomyces and • Can cause cutaneous disease Streptomyces are not acid-fast • • Specimen: Same All genera may produce granules, Actinomyces almost • Laboratory identification always produce granules • Macroscopic ○ Mold colony mature in 2 to 3 weeks Actinomyces Species ○ Flat, white colonies • Gram-positive obligate anaerobes ○ Yeast will form on enriched media at 37 C • Reside in the mouth and in the intestinal tract • Microscopic • ○ Form abscesses and swelling at site of infection Fine, septate, hyaline hyphae • ○ Diagnosis can be made by direct microscopy Conidiation absent on modified SDA • ○ Yellow sulfur granules: Bacterium and its waste Yeast form: Multiple thin-necked buds (mariner • Actinomyces israelii (most common), but several other wheel) bacteria in this genus are capable of causing disease

Sporothrix schenckii Nocardia Species • Found worldwide (soil saprobe) • Nocardiosis • Sometimes grouped with subcutaneous mycoses • Ubiquitous soil saprophytes • Organism is also a thermal dimorph • Route: Inhalation, direct inoculation • Occupational risks • Nocardia asteroides most common • Gardening: “Rose gardener disease” • Nocardia brasiliensis • Clinical disease • Most important in tropical areas • Sporotrichosis • Cutaneous infection with normal immune function ○ Chronic cutaneous and subcutaneous mycosis • 70% of cases are seen in immunocompromised characterized by ulcers and abscesses along lym- • Nocardiosis phatic channels • Generally: Immunocompromised population CHAPTER 2 Mycology, Virology, and Parasitology 65

• Nocardia may colonize the respiratory tract • Sometimes partially acid-fast ○ Immunocompetent individuals with compro- • Does not hydrolyze mised pulmonary function ○ Casein, xanthine, tyrosine ○ Pneumonia can disseminate • No branching on tap water agar § Kidney, skin, gastrointestinal (GI) tract, and brain are common targets Species • Laboratory identification Streptomyces • Gram-variable/modified acid-fast bacilli positive • Streptomyces griseus (found in soil) • Strictly aerobic • Musty smell • Filamentous and branching • Nonpathogenic • May be isolated on routine media • Forms colony in 3 to 5 days at 35C ○ Colonies usually form within 4 days • Waxy, white powdery top § May require up to 2 to 4 weeks • Gram-positive filamentous bacilli ○ Nocardia spp. can be difficult to isolate by culture • Non–acid-fast § Faster growing organisms may overgrow • Aerial, tertiary branching on tap water agar • Colony morphology • Hydrolyzes casein, xanthine, and tyrosine • Colonies smooth and moist or have a “moldlike,” gray-white, waxy or powdery appearance • Distinct, strong mildew odor Actinomadura Species • Microscopic • Eight reported species, two more common • Usually gram-variable or “beaded” appearance • Actinomadura madurae ○ Alternating gram-positive and gram-negative • Actinomadura pelletieri segments along a filament • Causes mycetoma • Nocardia under suboptimal conditions appears uni- • Found only in tropics formly gram-negative • Gram-positive filamentous bacilli • Modified Ziehl-Neelsen or Kinyoun acid-fast stain • Non–acid-fast ○ Nocardia organisms are acid-fast with these • Aerial, tertiary branching on tap water agar modified staining procedures • Hydrolyzes casein and tyrosine ○ Tests based on acid-fastness alone are not reliable for differentiation • Differentiation of Nocardia spp. Nocardiopsis Species • Tap water agar morphology • Soil saprophyte ○ Differentiate Nocardia spp. and other aerobic • Similar to Streptomyces and Actinomadura actinomycetes • Thermotolerant ○ Nocardia spp. have recursively branching • Grows at higher temperatures hyphae with aerial hyphae • Very rare case of mycetoma • Biochemical characteristics ○ Hydrolysis of casein, tyrosine, or xanthine • N. asteroides VIROLOGY ○ Does not hydrolyze casein, xanthine, or tyrosine • N. brasiliensis • Properties of viruses ○ Hydrolyzes casein and tyrosine • Small • Molecular identification • DNA or RNA, not both • Replicate or multiply on their own • Rhodococcus equi Intracellular • Replication is directed by viral nucleic acid • Soil saprophyte • Lack genes and enzymes necessary for energy • Associated with domestic farm animals production • Causes pulmonary infection that resembles tuberculosis • They depend on the machinery of the host cell for • Pneumonia that spreads to brain, liver, spleen protein and nucleic acid production • Opportunistic • Some are capable of inducing cancerous growth in • AIDS, transplants, Hodgkin’s lymphoma, lym- animals and culture phoma, and leukemia ○ Hepatitis B: Liver cancer • Laboratory identification • Characteristics of a typical virion • Colony forms in 2 to 4 days • Either DNA or RNA, single or double stranded • Glistening, smooth, pink to red ○ In contrast to eukaryotes and prokaryotes, • Gram-positive coccobacillus viruses do not contain both 66 CHAPTER 2 Mycology, Virology, and Parasitology

• A capsid or protein coat Clinical Virology: Culture Method • Enveloped viruses have an outer envelope com- • posed of lipids and polysaccharides 1937: Propagated yellow fever virus in chick embryos • • Important definitions Successfully produced an attenuated vaccine • • Capsid: Protein shell Influenza vaccine still produced in eggs • • Nucleocapsid: Nucleic acid+capsid Growth of viruses • • Capsomeres: Structural units of capsid Chicken embryos: Historical • • Envelope: Lipid membrane around nucleocapsid in Tissue explants: Research use only • some viruses Cell culture ○ ○ Stolen from cell: Essential for infectivity Primary cell culture ○ • Virion: Complete virus particle Diploid cell lines ○ • Characteristics used to classify viruses Continuous cell lines • • Type of genetic material (either DNA or RNA) they Primary cell cultures • contain Derived directly from donor (animal or human) • • Size and shape of the assembled virus Most common are kidney cells ○ • Presence or absence of an envelope Rhesus monkey, rabbit kidney • • Type of host that it infects One or two passages • • Type of disease produced Diploid cell line • • Taxonomy/nomenclature Prepared from animal tissues ○ • International Committee on Taxonomy of Viruses Usually fibroblasts from lung or foreskin • divides all viruses to families (-viridae) Terminally differentiated, postmitotic • • Subfamilies (-virinae) Limited to 20 to 50 passages • • Genera (-virus) Continuous cell lines • • Species or virus name Single cell type that can be propagated indefinitely • • Above the family level, orders (-virales) may be used Do not resemble the cell of origin • • http://www.virustaxonomyonline.com Often abnormal in chromosome morphology • Other viruslike things and number • • Prion Derived from tumors or mutagenic treatment of pri- ○ Proteinaceous infectious particle mary cell culture • ○ Structures that replicate through conversion of Unlimited passages • other host proteins Care and feeding of cells • ○ Exact mechanisms of action and reproduction Cells are usually in tubes or in shell vials ○ are unknown Flasks are used for some applications • ○ Transmissible spongiform encephalopathy In tubes, the cells are on the “down” side • § Scrapie Cells need to be fed or refed with appropriate § Kuru medium • § Creutzfeldt-Jakob disease (CJD) Tubes and vials are read using an inverted § Bovine spongiform encephalopathy (BSE): microscope • Mad cow disease Cytopathic effects (CPEs) • • Replication of virus particles CPEs can take a variety of forms ○ • Attachment Rounding up and detachment ○ ○ Specific cell receptor Cell lysis ○ ○ Responsible for varying cell tropism Swelling of nuclei ○ • Penetration Formation of fused cells termed syncytia • ○ Virus passing through cell membrane Induction time varies among viral agents • ○ May take cell membrane as protection Hemadsorption • • Uncoating Certain viruses produce hemagglutinin • ○ Removes all or part of the capsid Hemagglutinin binds erythrocytes ○ ○ Exposes the nucleic acid Human type O ○ • Biosynthesis Chicken ○ ○ Proteins, nucleic acids, and other components Guinea pig • ○ Some made in tremendous excess Hemadsorption performed on cell cultures from • Morphogenesis respiratory specimens • ○ Components assembled Fluorescent antibody tests • ○ Often uses enzymes encoded by virus Used for confirmation of CPEs • • Release Some can be used on patient samples • ○ Budding through membrane Examples include ○ ○ Lysis of membrane Cytomegalovirus (CMV) CHAPTER 2 Mycology, Virology, and Parasitology 67

○ Respiratory viruses ○ Vesicle fluid ○ Herpes simplex virus (HSV) ○ HSV, varicella-zoster virus (VZV) ○ Enterovirus groups ○ Skin scrapings: Papillomavirus, molluscum • Shell vial cultures contagiosum • Cells grown on coverslips in the bottom of a vial • Disadvantages with electron microscopy (shell vial) ○ Expensive equipment • Patient specimens added to vial and centrifuged to ○ Expensive maintenance “drive” the virus into the cell ○ Require experienced observer • Incubated and stained ○ Sensitivity often low • Time to positive usually much less than with con- • Nucleic acid amplification ventional tube cultures • Allows the amplification of specific target DNA • Used for several different agents sequences by a factor of approximately 106 ○ CMV • PCR is the most common method but other varia- ○ Herpes simplex and varicella-zoster tions are being developed and used ○ Respiratory viruses • Detection of the PCR product usually has been by ○ Chlamydia agarose gel electrophoresis, probe hybridization, • Some systems contain mixed cell types or DNA sequencing • Real-time PCR has streamlined the amplification and the detection processes Clinical Virology: Nonculture Methods • Advantages of PCR • Extremely high sensitivity • Direct examination • Fast turnaround time for real-time PCR • Antigen detection • Nucleic acid amplification ○ Immunofluorescence, enzyme-linked immuno- • Disadvantages of PCR sorbent assay (ELISA), etc. • Extremely liable to contamination • Electron microscopy • High degree of operator skill required ○ Morphology of virus particles • Not easy to set up a quantitative assay ○ Immune electron microscopy • A positive result may be difficult to interpret • Light microscopy • Serologic tests for viral infections ○ Histologic appearance • Classic techniques ○ Inclusion bodies ○ Complement fixation tests ○ Limited to pathology ○ Hemagglutination inhibition tests • Antigen detection ○ Immunofluorescence techniques • Enzyme immunoassay (EIA) ○ Counter-immunoelectrophoresis ○ Plate-based assays • Older techniques ○ Lateral flow technology ○ Radioimmunoassay • Nasopharyngeal aspirate • Newer techniques ○ Respiratory syncytial virus (RSV) ○ Enzyme-linked immune assay (EIA) ○ Influenza ○ Particle agglutination • Stool ○ Western blot ○ Rotavirus ○ Recombinant immunoblot assay ○ Adenovirus ○ Lateral flow “rapid” devices • Advantages and disadvantages • Serology • Advantages • Primary infection ○ Result available quickly, point-of-care testing ○ Fourfold rise titer of immunoglobulin G (IgG) or • Potential problems total antibody between acute and convalescent ○ Reduced sensitivity compared to cell culture or sera PCR: 40% to 80% ○ Presence of IgM ○ Labor intensive ○ A single high titer of IgG ○ Not well suited to a core laboratory • Reinfection • Electron microscopy ○ Fourfold or greater rise in titer of IgG or total • 106 virus particles/mL required for visualization antibody between acute and convalescent sera • Approximately 50,000 to 60,000 magnification ○ Absence or slight increase in IgM normally used • Use of serologic results • Original method to find many viruses • Disease dependent • Viruses may be detected in the following specimens ○ Rubella and hepatitis A ○ Feces: Rotavirus, adenovirus, noroviruses, astro- § Clinical symptoms coincide with antibodies virus, calicivirus § Detection of IgM or rise in IgG: Disease 68 CHAPTER 2 Mycology, Virology, and Parasitology

○ Respiratory and diarrhea viruses may cause dis- TABLE 2-2 Medically Important Viruses ease before antibody rise § RSV or influenza Groups Virus Families § Serologic diagnosis would be retrospective Major groups of DNA viruses ○ Adenoviridae HIV produces clinical disease months or years Hepadnaviridae after seroconversion Herpesviridae ○ Antibody: Definitive diagnosis Papillomaviridae • Some infections can be detected only by serology Parvoviridae ○ Bartonella Polyomaviridae • Limitations of serology Poxviridae • Long period of time required using paired sera Major groups of RNA viruses Arenaviridae ○ Extensive antigenic cross-reactivity Astroviridae ○ HSV and VZV Bunyaviridae ○ Japanese B encephalitis and dengue Caliciviridae ○ CMV and Epstein-Barr virus Coronaviridae Filoviridae • Immunocompromised patients often have reduced Flaviviridae or absent immune response Orthomyxoviridae ○ Late AIDS Paramyxoviridae • Patients with infectious mononucleosis or diseases Picornaviridae such as systemic lupus erythromatosus may react Reoviridae nonspecifically Retroviridae • Transfusion may give a false positive result because Rhabdoviridae of the transfer of antibody Togaviridae • Guidelines for selecting and collecting specimens for virology • Culture only infected sites • Polyomaviridae • Collect and send tissue or fluid • Poxviridae Adenoviruses • Do not use swabs • Send fluid in its original container/syringe • Double-stranded DNA (dsDNA), replicate in nucleus • Collect and send as much specimen as possible • Icosahedral, nonenveloped • Tools of the trade • 51 serotypes • Swabs • Causes respiratory disease, eye infections, and GI ○ Swab for skin lesion disease ○ Nasopharyngeal swabs from the nasopharynx • Can cause cancer in animals ○ All must go into viral transport medium Hepadnavirus • Viral/chlamydial culture transport media • dsDNA with a short single-stranded region ○ Supplied by the laboratory • Icosahedral core with envelope ○ Usually stored refrigerated • Human: Hepatitis B ○ Inoculate and send on ice • Two important antigens • Nasal wash kit • Surface Ag ○ Sterile saline included • Core Ag ○ Use for rapid virus tests and nucleic acid • Virus not isolated in culture amplification test • Serologic test ○ Use for culture by transfer to vial transport • Hepatitis medium or directly • Cirrhosis • Specimen labeling • Hepatocelluar carcinoma • The specimen must be labeled accurately and • Transmission through blood completely • Transfusion • Indicate exactly what the specimen is • Drug abuse • Sexual contact Major Groups of DNA Viruses • Vaccine • Engineered • Adenoviridae • Infants at birth, health care workers • Hepadnaviridae Family Herpesvidae • Herpesviridae Herpesviruses • Papillomaviridae • dsDNA, replicate in nucleus • Parvoviridae • Icosahedral nucleocapsid, envelope CHAPTER 2 Mycology, Virology, and Parasitology 69

• 120- to 200-nm diameter • Transient in normal individuals • Most prominent feature: Latency • In immunocompromised • Herpes simplex virus • Can be severe • Two types • Blood hemoglobin decreases and cannot recover ○ Cold sores • Not isolated in culture ○ Genital • Detected by molecular assays • Skin and mucous membrane infections Poxviruses • Encephalitis • dsDNA, replicate in cytoplasm of cell • Isolated easily in culture • Largest viruses (400250 nm) Varicella-Zoster Virus • Complex construction • Chickenpox and shingles • Brick shape, lipid envelope • Vaccine important in controlling outbreaks • Resistant to environmental factors • Shingles return • Smallpox: Variola • Tzanck stain: Giant cells • Eradicated by the World Health Organization • Isolation more difficult than HSV • Stocks in Atlanta and Moscow Cytomegalovirus • Good vaccine and no animal reservoir • Isolated from blood, urine, throat • Vaccinia • In adults: Syndrome similar to mononucleosis, may • Used to immunize against smallpox infect kidney (shed in urine) • Rare zoonotic • In immunocompromised: Kidney, eye, lung, often fatal • Africa • Laboratory tests • Others • Shell vial culture • Serology Major Groups of RNA Viruses Epstein-Barr Virus • Heterophile-positive infectious mononucleosis (85%) • Arenaviridae • Can produce tumors • Astroviridae • Not isolated in culture • Bunyaviridae • Serologic diagnosis • Caliciviridae • Early antigen • Coronaviridae • Viral capsid antigen: IgM and IgG • Filoviridae • Nuclear antigen • Flaviviridae Human Herpesvirus 6, 7, and 8 • Orthomyxoviridae • Human herpesvirus–6 • Paramyxoviridae • Exanthema subitum/roseola infantum • Picornaviridae • Sixth disease • Reoviridae • Spread by respiratory route • Retroviridae • Molecular assays used for detection • Rhabdoviridae • Human herpesvirus–7 • Togaviridae • Cause a small percentage of roseola Arenaviruses • Human herpesvirus–8 • ssRNA, enveloped • Found in Kaposi’s sarcoma • Helical capsid symmetry Papillomavirus • Lymphocytic choriomeningitis virus • dsDNA • Benign aseptic meningitis • Over 100 types of human (HPV) • Lassa fever virus: Africa • Infect skin and mucous membranes • Junin and Machupo viruses: South America • Causes • Rodent reservoir, greater than 50% mortality • Warts • Biosafety level 4 containment • Cervical cancer Bunyaviruses • Laryngeal carcinoma • ssRNA, enveloped • No culture: Molecular assays are used • Helical capsid symmetry Parvoviruses • La Crosse encephalitis virus • Single-stranded DNA (ssDNA), nucleus replication • Mouse host, mosquito vector • Small nonenveloped • Encephalitis • Human parvovirus B-19 • Hantaviruses • Erythema infectiosum (fifth disease) • Mouse host • Infects bone marrow cells (erythrocyte) • Respiratory infection • Causes aplastic crisis • Not isolated in the laboratory 70 CHAPTER 2 Mycology, Virology, and Parasitology

Calicivirus • Can be isolated in culture • ssRNA, nonenveloped ○ Subtle CPE • Icosahedral symmetry ○ Hemadsorption • Family Caliciviridae Paramyxoviruses • Sapporo • Helical and enveloped, larger than myxoviruses • Norovirus • Have only one long ssRNA genome ○ Prototype strain is Norwalk virus • No reassortment ○ Outbreaks of diarrhea • Replicate in both the nucleus and cytoplasm ○ Genetically and antigenically diverse • Five genera: Mumps, parainfluenza 1 to 4, measles, ○ Noncultivatable RSV, metapneumovirus Coronaviruses • Parainfluenza virus • ssRNA, enveloped ○ Four antigenic types • Pleomorphic/spherical capsid ○ Respiratory infections • Large club-shaped spikes on surface gives “corona” ○ Isolated from throat effect ○ Grow in cell culture Filoviruses ○ Hemadsorption for identification • ssRNA • Measles virus • Helical symmetry, long and slender ○ One serologic type • Marburg and Ebola viruses ○ Maculopapular rash, fever, respiratory disease • Monkey reservoir but transmitted to humans ○ Can be isolated in culture • More than 80% mortality ○ Hemadsorption for identification • Pan-organ effects • Mumps virus • Contact with blood ○ Infects parotid salivary glands • Biosafety level 4 containment ○ Can infect testis, ovaries, kidneys Flaviviruses ○ Isolated from throat swab or urine • ssRNA, enveloped ○ Identified by hemadsorption • Icosahedral symmetry • RSV • St. Louis encephalitis, West Nile virus, yellow fever, ○ Bronchiolitis, pneumonia in infants dengue, hepatitis C ○ Labile virus • Not isolated by culture ○ Produces typical CPE Hepatitis C Virus ○ Monoclonal antibody to confirm • Blood or sexual contact ○ Rapid testing available • No other vector • Metapneumovirus • Chronic liver infection ○ Acute respiratory tract infections worldwide in • Serology most common diagnosis children and adults • Screen with EIA ○ Annual epidemics in winter and spring • Western blot confirmation months • Also detected in blood by molecular assays ○ Two distinct human metapneumovirus groups Orthomyxoviruses with subgroups • ssRNA: Eight segments ○ PCR for diagnosis, for now • Helical symmetry capsid, enveloped Picornaviruses • Replicate in cytoplasm • ssRNA • Influenza viruses • Very small: Approximately 27 nm • Two important surface Ag • No envelope, icosahedral symmetry ○ Neuraminidase • Replicate in cytoplasm of cell ○ Hemagglutinin Enteroviruses • Segmented genomes: Heavy reassortment • Fecal-oral transmission ○ Antigenic shift: Large • Polioviruses: 3 types ○ Antigenic drift: Small • Coxsackievirus A: 24 types ○ Animal strains • Coxsackievirus B: 6 types ○ Three antigenic groups • Echoviruses: 34 types § A, B, C • Many cultivatable • Severe in elderly, immunocompromised Rhinoviruses • Pandemics • Common cold virus ○ 1918 • More than 100 serotypes ○ Avian flu • Can be isolated in culture ○ Swine flu • Acid-sensitive CHAPTER 2 Mycology, Virology, and Parasitology 71

• Limited to upper respiratory tract • Can be isolated in culture and mice • Limited growth at 37 C • Diagnosis by immunofluorescence of brain tissue Hepatitis A Virus Togaviruses • 25% of hepatitis • ssRNA • Usually fecal-oral transmission • Enveloped, icosahedral symmetry • Food • Family Togaviridae • Water • Genus alphavirus: Arboviruses • Needles • Genus rubivirus: Rubella • No growth in cell culture Arboviruses • Serology for diagnosis • Eastern and Western equine encephalitis Reovirus • Bird reservoir • Respiratory enteric orphan • Mosquito vectors • dsRNA, 60 to 80 nm • Symptoms include fever, encephalitis, rash • Nonenveloped, icosahedral symmetry • Cell culture possible, but serology most commonly used • Replicates in the cytoplasm Rubella virus • Rotaviruses • Transmitted by droplets • Fecal-oral • Infection in children mild • Causes infantile diarrhea • Congenital disease serious • Not isolated in laboratory, ELISA • Mother contracts rubella in first trimester • At least six serotypes • Vaccine developed for children because of congenital Retroviruses disease • ssRNA (may have two copies) • Cell culture possible but serology most common • Enveloped, icosahedral symmetry • All have reverse transcriptase • DNA made from RNA PARASITOLOGY • Integrates into genome • Many can cause tumors in animals • Techniques • Replicate in nucleus and cytoplasm • Specimen collection and processing • HIV 1 and 2, human T-lymphotropic virus 1 and 2 ○ Stool: Routine is three specimens, every other • HIV day within 10 days ○ 100 to 400 nm, cylindrical or conical core § Clean, watertight container with a tight lid ○ Two broad types: 1 and 2 § 5 g, not contaminated with water, urine, bar- § Several subtypes in type 1 ium, or other substances § Type 1, clade (subtype) B in United States § Liquid or near-liquid specimens examined § Type 2 mainly in Africa within 30 minutes of collection to preserve ○ Does not form tumors motile trophozoites ○ Infects CD4+ cells § Soft specimens examined within 60 minutes of § Lymphocytes, macrophage, brain cells, and collection dendritic cells § Formed specimens processed within 24 hours, § Destroys immune system may be refrigerated ○ Characteristic secondary diseases § Fixatives: Two-vial system, usually formalin § Pneumocystis pneumonia, CMV, Kaposi’s and polyvinyl alcohol (PVA) or one-vial sys- sarcoma tem of sodium acetate formalin (SAF) ○ ELISA and Western blot analysis § 3 parts fixative to 1 part stool Rhabdoviruses § 5% to 10% formalin, concentration • ssRNA, bullet shaped, enveloped methods, and iodine-stained mounts • Replicate in cytoplasm § PVA, concentration methods, and tri- • Vesicular stomatitis virus of horses chrome and other permanent stains • Rabies virus § SAF: Concentration methods and perma- • One serologic type nent stains • Encephalitis § PVA contains mercury, SAF is a mercury- ○ Bite of infected animal free alternative ○ Travels up sensory nerves to the central nervous § Concentration methods system (CNS) § Fresh or preserved stools ○ Incubation of 2 to 16 weeks § Sedimentation: Formalin-ethyl acetate ○ Allows time for vaccine § Floatation, zinc sulfate with specific grav- ○ 100% fatal if untreated ity of 1.18 to 1.20 72 CHAPTER 2 Mycology, Virology, and Parasitology

§ Macroscopic Major Medically Important § Consistency TABLE 2-3 Parasites—cont’d § Appearance, color § Contaminants Classification Genus and Species § Larva, proglottids Protozoa: Coccidia Microsporidia spp. § Microscopic Isospora belli § Calibrated ocular micrometer Cyclospora cayetanensis § Direct saline wet mount for motile Cryptosporidium parvum trophozoites Protozoa: Misc Toxoplasma gondii § Iodine-stained wet mount from processed Blastocystis hominis specimen Cestodes: Tapeworms Diphyllobothrium latum § Permanent stains from processed specimen Hymenolepis nana § Trichrome: Cytoplasm is blue-green, purple; Hymenolepis diminuta spp. nuclear structures, red to pink Taenia Taenia saginata § Iron hematoxylin: Primarily for intestinal pro- Taenia solium tozoa, cytoplasm is blue to purple, nuclear Dipylidium caninum structures are blue to black Echinococcus granulosus § Modified acid-fast: For coccidian protozoa, Trematodes: Flukes Clonorchis sinensis oocysts are red Fasciolopsis buski • Other specimen types Fasciola hepatica • Specimen of choice depending on organism in ques- Heterophyes heterophyes tion and clinical situation Metagonimus yokogawa • Duodenal contents Paragonimus westermani • Sigmoidoscopy specimens Schistosoma haematobium • Enterotest Schistosoma japonicum Schistosoma mansoni • Blood Blood parasites: Babesia Babesia microti in United • Urine States • CSF and other fluids Babesia divergens in Europe • Sputum Blood parasites: Malaria Plasmodium falciparum • Tissue specimens Plasmodium malariae Plasmodium ovale Plasmodium vivax Plasmodium knowlesi Blood parasites: Filariae Brugia malayi TABLE 2-3 Major Medically Important Parasites Loa loa Wuchereria bancrofti Classification Genus and Species Mansonella ozzardi Nematodes: Intestinal Ascaris lumbricoides Onchocerca volvulus Blood parasites: Enterobius vermicularis Leishmania braziliensis Hemoflagellates—Leishmania Necator americanus Leishmania donovani complex Ancylostoma duodenale Leishmania tropica complex Strongyloides stercoralis Leishmania mexicana complex Blood parasites: Trichuris trichiura Trypanosoma cruzi Hemoflagellates— Nematodes: Nonintestinal Dracunculus medinensis Trypanosoma brucei Trypanosomes Trichinella spiralis rhodesiense Protozoa: Amoeba Entamoeba histolytica Trypanosoma brucei Entamoeba coli gambiense Arthropods Entamoeba hartmanni Pediculus humanus humanus Endolimax nana Pediculus humanus capitis Iodamoeba butschlii Phthirus pubis Acanthamoeba spp. Ixodes scapularis Naeglaria fowleri Dermacentor andersoni Protozoa: Flagellates Giardia lamblia Dermacentor variabilis Trichomonas vaginalis Ornithodoros spp. Chilomastix mesnili Cimex lectularius Trichomonas hominis Ctenocephalides canis Dientamoeba fragilis Ctenocephalides felis Protozoa: Ciliate Balantidium coli Sarcoptes scabei Continued CHAPTER 2 Mycology, Virology, and Parasitology 73

Nematodes • Adult larvae 9 to 12 mm, hook at end of tail; male possesses copulatory bursa Intestinal • Filariform larvae penetrate skin, lymphatic system, to Ascaris lumbricoides bloodstream, to lung; penetrate alveoli, coughed up, • Roundworm swallowed; migrate to large intestine • Worldwide; most common intestinalhelminth infection • Buccal cavity has cutting plates • Ova in stool • Asymptomatic if light infection; GI symptoms, ane- • Ova are the infective stage mia, weight loss, breathing difficulty, bloody sputum, • Ova are diagnostic cough; GI symptoms more severe if worm burden • Ova 85 to 95 38 to 45 mm. Corticated or decorti- heavy; eosinophilia cated. Unfertilized ovoid, 40 to 7430 to 50 mm • Repeated infection can cause dermal irritation, • Embryonate in soil, resist environmental conditions ground itch • Adult larva largest intestinal nematode, 22 to 35 cm • Ova considered to be indistinguishable from those of in length Ancyclostoma duodenale • Larva emerge in small intestine, migration to blood- Ancyclostoma duodenale stream, liver, lung, to pharynx; swallowed, return to • Old World hookworm intestine • Europe, Far East, Asia, Africa typically • GI symptoms, fever, pulmonary or asymptomatic, • Ova considered to be indistinguishable from those of eosinophilia N. americanus, although A. duodenale ova are 55 to • 250,000 ova per day, so worm burden can be high 6040 mm Enterobius vermicularis • Buccal cavity has teeth • Pinworm • Other details are same as for N. americanus • Worldwide; most common intestinal helminth infec- Strongyloides stercoralis tion in the United States • Threadworm • Scotch tape preparation is specimen of choice, ova or • Worldwide adult larva • Larva in stool at rhabditiform stage are the diagnostic • Ova are infective stage stage, ova rarely seen • Ova and larva are the diagnostic stages • Infection caused by third-stage filariform larval pene- • Ova 48 to 60 20 to 35 mm; oval, thick shell; flat on tration of skin, typically the foot one side; developing larva folded inside • Ova 4835 mm, advanced cleavage state, indistin- • Adult larva female 7 to 14 mm, male 2 to 4 mm, white guishable from hookworm to light yellow • Rhabditiform larvae 15220 mm, short buccal cavity • Hatch in small intestine, adults in colon; migrate to and prominent genital primordium anus to deposit ova • Filariform larvae is third stage, long esophagus, • Severe anal itching, inflammation notched tail • Ova infective in 4 to 6 hours, deposit in clothes, bed • Adult female 2 mm, short buccal cavity,long esophagus linens, toys • Unique lifecycle, three mechanisms • Highly communicable • Direct: Same as that of hookworm • Retroinfection: Ova hatch in anus but migrate back • Indirect: Larva freely living in environment, pro- into colon to reproduce duce infective rhabditiform larva • Autoinfection: Infective ova are ingested, hand • Autoinfection: Filariform larva develop in host’s to mouth intestine, invade bloodstream • Human is only known host ○ Asymptomatic if light infection; GI symptoms, • Ova may carry Dientamoeba fragilis, dual infections malabsorption, weight loss, breathing difficulty, are seen bloody sputum, cough, eosinophilia Necator americanus ○ Repeated infection can cause dermal irritation, • New World hookworm ground itch • North and South America typically Trichuris trichiura • Ova are the diagnostic stage, larva can also be found in stool • Whipworm • Ova in stool • Infection caused by third-stage filariform larval penetration of skin, typically the foot • Ova are infective stage • Ova are diagnostic • Ova 60 to 7540 mm, cell cleavage can be seen, thin shell • Ova 50 to 55 25 mm, barrel shaped with bipolar hyaline plugs • Rhabditiform larvae 15270 mm, long buccal cavity and small genital primordium, cutting plates • Adult larva 2 to 5 cm, male smaller with curled tail; pos- teriorend is large,resembles awhip handle;anteriorend • Filariform larvae is third stage, short esophagus, pointed tail is smaller, resembles whip; can be found in stool 74 CHAPTER 2 Mycology, Virology, and Parasitology

• Larva emerge in small intestine, migrate to cecum • Asymptomatic carrier state or can cause dysentery, then to colon which can be severe, and abscesses in liver, spleen, • Asymptomatic with light infection; heavier worm bur- lung, brain den includes GI symptoms, weight loss, weakness, Entamoeba coli eosinophilia; children’s symptoms can include GI, • Commensal anemia, and, if untreated, prolapsed rectum • Cysts or trophozoites in stool are diagnostic Nonintestinal • Cysts: 10 to 30 mm, round, one to eight nuclei, more than four to differentiate from Entamoeba histolytica, Trichinella spiralis eccentric karyosome, uneven peripheral chromatin, • Trichina worm splintered end chromatoid bar • Worldwide • Trophozoites: 15 to 50 mm, slow motility, one nucleus • Laboratory diagnosis with eccentric karyosome, coarse cytoplasm • Histologic preparation of encysted tissue, typically Entamoeba hartmanni skeletal muscle • Commensal • Serologic methods • Cysts or trophozoites in stool are diagnostic • Elevated muscle enzymes • Cysts: Less than 10 mm, resemble those of E. • Ingesting undercooked contaminated meat: Pork, deer, histolytica bear, walrus • Trophozoites: Less than 12 mm, resemble those of E. • Encysted larva 1005 mm, coil in cyst in muscle histolytica • Ingestion of infected meat, larva excysts, and develops Endolimax nana in intestine; adult female deposits larva, which migrate • Commensal through bloodstream to skeletal muscle and encyst, • Cysts or trophozoites in stool are diagnostic which ends lifecycle • Cysts: 5 to 10 mm, one to four nuclei with blot-like • Asymptomatic or flulike symptoms; heavier infec- karyosome tion includes GI symptoms, weakness, fever, pain, • Trophozoites: 5 to 12 mm, one nucleus with blot-like edema, muscular pain; can be fatal during migra- karyosome tory phase Iodamoeba butschlii Dracunculus medinensis • Commensal • Guinea worm • Cysts or trophozoites in stool are diagnostic • Africa, India, Asia, Middle East • Cysts: 5 to 20 mm, one nucleus, large glycogen vacuole • Laboratory diagnosis • Trophozoites: 8 to 20 mm, one nucleus • Examination of emerging larva from the skin ulcer Acanthamoeba Species • Ingestion of infected copepods; larva emerge in intes- • Pathogen tine of host, develop and migrate to connective tissue • Granulomatous amoebic encephalitis (GAE) and body cavities, and to subcutaneous tissues, where • Worldwide adult female deposits larva; ulcer forms, from which • Trophozoites and cysts in CSF, in brain tissue, or at larva can emerge autopsy • Allergic reaction, skin ulcer • Trophozoites: 12-45 mm, one nucleus, slow motility • Cysts: 8-25 mm, double cell wall, one nucleus Protozoa • Environmental organism, enters nasal passages, bloodstream to CNS; swimming in ponds, lakes in Amoeba warm months Entamoeba histolytica • GAE: Stiff neck, headaches, seizures, can progress • Pathogenic rapidly • Amoebic dysentery, amoebic abscess • Eye involvement, contact lens fluid contamination, • Worldwide, a leading cause of parasitic death keratitis • Cysts are infective form • Keratitis: Ocular pain, vision impairment • Cysts or trophozoites in stool are diagnostic, or amoe- Naeglaria fowleri bic abscess fluid or sigmoidoscopy specimen • Pathogen • Cysts: 10 to 20 mm, round, one to four nuclei, central • Worldwide karyosome, fine and even peripheral chromatin, • Primary amoebic meningoencephalitis (PAM) rounded chromatoid bar; young cysts can contain • PAM: Stiff neck, headaches, seizures, can progress glycogen vacuole rapidly • Trophozoites: 12 to 60 mm, rapid and directional • Trophozoites in CSF, in brain tissue, or at autopsy motion, one nucleus with central karyosome; ingested • Trophozoites: 8-25 mm, one nucleus, slow motility red blood cells (RBCs) are diagnostic • Cysts: No known cyst form CHAPTER 2 Mycology, Virology, and Parasitology 75

• Environmental organism, enters nasal passages, then • Asymptomatic in men or urethritis, vaginitis with yel- bloodstream to CNS; swimming in ponds, lakes in low discharge warm months Ciliate Flagellates Balantidium coli Giardia lamblia • Pathogen • Pathogenic • Worldwide • Worldwide • Cysts or trophozoites in stool are diagnostic • Giardiasis, traveler’s diarrhea, • Cysts: 50 to 75 mm, round, two nuclei, small micronu- • Worldwide, natural water sources cleus, large kidney bean–shaped macronucleus, • Cysts are infective form cytosome, cilia • Cysts or trophozoites in stool are diagnostic or on sig- • Trophozoites: 50 to 100 mm, round, two nuclei, small moidoscopy specimen, shed irregularly micronucleus, large kidney bean–shaped macronu- • Immunologic methods, PCR cleus, cytosome, cilia • Cysts: 8 to 14 mm, one to four nuclei, four axonemes, • Asymptomatic or GI symptoms, mild dysentery, four median bodies, oval abscesses in intestinal mucosa, anemia • Trophozoites: 9 to 21 mm, two nuclei, two axonemes, eight flagella, sucking disk, tear-drop shape Coccidia • Asymptomatic or GI symptoms, can be severe, light- Cryptosporidium parvum colored stools, incubation period is 10 to 36 days, • Pathogen can be self-limiting • Worldwide Dientamoeba fragilis • Oocyst is infective stage • Pathogenic • Oocysts in stool are diagnostic, immunologic • Thought to be worldwide methods, modified acid-fast stain • Trophozoite is infective form • Oocysts: 4 to 6 mm, round, no sporocysts • Trophozoites in stool are diagnostic • Food-borne and water-borne illness • Cysts: No known cyst form • Mild GI symptoms, self-limiting, in compromised • Trophozoites: 7 to 12 mm, one or two nuclei, 80% patients more severe, can be fatal binucleate Isospora belli • Asymptomatic or GI symptoms, mild dysentery • Pathogen • Possible association with Enterobius vermicularis • Worldwide infection • Oocyst is infective stage Chilomastix mesnili • Oocysts in stool are diagnostic, immunologic • Commensal methods, modified acid-fast stain • Worldwide • Oocysts: 3012 mm, ovoid, one or two sporocysts, • Cysts and trophozoites in stool are diagnostic thin shell • Cysts: 5 to 10 mm, lemon shape, one nucleus, cytosome • Human is definitive host with fibrils • Asymptomatic or GI symptoms, mild, self-limiting • Trophozoites: 10 to 20 mm, one nucleus, four flagella, Cyclospora cayetanensis cytosome with fibrils • Pathogen Trichomonas hominis • Thought to be worldwide • Commensal • Oocysts in stool are diagnostic, modified acid- • Thought to be worldwide fast stain • Trophozoites in stool are diagnostic • Oocyst is infective form • Cysts: No known cyst form • Oocysts: 7 to 10 mm, round, two sporocysts • Trophozoites: 7 to 15 mm, one nucleus, four flagella, • Linked to water-borne and food-borne illness undulating membrane, axostyle • Mild GI symptoms Trichomonas vaginalis Microsporidia Species • Pathogenic • Pathogen • Worldwide • Thought to be worldwide • Trophozoite is infective form • Spores in stool are diagnostic, modified acid-fast stain, • Trophozoites in urine, vaginal secretions, urethral serologic methods secretions are diagnostic • Spore is infective form • Cysts: No known cyst form • Spores: 1 to 5 mm, round, two sporocysts • Trophozoites: 5 to 15 mm, one or two nuclei, four fla- • Enterocytozoon bieneusi is most commonly isolated gella, undulating membrane, axostyle, jerky motility species, seen in patients with HIV in fresh specimens • Mild GI symptoms, peritonitis, hepatitis 76 CHAPTER 2 Mycology, Virology, and Parasitology

Miscellaneous Protozoa • Ova are infective stage Blastocystis hominis • Ova in stool are diagnostic • Pathogen although pathogenicity is unclear • Ova: 4540 mm, typically round, hexacanth embryo • Thought to be worldwide with three pairs of hooklets, shell with bipolar thicken- • Cyst is infective stage ings and filaments in clear embryophore • Cysts in stool are diagnostic, immunologic methods • Scolex: Four suckers, short rostellum with hooks • Cysts: 8 to 30 mm, round, central vacuole surrounded • Proglottids: Rectangular by several peripheral nuclei • Ingestion of infective ova. Cysticercoid larva develop • Mild GI symptoms in intestine, scolex emerges and attaches to intestinal Toxoplasma gondii mucosa and further develops. Ova from the adult larva • Pathogen can pass in feces as an infective ovum or autoreinfect • Toxoplasmosis the human host • Worldwide, many animals harbor the organism • No intermediate host required • Oocyst is infective stage • Can be asymptomatic or GI symptoms, weight loss, • Immunologic methods abdominal pain • Oocysts: 10 to 15 mm, oval, two sporocysts Hymenolepis diminuta • Tachyzoites: 53 mm, crescent shape, one central • Rat tapeworm nucleus • Worldwide • Bradyzoites: 53 mm, crescent shape, one central • Ova are infective stage nucleus, form a packet in host cell that contains many • Ova in stool are diagnostic bradyzoites • Ova: 5585 mm, typically round, hexacanth embryo • Cat is definitive host, humans are accidental hosts with three pairs of hooklets, shell with bipolar thicken- by four mechanisms: Ingest oocyst from cat feces; ings but no filaments in clear embryophore ingest contaminated meat from cattle, pig, sheep; • Scolex: Four suckers, small projecting rostellum transplacental to fetus; blood transfusion without hooks • Asymptomatic or flulike symptoms in mild cases, • Proglottids: Rectangular but can be chronic; congenital results in fetal death, • Human is accidental host, primary host is rat. Inges- mental retardation, blindness, severe brain damage, tion of infective ova. Cysticercoid larva develop in or neonate is asymptomatic at birth intestine, scolex emerges and attaches to intestinal mucosa and further develops. Ova from the adult larva Cestodes: Tapeworms can pass in feces as an infective ovum or autoreinfect the human host Diphyllobothrium latum • No intermediate host required • Broadfish tapeworm • Can be asymptomatic or GI symptoms, weight loss, • Great Lakes area, Alaska, South America, Asia, Africa, abdominal pain Scandinavia Taenia Species • Ova are infective stage • Worldwide • Ova in stool are diagnostic, more rarely proglottids • Two species • Ova: 55 to 7540 to 55 mm, operculated, abopercular • Ova indistinguishable, 3525 mm, round, hexacanth knob, not a hexacanth embryo, coracidium sur- embryo with three pairs of hooklets, surrounded rounded by dark shell by striated embryophore, sunburst appearance, • Scolex: Almond shaped with two long sucking grooves nonoperculated • Proglottids wider than long, central uterine structure • Ova in stool are diagnostic, more rarely proglottids • Two intermediate hosts. Infection caused by ingestion • Infection caused by ingesting undercooked beef or of pleurocercoid in undercooked or raw fish. Scolex pork that contains cysticercus larva, larva emerge in emerges and attaches to intestinal mucosa. Ova passed small intestine where scolex attaches to intestinal in stool, further develops if deposited in water. Coraci- mucosa dium hatches, larva ingested by copepod. Develops Taenia saginata to procercoid in copepod, which is ingested by fresh- • Beef tapeworm water fish; procercoid develops to pleurocercoid larva • Scolex: 1 to 2 mm, four suckers in fish • Proglottids: greater than 1000, rectangular, 15 to 30 • Can be asymptomatic or GI symptoms, weight loss, uterine branches abdominal pain, vitamin B12 deficiency, pernicious Taenia solium anemia • Pork tapeworm Hymenolepis nana • Scolex: 1 to 2 mm, four suckers, rostellum, and hooks • Dwarf tapeworm • Proglottids: less than 1000, square, 7 to 15 uterine • Worldwide, most common tapeworm in United States branches CHAPTER 2 Mycology, Virology, and Parasitology 77

• Can cause cysticercosis after ingesting infective ovum. • Ova 3015 mm with miracidium, shoulders are large, Onchosphere migrates to organs, muscle; can invade operculated, terminal knob brain, potentially fatal • Metacercariae mature in liver, migrate to bile duct Dipylidium caninum • Can be asymptomatic or GI symptoms, weight loss, • Dog or cat tapeworm abdominal pain • Worldwide Fasciolopsis buski • Egg packets in stool, or proglottids are diagnostic • Large intestinal fluke • Larva are infective stage • Far East, India, Indonesia • Egg packets each containing 10 to 30 ova, ova • Ova in stool 4060 mm, oncosphere with three pairs of hooklets • Ova are infective stage • Scolex: Four suckers, small rostellum with several cir- • Ova in the stool are diagnostic cles of spines • Ova 128 to 14080 mm, oval, contain miracidium, • Proglottids: Seed shaped operculated • Human is accidental host, primary host is dog or • Adult larva: 51.5 cm, ovoid cat. Intermediate host is flea ingestion of larval stage, • Ingestion of infected water plants; miracidium develops to adult larva. Egg packets or proglottids develops, adult resides in small intestine of host passed in stool. Cycle continues if these are ingested • Abdominal pain, GI symptoms, jaundice, malabsorp- by flea tion syndrome, intestinal obstruction in severe cases • Flea is intermediate host Fasciola hepatica • Can be asymptomatic or GI symptoms, weight loss, • Sheep liver fluke abdominal pain • Worldwide, areas cattle and sheep ranching Echinococcus granulosus • Ova are infective stage • Hydatid tapeworm, or dog tapeworm • Ova in stool are diagnostic • West and southwest United States and Alaska, South • Ova 128 to 15080 mm, oval, contain miracidium, America, Africa, Asia, Australia, Middle East, areas operculated where dogs and sheep or cattle coexist • Adult larva: 31 cm, ovoid, possesses shoulders that • Laboratory diagnosis by examining hydatid cyst fluid, distinguish it from F. buski hydatid sand under microscope, scolices floating in • Human is accidental host, sheep definitive host. Inges- fluid; serological methods tion of infected water plants, miracidium develops, • Ova are not the diagnostic phase but are identical to adult resides in bile ducts of host ova of Taenia spp. • Abdominal pain in liver area, GI symptoms, jaundice • Hydatid cyst consists of daughter cysts surrounded by Heterophyes heterophyes a capsule. Brood capsules can form in the germinal • Heterophid fluke layer of the cyst. Daughter cysts and brood capsule • Africa, Far East, Near East, Egypt contain scolices, which can develop into adult larva • Ova are infective stage • Scolex: Four suckers and hooks • Ova in the stool are diagnostic • Larva: 5 mm, scolex, neck, and three proglottids • Ova 15 to 30 mm, oval, flask shaped, contain miracid- • Human is accidental intermediate and end host. Inges- ium, operculated, small shoulders, thick shell, can lack tion of infected ova. Larvae penetrate intestinal mucosa terminal knob and migrate to organs, usually lung, liver. Hydatid cyst • Ova of Heterophyes heterophyes, C. sinensis, and develops in the organ. Dog is definitive host, sheep Metagonimus yokogawa very similar intermediate host. Cysts form in sheep, dog ingests • Adult larva: 10.5 mm, pyriform, spines cover outside infected sheep viscera. Cyst develops into adult larva, • Ingestion of undercooked fish, miracidium develops, which reside in dog’s intestine, ova passed in feces adults reside in small intestine • Symptoms depend on location and size of cyst. Asymp- • Asymptomatic or heavier infections cause GI symptomatic until cyst enlarges. Pain in area. Can be fatal if toms, abdominal pain, eosinophilia cyst ruptures, because fluid can cause anaphylactic Metagonimus yokogawa shock. New cyst can form from ruptured cyst if scolex • Heterophid fluke is extruded • Far East, Europe, Siberia • Ova are infective stage Trematodes: Flukes • Ova in stool are diagnostic Clonorchis sinensis • Ova 15 to 30 mm, oval, flask shaped, contain miracid- • Chinese liver fluke ium, operculated, small shoulders, thin shell, can lack • Far East terminal knob • Ingesting undercooked fish containing infective • Adult larva: 11.5 mm, piriform, spines cover outside metacercariae • Ingestion of undercooked fish; miracidium develops, • Ova in stool are diagnostic adults reside in small intestine 78 CHAPTER 2 Mycology, Virology, and Parasitology

• Asymptomatic or heavier infections cause GI symp- Schistosoma mansoni toms, abdominal pain, eosinophilia • Manson’s blood fluke Paragonimus westermanni • Central and South America, Puerto Rico, West Indies • Oriental lung fluke • Ova in stool or rectal biopsy • Asia, Africa, India, South America • Cercariae penetrate skin to infect human host • Ova are infective stage • Ova 100 to 18540 to 75 mm, oblong, contain devel- • Ova in stool or in sputum are diagnostic oped miracidium, large lateral spine • Ova 78 to 12045 to 60 mm, oval, contain undeve- • Adult larva: 2 cm, oblong, male and female organisms loped miracidium, thin shell, operculated with shoul- • Cercariae penetrate skin to infect human host, migrate ders, thickening at terminal end to bloodstream to develop. Adult resides in blood ves- • Adult larva: 10.5 mm, oval, spines sels around intestinal tract. Ova from adult females are • Ingestion of undercooked crayfish or crab. Developing excreted in urine. If deposited in water, miracidium larva migrate to small intestine, into peritoneal cavity, infects snail where it develops into cercariae into diaphragm, then to lung tissue • Snail is intermediate host • Pulmonary symptoms, cough, bloody sputum, eosino- • Reservoir hosts include cattle, sheep, dog, cat, mon- philia, other symptoms if larvae migrate to other keys, rodents organs • Asymptomatic or skin irritation at penetration site, Schistosoma haematobium swimmer’s itch, abdominal pain, cough, fever, • Bladder fluke eosinophilia • Africa, Middle East, Iran, Iraq, Saudi Arabia • Ova in concentrated urine sample, immunodiagnostic methods Blood Parasites • Cercariae penetrate skin to infect human host • Ova 110 to 17040 to 70 mm, oblong, contain devel- Babesia oped miracidium, large terminal spine • Babesiosis • Adult larva: 2 cm, oblong, male and female • Babesia microti in United States organisms • Babesia divergens in Europe • Cercariae penetrate skin to infect human host, migrate • Giemsa-stained thick and thin blood smears, serologic, to bloodstream to develop. Adult resides in blood ves- PCR, blood smears collected at the patient’s bedside sels around urinary bladder. Ova from adult females • Small delicate ring form trophozoites, 1 to 2 mm are excreted in urine. If deposited in water, miracidium • Rings in single, double, or classic tetrad infects snail, where it develops into cercariae • Tetrad: Maltese cross • Snail is intermediate host • Fever, chills, sweating, myalgias, fatigue, hepatosple- • Reservoir hosts include cattle, sheep, dog, cat, mon- nomegaly, and hemolytic anemia keys, rodents Malaria • Asymptomatic, skin irritation at penetration site, • Tropical and subtropical worldwide swimmer’s itch, abdominal pain, cough, fever, eosino- • Five Plasmodium spp. infect humans philia, painful urination, hematuria • Lifecycle summary Schistosoma japonicum • Sporozoites injected during mosquito feeding • Blood fluke • Invade liver cells • Far East • Liver replication ! merozoites • Ova in stool or rectal biopsy • Merozoites invade RBCs • Cercariae penetrate skin to infect human host • Repeated erythrocytic schizogony • Ova 50 to 8540 to 60 mm, slightly oblong, contain • Gametocytes infect mosquito developed miracidium, small lateral spine • Fusion of gametes in gut • Adult larva: 2 cm, oblong, male and female organisms • Sporogony on gut wall sporozoites invade salivary • Cercariae penetrate skin to infect human host, migrate glands to bloodstream to develop. Adult resides in blood ves- • Giemsa-stained thick and thin blood smears, serologic, sels around intestinal tract. Ova from adult females are PCR; blood smears are collected at the patient’s excreted in urine. If deposited in water, miracidium bedside. infects snail, where it develops into cercariae • Anopheles mosquito is vector • Snail is intermediate host Plasmodium falciparum • Reservoir hosts include cattle, sheep, dog, cat, mon- • RBC normal size keys, rodents • Schuffner’s stippling: No • Asymptomatic or skin irritation at penetration site, • Merozoites in schizonts: 8 to 36, average 24 swimmer’s itch, abdominal pain, cough, fever, • Ring forms single or double chromatin dots eosinophilia • Multiple ring forms: Common CHAPTER 2 Mycology, Virology, and Parasitology 79

• Banana-shaped gametocyte Onchocerca volvulus • Accole´ forms • Blinding filaria, river blindness Plasmodium vivax • Tropical Africa and Central America • RBC normal size • Giemsa-stained skin snips • Schuffner’s stippling: Yes • No periodicity • Merozoites in schizonts: 12 to 24, average 16 • Simulium blackfly is vector • Ring forms single chromatin dot • No sheath, no nuclei in tail tip • Multiple ring forms: Occasional • Subcutaneous fibrous nodules • Ameboid trophozoites • Blindness if eye is affected Plasmodium malariae Mansonella ozzardi • RBC normal size • New World filaria • Schuffner’s stippling: No • North, Central, and South America • Merozoites in schizonts: 6 to 12, average 8, rosette • Giemsa-stained blood • Single chromatin dot • No periodicity • Multiple ring forms: Rare • Simulium blackfly is vector, or Culicoides midge • Band form trophozoite • No sheath, nuclei in tail but not to tip Plasmodium ovale • Asymptomatic, lymphadenopathy • RBC oval • Blindness if eye is affected • Schuffner’s stippling: Yes • Hemoflagellates • Merozoites in schizonts: 4 to 12, average 8 Leishmaniae • Single chromatin dot Leishmania braziliensis • Multiple ring forms: Occasional • Mucocutaneous leishmaniasis Plasmodium knowlesi • Mexico, Central and South America • Simian malaria form thought to be rarely found in • Giemsa-stained slides of the affected body sites, humans amastigotes • Forms resemble P. falciparum or P. malariae • Sandfly is vector, bite transfers promastigotes to the Filariae human host, promastigotes migrate to reticuloendo- Brugia malayi thelial cells and develop to amastigotes • Malayan filaria • Lesions in mucocutaneous tissues • Tropical and subtropical worldwide • Can be self-limiting • Giemsa-stained blood, Knott’s technique Leishmania donovani Complex • Nocturnal periodicity • Visceral leishmaniasis, dumdum fever, kala-azar • Aedes, Anopheles, Mansonia mosquitos are vectors, • Africa, India, Middle East, Far East intermediate host • Giemsa-stained slides of the affected body sites, amas- • Sheath, two nuclei in tail tip tigotes, serologic methods, Montenegro screening • Asymptomatic for months, years; granulomatous skin test lesions, fever, chills, lymphadenopathy • Affects visceral tissue • Elephantiasis • Sandfly is vector, incubation period of weeks to months Loa loa • Flulike symptoms resembling malariae, GI symptoms, • Eyeworm abdominal pain, hepatosplenomegaly • Africa • Darkening of the skin, black fever or kala-azar • Giemsa-stained blood, Knott’s technique • Can be fatal • Diurnal periodicity Leishmania mexicana Complex • Chrysops fly is vector • New World cutaneous leishmaniasis • Sheath, continuous nuclei in tail tip • Central and South America, Mexico • Inflammation at bite site, Calabar swellings at any • Giemsa-stained slides of the lesions, amastigotes body site • Affects skin Wuchereria bancrofti • Sandfly is vector • Bancroft’s filaria • Skin lesion, ulcer • Tropical and subtropical worldwide • Can be self-limiting • Giemsa-stained blood, Knott’s technique Leishmania tropica Complex • Nocturnal periodicity • Old World cutaneous leishmaniasis, Baghdad boil, • Aedes, Anopheles, Culex mosquito are vectors, Delhi boil intermediate host • Middle East, Northern Africa • Sheath, no nuclei in tail tip • Giemsa-stained slides of the lesions or fluid, amasti- • Fever, chills, lymphadenopathy gotes, serologic testing • Elephantiasis • Affects skin 80 CHAPTER 2 Mycology, Virology, and Parasitology

• Sandfly is vector • Vector for typhus (Rickettsia prowazekii), trench fever • Skin lesion, ulcer (Bartonella quintana), and relapsing fever (Borrelia • Can be self-limiting recurrentis) Trypanosomes • Spread from human to human Trypanosoma brucei gambiense • Body lice usually on the body and head • West African sleeping sickness • Crab lice usually in pubic region, spread to the arm- • West and Central Africa pits, facial hair, eyebrows, and eyelashes • Giemsa-stained slides blood or lymph nodes, CSF Pediculus humanus humanus studies • Body louse • Tsetse fly is vector; bite transfers trypomastigotes to Pediculus humanus capitis the human host, migrate to lymphatic system, eventu- • Head louse ally to CNS Phthirus pubis • Asymptomatic for a period. Chancre develops at bite • Crab or pubic louse site, flulike symptoms, rash, lymphadenopathy, Win- Ticks terbottom’s sign in neck area. Kerandel’s sign can Hard Ticks develop, delayed sensation to pain. CNS symptoms Ixodes scapularis in final stages, coma, and death • Deer tick Trypanosoma brucei rhodesiense • Main vector of Lyme disease • East African sleeping sickness • Ixodes pacificus in the U.S. West Coast states also able • East and Central Africa to transmit Lyme disease • Giemsa-stained slides of blood, CSF studies Dermacentor andersoni • Tsetse fly is vector; bite transfers trypomastigotes to • Rocky Mountain wood tick, western United States the human host, which migrate to lymphatic system, • Vector of many diseases, including Rocky Mountain eventually to CNS spotted fever, tularemia, Colorado tick fever, and • Virulent. Asymptomatic for a short period. CNS Q fever involvement early, weight loss, lethargy, confusion, Dermacentor variabilis Winterbottom’s sign in neck area may be present. In • American dog tick, eastern United States final stages, glomerulonephritis, myocarditis, coma, Soft Ticks and death • Ornithodoros spp. Trypanosoma cruzi • Parasitize mammals • Chagas disease • Transmit relapsing fever • Central and South America, Mexico, southern United Fleas States • Cat flea: Ctenocephalides felis • Giemsa-stained slides to blood, serologic methods • Dog flea: C. canis can be found on cats and dogs • Reduviid bug is vector; defecates near bite, transfers • Serve as intermediate host for tapeworms trypomastigotes to the human host. Amastigotes and • Feed on humans as well as pets trypomastigotes facilitate cell damage throughout • Cause a localized skin reaction body. Liver, brain, and heart muscle involved Mites • Can also be transmitted by blood transfusion, placenta • Sarcoptes scabei is the cause of scabies worldwide • Chagas can be asymptomatic, chronic, or acute. Cha- • Transmitted by contact goma develops at bite site, typically face. Eye area • Organisms burrow into the skin on the webbing swelling is Roman˜ a’s sign. Acute flulike symptoms. side of fingers, later spreading to the wrists, elbows Chronic disease early or years later. Myocarditis, and beyond mega colon, hepatosplenomegaly, brain damage, Bedbugs death • True insect • Children most at risk • Cimex lectularius • Arthropods • Preferential feeding host is human • Nocturnal blood meals Ectoparasites and Vectors of Disease • Symptoms occur days after bite Mosquitos • Lice, ticks, fleas, mites, bedbugs, mosquitos • True insect • Structures used in arthropod identification • Blood meals • Body parts, legs, wings, antenna, mouth parts • Transmit malariae, filariasis, dengue fever, yellow Lice fever, West Nile virus • Lice occur worldwide and in all socioeconomic classes • Species include Culex, Anopheles, Aedes, Mansonia CHAPTER 2 Mycology, Virology, and Parasitology 81

CERTIFICATION PREPARATION QUESTIONS a. Sporangium b. Blastoconidia For answers and rationales, please see Appendix A. c. Ascospores 1. Which of the following terms is best described as the d. Conidiophore process of reproduction in yeast that begins with a 6. A patient with a Wood’s lamp–positive, dermatophytic weakening and outpouching of the yeast cell wall infection has a skin scraping taken for culture. The and then formation of a cell wall septum between organism grows on SDA agar with a light-tan front the mother and daughter yeast cells? and salmon-colored reverse. Microscopically the a. Binary fission organism produces rare distorted macroconidia and b. Unisexual division rare microconidia. Additionally, there was no growth c. Budding on sterile rice media. What is the most likely organism? d. Outpouch germing a. Microsporum canis 2. The loose intertwined network of basic structural b. Microsporum gypseum units of the molds that penetrates the substrate from c. Microsporum audouinii which it obtains the necessary nutrients for growth is d. Epidermophyton floccosum called which of the following? 7. A KOH preparation of respiratory secretions of a 78- a. Hyphae year-old man reveals large, spherical, thick-walled b. Germ tubes yeast cells 8 to 15 mm in diameter, usually with a single c. Pseudohyphae bud that is connected to the parent cell by a broad base. d. Mycelium Which fungus will likely be isolated from the culture? 3. The term hyaline molds is used to describe which of a. Coccidioides immitis the following? b. Blastomyces dermatitidis a. Molds that have septate hyphae c. Histoplasma capsulatum b. Molds that have septate, nonpigmented hyphae d. Paracoccidioides brasiliensis c. The presence of pigmentation within the hyphae 8. Which of the following is a key characteristic of Coc- or the spores cidioides immitis? d. Molds with intercalated hyaline chlamydoconidia a. Has a higher dissemination rate in white females 4. Large,usuallymultiseptateandclub-shapedorspindle- b. Is endemic in the northeastern United States shaped spores are called which of the following? c. Produces endosporulating spherules in tissue a. Microconidia d. Forms foot cells b. Macroconidia 9. Using PAS to stain a respiratory specimen from a c. Conidiophores patient with lung disease, the technologists observed d. Phialides the organisms in the image. Based on the microscopic 5. A Scotch tape preparation is made from a mold morphology shown in the image, the most likely iden- growing on solid media in the mycology laboratory. tification of the dimorphic fungi is which of the The structure shown in the image is best described as following? which of the following?

FIGURE 2-2 (Courtesy Joel Mortensen, PhD. See also color plate 9.) a. Blastomyces dermatitidis FIGURE 2-1 (Courtesy Joel Mortensen, PhD. See also color plate 8.) b. Coccidioides immitis 82 CHAPTER 2 Mycology, Virology, and Parasitology

c. Histoplasma capsulatum 14. An immunocompromised patient exhibited fever, d. Sporothrix schenckii nonproductive cough, and shortness of breath. Rou- 10. A landscaper noticed a hard, unmovable lump under tine and fungal cultures did not grow. The respira- the skin of his index finger but decided to ignore it. A tory specimen was stained with a silver stain in month later, the lump ulcerated to present a necrotic anatomic pathology. Based on the microscopic mor- appearance, and two more lesions developed further phology in the image, what is the most likely identi- up the wrist and forearm. A histologic stain of mate- fication of this organism? rial from deep in the lesions showed elongated yeast cells resembling cigars. What disease is suspected?

FIGURE 2-4 (From Public Health Photo Library [PHL 960]. See also color plate 11.) FIGURE 2-3 (Courtesy Joel Mortensen, PhD. See also color plate 10.) a. Pneumocystis jiroveci b. Saccharomyces sp. c. Candida albicans a. Mycetoma d. Cryptococcus sp. b. Sporotrichosis 15. A significant amount of yeast was isolated from a c. Chromoblastomycosis vaginal culture of a patient in the teen clinic of your d. Blastomycosis hospital. It exhibited the following characteristics: 11. A germ tube–negative yeast is isolated in the labora- Microscopic: Clusters of blastoconidia along pseu- tory. The isolate is found to be negative for urease dohyphae, terminal chlamydoconidia and unable to assimilate dextrose, maltose, or Positive germ tube sucrose. CMT agar morphology showed blastoconi- Positive sucrose dia only. The organism is most likely: Which of the following is the most likely identifica- a. Candida albicans tion of this fungi? (Image from primary plate, b. Candida parapsilosis gram-stained smear, 40.) c. Torulopsis glabrata d. Geotrichum candidum 12. Which of the following is a key characteristic by which an unknown Cryptococcus spp. can be identified as Cryptococcus neoformans? a. Appearance of yellow colonies b. Positive urease test c. Presence of a capsule d. Positive niger seed agar test 13. Which of the following statements concerning the germ tube test is true? a. Using a heavy inoculum enhances the rapid production of germ tubes b. Germ tubes should be read after 2 hours of incubation at 25 C c. Candida albicans and Candida tropicalis can be FIGURE 2-5 (Courtesy Joel Mortensen, PhD. See also color used as positive and negative controls, plate 12.) respectively d. Serum will be stable for 1 year if stored at room a. Rhodotorula rubra temperature b. Candida albicans CHAPTER 2 Mycology, Virology, and Parasitology 83

c. Geotrichum candidum 18. A mold isolated in the laboratory displays a white d. Trichosporon beigelii cottony macroscopic morphology. On microscopic 16. The pharmacy at your hospital was concerned about evaluation, hyaline, septate hyphae, and “tooth- the hyperalimentation fluid they were preparing. The brush”-like conidiophres are seen. The most likely high lipid contact was a concern for contamination. organism is which of the following? A PAS stain of the suspect fluid is shown. Which of a. Aspergillus sp. the following organism would most likely demon- b. Acremonium sp. strate this morphology? c. Gliocladium sp. d. Scopulariopsis sp. 19. A mold is isolated in the laboratory that displays a velvety, gray-green colony morphology. On microscopic evaluation, flask-shaped conidiophores arranged in a brushlike formation are seen. The most likely organism is which of the following? a. Penicillium sp. b. Acremonium sp. c. Paecilomyces sp. d. Scopulariopsis sp. 20. A patient who underwent solid organ transplant appears to have systemic fungemia. The organism that has grown from the blood cultures macroscopically had a blue-green color to the colony, matured in 3 days, and grew well at 45 C. Microscopically, foot FIGURE 2-6 (Courtesy Joel Mortensen, PhD. See also color cells were seen and the phialides were uniserate with plate 13.) a round vesicle and columnar conidia. Which of the following is the most likely identification of this mold? a. Candida albicans b. Malassezia furfur c. Trichosporon cutaneum d. Scedosporium apiospermum 17. Several important types of conidiation of dematiaceous fungi exist. The image is an example of which one of these forms? (Lactophenol cotton blue stain.)

FIGURE 2-8 (Courtesy Joel Mortensen, PhD. See also color plate 15.)

a. Aspergillus fumigatus b. Aspergillus niger c. Scopulariopsis sp. d. Fusarium sp. 21. The protein coat that surrounds the nucleic acid of a virion is called which of the following? a. Capsomere FIGURE 2-7 (Courtesy Joel Mortensen, PhD. See also color b. Capsid plate 14.) c. Capsule d. Nucleocapsid a. Cladosporium type 22. During viral assembly, how are viral envelopes b. Phialophora type acquired? c. Rhinocladiella type a. By production of envelope constituents by host d. Rinderpest type cellular DNA 84 CHAPTER 2 Mycology, Virology, and Parasitology

b. As the virion buds from a host cell membrane c. Through replication of viral nucleic acid d. As host cell lysis produces many membrane fragments 23. Prions are best described by which of the following? a. Infectious viral RNA without capsid proteins b. Infectious protein with no associated nucleic acid c. Infectious viral DNA without capsid proteins d. Nonenveloped virus highly resistant to heat and chemical inactivation 24. The viral nucleocapsid always contains which of the following? a. Viral genome b. Virus-encoded glycoprotein c. Virus-encoded polymerase FIGURE 2-9 (Courtesy Joel Mortensen, PhD. See also color plate 16.) d. Viral envelope 25. Which of the following viruses are thought to predominately cause gastroenteritis? a. Herpes simplex virus a. Hepadnaviruses b. Adenovirus b. Filoviruses c. Cytomegalovirus c. Noroviruses d. Epstein-Barr virus d. Arboviruses 31. Trophozoite forms of amoebae are found in what 26. Which of the following groups contains the SARS type of stool specimen? virus? a. Formed a. Calicivirus b. Loose b. Coronavirus c. Soft c. Flavivirus d. Watery d. Filovirus 32. Which preservation method is most suitable and 27. Which of the following groups of virus is best the most widely used for subsequent fixed smear described as: preparation? ssRNA, enveloped, a. Formalin-ethyl acetate Pleomorphic/spherical capsid b. PVA Large club-shaped spikes on surface gives “corona” c. Trichrome effect d. MIF Causes approximately 15% of coldlike illness 33. If the ova of this parasite are ingested by humans, the a. Influenza A oncosphere form can migrate through the body via b. Influenza B the bloodstream, resulting in the condition known c. Coronaviruses as cysticercosis. Which of the following is correct? d. Pneumovirus a. Taenia solium 28. Which of the following is the specimen of choice for b. Entamoeba histolytica detecting rotavirus? c. Hymenolepis nana a. Throat swab d. Clonorchis sinensis b. Urine sample 34. Ova recovered from the stool are routinely used to c. Bronchoalveolar wash diagnose infections caused by all of the following d. Feces sample except? 29. The test of choice and most sensitive assay for use a. Necator americanus with CSF to diagnose aseptic meningitis caused by b. Ascaris lubricoides enterovirus is which of the following? c. Trichuris trichiura a. Cell culture d. Strongyloides stercoralis b. PCR 35. An MLS finds an E. coli cyst on a wet mount of a c. Antigenemia immunoassay fresh stool specimen. Which of the following should d. Shell vial assay be done? 30. A specimen from a genital lesion was inoculated into a. Request a second specimen a standard set of cells for virus isolation. On day 1 the b. Look for additional E. coli cysts human foreskin fibroblasts exhibited the CPE shown c. Examine the remaining area of the wet preparation in the figure. d. Generate a final report CHAPTER 2 Mycology, Virology, and Parasitology 85

36. Which of the following parasites have migration a. Ascaris lumbricoides through the lungs as part of their lifecycle? b. Ancyclostoma duodenale a. Necator americanus, Ancylostoma duodenale, c. Necator americanus Strongyloides stercoralis d. Trichuris trichiura b. Giardia lamblia, Wuchereria bancrofti, Brugia 39. The eggs in the figure below were found in the urine malayi of a Peace Corp worker who had just returned to the c. Enterobius vermicularis, Trichuris trichiura, Tri- United State after spending 2 years in the Middle chinella spiralis East. The eggs measured about 160 mm long60 mm d. Toxocara canis, Toxoplasma gondii, Blastocystis wide. Which of the following organisms is the most hominis likely identity? 37. The image below is of a suspected parasite seen on direct examination of material taken from a corneal scraping in an ophthalmology clinic. The most likely identification of the parasite in this specimen is which of the following?

FIGURE 2-12 (Courtesy the Centers for Disease Control and Pre- vention. See also color plate 19.)

a. Diphyllobothrium latum b. Schistosoma haematobium FIGURE 2-10 (Courtesy the Centers for Disease Control and Prevention. See also color plate 17.) c. Schistosoma japonicum d. Schistosoma mansoni a. Acanthamoeba sp. 40. A patient was diagnosed with cysts in his liver. He is b. Enterobius sp. originally from Australia, where he was involved in c. Paragonimus sp. a sheep herding operation. The adult parasite shown d. Naegleria sp. below was passed by his pet dog. It measured 5 mm. 38. The organism shown below was recovered from the What is the most likely identification of this organism? stool of a patient who resides in rural Texas. The most likely identification is which of the following?

FIGURE 2-11 (Photograph by Dr. Mae Melvin, courtesy the Cen- ters for Disease Control and Prevention, Public Health Image Library, FIGURE 2-13 (Courtesy the Centers for Disease Control and Pre- http://phil.cdc.gov/. See also color plate 18.) vention. See also color plate 20.) 86 CHAPTER 2 Mycology, Virology, and Parasitology

a. Diphyllobothrium latum a. Chilomastix mesnili b. Dipylidium caninum b. Cyclospora cayetanensis c. Echinococcus granulosus c. Giardia lamblia d. Taenia solium d. Iodamoeba butschlii 41. These trophozoites were found in a trichrome- 43. The image below is a cyst found in a human fecal stained slide of a stool sample, measuring an average smear. The cyst measured about 12 mm in length of 25 microns in diameter. Which of the following is and contained four nuclei and a rounded chromatoid the most likely identity of this organism? bar. The patient had severe diarrhea and some blood in the stool. What is the most likely identification of this organism?

FIGURE 2-14 (Courtesy the Centers for Disease Control and Pre- vention. See also color plate 21.)

a. Endolimax nana FIGURE 2-16 (Courtesy the Centers for Disease Control and Pre- b. Entamoeba coli vention. See also color plate 23.) c. Balantidium coli d. Dientamoeba fragilis 42. The cyst in the image below was observed in a stool sample of a child at a daycare center. The ovoid cyst a. Endolimax nana measures approximately 108 microns. Which b. Entamoeba coli organism is the most likely cause of the child’s c. Entamoeba histolytica diarrhea? d. Iodamoeba butschlii 44. Match the parasite with the most appropriate description. _____ Plasmodium falciparum _____ Plasmodium malariae _____ Plasmodium ovale _____ Plasmodium vivax a. RBC enlarged, oval, Schuffner’s dots, gametocytes seen by day 4 to 18 b. Large RBC, troph irregular, multiple phases seen, gametocytes appear early c. Delicate ring forms, multiple rings per cell, crescent-shaped gametocytes after 7 to 10 days d. RBC normal in size and color, troph compact, band forms may be seen, gametocytes seen after weeks 45. Microfilariae found in the blood that have a sheath, demonstrate nocturnal periodicity and exhibit nuclei FIGURE 2-15 (Courtesy the Centers for Disease Control and Pre- that do not extend to the tail tip are which of the vention. See also color plate 22.) following? CHAPTER 2 Mycology, Virology, and Parasitology 87

a. Blastocystis hominis b. Cyclospora cayetanensis c. Isospora belli d. Balantidium coli 48. Match the scientific name with the corresponding common name. _____ Sarcoptes scabei _____ Ixodes scapularis _____ P. humanus humanus _____ Cimex lectularius a. Body louse b. Bedbug c. Scabies d. Lyme disease 49. The only known human tapeworm with an opercu- lum is: FIGURE 2-17 (Courtesy the Centers for Disease Control and Pre- vention. See also color plate 24.) a. Diphyllobothrium latum b. Hymenolepis nana c. Giardia lamblia a. Brugia malayi d. Schistosoma haematobium b. Onchocerca volvulus 50. Identify the following organism as it appears in this c. Loa loa peripheral blood smear. d. Wuchereria bancrofti 46. Necator americanus rhabditiform larvae can be differentiated from Strongyloides stercoralis rhabditiform larvae by: a. Length of the notched tail b. Length of the head region c. Segmentation d. Size of the genital primordium 47. The image below is from a fecal smear of an individual complaining of diarrhea and intestinal discomfort. The parasites were numerous and quite variable in size, but the majority measured about 15 to 20 mm in diameter. What is the most likely identification of this organism?

FIGURE 2-19 (Courtesy the Centers for Disease Control and Pre- vention. See also color plate 26.)

a. Trypanosoma sp. b. Leishmania sp. c. Wuchereria bancrofti d. Loa loa

FIGURE 2-18 (Courtesy the Centers for Disease Control and Pre- vention. See also color plate 25.) 88 CHAPTER 2 Mycology, Virology, and Parasitology

SELF-ASSESSMENT

Content Area: ______

Score on Practice Questions: ______

List the specific topics covered in the missed questions:

List the specific topics covered in the correct questions: CHAPTER 2 Mycology, Virology, and Parasitology 89

NOTES CHAPTER 3 Hematology

Sandy Cook

HEMATOPOIESIS Skull • Regulated process of blood cell production • Hematopoietic stem cells give rise to red blood cells (RBC), white blood cells (WBC), and platelets • Most hematopoiesis occurs in the bone marrow of adults; however, some changes in cell production Proximal end of occur from conception to adulthood large bones ○ Bone marrow Sternum § Tissue present in the cavities of cortical bones Vertebrae § Red marrow: Hematopoietically active marrow located in most bones in early fetal Axial skeleton and childhood development but transitions to fewer locations as an adult Iliac crest § Adults have red marrow in the proximal ends of long bones, sternum, skull, scapu- lae, ribs, and pelvis, with approximately equal amounts of red and yellow marrow (Figure 3-1) § Yellow marrow: Hematopoietically inactive marrow, consisting primarily of fat cells • Sites of hematopoiesis (Figure 3-2 and Table 3-1)

Cell Lines Produced

• The hematopoietic stem cells give rise to the different cell lines. Progenitor cells commit to various states of maturation, with the common lymphoid progenitor producing lymphoid cells and the common myeloid progenitor leading to the production of the neutrophil, monocyte, erythrocytic, and megakaryocytic cell lines (Figure 3-3) FIGURE 3-1 The adult skeleton, in which darkened areas depict active red marrow hematopoiesis. (From Rodak BF, Fritsma GA, RED BLOOD CELL PRODUCTION AND Keohane E: Hematology: clinical principles and applications, ed 4, DESTRUCTION St Louis, 2012, Saunders.)

• Pronormoblasts divide and progress to form mature erythrocytes § (1) Pronormoblast, (2) basophilic normoblast, • Production of RBCs from the multipotential stem cell (3) polychromatophilic normoblast, (4) ortho- ○ BFU-E: Burst-forming unit–Erythroid chromic normoblast, (5) reticulocyte, (6) ○ CFU-E: Colony-forming unit–Erythroid mature erythrocyte ○ Pronormoblast dividing and progressing to ○ As cells divide and mature, cell size gradually become a mature erythrocyte decreases and the nucleus is eventually extruded § Stages from most immature to mature at maturity (Table 3-2)

90 CHAPTER 3 Hematology 91

Cellularity (%)

100

Bone marrow 80 Vertebra Yolk sac Liver 60 Sternum 1 2 3 40 Femur Sites of hematopoiesis Rib Tibia 1 Mesoblastic 20 2 Hepatic Spleen Lymph nodes 3 Myeloid 0 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 Fetal months Birth Age in years FIGURE 3-2 Sites of hematopoiesis. (From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.)

TABLE 3-1 Sites of Hematopoiesis

Age Location Product Produced Other Embryo Aorta-gonad-mesonephros Hematopoietic stem cells, (AGM) region/yolk sac primitive erythroblasts, and fetal hemoglobins Fetal Liver Erythroblasts, granulocytes, Liver is the primary site through first trimester Other sites (spleen, kidney, monocytes Bone marrow becomes active around the fifth month of thymus, lymph nodes) gestation Bone marrow Birth through Bone marrow All hematopoietic cell lines Bone marrow is the primary site adult Some involvement from lymph Babies and children have more red marrow activity, whereas nodes, spleen, liver, kidney, normal adult marrow has an approximately equal thymus composition of red and yellow marrow Thymus activity decreases after childhood

○ RBC synthesis is stimulated by erythropoietin, ○ Each heme molecule can combine reversibly with which is produced primarily in the kidney in one molecule of oxygen response to hypoxia • The globin portion contains two pairs of ○ RBCs normally have a lifespan of approximately globin chains, two a chains and two non-a chains 120 days. At the end of their life, they are ○ a and z chain production is controlled by genes removed by intravascular or extravascular on chromosome 16 hemolysis, and the contents of the cell are ○ b, g, d, and e chain production is controlled by recycled or excreted. genes on chromosome 11 ○ Chain production is turned on and off through HEMOGLOBIN (HGB) PRODUCTION stages of development, leading to production of mainly a and b chains with maturity • Occurs from pronormoblast to reticulocyte stage of (Table 3-4) RBCs • Hgb is used for the transport of gases • Heme is synthesized in the mitochondria, so mature ○ O2 affinity of Hgb is low at a low O2 tensioninthe RBCs are unable to produce heme because of loss of body and affinity is high at a high O2 tension. This mitochondria with maturation is demonstrated by the O2 dissociation curve • Normal Hgb is composed of four heme molecules (Figure 3-4) nested in four globin molecules • Oxyhemoglobin is the primary Hgb for gas • Heme is composed of a protoporphyrin IX ring with transport in the body, but other Hgb variants may Fe+2 at its center be seen 92 CHAPTER 3 Hematology

Long-term self- Short-term self- renewing stem cell renewing stem cell

Multipotent progenitor hematopoietic stem cell

Common Common myeloid progenitor lymphoid progenitor

Granulocyte-monocyte Eosinophil-basophil Megakaryocyte-erythrocyte Dendritic Pre-B Pre-T Natural progenitor progenitor progenitor cell killer cell

Myeloblast Monoblast Myeloblast Myeloblast Pronormoblast Megakaryoblast B lymphoblast T lymphoblast

Neutrophil Monocyte Eosinophil Basophil Erythrocyte Megakaryocyte B cell T cell

Macrophage Mast cell Platelets Plasma cell FIGURE 3-3 Diagram of hematopoiesis shows derivation of cells from the multipotent stem cell. (From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.)

TABLE 3-2 Red Blood Cell Maturation

Nucleus-to- % in Bone Bone Marrow Transit Cell or Stage Diameter Cytoplasm Ratio Nucleoli Marrow Time (hr) Pronormoblast 12-20 mm 8:1 1-2 1 24 Basophilic normoblast 10-15 mm 6:1 0-1 1-4 24 Polychromatic normoblast 10-12 mm 4:1 0 10-20 30 Orthochromic normoblast 8-10 mm 1:2 0 5-10 48 Shift (stress) reticulocyte 8-10 mm No nucleus 0 1 48-72* (polychromatic erythrocyte) Polychromatic erythrocyte 8-8.5 mm No nucleus 0 1 24-48* From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders. *Transit time in peripheral blood. CHAPTER 3 Hematology 93

• Maintain the RBC membrane flexibility 100 • Supply offshoot pathways ○ Hexose monophosphate shunt 80 § Protection of the RBC from oxidant damage Myoglobin pH 7.6 by production of reduced glutathione B ○ Methemoglobin reductase pathway 60 § pH 7.2 Maintaining iron in the ferrous form for Hgb P50 to limit the production of methemoglobin pH 7.4 saturation 2 40 C ○ Rapoport-Luebering pathway A § % O Regulation of O2 delivery to tissues by the production of 2,3 bisphosphoglycerate (2,3 Hemoglobin 20 BPG) (Figure 3-5) WHITE BLOOD CELLS

0 20 40 60 80 100 • Neutrophils PO (mm Hg) 2 • Phagocytic cells present in the peripheral circula- Oxygen dissociation curve. Normal dissociation FIGURE 3-4 A, tion destroy foreign substances and microorganisms curve. B, Left-shifted curve with reduced P50 caused by a decrease in 2,3-bisphosphoglycerate (2,3-BPG), partial pressure of • Constitute the majority of circulating WBCs in adults + carbon dioxide (PCO2), temperature, and H ions (raised pH). A • Development (Table 3-3) left-shifted curve is also seen with hemoglobin variants that have ○ Stages from most immature to mature include increased oxygen affinity. C, Right-shifted curve with increased + 1. myeloblast 2. promyelocyte 3. myelocyte 4. P50 caused by an elevation in 2,3-BPG, PCO2, temperature, and H ions (lowered pH). A right-shifted curve is also seen with metamyelocyte 5. band 6. segmented neutrophil (Table 3-3). hemoglobin variants that have decreased O2 affinity. (From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and • Normal neutrophil function applications, ed 4, St Louis, 2012, Saunders.) ○ Cells move to a site of inflammation, and granules are released to assist in travel and adhesion. Hemoglobin Variants Once at their target site, cells work to eliminate the foreign material by phagocytosis Granule Methemoglobin Hgb containing Fe+3 instead of the normally contents and neutrophil extracellular traps +2 reduced Fe are used to trap and kill microorganisms Formed normally as a result of oxidation; (Boxes 3-1 to 3-3) however, it is usually kept from • Location overproduction because of the ○ methemoglobin reductase pathway, an Present in a circulating pool in which neutrophils offshoot of the Embden-Meyerhof pathway travel throughout the peripheral circulation Sulfhemoglobin Hgb formed as a result of oxidation of Hgb by and a marginating pool in which neutrophils materials containing sulfur line the walls of the vasculature, waiting to be Process is irreversible, so once Hgb has become called into use. In the bone marrow, before sulfhemoglobin, it will remain for the life of release to the peripheral vasculature, a storage the cell pool and mitotic pool are present Unable to transport O2, leading to cyanosis • Eosinophils Carboxyhemoglobin Hgb resulting from heme iron-binding carbon • Maturation is similar to neutrophil maturation, monoxide although granule contents are different; eosinophils CO has a high O affinity and does not give up 2 have a variety of substances in their granules (see O2 to the tissues easily Small amounts of carboxyhemoglobin are Box 3-3) produced within the cells, but most problems • Normal function ○ occur as a result of environmental exposures Cells serve in immune regulation, from antigen presentation to initiation of immune response RED BLOOD CELL METABOLISM AND ○ Primarily increased in parasitic infection (especially PHYSIOLOGY helminths) and allergic disorders • Basophils • Mature RBCs have no mitochondria, so rely on anaerobic • Maturation is similar to eosinophil and neutrophil glycolysis for energy via the Embden-Meyerhof pathway maturation. Basophils have several different types • Energy is needed for the main Embden-Meyerhof of granules (Box 3-4) pathway • Cells have immunoglobulin E (IgE) receptors that • Maintain cation gradients lead to their effectiveness in allergic and hypersen- ○ Keeps potassium inside and sodium outside sitivity reactions. They also play a role in initiating the RBC the immune response Embden-Meyerhof Pathway (Anaerobic Pathway of Glucose Metabolism) H2O2 H2O Hexose Monophosphate Pathway

Glutathione peroxidase

GSH GSSG

Glucose Glutathione reductase ATP Hexokinase (–1 ATP) ADP NADP NADPH Glucose 6-phosphate 6-phospho-gluconate Glucose-6-phosphate dehydrogenase Glucose phosphate isomerase

Fructose 6-bisphosphate Pentose phosphate ATP Phosphofructokinase (–1 ATP) ADP Fructose 1,6-bisphosphate aldolase Dihydroxyacetone Glyceraldehyde phosphate 3-phosphate Triose phosphate isomerase Methemoglobin Glyceraldehyde NAD reductase +H Methemoglobin 3-phosphate dehydrogenase NADH Methemoglobin Hemoglobin reductase

1,3-Bisphosphoglycerate Methemoglobin Bisphosphoglycerate Reductase Pathway mutase ADP (+2 ATP) ATP Rapaport-Luebering Phosphoglycerate Pathway kinase 2,3-Bisphosphoglycerate (2,3-BPG) 3-Phosphoglycerate Bisphosphoglycerate phosphatase

Phosphoglyceromutase

2-Phosphoglycerate

Enolase

Phosphoenolpyruvate

ADP Pyruvate (+2 ATP) kinase ATP

Pyruvate

NADH

NAD

Lactate FIGURE 3-5 Glucose metabolism in the erythrocyte. ADP, Adenosine diphosphate; ATP, adenosine triphosphate; G6PD, glucose-6-phosphate dehydrogenase; NAD, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide (reduced form); NADP, nicotinamide adenine dinucleotide phosphate (oxidized form). (From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.) CHAPTER 3 Hematology 95

TABLE 3-3 Neutrophil Development

Stage Description Image

Myeloblast Earliest recognizable stage; cells are large with large nuclei and loose chromatin, some primary granules may be seen

(From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders. See Color Plate 27.)

Promyelocyte Cell begins to decrease in size, and chromatin begins to compact, with nucleus often appearing as eccentric; primary granules are prominent

(From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders. See Color Plate 28.)

Myelocyte Cell continues to decrease in size, and chromatin continues to compact into a round nucleus; last stage capable of mitosis; secondary granules are formed

(From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders. See Color Plate 29.)

(Continued) 96 CHAPTER 3 Hematology

TABLE 3-3 Neutrophil Development—cont’d

Stage Description Image

Metamyelocyte Cell continues to decrease in size, chromatin continues to compact into a kidney bean shape; secondary and tertiary granules are formed

(From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders. See Color Plate 30.)

Band neutrophil Nucleus shows compact chromatin that is shaped into a horseshoe form; tertiary and secretory granules are formed

(From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders. See Color Plate 31.)

Segmented Nucleus begins to segment into three to four neutrophil lobes, each attached by a threadlike nuclear filament; secretory granules are formed

(From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders. See Color Plate 32.) CHAPTER 3 Hematology 97

• NOTE: Mast cells are somewhat related to baso- • Monocytes phils; however, they are tissue cells used in allergic • Maturation starts with a monoblast and con- reactions and inflammation tinues to the promonocyte and mature monocyte stages • Cell appearance shows large cells, the largest in peripheral circulation, with finely granular cyto- Normal Hemoglobins and Globin plasm (“ground-glass” appearance) and a nucleus TABLE 3-4 Chains with relatively loose, lacy chromatin, with the occa- Embryonic Fetal Adult sional presence of folding or indentation. Cyto- plasm may show vacuolization Gower 1: Hgb F: Hgb A: z2e2 a2g2 a2b2 • Functions 50%-90% 97% ○ Cells are used in both innate and adaptive immu- Gower 2: a22e2 Hgb A: a2b2 Hgb A2: a2d2 10%-40% 1.5%-3.5% nity. They can recognize and phagocytize foreign materials; in addition they can serve as antigen- Portland: z2g2 Hgb A2: a2d2 <2% Hgb F: a2g2 <2% presenting cells to initiate T and B cells they

BOX 3-1 Neutrophil Granules

Primary (Azurophilic) Granules Tertiary Granules Formed during the promyelocyte stage Formed during metamyelocyte and band stages Last to be released (exocytosis) Second to be released Contain Contain Myeloperoxidase Gelatinase Acid b-glycerophosphatase Collagenase Cathepsins Lysozyme Defensins Acetyltransferase

Elastase b2-Microglobulin Proteinase-3 Secretory Granules (Secretory Vesicles) Others Formed during band and segmented stages Secondary (Specific) Granules First to be released (fuse to plasma membrane) Formed during myelocyte and metamyelocyte stages Contain (attached to membrane) Third to be released CD11b/CD18 Contain Alkaline phosphatase

b2-Microglobulin Vesicle-associated membrane-2 Collagenase CD10, CD13, CD14, CD16 Gelatinase Cytochrome b558 Lactoferrin Complement 1q receptor Neutrophil gelatinase–associated lipocalin Complement receptor-1 Others

From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.

BOX 3-2 Phagocytosis

Recognition and attachment Oxygen independent • Phagocyte receptors recognize and bind to certain foreign molec- • The pH within the phagosome becomes alkaline and then neutral, ular patterns and opsonins such as antibodies and complement the pH at which digestive enzymes work. components. • Primary and secondary lysosomes (granules) fuse to the phago- Ingestion some and empty hydrolytic enzymes and other bacteriocidal mol- • Pseudopodia are extended around the foreign particle and enclose ecules into the phagosome. it within a phagosome (engulfment). Formation of neutrophil extracellular traps • The phagosome is pulled toward the center of the cell by polymer- • Nuclear and organelle membranes dissolve, and activated cyto- ization of actin and myosin and by microtubules. plasmic enzymes attach to DNA. Killing and digestion • The cytoplasmic membrane ruptures and DNA with attached Oxygen dependent enzymes is expelled, so that the bacteria are digested in the exter- • Respiratory burst through the activation of nicotine adenine nal environment.

diphosphate oxidase (reduced form). H2O2 and hypochlorite are produced.

From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders. 98 CHAPTER 3 Hematology

BOX 3-3 Eosinophil Granules

Primary Granules Others Formed during promyelocyte stage Small lysosomal granules Contain Acid phosphatase Charcot-Leyden crystal protein Arylsulfatase B Secondary Granules Catalase Formed throughout remaining maturation Cytochrome b558 Contain Elastase Major basic protein (core) Eosinophil cationic protein Eosinophil cationic protein (matrix) Lipid bodies Eosinophil-derived neurotoxin (matrix) Cyclooxygenase Eosinophil peroxidase (matrix) 5-Lipoxygenase Lysozyme (matrix) 15-Lipoxygenase Catalase (core and matrix) Leukotriene C4 synthase b-Glucuronidase (core and matrix) Eosinophil peroxidase Cathepsin D (core and matrix) Esterase Interleukins-2, -4, and -5 (core) Storage vesicles Interleukin-6 (matrix) Carry proteins from secondary granules to be released into the Granulocyte-macrophage colony-stimulating factor (core) extracellular medium

From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.

BOX 3-4 Basophil Granules TABLE 3-5 Normal Range Values in Adults

Secondary Granules Adult Adult Histamine Male Female Platelet activating factor WBC (109/L) 4.5-11.5 4.5-11.5 12 Leukotriene C4 RBC (10 /L) 4.60-6.00 4.00-5.40 Interleukin-4 Hgb (g/dL) 14.0-18.0 12.0-15.0 Interleukin-13 Hct (%) 40-54 35-49 Vascular endothelial growth factor A MCV (fL) 80-100 80-100 Vascular endothelial growth factor B MCH (pg) 26-32 26-32 Chondroitin sulfates (e.g., heparin) MCHC (g/dL) 32-36 32-36 RDW (%) 11.5-14.5 11.5-14.5 Platelet (109/L) 150-450 150-450 are and can be used for housekeeping purposes to MPV (fL) 6.8-10.2 6.8-10.2 remove dead cells and debris Neutrophils (%) 50-70 50-70 ○ NOTE: Once monocytes migrate to tissues, they Lymphocytes (%) 18-42 18-42 serve as tissue macrophages with similar functions Monocytes (%) 2.0-11 2.0-11 • Lymphocytes Eosinophils (%) 1.0-3 1.0-3 Basophils (%) 0-2 0-2 • The maturation stages are 1. lymphoblast 2. pro- Segmented neutrophils (109/L) 2.3-8.1 2.3-8.1 lymphocyte 3. mature lymphocyte. There are three Band neutrophils (109/L) 0-0.6 0-0.6 major subgroups of lymphocytes: T cells, B cells, Lymphocytes (109/L) 0.8-4.8 0.8-4.8 and natural killer (NK) cells. Monocytes (109/L) 0.45-1.3 0.45-1.3 • Lymphocytes can be produced in both the bone mar- Eosinophils (109/L) 0-0.4 0-0.4 row and the lymphoid tissues. Cells can return from Basophils (109/L) 0-0.1 0-0.1 an inactive/resting form into active blasts, as needed • Hct, Hematocrit; Hgb, hemoglobin; MCH, mean corpuscular hemoglobin; Functions mean corpuscular volume; mean platelet volume; red blood ○ MCV, MPV, RBC, B cells produce antibodies and also play a role cell; RDW, red cell distribution width; WBC, white blood cell. in antigen presentation to the T cells ○ T cells mediate the immune response are relatively similar, some slight variations may occur LABORATORY CONSIDERATIONS based on a specific laboratory’s population (Table 3-5)

Normal Ranges Staining of Blood and Bone Marrow Samples

• Patient values are compared against established normal • Smears are made of blood or bone marrow to provide ranges, which can varybased onage,gender,population, smears with the best possible distribution of cellular and geographic distribution. Although normal ranges elements, leaving a “critical area” or examination area CHAPTER 3 Hematology 99

where a single layer of cells is evenly dispersed, allowing • Measured by placing whole blood in capillary tubes visualization of individual cellular elements and centrifuging to read the packed cell volume on a • Wright or Wright-Giemsa stains (Romanowsky-type manual microhematocrit reading device stains) are polychrome stains used to stain slides • Hct values are available on automated general hematol- of peripheral blood and bone marrow, which gives ogy cell counters; however, values are derived by using a elements their characteristic colors calculation as opposed to a physical measurement • Slides are fixed with a methanol fixative, followed by staining with a solution or solutions containing eosin and methylene blue to impart color to cellu- Reticulocyte Count lar elements • • Eosin is acidic, so it will stain basic elements, such as Reticulocytes are the final stage before an RBC Hgb and basic proteins found in some cell granules reaches maturity. Reticulocytes may be counted • Methylene blue is basic and will stain acidic elements, manually to determine the erythrocyte production such as cell nuclei and immature cell cytoplasm and release from the bone marrow • • Stained slides can be used for counting the 100 cell Manual counts are performed by incubating ethylene- count WBC differential and examining RBC and diametetraacetic acid (EDTA) whole blood with a platelet morphology. In the case of bone marrow, supravital stain, usually new methylene blue. If any aspirate and core biopsy slides can be stained for RNA or residual organelles are present, they will take a bone marrow differential, myeloid-to-erythroid up the supravital stain and are visible microscopically • (M/E) ratio, or sample cellularity Various methods are used for the manual reticulocyte count, including the Miller ocular and other Red Blood Cell Morphology techniques to determine the total percentage of reticulocytes present See Tables 3-6 and 3-7 • Automated reticulocyte counts are now commonly performed Red Blood Cell Indices • Various methods are used for automated counts, including treating RBCs with a stain or fluorescent • Red blood cell indices may be calculated manually or dye to identify the reticulocyte by optical methods derived from automated instrumentation. or flow cytometry. Automation allows for a larger See Table 3-8 number of cells to be examined. Manual and Semiautomated Techniques Sickle Cell Testing • Most hematologic testing is performed using automated instrumentation; however, a few manual tech- • Samples may be screened for the presence of abnormal niques may be used on occasion Hgb because of the different solubility properties of various Hgb Hemacytometer • Hgb S exhibits decreased solubility when deoxygen- ated, as opposed to the more soluble Hgb A and A • 2 Counting chamber used for manual cell counting, cur- • Screening tests use a lysing agent and a dithionite rently most frequently used for body fluid cell counting solution, or other reducing agent, to induce deoxy- • Based on the use of a known counting area of genation of Hgb. Abnormal Hgbs have decreased nine squares each with an area of 1 mm2 and a total 3 3 solubility and will precipitate in the solution, volume of 0.9 mm for a total area of 9 mm .Astan- appearing cloudy on observation dard formula is used to determine a total cell count/m • Positive sickle cell screens require follow-up with a 3 ¼ L(1mm 1 mL) (Figure 3-6) more definitive test, such as Hgb electrophoresis, high-performance liquid chromatography (HPLC), Total cell countðÞ¼ cells= L 0 m 1 or isoelectric focusing B Cells counted C B C Reciprocal dilution @ Number of squares counted A area of squares counted depth Erythrocyte Sedimentation Rate (ESR) • Screening test used to screen or monitor for various Manual hematocrit (Hct, also known as inflammatory states. The ESR looks at how much microhematocrit) RBC settling will occur in a well-mixed whole blood sample over a 1-hour period • Hct is the volume of packed RBCs occupying a specific • RBCs normally have a net negative charge, causing volume of whole blood, expressed as a percentage or in them to repel each other in a whole blood sample, liters per liter leading to slow settling of the RBCs over time 100 CHAPTER 3 Hematology

TABLE 3-6 Description of Red Blood Cell Abnormalities and Commonly Associated Disease States

RBC Abnormality Cell Description Commonly Associated Disease States Anisocytosis Abnormal variation in RBC volume or diameter Hemolytic, megaloblastic, iron-deficiency anemia Macrocyte Large RBC (>8 mm in diameter), MCV >100 fL Megaloblastic anemia Myelodysplastic syndrome Chronic liver disease Bone marrow failure Reticulocytosis Oval macrocyte Large, oval RBC Megaloblastic anemia Poikilocytosis Abnormal variation in RBC shape Severe anemia Certain shapes helpful diagnostically Spherocyte Small, round, dense RBC with no central pallor Hereditary spherocytosis Immune hemolytic anemia Extensive burns (along with schistocytes) Elliptocyte, ovalocyte Elliptical (cigar shaped), oval (egg shaped), RBC Hereditary elliptocytosis or ovalocytosis Iron-deficiency anemia Thalassemia major Myelophthisic anemias Stomatocyte RBC with slitlike area of central pallor Hereditary stomatocytosis Rh deficiency syndrome Acquired stomatocytosis (liver disease, alcoholism) Artifact Sickle cell Thin, dense, elongated RBC pointed at each end; may be curved Sickle cell anemia Sickle cell–b-thalassemia Hgb C crystal Hexagonal crystal of dense Hgb formed within the RBC membrane Hgb C disease Hgb SC crystal Fingerlike or quartzlike crystal of dense Hgb protruding from the Hgb SC disease RBC membrane Target cell (codocyte) RBC with Hgb concentrated under and around the periphery Liver disease resembling a target Hemoglobinopathies Thalassemia Schistocyte (schizocyte) Fragmented RBC resulting from rupture in the peripheral circulation Microangiopathic hemolytic anemia* (along with microspherocytes) Traumatic cardiac hemolysis Extensive burns (along with microspherocytes) Helmet cell (keratocyte) RBC fragment in shape of a helmet Same as schistocyte Folded cell RBC with membrane folded over Hb C disease Hb SC disease Acanthocyte (spur cell) Small, dense RBC with few irregularly spaced projections of varying Severe liver disease (spur cell anemia) length Neuroacanthocytosis (abetalipoproteinemia, McLeod’s syndrome) Burr cell (echinocyte) RBC with blunt or pointed, short projections that are usually evenly Uremia spaced over the surface of cell; present in all fields of blood film Pyruvate kinase deficiency but in variable numbers per field{ Teardrop cell RBC with a single pointed extension resembling a teardrop or pear Primary myelofibrosis (dacryocyte) Myelophthisic anemia Thalassemia Megaloblastic anemia

From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders. *Such as thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, disseminated intravascular coagulation. {Cells with similar morphology that are unevenly distributed in a blood film (not present in all fields) are likely the result of a drying artifact in blood film preparation; these artifacts are sometimes referred to as crenated RBCs. Hgb, Hemoglobin; MCV, mean cell volume; RBC, red blood cell; SC, sickle cell.

• When a change to the charge occurs, usually resulting Hematology Instrumentation from increases in plasma proteins, the cells become attracted to each other, leading to increased settling • Cell counters speeds of the RBCs • Automated method for performing complete blood • Tests are performed with manual Westergren and cell counts Wintrobe procedures or automated analyzers to • Count cells using impedance and optical allow for a faster reading measurements CHAPTER 3 Hematology 101

Erythrocyte Inclusions: Description, Composition, and Selected Commonly Associated TABLE 3-7 Disease States

Appearance in Inclusion Associated Diseases Inclusion Supravital Stain Appearance in Wright Stain Composition and Conditions Diffuse basophilia Granules and Bluish tinge throughout cytoplasm; RNA Hemolytic anemia filaments also referred to as After treatment for iron,

polychromasia vitamin B12, or folate deficiency Basophilic Granules and Blue-purple granules distributed Precipitated RNA Lead poisoning stippling filaments throughout cytoplasm Thalassemia (punctate Hemoglobinopathies basophilia) Abnormal heme synthesis Howell Jolly body Dense, round, granule Dense, round, blue or purple DNA (nuclear Hyposplenism granule; usually one per cell; fragment) After splenectomy occasionally multiple Megaloblastic anemia Hemolytic anemia Heinz body Round granule Not visible Denatured Glucose-6-phosphate attached to inner hemoglobin dehydrogenase membrane deficiency Unstable hemoglobins Oxidant drugs/chemicals Pappenheimer Clusters of small Clusters of small, light blue Iron Sideroblastic anemia bodies* granules granules, often near periphery Hemoglobinopathies of cell Hyposplenism Megaloblastic anemia Cabot ring Rings or figure-eights Blue rings or figure-eights Remnant of mitotic Megaloblastic anemia spindle Myelodysplastic syndromes Hgb H Fine, evenly dispersed Not visible Precipitate of b Hgb H disease granules chains of hemoglobin

From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders. *Blue (siderotic) granules observed in Prussian blue stain. Hgb, Hemoglobin.

TABLE 3-8 Red Blood Cell Indices

Normal Definition Manual Calculation Range Mean corpuscular volume (MCV) Average volume of an individual RBCAnalyzer: (Hct [%]/RBC) 10 80-100 fL Measures directly or Mean corpuscular hemoglobin (MCH) Average weight of hemoglobin in an individual (Hgb [g/dL]/RBC) 10 28-34 pg RBCAnalyzer: Measures directly or Mean corpuscular hemoglobin Ratio of hemoglobin mass to the cell volume (Hgb [g/dL]/Hct [%]) 100 32-36 g/dL concentration (MCHC) Red cell distribution width (RDW) Variation of RBC volume used to help identify 11.5%-14.5% the presence of anisocytosis Analyzer calculation ¼ Standard Deviation of MCV100 Mean MCV

○ Impedance: Uses changes in electrical charge as § Pulses formed by electrical resistance are cells, which have low conductivity, move counted through an electrically conductive fluid to deter- § The number of pulses recorded is propor- mine cell count tional to the cell count 102 CHAPTER 3 Hematology

1 mm 1 mm

side view

cover slip 0.1 mm moat moat

FIGURE 3-6 Hemacytometer and close-up view of the counting areas as seen under the microscope. The areas for the standard white blood cell count are labeled W, and the areas for the standard red blood cell count are labeled R. The entire center square is used for counting platelets. (From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.)

§ Pulse size is proportional to cell size § NOTE: If a corrected WBC count is needed, § Can be used to count WBC, RBC, and platelets it should be lower than the original WBC ○ Optical counts: Use of optical light scatter to deter- count from the analyzer mine the size and complexity of cells present as a single cell moves through a focused light source Bone Marrow Collection § Forward and side scatter are used to determine an automated differential count • Bone marrow samples may be needed for evaluation of • Hgb measurement the patient’s hematologic condition. Bone marrow ○ Determined using a cyanmethemoglobin reagent samples, including a core biopsy and aspirate, are col- to quantitate the amount of Hgb spectrophoto- lected by a physician, and the samples obtained are metrically within the instrument processed in the laboratory • Calculations • Samples are collected and analyzed for various reasons, ○ Automated calculations performed from mea- including diagnosis and staging hematopoietic malig- sured parameters provide values for Hct, mean nancies, diagnosis and evaluation of unexplained corpuscular volume (MCV), mean corpuscular cytopeniasorsystemicdisorders,follow-upafterpatients hemoglobin (MCH), mean corpuscular hemo- undergo various treatments (chemotherapy, radiation, globin concentration (MCHC), and red blood transplants), determining sources of otherwise undiag- cell distribution width (RDW) nosed infections, and other miscellaneous purposes • Corrected WBC count • Bone marrow samples are collected from sites ○ In the case of elevated numbers of nucleated RBCs, where active red marrow is more prevalent, usually the nucleated red blood cells (NRBCs) should the posterior superior iliac crest; however, the ster- be counted independently of the 100 cell count num or anterior iliac crest may be used in adult WBC differential. Some analyzers automatically patients. Sometimes tibia samples are obtained in correct for the presence of NRBCs; however, young children sometimes the WBC count must be corrected • Aspirate samples are used to assess morphology and manually perform a differential count and M:E ratio ○ Calculated WBC Correction for NRBCs ○ The M:E ratio looks at the number of myeloid § Corrected WBC count¼Analyzer WBC cells compared to the numbers of nucleated ery- count100/100+number of NRBCs per throid precursors 100 WBCs counted § Lymphocytes are not included in this ratio CHAPTER 3 Hematology 103

§ A normal M:E ratio is usually in the range of Lineage-Associated Markets 2:1 to 4:1 Commonly Analyzed in Routine Flow • Core biopsy samples are used to determine bone mar- TABLE 3-9 Cytometry row cellularity • Helps assess if the sample is normocellular, hypocel- Lineage Markers lular, or hypercellular Immature CD34 • Cytochemical stains, flow cytometry, and other spe- CD117 cialized testing may be performed on bone marrow Terminal deoxynucleotidyl transferase samples Granulocytic/monocytic CD33 CD13 CD15 Flow Cytometry CD14 Erythroid CD71 • Flow cytometry is an automated method of sorting Glycophorin A cells. It uses a cell suspension injected into a stream Megakaryocytic CD41 sheath fluid to determine specific characteristics of CD42 the cell, including size, complexity, immunopheno- CD61 type, and cytochemistry B lymphocytes CD19 • Hydrodynamic focusing: Cells in suspension CD20 CD22 move through sheath fluid in a single-file line, allow- k Light chain ing each to individually pass through a laser light λ Light chain ○ Allows specific characterization of each cell T lymphocytes CD2 § Forward scatter for size determination CD3 § Side scatter for complexity of the cell CD4 • Gating: Electronic boundaries created to separate a CD5 specific population of interest CD7 ○ Performed during or after initial analysis CD8 • Fluorescently tagged antibodies can be used to determine cell lineage and aid in characterizing patient immunophenotype (Table 3-9) ○ Glossitis ○ Splenomegaly ○ Neurologic symptoms Special Stains and Cytochemistry • Common tests for initial anemia evaluation • • Special staining techniques may be performed to Complete blood count (CBC) with peripheral smear help differentiate cell types, particularly in cases of leu- review ○ kemia (Table 3-10) Determines RBC, Hgb, Hct, and RBC indices • Flow cytometry has decreased the use of cytochem- Normal Range Resulting Morphology istry in differentiating leukemias; however, ele- (If Abnormal) ments are incorporated into flow cytometry or MCV 80-100 fL <80 fL: Microcytic still performed as stand-alone testing to assist in a >100 fL: Macrocytic definitive diagnosis MCH 28-34 pg MCHC 32-36 g/dL <32 g/dL: Hypochromic 36-40 g/dL: Normochromic ANEMIA AND RED BLOOD CELL >40 g/dL: Results may be invalid DISORDERS ○ Smear examination will reveal the appearance • Usually defined as decreased ability of blood to carry of RBCs (anisocytosis and poikilocytosis and O or as a decrease in RBCs/Hgb from an established 2 inclusions) reference range • Reticulocyte count • Common physical symptoms ○ Shows the bone marrow response to decreases • Fatigue in RBCs • Shortness of breath • • Pallor Anemias may be classified by combinations of differ- • Cardiac issues ent criteria • Other symptoms may be more related to specific • Morphology causes of anemia ○ RBC indices are used to gauge size and ○ Jaundice hemoglobinization ○ Pica § Normocytic/normochromic 104 CHAPTER 3 Hematology

TABLE 3-10 Staining Techniques

Negative Cytochemical Stain Element Stained Positive Reaction Reaction Diagnostic Utility Myeloperoxidase (MPO) Enzyme found in the Myeloblasts and promyelocytes, Lymphoblasts Differentiating AML versus ALL primary granules although some weak activity of granulocytic may be seen in monocytes cells Sudan black B (SBB) Lipids found in Myeloblasts and promyelocytes, Lymphoblasts Differentiating AML versus ALL primary and although some weak activity secondary may be seen in monocytes granules and in lysosomal granules of monocytes Specific esterase (naphthol Esterase enzymes Myeloblasts Lymphoid cells Identifying cells of myeloid AS-D chloroacetate found in origin esterase) neutrophils Nonspecific esterase Esterase enzymes Monoblasts and monocytes Granulocytes AML of myeloid versus (a-naphthyl acetate or found in and lymphoid monocytic origin butyrate esterase) monocytes cells Tartrate-resistant acid Isoenzymes of acid Hairy cell lymphocytes (in hairy All cells except Positive diagnosis of hairy cell phosphatase (TRAP) phosphatase cells, isoenzyme 5 of acid hairy cell leukemia phosphatase is not inhibited lymphocytes with the addition of tartrate, leaving a positive reaction) Periodic acid–Schiff (PAS) Glycogen, Multiple cell types Normal AML of erythroid or mucoproteins erythroblasts megakaryoblastic origin and and some cases of ALL high-molecular- based on staining pattern weight (leukemic lymphoblasts carbohydrates may have a coarse blocklike pattern, whereas erythroid precursors have a coarse and granular staining pattern Leukocyte alkaline Enzyme in the Neutrophils (activity is scored in Differentiation of CML (low phosphatase (LAP) secondary mature bands and activity) and leukemoid granules of segmented neutrophils on a reaction (high activity) mature 0-4 rating scale) neutrophils Terminal deoxynucleotidyl DNA polymerase in Lymphoblasts Myeloblasts and Identification of lymphoblasts transferase (TdT) cell nuclei monoblasts in ALL

ALL, Acute lymphoblastic leukemia; AML, acute myelogenous leukemia; CML, chronic myelogenous leukemia.

§ Microcytic/hypochromic IRON AND HEME DISORDERS § Macrocytic/normochromic • Iron-deficiency anemia (IDA): Lack of iron to make • Function adequate heme ○ Defects leading to RBC decreases • Sideroblastic anemia: Adequate/excess iron that is not § Proliferation: RBCs are not produced at able to be effectively incorporated into heme normal rates • Anemia of chronic disease/inflammation: Adequate § Maturation: RBCs are produced in the mar- iron stores that have impaired release for incorpora- row but may not mature appropriately tion into heme/RBCs § Survival: RBCs are produced appropriately • Hemochromatosis: Iron disorder that is not anemia, but are lost/destroyed prematurely with excess iron absorption and stores CHAPTER 3 Hematology 105

Iron Depletion

Normal Iron Status Stage 1 Stage 2 Stage 3

Storage Iron Transport Iron Functional Iron Depletion Depletion Depletion (Iron Deficiency Anemia)

Iron Storage Compartment

Iron Transport Compartment

Functional Iron Compartment

Laboratory test values Hemoglobin N N N

Serum iron N N

TIBC N N

Ferritin N

FIGURE 3-7 Development of iron deficiency anemia. ↑, Increased; #, decreased; N, normal; TIBC, total iron-binding capacity. (Modified from Suominen P, Punnonen K, Rajama¨ki A, et al: Serum transferrin receptor and transferrin receptor–ferritin index identify healthy subjects with subclinical iron deficits, Blood 92:2934-2939, 1998; reprinted with permission.)

Iron-Deficiency Anemia ○ Parasite related • Laboratory diagnosis of IDA (Figure 3-7) • Iron intake and stores do not meet the body’s needs for • CBC RBC production ○ Varies with the severity of depletion • Inadequate intake ○ RBC, Hgb, Hct may be decreased ○ Daily intake does not meet daily loss ○ MCV and MCH often are decreased, resulting in ○ Nutritional deficiencies microcytic/hypochromic cells • Increased requirements § RBCs tend to be small with increased ○ Rapid growth periods central pallor because of the lack of avail- ○ Menstruating women able Hgb ○ Pregnancy and lactation • Iron studies • Absorption issues ○ Serum iron: Decreased (may be normal in early ○ Enterocytes are unable to absorb iron stages) § Celiac disease ○ Ferritin: Decreased § Bariatric surgeries ○ Total iron-binding capacity (TIBC): Increased § Medications ○ % Saturation: Decreased § Other diseases impairing absorption • Treatment and follow-up § Loss of gastric absorption with age • Determine and treat any underlying condition § Parasitic infections • Oral iron supplements • Chronic RBC loss • RBC transfusions only if Hgb is critically low • Chronic GI bleeding • Response to therapy regimen monitored by • Prolonged menorrhagia ○ Reticulocyte counts increase within the 2 weeks • Other chronic bleeds of supplementation ○ Aspirin related ○ CBC and Hgb increases 2 to 3 weeks after ○ Alcohol related supplementation 106 CHAPTER 3 Hematology

Sideroblastic Anemia • CBC ○ Anemia is usually mild • Anemia characterized by the presence of normal or ○ RBC, Hgb, Hct may be slightly decreased (Hgb increased iron that is not effectively incorporated into 9-11 g/dL) heme ○ MCV and MCH are usually normal • Hereditary ○ Reticulocytes are normal to decreased • X-Linked or autosomal • Iron studies • Acquired ○ Serum iron: Increased • Refractory anemia (as seen in myelodysplastic ○ Ferritin: Increased (or may be normal) syndromes) ○ TIBC: Decreased • Drugs and toxins ○ % Saturation: Decreased ○ Lead • Bone marrow will show increased iron stores in ○ Alcohol macrophages ○ Other varied drugs or toxins • Treatment and follow-up • Laboratory diagnosis of sideroblastic anemia • Underlying condition may be treated • CBC • In some cases erythropoietin or iron may be admin- ○ Varies istered for the patient ○ RBC, Hgb, or Hct may be decreased ○ MCV and MCH may be decreased (microcytic and hypochromic cells) § Cells are often normocytic/normochromic in Hemochromatosis cases of lead poisoning. § Occasionally, siderotic granules are seen • Iron problem that does not involve anemia ○ Coarse basophilic stippling is seen in lead poi- • Increased iron stores (absorption greater than loss) soning, although it can also be seen in other • Stored as ferritin and hemosiderin conditions ○ Often stored around organs (heart, liver, • Iron studies pancreas) ○ Serum iron: Increased • Acquired ○ Ferritin: Increased • Transfusion related ○ TIBC: Decreased ○ In cases of chronic transfusion, the body recycles ○ % Saturation: Decreased or normal the iron from transfused RBCs, in addition to its • Bone marrow own senescent RBCs ○ Sometimes performed to reveal presence of • Chronic liver disease increased iron/sideroblasts • Alcoholism • Treatment and follow-up • Supplemental or dietary iron overload • Hereditary forms may require medications to • Inherited stimulate heme synthesis • Relatively high frequency in people of northern • Acquired forms may require the removal of the European descent (1/200) offending toxin or problem • Several known mutations ○ Classic hereditary hemochromatosis, associated with the HFE gene Anemia of Chronic Inflammation (Disease) ○ Hepcidin mutations, associated with the • Acquired anemia characterized by abundant iron HAMP gene ○ stores, yet iron cannot be readily incorporated into Hemojuvelin mutations, associated with the serum or RBCs for use HJV gene • Occurs as a result of increases in various acute phase reactants present with inflammation which slows iron release that is needed by developing cells MACROCYTIC ANEMIAS • Hepcidin decreases iron release from macrophages and hepatocytes • Megaloblastic anemia results from defective DNA • Lactoferrin competes with transferrin for plasma synthesis iron, but RBCs cannot incorporate this because of • Vitamin B12 and folic acid deficiencies lack of lactoferrin receptors • Nonmegaloblastic macrocytic anemia results from • Ferritin binds iron, but developing RBCs lack other causes ferritin receptors and cannot incorporate into the • Liver disease erythroid precursors • Alcoholism • Laboratory diagnosis of anemia of chronic • Hypothyroidism inflammation • Reticulocytosis CHAPTER 3 Hematology 107

Megaloblastic Anemia • Increases in need ○ Pregnancy • Impairment of DNA synthesis leads to large, abnormal ○ Lactation cells. ○ Growing children • Most commonly caused by lack of vitamin B12 • Impaired absorption and use and/or folic acid, although some other conditions ○ Inability to obtain vitamin B12 from food in the may show megaloblastic changes stomach ○ All rapidly dividing, nucleated cells, including § Gastric issues RBCs, are affected § Inability to produce hydrochloric acid, gas- ○ Ineffective erythropoiesis, with cells showing tric bypass, drugs for lowering gastric nuclear-cytoplasmic asynchrony as they mature acidity ○ Vitamin B12 and folic acid are needed for DNA § Lack of intrinsic factor synthesis (Figure 3-8) ○ Autoimmune disease, Helicobacter • Causes for folate deficiency pylori infection, gastrectomy • Poor dietary intake § Competition for vitamin B • 12 Increases in need ○ Diphyllobothrium latum ○ Pregnancy ○ Intestinal bacteria in blind loop ○ Lactation syndrome ○ Growing children • Excessive loss • Impaired absorption and use ○ May occur in renal dialysis patients, so ○ Intestinal diseases, including celiac disease and patients are supplemented with folic acid (see sprue Figure 3-8) ○ Intestinal surgery • Physical examination ○ Medications • General anemia symptoms • Excessive loss • Glossitis ○ May occur in renal dialysis patients, so patients • Gastrointestinal (GI) symptoms are often supplemented with folic acid • Vitamin B deficiency also may result in neurologic • 12 Causes for vitamin B12 deficiency symptoms • Poor diet ○ Memory loss, balance, and gait abnormalities, ○ Lack of dietary vitamin B12 or folic acid personality changes

Uridine Thymidine dATP dCTP dGTP

3 dUMP dTMP dTTP DNA

5,10-methylene THF DHF Glycine 2 4

Serine THF* *Polyglutamation of THF (addition of 1-6 more glutamic acid residues) is essential for its retention in cell Methionine

Vitamin B 1 Site of 12 folate trap Homocysteine 5-methyl THF 1 Methionine synthase and vitamin B (in methylcobalamin form) Cytoplasm 12 2 Serine hydroxymethyl transferase Plasma and vitamin B6 5-methyl THF 3 Thymidylate synthetase 4 Dihydrofolate reductase

FIGURE 3-8 Role of folate and vitamin B12 in DNA synthesis. Folate enters the cell as 5-methyltetrahydrofolate (5-methyl THF). In the cell, a methyl group is transferred from 5-methyl THF to homocysteine, converting it to methionine and generating tetrahydrofolate (THF). This reaction is catalyzed by methionine synthase and requires vitamin B12 as a cofactor. THF is then converted to 5,10-methylene THF by the donation of a methyl group from serine. The methyl group of 5,10-methylene THF is then transferred to deoxyuridine monophosphate (dUMP), which converts it to deoxythymidine monophosphate (dTMP) and converts 5,10-methylene THF to dihydrofolate (DHF). This reaction is catalyzed by thymidylate synthetase. dTMP is a precursor of deoxythymidine triphosphate (dTTP), which is used to synthesize DNA. THF is regenerated by the conversion of DHF to THF by the enzyme DHF reductase. A deficiency of vitamin B12 prevents the production of THF from 5-methyl THF; as a result, folate becomes metabolically trapped as 5-methyl THF. This constitutes the “folate trap.” (From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.) 108 CHAPTER 3 Hematology

§ In vitamin B12 deficiencies, prolonged/severe § Although a classic means of determining the cases may have demyelination of the neurons; cause of vitamin B12 deficiency, the Schilling folic acid deficiency does not have neurologic test is no longer performed regularly in the involvement United States. Homocysteine and MMA are • Laboratory diagnosis of megaloblastic anemias replacing this test, because they are better • CBC indicators of the deficiency ○ Decreases in RBC count, WBC, platelet, Hgb, • Treatment of megaloblastic anemia and Hct; elevated MCV • Once the underlying cause is established, specific § RBCs include oval macrocytes, and inclusions treatment can be determined may be present (Howell-Jolly bodies, NRBCs, ○ Remove/repair underlying problem Cabot rings) ○ Supplement with appropriate deficient vitamin, § Neutrophils may appear hypersegmented either vitamin B12 or folic acid • Bone marrow § In cases of intrinsic factor problems, ○ May be used to confirm presence of megaloblas- intramuscular injection of vitamin B12 can tic anemia be used § Megaloblastic changes are apparent (nucleus- § If neurologic issues occur with vitamin B12 to-cytoplasm [N:C] asynchrony) deficiency, they may be irreversible based on § Hypercellularity with increased, abnormal the extent of the damage RBC precursors • Follow-up testing § Giant bands and metamyelocytes • CBC and reticulocyte count • Other laboratory testing ○ Reticulocyte response may be seen within a week ○ Serum vitamin B12 and folic acid assays of treatment § Decreases appear with deficiencies ○ Hypersegmented neutrophils are replaced by ○ Methylmalonic acid (MMA) normal neutrophils in about 2 weeks § Increased in vitamin B12 deficiency, because it ○ CBC may return to overall normal state in 3 to is needed for conversion of methylmalonyl 6 weeks coenzyme A (CoA) in the pathway ○ Homocysteine Nonmalignant Macrocytic Anemias § Increased in vitamin B12 and folic acid deficiency, because vitamin B12 is needed for con- • Macrocytosis without megaloblastic changes can verting homocysteine to methionine in the occur pathway • Normal babies ○ Intrinsic factor antibodies ○ After delivery, macrocytosis and reticulocytosis ○ Parietal cell antibodies are normal in newborns ○ Ova and parasite examination in cases of sus- § MCV may increase up to 123 fL pected vitamin B12 deficiency resulting from D. • Liver disease latum infection • Alcoholism ○ Schilling test • Hypothyroidism § Classic two-part test used to determine if the cause of vitamin B12 deficiency is malabsorption, HYPOPROLIFERATIVE DISORDERS dietary deficiency, or a lack of intrinsic factor § Part I: Patients are dosed orally with radi- • Disorders occurring as a result of decreased or olabeled vitamin B12, followed by a flush- absent production of hematopoietic cells in the bone ing dose of unlabeled vitamin B12. Excess marrow vitamin B12 is filtered by the kidney, and • Includes aplastic anemia, both inherited and urine is measured for radioactivity. If radi- acquired, in addition to other disorders resulting olabeled vitamin B12 levels are elevated, the from decreases in bone marrow production patient is likely deficient in vitamin B12 and • Other disorders resulting from decreases in bone unable to absorb the vitamin marrow production include § Part II: If the vitamin B12 excretion in part I ○ Pure red cell aplasia is decreased, the patient receives an oral ○ Congenital dyserythropoietic anemia dose of radiolabeled vitamin B12 and a dose ○ Myelophthisic anemia of intrinsic factor. If the radiolabeled vita- ○ Anemia resulting from chronic kidney disease min B12 levels are increased from those in part I, the patient is lacking intrinsic factor Aplastic Anemia and has pernicious anemia. If the levels are abnormal, the patient may have another • Rare disorders characterized by pancytopenia in the defect leading to malabsorption peripheral circulation. Result from decreased bone CHAPTER 3 Hematology 109

marrow production of RBC, WBC, and platelets ○ Hypocellular with increased fat cells because of deficiency or damage of hematopoietic § Biopsy is needed for accurate diagnosis stem cells ○ Decreased granulocytes, RBCs, and platelets • Acquired aplastic anemia ○ Treatment • Most cases identified are acquired, the majority of § Eliminate the problem causing bone marrow which are idiopathic failure, if it can be identified ○ Cause of the marrow failure in idiopathic cases is § Transfusions (RBCs and platelets) can be currently unknown given, as needed ○ Other acquired cases have been linked to various § Immunotherapy may be used drugs/chemicals, radiation, viral infections, and § Bone marrow/stem cell transplant can be per- other miscellaneous causes formed for severe cases, if a suitable donor is • Inherited aplastic anemia available and the recipient meets certain • A smaller number of cases are inherited, including criteria Fanconi’s anemia, dyskeratosis congenita, and § Treatments vary based on the specific case, Shwachman-Diamond syndrome disorder, and suspected cause • Fanconi’s anemia ○ Chromosomes are susceptible to breakage and Pure Red Cell Aplasia the cell may not be able to repair DNA damage. Cells also show accelerated telomere shortening • Bone marrow exhibits decreased production of RBCs and apoptosis. and RBC precursors, whereas other cell lines are pre- § This property is used to help diagnose sent and produced normally Fanconi’s anemia • Acquired ○ Genetic mutations in one of 13 genes • Primary is idiopathic or autoimmune § FANCA mutations occur most frequently. • Secondary is usually associated with tumors, infec- Inheritance is autosomal recessive in all of tions, drugs or chemicals, other disorders the associated genes except FANCB, which ○ Transient erythroblastopenia of childhood (TEC) is an X-linked gene. ○ Pure red cell aplasia (PRCA) seen in children, ○ Appears at birth or in early childhood often related to viral infection § Symptoms resulting from pancytopenia may § Treated by transfusion, as needed, although be apparent early on or manifest later in life most patients eventually restore their ability ○ Skeletal abnormalities may be seen in many, in to produce RBCs addition to abnormalities in skin pigmentation, • Congenital and organ problems • Diamond-Blackfan anemia ○ Patients may have a higher risk for malignancies, ○ Mutations are usually autosomal dominant; in addition to the bone marrow failure however, they also may occur sporadically • Dyskeratosis congenita ○ Many patients show symptoms before 1 year of ○ Very rare disorder in which chromosomes have age, although some are asymptomatic short telomeres ○ Many patients have physical issues, including ○ Inheritance is autosomal dominant, X-linked bone malformations recessive, and autosomal recessive, with various • Congenital dyserythropoietic anemia (CDA) mutations present ○ Group of inherited rare disorders leading to inef- ○ Patients may show abnormalities in skin pigmen- fective erythropoiesis tation and nails, in addition to a variety of § Patients have varying degrees of refractory abnormalities anemia and symptoms resulting from the inef- • Shwachman-Diamond syndrome fective erythropoiesis, in addition to variable ○ Autosomal recessive disorder leading to neutro- physical effects penia and/or anemia and thrombocytopenia ○ Mutations are usually autosomal recessive; how- ○ Patients have decreased pancreatic enzymes, ever, rare cases of an autosomal dominant inher- skeletal anomalies, and increased risk of itance have been reported infection ○ Symptoms usually appear in childhood or • General CBC findings in aplastic anemia adolescence • Pancytopenia, with decreases in one or more cell lines • Anemia of chronic kidney disease ○ Anemia with normocytic, normochromic RBCs ○ Individuals are unable to produce erythropoie- ○ Decreased granulocytes with normal to slightly tin, leading to decreased production of RBCs decreased lymphocytes • Therapy for PRCA ○ Decreased platelets • Therapy is variable based on each specific anemia, ○ Decreased reticulocyte production including supportive therapy, transfusion therapy, • Bone marrow corticosteroids, treating the underlying problem 110 CHAPTER 3 Hematology

HEMOGLOBINOPATHIES ○ Patients are usually normal and asymptomatic unless in extreme conditions of hypoxia • Disorders resulting from genetic mutations that lead to • Laboratory diagnosis structural changes in the Hgb molecule • CBC • Most occur because of amino acid substitutions in ○ Generally normal, although some target cells the b-globin chains may be present • Numerous Hgb variants exist; however, several are • Sickle screen more common than others ○ Positive • Common abnormal Hgbs ○ Hgb Electrophoresis and HPLC • Hgb S • Hgb A and Hgb S are present ○ Usually affects African patients or patients of African descent Hemoglobin C ! ○ Mutation: b6(Glu Val) • Usually affects patients of West Africa or West African § Hgb S polymerizes into long, thin polymers descent to form sickles when O saturation decreases ! 2 • Mutation: b6(Glu Lys) • Hgb C polymerizes into thick crystals when O2 sat- Sickle Cell Disease uration decreases • RBC shape alteration is less extreme than in Hgb S • Homozygous for Hgb S ○ Crystals look like bars of gold or the Washington • Majority of Hgb present is Hgb S Monument • Clinical symptoms • Diagnosis • Chronic hemolytic anemia, autosplenectomy, • CBC vasoocclusion, vasoocclusive crises, susceptibility ○ Mild-to-moderate anemia to bacterial infections, acute chest syndrome, pul- ○ Increased target cells and Hgb C crystals monary hypertension, myocardial infarction, and may appear numerous other complications ○ Some polychromasia and NRBCs may be present • Laboratory diagnosis of sickle cell disease • Solubility testing is negative • CBC • Hgb electrophoresis and HPLC ○ Normocytic, normochromic anemia ○ Positive for Hgb C if homozygous and positive ○ Various poikilocytes present for Hgb A and C if heterozygous for the mutation § Drepanocytes (sickle cells) • Treatment is usually not needed and prognosis § Target cells, NRBC, Howell-Jolly bodies, is good polychromasia are commonly seen • NOTE: Hgb C-Harlem is characterized by a double ○ WBC count usually elevated, with neutrophils ! ! mutation(b6(Glu Val) andb73(Asn Asp) andisclinically predominating significant if inherited in combination with Hgb S • Reticulocyte count is elevated • Bone marrow is not indicated Hemoglobin E • Other tests ○ Dithionite solubility (sickle screen) is positive • Usually affects patients from Southeast Asian, particu- ○ Hgb electrophoresis is positive for Hgb S and larly Thailand ! NO Hgb A is present • Mutation: b26(Glu Val) ○ HPLC or isoelectric focusing may also be used to • Diagnosis demonstrate Hgb S • CBC • Treatment and follow-up ○ Mild anemia • Supportive therapy and prognosis ○ Microcytes and target cells ○ Transfusions, prophylactic antibiotics, avoidance • Solubility testing is negative of situations leading to low O2 saturation, and • Hgb electrophoresis and HPLC dehydration ○ Positive for Hgb E ○ Hydroxyurea to help retain Hgb F production • Therapy is not usually needed and prognosis is good ○ Bone marrow transplant may be an option if a matched donor is available Hemoglobin SC ○ Appropriate therapy can lead to a life span of • Combination disorder with inheritance of mutations approximately 50 years for both Hgb S and Hgb C Sickle Cell Trait • Most common of the compound disorders • Similar clinical picture to sickle cell disease; however, it • Heterozygous for Hgb S does not usually manifest clinically until teenage years • Majority of Hgb present is Hgb A and tends to be slightly less severe than Hgb S CHAPTER 3 Hematology 111

• Laboratory diagnosis • Hgb H disease ○ CBC ○ Three gene deletion (/a) § Mild anemia § Unpaired b chains will form tetramers of § Target cells and Hgb SC crystals may be present Hgb H § Hgb SC polymerizes into crystalline struc- § Other Hgb present are Hgb A2, some tures that have features of both Hgb S and Hgb Bart Hgb C, often described to look like birds or ○ Clinical symptoms fingers § Mild-to-moderate chronic hemolytic anemia ○ Solubility testing is positive because of the pres- § Splenomegaly ence of Hgb S § Other variable findings may occur ○ Hgb electrophoresis and HPLC ○ CBC § Hgb S and Hgb C are present § Decreased RBC and Hgb ○ Therapy and prognosis is similar to sickle cell § Microcytic, hypochromic RBCs, target cells, disease, although expected life span is longer and other poikilocytes may be present § Hgb H inclusions (precipitated Hgb H) may Combination Disorders be seen when using a supravital stain, such as new methylene blue • Mutations may occur in combination because of inher- • a-Thalassemia minor itance of different genetic mutations from each parent ○ Two gene deletion (/aa)or(a/a) • Each compound disorder has variable symptoms and § Asymptomatic presentation severities § Usually no therapy is needed • Some examples include Hgb SC disease, Hgb S–b thal- ○ CBC assemia, Hgb C–Harlem § Mildly decreased Hgb and Hct, with microcytic, hypochromic cells • Silent carrier THALASSEMIAS ○ One gene deletion (a/aa) ○ No clinical symptoms, because globin chain ratio • Disorders caused by genetic mutations that lead to is almost normal quantitative changes in the amount of globin chains ○ No therapy is needed produced, resulting in an imbalance of globin chain ○ CBC synthesis § No hematologic abnormalities • Types and severity of thalassemia depends on the ○ Diagnosis globin gene mutated (a or b) and the number of § Diagnosis is by genetic analysis genes affected by the mutation • a-Globin is coded for on chromosome 16 ○ A normal genotype is designated as aa/aa b-Thalassemia • b-Globin is coded for on chromosome 11 • Thalassemias caused by mutations or deletions in the ○ A normal genotype is designated as b/b b-globin genes § b+ Designates decreased production of b • Four main groups with varying clinical chains; b0 shows the absence of b chains presentations • a-Thalassemia a-Thalassemia major ○ Occurs with genotypes (b+/b+), (b+/b0),(b0/b0) • Thalassemias caused by mutations or deletions in the ○ Also called Cooley’s anemia a-globin genes ○ Severe hemolytic anemia, usually diagnosed at • Four main groups approximately 6 months of age when Hgb F levels • Hydrops fetalis/Hgb Bart normally decrease (the patient cannot make Hgb A) ○ Four gene deletion (/)sonoa chains are ○ Clinical picture shows hepatosplenomegaly and produced distinct bone changes, if untreated, in addition § Hgbs present are Bart (g4) with small amounts to other physical issues of Hgb Portland and Hgb H (b4) § Pathologic fractures and abnormalities of the ○ Not compatible with life as the blood is unable to skull are frequently seen as a result of ery- oxygenate tissues because of high O2 affinity of throid hyperplasia in the bone marrow Hgb Bart ○ CBC § Fetus will either be delivered prematurely and § Severe anemia die shortly after birth or will be delivered still- § RBC count is slightly elevated, but pro- born in the third trimester nounced hypochromic/microcytic cells and § Clinically exhibit severe anemia, cardiac target cells are present, in addition to other defects, and hepatosplenomegaly varied morphologies and NRBCs 112 CHAPTER 3 Hematology

○ Bone marrow HEMOLYTIC ANEMIA § Usually not performed, but would show erythroid hyperplasia and ineffective erythropoiesis • Disorders of premature RBC destruction, leading to ○ Hgb electrophoresis and HPLC anemia § Increased Hgb F, slightly increased Hgb A2, • Classified in several different ways little or no Hgb A • Intrinsic versus extrinsic defects ○ Therapy and prognosis • Acquired versus hereditary defects § Supportive therapy with regular transfusions • Intravascular versus extravascular hemolysis § Iron chelation therapy to help avoid iron • General features overload • Clinical presentation § Bone marrow transplant if a good match is ○ General anemia symptoms when hemolysis leads available to anemia • b-Thalassemia intermedia ○ Jaundice may be present, variable depending ○ Occurs with genotypes (bsilent/bsilent), (db0/db0), on cause (b0/db0) ○ Splenomegaly in cases of extravascular § Clinical symptoms and CBC hemolysis § Varies because of multiple genotypic ○ Gallstones in cases of chronic hemolysis presentations • Laboratory testing § Usually not transfusion dependent • Increased bilirubin: RBCs are breaking down ○ Anemia varies between that of b-thalassemia • Decreased haptoglobin: Free Hgb from intravascu- minor and b-thalassemia major lar hemolysis may exceed haptoglobin’s binding • b-Thalassemia minor capacity ○ Occurs with genotypes (b+/b), (b0/b) • Increased reticulocyte count § Clinical symptoms and CBC • Variable anemia depending on degree and fre- § Usually mild anemia quency of hemolysis § Normal to elevated RBC with decreased ○ Anisocytosis and poikilocytosis on the CBC, usu- Hgb and Hct ally including some macrocytosis and polychro- ○ Poikilocytosis (target cells and some masia (reticulocyte production) and others) spherocytes and/or schistocytes depending on § Hgb electrophoresis and HPLC the cause of the anemia § Elevated Hgb A2 and F ○ Silent carrier silent Hemolytic Anemias Resulting from Intrinsic § Occurs with genotypes (b /b) Defects § No clinical symptoms, because globin chain ratio is almost normal Abnormalities in Red Blood Cell Membrane § No therapy is needed Hereditary Spherocytosis § CBC does not show hematologic • Incidence is 1/3000 of northern European ancestry abnormalities • Autosomal dominant inheritance in most cases § No hematologic abnormalities • Mutations affect genes coding for membrane pro- § Diagnosis is by genetic analysis teins, leading to changes in the membrane skeleton and decreased survival because of decreased Other Related Disorders deformability • Symptoms are variable, but a symptomatic clinical • Hereditary persistence of fetal hemoglobin (HPFH) picture shows anemia, jaundice, and splenomegaly • Deletion in the b-globin gene leading to increased ○ CBC production of Hgb F (g chains) § Decreased RBC, Hgb, and Hct, increased ○ Patients are usually asymptomatic; abnormali- MCHC and RDW ties will show up in Hgb electrophoresis/HPLC § Spherocytes, polychromasia where increases in Hgb F are present ○ Other testing § Hgb Lepore § Family history to look for evidence of § db fusion gene inheritance § Shows anemia similar to b-thalassemia § Direct antiglobulin test (DAT) is negative, minor which rules out immune-mediated cause § Combination disorders § Osmotic fragility is increased because of § Hgb S–thalassemia decreased membrane deformability § Hgb C–thalassemia § Autohemolysis tests and membrane protein § Hgb E–thalassemia studies may be performed CHAPTER 3 Hematology 113

○ Treatment and prognosis • Cells lack glycoslyphosphatidlyinositol-anchored pro- § Many are asymptomatic, but those with teins, including CD55 and CD59 severe hemolysis may require splenectomy • RBCs are susceptible to complement lysis, because or transfusion therapy CD55 and CD59 inhibit complement and are absent, Hereditary Elliptocytosis cells may lyse spontaneously • Mutations in genes coding for spectrin or band 4.1, • Clinical presentation leading to disruption of the cell shape in circulation • Usually manifests in young adults, but can occur at • Incidence is 1 in 2000 to 4000, although it is any age more common in Africa and the Mediterranean • Variable symptoms related to the hemolysis, throm- and those descended from the area bosis resulting from thrombophilia, and bone mar- • Several variants occur, including hereditary row failure may occur pyropoikilocytosis • Laboratory findings • Many cases are asymptomatic, and disorder is discov- • General signs of intravascular hemolysis, including ered incidentally; however, some exhibit more pro- hemoglobinuria nounced hemolysis • Reticulocytes show a slight increase • CBC • Bone marrow examination may be done to look for • Normal (in asymptomatic cases) to decreased RBC, underlying marrow failure or cytogenetic Hgb, and Hct (in hemolytic cases) abnormalities • Increased elliptocytes, although morphology is • Flow cytometry shows deficiencies of CD55 and more extreme in hereditary pyropoikilocytosis, CD59 which also may show schistocytes and micro- ○ CD24 and CD15 also may be deficient spherocytes • Therapy and prognosis • Other testing • Eculizumab can be used for hemolysis to decrease • Thermal sensitivity may be increased in hereditary complement activity elliptocytosis with spectrin mutations • Supportive transfusion, prophylactic antibiotics, • Molecular testing may be done to look for vitamin supplementation to counter loss through mutations kidneys, anticoagulants are used if there are clotting • Treatment and prognosis complications, and stem cell transplant may be an • Symptomatic cases with anemia may require trans- option if a suitable donor is available fusion therapy and sometimes splenectomy Abnormalities in Enzymes • Usually asymptomatic cases require no treatment Glucose-6-Phosphate Dehydrogenase Deficiency and have a good prognosis • RBCs are unable to reduce glutathione, which is Acanthocytosis needed to battle oxidant damage in the cell, leading • Spur cell anemia to oxidation of Hgb into Heinz bodies, which are then • Defects in RBC membrane lipid balance, often removed from circulation resulting from liver issues • X-linked disorder • Seen in severe liver disease as a result of excess • Multiple mutations and enzyme a presentation of free plasma cholesterol that accumulates on chronic hemolytic anemia to patients with few or the RBC membrane, leading to deformation of no abnormalities. the cell within the spleen, if cells are not • Tends to present in patients in Africa and the Mid- hemolyzed East, and their descendants • CBC • Most common RBC enzyme deficiency, with a prev- ○ Moderate anemia with acanthocytes alence of up to 5% worldwide • Clinical presentation • Clinical presentation ○ Splenomegaly, jaundice • Most patients are asymptomatic unless exposed • Therapy and prognosis to something that will trigger hemolytic episodes ○ Prognosis is poor unless a patient can success- • Oxidative drugs, including antimalarial medica- fully undergo a liver transplant tions, infections, and fava beans are main trig- Neuroacanthocytosis gers of hemolysis • Rare inherited disorders with neurologic symptoms ○ Leads to transient hemolytic episodes within sev- and acanthocytosis eral hours of exposure and may begin the return • Abetalipoproteinemia, McLeod’s syndrome to normal once the offending trigger is removed Paroxysmal Nocturnal Hemoglobinuria or resolved • Rare acquired disorder resulting from stem cell muta- ○ Hemoglobinuria may be one of the first clinical tion in the PIGA gene clues • Defect in platelets and WBCs, as well • Laboratory testing • Severity is variable depending on the phenotype • CBC is normal unless patient has an episode 114 CHAPTER 3 Hematology

○ With hemolytic episodes, patients exhibit a nor- Disseminated Intravascular Coagulation (DIC) mocytic or normochromic anemia and Hgb may • Activation of all parts of the hemostatic systems lead- drop quickly, but response can be variable ing to the production of fibrin clots, the consumption ○ Bite and blister cells may occur on Wright’s stain, of platelets and coagulation proteins, and degradation and Heinz bodies are often visualized when using of fibrin. Clotting and bleeding both occur a Heinz body stain or other supravital stain. • Acute or chronic, both secondary to other underlying Other morphologic findings may be present conditions • Glucose-6-phosphate dehydrogenase (G6PD) enzyme • Clinical presentation activity screens and quantitative assays • Variable presentation, because patients show • May be used to screen or to assess the degree of symptoms consistent with the underlying disorder severity that has prompted disseminated intravascular • Therapy and prognosis coagulation • Usually involves avoiding or removing the trigger • Underlying causes may include infections, malig- for hemolysis nancies, obstetric complications, venom exposure, • Most cases require no treatment and resolve on and chronic inflammation, among others their own, but some severe cases may require trans- • CBC fusion therapy • Anemia and decreased platelet count, WBC may be Pyruvate Kinase Deficiency elevated • • Autosomal recessive disorder, relatively rare Schistocytes and apparent thrombocytopenia • • Leads to adenosine triphosphate (ATP) depletion and Laboratory findings • increase in 2,3 BPG Coagulation tests are abnormal ○ Elevated prothrombin time (PT), activated par- • Clinical findings tial thromboplastin time (aPTT) • Variable from asymptomatic to chronic hemolytic ○ D-dimer/fibrin degradation products (FDPs), and episodes • Laboratory findings decreased fibrinogen • • CBC Therapy and prognosis • Underlying disorder should be treated, in addition ○ Variable RBC and Hgb to supportive therapies. In cases of acute DIC, ther- ○ Increased echinocytes with other variable morphologic findings apies may be more aggressive to try to stem organ failure. Heparin can be used carefully to try to stop • Pyruvate kinase enzyme activity can be measured activation of the coagulation cascade. Blood prod- spectrophotometrically. Activity is decreased. • Treatment and prognosis ucts may also be used, including frozen plasma to • Supportive treatment with transfusions, as replace consumed coagulation proteins and replace indicated blood volume, platelet transfusions may be admin- • istered if thrombocytopenia is severe Some severe cases may require splenectomy Thrombotic Thrombocytopenic Purpura (TTP) • Patients have long von Willebrand factor (vWF) multimers that bind vascular endothelium and platelets, trig- Hemolytic Anemias Resulting from gering platelet aggregation. Platelets are used up in this Extrinsic Defects process and microclots block small blood vessels, which leads to shearing of the RBCs in circulation • A variety of different conditions can cause mechanical • Disorder is acquired or inherited destruction of the circulating RBCs; these are not • Several subtypes are present immune mediated hemolytic anemias • Most patients have a decrease or mutation in the Microangiopathic Hemolytic Anemia ADAMTS 13 gene, which is normally used to cleave • Group of disorders characterized by intravascular long vWF multimers into smaller fractions, helping fragmentation of RBCs as they move through blood to avoid excess platelet adhesion to the endothelium vessels obstructed by microclots or endothelial • Clinical findings damage • Usually characterized by a combination of symp- • CBC shows decreases in Hgb and Hct with schisto- toms, including microangiopathic hemolytic ane- cytes usually appearing on the peripheral smear, in mia, thrombocytopenia, neurologic symptoms, addition to possible other morphologies renal dysfunction, and fever • Other laboratory testing • CBC • Bilirubin (unconjugated) is increased • Characterized by decreased Hgb (usually <10 g/dL) • Haptoglobin is decreased and platelet count (<20109/L), with schistocytes • Hemostasis testing varies based on the specific on the peripheral smear disorder • Laboratory testing CHAPTER 3 Hematology 115

• Coagulation tests (PT, APTT) are usually normal • Mild hemolysis resulting from RBCs flowing • vWF multimer analysis is abnormal around the implanted valves • Therapy and prognosis • Patients are often asymptomatic, but severe cases • Plasma exchange therapy to remove large vWF mul- may present with noticeable anemia timers and providing the missing ADAMTS 13 pro- • CBC may show the presence of schistocytes tease can lead to favorable prognosis • If anemia is severe, patients may require transfusion • Immunosuppressive therapy may also be used therapy and surgical repair of the prosthetic valve Hemolytic Uremic Syndrome (HUS) • March hemoglobinuria (exercise induced) • Microangiopathic hemolytic anemia with thrombocy- • Condition occasionally seen in long-distance run- topenia and renal involvement as a result of clots form- ners or others who engage in intense exercise ing in the microvasculature of the kidney • Although hemolysis may occur, patients usually do • Acquired disorder, usually found in young children not have anemia unless hemolysis is recurrent with a history of hemorrhagic Escherichia coli or Shi- • Hemoglobinuria may be present, in addition to gella dysenteriae infections, although it may be found decreased haptoglobin levels in adults after exposure to immunosuppressive agents • Therapy includes minimizing physical trauma or or chemotherapy discontinuing the activity that leads to hemolysis • Clinical presentation Infectious Agents • Children often present with a bloody diarrhea, CBC • Malaria (Plasmodium spp.) abnormalities, and renal issues, whereas adults tend • Caused by infection with one of the major species of to present with renal issues and CBC abnormalities Plasmodium without bloody diarrhea • Malarial parasites may lyse RBCs as they use Hgb, • CBC in addition to the destruction of infected cells by • Decreased RBC, Hgb (<10 g/dL), Hct, and platelets extravascular hemolysis; additionally, inflamma- • Smear shows schistocytes and decreased platelets tory response can lead to inhibited and ineffective • Laboratory testing erythropoiesis • Blood urea nitrogen and creatinine are elevated • Clinical symptoms vary but often include fever, • Culture results may be positive for E. coli or S. chills, headache, and other physical manifestations dysenteriae • Organism presence can be confirmed by visualiza- • Urinalysis shows elevated protein, blood, and casts tion of organism, intracellular or extracellular, on • Therapy and prognosis a peripheral smear • Supportive therapy, as needed; prognosis is usually • Treatment using chloroquine drugs is administered favorable unless patient harbors organism from areas known Other Causes for chloroquine resistance, where other drugs need HELLP Syndrome to be used. Transfusion therapy may be used, too, if • Hemolysis, elevated liver enzymes, and low platelet anemia is severe count • Babesia • Relatively uncommon complication of pregnancy, • Babesia microti is the most common cause of infection although it is more likely to affect patients with pre- • Some patients are asymptomatic, whereas others eclampsia toward the end of their pregnancy show mild-to-severe anemia and generalized flulike • Vascular insufficiency in the placenta can lead to dys- symptoms function in the maternal endothelium, causing platelet • Diagnosis is confirmed by the visualization of activation and fibrin deposition in the small vessels organism (ring forms or tetrads) on peripheral • Laboratory testing smear, in addition to antibody testing for organism • CBC shows decreased platelet count and lactate • Bacteria dehydrogenase is elevated • Toxin-producing microorganisms can occasionally • Therapy and prognosis lead to hemolysis • Relatively good with supportive therapy and deliv- • Clostridium perfringens ery of the fetus and placenta ○ a-Toxin is produced and can hydrolyze mem- Hypertensive Crisis and Malignant Hypertension brane phospholipids, rendering changes in RBC • Severe increase in blood pressure, leading to acute shape and deformability organ damage ○ Hemolysis is severe and can lead to DIC and • Endothelial cells are damaged, leading to activation of renal failure; prognosis is poor the hemostatic system Additional Causes of Mechanical Hemolysis • Platelets return to normal once the blood pressure is • Drugs controlled • Chemicals Mechanical Damage • Venoms • Prosthetic heart valves • Thermal injury 116 CHAPTER 3 Hematology

Immune-Mediated Hemolytic Anemia • CBC • RBC life span is shortened because of presence of anti- • Mild-to-severe anemia with polychromasia and bodies, usually IgG or IgM, on RBC surfaces spherocytes • Autoimmune or alloimmune causes • Laboratory testing • IgM antibodies usually activate complement, leading • DAT is positive in majority of cases to intravascular and extravascular hemolysis • Treatment and prognosis • IgG antibodies can occur with or without the pres- • In symptomatic cases, prednisone therapy may be ence of complement, and most removal attempts used are extravascular, leading to hemolysis or the • Immunosuppressive therapy may be used, but increased presence of spherocytes leaves the risk for side effects • General laboratory findings in immune-mediated • Transfusion therapy can be used in cases with severe hemolytic anemia anemia • CBC Cold Agglutinin Disease ○ Decreased RBC, Hgb, and Hct with macrocytes, • Usually caused by IgM antibodies that react best at 4 C spherocytes, and polychromasia; increased • Usually do not react at temperatures above 30 C reticulocyte count • IgM antibodies bind to the RBC after exposure to • Laboratory testing colder temperatures, and they can activate the comple- ○ Increased bilirubin and lactate dehydrogenase, ment cascade as they move through the cooler extrem- decreased haptoglobin ities. When cells return to warmer areas, the IgM is no ○ DAT is positive and can further be tested for IgG longer a factor; however, complement remains and is and C3d removed via extravascular hemolysis or in some cases, intravascularly Autoimmune Hemolytic Anemia • Can occur in acute or chronic state • Caused by autoantibodies that attach to the RBC sur- • Chronic cold agglutinin disease (CAD) is rare and face (Table 3-11) may be idiopathic or secondary to lymphoid Warm Autoimmune Hemolytic Anemia malignancy • Warm autoimmune hemolytic anemia is the most • Clinical presentation common autoimmune anemia (AIHA), occurring as • Symptoms are variable, because patients have vari- idiopathic or secondary disease able anemia (mild to severe). Depending on the • Secondary particularly in B-cell lymphoid malig- severity, patients may show general anemia symp- nancies, such as chronic lymphocytic leukemia toms and acrocyanosis (CLL), solid tumors, autoimmune disorders, and • Acute CAD can occur secondary to Mycoplasma viral infections pneumoniae and viral infections • Usually caused by IgG autoantibodies that react the • CBC best at 37 C • If blood has cooled before analysis, values will not • Usually extravascular hemolysis occur in their normal proportions, leading to

TABLE 3-11 Characteristics of Autoimmune Hemolytic Anemias

Mixed-Type Warm Autoimmune Cold Aggultinin Paroxysmal Cold Autoimmune Hemolytic Hemolytic Anemia Disease Hemoglobinuria Anemia Immunoglobulin class IgG (rarely IgM, IgA) IgM IgG IgG, IgM Optimum reactivity 37 C4 C; reactivity extends 4 C4-37 C temperature of to >30 C autoantibody Sensitization detected by IgG or IgG+C3d; only C3d C3d C3d IgG and C3d direct antiglobulin test uncommon Complement activation Variable Yes Yes Yes Hemolysis Extravascular primarily Extravascular; rarely Intravascular Extravascular and intravascular intravascular Autoantibody specificity Panreactive or Rh complex; I (most), i (some), Pr P Panreactive; unclear rarely specific Rh or other (rare) specificity antigen

From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders. Ig, Immunoglobulin. CHAPTER 3 Hematology 117

decreases in the RBC and Hct, with a dispropor- • Drug is discontinued and should be avoided in the tional (normal) Hgb and grossly abnormal future increases in the MCV, MCH, and MCHC. The • If anemia is severe, the patient may require transfu- Hgb value is normal because the method of mea- sion therapy surement requires lysis of cells before analyzing Alloimmune Hemolytic Anemias Hgb. Agglutinates may be seen on peripheral • Hemolytic anemia resulting from immune incompati- smear in high-titer cold agglutinins. Warming sam- bility of donor and recipient (or immune incompatibil- ple before reanalyzing may resolve the agglutina- ity of mom and baby) tion or keeping sample warm until the time of • Antibodies can be IgM or IgG with intravascular and/ analysis may also help or extravascular hemolysis • Laboratory testing • Onset can be immediate or delayed • Cold agglutinin titer is increased • Therapy and prognosis Transfusion Reactions • Patients do not often require transfusions unless Acute Hemolytic Reaction hemolysis leads to a severe anemia. Patients are usu- • Occurs within hours of transfusion of incompatible ally instructed to avoid cold temperatures blood products Paroxysmal Cold Hemoglobinuria • Most commonly caused by ABO incompatibility, • Acute cold AIHA associated with the Donath- because recipients produce natural IgM antibodies to Landsteiner antibody (anti-P autoantibody) the incompatible antigen, leading to complement- • Donath-Landsteiner antibody is a biphasic anti- mediated intravascular hemolysis. • body that can bind to RBCs and partially activate Clinical presentation • complement at low temperatures (optimal binding Various symptoms occur, including fever, at 4 C) but full-blown complement activation chills, urticaria, chest pain, back pain, shock, and hemolysis occur at 37 C cardiac symptoms, and bleeding (if DIC is • Clinical presentation present) • • Mainly affects children, although it can occur in CBC • adults Hgb is decreased • • Symptoms include fever, malaise, and extremity and Laboratory tests • back pain that usually manifests 1 to 2 weeks after a Hemoglobinuria, hemoglobinemia • respiratory infection. Patients often have a rapid DAT is usually positive on posttransfusion specimen • onset of hemolysis and hemoglobinuria, leading Haptoglobin is decreased • to a severe anemia Coagulation tests may be abnormal if DIC occurs • CBC • Therapy and prognosis • • Severe anemia after hemolytic episodes (Hgb often Transfused unit must be stopped as soon as <5 g/dL) with morphology showing polychromasia possible, and treatment to minimize or correct the and spherocytes, in addition to various other clinical symptoms is undertaken quickly morphologies Delayed Hemolytic Transfusion Reaction • Laboratory tests • Reaction may occur days to weeks after initial transfu- • DAT is positive for C3d (auto–anti-P usually disso- sion, because the recipient’s antibody titer may take ciates from RBCs at 37 C) time to increase • Donath-Landsteiner test is positive • Antibodies implicated are usually IgG and can bind to • Therapy and prognosis transfused RBCs, which are then removed by extravas- • Transfusion therapy is needed with severe hemo- cular hemolysis lytic episodes; however, the disorder is usually • CBC self-limiting and has a favorable prognosis • May not show adequate posttransfusion increase in Hgb Drug-Induced Hemolytic Anemia • Laboratory tests • Characterized by a sudden onset of anemia with hall- • DAT is positive marks of hemolytic anemia after a patient is exposed to • Bilirubin is usually indirect fraction may be a medication increased • Patients produce antibodies to the medication, which are either drug dependent or drug independent Hemolytic Disease of the Fetus and Newborn • Laboratory testing • Rhesus (Rh) hemolytic disease of the fetus and new- • DAT is positive born (HDFN) occurs when maternal IgG antibodies • CBC cross the placenta and enter fetal circulation, binding • Anemia of varying severity to fetal RBCs positive for the corresponding antigen, • Therapy and prognosis leading to extravascular hemolysis 118 CHAPTER 3 Hematology

• Clinical presentation • Congenital neutropenia may occur in several disor- • Fetus may show erythroid hyperplasia in the mar- ders. The disorders are relatively rare row and extramedullary hematopoiesis to compen- • Acquired neutropenia occurs more commonly. It is sate for hemolysis usually as a result of decreased production in the • Laboratory testing bone marrow, anti-neutrophil antibodies, chemo- • Maternal samples are tested for ABO and Rh to therapy and radiation, and severe infections. Cell determine the need for RhIG to help prevent alloim- production is unable to keep up with cell munization to fetal D antigen consumption • Antibody titers may be used to help monitor the Eosinophilia patient and determine if other methods are needed • Absolute counts are increased above 0.40109/L for monitoring the patient • Frequently seen in parasitic infections and allergies • CBC (fetus) • May also be increased in some myeloproliferative neo- • Decreased Hgb with polychromasia and NRBCs on plasms, including chronic myelogenous leukemia the peripheral smear (CML) and chronic eosinophilic leukemia • Reticulocyte count is increased Basophilia • Bilirubin is usually increased • 9 • DAT is positive Absolute counts are increased above 0.15 10 /L • • Therapy and prognosis Can be seen in some hypersensitivity reactions • • In severe cases, intrauterine transfusion may be May be increased in malignancies, particularly myelo- indicated or exchange transfusions may be adminis- proliferative neoplasms, including CML Monocytosis tered after delivery 9 • Phototherapy can help reduce bilirubin after delivery • Absolute counts are increased above 1.1 10 /L • • NOTE: ABO HDFN also can occur and is more common Often seen as patients are recovering from infections than Rh HDFN; however, it is usually asymptomatic • May be increased in some solid tumors and hemato- or produces mild anemia with spherocytes and poly- logic malignancies, including acute monocytic leuke- chromasia in addition to hyperbilirubinemia. IgM anti- mia, acute myelomonocytic leukemia or chronic bodies are unable to cross the placenta myelomonocytic leukemia Lymphocytosis • Absolute counts are increased above 10.0109/L 9 NONMALIGNANT WHITE BLOOD (children) or above 4.810 /L (adults) CELL DISORDERS • Reactive lymphocytes are typically present in infectious monocytosis and other viral infections Quantitative White Blood Cell Disorders • A more normal lymphocyte morphology is seen in disorders such as Bordetella pertussis infection Neutrophilia Lymphocytopenia Causes of Neutrophilia • Absolute counts are decreased below 2.0109/L (chil- • Increase in relative and/or absolute numbers of cells dren) or below 1.0109/L (adults) (>8.7109/L or >70%) • Decreased counts are often seen in immunodefi- • Neutrophils can move from marginating to circulat- ciencies, particularly human immunodeficiency virus ing pool or increased need can lead to additional infection and also during steroid treatment release from bone marrow into circulation • Physiologic neutrophilia, also known as shift or Changes in White Blood Cell Morphology transient neutrophilia occurs when the body is under stress. Cells move from the marginating to • Abnormalities (changes) seen in neutrophils the circulating pool. Counts will return to normal • Do¨ hle bodies: Pale bluish inclusions in the cyto- levels plasm composed of rough endoplasmic reticulum, • Infections, particularly bacterial usually associated with bacterial infections and • Inflammation inflammation • Medications • Toxic granulation: Large bluish-black granules • Increases may be caused by some neoplasms, partic- appearing in the cytoplasm, usually present in ularly myeloproliferative disorders inflammation • Numerous others • Vacuolization: Vacuoles within the cytoplasm that are often indicative of phagocytosis. Vacuoles can Neutropenia be seen in bacterial or fungal infections, but also Causes of Neutropenia may appear as artifact in old samples • Decrease in the absolute numbers of cells • Hypersegmentation: Nuclei have more than five (<2.0109/L) segments, usually seen in infection. This may also CHAPTER 3 Hematology 119

be seen as a nuclear abnormality without infection granulomatous disease test negative for the ability in patients with megaloblastic anemia to reduce the substance • Changes seen in lymphocytes • Leukocyte adhesion disorder (LAD) • Reactive/variant: Mature lymphocytes showing • Mutations in the genes needed to form cell adhesion nuclear and cytoplasmic changes after stimulation molecules, particularly b integrins by antigens. Cells tend to be large with abundant • Three subtypes of LAD, all affecting neutrophil cytoplasm with slightly less chromatin clumping adhesion than resting lymphocytes • Patients have difficulties with recurrent infections

Inherited Abnormalities of Neutrophils HEMATOPOIETIC NEOPLASMS • Pelger-Huet anomaly • Autosomal dominant mutation of the lamin B Myeloproliferative Neoplasms receptor • Neutrophils are hyposegmented, with nuclei • Clonal hematopoietic stem cell disorders that result in showing mature chromatin. Nuclei shapes are the overproduction and accumulation of cells in the round/oval, bands, or bilobed and separated by a thin granulocytic, RBC, and platelet cell lines, leading to filament chronic neoplasms • Cells function normally, as granule function is not • Disorders often have an insidious onset and progress impaired slowly through chronic stages, usually terminating in • May-Hegglin anomaly an aggressive acute stage • Autosomal dominant mutation of the MYH9 gene • Disorders can occur at any age, but most patients are • Do¨ hle-like inclusions are found in neutrophils, over 40 years of age eosinophils, basophils, and monocytes, in addition • WHO has classified myeloproliferative neoplasms to the presence of thrombocytopenia and giant (MPNs) into four major categories, in addition to sev- platelets eral less common categories • Usually asymptomatic, although patients may • CML exhibit bleeding as a result of thrombocytopenia; • Polycythemia vera WBCs function normally • Primary myelofibrosis • Alder-Reilly anomaly • Essential thrombocythemia • Autosomal recessive disorder leading to the inabil- • Additional, less common categories include chronic ity to fully degrade mucopolysaccharides neutrophilic leukemia, chronic eosinophilic leuke- • Cells are filled with large, prominent granules com- mia, mast cell disease, and unclassified posed of mucopolysaccharides Chronic Myelogenous Leukemia • WBCs function normally • Characterized by production and accumulation of • Chediak-Higashi syndrome neutrophils in all stages of maturation • Autosomal recessive mutation of the LYST • Chronic disorder that can lead to an acute/accelerated gene that affects all cells with lysosomal phase several years after onset if the disorder is not organelles treated • All WBCs may show presence of large lysosomal • Acute phase usually terminates in an acute leukemia granules • Clinical presentation • Patients tend to die early in life as a result of bacte- • Anemia, bleeding, infection, sometimes splenomegaly rial infections, because cells do not function • CBC normally • Elevated WBC, often greater than 100109/L • Patients also may exhibit bleeding, albinism, and • Elevations of all granulocytic cells, showing all neurologic issues stages of maturation • Chronic granulomatous disease ○ Left shift through the promyelocyte stage • Mutations, either X-linked recessive or autosomal ○ Predominant WBCs tend to be segmented neu- recessive, in the proteins coding for NADPH trophils, bands, metamyelocytes, and myelocytes oxidase ○ Myeloblasts and promyelocytes are present in • Phagocytic cells produce superoxide, which is the chronic stage, although they are less than needed for the kill mechanism that targets many 5% of the differential bacteria and fungi ○ Eosinophils and basophils tend to be elevated • Patients tend to have frequent infections • Bone marrow • Cells look normal but are unable to kill many bac- • Hypercellular with an elevated M:E ratio teria or fungi, leading to frequent infections • Erythroid cells are often decreased • Testing for the disorder uses the nitroblue tetrazo- • Megakaryocytes are present in normal to increased lium reduction test, in which patients with chronic numbers 120 CHAPTER 3 Hematology

• Laboratory testing § Additional chromosomal abnormalities may • Uric acid is increased as a result of elevated cell be present turnover § Patients present as they would with an acute • Leukocyte alkaline peroxidase (LAP) score is leukemia decreased Polycythemia Vera ○ A low score can help differentiate CML from leu- • Characterized by increased RBCs, granulocytes, and kemoid reaction, where the LAP score is increased platelets in the peripheral blood, with notable • Cytogenetics and molecular testing increases in RBC and Hgb, while erythropoietin levels • Karyotyping remain normal to decreased ○ Philadelphia chromosome is present, which is • Patients present with a JAK2 V617F mutation, which required for diagnosis affects the cellular response to erythropoietin, in addi- § Formed by the translocation of the long arms tion to decreasing normal apoptosis of chromosomes 9 and 22, also leading to the • Clinical presentation production of the BCR-ABL fusion gene. • Patients may show symptoms related to increased § BCR-ABL codes for protein 210 (p210), RBC mass, including headaches and ruddy cyanosis which results in an increase of tyrosine from the increase in circulating RBCs kinase activity, and prognosis is more • CBC favorable if p210 is present • Elevated Hgb and Hct, with normal RBC morphol- ○ If protein 190 (p190), another protein ogy, increases in WBC and platelets with increased tyrosine kinase activity, • Bone marrow is present, the prognosis is poor • Hypercellular with increases in all three cell lines • Fluorescence in situ hybridization (FISH) testing during the initial chronic phase ○ Identifies the presence of the BCR-ABL fusion • Some patients will progress to a “spent” phase, in gene used in diagnosis which bone marrow becomes fibrotic and spleno- • Reverse-transcriptase polymerase chain reaction megaly becomes prominent, often as a result of (RT-PCR) extramedullary hematopoiesis ○ Used to monitor cytogenetic and molecular • Laboratory testing remission • Erythropoietin level is decreased • Therapy and progression • RBC mass is increased • Therapy • LAP is normal to increased ○ Imatinib mesylate (Gleevic) therapy has been • Cytogenetics and molecular testing favorable in leading to remissions • JAK2 V617F mutation present in the majority of § Tyrosine kinase inhibitor working to block patients the tyrosine kinase activity • Other JAK2 mutations may be present § Response can lead to complete remission • Therapy and progression § Relapse may occur in some patients who • Therapeutic phlebotomy is used to decrease RBC develop imitanib resistance counts and provide relief from symptoms ○ Previous to imatinib, various therapies were used • Myelosuppressive therapy also may be used to con- to decrease the tumor burden, from chemother- trol cell burden apy to interferon-a (IFN-a) • Progression to an acute leukemia may occur in ○ Bone marrow transplant from a matched donor, approximately 15% of patients particularly in younger patients, can also be used Essential Thrombocythemia • Progression • Characterized by increased platelets and megakaryo- ○ Before the use of imitanib therapy,a chronicperiod poiesis; however, platelets may not function of the disease would occur and then it would tran- normally sition to an acute leukemia (blast phase) • Clinical presentation § Often, before the blast stage, the disease • Patients may present with symptoms related to would transition through an accelerated stage bleeding and clotting, including neurologic symp- in which clinical presentation and laboratory toms, myocardial infarction, headache, mucous values would begin to deteriorate as the blast membrane bleeds, and others count increased • May be asymptomatic but incidental finding of ○ Blast crisis: Terminal phase of CML increased platelets may lead to further workup § Increased blasts in the peripheral blood and • Diagnosis requires the ruling out of reactive throm- bone marrow (>20%) bocytosis or other MPNs § Blast origin may be myeloid or lymphoid • CBC § Extramedullary hematopoiesis also may • Increased platelet counts, >450109/L with sus- occur tained elevations CHAPTER 3 Hematology 121

○ Platelets may be of variable size and granularity • Disorder similar to CML; however, the majority of the • Slight decreases in Hgb and Hct cells are mature (>90%), and fewer than 10% are • Bone marrow immature neutrophils • Megakaryopoiesis without increased erythropoiesis • Differential diagnosis requires the absence of the Phil- or granulopoiesis adelphia chromosome, in addition to ruling out any ○ Megakaryocytes may be larger than normal other causes of reactive neutrophilia • Laboratory testing Chronic Eosinophilic Leukemia • Little additional testing is currently used for • Chronic disorder showing an elevated absolute eosin- diagnosis ophil count (>1.5109/L) with no evidence of reac- • Cytogenetics and molecular testing tive eosinophilia (parasitic infections, allergic • JAK2 V617F mutation may be present reactions, etc.) or other malignancies that feature • Therapy and progression increased eosinophils • Prevention of bleeding and clotting episodes • Abnormalities and immature forms of eosinophils are • Myelosuppression to suppress platelet present in the peripheral circulation and bone marrow production Mastocytosis • Patients usually survive longer than 10 years • Group of chronic disorders with accumulations of mast cells within the organ systems. Classified into Primary Myelofibrosis seven subcategories by the WHO • Other names have been used for this disorder, includ- • Clinical presentation usually includes urticarial lesions ing chronic idiopathic myelofibrosis and myelofibrosis and a variety of other symptoms based on the sub- with myeloid metaplasia group of the disorder • Characterized by bone marrow fibrosis, extramedul- • Mast cells may be elevated in the bone marrow or skin lary hematopoiesis, and increases in megakaryocytes lesions • Clinical presentation • KIT mutations occur in many with systemic • Nonspecific symptoms including fatigue, weak- mastocytosis ness, shortness of breath, in addition to • Prognosis is variable from benign to aggressive splenomegaly Unclassifiable • CBC • Catch-all group of MPNs to classify disorders that are • Normal to decreased Hgb and Hct with RBC and consistent with MPN diagnosis, including those that platelet abnormalities do not meet the WHO diagnostic criteria for diagnosis ○ Teardrops, other morphologies, polychromasia, or disorders that occur in conjunction with another and NRBC disorder ○ Platelet counts are variable, as are platelet morphologies. Circulating micromegakaryocytes MYELODYSPLASTIC SYNDROMES may be seen • Bone marrow • Group of neoplastic disorders characterized by periph- • Areas of fibrosis with hypercellularity of granulo- eral blood cytopenias and dyspoiesis that occur in one cytes and megakaryocytes or more cell lines • Cytogenetics and molecular testing • Cytopenias do not respond to most usual therapies • JAK2 mutation may be seen • Most cases arise de novo or related to other therapy • Therapy and progression (usually chemotherapy or radiation) • Survival is approximately 5 years after diagnosis, • Usually affects patients older than 50 years of age, with although some patients have lived longer a median age of diagnosis of 70 years of age • Multiple therapies may be used, including RBC and • Clinical symptoms chemotherapy and immunotherapy • Clinical symptoms parallel the dyspoietic cell line(s) • Treatment for anemia ○ RBC: Anemia symptoms • Radiation or splenectomy may be used to target ○ Platelets: Bruising/bleeding splenomegaly and spleen pain or hyperfunction ○ Granulocytes: Increased infections • Bone marrow transplant may be used in younger • CBC patients • Cytopenias may occur in one or more cell lines. Dysplasia may be present Other Myeloproliferative Neoplasms • Bone marrow Chronic Neutrophilic Leukemia • Cells present are dyspoietic and have any number • Chronic disorder characterized by an elevated WBC of abnormalities from abnormal nuclei, abnormal count (>25109/L) with a proportional increase in granulation of cytoplasm, N/C asynchrony, and neutrophils and their precursors in the bone other abnormalities, and they may not function marrow normally 122 CHAPTER 3 Hematology

○ Examples of dyspoietic cell appearance ○ Abnormalities most frequently occur in chromo- § Dyserythropoieis: Oval macrocytes, multi- somes 5, 7, 8, 11, 13, 20 nucleate NRBCs, nuclear bridging, other • Treatment and prognosis abnormal nuclear shapes, inclusions • Treatment varies between the different disorders in (Howell-Jolly bodies, siderotic granules, baso- the group; however, supportive therapy tends to be philic stippling) used, often because of patient age and presence of § Dysmyelopoiesis: Abnormal granulation other coexisting diseases (lacking granules, decreased granule presence, ○ Transfusion therapy, prophylactic antibiotics, or large granules), N/C asynchrony, abnormal growth factors to stimulate bone marrow nuclear shape, multinucleate WBCs response, or immunosuppressive therapy § Dysmegakaryopoiesis: Giant platelets, agra- ○ Patients <70 years of age may be candidates for nular platelets, circulating micromegakaryoc- stem cell transplant tyes, unusual nuclei • Prognosis is variable based on subgroup, because • Laboratory testing levels of risk are based on the types of dyspoiesis, • Chromosomal abnormalities are present in approx- number of blasts present, and cytogenetic imately half of the cases of MDS findings ○ Karyotyping can help determine treatments and • WHO (2008) Classification (Table 3-12) their predicted response • Refractory Cytopenia with Unilineage Dysplasia • Refractory Anemia with Ringed Sideroblasts

TABLE 3-12 Peripheral Blood and Bone Marrow Findings in Myelodysplastic Syndromes

Disease Blood Findings Bone Marrow Findings Refractory cytopenia with unilineage dysplasia Unicytopenia* Unilineage dysplasia: 10% of cells in one myeloid lineage (RCUD), refractory anemia (RA), refractory No or rare blasts (<1%){ <5% blasts neutropenia (RN), refractory <15% of erythroid precursors are ringed sideroblasts thrombocytopenia (RT) Refractory anemia with ringed sideroblasts (RARS) Anemia 15% of erythroid precursors are ringed sideroblasts No blasts Erythroid dysplasia only <5% blasts Refractory cytopenia with multilineage dysplasia Cytopenia(s) Dysplasia in 10% of cells in two or more myeloid lineages (RCMD) No or rare blasts (<1%){ (neutrophil and/or erythroid precursors and/or No Auer rods megakaryocytes) <1109/L monocytes <5% blasts in marrow No Auer rods 15% ringed sideroblasts Refractory anemia with excess blasts 1 (RAEB-1) Cytopenia(s) Unilineage or multilineage dysplasia <5% blasts 5%-9% blasts{ No Auer rods No Auer rods <1109/L monocytes Refractory anemia with excess blasts 2 (RAEB-2) Cytopenia(s) Unilineage or multilineage dysplasia 5-19% blasts 10-19% blasts{ Auer rods{ Auer rods{ <1109/L monocytes Myelodysplastic syndrome, unclassified (MDS-U) Cytopenia(s) Unequivocal dysplasia in <10% of cells in one or more myeloid 1% blasts{ cell lines when accompanied by a cytogenetic abnormality considered as presumptive evidence for a diagnosis of MDS <5% blasts MDS associated with isolated del(5q) Anemia Normal to increased megakaryocytes with hypolobulated nuclei Usually normal or <5% blasts increased platelet Isolated del(5q) cytogenetic abnormality count No Auer rods No or rare blasts (<1%)

From Swerdlow SH, Campo E, Harris NL, et al, editors: WHO classification of tumours of haematopoietic and lymphoid tissues, ed 4, Lyon, France, 2008, IARC Press. *Bicytopenia may occasionally be observed. Cases with pancytopenia should be classified as MDS-U. {If the marrow myeloblast percentage is <5% but there are 2%-4% myeloblasts in the blood, the diagnostic classification is RAEB-1. Cases of RCUD and RCMD with 1% myeloblasts in the blood should be classified as MDS-U. {Cases with Auer rods and <5% myeloblasts in the blood and <10% myeloblasts in the marrow should be classified as RAEB-2. CHAPTER 3 Hematology 123

• Refractory Cytopenia with Multilineage Dysplasia • RAEB-2: 10% to 19% blasts, Auer rods may be • Refractory Anemia with Excess Blasts present • Myelodysplastic Syndrome with Isolated 5q • Prognosis Deletion • Disease is more aggressive because more blasts are • Myelodysplastic Syndrome, Unclassifiable present, leading to a higher number of cases that can • Childhood Myelodysplastic Syndromes transform into AML

Refractory Cytopenia with Unilineage Dysplasia (RCUD) Myelodysplastic Syndrome with Isolated 5qDeletion (5q-Syndrome) • CBC • CBC • Dysplasia occurs in more than 10% of one cell line • Refractory anemia and other parameters are • Less than 1% blasts in peripheral blood relatively normal • Bone marrow • Other parameters are relatively normal • <5% blasts in bone marrow • Bone marrow • Evidence of dyspoiesis • Less than 5% blasts • Prognosis • Abnormal megakaryocytes and erythroid • Survival of 2 to 5 years after diagnosis hyperplasia • Other testing Refractory Anemia with Ringed • Cytogenetics showing a 5qdeletion Sideroblasts (RARS) • Prognosis • Survival of approximately 12 years, disease tends to • CBC be stable • Anemia, possibly dimorphic population of normal and hypochromic cells, dyserythropoiesis • No blasts in peripheral blood Myelodysplastic Syndrome, Unclassifiable • Bone marrow • <15% ringed sideroblasts • Cytopenias and some marrow dysplasia are present; • Prognosis however, it may not meet all criteria to be classified • Survival of 6 to 10 years after diagnosis into one of the specific groups of MDS

Refractory Cytopenia with Multilineage Dysplasia MYELOPROLIFERATIVE AND MYELODYSPLASTIC NEOPLASMS • CBC • One or more cytopenias is present • Category of disorders with features of both myelopro- • Dysplasia in two or more lines liferative and myelodysplastic neoplasms (MDS/MPN) • Less than 1% blasts in peripheral blood • Bone marrow Chronic Myelomonocytic Leukemia (CMML) • Less than 5% blasts, some ringed sideroblasts may be present • Two subgroups, CMML-1 and CMML-2, can occur in • Prognosis adults • Disease course is more aggressive • Elevated WBC count with monocytosis (>1.5109/L) and less than 20% blasts and promonocytes in the peripheral blood and bone marrow Refractory Anemia with Excess Blasts • (RAEB) Dysplasia in one or more myeloid line, particularly dysgranulopoiesis • Two subtypes occur: RAEB-1 and RAEB-2 • Prognosis is variable; however, CMML-1 has fewer • CBC blasts, leading to a more favorable prognosis than • Cytopenias in all three cell lines, in addition to dys- CMML-2, in which more blasts are present myelopoiesis and/or dysmegakaryopoiesis • RAEB-1: 2% to 4% blasts, decreased monocytes Juvenile Myelomonocytic Leukemia (JMML) • RAEB-2: 5% to 19% blasts, decreased monocytes ○ Some blasts may have Auer rods • Similar to CMML, but it occurs in children up to age 14 • Bone marrow • These patients are usually candidates for stem cell • RAEB-1: 5% to 9% blasts transplant 124 CHAPTER 3 Hematology

Atypical Chronic Myeloid Leukemia ○ Small lymphoblasts, which are up to two times larger than a normal lymphocyte, with small • Blood picture is similar to that of CML, but cells amounts of cytoplasm exhibit dysplasia, particularly in the granulocyte line ○ Nucleoli are present but may not be prominent • Philadelphia chromosome BCR/ABL1 negative ○ Large lymphoblasts, which are 2 to 3 times larger • Prognosis is poor than a normal lymphocyte with prominent nucleoli and abnormalities in the nuclear membrane Myeloproliferative and Myelodysplastic • Other laboratory tests Neoplasms Unclassifiable • Flow cytometry is used to determine specific precursor cells present based on CD markers present • Cytopenias and some marrow dysplasia is present, ○ Breaks ALL into subtypes along with features of myeloproliferative disease; 1. Early B-cell (pro-B) ALL: TdT+, CD34+, however, it may not meet all criteria to be classified CD19+, CD20, CD22+ into one of the specific groups of MDS/MPN 2. Intermediate pre–B-cell ALL: TdT+, CD34 +/, CD19+, CD20+/, CD10+ 3. Pre–B-cell ALL: TdT+, CD19+, CD20+, ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) CD22+, CD10+/ • Acute leukemia characterized by the presence of more 4. Pre–T-cell ALL: TdT+/ , CD34+/ , CD2+, than 20% lymphoid blasts in the peripheral blood and/ CD3+, CD7+, CD10+/ or bone marrow • Therapy • Primarily affects children, but is also seen in older • Treatment varies based on age and risk ○ adults Low risk: Children 1 to 10 years of age with a • WHO classifies into B lymphoblastic leukemia/lym- lower WBC count, no extramedullary disease ○ phoma (B-ALL) and T lymphoblastic leukemia/lym- High risk: Adults, children younger than 1 year phoma (T-ALL). Both are divided into several of age and those with extramedullary disease subgroups based on recurrent genetic abnormalities. • Treatment usually begins with induction therapy, The subtype can influence treatment and prognosis followed by consolidation therapy, and continua- • Genetic abnormalities in B cell lymphoblastic tion therapy ○ leukemia/lymphoma include Intrathecal therapy may be used if there is evi- ○ t(9;22)(q34;q11.2); BCR-ABL1 dence of blasts infiltrating the CNS ○ t(11q;23); MLL • For patients not responding to initial therapy or ○ t(12;21) (p13;q22); TEL-AML1(ETV6- patients no longer in remission, stem cell trans- RUNX1) plants can be used if a matched donor is present ○ Hypodiploidy • Prognosis ○ Hyperdiploidy • Varies based on age, number of blasts, immunophe- ○ t(5;14)(q31;q32); IL3-IGH notype, and genetic mutations ○ ○ (1;19)(q23;p13.3); E2A-PBX1 (TCF3-PBX1) Prognosis tends to be better in cases in which § • T-cell lymphoblastic leukemia/lymphoma may exhibit Patient is younger § abnormal karyotypes, but they are not as defined as Blast counts are lower § seen in B-cell acute lymphoblastic leukemia (B-ALL) Immunophenotype fits the regular criteria § • Clinical presentation Chromosomal translocations have variable • Generalized symptoms including fatigue and fever,in prognosis § addition to bleeding from mucous membranes If patient is Philadelphia chromosome pos- • Lymphadenopathy and bone pain may be present itive, they tend to have a poorer prognosis § because of leukemic cells Hyperdiploidy in children is favorable but • Malignant cells may also infiltrate the central ner- not in adults § vous system (CNS), leading to blast presence in Hypodiploidy has a poor prognosis overall the spinal fluid • CBC ACUTE MYELOID LEUKEMIA (AML) • Variable WBC count; counts can range from low to elevated numbers • Acute myeloid leukemia (AML) is characterized by the • Usually see anemia, thrombocytopenia, and presence of greater than 20% blasts (WHO 2008 Clas- neutropenia sification System) in the peripheral blood and/or bone • Not all patients have circulating lymphoblasts; marrow however, the lymphoblasts exhibit two main mor- • Most common group of leukemias in adults and chil- phologies when they are seen dren younger than 1 year of age • Clinical presentation CHAPTER 3 Hematology 125

• Patients often present with nonspecific symptoms, • CBC and bone marrow show myeloblasts, mono- related to the bone marrow takeover by the abnor- blasts, and promonocytes, and may see dysplastic mal clone of malignant cells, which may crowd out eosinophils in marrow normal cells • Extramedullary involvement may be present, par- ○ Anemia: The malignant clone decreases space ticularly in the CNS available for erythroid precursors, leading to • Prognosis often shows a good remission rate typical anemia symptoms, such as fatigue, • AML with t(15;17)(q22;q12); (PML/RARA); also pallor, etc. referred to as acute promyelocytic leukemia (APL) ○ Thrombocytopenia: The malignant clone • All ages, but usually seen in younger adults, often decreases space available for megakaryocyte females production, leading to symptoms of bleeding • CBC and bone marrow show myeloblasts/abnormal and bruising promyelocytes, Auer rods usually present singly or ○ Neutropenia: Fewer mature cells are present in bundles because of the hyperproliferation of immature • Complications can include DIC, because primary myeloid cells. May lead to infections granules in promyelocytes can serve as procoagulants and fever • Cells have a maturation block at the promy- • CBC elocyte stage, and all-trans retinoic acid (ATRA) • WBC counts are variable, usually between 5 and therapy can be used to make cells continue maturation 30109/L, but can range from 1 to 200109/L • Remission may be achieved with ATRA therapy • Platelet and RBC counts may be decreased because unless patients do not respond to ATRA. of heavy leukemic clone involvement in the bone • AML with t(9;11)(p22;q23); (MLLT3-MLL) marrow • Rare, usually occurring in children • Blasts may be present on the peripheral smear • Clinical signs may involve gingival bleeding, DIC, • Specific smear abnormalities are present in each and skin effects specific subtype • CBC and bone marrow show increased monoblasts • Bone marrow and immature monocytes • Hypercellular marrow with more than 20% blasts • Prognosis is moderate to poor of myeloid origin present • AML with t(6;9)(p23;q34); (DEK-NUP214) • Specific smear abnormalities are present in each • Rare, usually in teenagers or younger adults specific subtype • CBC often exhibits pancytopenia; blasts often are • Other laboratory tests monocytic or have Auer rods • Uric acid is increased because of high cell turnover • Prognosis is usually poor • Lactate dehydrogenase is increased because of high • AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); cell turnover (RPN1-EVI1) • Calcium may be decreased • Rare, usually occurs in adults • Phosphate may be increased • CBC shows variable blast morphology, dyspoietic • Potassium may be decreased cells, platelet abnormalities • Therapy and prognosis • Bone marrow shows increased and abnormal • Variable, based on specific classifications megakaryoblasts • WHO 2008 classifies acute leukemia subgroups • Prognosis is poor based on molecular features and cytogenetics, in addi- • AML (megakaryoblastic) with t(1;22)(p13;q13); tion to traditional cell counts, morphologic appear- (RBM15-MKL1) ance, and bone marrow examination. Specific • Rare, usually occurs in infants, especially those with molecular and cytogenetic features can influence treat- Down syndrome ment and prognosis • Clinical symptoms of organomegaly, anemia and thrombocytopenia, megakaryoblasts and micro- Acute Myeloid Leukemia with Recurrent megakaryocytes Genetic Abnormalities • Marrow may show fibrotic areas • Prognosis has shown improvement with intense • AML with t(8;21)(q22;q22); (RUNX1-RUNX1T1) chemotherapy regimens • Children and young adults • CBC and bone marrow show myeloblasts with Auer Acute Myeloid Leukemia with rods and some maturation; some dysplasia Myelodysplasia-Related Changes • Prognosis is usually favorable • AML with inv(16)(p13.1q22) or t(16:16)(p13.1;q22); • Usually affects older adults (CBFB-MYH11) • Patients have a history of MDS or other similar • All ages, although usually seen in younger patients disorders 126 CHAPTER 3 Hematology

• More than 20% blasts, dysplastic cell morphology in Acute Erythroid Leukemia one or more cell lines • Divided into acute erythroleukemia or pure erythroid • Usually has a poor prognosis leukemia • Both show more than 50% normoblastic cells in the bone marrow; however, acute erythroleuke- Therapy-Related Myeloid Neoplasms mia additionally shows more than 20% myeloblasts • Myeloid neoplasms associated with treatment with • CBC and bone marrow show immature RBCs with chemotherapy and/or radiation obvious dysplastic changes • Prognosis tends to be poor • Cytochemistry shows normoblasts staining with a diffuse or block pattern with periodic acid–Schiff (PAS) stain • Prognosis is poor Acute Myeloid Leukemia, Not Otherwise Acute Megakaryoblastic Leukemia Specified • Characterized by cytopenias, with some cases showing • Contains disorders that have not yet been recognized thrombocytosis. Dysplastic cells are present in all to have a common genetic abnormality cell lines • Characterized by morphology, flow cytometry, and • CBC and bone marrow shows more than 20% blasts, cytochemistry system with over 50% megakaryoblasts Acute Myeloid Leukemia with Minimal Differentiation • Flow cytometry is positive for CD41, CD42b, CD61 • Usually affects infants and older adults Acute Anemias of Ambiguous Lineage • Flow cytometry is positive for CD13, CD33, CD34, • Some leukemias may have characteristics, both by CD117 morphology and immunophenotyping, of more than • CBC and bone marrow show blasts with no evidence one cell type of maturation • These may be designated as biphenotypic or bilineage • Cytochemistry is negative for myeloperoxidase and acute leukemias Sudan black B Acute Myeloid Leukemia Without Maturation CHRONIC LYMPHOID NEOPLASMS • Usually affects adults but can occur at any age • Neoplasms of mature lymphoid cells, both B cells and • Flow cytometry is positive for CD13, CD33, CD117, T cells and frequently CD34 • Increases in mature lymphoid cells in peripheral circu- • CBC and bone marrow show numerous blasts, many lation (leukemic) or in masses in the tissues with Auer rods, and less than 10% of WBCs do not (lymphoma) mature beyond promyelocyte stage • Therapies and prognosis vary based on the specific • Cytochemistry is positive for myeloperoxidase and disorders and mutations present (Table 3-13) Sudan black B Acute Myeloid Leukemia with Maturation • Chronic Lymphocytic Leukemia (CLL)/Small May occur in all age groups Lymphocytic Lymphoma • CBC and bone marrow show more than 20% blasts, more than 10% maturing cells are neutrophilic. Cells • Mature B-cell disorder with an indolent course may show Auer rods and other dysplastic features • CBC shows elevated WBC counts with a predomi- Acute Myelomonocytic Leukemia nance of small lymphoid cells, usually with dense, • Flow cytometry is positive for CD13, CD33, CD14, hypermature nuclei and little cytoplasm, and smudge CD4, CD11b, CD11c, CD64, CD36 cell are frequently seen • CBC and bone marrow show increased myeloid and • Bone marrow shows decreased M:E ratio because of monocytoid cells in the blood and bone marrow elevated lymphoid cells. • More than 20% of marrow cells are monocytoid • Flow cytometry is positive for CD5, CD19, Acute Monoblastic and Monocytic Leukemia CD20, CD23 • Usually seen in younger patients • Usually affects the elderly with a slowly progressing • Clinical symptoms may include skin and gingival disease issues, in addition to bleeding problems • Survival is currently around 10 years after diagnosis • Flow cytometry is positive for CD14, CD4, CD11b, CD11c, CD36, CD64, CD68 Prolymphocytic Leukemia • CBC shows increases in monocytes, and >80% of marrow cells are of monocytic origin • Rare B-cell disorder that affects both T-cell and B-cell • Cytochemistry is positive for nonspecific esterase lines, looking similar to CLL but with the presence of positive prolymphocytes CHAPTER 3 Hematology 127

• CBC shows elevated WBC counts with lymphocytosis, may be seen, but they do not tend to circulate in including prolymphocytes with prominent nucleoli large numbers • Bone marrow • Bone marrow • Increased presence of prolymphocytes • Often exhibits areas offibrosis, leading to a drytap on • Flow cytometry is positive for CD19, CD20, FMC7, aspiration. Hairy cells may be visible on core biopsy and sometimes CD5 • Flow cytometry is positive for CD19, CD20, CD22, • Prognosis CD11c, CD25, CD103 • Disease progression is more aggressive than in CLL • Cytochemistry (tissue samples, including bone mar- • T-cell varieties are more aggressive than B-cell row core biopsy) varieties • Positive for tartrate-resistant acid phosphatase (TRAP) stain ○ Hairy lymphocytes may be seen Hairy Cell Leukemia (HCL) • Annexin A1 is the most specific marker for HCL, in addition to being positive for DBA-44 • Chronic B-cell neoplasm with lymphocytes showing • Prognosis threadlike or “hairy” projections • Patients may achieve long remissions with therapy, • Relatively rare, usually occurring in middle-aged including IFN-a or purines adults, predominantly males • Clinical presentation • Patients often present with massive splenomegaly Plasma Cell Neoplasms • CBC • The CBC is often relatively normal, although it • B cell disorders leading to increases in plasma cells may show pancytopenia. Neoplastic “hairy cells” • Typically affects older adults

TABLE 3-13 Morphologic and Immunophenotypic Features of Mature B-Cell Lymphomas

Immunophenotype/ Cell of Subtype Architectural Features Cytologic Characteristics Cytogenetics Origin Chronic lymphocytic Diffuse lymphocytic Small lymphoid cells CD20+, CD19+, CD5+, Naive or leukemia/small proliferation with growth CD23+ memory lymphocytic lymphoma lefts B cells B-cell prolymphocytic Diffuse proliferation Medium-sized lymphoid cells with CD20+, CD19+, FMC7 Unknown leukemia distinct “punched-out” nucleoli +, CD5+/ mature and abundant cytoplasm B cell Mantle cell lymphoma Diffuse, nodular, or mantle Medium-sized lymphocytes with CD20+, CD19+, CD5+, Mantle zone pattern irregular nuclei FMC7+, cyclin D1+, zone cell t(11;14) Follicular lymphoma Follicular pattern Medium-sized lymphocytes with CD20+, CD19+, CD10 Germinal indented nuclei and variable +, BCL-6+, BCL-2+ center numbers of large lymphoid cells t(14;18) cell Extranodal marginal zone Diffuse lymphoid Medium-sized lymphocytes with CD20+, CD19+, CD43 Marginal lymphoma of mucosa- proliferation, occasionally irregular nuclei and clear +/– zone cell associated lymphoid marginal zone or nodular abundant cytoplasm tissue pattern Plasma cell myeloma, Sheets or large aggregates of Plasma cells, frequently with CD20–, CD19+/–, CD38 Plasma cell plasmacytoma plasma cells cytologic atypia +, CD138+, cytoplasmic light chain+ Diffuse large B-cell Diffuse proliferation Large lymphoid cells CD20+, CD19+, CD10 Different lymphoma +/–, BCL-6+/–, BCL- stages 2+/–, CD5+/– of mature B cells Burkitt lymph Diffuse lymphoid proliferation Medium-sized lymphocytes with CD20+, CD19+, CD10 Germinal ^_frp_secowid=85.4046 with “starry sky” pattern evenly distributed chromatin, +, BCL-6+, BCL-2–, center oma inconspicuous nucleoli t(8:14) cell

From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders. 128 CHAPTER 3 Hematology

Plasma Cell Myeloma • Characterized by clefted cells (butt cells) in the periph- • Clinical presentation eral circulation or cerebrospinal fluid • Variable based on type of myeloma. Multiple myeloma is characterized by bone involvement, CERTIFICATION PREPARATION QUESTIONS whereas Waldenstro¨ m’s macroglobulinemia does not show bone involvement. Plasma cells may infil- For answers and rationales, please see Appendix A. trate other tissues, such as the CNS 1. A physician wants to obtain a measure of a patient’s • CBC iron stores. Which of the following tests would be the • WBC counts may appear normal or increased, with most suitable? circulating plasma cells or plasmacytoid lympho- a. Serum iron cytes depending on disease progression b. Serum transferrin (TIBC) • Bone marrow c. Serum ferritin • Sheets of plasma cells may be seen in the marrow d. Transferrin saturation • Laboratory testing 2. A 68-year-old woman visited her physician with • Total protein tends to be elevated reports of fatigue and weakness. A CBC was ordered, • Surface Ig is positive and the patient’s results were as follows: • Protein and immunoelectrophoresis is positive for 12 the protein(s) present in excess RBC 2.5010 /L Hct 18.8% MCH 24.8 pg Hgb 6.2 g/dL MCV 75.2 fL MCHC 33% • Flow cytometry is positive for CD 19, CD20, CD138, CD38, and monoclonal cytoplasmic Ig, but is negative for surface Ig Which of the following would be a plausible diagno- • Prognosis sis for this patient? • Variable depending on the specific plasma cell mye- a. Iron-deficiency anemia loma present; however, multiple myeloma is often b. Vitamin B12 deficiency quickly progressive c. Anemia of chronic inflammation d. Hemochromatosis 3. A peripheral smear shows a decreased RBC count Lymphoma with microcytic, hypochromic cells with small grapelike inclusions in the RBCs on both Wright stain and Lymphoid Neoplasms Manifesting as Solid Tumors Prussian blue stain. This is consistent with: Hodgkin Lymphoma a. Iron-deficiency anemia • Localized tumor of the lymph nodes, tumor cells do b. Sideroblastic anemia not enter the peripheral blood c. Pernicious anemia • Manifests with the presence of Reed-Sternberg cells in d. b-Thalassemia minor the tumor 4. Given the following results of iron studies, which dis- • Reed-Sternberg cells are described as having the order is the most likely? appearance of “owl eyes” or “popcorn” Non-Hodgkin Lymphoma # Serum iron ↑ TIBC # Ferritin # % Saturation • B-cell disorder is most common, although T-cell or NK-cell lymphomas may occur a. Iron-deficiency anemia • Se´zary syndrome (mycosis fungoides) is an example of b. Sideroblastic anemia a T-cell lymphoma. The Se´zary cells are small to c. Anemia of chronic inflammation medium sized lympoid cells with convoluted or cere- d. Hemochromatosis briform nuclei. The disorder has skin involvement 5. Acquired sideroblastic anemia may be present in all and may disseminate throughout the body of the following except: • Multiple types of non-Hodgkin lymphoma exist, a. Alcoholism including follicular, mantle cell, diffuse large B cell, b. Lead poisoning among many others c. Malabsorption • Flow cytometry for immunophenotyping is often d. Myelodysplastic syndromes used along with cytology for diagnosis 6. A patient has a macrocytic anemia, and the physician Circulating Lymphoma suspects pernicious anemia. Which test would best • Some cases of non-Hodgkin lymphoma may have rule in a definitive diagnosis of pernicious anemia? peripheral blood involvement when tumor cells enter a. Homocysteine the peripheral circulation b. Intrinsic factor antibodies CHAPTER 3 Hematology 129

c. Ova and parasite examination for D. latum c. Microcytic, normochromic cells with increased d. Bone marrow examination poikilocytosis 7. Megaloblastic anemias result from which of the d. Macrocytic, hypochromic cells with increased following? polychromasia a. Deficiencies in free erythrocyte protoporphyrin 15. Which of the following disorders does not have a b. Deficiencies in Vitamin B12 and folic acid hemolytic component? c. Increases in iron and hepcidin a. Sickle cell anemia d. Decreases in liver function b. Autoimmune hemolytic anemia 8. A patient’s bone marrow showed erythroid hyperpla- c. Glucose-6-phosphate dehydrogenase deficiency sia with signs of dysplastic maturation, particularly d. Anemia of chronic disease in the RBC precursors. This is consistent with which 16. A patient presents with evidence of a hemolytic of the following? anemia. Spherocytes, polychromasia, and macro- a. Sickle cell anemia cytosis are observed. Which of the following b. b-Thalassemia major would best help to distinguish the cause of the c. Pernicious anemia anemia? d. G6PD deficiency a. Osmotic fragility 9. The CBC for a 57-year-old man had the b. DAT following results. Which tests would be best to c. G6PD activity assay order next? d. Vitamin B12 level 17. RBC 2.501012/L Hct 26.0% MCH 34 pg Paroxysmal nocturnal hemoglobinuria is character- Hgb 8.5 g/dL MCV 104 fL MCHC 33% ized by flow cytometry results that are: a. Negative for CD55 and CD59 a. Iron studies b. Positive for CD55 and CD59 b. Vitamin B12 and folic acid levels c. Negative for CD4 and CD8 c. Bone marrow examination d. Positive for all normal CD markers d. Intrinsic factor antibodies 18. G6PD deficiency episodes are related to which of the 10. The majority of acquired aplastic anemia cases usu- following? ally results from which of the following? a. Exposure to oxidant damage a. Unknown causes b. Defective globin chains b. Pregnancy c. Antibodies to RBCs c. Chloramphenicol exposure d. Abnormal protein structures d. Radiation exposure 19. Which of the following disorders is not classified as a 11. Which of the following values is the most likely to be microangiopathic hemolytic anemia? normal in a patient with aplastic anemia? a. Disseminated intravascular coagulation a. RBC count b. Hemolytic uremic syndrome b. Absolute neutrophil count c. Traumatic cardiac hemolytic anemia c. Absolute lymphocyte count d. Thrombotic thrombocytopenic purpura d. Platelet count 20. A previously healthy 36-year-old woman with 12. Fanconi’s anemia is an inherited aplastic anemia with visited her physician because of a sudden onset of easy mutations that lead to: bruising and bleeding. Of the following, which is the a. Increased chromosome fragility most likely cause of her laboratory results?

b. Myelophthisic anemia 9 12 c. Pancreatic issues WBC 10.510 /L RBC 3.0010 /L Hgb 8.0 g/dL Hct 25.0% MCV 83 fL MCH 26 pg d. RBC enzymatic defects 9 13. MCHC 32% Platelets 1810 /L Differential: Which of the following is decreased in cases of intra- Normal WBCs vascular hemolytic anemia? with moderate a. Bilirubin schistocytes b. Urine hemosiderin and c. Haptoglobin polychromasia d. Plasma hemoglobin PT: 12.8 seconds aPTT: 34 seconds 14. Typical CBC findings in hemolytic anemia include: a. Microcytic, hypochromic cells with increased a. Sickle cell anemia poikilocytosis b. Chronic myelogenous leukemia b. Macrocytic, normochromic cells with increased c. Disseminated intravascular coagulation polychromasia d. Thrombotic thrombocytopenic purpura 130 CHAPTER 3 Hematology

21. Warm autoimmune hemolytic anemia is usually b. This patient has infectious mononucleosis and caused by which of the following? warm autoimmune hemolytic anemia a. IgA antibodies c. This patient is likely to have b-thalassemia b. IgG antibodies minor c. IgM antibodies d. There is a specimen quality issue because of a cold d. Complement agglutinin 22. Which of the following conditions is not associated 29. Hemoglobin H disease is described as: with secondary warm autoimmune hemolytic anemia? a. /a a. CLL b. a/a b. Idiopathic onset c. /bb c. Rheumatoid arthritis d. b/b d. Viral infections 30. A 3-year-old female patient is seen in the hematology 23. The mutation seen in sickle cell anemia is: clinic to investigate the cause of her persistent ane- ! a. b6Glu Val mia. Hemoglobin electrophoresis was ordered, and ! b. b6Glu Lys results showed an elevation in Hgb F, with a small 26Glu!Lys c. b increase in Hgb A2. What is the most likely disorder ! d. b63Glu Arg based on these results? 24. The majority of hospitalizations associated with a. a-Thalassemia major sickle cell anemia are due to: b. b-Thalassemia major a. Cardiomegaly c. a-Thalassemia minor b. Cholelithiasis d. Hemoglobin H disease c. Pneumonia 31. A 36-year-old male patient has a CBC performed as d. Vasoocclusion part of a routine work physical. The WBC count was 25. Patients with sickle cell trait usually have RBC mor- 6.5109/L with a differential count of 48% neutro- phology that includes which of the following? phils, 40% lymphocytes, 8% monocytes, 3% eosin- a. Normocytic, normochromic RBCs with occa- ophils, and 1% basophils. The majority of the sional target cells neutrophils were mature but hyposegmented, show- b. Normocytic, normochromic RBCs with rare ing bandlike or single nuclei. What disorder would be sickle cells suspected? c. Hypochromic, microcytic RBCs with moderate a. Alder-Reilly anomaly target cells b. Leukocyte adhesion deficiency d. Macrocytic, normochromic cells with c. Pelger-Huet anomaly occasional NRBCs d. Reed Sternberg syndrome 26. Which laboratory test is best used for definitive diag- 32. A 38-year-old male patient has the following CBC nosis of sickle cell anemia? results: a. Solubility testing b. Hemoglobin electrophoresis WBC RBC Hgb 16.0 g/dL c. Peripheral smear review for sickle cells 32.5109/L 5.501012/L d. Bone marrow analysis Hct 48.0% Platelet Differential: 49% 9 27. A peripheral smear review shows mildly anemic sam- 22510 /L segmented ple with target cells and oblong hexagonal crystal- neutrophils, 9% bands, 25% loids. What is a possible identity for the crystalloids? lymphocytes, 9% a. Hemoglobin S monocytes, 1% b. Hemoglobin C eosinophils, 4% c. Hemoglobin SC metamyelocytes, 3% d. Hemoglobin E myelocytes; RBC and 28. An 18-year-old man has a CBC done when visiting platelet morphology his physician for a persistent sore throat. He has appear normal the following results: WBC 12.5109/L RBC 6.001012/L Hgb 10.0 g/dL Hct 30.0% MCV 60 fL MCH 20 pg Which of the following conditions is the most likely MCHC 33% Platelet 218109/L cause of these results? a. Bacterial infection Which of the following is most likely? b. CML a. This patient is normal with a slightly elevated c. Refractory anemia WBC count because of his sore throat d. Viral infection CHAPTER 3 Hematology 131

33. Which of the following cytochemical stains is best CD79a(+), TdT(+). Which of the following diagno- used to distinguish cells of monocytic origin? ses is the most likely? a. a-Naphthyl acetate esterase a. Intermediate B-cell ALL b. Naphthol AS-D chloroacetate esterase b. Pre–B-cell ALL c. Myeloperoxidase c. T-cell ALL d. Periodic acid–Schiff d. Pre–T-cell ALL 34. A positive tartrate-resistant acid phosphatase 41. Which of the following may predict a better progno- (TRAP) stain is indicative of: sis in patients with ALL? a. Burkitt’s lymphoma a. The patient is a child b. Chronic myelogenous leukemia b. Peripheral blood blast counts greater than c. Hairy cell leukemia 30109/L d. Multiple myeloma c. The Philadelphia chromosome is present 35. Which mutation is shared by a large percentage of d. The patient is hypodiploid patients with polycythemia vera, essential thrombo- 42. A 28-year-old female patient presented to the emer- cythemia, and primary myelofibrosis? gency department with symptoms suggestive of a. BCR/ABL DIC. A CBC and coagulation studies were ordered. b. JAK2 V617F The peripheral smear showed blasts and immature c. PDGFR cells with heavy granulation and Auer rods. Which d. RUNX1 of the following disorders would be the most likely? 36. A patient has a CBC and peripheral smear with an a. AML with t(9;11)(p22;q23); MLLT3-MLL elevated WBC count and left shift, suggestive of a b. AML with t(15;17)(q22;q12); PML-RARa diagnosis of CML. Which of the following tests c. ALL with t(12;21)(p13;q22); ETV6-RUNX1 would be the most helpful in confirming the sus- d. ALL with t(9;22)(q34;q11.2); BCR-ABL1 pected diagnosis? 43. A patient presents with an elevated WBC count, a. Cytochemical staining for myeloperoxidase increased monocytes, and blasts present on the and LAP differential. Flow cytometry is performed with the b. Karyotyping for the Philadelphia chromosome following results: CD4+, CD11b+, CD11c+, CD13+, c. Flow cytometry for myeloid cell markers CD14+, CD33+, CD36+, CD64+. Which of the follo- d. Lymph node biopsies for metastasis wing diagnoses is the most likely? 37. A patient has a splenomegaly, and his CBC shows a a. AML with minimal differentiation left shift; bizarre RBCs, including dacryocytes; and b. AML with maturation notable platelet abnormalities. Which of the follow- c. Acute myelomonocytic leukemia ing would be the most helpful in determining the d. Acute monoblastic leukemia patient’s diagnosis? 44. A 75-year-old male patient visits his physician for an a. Bone marrow biopsy annual checkup. His CBC showed an elevated WBC b. LAP staining count with numerous small lymphocytes and smudge c. Karyotyping for the Philadelphia chromosome cells, and a subsequent bone marrow biopsy and d. Spleen biopsy aspirate showed hypercellularity with increased lym- 38. Which of the following peripheral blood findings phoid cells. What is a presumptive diagnosis based would not be expected in a patient with a myelodys- on this information? plastic syndrome? a. Acute lymphoblastic leukemia a. Hypogranular neutrophils b. Chronic lymphocytic leukemia/small cell lym- b. Binucleate neutrophils and NRBCs phocytic lymphoma c. Circulating micromegakaryocytes c. Hairy cell leukemia d. Therapy-related acute myelogenous leukemia d. Decreased vitamin B12 and folic acid 45. Which of the following is not considered a disorder 39. The WHO system classifies this disorder as a Myelo- of plasma cells? proliferative/Myelodysplastic syndrome. a. Monoclonal gammopathy of undetermined a. Refractory Anemia with Ringed Sideroblasts significance b. 5qSyndrome b. Multiple myeloma c. Chronic Myelomonocytic Leukemia c. Se´zary syndrome d. Refractory Anemia with Multilineage Dysplasia d. Waldenstro¨ m’s macroglobulinemia 40. A 4-year-old male patient presents with a slightly ele- 46. Which of the following sets of CD markers are asso- vated WBC count, and occasional blasts are present ciated with T lymphocytes? on the differential. Flow cytometry is performed with a. CD2, CD3, CD4 the following results: CD10(+), CD19 (+), CD22(+), b. CD13, CD14, CD15 132 CHAPTER 3 Hematology

c. CD19, CD20, CD22 c. Immunosuppressed d. CD34, CD71, CD117 d. A patient with leukemia 47. Bone marrow cellularity is most often estimated by 55. Which of the following cell types exhibit IgE recep- examining which of the following? tors on their surface membranes? a. Aspirate a. Basophils b. Buffy coat b. Eosinophils c. Core biopsy c. Band neutrophils d. Crush preparations d. Monocytes 48. A dry tap may be seen in bone marrow aspirations in 56. A 62-year-old female patient’s CBC showed the fol- all of the following conditions except: lowing results: total WBC count of 14.0109/L, a. Aplastic anemia RBC count of 3.951012/L, and platelet count of b. Hairy cell leukemia 245109/L. The differential showed 65% seg- c. Multiple myeloma mented neutrophils, 10% bands, 15% lymphocytes, d. Primary myelofibrosis and 10% monocytes. Toxic granulation and Do¨ hle 49. The largest hematopoietic cells present in the bone bodies were seen in many of the neutrophils. Which marrow are: of the following is most likely? a. Lymphoblasts a. The patient had just finished running a half b. Megakaryocytes marathon c. Osteoblasts b. The patient has a bacterial infection d. Pronormoblasts c. The patient is normal 50. Hemoglobin A contains which of the following con- d. The patient has a helminth infection figurations of globin chains? 57. A CBC on a patient with Chediak-Higashi syndrome a. a2b2 is expected to exhibit which of the following? b. a2d2 a. Giant platelets and Do¨ hle-like inclusions in the c. a2g2 cytoplasm of all granulocytes d. a2e2 b. Large, darkly staining cytoplasmic granules in 51. Which of the following locations is not a site of extra- all WBCs medullary hematopoiesis? c. Giant fused granules and lysosomes in WBC a. Bone marrow cytoplasm b. Liver d. Leukocytosis and bilobed eosinophils c. Spleen 58. Patients with infectious mononucleosis often have d. Thymus the following CBC results: 52. Patients with renal failure often exhibit compro- a. Lymphocytosis, including increased variant/ mised hematopoietic activity because of which of reactive lymphocytes the following? b. Lymphocytopenia with numerous small a. Concurrent depression of thyroid lymphocytes hormones c. Neutrophilia, including a predominant shift to b. Decreased production of erythropoietin the left c. Decreased production of GM-CSF d. Neutropenia with a distinct predominance of d. Bone marrow suppression caused by medica- toxic granulation tions 59. Flow cytometry for monitoring a patient with acqui- 53. Which of the following best describes the function of red immunodeficiency syndrome should include the Rapoport-Luebering pathway? markers for which of the following? a. It produces ATP to help maintain RBC membrane a. CD30 and CD42 deformability b. CD4 and CD8 b. It results in the reduction of glutathione c. CD34 and CD33 c. It produces 2,3 diphosphoglycerate d. CD21 and CD22 (2,3 DPG) 60. Which of the following disorders is classified as a d. It produces cytochrome b reductase myelodysplastic/myeloproliferative disease? 54. A 3-year-old male patient visits the pediatrician a. Acute promyelocytic leukemia for a well-child checkup and routine CBC. He has b. Chronic lymphocytic leukemia a total WBC count of 5.0109/L, RBC count of c. Atypical chronic myelogenous leukemia 3.81012/L, and platelet count of 225109/L. The d. Essential thrombocythemia differential showed 25% segmented neutrophils, 61. All of the following cells are derived from 62% lymphocytes, 10% monocytes, and 3% eosino- CFU-GEMM, common myeloid progenitor cells phils. This patient is likely: except: a. A normal child a. Basophils b. Suffering from an acute bacterial infection b. Lymphocytes CHAPTER 3 Hematology 133

c. Neutrophils 68. Polycythemia vera can be differentiated from d. RBCs secondary polycythemia because of polycythemia 62. A patient’s differential count shows an elevated vera presenting with which of the following? eosinophil count. This is consistent with which of a. Elevated hemoglobin results the following? b. Decreased erythropoietin levels a. Aplastic anemia c. Normal to decreased WBC counts b. Bacterial infection d. Erythroid hyperplasia in the marrow c. Parasitic infection 69. The genetic mutation associated with CML is: d. Viral infection a. t (15;17)(q22;q12) 63. Antibodies are produced by which of the following: b. t(11;14)(p15;q11) a. Macrophages c. t(9:22)(q34;q11.2) b. T lymphocytes d. t(8:21)(q22;q22) c. Plasma cells 70. Which of the following is not classified as a myelo- d. Basophils proliferative neoplasm? 64. The nitroblue tetrazolium reduction test is used to a. Chronic eosinophilic leukemia assist in the diagnosis of: b. Essential thrombocythemia a. Leukocyte adhesion disorders (LADs) c. Mastocytosis b. Chronic granulomatous disease (CGD) d. Waldenstro¨ m’s macroglobulinemia c. May-Hegglin anomaly 71. What is the minimum percentage of ringed sidero- d. Pelger-Huet anomaly blasts present in the bone marrow for a diagnosis 65. A newly diagnosed patient has an acute leukemia. of refractory anemia with ringed sideroblasts? Which of the following would initially be the a. 10% most useful in determining the origin of the blasts seen? b. 15% a. Leukocyte alkaline peroxidase (LAP) and nonspe- c. 20% cific esterase (NSE) d. >25% b. Periodic acid–Schiff (PAS) and tartrate-resistant 72. All of the following are considered to be signs of dys- acid phosphatase (TRAP) erythropoiesis except: c. Myeloperoxidase (MPO) and terminal dexoynu- a. Multinucleate RBCs cleotidyl transferase (TdT) b. Basophilic stippling d. Sudan black B and brilliant cresyl blue c. Do¨ hle bodies 66. Therapy for CML often includes the use of a targeted d. Oval macrocytes tyrosine kinase inhibitor, such as: 73. Features of dysmyelopoiesis and dysmegakaryopoi- a. Imatinib mesylate esis seen on a peripheral smear or bone marrow in b. All-trans retinoic acid cases of myelodysplastic syndromes include all of c. Ablative chemotherapy the following except: d. 2-CDA/cladribine a. Pelgeroid neutrophils 67. A 58-year-old female was seen by her physician for b. Neutrophils showing hypogranulation increasing fatigue. Her CBC shows the following c. Giant abnormal platelets with abnormal gran- results: ules WBC RBC Hgb 17.5 g/dL d. Siderotic granules 15.5109/L 5.901012/L 74. The peripheral blood and bone marrow picture Hct 53.0% Platelet Differential: 55% sometimes will look similar in myelodysplastic syn- 425109/L segmented neutrophils, dromes and some RBC disorders. Which of the fol- 3% bands, 30% lowing RBC disorders tends to have a peripheral lymphocytes, 9% smear appearance similar to cases of myelodysplastic monocytes, 1% syndromes? eosinophils, 2% a. Iron deficiency anemia metamyelocytes; RBC b. a-Thalassemia minor and platelet c. Megaloblastic anemia morphology appear normal d. Warm autoimmune hemolytic anemia 75. Most of the chromosome abnormalities seen in myelodysplastic syndrome involve which of the follow- Which of the following conditions is the most likely ing chromosomes? cause of these results? a. 5, 7, 8, 11, 13, 20 a. Chronic myelogenous leukemia b. 2, 3, 9, 15, 16, 26 b. Polycythemia vera c. 3, 6, 10, 14, 21 c. Acute bacterial infection d. 1, 4, 15, 17, 21 d. The patient is normal 134 CHAPTER 3 Hematology

76. Whichofthe following isnotone oftherecurrentgenetic What is the most likely reason that the physician abnormalities seen in cases of acute myeloid leukemia? ordered a lumbar puncture after receiving the a. AML with t(8;21)(q22;q22); AML1(CBFa)/ETO CBC results? b. AML with t(15;17)(q22;q12); (PML/RARa) a. To rule out an acute case of meningitis c. AML with inv(16)/p(13;q22); (CBFb/MYH11) b. To look for leukemia cells in the spinal fluid d. AML with t(1;19)(q23;q13); (E2A/PBX1) c. To rule out infectious mononucleosis 77. AML with 11q23 (MLL) abnormalities are associ- d. To rule out multiple sclerosis ated with which cell line? 82. A 78-year-old man was previously diagnosed with a. Eosinophil chronic lymphocytic leukemia (CLL). Periodic b. Erythrocyte CBCs were ordered, and several months of CBCs c. Monocyte maintained an appearance consistent with cases d. Neutrophil of CLL. 78. WBC 58.5 RBC 3.901012/L Hgb 12.0 g/dL T-cell ALL most commonly affects which of the 12 following? 10 /L Hct 36.0% MCV 92 fL MCH 3 pg a. Infants MCHC 33% Platelet 132109/L Differential: 70% b. Teenaged males lymphocytes, 8% c. Adult females segmented d. Elderly males neutrophils, 2% 79. Which of the following disorders is considered to be monocytes, 20% classified by WHO as an AML, not otherwise unidentified cells with classified? lymphoid appearance a. Acute erythroid leukemia and a prominent nucleolus b. Acute megakaryoblastic leukemia c. Acute promyelocytic leukemia Which of the following is most likely? d. AML without maturation a. The patient has developed Se´zary syndrome 80. A 69-year-old female patient presented with symp- b. The patient has developed prolymphocytic leukemia toms of fatigue and easy bruising. A CBC was ordered. c. The patient has developed multiple myeloma The peripheral smear showed a large number of d. The patient now has a concurrent case of CLL blasts, anemia, and thrombocytopenia. A bone mar- and ALL row examination was performed, revealing hypercel- 83. Multiple myeloma exhibits laboratory features lularity and a blast appearance similar to that of the except which of the following? peripheral smear. Flow cytometry revealed cells posi- a. Occasional plasma cells in the peripheral blood tive for CD 13, CD 33, CD 34, CD 38, CD 117, and b. Rouleaux HLA-DR. Cells were negative for TdT, myeloperoxi- c. Hypercalcemia dase, and nonspecific esterase. Based on this informa- d. Decreased immunoglobulin tion, which of the following is most likely? 84. The diagnostic cell type seen in Hodgkin lymphoma a. AML with minimal differentiation is: b. AML without maturation a. Binucleate plasma cell c. B-cell ALL without maturation b. Reed Sternberg cell d. Acute monoblastic leukemia c. Bence Jones lymphocyte 81. A 3-year-old female patient was having symptoms of d. Burkitt lymphocyte lethargy and bruising and reported pain in her legs. 85. Which of the following appearances describes the Her mother also mentioned noticing several swollen types of cells seen in Se´zary syndrome? lymph nodes when bathing the child. The pediatri- a. Plasma cells containing immunoglobulin deposits cian ordered a CBC, which had the following results. b. Large circulating micromegakaryocytes WBC 18.51012/L c. Lymphocytes with convoluted, cerebriform RBC 3.001012/L nuclei Hgb 9.0 g/ d. Prolymphocytes with prominent azurophilic dL granules Hct 27.0% MCV 90 fL MCH 30 pg 86. Which of the following best describes the function of 9 MCHC 33% Platelet 5810 /L the hexose-monophosphate pathway? Differential: blastocytes, 6% segmented a. It produces ATP to help maintain RBC membrane 80% neutrophils, 8% lymphocytes, deformability 6% monocytes. RBC b. It results in the reduction of glutathione morphology was normal, and platelets were markedly c. It produces 2,3 diphosphoglycerate (2,3 DPG) decreased d. It produces cytochrome b reductase CHAPTER 3 Hematology 135

87. A patient has a reticulocyte count of 3.5%. This c. Increased RBC count with normal RBCs shows which of the following? d. Increased RBC count with microcytic/ a. Bone marrow response in producing more RBCs hypochromic RBCs because of increased need 94. Patients with sickle cell anemia and b-thalassemia b. A normal reticulocyte count major may not show clinical symptoms until the c. Patient transfusion of whole blood patient is at least 6 months of age because of which d. Lack of response to vitamin therapy after a diag- of the following? nosis of iron-deficiency anemia a. The mutations are acquired after the child is born 88. Which of the following cases does not warrant a bone b. The mutations are activated by dietary and marrow examination? maternal factors a. Presence of blasts on the peripheral smear c. The mutations may not manifest clinically at birth b. Postchemotherapy assessment for minimal resid- because the presence of hemoglobin F decreases ual disease d. The mutations lead to elevations in a genes that c. Diagnosis of iron-deficiency anemia compensate for the decreased gene expression d. Diagnosis of suspected systemic fungal infection 95. The thymus is a site used as a maturation compart- 89. A bone marrow sample for a patient with newly diag- ment for: nosed chronic myelogenous leukemia would often be a. B cells expected to have an M/E ratio of: b. T cells a. 1:1 c. Megakaryocytes b. 2:1 d. Monocytes c. 1:2 96. A manual hemocytometer count was required to d. 10:1 check a patient’s total WBC count. A 1:20 dilution 90. Which of the following is not implicated as a cause of was made and used when the four large “W” nonmegaloblastic macrocytic anemia? squares were counted on both sides of the hemacy- a. Alcoholism tometer. A total of 105 cells were counted between b. Hemochromatosis the two sides. What was the patient’s total WBC c. Hypothyroidism count? 9 d. Liver disease a. 0.3310 /L 9 91. Which of the following results is consistent with a b. 2.1 10 /L 9 diagnosis of aplastic anemia? c. 2.6 10 /L 9 a. Hypocellular bone marrow, absolute neutrophil d. 5.3 10 /L 97. count of 0.5109/L, platelet count of 40109/L, Hereditary elliptocytosis results from defects in Hgb 8 g/dL which of the following? b. Hypocellular bone marrow, absolute neutrophil a. Ankyrin count of 2.5109/L, platelet count of 75109/L, b. Band 3 protein Hgb 10 g/dL c. Spectrin c. Hypercellular bone marrow, absolute neutrophil d. Pyruvate 98. count of 1.5109/L, platelet count of Primary neutrophil granules contain: 100109/L, Hgb 14 g/dL a. Acetyltransferase, collagenase, gelatinase, lyso- d. Hypocellular bone marrow, absolute neutrophil zyme, b2-microglobulin count of 0.5109/L, platelet count of 90109/L, b. Alkaline phosphatase, cytochrome b558, com- Hgb 11 g/dL plement receptor 1, complement 1q receptor, 92. The following statement is true of mutations in a- vesicle-associated membrane-2 thalassemia compared to those seen in b- c. b2-Microglobulin, collagenase, gelatinase lacto- thalassemia: ferrin, neutrophil gelatinase-associated lipocalin a. Mutations in a-thalassemia occur as a result d. Acid b-glycerophosphatase, cathespins, defen- of reduced or absent expression of the globin gene sins, elastase, myeloperoxidase, proteinase-3 99. b. Mutations in a-thalassemia occur as a result of A 36-year-old man visited the emergency depart- the deletion of one or more globin genes ment because of alternating episodes of fever and c. The a-globin gene is expressed on chromosome 11 chills that persisted over several days. The patient d. The b-globin gene is expressed on chromosome 16 stated he had not felt well since returning from a mission trip to Africa. The physician ordered a 93. A patient’s genotype isa/a. This patient will have a CBC that shows which of the following? CBC with the following results. a. Decreased RBC count with numerous target cells WBC RBC Hgb 12.0 g/dL b. Decreased RBC count with microcytic/ 3.5109/L 3.801012/L hypochromic RBCs Hct 36.0% MCV 95 fL MCH 32 pg 136 CHAPTER 3 Hematology

MCHC 33% Platelet Differential: Normal WBC 100. Patients with suspected paroxysmal cold hemoglo- 145109/L distribution, normocytic binuria can be confirmed by performing which of normochromic RBCs the following? with some inclusions a. Direct antiglobulin test (DAT) present and several b. Donath-Landsteiner test abnormal platelet-like c. Osmotic fragility test structures shaped like d. G6PD activity assay boomerangs

What should be done with this sample next? REFERENCE a. Rerun the sample to make sure it is not Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles clotted and applications, ed 4, St Louis, 2012, Saunders. b. Clean the stainer and make another slide to examine c. Refer the sample to the pathologist for further identification d. Report the results, because the results are normal CHAPTER 3 Hematology 137

SELF-ASSESSMENT

Content Area: ______

Score on Practice Questions: ______

List the specific topics covered in the missed questions:

List the specific topics covered in the correct questions: 138 CHAPTER 3 Hematology

NOTES CHAPTER 4 Hemostasis

Charity E. Accurso

STEPS OF HEMOSTATIC RESPONSE DISORDERS OF VASCULATURE AFFECTING HEMOSTASIS Injury to endothelium (or vessel) # Hereditary Primary hemostasis (formation of primary hemostatic plug, platelets have the main role) • Hereditary hemorrhagic telangiectasia (also called # Osler-Weber-Rendu disease): Abnormal formation of Secondary hemostasis (formation of fibrin clot, coagula- vessels in which arterial blood may flow directly into tion proteins are the major contributor) a vein without passing through a capillary. The con- # necting area is often fragile and ruptures easily, result- Fibrinolysis (removal of clot) ing in bleeding and bruising • Ehlers-Danlos syndrome: Connective tissue disorder caused by mutation in collagen synthesis; resulting SYSTEMS OF HEMOSTASIS blood vessels are fragile and easily broken

1. Vasculature 2. Platelets Acquired 3. Clot formation Type of Purpura (More Common Types) 4. Fibrinolytic • Decreased connective tissue • Senile purpura: Degeneration of skin matrix resulting in weak capillaries ROLE OF VASCULATURE • Excess glucocorticoid: Cushing’s syndrome and • Hemostasis usually occurs in the arterioles and venules therapeutic glucocorticoids can result in vessel • Endothelial cells line lumen fragility • • Luminal side coated by glycocalyx (carbohydrates Vitamin C deficiency (scurvy): Vessel fragility and proteins) resulting from disruption of collagen production • • Abluminal side is attached to basement membrane Paraprotein disorders • (type IV collagen and proteins) Amyloidosis: Deposition of amyloid material in ves- • Vessels are nonthrombotic under normal sels leading to fragility; thrombosis is also possible • circumstances Paraproteins: Many different effects, depending on • Negatively charged surfaces repel (endothelium and malignancy • platelets) Vasculitis: Inflammation of blood vessels leading to complement activation; immune complex deposition • Inhibit platelet activation: Prostacyclin (PGI2) and nitric oxide (NO) synthesis and secretion, ADPase also leads to activation and aggregation of platelets • • Inactivation of thrombin: Heparin sulfate, Henoch-Scho¨ nlein purpura: Considered to be thrombomodulin another form of vasculitis more commonly affecting • Damaged vessels are prothrombotic children • Exposure of subendothelium: Collagen—platelet activation ROLE OF PLATELETS • Secretion of platelet activating factor • Secretion of von Willebrand factor (vWF): Platelet • Characteristics adhesion • Circulate as inert cell fragments • Release of tissue factor: Aids in secondary hemosta- • Repel each other and endothelial lining (nonthrom- sis activation botic property)

139 140 CHAPTER 4 Hemostasis

TABLE 4-1 Platelet Components, Functions, and Structure

Major Zones Location Within Platelet Significant Components and Major Functions Peripheral Glycocalyx Factor V: Component of prothrombinase complex, attachment site for factor X on platelet zone Cytoplasmic membrane surface Open canalicular system vWF: Transports factor VIII, mediates adhesion between platelets via GPIb/IX Submembranous area Fibrinogen: Converted to fibrin in final clot formation stages GPIb/IX: Platelet receptor for vWF GPIIb/IIIa: Platelet receptor for fibrinogen (and others) Others: Glycolipids, phospholipids, proteins, mucopolysaccharides Structural Circumferential and throughout the Microtubules zone platelet Microfilaments Intermediate filaments All involved in maintenance of shape and shape change on platelet activation Organelle Internally located Granules zone a (50-80 per platelet): See Table 4-2 Dense (3-8 per platelet): See Table 4-2 Lysosomal granules: Hydrolytic Peroxisomes Mitochondria Glycogen particles Membrane Surface connected open canalicular SCCS: Interior of platelet and connects to platelet surface; allows substances to enter platelet systems system (SCCS, OCS) and others to exit; important in storage and secretion; serves as source of surface Dense tubular system (DTS) membrane after activation DTS: Does not connect to platelet surface, primarily a source of ionized calcium, site of prostaglandin and thromboxane synthesis

GP, Glycoprotein; vWF, von Willebrand factor.

• Become activated after an injury • After activation, platelets interact with other platelets and the damaged vessel wall platelet vWF

GPIb/IX receptor collagen from Platelet Ultrastructure basement membrane FIGURE 4-1 Platelet adhesion. • The platelet is divided into arbitrary zones described by location and function (Table 4-1) • vWF: Stored in a-granules in platelets and Weibel- Palade bodies in endothelial cells Platelet Functions • Important step that triggers several events leading to platelet activation • Passive surveillance: Monitor vessel lining for small holes or gaps, platelets plug holes without activation Platelet Activation of coagulation system • Formation of primary hemostatic plug • Triggered after platelet adhesion or exposure to • Provides phospholipid surface for secondary agonist hemostasis • Results: Shape change, altered orientation of phos- • Promotion of healing by stimulation of smooth muscle pholipids, new receptor expression, changes in cells and fibroblasts biochemistry • Platelet agonists: Collagen, adenosine diphosphate PRIMARY HEMOSTASIS (ADP), thrombin, epinephrine, thromboxane A2 (TXA2), arachidonic acid ○ Platelet Adhesion TXA2: Synthesized from arachidonic acid by cyclooxygenase and thromboxane synthase, • Major interaction is the binding of platelet receptor stimulates platelet granule secretion, enhances glycoprotein Ib (GPIb)/IX to vWF, which binds to col- vasoconstriction; if blocked, secretion is lagen (Figure 4-1) impaired; aspirin blocks cyclooxygenase CHAPTER 4 Hemostasis 141

Major Components (and Select TABLE 4-2 Functions) of Platelet Granules

Dense Bodies a-Granules Shape change. FIGURE 4-2 ADP: Platelet agonist— Factor V: Fibrin formation positive feedback to ○ Collagen and thrombin are strong agonists enhance platelet ○ ADP and epinephrine are weak agonists response and recruitment § Required presence of TXA2 and platelet ATP: Activation of Ca2+ Factor XI: Fibrin formation aggregation channel, agonist for other cells Shape Change Calcium: Secondary Fibrinogen: Converted to fibrin, hemostasis platelet aggregation • Occurs after agonist stimulation, appearance of pseu- Serotonin: Platelet Protein S: Regulation of fibrin dopods, will convert to original shape if stimulus is not agonist, vasoconstriction formation via protein C pathway sufficient (Figure 4-2) TFPI: Regulation of fibrin formation by • Microtubules, microfilaments, and intermediate fil- inhibiting factor VII/tissue factor aments reorganize so that organelles are centrally complex located in the activated platelet vWF: Binding of platelets to collagen ○ Phospholipid orientation: Large surface avail- PAI-1: Inhibitor of fibrinolysis able for biochemical reactions in secondary PF4: Heparin neutralizing, chemoattractant hemostasis ○ Receptor expression: GPIb/IX on surface, increase ß-Thromboglobulin: Chemoattractant Thrombospondin: Stabilization of in number of GPIIb/IIIa receptors on surface platelets

Platelet Secretion ADP, Adenosine diphosphate; ATP, adenosine triphosphate; PAI-1, plasminogen activator inhibitor–1; PF4, platelet factor–4; TFPI, tissue • Requires adenosine triphosphate (ATP); open canalic- pathway factor inhibitor; vWF, von Willebrand factor. ular system fuses with granular membrane, and contents of a-granules and dense bodies are released to the outside of the platelet (Table 4-2) • Agonists released further activate platelets Summary of Major Biochemical • Calcium released for use in secondary hemostasis TABLE 4-3 Mediators of Activation (Table 4-3) 2+ Arachidonic Increased cytoplasmic Ca : Results in Platelet Aggregation pathway phospholipase A2 activation Phospholipase A2: Hydrolyzes arachidonic acid • vWF binding to GPIb/IX activates an intracellular sig- Cyclooxygenase: Synthesizes thromboxane A2 naling pathway that results in the activation of GPIIb/ from arachidonic acid (thromboxane IIIa, which then binds to fibrinogen synthesis involved) TXA2: Platelet agonist, required for secondary vWF binding GPIb/IX aggregation #Intracellular signaling Aspirin: Inhibits cyclooxygenase (lifetime of platelet) GPIIb/IIIa activation and binding to fibrinogen Ca2+ Required for several • Fibrinogen forms bridges to other GPIIb/IIIa recep- Intracellular signaling: reactions, including secondary hemostasis, tors on other activated platelets, resulting in platelet activation of some cellular enzymes aggregates; Ca2+ is needed for aggregation CAMP Negative regulator of platelet activation, • Fibrinogen and Ca2+ are delivered locally from CAMP: production of CAMP inhibits protein kinase granules and dense tubular system which inhibits aggregation • Primary aggregation versus secondary aggregation: ADP: Inhibits adenyl cyclase In vitro ○ Phospholipase Activation of several reactions leads to calcium Primary: Loose aggregation, reversible if stimu- C mobilization, granule secretion, and lus is not sufficient fibrinogen receptor expression ○ Secondary: Irreversible provided sufficient G proteins Platelet agonists bind to G protein receptors, stimulus; occurs after internal ADP release, intracellular messaging system TXA2 synthesis and release, further stimulation then occurs ADP, Adenosine diphosphate; CAMP, cyclic adenosine monophosphate. 142 CHAPTER 4 Hemostasis

SECONDARY HEMOSTASIS • Substrate fibrinogen: Acted on by thrombin • Enzymes: Circulate as zymogens • Through a series of enzymatic reactions, the primary • Activation: Two routes platelet plug is reinforced by fibrin ○ Conformational change • Secondary hemostasis is a complex system of procoa- ○ Proteolytic cleavage gulant activities and control activities to contain and limit clot formation • Zymogens are inactive precursors of coagulation Intrinsic Pathway factors • Activation of contact factors when they come into con- • Zymogens serve as substrates for previous enzy- tact with negatively charged surfaces matic reaction in the coagulation cascade • Glass, kaolin, ellagic acid • Vitamin K–dependent factors: II, VII, IX, X, protein C, • Not dependent on calcium and protein S • Deficiency of contact factors (XII, PK, and HK) • Vitamin K–dependent factors are not functional does not lead to in vivo bleeding issues. Deficiency unless an additional carboxyl group (COOH) is added of XI is associated with bleeding abnormalities in to the g-carbon of the glutamic acid residues. This approximately 50% of individuals reaction is called g-carboxylation and is dependent • Contact factors are involved in activation of fibri- on vitamin K. The factors will be formed in the absence nolysis, complement activation, kinin formation, of Vitamin K but will not be functional because this inflammation, and angiogenesis (Figure 4-3) modification is required for binding to a negative phospholipid surface • The coagulation cascade has two pathways—intrinsic Extrinsic Pathway and extrinsic—and shares a common final pathway, the common pathway • Damage to the vessel results in the exposure of tissue • The end-point of the common pathway is the forma- factor on the surface of nonvascular cells tion of a fibrin clot that reinforces the platelet plug • VII and VIIa bind to tissue factor in the presence of cal- • The concept of the three pathways was derived from in cium to form the VIIa/tissue factor complex, also vitro experiments; physiologically, hemostasis occurs called extrinsic Xase, and the extrinsic pathway is thus through one pathway—the tissue factor pathway activated. Extrinsic Xase also can activate IX in the intrinsic pathway (Figure 4-4) Grouping of Coagulation Factors

See Tables 4-4 and 4-5 Common Pathway • Cofactors enhance activity of enzymes • Begins with the activation of X by either the intrinsic • Va is a cofactor for Xa, no enzymatic activity alone or extrinsic pathway (Figure 4-5) • VIIIa is a cofactor for IXa, no enzymatic • End result: Formation of fibrin clot (see Figure 4-5) activity alone • Important notes about fibrin formation (Figure 4-6) • High-molecular-weight kininogen (HK) is a cofac- • Thrombin cleaves fibrinopeptides from fibrinogen, tor for XIIa and Xia forming a fibrin monomer. Fibrin monomers associ- • Protein S is a cofactor for activated protein C ate in half-staggered overlap pattern between the D • Tissue factor is a cofactor for VIIa and E domains

TABLE 4-4 Grouping of Coagulation Factors

Contact Group Fibrinogen Group Prothrombin Group Factors HK, PK, XII, XI XIII, VIII, V, I X, IX, VII, II Consumed in clot No Yes No, except for II Molecular weight 80,000-173,000 Da 300,000-350,000 Da 50,000-100,000 Da Critical to hemostasis XI is essential to hemostasis Yes Yes XII, PK, HK do not play a major role in hemostasis (in vivo), so deficiency does not cause bleeding Other important information Activation of fibrinolytic, kinin, and Thrombin acts on all factors Vitamin K dependent complement systems; role in inflammation

HK, High-molecular-weight kininogen. TABLE 4-5 Important Factors in Hemostasis

Factors Activation and Functions Important Notes Contact Factors XII (Hageman factor) Activated to XIIa by kallikrein, plasmin, or autoactivation (from contact Deficiency not associated with bleeding with negatively charged surface) condition XIIa cleaves PK to kallikrein XIIa (+ cofactor HK) converts XI to XIa Activates fibrinolytic and complement systems XI Activated by XIIa, thrombin, and XIa Hemophilia C Activates IX in the presence of calcium Binds to surface of activated platelets Prekallikrein (PK) Activated to kallikrein by XIIa Majority circulates bound to HK Cleaves HK in smaller fragments—kinin Chemoattractant Activates plasminogen to plasmin conversion Deficiency not associated with bleeding Conversion of scuPA to uPA condition High-molecular-weight Kinin source Located in endothelial cells, platelets, kininogen (HK) Accelerates XII and PK activation granulocytes Deficiency not associated with bleeding condition Prothrombin Group: Vitamin K Dependent II (prothrombin) Cleaved to thrombin by prothrombinase Thrombin: Many functions (both procoagulant and anticoagulant; other functions described in section on regulation) Cleaves fibrinopeptides from fibrinogen to form a fibrin monomer Further activates Va, VIIIa, and XIIIa Activates platelets Stimulates release of vWF and PAI-1 from endothelial cells and expression of tissue factor VII Small amount circulates as VIIa, which further activates VII after binding to tissue factor Component of extrinsic Xase (with tissue factor and Ca2+) Activates X and IX as part of complex IX Activated by XIa in the presence of Ca2+ Binds to activated platelets Additional activation by VIIa/tissue factor complex Hemophilia B Component of intrinsic Xase (with VIIIa and Ca2+) X-Linked inheritance Activates X (as part of Xase) X Activated by extrinsic Xase (VIIa, VIIIa, tissue factor, Ca2+) or intrinsic Xase (IXa, VIIIa, Ca2+) Complexes with Va, Ca2+ and phospholipids to form prothrombinase Fibrinogen Group VIII Activated by Xa or thrombin Circulates associated with vWF Cofactor for IXa Hemophilia A Component of intrinsic Xase (with IXa and Ca2+) X-Linked inheritance V Activated by thrombin and Xa 25% in a-granules of platelets Binds to activated platelet Component of prothrombinase (with Xa, Ca2+, platelet) Fibrinogen Converted to fibrin by cleavage of fibrinopeptides A and B from fibrinogen Glycoprotein in plasma and platelet Fibrin monomers polymerize in half-staggered overlap with other a-granules monomers forming fibrin polymer (see Figure 4-6) Most abundant coagulation protein XIII Stabilizes fibrin monomer by cross-linking D-domains Transglutaminase Others vWF Mediates adhesion to vessel wall Synthesized and stored in endothelial Binds to GPIb/IX on platelets cells and megakaryocytes Binds to collagen and elastin in subendothelium Promotes platelet aggregation by binding to GPIIb/IIIa on platelets Circulates as vWF/VIII complex in plasma, stabilizes and protects VIII Tissue factor Cofactor for VII and VIIa Lipoprotein not normally expressed on Attracts Ca2+ and facilitates procoagulant complex formation vessel surfaces Expressed under variety of conditions (e.g., site of injury, toxins, immune complexes, interleukin-1)

GP, Glycoprotein; PAI-1, plasminogen activator inhibitor; scuPA, single-chain plasminogen activator; uPAa, urokinase plasminogen activator; vWF, von Willebrand factor. 144 CHAPTER 4 Hemostasis

○ Contains lysine-binding sites (kringle domains) Contact collagen ○ Transported in eosinophils K ○ Incorporated into fibrin clot HMWK XII XIIa • Plasmin HMWK ○ Serine protease with broad specificity PK § Degrades proteins susceptible to trypsin XI XIa degradation 2+ Ca § Relevant hemostatic targets: Fibrin, fibrino- IX IXa gen, factors V and VIII Ca2+ • Activation of plasminogen to plasmin conversion VIII PF3 ○ Physiologic plasminogen activators § Common Pathway Tissue type plasminogen activator (tPA) § Produced by vascular endothelial cells, cir- FIGURE 4-3 Intrinsic pathway. HMWK, High molecular weight culates in active form kininogen; kallikrein; Platelet factor 3; Pre-kallikrein. K, PF3, PK, § Increased activation ability when plasminogen is bound to fibrin § Stimuli: Thrombin, bradykinin, histamine, Vessel Injury venous stasis, desmopressin (DDAVP) administration § Urokinase type plasminogen activator (uPA) Tissue thromboplastin, § tissue factor, factor III Produced by renal tubular and vascular epi- VIIa VII thelium, circulates as a single-chain molecular Ca2+ plasminogen activator (scuPA) with little activity, converted by plasmin, factor XIIa, Common Pathway and kallikrein to two-chain form FIGURE 4-4 Extrinsic pathway. § Found in plasma and urine § Digests extracellular matrix § uPA receptor localizes • Factor XIII covalently cross-links D domains to § Exogenous plasminogen activators form urea-insoluble fibrin clot ○ Streptokinase ○ If factor XIII is deficient, clot will form but will § Causes conformational change to plasmin- be soluble in urea ogen when bound, allowing conversion to plasmin Fibrinolysis § Not localized to fibrin ○ Staphylokinase • Process of breaking down fibrin clot through hydroly- § Fibrin required for activation of plasmino- sis of fibrin gen (Figure 4-7) • Activation of system occurs when the intrinsic path- • Outcomes: Fibrin degradation products (FDPs) and way is activated D-dimers • Components • FDPs • Plasminogen: Zymogen for plasmin ○ Fragment X: Limited binding to thrombin

Intrinsic Pathway Extrinsic Pathway VIII X Xa Ca2+ PF3 XIII V Prothrombin Thrombin XIIIa Soluble Stable Fibrinogen Fibrin Fibrin Monomer Polymer

FIGURE 4-5 Common pathway. PF3, Platelet factor 3. CHAPTER 4 Hemostasis 145

A A BB

Fibrinogen D ED Thrombin cleaves fibrinopeptides A and B Fibrin monomer D ED

Spontaneous polymerization

D ED Fibrin polymer D EDD ED XIIIa cross-links D domains

Stabilized fibrin DEEDD D D EDD EDD ED DEEDD D FIGURE 4-6 Formation of stabilized fibrin clot. Thrombin cleaves fibrinopeptides A and B to form fibrin monomer. Fibrin monomers polymerize because of the affinity of thrombin-cleaved positively charged E domains for negatively charged D domains of other monomers. Factor Xllla catalyzes the covalent cross-linking of g chains of adjacent D domains to form a urea-insoluble stable fibrin clot. (From McKenzie SB, Clinical Laboratory Hematology, ed 2, 2010. Printed and electronically reproduced by permission of Pearson Education, Inc, Upper Saddle River, New Jersey.)

Fibrinogen Fibrin

DDDDE E DDE D E DD E DD E D Plasmin DDDDEE Plasmin Fragment X D E D DD Plasmin EE DDE DD Fragment Y D YY and E D DXD Plasmin complex

Plasmin

D DD Fragments E DED complex, D and E EED E, and D-dimer

DD FIGURE 4-7 Degradation of fibrinogen and fibrin by plasmin. Plasmin systematically degrades fibrinogen and fibrin by digestion of small peptides and cleavage of DE domains. Fragment X consists of a central E domain with two D domains (D-E-D); further cleavage produces fragment Y (D-E), with eventual degradation to D and E domains. From cross-linked fibrin, plasmin digestion produces fragment complexes from one or more monomers. D-Dimer consists of two D domains from adjacent monomers that have been cross-linked by Xlla in the process of fibrin formation (thrombosis). (From Rodak B, Fritsma G, Keohane E: Hematology: clinical principles and applications, ed, Philadelphia, 2012, Saunders.)

○ Fragments Y, D, and E: Inhibit fibrin polymeriza- • Plasminogen activator inhibitors (PAIs) tion and platelet aggregation ○ PAI-1: Significant role in plasminogen activation • D-Dimers inhibition inhibits tPA, scuPA, uPA ○ Two D domains linked through factor XIII § Produced by endothelial cells and several action other cell types ○ Significance: Indicative of clot formation § 70% of tPA circulates bound to PAI-1, incor- • Fibrinolysis inhibitors porated into fibrin matrix 146 CHAPTER 4 Hemostasis

§ Acute phase reactant • Heparin cofactor II § Deficiency: Excessive, unregulated fibrinoly- • Similar to antithrombin sis results in bleeding • Inhibits thrombin: Faster when bound to heparin ○ PAI-2: Located in placenta and macrophages, • Proteins C and S: Major anticoagulant system, other inhibits tPA and uPA involved components include thrombin and • Thrombin-activatable fibrinolysis inhibitor (TFPI) thrombomodulin ○ Inhibits binding and activation of plasminogen • Protein C: Vitamin K dependent, activated by ○ Short half-life thrombin • a2-Antiplasmin • Protein S: Vitamin K dependent, cofactor for pro- ○ Inhibitor of circulating plasmin, limiting sys- tein C, circulate bound to C4 binding protein temic fibrinolysis • Thrombomodulin: Endothelial cell protein; when ○ Located in a-granules of platelets bound to thrombin, activates protein C with protein S ○ Small amount incorporated into clot • Activated protein C (APC) inactivates factors Va • a2-Macroglobulin and VIIIa in the presence of protein S and calcium ○ Wide specificity against many proteases ○ Useful after a2-antiplasmin exhausted Disorders of Primary Hemostasis

CONTROL OF HEMOSTASIS • Quantitative disorders: Relates to number of platelets (increase versus decrease) (Table 4-6) Blood Flow • Qualitative disorders: Relates to function of platelets (Table 4-7) • Vasoconstriction to slow blood flow for accumulation of coagulation factors and platelets, allow time for clot formation Other Causes of Thrombocytopenia • Return to normal blood flow limits clot formation and removes excess activated factors • Conditions that result in decreased production or ineffective thrombopoiesis • Hereditary thrombocytopenia: Bernard-Soulier syn- Liver Clearance drome, May-Hegglin anomaly, Wiskott-Aldrich syndrome (described later in more detail) • Production site for many coagulation proteins • Increase sequestration by the spleen resulting from • Removal of activated factors, factors bound to other conditions inhibitors, FDPs • Dilutional thrombocytopenia Positive Feedback Amplification Acquired Platelet Function Disorders • Thrombin: Activator of platelets, promotes release of platelet factor Va, exposure of negatively charged sur- • Aspirin: Irreversibly inactivates cyclooxygenase; non- face, activation of factors Va and VIIIa functional platelets • Factor Xa: Feedback to further activate factor VII, • Alcohol: May eventually lead to platelet dysfunction, small amount of factor VIII activation possibly as a result of prostaglandin synthesis • Antibiotics: Penicillins and cephalosporins, antibiotic coats platelet receptors for ADP and epinephrine, Negative Feedback Amplification may be serious • • Thrombin: Involved in activated protein C pathway Cardiopulmonary bypass surgery: Activation of • TFPI inactivated factor Xa platelets • • Fibrin binding to thrombin limits further conversion Chronic renal failure: Cause unknown, decreased • FDPs interfere in fibrin formation and polymerization platelet aggregation, may be severe • Hematologic disorders: Several can affect platelets, such as multiple myeloma, macroglobulinemia, acute Biochemical Mediators leukemia • Antithrombin: Serine protease inhibitor • Produced by hepatocytes and endothelial cells Thrombocytosis • Major inhibitor to thrombin and factor Xa, also inhibits factors XIIa, XIa, IXa, kallikrein, and • Primary thrombocytosis: Uncontrolled/autonomous plasmin proliferation of megakaryocytes, hemorrhagic or • Heparin significantly accelerates activity thrombotic episodes, platelet count greater than TABLE 4-6 Major Quantitative Disorders

Prognosis/Other Name Pathophysiology Symptoms Diagnosis Treatment Important Information Neonatal alloimmunoe Maternal alloantibodies against Mucosal bleeding Symptoms Self-limiting – 2-6 weeks thrombocytopenia platelet antigens Purpura Identification of Platelet transfusion is controversial (NAIT) antibody Immune thrombocytopenic Autoantibodies bind to platelets Varies Of exclusion Acute: spontaneous remission (2-6 weeks) Acute - Associated conditions: purpura (ITP) Shortened platelet lifespan Asymptomatic to Thrombocytopenia Prevent trauma, monitor symptoms, upper respiratory infections, severe mucosal (<20x109/L) replacement therapy if serious situation chickenpox, rubella, CMV, bleeding viral hepatitis Acute ITP: children; 2-4 Acute: petechiae, Acute: may see Chronic: spontaneous remission is rare Chronic: may see relapses years of age, often abrupt onset increase in follows viral infection lymphocytes, mild eosinophilia Chronic ITP: adults; Chronic: insidious Administer components if count <30x109/ increased female onset, lasts 6-12 L, corticosteroid, splenectomy if incidence months nonresponsive to treatment, other treatments available Thrombotic Ultralarge VWF multimers present Petechiae, Reduced levels of Platelet infusion of ADAMTS-13 thrombocytopenic in circulation which induce microangiopathic ADAMTS-13 HPE 4 CHAPTER purpura (TTP) platelet agglutination hemolytic anemia, renal failure Heparin induced Heparin-dependent IgG Bleeding Continual reduction Discontinue heparin thrombocytopenia antibodies against platelets Occasionally in platelet count Supportive therapy

(HIT) that complex to platelets and thrombotic after heparin Hemostasis enhance clearance complications administration 147 148

TABLE 4-7 Major Qualitative Disorders HPE 4 CHAPTER Prognosis/Other Important Name Pathophysiology Symptoms Diagnosis Information Bernard-Soulier Syndrome Disorder of adhesion Usually no significant Moderate to severe thrombocytopenia Sometimes called giant platelet Rare, autosomal recessive bleeding issues Prolonged bleeding time syndrome GPIb/IX complex: decreased or abnormal ADP: normal Hemostasis (>600 genetic mutations possible) Collagen: normal ! Affects binding of platelet to collagen Epinephrine: normal via vWF Ristocetin: abnormal Distinguish from vWD by performing ristocetin agglutination in the presence of added vWF Glanzmann’s Thrombasthenia Disorder of aggregation Usually no significant Prolonged bleeding time Type I: <5% GPIIb/IIIa Rare, autosomal recessive bleeding issues Abnormal clot retraction receptors GPIIb/IIIa receptor deficiency ADP: no response Type II: 15% of normal number !Affects binding of platelets via fibrinogen Collagen: no response of receptors to initiate aggregation Epinephrine: no response Type III: 50-100% of normal Ristocetin: normal number of receptors △-Storage Pool Disease Decreased or absence of dense granules Moderate to mild Prolonged bleeding time Dense granule deficiencies also in: bleeding ADP: no secondary aggregation Chediak-Higashi, Hermansky Collagen: no response Pudlak syndrome, Wiscott- Epinephrine: no secondary aggregation Aldrich syndrome, TAR Ristocetin: normal syndrome Gray Platelet Syndrome Decreased or absence of alpha granules Mild bleeding Possible prolonged bleeding time Also called: Possible thrombocytopenia A-Storage Pool Disease Normal platelet aggregations

Defective TXA2 Synthesis Three possible causes: Mild bleeding Possible prolonged bleeding time Similar to △-Storage Pool Disease Phopholipase A2 deficiency ADP: no secondary aggregation Cyclooxygenase deficiency Collagen: no response Thromboxane synthase deficiency Epinephrine: no secondary aggregation Ristocetin: normal Von Willebrand Disease (VWD) Qualitative or quantitative deficiency of von Mild to moderate Abnormal platelet aggregation to ristocetin Several types and subtypes of Willebrand factor bleeding APTT: normal to increased VWD Some asymptomatic VWF functional test: decreased Therapy dependent on type VWF antigen: decreased

ADP, Adenosine diphosphate; GP, glycoprotein; TAR, thrombocytopenia with absent radius; TXA, thromboxane; vWF, von Willebrand factor. CHAPTER 4 Hemostasis 149

1000 109/L, abnormal epinephrine (possibly ADP • Liver disease: Site of synthesis for most hemostatic and collagen) platelet aggregation proteins • Reactive (secondary) thrombocytosis: Result of • Expected laboratory results: Increased PT and another condition, variable causes and platelet counts aPTT, decreased fibrinogen, antithrombin, proteins C and S MAJOR DISORDERS OF SECONDARY • Disseminated intravascular coagulation (DIC): HEMOSTASIS Uncontrolled bleeding and clotting, coagulation occurs systemically; condition results in a consumptive See Table 4-8. process in which hemostatic proteins, platelets, and regulatory factors are consumed at an increased rate, Acquired Disorders with Bleeding resulting in deficiencies • Vitamin K deficiency: Vitamin K needed for • Causes: Infections, pregnancy complications, neo- g-carboxylation step to form functional factors II, plasms, trauma or massive injury, snake bites, heat VII, IX, X, proteins C and S strokes • If absent, factor is formed but nonfunctional, bleed- • Diagnosis made by reviewing several different lab- ing may result if less than 30% functional oratory tests and patient history ○ • Causes: Dietary deficiency, decreased gastrointesti- Increased PT, aPTT, and D-dimer and decreased nal flora needed to synthesize vitamin K because of fibrinogen and platelet count • antibiotic therapy, immaturity of the liver Fibrinogenolysis: Plasminogen active without throm- • Expected laboratory results: Increased prothrombin bin generation, results in degradation of fibrinogen time (PT), normal activated thromboplastin time and several coagulation hemostatic factors • (aPTT) (increases with severity of deficiency), Differentiate from DIC by negative D-dimer and decreased proteins C and S normal platelet count

TABLE 4-8 Major Disorders of Secondary Hemostasis

Disorder Diagnosis Prognosis/Other Important Information Factor VIII deficiency APTT ! ↑ X-linked recessive Hemophilia A Factor VIII activity !# Severity and risk of bleeding dependent on severity of deficiency Some patients (5%-20% of affected individuals) develop inhibitors to FVIII due to treatment Factor IX deficiency APTT ! ↑ X-linked recessive Hemophilia B Factor IX activity !# Some patients (1%-3% of affected individuals) develop inhibitors to FIX due to Christmas disease treatment Factor XI deficiency APTT ! ↑ Increased frequency in the Ashkenazi Jewish population Hemophilia C (may be normal in mild deficiency) Factor XI activity !# Afibrinogenemia PT ! ↑ Complete absence of fibrinogen—homozygous mutation APTT ! ↑ Some have severe bleeding; most have milder challenges than hemophiliacs TT ! ↑ Fibrinogen ! absent Hypofibrinogenemia PT ! Normal 50% of normal plasma levels—heterozygous mutation APTT ! Normal Mild bleeding TT ! ↑ Fibrinogen ! Normal Dysfibrinogenemia – PT ! Normal Structural abnormality—usually heterozygous structural abnormality APTT ! Normal Complications: 50% of affected have no clinical bleeding; 25% of affected have TT ! ↑ clinical bleeding; 25% of affected have thrombosis Fibrinogen ! Variable Other factor deficiencies Bleeding deficiencies ! factor deficiencies of factors II, V, and X have also been reported. Diagnosis is made by observing prolongation of PT and/or APTT, and measurement using a specific factor assay. Factor XIII deficiency ! Rare disorder associated with delayed wound healing as factor XIII’s actions occur after fibrin formation. Non-bleeding deficiencies ! factor deficiencies of factors XII, pre-kallikrein, and high molecular weight kininogen have been reported. Clinical bleeding is not observed, however the APTT is prolonged. aPPT, Activated partial thromboplastin time; PT, prothrombin time; TT, thrombin time. 150 CHAPTER 4 Hemostasis

Thrombotic Conditions • Complications: excessive bleeding; heparin induced thrombocytopenia • Thrombotic conditions can occur when balance between procoagulation and anticoagulation is tilted Thrombolytic therapy ! administered after throm- toward procoagulation botic embolism • Major causes • Major forms – classified as plasminogen activators ○ Factor V Leiden: Mutation in factor V causes (PA) inability to inactivate factor Va in the presence 1. Streptokinase (SK) of APC 2. Urokinase (UK) ○ Deficiency of regulatory factors 3. Alteplase (tPA) § Protein C • Monitoring: thrombin time, fibrin(ogen) degradation § Protein S productions, D-dimers § Antithrombin § Heparin cofactor II Antiplatelet therapy ! goal to reduce or block platelet ○ Prothrombin 20210 mutation function or action ○ Decreased fibrinolytic activity 1. Aspirin: inhibits cyclooxygenase (COX-1) thereby § Plasminogen deficiency blocking prostaglandin (TXA ) synthesis; lasts § 2 Decreased a2-antiplasmin lifespan of platelet § Increased PAI 2. ADP receptor antagonists: block ADP receptor in ○ Antiphospholipid antibodies platelets ○ Hyperhomocysteinemia 3. Inhibitors to GPIIb/IIIa: affect interaction of fibrinogen and vWF

Anticoagulant Therapy MAJOR HEMOSTASIS Coumadin ! oral anticoagulant LABORATORY TESTS • Action: Vitamin K antagonist – interferes with vitamin See Table 4-9. K-dependent g-carboxylation step regulating the production of non-functional factors (II, VII, IX, X, Major Tests to Assess Thrombosis Protein C, Protein S); therapeutic levels reached 4- 5 days after initial dose • Antithrombin: Previously called antithrombin III • Monitoring: PT in combination with INR • Chromogenic assay: Functional activity assessed in • Complications: bleeding two-part assay ○ Patient plasma mixed with excess of thrombin in Heparin ! intravenous or subcutaneous the presence of heparin: Antithrombin in patient administration will neutralize added thrombin • Major forms ○ Residual thrombin is measured in assay assessing 1. UFH – unfractionated heparin – heterogenous ability to convert fibrinogen to fibrin mixture of molecules of 5000-30,000 Daltons ○ Comparison to standard curve: Residual anti- in size thrombin (what was not neutralized by anti- 2. LMWH – low molecular weight heparin – more thrombin) is inversely proportional to patient homogenous mixture of molecules of 4500-5000 antithrombin level Daltons in size ○ Normal range: 80% to 120% • Action: binds to antithrombin (AT) enhancing neutral- • Protein C: Functional and antigenic measurements ization of serine proteases • Functional: Patient plasma mixed with protein 1. UFH – enhances activity against thrombin, FXa C–deficient plasma, aPTT reagent (with an activa- and FIXa tor) and calcium chloride are added, time to clot 2. LMWH – enhances activity against FXa is measured • Monitoring: monitoring needed as individuals respond ○ If functional, APC will inactivate factors Va differently and VIIIa causing prolongation of aPTT result; 1. FH: prolongation of APTT; therapeutic dosage time compared to standard curve to determine 1.5-2.5 times patient’s baseline APTT prior to percent function treatment ○ Normal range: 60% to 150% 2. LMWH: anti-Xa assay (prolongation of APTT not ○ Chromogenic functional assay available predictable) • Antigenic measurements: Immunologic assays TABLE 4-9 Major Hemostasis Laboratory Tests

Test Principle Interpretation Additional Information Assessment of Primary Hemostasis Bleeding time (BT) A small incision is made in the arm, and the time needed for Normal range is 1-9 min. Abnormal bleeding is Test is rarely performed because it is the platelets to stop bleeding is measured in minutes. >9 min. affected by many factors—depth of incision, platelet count, pressure on arm. Platelet function analyzer Platelet function is assessed in whole blood by passing blood A longer closure time in the presence of specific Test requires special instrument. (PFA) through cartridge containing agonists (collagen/ agonists may indicate a platelet disorder. epinephrine and collagen/ADP). The time needed for the platelets to occlude the aperture is measured. Platelet aggregation The ability of platelets to aggregate in the presence of specific Aggregation is measured by a decrease in optical Patients must refrain from aspirin- agonists (ADP, arachidonic acid, collagen, epinephrine, density after addition of an agonist to platelet-rich containing products for 7-10 days ristocetin) is measured using a photooptical instrument. plasma. before testing. Normal or abnormal aggregation in response to Specimen must be tested within 4 hr of agonists is compared to expected results in platelet collection. disorders. Assessment of Secondary Hemostasis Prothrombin time (PT) Clot formation in citrated plasma measured in seconds after Reported with INR normalized ratio PT is also prolonged in the presence of addition of thromboplastin (tissue factor+calcium). INR¼(patient PT/mean normal PT)ISI. deficiency of vitamin K–dependent ISI: International sensitivity index—correction factor factors. from WHO for each lot of reagent. Test is used to monitor oral anticoagulant Prolonged clotting time may be associated with factor therapy. deficiency, extrinsic (VII) or common (X, V, II, I) pathway, presence of inhibitor, or circulating anticoagulant. Activated partial Clot formation in citrated plasma measured in seconds after Prolonged clotting time may be associated with factor Test is used to monitor heparin therapy. thromboplastin time addition of partial thromboplastin reagent and calcium. deficiency, intrinsic (XII, XI, IX, VIII) or common (X, (aPTT) V, II, I) pathway, presence of inhibitor, or circulating

anticoagulant. 4 CHAPTER Fibrinogen (FGN) Clauss-based method: Measurement of time to clot after Inverse relationship. Decreased FGN: DIC, primary and addition of thrombin (bypasses rest of cascade). Longer clotting time¼lower fibrinogen level. secondary fibrinolysis, Patient 1:10 plasma dilution measured against standard curve. dysfibrinogenemia, afibrinogenemia, liver disease Acute phase reactions,

Increased FGN: Hemostasis pregnancy, hormone therapy Thrombin time (TT) Excess of thrombin is added and fibrinogen to fibrin Evaluates formation of fibrin. Measurement is affected in low FGN levels, conversion is measured. dysfibrinogenemia, FDPs presence, heparin therapy. Mixing studies Equal parts patient plasma and normal pooled plasma are Correction of clotting time¼suspected factor Studies are useful to differentiate between mixed and measured using PT or aPTT. deficiency (50% of a factor is needed for normal factor deficiency and circulating PT or APTT result). inhibitor.

Continued 151 152

TABLE 4-9 Major Hemostasis Laboratory Tests—cont’d

Test Principle Interpretation Additional Information HPE 4 CHAPTER Specific factor assays Patient plasma and factor-deficient plasma are mixed and Normal range: 5%-150% Tests are functional-based assays. measured using PT or PTT (depending on factor suspected). Results measured against factor-specific standard curve to determine percentage of factor present.

Factor XIII assay – Urea Clot solubility is measured in the presence of urea or Clot dissolution corresponds to factor XIII level of Symptoms of deficiency are delayed Hemostasis Solubility Test monocholoroacetic acid in 24 hr. 1%-2%. bleeding or increased bruising. Von Willebrand factor Functional test: Platelet aggregation test assessing ability of Fluctuations over time are possible, so testing is often Important to assess functional and (vWF) patient’s platelets to aggregate in the presence of repeated. antigenic levels to accurately diagnose ristocetin; slope of aggregation compared to slopes of Results assessed in conjunction with other and treat. standard curve dilutions; also called ristocetin cofactor measurements: aPTT, vWF multimers, ADAMS-13. assay (RCoF). Antigen test: Levels measured using antibody against vWF; methodologies include ELISA, EIA, immunoturbidometric. Assessment of Fibrinolysis Fibrin(ogen) degradation Patient specimen is collected into special tube containing Agglutination: Positive response Other conditions with positive result are products (FDPs) thrombin and a fibrinolytic inhibitor (prevents in vitro Nonspecific for fibrinogen or FDPs. liver disease, DVT, DIC, kidney disease, fibrinolysis). Patient specimen is mixed with latex beads Confirmatory testing needed to identify FDPs. carcinoma. coated with antibodies for FDPs. D-Dimers Patient plasma is mixed with latex beads coated with a Agglutination: Positive response Elevated in DIC, emboli, thrombi. monoclonal antibody for D-dimers. Positive result indicates that clotting is occurring. Assessment of Inhibitors Bethesda titer (factor Patient plasma and normal pooled plasma are mixed (several Presence if inhibitor will inactivate patient factor, Used to assess inhibitor levels for patients inhibitors) different dilutions) and incubated at 37 C for 2 hr, and resulting in decreased activity. receiving factor concentrates; most factor level is measured. often for patients with severe hemophilia (factors VIII or IX). Inhibitor suspected when patient fails to respond to treatment. Lupus-like anticoagulants Diagnosis based on several tests because no single test is Criteria for LAs: Several other tests may be used to identify, (LAs) diagnostic: 1. Prolongation of phospholipid-dependent but DRVVT is most common. 1. aPTT coagulation reaction LAs in children are often transient. 2. Mix with normal pooled plasma 2. Demonstration that it is an inhibitor and not a factor 3. Dilute Russell viper venom test (DRVVT): In the presence of deficiency lupus anticoagulant, the DRVVT result will be prolonged 3. Demonstration of inhibitor against phospholipid because of phospholipids in the reagent. inhibitor (DRVVT)

ADP, Adenosine diphosphate; DIC, disseminated intravascular coagulation; DVT, deep vein thrombosis; EIA, enzyme immunoassay; ELISA, enzyme-linked immunosorbent assay; INR, international normalized ratio; WHO, World Health Organization. CHAPTER 4 Hemostasis 153

• Protein S: Functional and antigenic measurements 4. Which of the following factors binds to platelets via • Protein S circulates in free and bound forms the glycoprotein IIb/IIIa receptor? • Functional measurement: Free portion measured by a. Von Willebrand factor functional assay—ability of protein S to serve as b. Factor II cofactor for protein S determined c. Fibrinogen ○ Patient plasma is mixed with protein S–deficient d. Thrombin plasma, APC, factor Va, and calcium chloride 5. Which of the following is not an agonist of platelet ○ Time for clot formation is compared to aggregation? standard curve a. Saline • Antigenic measurements: Immunologic assays b. ADP • Activated protein C resistance (APCR): Screening test c. Collagen for factor V Leiden mutation d. Epinephrine • Patient tested in the presence and absence of APC 6. Which enzyme is blocked by the presence of aspirin? ○ Normal patients: Addition of APC will cleave a. Phospholipase A2 and inactivate factors Va and VIIIa b. Cyclooxygenase ○ Affected patients: Clotting time will be shortened c. Protein kinase because factor Va is not cleaved and will not be d. ATPase inactivated 7. Secondary hemostasis occurs when a sufficient stim- ○ Ratio: Partial thromboplastin time (PTT) ulus is present to cause the release of internal ADP, (+APC)/PTT synthesis and release of thromboxane A2, and § Normal greater than 1.0 increased calcium release. § Less than 1.0 suggestive of factor V Leiden a. True defect b. False § Perform confirmatory testing: Molecular 8. Which of the following factors is called testing prothrombin? • Prothrombin 20210 mutation: Molecular testing to a. Fibrinogen identify presence of mutation b. Factor II c. Factor X d. Factor XIII 9. Which of the following factors usually results in no CERTIFICATION PREPARATION QUESTIONS clinical bleeding when deficient? a. Factor XII For answers and rationales, please see Appendix A. b. Factor IX 1. Which of the following lists the steps of the hemo- c. Factor VIII static response in the correct order? d. Factor VII a. Fibrinolysis ! injury ! secondary hemostasis ! 10. The step necessary for the functionary factors II, VII, primary hemostasis IX, and X is called the ______step. b. Injury ! primary hemostasis ! secondary hemo- a. Oxidation stasis ! fibrinolysis b. Hydrolysis c. Injury ! secondary hemostasis ! primary hemo- c. Cleavage stasis ! fibrinolysis d. g-Carboxylation d. Injury ! fibrinolysis ! primary hemostasis ! 11. Monitoring of the intrinsic pathway is accomplished secondary hemostasis by performing which of the following analytical 2. Which of the following properties renders the vessel tests? wall prothrombotic? a. PT a. Negatively charged surface b. PTT b. Production of prostacyclin and nitric oxide c. Thrombin time c. Release of tissue factor d. Fibrinogen assay d. Inactivation of thrombin 12. Monitoring of the extrinsic pathway is accomplished 3. Which of the following is not true regarding by performing which of the following analytical platelets? tests? a. Platelets are not affected by aspirin a. PT b. Platelets have a life span of 7 to 10 days b. PTT c. Platelets undergo shape change and develop pseu- c. Thrombin time dopods when activated d. Fibrinogen assay d. Von Willebrand factor serves as a bridge between 13. Which of the following cleaves the fibrinopeptides platelets and collagen from fibrinogen? 154 CHAPTER 4 Hemostasis

a. Factor VIII 22. Which of the following are fibrin degradation b. Thrombin products? c. Tissue factor a. Fragment X d. Factor XIII b. Fragment Y 14. Activation of factor VII after the release of tissue fac- c. Fragment D tor initiates which of the following pathways? d. All of the above a. Intrinsic pathway 23. Which of the following describes the role of PAI-1 in b. Extrinsic pathway hemostasis? c. Common pathway a. Plasminogen activator inhibitor–1 limits the acti- d. Fibrinolytic pathway vation of plasminogen 15. Which of the following is not true? b. Plasminogen activator inhibitor–1 stimulates the a. Factor VIII is a cofactor for factor IXa activation of plasminogen b. Factor V is a cofactor for factor Xa c. Plasminogen activator inhibitor–1 is involved in c. Protein K is a cofactor for protein C limiting clot formation in vessels d. High-molecular-weight kininogen is a cofactor d. Plasminogen activator inhibitor–1 blocks platelet for factor XIIa binding to the fibrin clot 16. Ifadeficiencyofthisfactorispresent,thecross-linking 24. Which of the following fibrinolytic inhibitors is of fibrin will not occur. useful when a2-antiplasmin activity has been a. Factor II exhausted? b. Factor V a. PAI-1 c. Factor XI b. Thrombin-activatable fibrinolysis inhibitor d. Factor XIII c. a2-Macroglobulin 17. Factor VIII is protected from degradation when cir- d. Plasminogen culating in the plasma by its carrier protein 25. Positive feedback control of the hemostatic response ______. is accomplished by: a. Factor IX a. Fibrin binding to thrombin to limit further b. Thrombin activation c. Von Willebrand factor b. Fibrin degradation products interfere with fibrin d. Glycoprotein IIb/IIIa formation and polymerization 18. Which of the following factors is associated with c. Thrombin activates platelets and promotes the hemophilia B? release of platelet factor V a. Factor VIII d. Thrombin initiates activation of the protein C b. Factor IX pathway c. Factor XI 26. Which of the following descriptions best describes d. Fibrinogen the actions of protein S? 19. The activation of plasmin results in which of the a. Protein S inactivates factors Va and VIIIa following? b. Protein S is involved in the activation of thrombin a. The formation of a fibrin clot c. Protein S serves as a cofactor for protein C b. The formation of the bridge between platelets and d. None of above are functions of protein S the vessel wall 27. A child presents to the pediatrician after having recov- c. The start of the process to break down a fibrin clot ered from a viral infection, because the child now has d. The point at which the intrinsic and extrinsic petechiae. The pediatrician orders laboratory testing, pathways feed into the common pathway and the results reveal that the platelets are decreased. 20. Which of the following proteins is degraded by An increase in lymphocytes and eosinophils is also plasmin? found. What is the probable diagnosis? a. Fibrin a. Acute ITP b. Fibrinogen b. Chronic ITP c. A and B c. NAIT d. None of the above d. Medication reaction 21. Streptokinase differs from urokinase plasminogen 28. Which of the following tests would help to differen- activator (uPA) in that: tiate between Bernard-Soulier syndrome and Glanz- a. Streptokinase activates plasminogen to plasmin mann’s thrombasthenia? conversion, whereas uPA inhibits the conversion a. Bleeding time b. uPA is effective only when given as a medication b. Platelet count c. Streptokinase is an exogenous activator, whereas c. PT uPA is a physiologic activator d. Response to ADP, collagen, and epinephrine in an d. No difference exists between streptokinase and uPA aggregation assay CHAPTER 4 Hemostasis 155

29. Glanzmann’s thrombasthenia is best described as a: a. Protein C deficiency a. Platelet deficiency b. Antithrombin deficiency b. Deficiency of glycoprotein Ib/IX c. Factor V Leiden mutation c. Deficiency of glycoprotein IIb/IIIa d. Factor V deficiency d. Deficiency of dense granules 36. Which of the following accelerates the activity of 30. The lack of a secondary wave of platelet aggregation antithrombin? in response to ADP is associated with which of the a. Coumadin following disorders? b. Aspirin a. Bernard-Soulier syndrome c. Heparin b. Gray platelet syndrome d. tPA c. Glanzmann’s thrombasthenia 37. Which of the following tests is helpful in differentiat- d. D-Storage pool disease ing fibrinogenolysis from DIC? 31. Ayoungboy istakentohis pediatricianbecausehis par- a. PT ents noticed that he seems to bleed easily and has swol- b. aPTT len knees. The following test results were obtained: c. Fibrinogen d. D-Dimer PT¼Normal aPTT¼Prolonged 38. Fibrinogen¼Normal aPTT with normal pooled A patient who has been receiving a broad-spectrum Platelet count¼Normal plasma¼Corrected the aPTT antibiotic is found to have a prolonged PT. After running a couple of factor assays, you conclude that both Which of the following statements best describes the the factor X and factor VII levels are decreased. The next steps? PT corrected when mixed with normal pooled a. The pediatrician should order factor assays for plasma. What is a possible cause? factors VIII and IX a. Inherited factor deficiency b. The pediatrician should order factor assays for b. Circulating anticoagulant factors X and V c. Vitamin K deficiency c. The pediatrician should order platelet aggrega- d. Effect resulting from antibiotic presence in tion testing plasma d. The pediatrician should request a molecular test 39. Which of the following descriptions best describes for the factor V Leiden defect the principle of platelet aggregation? 32. Which of the following results would be expected in a. The decrease in optical density is observed a patient with dysfibrinogenemia? after the addition of an agonist in platelet-poor a. Normal PT, normal aPTT, prolonged plasma thrombin time b. The increase in optical density is observed after b. Abnormal PT, normal aPTT, prolonged the addition of an agonist in platelet-rich plasma thrombin time c. The decrease in optical density is observed c. Abnormal PT, abnormal aPTT, normal after the addition of an agonist in platelet-rich thrombin time plasma d. Normal PT, normal aPTT, normal thrombin time d. The increase in optical density is observed after 33. In factor deficiencies, normal PT and aPTT results the addition of an agonist in platelet-poor plasma may be recorded until a factor level is ______. 40. When performing platelet aggregation assays, which a. Less than 30% of the following is an important preanalytical factor? b. Less than 50% a. The patient should have fasted overnight c. Less than 75% b. The patient must refrain from aspirin-containing d. Less than 100% products for 7 days before testing 34. The following results were obtained from a patient c. After collection, the specimen can be frozen who recently underwent major surgery. before transport to the laboratory PT¼Prolonged APTT¼Prolonged d. All of the above are important Fibrinogen¼Decreased Platelet count¼Decreased 41. Which of the following conditions will cause an D-Dimer¼Positive increase in fibrinogen levels? a. DIC Which of the following conditions is likely? b. Afibrinogenemia a. Fibrinogenolysis c. Acute phase reactions b. Fibrinogen deficiency d. Liver disease c. Disseminated intravascular coagulation 42. Which of the following describes the principle of the d. Vitamin K deficiency thrombin time test? 35. Which of the following conditions is not usually a. After the addition of thromboplastin, the time associated with thrombosis? needed for plasma to form a clot is measured 156 CHAPTER 4 Hemostasis

b. Patient plasma is mixed with thrombin-deficient laboratory in Italy, his PT was 20.2 seconds and his plasma, and the time to clot is measured INR was 1.74. Which of the following descriptions c. An excess of thrombin is added to patient plasma, best describes the situation? and the time to clot is measured a. The patient is taking excessive anticoagulation d. Clot solubility is assessed using 5 M urea medication, causing his PT to be prolonged 43. When performing a factor assay for factor VIII, the b. An error is expected in the results from the labo- MLS accidentally added factor IX–deficient plasma ratory in Italy to the patient specimens. Which of the following best c. The result is not concerning because the INR describes the expected results? results are essentially the same a. The test will not be affected because the correct d. He should immediately return to the United factor-deficient plasma was added States for further assessment b. The test will not be affected because factor- 48. During presurgical testing, the aPTT for a patient deficient plasma is not needed in a factor assay was longer than 120 seconds. The patient’s history c. The test results will not be an assessment of factor for bleeding is negative. The result was corrected VIII levels because factor VIII is present in the fac- after mixing with normal pooled plasma. The sur- tor IX–deficient plasma gery is delayed because the surgeon is concerned d. The test results will be an assessment of factor IX about bleeding. Which of the following descriptions levels and can be calculated using the factor VIII best describes this situation? standard curve a. The patient is at risk for a bleeding incident if the 44. What are the reagents needed to perform the aPTT surgery proceeds test? b. The patient likely has a circulating inhibitor a. Calcium chloride c. The patient likely has a deficiency of a contact b. Partial thromboplastin factor that is not associated with bleeding c. A and B d. The sample was likely contaminated with heparin d. None of the above 49. A 30-year-old woman with a history of miscarriages 45. Which of the following tests is reported in conjunc- is being seen by an obstetrician/gynecologist. Which tion with the INR? of the following tests would be included in a panel to a. PT assess her condition? b. APTT a. DRVVT c. Thrombin time b. PT and PTT d. Fibrinogen assay c. PT and PTT using normal pooled plasma mix 46. The following test results were obtained on a patient d. All of the above who is being seen for easy bruising. 50. A patient presents to the emergency department with Bleeding time¼Increased PT¼Normal symptoms consistent with a DVT. Which of the fol- aPTT¼Prolonged (mix with normal Platelet count¼Normal lowing tests is helpful as a screening test for the factor pooled plasma corrected aPTT) V Leiden mutation? ADP, collagen, epinephrine platelet Ristocetin platelet a. Protein S assay aggregation¼Normal aggregation¼Absent b. Antithrombin assay c. Prothrombin 20210 molecular test Which of the following conditions is expected? d. APCR test a. Hemophilia A b. Hemophilia B BIBLIOGRAPHY c. Von Willebrand’s disease d. Factor XII deficiency Marder VJ, Aird WC, Bennett JS, Schulman S, White GC: Hemosta- 47. During a lengthy overseas trip, a 60-year-old man sis and thrombosis: basic principles and clinical practice, ed 6, went to a laboratory in Italy to have PT measured Philadelphia, 2012, Lippincott Williams & Wilkins. to assess his dosage of Coumadin. Typically, in the McKenzie SB, Williams JL: Clinical laboratory hematology, ed 2, Upper Saddle River, NJ, 2010, Pearson Education. United States, his results are in the range of 17 to Rodak BF, Fritsma GA, Keohane EM: Hematology: clinical princi- 18 seconds, with an average INR of 1.75. In the ples and applications, ed 4, St. Louis, 2012, Saunders. CHAPTER 4 Hemostasis 157

SELF-ASSESSMENT

Content Area: ______

Score on Practice Questions: ______

List the specific topics covered in the missed questions:

List the specific topics covered in the correct questions: 158 CHAPTER 4 Hemostasis

NOTES CHAPTER 5 Clinical Fluid Analysis

Sue King Strasinger

URINALYSIS • Bilirubin: Oxidation to biliverdin • Urobilinogen: Oxidation to urobilin Types of Common Urine Specimens

• Random Urine Volume • Routine screening • May require confirmatory testing based on diet and • Normal: 600 to 2000 mL/day exercise • Oliguria: Decreased output, less than 400 mL/day • First morning • Anuria: No urine output • Collected immediately on arising • Nocturia: Increased urine output at night • Routine screening/confirmatory testing • Polyuria: Increased urine output greater than 2.5 L/day • Orthostatic proteinuria • Diabetes mellitus: Increased urine output to excrete • Pregnancy tests excess urine glucose • Midstream clean catch • Diabetes insipidus: Increased urine output caused • Requires patient to cleanse the genital area by lack of dysfunction of antidiuretic hormone • Void first into the toilet, then collect specimen and (ADH) finish voiding into the toilet • Results in polydipsia • Bacterial cultures • Catheterized • Collected from a catheter passed into the bladder PHYSICAL EXAMINATION OF URINE • Bacterial cultures • NOTE: When a routine urinalysis and a culture are Urine Color both ordered, perform the culture first • 24-Hour (timed) • Normal urine is yellow. Shades of yellow are based on • Patient voids into the toilet and then begins timing fluid consumption and vary from pale yellow (dilute) • Collects all urine during the designated period to dark yellow (concentrated) (Table 5-1) • Voids and collects urine at the end of the period • Specimens can provide quantitative results • Drug screening Urine Clarity • Strictly follow chain-of-custody form requirements • Terminology: Clear, hazy, cloudy, turbid, milky • Preservation of urine specimens • Freshly voided normal urine is clear • Test specimens within 2 hours of collection • Refrigerated normal urine • Refrigerate specimens that cannot be tested within • White turbidity in urine with an alkaline pH from 2 hours amorphous phosphates and carbonates • Pink turbidity in urine with an acid pH from amor- Changes in Unpreserved Urine phous urates • Nonpathologic turbidity • Increased results • Squamous epithelial cells • pH • Mucus • Nitrite • Amorphous phosphates, carbonates, and urates • Bacteria • Semen • Decreased results • Feces • Glucose: Glycolysis • Radiographic contrast media • Ketones: Oxidation • Powder and creams

159 160 CHAPTER 5 Clinical Fluid Analysis

TABLE 5-1 Clinical Correlation of Urine Color COOH COOH COOH COOH COOH COOH Color Cause Correlation Orange Bilirubin Produces yellow foam when POLYELECTROLYTE ON REAGENT STRIP shaken, abnormal liver function + + – + – Pyridium Produces thick orange pigment that – – + + – can interfere with reagent strip + – + – – + tests Red RBCs Cloudy urine, positive test for Ions in urine with Ions in urine with blood, microscopic RBCs low specific gravity high specific gravity Hemoglobin Clear urine, positive test for blood COOH COOH COO–H+ COO–H+ Myoglobin Clear urine, positive test for blood, COO–H+ COO–H+ COO–H+ COO–H+ needs further testing COOH COOH COO–H+ COO–H+ Porphyrins Negative test for blood, needs INITIAL REACTION ON REAGENT STRIP further testing + + Black Oxidized RBCs, Clear urine, positive test for blood 2H - Bromothymol blue 6H - Bromothymol blue denatured hemoglobin Melanin Clear urine, darkens on standing Blue-green alkaline pH Yellow-green acid pH

RBCs, Red blood cells. SECONDARY REACTION ON REAGENT STRIP FIGURE 5-1 Reagent strip specific gravity reaction. (From Strasinger SK, Di Lorenzo MS: Urinalysis and body fluids, ed 5, Philadelphia, 2008, FA Davis). • Pathologic turbidity • Red blood cells (RBCs) • The refractometer uses a prism to direct a wave length • White blood cells (WBCs) of light through the urine; the angle of the light can • Yeast be read on a scale calibrated with distilled water (1.000) • Urothelial and renal tubular epithelial cells Osmolarity • Abnormal crystals • Considered more representative of renal concentrating • Lipids (milky) ability than specific gravity because it measures only the number of particles and their size is not relevant Specific Gravity • Measurement is the number of particles into which 1 g molecular weight of a substance dissociates. Example: • Screening test for renal tubular reabsorption of essen- Nonionizing urea (molecular weight [MW] 60)¼1 tial elements filtered by the glomerulus particle, ionizing NaCl (MW 58.5)¼2 particles • Based on the fact that the glomerular filtrate has a spe- • Reported in milliosmoles (mOsm) cific gravity of 1.010 • Colligative properties measured in the clinical • Comparison of the density of urine to the density of laboratory distilled water (1.000) • Freezing point depression • Urine contains dissolved substances that produce den- ○ One mole of a nonionizing substance will lower sity by their size and number the freezing point 1.86 C Tests ○ Volatile substances such as alcohol can interfere Reagent strip • Vapor pressure depression • Primary test for routine urinalysis is the reagent ○ Actual measurement is the dew point (tempera- strip test ture at which vapor condenses to a liquid) of • Principle is based on the number of hydrogen ions (H+) the urine sample released from a polyelectrolyte (pKa) is proportional to ○ Uses microsamples on filter paper discs. Care the number of ions in the urine must be taken to avoid evaporation • Increased urine concentration¼increased H+ ○ No interference from volatile substances released¼low pH Clinical Significance • The indicator on the strip is bromothymol blue • Normal serum osmolarity: 275 to 300 mOsm • Reaction¼yellow-green (acid) ! green-blue (alka- • Fluid intake influences urine osmolarity line) (Figure 5-1) • Random serum-to-urine ratio is 1:1 Refractometer • Controlled fluid intake should reach 3:1 • Principle: The concentration of dissolved particles in a • Used to determine ADH production or tubular solution determines the velocity and angle of light response to ADH for diabetes insipidus passing through a solution • Harmonic oscillation density CHAPTER 5 Clinical Fluid Analysis 161

• Automated instrumentation passes a sound wave Reagent Strip Sources of Error and through the urine and records the change in frequency TABLE 5-2 Correlations—cont’d of the sound wave, which is proportional to the urine density Test Test Sources of Error Correlations Glucose Oxidizing agents + Ketones CHEMICAL EXAMINATION OF URINE Detergents + Protein Increased ascorbic acid Reagent Strips Low temperature Care and Quality Control Old specimens Ketones Red urine + Glucose • Store in tightly closed opaque bottles Sulfhydryl medications + • Remove immediately before use Levodopa + • Do not refrigerate (room temperature below 30 C) Old specimens • Run positive and negative controls every 24 hours Blood Oxidizing agents + Color • Run controls when a new bottle is opened Bacterial peroxidases + Microscopic • Observe expiration dates Menstrual contamination + • Observe discolored reagent pads Crenated red blood cells Technique Increased ascorbic acid • Thoroughly mix specimens (detection of RBCs Increased nitrite Unmixed specimens and WBCs) Bilirubin Pyridium + Urobilinogen • Warm refrigerated specimens (enzyme reactions) Indican + Color • Briefly dip reagent strips (prevent leaching of reagents Lodine + from strip) Specimen exposed to light • Blot strip while removing from urine (prevent runover) Increased ascorbic acid • Observe manufacturer timing instructions (reaction Increased nitrite color changes) Urobilinogen Multistix Bilirubin • Relate chemical with physical and microscopic results Porphobilinogen + (Table 5-2) Ehrlich’s reactive compounds + Highly pigmented urine + Old specimens KEY POINTS Preservation in formalin Chemstrip pH Highly pigmented urine + Preservation in formalin • The pH of fresh urine does not reach 9.0 Increased nitrates • A reading of 9.0 indicates an old specimen that should Nitrite Old specimens + Protein be recollected; the normal value is 4.5 to 8.0 Highly pigmented urine + Leukocytes • Reagent strip principle: Double indicator (methyl red Non–reductase-containing Microscopic and bromothymol blue) bacteria Early infection No urinary nitrate Increased bacteria converting Reagent Strip Sources of Error and nitrite to nitrogen TABLE 5-2 Correlations Antibiotics Increased ascorbic acid Test High specific gravity Test Sources of Error Correlations Leukocyte Oxidizing agents + Protein pH Runover from adjacent strips Nitrites esterase Formalin + Nitrite Old specimens + Leukocyte Highly pigmented urine + Microscopic esterase Nitrofuration + Microscopic Increased ascorbic acid Protein Highly buffered alkaline urine + Blood High protein and glucose Detergents + Nitrites Antibiotics Pyridium + Leukocyte Inaccurate timing Chlorhexidine + esterase Specific Increased protein + High specific gravity + Microscopic gravity Highly alkaline urine – Microalbuminuria Add 0.005 to any urine with a pH of 6.5 or higher Continued 162 CHAPTER 5 Clinical Fluid Analysis

• Clinical significance • Clinical significance: Diabetes mellitus, monitoring of • Detection of systemic acid-base disorders insulin therapy, starvation, malabsorption, and loss of • Identification of urinary crystals carbohydrates (vomiting)

Protein Blood

• Reagent strips measure primarily albumin • Positive reactions are seen with hematuria, hemoglo- • The normal value is less than 10 mg/dL binuria, and myoglobinuria • A result of 30 mg/dL or greater is considered clinical • Principle: Pseudoperoxidase activity of hemoglobin proteinuria • Clinical significance: Both hemoglobinuria and myo- • Reagent strip principle: The protein error of indicators globinuria can cause acute renal failure • Clinical significance • Hematuria: Bleeding within the genitourinary system, • Clinically significant proteinuria is primarily caused including renal calculi, trauma, anticoagulants, glo- by glomerular or tubular disorders merulonephritis, and pyelonephritis • Benign orthostatic proteinuria testing requires a • Hemoglobinuria: Intravascular hemolysis/transfusion first morning specimen and a specimen after the reactions, lysis of old RBCs by dilute alkaline urine patient has been active for 2 hours. The first speci- • Myoglobinuria: Rhabdomyolysis caused by muscle- men should be negative and the second specimen wasting disorders, crush injuries, prolonged coma, positive and cholesterol statin drugs Microalbuminuria Bilirubin

• Requires a different reagent strip capable of testing for • Both bilirubin and urobilinogen are products of hemo- only albumin at levels below 10 mg/dL globin degradation (Figure 5-2) • Provides early detection of renal disease, particularly • Principle: Diazo reaction in patients with diabetes • Clinical significance: Conjugated bilirubin enters the • The Mulitsix PRO 11 reagent strip tests for microalbu- urine as a result of leakage from a damaged liver or min and creatinine, along with all other routine strip blocked bile duct tests, except urobilinogen • The kidneys cannot filter unconjugated bilirubin • Albumin-to-creatinine ratio corrects for hydration in a • Patients will appear jaundiced random sample to provide an estimate of the 24-hour microalbumin level

Glucose Red blood cells • Principle: Glucose oxidase test (specific for glucose) • The renal threshold for glucose is 160 to 180 mg/dL Hemoglobin • Clinical significance • Diabetes mellitus, gestational diabetes (placental hormones blocking insulin) Protoporphyrin • Hormonal disorders and stress block insulin production and actions Bilirubin Blood (unconjugated) • Renal tubular disorders prevent tubular reabsorption of glucose Bilirubin Liver • Clinitest (conjugated) • Principle: Reducing substances including glucose and other sugars can reduce copper sulfate (blue- Bile duct Blood Kidney green) to cuprous oxide (orange-red) • May be used to test newborn urine for galactose Intestine • High levels will pass through the reaction and go from blue-green to orange-red to blue-green • Carefully observe the reaction Stercobilinogen Urobilinogen

Ketones Urobilin

• Intermediate metabolites of fat, acetoacetic acid, Feces acetone, and b-hydroxybutyric acid FIGURE 5-2 Processing of bilirubin and urobilinogen in • Principle: Reaction of acetoacetic acid or acetone hemoglobin degradation. (From Strasinger SK, Di Lorenzo MS: (with glycine) with sodium nitroprusside/ferricyanide Urinalysis and body fluids, ed 5, Philadelphia, 2008, FA Davis). CHAPTER 5 Clinical Fluid Analysis 163

Urobilinogen MICROSCOPIC EXAMINATION OF URINE • Some of the conjugated bilirubin is converted to urobilinogen in the intestine Casts • Then it circulates in the blood to the liver and passes See (Table 5-3). through the kidneys • A small amount of urobilinogen (1 mg/dL) is found in • Composed of Tamm-Horsfall (uromodulin) protein normal urine excreted by renal tubular epithelial cells • Principle: Multistix¼Ehrlich’s reaction, Chem- • When other urinary constituents are present they strip¼diazo reaction become enmeshed in the cast matrix or attached to • Clinical significance: Early detection of liver disease the matrix and hemolytic disorders, constipation • Formed in the distal convoluted tubule and collecting ducts (wider casts) Nitrite • Reported as the number per low-power field • Detects the presence of reductase-producing bacteria Cells that can convert urinary nitrate to nitrite • Principle: Diazo reaction Red Blood Cells • Clinical significance: Early detection of urinary tract • Appearance infection • Small, nonnucleated discs • A positive nitrite test should be accompanied by a • Appear crenated in concentrated urine positive leukocyte esterase test • Appear as larger empty cells in dilute urine (ghost • May be used to screen specimens for microbiology cells) testing • Regular (dysmorphic) shapes indicate glomerular bleeding Leukocyte Esterase • Sources of error • Oil droplets • Detects the presence of granulocytic WBCs, including • Air bubbles lysed WBCs • Yeast cells (look for budding) • Principle: Diazo reaction; the leukocyte esterase • Clinical significance reagent strip reaction should be read 2 minutes after • Glomerular membrane damage urine exposure • Bleeding in the urinary tract • Clinical significance: Urinary tract infections, includ- • Renal calculi ing with non–reductase-containing bacteria and para- • Malignancy sitic and fungal organisms that would have a negative • Urinalysis correlations nitrite test • A clear red urine with a positive reagent strip RBC • NOTE: Specific gravity is covered in the discussion of and no RBCs in the microscopic analysis indicates physical examination of urine hemoglobinuria or myoglobinuria

TABLE 5-3 Summary of Urinary Casts

Type Appearance Sources of Error Clinical Significance Hyaline Colorless Mucus, fibers, increased light Glomerulonephritis, pyelonephritis, congestive heart failure, stress, exercise RBC Orange/red–containing RBCs RBC clumps Glomerulonephritis, strenuous exercise (look for cast matrix) WBC WBCs in cast matrix WBC clumps Pyelonephritis, acute interstitial nephritis (look for cast matrix) Epithelial cell RTE cells attached to cast matrix WBC casts Renal tubular damage Bacterial Bacteria attached to cast matrix Granular casts Pyelonephritis Granular Coarse or fine granules in the matrix Clumps of small crystals, Glomerulonephritis, pyelonephritis, stress, exercise columnar RTE cells Waxy Highly refractile, jagged edges and Fibers, fecal material Stasis of urine flow, chronic renal failure notches Fatty Fat droplets and oval fat bodies Fecal material Nephrotic syndrome, diabetes mellitus, crush injuries attached to cast matrix Broad Wider than normal Fecal material, fibers Extreme urine stasis, renal failure

RBCs, Red blood cells; RTE, renal tubular endothelial (cells); WBCs, white blood cells. 164 CHAPTER 5 Clinical Fluid Analysis

White Blood Cells • Oval fat bodies • Appearance • Renal tubular epithelial cells that have absorbed • Larger than RBCs and contain a nucleus lipids • Neutrophils have multilobed nuclei and granules • Highly refractile • Eosinophils have red granules when stained with • Seen in conjunction with free-floating lipids Wright or Hansel stains • Confirm by staining with oil red O, Sudan III, or • Glitter cells are neutrophils that have swollen in polarized microscopy dilute urine, resulting in Brownian movement of • Clinical significance: Nephrotic syndrome, diabetes the granules in the cytoplasm mellitus, and crush injuries • Sources of error • Renal tubular epithelial cells, mononuclear lym- Bacteria phocytes, and monocytes • • Urinalysis correlations Small spheres (cocci) and rod-shaped organisms • • Leukocyte esterase, nitrite, pH, and specific gravity Should be accompanied by WBCs • (glitter cells) Sources of error: Amorphous urates, phosphates, and • Clinical significance old specimens with a high pH • • Urinary tract infection (neutrophils) Clinical significance • • Drug-induced interstitial nephritis (eosinophils) Urinary tract infection • Malignancy (mononuclear cells) Yeast Epithelial Cells Squamous Cells • Oval structures with buds or mycelia • Largest cells in the urine sediment • Should be accompanied by WBCs • Represent normal sloughing of old lower genitouri- • Associated with acidic urine from patients with diabe- nary tract cells tes mellitus • Folded squamous cells may resemble urinary casts • Sources of error (look for a centrally located nucleus) • RBCs • Clue cells are squamous epithelial cells covered with • Clinical significance Gardnerella vaginalis bacteria, indicating a vaginal • Diabetes mellitus, immunocompromised patients, infection vaginal infections Transitional (Urothelial) Cells • Found in the renal pelvis, ureters, bladder, and male Parasites urethra • Most common is Trichomonas vaginalis, which • Normally seen after catheterization procedures (often exhibits rapid flagellar movement in wet preparations seen in clumps) • Sources of error • Three different forms: Spherical, caudate, and • WBCs and renal tubular epithelial cells polyhedral • Clinical significance • Spherical cells resemble renal tubular cells, except they • Sexually transmitted disease that is asymptom- have a centrally located nucleus atic in males and causes a vaginal infection in • Increased transitional cells may indicate malignancy females Renal Tubular Epithelial Cells • Other parasites • Found in the renal tubules and collecting duct • Schistosoma haematobium (urine parasite) and • Cell shape varies with location Enterobius vermicularis (fecal contamination) • Convoluted tubule cells are rectangular with coarse granules and may resemble a cast (look for a nucleus) Mucus • Distal convoluted tubule cells are small and round, may resemble spherical transitional cells but have an • Strands of protein secreted by glands and renal tubular eccentric nucleus epithelial cells • Cells from the collecting duct are cuboidal with at least • Major protein is Tamm-Horsfall (uromodulin) protein one straight edge and are frequently seen in clumps • Sources of error • More than two renal tubular epithelial cells per high- • Clumps may resemble hyaline casts (look for the power field is significant consistent shape of a cast) • Clinical significance • Mucus is of no clinical significance • Tubular necrosis, often from poisoning or viral infections Crystals • Renal tubular epithelial cells absorb filtrate and may be bilirubin stained (liver damage), contain hemosid- • Precipitation of urine solutes affected by temperature, erin granules (hemoglobin) or lipids solute concentration, and pH CHAPTER 5 Clinical Fluid Analysis 165

• Crystals are more abundant in refrigerated urine ○ An inherited disorder that inhibits the reabsorp- samples tion of cystine by the renal tubules (cystinuria); • Polarized microscopy aids in their identification renal calculi form at an early age • Abnormal crystals are found only in acidic or • Cholesterol crystals normal urine • Appearance Normal Crystals Seen in Acidic Urine ○ Rectangular plates with notched corners, highly • Uric acid crystals birefringent under polarized light • Appearance • Seen in refrigerated urine and accompanied by fatty ○ Yellow-brown, flat-sided rhombic plates, casts and oval fat bodies wedges, and rosettes • Clinical significance • Sources of error ○ Nephrotic syndrome ○ Cystine crystals (uric acid crystals polarize and • Tyrosine crystals cystine crystals do not polarize) • Appearance • Clinical significance ○ Yellow needle-shaped forms in clusters or rosettes ○ Patients receiving chemotherapy • Clinical significance ○ Lesch-Nyhan disease ○ Severe liver disease • Amorphous urates • Leucine crystals • Appearance • Appearance ○ Small spheres producing brick-dust (uroerythrin) ○ Yellow-brown spheres with concentric circles or yellow-brown sediment • Seen in conjunction with tyrosine crystals • Calcium oxalate crystals • Clinical significance • May also be seen in alkaline urine ○ Severe liver disease • The dihydrate form is envelope shaped; clumps in • Bilirubin crystals fresh urine may indicate renal calculi • Appearance • The monohydrate form is oval or dumbbell shaped; ○ Bright yellow clumped needles and granules presence of this form indicates ethylene glycol (anti- • Clinical significance freeze) ingestion ○ Liver damage often from viral infections that Normal Crystals Seen in Alkaline Urine damage the renal tubules, preventing reabsorp- • Triple phosphate crystals tion of bilirubin • Coffin-lid shaped • Sulfonamide crystals • Associated with a very high pH and bacteria found • Appearance in old specimens ○ Needle, rosette, and rhombic shapes • Amorphous phosphate crystals • Clinical significance • Produce a white precipitate after refrigeration ○ Inadequately hydrated patients taking sulfon- • Calcium carbonate crystals amide medications • Dumbbell and spherical shapes • Ampicillin crystals • Produce gas with acetic acid • Appearance • Ammonium biurate crystals ○ Colorless needles that form clumps after • Yellow-brown thorny apple-shaped crystals refrigeration • Associated with old specimens with bacteria • Clinical significance Abnormal Crystals ○ Inadequately hydrated patients taking ampicillin • Cystine crystals • Appearance Artifacts ○ Hexagonal flat plates • Clinical significance See (Table 5-4).

TABLE 5-4 Artifacts

Artifact Appearance Sources of Error Comments Starch granules Refractile spheres with concentric circles RBCs Negative reagent strip for blood Oil droplets Refractile spheres RBCs Negative reagent strip for blood Air bubbles Refractile spheres RBCs Negative reagent strip for blood Pollen grains Spheres with concentric circles Red blood cells Large size often makes them out of focus Fibers Elongated strips Casts Check with polarized light, because only fatty casts polarize light

RBCs, Red blood cells. 166 CHAPTER 5 Clinical Fluid Analysis

RENAL PHYSIOLOGY • Renal tubular concentration • Takes place in the ascending and descending loops Nephrons of Henle (Figure 5-4) • Renal tubular secretion • Controls the ability of the kidney to clear waste products • Removes nonfiltered waste products from the blood and maintain the body’s water and electrolyte balance to the filtrate and maintains the acid-base balance in • Types of nephrons the body • Cortical nephrons are located in the cortex of the • Substances such as medications are bound to plasma kidney and remove waste products and reabsorb carrier proteins and are too large to be filtered nutrients • In the tubules they disassociate from the carrier pro- • Juxtaglomerular nephrons extend into the medulla tein and are then secreted into the filtrate of the kidney and concentrate the urine • Small hydrogen molecules (H+) are easily filtered and must be returned to the blood. In the filtrate they combine with phosphate ions or ammonia Nephron Functions in the Production secreted by the renal tubular cells and are secreted of Urine back to the blood • Small bicarbonate ions (HCO ) needed for the • Renal blood flow 3 acid-base buffering system are also easily filtered. • Renal artery ! afferent arteriole ! efferent arteri- They combine with the hydrogen molecules, pro- ole ! proximal convoluted tubule capillaries ! ducing bicarbonate (H CO ) that can be secreted vasa recta/loop of Henle ! distal convoluted tubule 2 3 back into the blood capillaries ! renal vein • Normal renal blood flow is approximately 1200 mL/min Renal Function Tests • Normal plasma flow is approximately 600 to • Glomerular filtration rate (GFR) 700 mL/min • Normal value is approximately 120 mL/min, with • Glomerular filtration some variance for body size • Nonselective filtration of plasma substances with • Classic test is creatinine clearance, which requires a MWs less than 70,000 (MW of albumin is 67.000) timed urine specimen (usually 24 hours) • Filtration pressure is controlled by the renin- • Formula angiotensin-aldosterone system (RAAS) (Figure 5-3) UV • Renal tubular reabsorption C ¼ • Passive transport reabsorbs water and urea P • Active transport reabsorbs glucose, amino acids, • Calculated GFR estimates (eGFR) do not require a chloride, and sodium timed urine specimen

Low blood pressure Low plasma sodium

Renin secretion

Angiotensinogen

Angiotensin I Angiotensin converting enzymes Angiotensin II

Vasoconstriction Proximal convoluted tubule Aldosterone ADH Sodium reabsorption

Distal convoluted tubule Collecting duct Sodium reabsorption Water reabsorption

FIGURE 5-3 The renin-angiotensin-aldosterone system. ADH, Antidiuretic hormone. (From Strasinger SK, Di Lorenzo MS: Urinalysis and body fluids, ed 5, Philadelphia, 2008, FA Davis). CHAPTER 5 Clinical Fluid Analysis 167

Aldosterone-controlled Na+ reabsorption

H2O

Cortex Glucose, Na+ H2O amino acids ADH-controlled H2O reabsorption 300 mOsm

300 mOsm

600 mOsm + Medulla H2O Na 900 mOsm

Cl– H2O

1200 mOsm FIGURE 5-4 Renal concentration. ADH, Antidiuretic hormone. (From Strasinger SK, Di Lorenzo MS: Urinalysis and body fluids, ed 5, Philadelphia, 2008, FA Davis).

• Formulas may use serum creatinine and combina- • The free water clearance test measures the ability of tions of age, sex, ethnicity, blood urea nitrogen the kidney to respond to body hydration (BUN), and serum albumin • Formula • Measurement of serum markers U V • Cystatin C is a small molecule produced at a con- C ¼ osm osm P stant rate by all nucleated cells osm • It is completely filtered, reabsorbed, and then bro- • Tubular secretion tests ken down by the renal tubular cells. Serum level • Titratable acidity detects the inability of the proxi- remains constant unless the GFR decreases causing mal convoluted tubule to secrete hydrogen the serum level to rise molecules • Renal tubular reabsorption • Urinary ammonia detects the inability to produce • Primary tests are serum and urine osmolarity (see ammonia in the proximal and distal convoluted discussion of specific gravity) tubules (Table 5-5)

TABLE 5-5 Renal Disease and Urinalysis Correlations

Disorder Urinalysis Results Comments Glomerular Disorders Acute glomerulonephritis Macroscopic hematuria, Poststreptococcal infection proteinuria, RBC casts Goodpasture’s syndrome Macroscopic hematuria, proteinuria, RBC casts Anti–glomerular basement membrane antibodies Wegner’s granulomatosis Macroscopic hematuria, proteinuria, RBC casts Antineutrophil cytoplasmic antibody Henoch-Scho¨ nlein purpura Macroscopic hematuria, proteinuria, RBC casts Primarily seen in children after respiratory infections Membranous glomerulonephritis Microscopic hematuria, proteinuria Autoimmune disorders Chronic glomerulonephritis Hematuria, proteinuria, glycosuria, cellular, waxy Progression of previous disorders and broad casts IgA nephropathy Hematuria, proteinuria, glycosuria, cellular, waxy, Immune IgA complexes deposited on the glomerular broad casts membrane Nephrotic syndrome Heavy proteinuria, hematuria, RTE cells, oval fat Circulatory disruption decreasing blood flow to the bodies, fatty and waxy casts kidney, increased serum lipids Minimal change disease Heavy proteinuria, light hematuria fat droplets Seen in children after allergic reactions, heavy edema, good prognosis

Continued 168 CHAPTER 5 Clinical Fluid Analysis

TABLE 5-5 Renal Disease and Urinalysis Correlations—cont’d

Disorder Urinalysis Results Comments Focal segmental Hematuria, proteinuria Drugs of abuse and HIV glomerulosclerosis Tubular Disorders Acute tubular necrosis RTE cells and casts, positive blood Hemoglobinuria, myoglobinuria, and reagent strip antibiotics Fanconi’s syndrome Glycosuria, proteinuria General failure of tubular reabsorption Renal glycosuria Glucose Failure of active transport of only glucose Tubulointerstitial Disorders Cystitis Positive LE, nitrite, elevated pH, WBCs, bacteria Bladder infection Acute pyelonephritis Positive LE, nitrite, and WBC casts Tubular infection indicated by WBC casts Chronic pyelonephritis Positive LE, nitrite, WBCs, granular, waxy and broad Structural abnormalities that affect normal tubular casts emptying, often in children Acute interstitial nephritis Hematuria, proteinuria, and WBC casts Urine eosinophils, no bacteria, reaction to toxic medications Renal lithiasis (calculi) Microscopic RBCs, high specific gravity Patient with severe back pain; pH varies with type of calculi

HIV, Human immune deficiency virus; IgA, immunoglobulin A; LE, leukocyte esterase; RBC, red blood cell; WBC, white blood cell.

BODY FLUIDS Cerebrospinal Fluid Appearance TABLE 5-6 and Clinical Significance Cerebrospinal Fluid Appearance Cause Clinical Significance Physiology • Cerebrospinal fluid (CSF) is formed in the choroid Crystal clear Normal Hazy, cloudy WBCs Meningitis plexuses Microorganisms Blood-brain barrier disorders • Circulates through the arachnoid space of the menin- Protein IgG production in the CNS ges lining the brain and spinal cord and is reabsorbed Bloody RBCs Hemorrhage in the arachnoid granules of the brain Traumatic tap • The tight-fitting endothelial cells in the choroid plex- Xanthochromia Hemoglobin Old hemorrhage uses make the blood-brain barrier that protects the Bilirubin Elevated serum bilirubin brain from toxic substances Protein Blood-brain barrier disorders Collection and Distribution of Cerebrospinal Carotene Elevated serum levels Fluid Specimens Pellicle Clotting factors Refrigerated tuberculosis meningitis specimens • CSF is collected in three or four sterile tubes numbered in the order of collection CNS, Central nervous system; IgG, immunoglobulin G; RBCs, red blood cells; • Three tube distribution WBCs, white blood cells. • Tube 1: Chemistry/immunology—least affected by blood or bacteria contamination from the spinal tap Hematology Tests • Tube 2: Microbiology • Tube 3: Hematology—less contamination of cell Cell Counts count from the puncture • Perform counts immediately because RBCs and gran- • Four tube distribution ulocytes lyse within 1 hour • Tube 1: Hematology—provides a comparison • The adult normal CSF WBC count is 0 to 5/mL between the cells from the puncture with the cells • Newborns and children may have slightly higher in the fourth tube collected counts and more monocytes • Tube 2: Chemistry • Cell counts are performed using a Neubauer counting • Tube 3: Microbiology—less chance of outside chamber contamination • Formula • Tube 4: Hematology—results compared with those of tube 1 for possible outside interference from the Number of cells counted dilution ¼ cells=mL tap (Table 5-6) Number of squares counted volume of 1 square CHAPTER 5 Clinical Fluid Analysis 169

TABLE 5-7 Appearance and Significance of Cerebrospinal Fluid Cells

Cell Clinical Significance Appearance Lymphocytes Normal, viral, tubercular, fungal meningitis, multiple sclerosis All developmental stages Neutrophils Bacterial meningitis, early stages of other types of meningitis, Granules may be less prominent cerebral hemorrhage Monocytes The same as lymphocytes Seen mixed with lymphocytes Macrophages RBCs in the CSF May contain phagocytized RBCs, hemosiderin granules, hematoidin crystals Blast forms Acute leukemias Myleoblasts, lymphoblasts, monoblasts Plasma cells Multiple sclerosis Traditional forms Ependymal, choroidal, Diagnostic procedures Seen in clusters with distinct nuclei and cell walls spindle-shaped cell Malignant cells Metastatic cancers, primary CNS carcinomas Seen in clusters with fusing nuclei and cell walls

CSF, Cerebrospinal fluid; RBCs, red blood cells.

• WBC counts require lysis of RBCs using percent gla- • Detection of the t transferrin protein identifies a cial acetic acid fluid as CSF • Differential • Myelin basic protein ○ Concentration by cytocentrifugation is recom- • The presence of myelin basic protein indicates mended to ensure an adequate number of cells destruction of the myelin sheath that protects the for evaluation (Table 5-7) axons of the neurons • Monitors the course of multiple sclerosis Chemistry Tests • CSF glucose • Normal values: 60% to 70% of the blood glucose • Normal values differ from plasma values because of • Compare with a blood glucose drawn approxi- the selective filtration by the blood-brain barrier mately 2 hours before the spinal tap Cerebrospinal Fluid Protein • Elevated CSF levels occur when the serum level is elevated • Normal value: 15 to 45 mg/dL (plasma protein is mea- • Clinical significance sured in grams per deciliter) ○ Helpful in the differentiation of types of meningi- • The t transferrin protein is found only in CSF tis. Decreased levels indicate damage to transport • Clinical significance of glucose across the blood-brain barrier and • Elevated results: Damage to the blood-brain barrier increased use of glucose by the brain cells (meningitis, tumors, hemorrhage), multiple sclero- • CSF lactate sis (increased immunoglobulin G [IgG] produced • Increased by tissue destruction caused by lack of in the central nervous system [CNS]) oxygen • Decreased results: CSF leakage and trauma • Normal value: Less than 25 mg/dL • CSF serum/albumin index • Clinical significance ○ Detects damage to the blood-brain barrier ○ Aids in the differentiation of meningitis ○ An index value less than 9 is normal organisms ○ Formula • CSF glutamine CSF albuminðÞ mg=dL • The normal CSF value is 8 to 18 mg/dL CSF=serum albumin index ¼ Serum albuminðÞ g=dL • Increased glutamine indicates increased ammonia in the CNS • CSF IgG index • Values greater than 35 mg/dL disrupt consciousness ○ Detects production of IgG within the CNS • Elevated values are seen in children with Reye’s syn- ○ Normal values are approximately 0.70 or lower drome that affects the liver (Table 5-8) ○ Formula CSF IgGðÞ mg=dL =serum IgGðÞ g=dL IgG index ¼ CSF albuminðÞ mg=dL =serum albuminðÞ g=dL Microbiology Tests • CSF electrophoresis • Gram stains are routinely performed on concentrated • Detects oligoclonal bands that represent inflamma- specimens tion in the CNS • Cryptococcus neoformans produces a starburst Gram • Primary testing is for multiple sclerosis. Two or more stain pattern. India ink preparations and latex aggluti- bands are seen in the CSF and no bands in the serum; nation tests may be performed Other disorders have banding in the serum • Immunologic tests are available for bacterial antigens 170 CHAPTER 5 Clinical Fluid Analysis

TABLE 5-8 Differential Diagnosis of Meningitis

Bacterial Viral Tubercular Fungal Elevated WBCs Elevated WBCs Elevated WBCs Elevated WBCs Neutrophils Lymphocytes Lymphocytes and monocytes Lymphocytes and monocytes Markedly decreased glucose Normal glucose Decreased glucose Normal to decreased glucose Marked protein elevation Moderate protein elevation Moderate-to-marked protein elevation Moderate-to-marked protein elevation Lactate >35 mg/dL Lactate <25 mg/dL Lactate >25 mg/dL Lactate >25 mg/dL

WBCs, White blood cells.

SEMINAL FLUID • pH • Normal: 7.2 to 8.0 Physiology • Increased pH indicates infection • Decreased pH indicates increased prostatic fluid • Semen consists of four components • Sperm concentration and count • Spermatozoa: Germ cells • Sperm concentration • Seminal fluid: Provides nutrients and fructose for ○ Normal: Greater than 20 million/mL sperm energy ○ Test is performed in a counting chamber • Prostate fluid: Provides substances for coagulation ○ Fluid must be diluted with specified diluting fluid and liquifaction to immobilize the sperm • Bulbourethral gland fluid: Provides alkaline mucus to ○ Counts must be corrected from microliters to neutralize the acidity of prostate fluid and the vagina milliliters ○ Spermatids and WBCs are counted separately; Specimen Collection more than 1 million spermatids indicates dis- rupted spermatogenesis, more than 1 million • The majority of the sperm are in the first part of the WBCs indicates infection ejaculate, so a complete collection is essential • Sperm count • Collect after 2 to 3 days but not more than 5 days of ○ The number of sperm per milliliter times the abstinance specimen volume¼the sperm count • Collect in warm glass or plastic containers (no routine • Sperm motility condoms) ○ Evaluated by the speed and direction of the • Keep the specimen at room temperature and deliver to sperm movement the laboratory within 1 hour ○ Criteria: Rapid, straight line; slower speed, some • Record the time of collecton and delivery at the lateral movement; slow forward movement, laboratory noticeable lateral movement; no forward move- Semen Analysis ment; no movement • Sperm morphology • Appearance ○ Head: Contains the acrosome with enzymes for • Normal: Gray-white, translucent ovum penetration; double and irregularly shaped • White turbidity: WBCs, infection heads interfere with penetration • Red: RBCs ○ Neck piece: Attaches the head to the midpiece; a • Yellow: Urine contamination (urine is toxic to neckpiece too long causes a bent head that inter- sperm) feres with motility • Liquifaction ○ Midpiece: Contains mitochondria to provide • Fresh semen is clotted energy for the tail • Normal liquifacton takes place within 30 to ○ Tail: Acts as a flagella to propel the sperm, 60 minutes double and coiled tails interfere with motility • Abnormal liquifaction indictes a deficiency of pros- ○ Normal values: Greater than 30% normal sperm tate enzymes using routine criteria and greater than 14% using • Volume strict criteria • Normal: 2 to 5 mL • Sperm viability • Viscosity ○ Stain smears with eosin-nigrosin, nonviable • Normal: Ability to form discrete droplets from a sperm absorb the stain and appear red pipette ○ Viable sperm do not stain • Increased viscosity and delayed liquifaction impede ○ Normal value: 75% normal sperm per 100 cells sperm motility counted CHAPTER 5 Clinical Fluid Analysis 171

Postvasectomy Semen Analysis • Primary cells are monocytes and macrophages, followed by neutrophils at less than 25% and lympho- • Carefully evaluate a wet preparation for viable and cytes at less than 15% and occasional synoviocytes nonviable sperm • Crystal identification • Report any sperm seen • Primary crystals are monosodium, urate seen in • Negative specimens may be centrifuged and reexamined gout, and calcium pyrophosphate dihydrate, seen SYNOVIAL FLUID in pseudogout • Identify using polarized and compensated polarized Physiology light • Monosodium urate crystals • Synovial fluid is located in the cavities between the ○ Needle-shaped, strongly birefringent under moveable joints polarized light • Synoviocytes secrete hyaluronic acid, a large molecule ○ Yellow when aligned with the slow vibration of that produces the viscosity of the fluid compensated polarized light and blue when ver- • Damage to the joints produces arthritis (Table 5-9) tical to the slow vibration ○ Elevated serum uric acid levels aid in the Laboratory Testing identification • Calcium pyrophosphate dihydrate crystals • Color ○ Rhombic, often seen intracellularly in neutrophil • Normal is colorless to pale yellow vacuoles (phagocytized monosodium urate crys- • Viscosity tals puncture the cell membrane) • The mucin clot test detects and measures ○ Blue when aligned with the slow vibration of hyaluronic acid compensated polarized light and yellow when • Addition of acetic acid to normal synovial fluid will vertical to the slow vibration form a firm mucin clot surrounded by clear liquid; clots become less firm as the viscosity decreases Microbiology • Addition of acetic acid to a questionable fluid iden- • Gram stains and cultures are routinely run on synovial tifies it as synovial fluid if a clot forms fluid specimens • Cell counts • Cultures require enriched agar (chocolate) for detection • Do not use normal WBC diluting fluid (acetic acid), of possible Haemophilus sp. and Neisseria gonorrhea use normal saline • Normal WBC count is less than 200 cells/mL • Differential SEROUS FLUID • Incubate fluid with hyaluronidase before slide preparation Physiology • Serous fluid is located between the parietal and visceral TABLE 5-9 Classification of Arthritis membranes that line the closed body cavities, mesothelial cells line the membranes Class Pathology Laboratory Results • Cavities: Pleural, pericardial, and peritoneal • Serous fluid is normally produced and reabsorbed at a Noninflammatory Osteoarthritis Clear, yellow, good viscosity constant rate; disruption of this process produces an WBCs <1000/mL, neutrophils <30% effusion Inflammatory/ Autoimmune Cloudy, yellow, poor Transudates and Exudates. (See Table 5-10) immunologic disorders viscosity • Effusions caused by systemic disorders are transudates WBCs 2000 to 75,000/mL, • Effusions caused by membrane disorders are exudates neutrophils >50% Inflammatory/ Gout, Clear or milky, low viscosity, Differentiation Between crystals pseudogout WBCs up to 100,000/mL, neutrophils <75% TABLE 5-10 Transudates and Exudates Septic Infection Cloudy, yellow-green, low viscosity, Test Transudates Exudates WBCs 50,000-100,000/mL, Appearance Clear Cloudy neutrophils >75% Fluid-to–serum protein ratio <0.5 >0.5 Hemorrhagic Trauma, Cloudy, red, low viscosity, Fluid-to–serum LD ratio <0.6 >0.6 coagulation WBCs and neutrophils equal White blood cell count <1000/mL >1000/mL disorders to blood values Spontaneous clotting No Yes

WBCs, White blood cells. LD, Lactate dehydrogenase. 172 CHAPTER 5 Clinical Fluid Analysis

Pleural Fluid Transudates and Exudates • The serum-ascites albumin gradient is the recom- • Collected by thorocentesis mended method for differentation • Appearance • Measure serum and ascites albumin levels • Milky: Thoracic duct leakage (chylous effusion), • Serum albumin fluid albumin¼1.1 or higher is a chronic infection (pseudochylous effusion) transudate • Bloody: Hemothorax, hemorrhagic effusion (embo- • Serum albumin fluid albumin¼1.1 or lower is an lus, tuberculosis, malignancy) exudate • Viscous: Malignant mesothelioma producing hya- • Appearance luronic acid • Turbid: Infection • Hematology: Differential count • Green: Gallbladder or pancreas disorder • Neutrophils: Pneumonia, pancreatitis • Blood-streaked: Trauma, infection, malignancy • Lymphocytes: Tuberculosis, viral infections • Milky: Lymphatic trauma or blockage • Eosinophils: Pneumothorax • Hematology • Mesothelial cells: Normal, decreased with • Normal WBC count: Less than 350/mL tuberculosis • Absolute neutrophil count distinguishes between • Plasma cells: Tuberculosis cirrhosis and peritonitis • Malignant cells: Small cell and adenocarcinoma, • More than 250 neutrophils/mL or 50% of the differ- metastatic carcinoma cells ential indicates peritonitis • Chemistry • Differential count • pH less than 7.0 indicates the need for chest tube • Additional cells seen include abundant mesothelial drainage cells, lipophages, yeast, Toxoplasma gondii, and • pH less than 6.0 indicates esophageal rupture malignant colon, prostate, and ovarian cells • Pleural fluid cholesterol: equal to or less than 45 to • Chemistry 60 mg/dL (transudate), higher (exudate) • Glucose: Decreased in infection and malignancy • Pleural fluid: Serum cholesterol ratio: Less than 0.3 • Amylase: Elevated in pancreatitis and gastrointesti- (transudate), higher (exudate) nal (GI) perforations • Pleural fluid: Serum bilirubin ratio: Less than 0.6 • Alkaline phosphatase: Elevated in intestinal (transudate), higher (exudate) perforation • Microbiology • BUN and creatinine: Bladder rupture or puncture • Acid-fast stains • Tumor markers: Carcinoembryonic antigen and CA125 • Microbiology Pericardial Fluid • Gram stains and cultures for both aerobic and anaerobic organisms • Collected by pericardiocentesis • Blood cultures aid in the detection of anaerobic • Appearance organisms • Cloudy, blood-streaked: Infection, malignancy • Grossly bloody: Cardiac puncture, anticoagulant medications • Hematology AMNIOTIC FLUID • Increased neutrophils are seen in bacterial endocarditis Specimens • Refer metastatic malignant cells for cytologic examination • Collected by amniocentesis • Microbiology • Hemolytic disease of the newborn (HDN) specimens • Gram stains and cultures are performed on concen- must be protected from light trated specimens • Specimens for fetal lung maturity (FLM) are delivered • Acid-fast stains for tuberculosis are associated with on ice and refrigerated or frozen acquired immunodeficiency syndrome • Specimens for cytogenic testing are kept at room temperature and delivered immediately • Appearance Peritoneal Fluid • Colorless: Normal • Blood-streaked: Trauma, traumatic tap • Often called ascitic fluid, effusion is ascites • Yellow: Bilirubin, HDN • Effusions are caused by liver disorders (cirrhosis), • Dark green: Meconium intestinal infection (peritonitis), and malignancy • Dark red-brown: Fetal death CHAPTER 5 Clinical Fluid Analysis 173

Tests for Fetal Distress FECAL ANALYSIS • HDN: Spectrophotometric analysis at optical densities between 365 and 550 nm (Figure 5-5) Physiology • Neural tube defects • Final digestion and reabsorption of food takes place in • Detected first by increased maternal serum a- the small intestine with the aid of enzymes from the fetoprotein pancreas and bile salts from the liver • Amniotic fluid levels are measured first between • The large intestine receives the digestive products and gestational weeks 12 to 15 and compared to mater- water from the digestive process nal serum levels • Much of the water is reabsorbed • Report both serum and fluid levels in multiples of • Additonal water results in diarrhea, and too little the mean (MOM) water produces constipation • Abnormal value: Two times the laboratory median • Types of diarrhea value in both serum and fluid • Secretory caused primarily by bacterial, viral, or • Positive tests are followed by measuring amniotic protozoan infection; fecal WBCs are seen in the acetylcholinesterase stool • Osmotic caused by incomplete breakdown or reabsorption in the small intestine and retention of Tests for Fetal Maturity water in the large intestine

• Respiratory distress syndrome is caused by decreased lung surfactant • Primary lung surfactants are lecithin, sphingomyelin, Laboratory Tests and phosphatidylglycerol • Aminostat-FLM • Color and appearance • Immunologic agglutination test for phosphatidyl- • Normal: Color is brown, caused by urobilin glycerol • Black: Upper GI bleeding, iron therapy, and • Not affected by meconium and blood contamina- antacids tion • Red: Lower GI bleeding, food coloring • Lamellar bodies • Pale yellow/white: Bile duct obstruction, barium • The storage form of phospholipids in the lungs. tests Secreted by the type II pneumocytes and enter the • Bulky/frothy: Bile duct obstruction, steatorrrhea amniotic fluid at 26 weeks of gestation • Blood-streaked mucus: Colitis, dysentry, malignancy • Fluid can be run through the platelet counting chan- • Microscopic tests nel of automated cell counters • Fecal leukocytes: Detects invasive non–toxin- • Normal values are 32,000 to 35,400 based on the producing microorganisms instrument • Muscle fibers: Detects undigested striated fibers • An optical density measurement of the fluid at seen with pancreatic insufficiency 650 nm of 0.150 is also normal • Only undigested fibers are counted; fiber classification ○ Digested fibers: No visible striations ○ Partially digested fibers: Striations are in only 0.6 Oxyhemoglobin Bilirubin peak at 410 peak at 450 one direction 0.5 ○ Undigested fibers: Vertical and horizontal fibers visible 0.4 • Qualitative fecal fats: Monitoring of malabsorption 0.3 Normal disorders scan • Neutral fats (triglycerides) stain easily with Sudan Absorbance 0.2 III in alcohol 0.1 • Soaps, fatty acids and cholesterol require application of heat to the slide • Chemical tests 365 410 450 500 550 • Occult blood: Screening for colorectal cancer Wavelength (nm) ○ Classic test (fecal occult blood test [FOBT]) FIGURE 5-5 Spectrophotometric scan of fetal bilirubin. (From uses the pseudoperoxidase activity of hemo- Strasinger SK, Di Lorenzo MS: Urinalysis and body fluids, ed 5, globin with gum guiac as the indicator Philadelphia, 2008, FA Davis). (Figure 5-6) 174 CHAPTER 5 Clinical Fluid Analysis

Hemoglobin H2O2 Guaiac Oxidized guaiac + H2O 2. In an unpreserved urine specimen left at room temperature overnight, which of the following will have Peroxidase (Blue color) increased? FIGURE 5-6 Measurement of hemoglobin using pseudoperoxidase and guiac. a. Bacteria and nitrite b. Specific gravity and bilirubin c. Glucose and ketones d. Urobilinogen and protein 3. A first morning specimen would be requested to con- • Patients must follow dietary instructions avoiding firm which of the following? meat, fish, vegetables that contain pseudoperoxi- a. Diabetes insipidus dase, aspirin, and vitamin C b. Fanconi’s syndrome • Immunologic FOBTs (iFOBTs) use antihuman c. Urinary tract infection hemoglobin antibodies that are specific for the glo- d. Orthostatic proteinuria bin portion of hemoglobin 4. Failure to collect the last specimen of a timed urine • Advantages: Do not require dietary and medication collection will: restrictions. Detects lower GI bleeding, which is a. Cause falsely increased results more indicative of colorectal cancer. Blood from b. Affect the preservation of glucose upper GI bleeding degrades before reaching the c. Cause falsely decreased results large intestine d. Adversely affect reagent strip results • Procedure: Stool samples should be taken from the 5. Which of the following is the principle of the reagent center of the stool to avoid contamination. Two strip test for pH? samples from negative samples from three different a. A double indicator reaction stools confirms a negative b. The protein error of indicators • Quantitative fecal fats c. The diazo reaction • Confirmatory test for fecal fats d. A dye-binding reaction • Requires collection of a timed 3-day specimen and 6. Which of the following best describes the chemical maintenance of a 100 g/day fat diet principle of the protein reagent strip? • Testing methods: Titration, acid steatocrit test, and a. Protein reacts with an immunocomplex on the pad near-infrared reflectance spectrophotometry b. Protein causes a pH change on the reagent strip pad • Fecal enzymes c. Protein accepts hydrogen ions from an indicator • Elastase I is pancreatic-specific enzyme measured dye by immunoassay using monoclonal antibodies d. Protein causes protons to be released from a against human pancreatic elastase I polyelectrolyte • This differentiates pancreatic steatorrhea from non- 7. Which of the following is the principle of the reagent pancreatic causes of steatorrhea strip test for glucose? • Fecal carbohydrates a. A double sequential enzyme reaction • Increased fecal carbohydrates are caused by failure b. Copper reduction to reabsorb carbohydrates (celiac disease) or lack of c. The peroxidase activity of glucose digestive enzymes (lactose intolerance) d. Buffered reactions of mixed enzyme indicators • Clinitest is the common screening test, followed by 8. Glucosuria not accompanied by hyperglycemia can carbohydrate intolerance tests be seen with which of the following? • Measuring stool pH can be used to test infant diar- a. Hormonal disorders rhea. Normal stool pH is 7 to 8, pH levels below 5.5 b. Gestational diabetes indicate carbohydrate disorders c. Diabetes mellitus d. Renal disease 9. Which of the following will cause ketonuria? a. Ability to use carbohydrates CERTIFICATION PREPARATION QUESTIONS b. Adequate intake of carbohydrates c. Decreased metabolism of carbohydrates For answers and rationales, please see Appendix A. d. Excessive loss of carbohydrates 1. The specific gravity of the glomerular ultrafiltrate is 10. Reagent strip reactions for blood are based on which ______. of the following? a. 1.000 a. Pseudoperoxidase activity of hemoglobin b. 1.010 b. Oxidation of hemoglobin peroxidase c. 1.025 c. Reaction of hemoglobin with bromothymol blue d. 1.040 d. Reduction of a chromogen by hemoglobin CHAPTER 5 Clinical Fluid Analysis 175

11. Myoglobinuria may be caused by which of the c. Acid pH and a positive glucose following? d. Alkaline pH and a positive protein a. Decreased glomerular filtration 17. The presence of dysmorphic red blood cells in the b. Incompatible blood transfusions urine sediment is indicative of which of the following? c. Strenuous exercise a. A coagulation disorder d. Biliary obstruction b. Menstrual contamination 12. A patient with severe back pain comes to the emer- c. Urinary tract infection gency department. A urine specimen has a 1+ reagent d. Glomerular bleeding strip reading for blood and a specific gravity of 18. The location of epithelial cells in the urinary tract in 1.030. This can aid in confirming a diagnosis of descending order is: ______. a. Squamous, transitional, renal tubular a. Pyelonephritis b. Transitional, renal tubular, squamous b. Appendicitis c. Renal tubular, transitional, squamous c. Renal calculi d. Squamous, renal tubular, urothelial d. Multiple myeloma 19. Urinary casts are formed in which of the 13. When a reagent strip is positive for bilirubin, it can be following? assumed that the bilirubin: a. Distal tubules and collecting ducts a. Is conjugated b. Distal tubules and loops of Henle b. Has passed through the small intestine c. Proximal and distal tubules c. Is attached to protein d. Proximal tubules and loops of Henle d. Is unconjugated 20. These crystals were seen in the urine of a child who 14. Which of the following results would be seen in urine had ingested antifreeze. They are: from a patient with autoimmune hemolytic anemia? a. Bilirubin¼negative, urobilinogen¼negative b. Bilirubin ¼positive, urobilinogen¼positive c. Bilirubin ¼positive, urobilinogen¼negative d. Bilirubin ¼negative, urobilinogen¼positive 15. Which of the following is the principle of the reagent strip test for specific gravity? a. Disassociation of the indicator bromothymol blue, producing a pH change b. Ionization of a polyelectrolyte, producing a pH change detected by bromothymol blue c. Disassociation of polyelectrolyte, producing a pH change detected by bromothymol blue d. Change in the pK of bromothymol blue to produce a pH change 16. These constituents are primarily seen in urine with an: FIGURE 5-8 (From Strasinger SK, Di Lorenzo MS: Urinalysis and body fluids, ed 5, Philadelphia, 2008, FA Davis.)

a. Triple phosphate b. Calcium oxalate dihydrate c. Calcium oxalate monohydrate d. Calcium phosphate 21. The test for which of the following results should be repeated? a. Positive blood and protein b. pH 7.0 with ammonium biurate crystals c. Positive nitrite and leukocyte esterase d. pH 5.0, WBCs, and triple phosphate crystals 22. Anti–glomerular basement antibody is seen with: a. Wegener’s granulomatosis b. IgA nephropathy FIGURE 5-7 (From Strasinger SK, Di Lorenzo MS: Urinalysis and c. Goodpasture’s syndrome body fluids, ed 4, Philadelphia, 2001, FA Davis.) d. Diabetic nephropathy 23. The most common composition of renal calculi is: a. Acid pH and a positive protein a. Calcium oxalate b. Alkaline pH and bacteria b. Magnesium ammonium phosphate 176 CHAPTER 5 Clinical Fluid Analysis

c. Cystine b. A traumatic or bloody tap and in all likelihood of d. Uric acid no pathogenic significance 24. Pyelonephritis can be differentiated from cystitis by c. The pathologic presence of RBCs and reported to the presence of ______. your supervisor immediately a. Eosinophils d. A pathologic presence of RBCs, but because the b. Hyaline casts RBC morphology is normal, the importance is c. White blood cell casts minimal d. Bacteriuria 31. An IgG index greater than 0.80 is indicative of which 25. Children develop a form of nephrotic syndrome called: of the following? a. IgA nephropathy a. Synthesis of IgG within the CNS b. Henoch-Scho¨ nlein purpura b. Alterations in the blood-brain barrier c. Minimal change disease c. Active demyelination of neural tissue d. Acute glomerulonephritis d. Increased reabsorption of IgG from the 26. Focal segmental glomerular nephritis is associated peripheral blood with which of the following? 32. Which of the following can decrease CSF protein? a. Untreated streptococcal infections a. Fluid leakage b. Heroin abuse b. Meningitis c. Diabetes mellitus c. Multiple sclerosis d. Autoimmune disorders d. Hemorrhage 27. Which of the following would be most characteristic 33. CSF lactate is used to verify cases of which of the of chronic glomerulonephritis versus acute glomeru- following? lar nephritis? a. Multiple sclerosis a. Red blood cells and red blood cell casts b. Bacterial meningitis b. Hyaline casts and mucus c. Reye’s syndrome c. Waxy and broad casts d. Tertiary syphilis d. Proteinuria 34. Which of the following can be used to identify a fluid 28. Which of the following results is not consistent with as CSF? cystitis? a. Oligoclonal bands Color: Yellow Protein: 1+ Blood: Trace b. Xanthochromia Clarity: Hazy Glucose: Negative Urobilinogen: c. Transferrin t protein 1.0 EU d. Absence of glucose Specific gravity: 1.015 Ketones: Negative Nitrite: Positive 35. Oligoclonal bands are significant in the diagnosis of pH: 7.0 Bilirubin: Negative Leukocyte multiple sclerosis when: esterase: ++ a. They are seen in both the serum and CSF 80-100 WBC/hpf 10- 5-10 red blood Many bacteria b. At least five bands are seen in the CSF 15 renal tubular cells/hpf c. They are seen in the CSF and not in the serum epithelial cells/hpf d. They appear in both the albumin and globulin fractions of serum and the CSF a. pH 36. Calculate the sperm count on a 3-mL semen speci- b. Protein men with a concentration of 12,000/mL. c. 5 to 10 RBC/hpf a. 4000/mL d. 10 to 15 renal tubular epithelial cells/hpf b. 12,000/mL 29. Cerebrospinal fluid is produced primarily by which c. 20,000/mL of the following? d. 36,000/mL a. Secretion by the choroid plexus cells 37. The most important sugar found in semen is b. Diffusion from the plasma into the central ner- ______. vous system a. Sucrose c. Ultrafiltration of plasma in the choroid plexuses b. Maltose d. Excretions from the ependymal cells lining the c. Fructose central nervous system d. Lactose 30. Three tubes of cerebrospinal fluid are submitted to the 38. The mucin clot test determines the presence of syno- laboratory. They are numbered l, 2, and 3 and show vial fluid ______. blood in all tubes but decreasing in amount in tubes a. Protein l through 3. This observation should be interpreted as: b. Glucose a. The tubes were numbered in wrong sequence, c. Fibrinogen because an increasing amount of blood would d. Hyaluronic acid be expected CHAPTER 5 Clinical Fluid Analysis 177

39. What is added to synovial fluid to determine the c. Thoracentesis viscosity? d. Pulmonary puncture a. Sodium hydroxide 45. Which of the following tests is used to differentiate b. Acetic acid between an effusion caused by cirrhosis and one c. Hydrochloric acid caused by peritonitis? d. Hyaluronic acid a. Absolute neutrophil count 40. Crystals that appear needle-shaped under polarized b. Fluid-to–serum bilirubin ratio light and are yellow when aligned with the slow c. Serum-ascites gradient vibration of compensated polarized light are d. Serum to LD ratio ______. 46. Amniotic fluid for fetal lung maturity testing should a. Monosodium urate be preserved ______. b. Calcium pyrophosphate a. In the refrigerator c. Hydroxyapatite b. At room temperature d. Corticosteroid c. In a dark container 41. The fluid that builds up between the serous mem- d. At 37 C branes is ______. 47. Which of the following results of a test on the mother a. A transudate would suggest apossible neuraltubedefectinthe fetus? b. An abscess a. A positive antibody screen c. An exudate b. A glucose value of 140 mg/dL d. An effusion c. An a-fetoprotein result of 0.1 MOM 42. Which of the following sets of results most closely d. An a-fetoprotein result of 3.0 MOM indicates a transudate? 48. A pale, frothy stool is indicative of which of the a. Clear, fluid-to–serum LD ratio: 0.8, fluid-to– following? serum protein ratio: 0.7, WBC count: 1000/mL a. Barium testing b. Cloudy, fluid-to–serum LD ratio: 0.5, fluid- b. Osmotic diarrhea to–serum protein ratio: 0.6, WBC count: c. Steatorrhea 1200/mL d. Excess carbohydrates c. Cloudy, fluid-to–serum LD ratio: 0.8, fluid- 49. The acid steatocrit test is performed to analyze which to–serum protein ratio: 0.7, WBC count: 2500/mL of the following? d. Clear, fluid-to–serum LD ratio: 0.45, fluid-to– a. Grossly bloody stools serum protein ratio: 0.40, WBC count: 800/mL b. Qualitative fecal fats 43. The most likely cause of increased neutrophils is a c. Carbohydrate reabsorption pericardial fluid exudate is ______. d. Quantitative fecal fats a. Tuberculosis 50. The most sensitive fecal enzyme test for the diag- b. Bacterial endocarditis nosis of pancreatic insufficiency measures c. Cardiac puncture ______. d. Pneumonia a. Lipase 44. Pleural fluid is obtained by which of the following? b. Trypsin a. Paracentesis c. Elastase I b. Pneumocentesis d. Chymotrypsin 178 CHAPTER 5 Clinical Fluid Analysis

SELF-ASSESSMENT

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NOTES CHAPTER 6 Immunology and Serology

Elizabeth G. Hertenstein

INNATE IMMUNITY • Turmoricidal effects • Antigen presentation The Natural Barriers of Immunity • Toll-like receptors: Also known as pattern-recognition receptors; the receptors recognize patterns in micro- • Mucous membranes bial cell walls or membranes and enhance recognition • Skin by phagocytic neutrophils and monocytes • Cough reflex • Cilia • pH of secretions Humoral Components of the Innate System • Enzymes in saliva and tears • • Normal flora competes for space and nutrients C-Reactive protein • Increases rapidly after infection or trauma, with a rapid decline Cells of the Innate Immune System • Can act as an opsonin and activate the classical complement pathway • Neutrophils: Early responders to foreign particles, par- • Complement ticularly bacteria, and move through vessel walls by • A series of proteins that mediate lysis of bacteria diapedesis to site of injury; response is phagocytosis and foreign particles via the mannose-binding lectin and inflammation enhanced by cytokines (MBL) pathway of complement activation (see later • Eosinophils: Responder/effector cells in allergic and discussion) parasitic infections; can be involved in phagocytosis but not to large degree because the numbers of eosin- Innate System Processes ophils in response is low • Basophils: Effector cells in immediate hypersensitivity • Phagocytosis: Stages of phagocytosis reactions also can be involved in phagocytosis, but to a • Chemotaxis: Phagocytic cell is attracted to the small degree because the number of circulating baso- invading organism by chemical mediators secreted phils is low by other immune system cells • Mast cells: Similar to basophils but have a longer life • Physical contact: Phagocytic cell and invading span, play a role in hypersensitivity reactions, bind organism attach immunoglobulin E (IgE) • Engulfment: Phagocytic cells engulf the particle • Dendritic cells: Have long membranous extensions, • Phagosome formation: Organism is enclosed in a are antigen-presenting cells and phagocytic, present vacuole to T cells in bloodstream or lymphoid tissue • Formation of phagolysosome: Vacuole fuses with • Monocytes: Large lobular phagocytic cells, in tissues granules in the cytoplasm to release contents are called macrophages • Digestion: Enzymes break down contents of • Tissue macrophages: In tissue, macrophages are phagolysosome named by their location • Excretion: By-products of digestion are transported • Liver: Kupffer cells to cell membrane and released outside the cell • Lung: Alveolar macrophage • Inflammation • Spleen: Splenic macrophage • Both cellular and humoral response aids healing, • Brain and central nervous system (CNS): but can also cause damage to tissue around the site Microglial cells of inflammation • Monocyte-macrophage: Important regulators • Effectors • Phagocytosis of bacteria and parasites ○ Neutrophils and macrophages are attracted to • Secret mediators such as cytokines the site

180 CHAPTER 6 Immunology and Serology 181

○ Secretion of chemoattractants and chemical • Recognition of self-antigens, negative signals sent to mediators nucleus will stop maturation, and cell dies by apoptosis ○ Complement acts as opsonin • CD 21, CD 40, and major histocompatibility com- • Physical hallmarks of inflammation are redness, plex (MHC) molecules appear pain, swelling, and fever and are due to increased • CD20 found on surface, marker for later stages of blood supply to site, increased capillary perme- B-cell development ation, migration of white blood cells (WBCs) to • Mature B cell inflamed area • Loss of CD10 on cell surface (marker for early ○ Localized inflammation: At site of injury stages of B-cell development) ○ Systemic inflammation: System-wide; if persists, • Leaves the bone marrow, travels to secondary adaptive immunity is activated organs to await activation ○ Chronic: Infection cannot be eradicated, inflam- • IgM and IgD on cell surface, thought to act as acti- matory process remains activated vating signalers for antigen contact B-Lymphocyte Maturation: Antigen-Dependent Phase ADAPTIVE IMMUNITY • Plasma cells • Immature B cells are activated to plasma or Organs of the Lymphatic System memory cells • Produce and secrete antibody • Primary: Maturation sites • No further maturation, these cells die within days of • Bone marrow: B-lymphocyte maturation site producing antibody • Thymus: T-lymphocyte maturation site • Memory cells • Secondary: Activation sites • Antigen-stimulated B cells waiting activation from • Spleen second exposure of specific antigen • Lymph nodes T-Lymphocyte Maturation • Outer cortex: B cells in primary and secondary follicles • Precursors released from bone marrow and mature in (or germinal center), few plasma cells the thymus • Paracortex: Area of T cells • Twomain subsets of T cells to mature: CD4+ and CD8+ • Inner medulla: Plasma cells with few T cells • CD4+: Helper T cells that indirectly protect from • Tonsils fungi and bacteria by secretion of cytokines to acti- • Peyer’s patches: Cluster of lymphocytes found vate B cells to maturity and produce antibody around the small intestine • CD8+: Cytotoxic T cells that directly protect from viral infection by cell lysis Cells of Adaptive Immunity • Double-negative stage of T-cell maturation • CD4 and CD8 are not present on cell B-Lymphocyte Maturation: Antigen-Independent • Cells possess pre–T-cell receptor (pre-TCR), which Phase is made of CD3 plus functional b chains • Pro–B cell • Signaling by b chains enhances maturation of the • Shows first rearrangement of immunoglobulin genes cell to express both CD4 and CD8 • Expresses CD 19 (acts as a coreceptor), 10, 45 • Double-positive stage (signaling) • T cell expresses both CD 4 and CD8 on surface • Successful rearrangement of immunoglobulin • T-cell receptor (TCR) is composed of CD3 plus a heavy chains leads to next stage or pre–B cell and b chains • Pre–B cell • Positive selection: Only cells with TCR, CD4, CD8 • m is first heavy chain produced survive • CD10 and CD19 expressed on surface • Negative selection: T cells that react strongly with • Pre–B-cell receptor (pre-BCR) on surface of B cell is self-antigens are eliminated made of m chain, surrogate light chains, and two • Mature T cell transmembrane molecules • Survivors of selection express either CD4 or CD8 on • A functional pre-BCR does not bind antigen, but surface rather signals the cell nucleus for further • Released from thymus to secondary lymphoid organs maturation • Become activated when antigen is presented by • Immature B cell antigen-presenting cell • Expresses IgM on surface (different from previous • CD4+ T cells stages) ○ Produce cytokines helping B cells to produce • B-cell receptor (BCR) on surface is IgM plus two antibody and stimulate hematopoiesis transmembrane molecules ○ Kill tumors • BCR is capable of binding antigen ○ Involvement in graft rejection 182 CHAPTER 6 Immunology and Serology

• CD8+ T cells • Redundant: The same cytokine can be produced by ○ Kill virally infected or diseased cells by different cells cytotoxicity • Autocrine effects: Affect the cell it was secreted from • Natural killer (NK) cell • Paracrine effects: Affect a nearby cell • Considered a bridge between the innate and adaptive • Endocrine effects: Have a systemic affect (Table 6-1) immune systems because it is thought to have the Complement. See (Figure 6-1) same precursor as a lymphocyte but can respond • Series of proteins that result in lysis of foreign particles, to diseased cells without prior exposure to antigen particularly bacteria, when activated • Thought to act by recognizing a lack of MHC class I • Proteins are found normally circulating in plasma proteins on diseased or cancerous cells • Complement regulator proteins present to keep from • The lack of these proteins along with signalers uncontrolled activation enhances the NK cell to cytolytic action against • Three known pathways of complement activation: the damaged cell Classical, alternative, and MBL pathways • Also act by way of antibody-dependent cell cytotox- • Classical pathway of complement activation icity (ADCC), or recognition and lysis of antibody- • Activated by antigen–antibody complexes coated infected cells • The “recognition unit”: C1 is made of three subunits designated as C1q, C1r, and C1s Humoral Components of the Adaptive • C1q binds to Fc portion of antibody, two molecules Immune System of C1q needed to bind for activation • At this step, two IgG molecules are necessary; how- Cytokines ever, only 1 IgM is needed because of its • Regulate growth and differentiation of immune system pentameric shape effector cells • C1r and C1s become enzymatically active and • Pleotropic, with many different effects cleave C4 and C2

TABLE 6-1 Selected Cytokines and Activity

Cytokine Family Cytokine Produced By Target Biologic Activity Interferon INF-a Virally infected dendritic NK cells Antiviral activity, mediates MHC class I and II INF-b cells expression INF-g T-helper cells Macrophages, cytotoxic T cells, NK cells Tumor necrosis TNF-a Macrophages, gram- Macrophages, mast Mediates inflammation by recruiting and factors negative bacteria cells activating phagocytes TNF-b Epithelial cells T cells and B cells Also known as lymphotoxin Interleukins IL-1 superfamily Monocytes, macrophages Hypothalamus, Acute phase reactions, mediate cell adhesion epithelial cells for diapedesis IL-2 T-helper cells T cells and B cells, Growth and differentiation of T cells and B macrophages cells, activates monocytes, macrophages, and NK and cytotoxic cells IL-4 Naive T cells and T-helper T cells Induces growth of T-helper cells, mediates cells humoral immunity IL-5 T-helper cells Eosinophils, B cells Growth of eosinophils, mediator of type 1 hypersensitivity reactions IL-6 T cells, B cells, monocytes, B cells, T cells Acute phase reactions, antibody production, macrophages, other stimulates B cells to plasma cells nonimmune cells IL-10 Monocytes, macrophages, T T-helper cells, antigen- Antiinflammatory and suppressive effect cells presenting cells Transforming TGF-b T-regulatory cells T cells, B cells Activates and inhibits proliferation, regulates growth factor inflammatory response Colony-stimulating CSFs T cells Bone marrow stem Stimulate hematopoietic stem cell factors cells differentiation of granulocytes, macrophages

CSF, Cerebrospinal fluid; MHC, major histocompatibility complex; NK, natural killer CHAPTER 6 Immunology and Serology 183

• Effector Alternative pathway of complement activation functions • Is triggered by polysaccharide components of bacte- Alternative Classical Lectin rial cell walls, parasites, fungi, tumor cells, viruses, pathway pathway pathway yeast Antibody Mannose- Initiation of • Inactive C3 in normal plasma is activated to C3b Microbe binding complement lectin • C3b binds with factor B to microbial cell wall activation • Enzymatically active factor D cleaves cell-bound factor B to Ba and Bb • Ba is released to plasma C3 C2 C4 • The resulting C3aBb¼C3 convertase, capable of B C4 C2 cleaving additional C3 to C3a and C3b C3b • More C3b is bound to complex, forming C3 C3 Early steps C3b C3b C3bBb3b ¼C5 convertase C3 C3a: • C5 convertase cleaves C5 to C5a and C5b C3a Inflammation • C5b binds to antigen and begins the first part of the MAC, same as in the classical pathway • Properdin is a protein of the alternative pathway C3b C3b: C3b is Opsonization that stabilizes the C3 convertase complex, allowing deposited C3b and the complex to function properly on microbe phagocytosis • Factor H prevents the binding of C3b to factor B in the early steps of this pathway, preventing uncon- C5b trolled activation C5 C5b • MBL pathway of complement activation C5a: • Activated when MBL binds to certain sugars found C5a Inflammation in cell walls and membranes of bacteria, yeasts, and Late steps some parasites • MBL is similar to C1q Complement MAC proteins form Lysis of • When bound to cell surface, a series of MBL- membrane microbe attack complex associated serine proteases cleave C2 and C4 • The rest is the same as in the classical pathway FIGURE 6-1 Pathways of complement activation. MAC, (Table 6-2) Macrophage attack complex. (From Abbas A, Lichtman A, Pillai S: Antibodies. See (Figure 6-2) Basic immunology: functions and disorders of the immune system, • Two heavy chains ed 4, Philadelphia, 2013, Saunders.) • Heavy chains are designated as m (IgM), d (IgD), g (IgG), a (IgA), and e (IgE) • C4 is cleaved to C4a and C4b, C2 is cleaved to C2a • Each heavy chain is made of three constant domains, 1 2 3 and C2b CH ,CH ,CH , and one variable (V) domain, VH • C4a and C2b are released, C4a has anaphylatoxic • Two light chains activity • Light chains are designated as k or l • C4b and 2a bind to antigen ¼C4b2a¼C3 conver- • Each light chain is made of one constant domain, CL tase enzyme and one variable domain, VL • C3 convertase cleaves C3 to C3a and C3b • The constant domains are unique to each class of anti- • C3a released has anaphylatoxic activity body and determine the isotype—m, g, d, a,ore • C3b binds to complex¼C4b2a3b¼C5 convertase • Minor variations of amino acid sequences in the con- • C5 convertase cleaves C5 to C5a and C5b stant domains of the molecule between individuals are • C5b deposits on cell membrane of antigen known as allotypes • C5b plus C6, C7, C8 make up the membrane attack • Immunoglobulin molecule variations in the variable complex (MAC); on the surface of a cell, this com- domains are known as idiotypes plex bound together causes a small pore in the • The constant domain is where biological activity of the membrane molecule occurs • C9 binds and stabilizes complex, leading to larger • Agglutination pore and ultimately cell lysis • Complement fixation • C1 Inhibitor is a complement protein that keeps this • Antigen attachment to aid opsonization system from uncontrolled activation • ADCC • Additional activities of complement proteins include • Viral neutralization anaphylatoxic reactions of C4a, C3a, and C5a • The variable region is unique to each antibody mole- • Released C3b can also act as opsonin cule and makes up the antigen-binding site 184 CHAPTER 6 Immunology and Serology

TABLE 6-2 Complement Components, Pathways, and Related Activity

Complement component Pathway Activity Other information C1qrs Classical Binds antibody and cell surface Recognition unit C4 and C2 Classical C4a and C2b released into plasma Activation C4b2a Classical Cleaves C3 C3 convertase enzyme C3 Classical, alternative, and C3a released in plasma, C3b combines with Activation mannose binding C4b2a in classical and factor B in alternative Factor B Alternative Is split into Ba and Bb, combine with C3 Activation C3bBb Alternative Cleaves C3 C3 Convertase enzyme C4b2a3b Classical, mannose binding Cleaves C5 C5 convertase enzyme C3bBb3b Alternative, mannose binding Cleaves C5 C5 convertase enzyme C5-C9 Classical, alternative, and Cell lysis Membrane attack mannose binding complex

• IgA: Primary protective antibody in secretions and Light chain VL intestinal mucosa • IgE: Primary antibody in allergic responses, able to C L bind basophils and mast cells VH Fc Disulfide • IgD: Unknown function but may play a role as B-cell bond signaler and/or coreceptor Heavy chain CH1 Hinge region CH2 CH3 Antigens and Immunogens

Heavy chain • Antigen: Foreign particles that may elicit an immune

Antigen binding sites 450 residues response • Immunogen: Any foreign particle that elicits an Carbohydrate immune response • Light chain All immunogens are antigens, but not all antigens are 212 residues immunogens • Traits that make good immunogens • Large molecular weight VL and VH = Variable regions CL and CH = Constant regions • Chemical complexity: The higher the complexity, or variety of makeup, the more immunogenic FIGURE 6-2 Basic structure of immunoglobulin G. (Modified from Turgeon ML: Fundamentals of immunohematology, ed 2, Baltimore, • Foreignness 1995, Williams & Wilkins.) • Stability: The ability for the immunogen to be digested and processed is greater if it is less stable • Proteins make good immunogens because of their • Hinge region complexity • Area that gives molecule flexibility allowing for • Carbohydrates such as glycolipids or glycoproteins multiple binding sites are less immunogenic because they consist of fewer 1 2 • Located between the CH and CH domains molecules and are of lesser complexity • Enzyme papain cleaves the antibody molecule • Epitopes: Sites of the antigen that can vary above the hinge region • Different epitopes of the same antigen causes ○ Leaves three fragments: Two Fab fragments and polyclonal immune response one Fc • Recognition of epitopes differs for B- or T-cell • Enzyme pepsin cleaves the antibody molecule activation below the hinge region • B-cell activation: Epitopes are capable of cross- ○ Leaves one F(ab)2 (capable of binding antigen) linking with immunoglobulin ○ Fc fragment is destroyed (Table 6-3) • T-cell activation: Immunogen epitope is presented to the • IgM: Large macroglobulin, first immunoglobulin pro- T cell in the form of MHC proteins (see later discussion) duced in the primary immune response • Haptens • IgG:Predominantimmunoglobulin inthe body, primary • Small molecules considered nonimmunogenic by immunoglobulin in the secondary immune response themselves CHAPTER 6 Immunology and Serology 185

TABLE 6-3 Summary of Immunoglobulin Structure and Function

Feature IgA IgD IgE IgG IgM Structure Monomer and Monomer Monomer Monomer Pentamer with J (monomer, dimer with J chain dimer or chain pentamer, include subunits)

Subclasses 2 (a1,a2) None None g1-4 None Found in Secretions (saliva, On surface of B cells Blood Blood: Intravascular and Blood: tears, breast extravascular spaces Intravascular milk), spaces intestinal fluids, and serum Summary of In secretions it Basically unknown, but thought Binds to mast cells Neutralize toxins, Complement biologic protects to play a role in antigen and basophils activates activation activity against foreign binding in association with mediating release complement, crosses and antigens IgM and B-cell activation of histamine and placenta, agglutination heparin agglutination, Plays a role in opsonization helminth infections

• Are immunogenic when coupled with a larger mol- Major Histocompatibility Complex ecule to produce an immune response • • Can react with specific antibody alone, but cannot Main function is to present antigen, or digested anti- produce a visible reaction such as precipitation or gen molecules, to T cells for activation • agglutination Class I MHC • • When coupled with a carrier, can react with anti- On all nucleated cells in the body • body and produce a visible reaction Presents endogenous antigens (antigens inside infected cells) to CD8+ cytotoxic T cells IMMUNE RESPONSE TO ANTIGEN • Endogenous antigens are those made in the infected cells, such as proteins and molecules synthesized • Primary response from viruses • After exposure to antigen a lag phase occurs • For protection against viruses and parasites • IgM is typically first antibody produced • Tumor cells and some viruses cause a disruption to • IgG secretion begins shortly after IgM the creation of MHC class molecules from forming • Both levels decline after antigen has been eliminated on the surface, decreasing the cytotoxic effect of • Secondary or anamnestic response CD8+ cells • After second exposure to same antigen • These MHC class I–deficient diseased cells trigger • More rapid response because of activation of mem- and activate NK cells ory B cells • Class II MHC • Rapid rise in levels of antigen-specific IgG antibody • Mainly on antigen-presenting cells: B cells, mono- • Some IgM may be produced but in lesser quantities cytes, macrophages, and dendritic cells in comparison to levels of IgG • These cells predominantly take up exogenous anti- • Antibody responses are typically polyclonal—that is, gens or antigens that exist outside the host cell and the antibody response is directed against more than are engulfed by phagocytosis one epitope of the specific antigen • Presentation is to CD4+ helper T cells for produc- • Monoclonal antibodies are produced artificially by tion of cytokines that enhance the production and hybrid cell lines called hybridomas. The myeloma secretion of antibody from B cells fused cell lines produce antibody from a single clone • For protection against bacteria, viruses, and other and are used in a variety of immunologic arrays exogenous antigens 186 CHAPTER 6 Immunology and Serology

ANTIGEN–ANTIBODY REACTIONS AND • The rocket lines are formed as antigen concentra- BASIC PRINCIPLES tion decreases • Immunoelectrophoresis: Simple, two-step assay • Affinity: Force between a one-Fab site on the immuno- • Serum, containing antigen, is placed in wells and globulin and one epitope of the antigen electrophoresed to separate proteins • Avidity: Total of all forces between the immunoglobu- • The second step involves adding antibody in troughs lin and epitopes of the antigen cut in the gel alongside the electrophoresis trail • Zone of equivalence: Area where antigen and antibody • The antigen and antibody are allowed to diffuse are approximately equal and visualization of the reac- • The location, shape, and intensity of the precipitation is optimized by either precipitation or agglutination tion lines are measured • Lattice formation: Antigen and antibody together in • Used to measure many serum proteins approximately equal proportions, the immunoglobu- • Immunofixation electrophoresis lin molecules cross-link with the specific antigen • Sample containing antigen is placed in a well and • Precipitation: Visible reaction between antibody and electrophoresed, separating proteins soluble antigen • Reagent antibody on a cellulose or agarose gel tem- • Agglutination: Visible reaction between antibody and plate is placed over the electrophoresed sample particulate antigen • Reactions between antibody and antigen occur and • Prozone phenomenon: Concentration of antibody are made visible by staining exceeds that of the antigen in solution, and lattice for- • Electrophoretic errors can occur when current is too mation does not happen strong or too weak, current is applied backward, • Postzone phenomenon: Concentration of antigen is in buffer is not at correct pH, or time to run is too long excess and lattice formation does not occur or too short • Both prozone and postzone can cause false-negative serologic reactions and erroneous titer results AGGLUTINATION TESTS AND ASSAYS PRECIPITATION TESTS AND ASSAYS • Agglutination is the visible reaction of antibody with particulate antigen • Nephelometry: Measures the amount of light scattered • Particulates in agglutination can be synthetic latex in a solution containing antibody–antigen complexes beads or organic red blood cells (RBCs) • Can be used to measure both antibody and antigen • Two steps to agglutination: Sensitization and lattice • Is widely used to quantify immunoglobulins formation • Turbidimetry: Measures the decrease in light intensity • Sensitization occurs when the antibody and spe- in a solution of antibody–antigen complexes cific antigen (on the surface of the particulate) • The lower the light intensity, the higher the concen- combine tration of complexes ○ The size and class of the antibody and the nature • Radial immunodiffusion: Gel containing antibody of the antigen epitope can affect this step with cut small holes as wells • Lattice formation occurs when antibody–antigen • Sample containing the antigen is placed in the well complexes cross-link • Line of precipitation forms in a circle around the well • The particulates must be somewhat neutral in • Diameter of the circle is proportionate to the con- charge for the complexes to link centration of antigen in the sample • RBCs carry a net negative charge that acts as a • Ouchterlony diffusion repellent (zeta potential) • Wells are cut in gel with multivalent antibody is ○ Low ionic strength saline or albumin is used to placed in a center well and antigen in remaining wells lower the zeta potential (enhance the lattice for- • Precipitation lines that form will identify relation- mation when RBCs are used as the particulate) ship between antigens • This stage is influenced by ionic strength of solu- • Noidentity: Precipitation lines formed will crisscross, tion, pH, and temperature as an X, indicating no identity between antigens • Latex agglutination • Partial identity:Precipitationlines formedwill appear • Use latex beads (as the particulate) coated with anti- partially crossed, as a line with a spur on the end, indi- gen to directly agglutinate patient serum containing cating cross reactivity between the antigens specific antibody • Identity: Precipitation lines meet but do not cross, no • Agglutination inhibition spurs,indicating no relationshipbetweenthe antigens • Assays based on the principle of no agglutination as • Rocket electrophoresis a positive result • Antibody in gel and antigen pipetted into a cutout well • Serum (containing antigen) is mixed with beads • Electrical current is applied and resulting precipita- coated with antigen tion lines form narrow triangles, like a rocket • If agglutination occurs, the antigen is not present CHAPTER 6 Immunology and Serology 187

• Hemagglutination antibody and same patient, a result of more than • Assays that use RBCs as the antigen particulate fourfold or two-tube difference is considered reacting with antibody diagnostic • Reactions can occur either in vivo or in vitro ○ Titers are generally not performed on infant sam- depending on the test ples because it is not known if the antibody was • Direct antiglobulin test (DAT) identifies in vivo produced by the infant or is of maternal origin reactions of RBC antigen with specific antibody • Indirect antiglobulin test (IAT) identifies reactions INFECTIOUS DISEASES AND SEROLOGY of reagent RBC antigen with specific antibody when the reaction occurs in vitro Bacteria

OTHER IMMUNOASSAYS • Bacteria evade host defenses by avoiding phagocytosis and inactivating or blocking complement • Noncompetitive assay • Streptococci • Capture or sandwich assays • Bacteria divided into serotypes based on the M and • Antibody is bound to solid phase and allowed to T proteins in the cell wall react with patient sample containing antigen • Some streptococci possess a hyaluronic acid capsule • Amount of reactivity is proportional to concentra- that helps the organism evade phagocytosis tion of antigen • Beneath the cell wall is a carbohydrate that is • Competitive assays divided further into Lancefield groups A to H • Labeled antigen and patient antigen are mixed with and K to V antibody at the same time • The main virulence factor of group A streptococci is • Antigens compete for the limited amount of anti- the M protein, which helps the organism evade body binding sites phagocytosis and inactivates complement • Concentration of bound label is inversely propor- • Other virulence factors include exoantigens pro- tional to concentration of antigen in the sample duced by the organism during the infection • Multiple detection methods for these assays • The most common exoantigens tested for include • Use of radioisotopes (radioimmunoassay) DNase B and streptolysin O • Color reaction from enzyme and substrate (enzyme • Acute pharyngitis is typically detected by a throat immunoassay [EIA]) swab and culture • Heterogeneous assays: Those that require a step to sep- • Throat swabs chemically or enzymatically remove arate bound and free analyte antigen and are tested by a rapid enzyme assay or • Homogenous assays: Those that do not require a agglutination separation step • Anti–streptolysin O (ASO) titers may be performed • Direct fluorescent assays if rheumatic fever is suspected • Antibody tagged with a fluorescent dye to react ○ In a traditional ASO titer, the patient sample con- directly with patient specimen containing antigen taining ASO inhibits red blood cell lysis • Typically prepared on a slide and viewed under a ○ In a serial dilution, a titer is determined fluorescent microscope ○ A high titer indicates a recent infection and • Indirect fluorescent assays (IFAs) are two-step assays declines to a low titer later in the infection, but • Patient serum is incubated with known antigen can remain positive • Antihuman globulin tagged with fluorescent dye • Anti–DNase B titers are useful in poststreptococcal is added glomerulonephritis and skin infections • Chemiluminescence assays • Most bacteria or bacterial infections are identified by • Light is produced during a chemical reaction and culture, polymerase chain reaction (PCR) testing, and/ measured or serologic tests • Used widely in immunochemical instrumentation • Serologic assays Rickettsia • Technique called a titer to semiquantitate antibody levels in serum • Suspected rickettsial infections are detected primarily ○ Titers are performed using serial dilutions (see by serologic test using immunofluorescence, EIAs, discussion of serial dilutions in Chapter 11) and molecular techniques ○ Titer results are used to evaluate immunity in • Weil-Felix test terms of exposure, acute phase, or convalescence • An agglutination test that detects antibodies in of an organism or disease patients with rickettsial infections ○ When comparing initial titer results to subse- • Antibodies cross react with antigens present on quent or convalescent titer results for the same certain strains of Proteus spp. 188 CHAPTER 6 Immunology and Serology

Parasites the antibody is passively acquired from the mother or is from the infant • Parasites evade hosts by • Best to use a test that detects IgM antibodies (EIA or • Living within host cells to avoid the immune Western blot) response • Detection of IgM antibodies in an infant is an indi- • Can change surface antigens to confuse the host’s cation that the antibodies are produced by the immune response infant’s immune system • Acquire host’s antigens, masking themselves as self- Lyme disease antigens • Vector-borne illness, typically carried by a tick • Immunologic response to parasites include the produc- • Caused by the spirochete Borrelia burgdorferi tion of cytokines to induce • Symptoms • Migration of cytotoxic cells • Rash, initially localized known as erythema • Proliferation and concentration of neutrophils and migrans (bulls’ eye or target) macrophages • Joint involvement • Antibody production • Neurologic and heart involvement if left untreated • IgE and mast cells activate eosinophils to release the ○ A classic sign of neurologic involvement includes contents of cellular granules facial palsy • Early clinical symptoms are most useful in early Spirochetes diagnosis of Lyme disease • Early serologic tests are not useful Syphilis • Antibody response to Lyme disease does not occur • Causative organism: Treponema pallidum for approximately 3 to 6 weeks • Four stages of syphilis • IgM and IgG response in Lyme disease is somewhat • Primary stage atypical ○ Characterized by a chancre ○ Both antibody responses, when detected, can be • Secondary stage of syphilis detected together ○ Characterized by lymphadenopathy, rash, fever, • When patient diagnosis occurs early and antibiotic and pharyngitis treatment has begun, serologic findings may be • Latent stage limited ○ Characterized by no symptoms of the disease • Centers for Disease Control and Prevention recom- • Tertiary stage mends specific algorithm for laboratory diagnosis of ○ Seen later in the disease with cardiac and nervous Lyme disease system involvement • First step - test patient serum using IFA or EIA for • Nontreponemal tests for syphilis: antibodies as a screening measure • Rapid plasma reagin (RPR) • Second step - confirmation must be made using a ○ Reagents contain charcoal, which allows for Western blot test (because of cross reactivity using macroscopic visualization these tests) • The Venereal Disease Research Laboratory (VDRL) test is viewed under a microscope and interpreted Viruses microscopically ○ A positive VDRL result obtained using spinal • Innate, humoral, and cell-mediated defenses protect fluid is indicative of neurosyphilis against viruses • Both RPR and VDRL detect reagin in patient’s serum • Cells infected by virus produce interferon, which • Reagin is an antibody formed against material from enhances activity of NK cells cells damaged by the spirochete • Antibodies produced and involved in • A positive nontreponemal result must be confirmed ○ Viral neutralization with a test to directly detect treponemal organisms ○ ADCC • Fluorescent treponemal antibody absorption test ○ Activation of complement (FTA-ABS) • Ways viruses avoid the immune system • T. pallidum microhemagglutination assay (MHA-TP) • Fast replication: Evolve and mutate quickly • Darkfield microscopy • Alter the function of immune system cell by insert- • EIAs used to detect antibodies to the treponemal ing their genetic material into a host cell spirochete • TORCH testing is a panel of serologic procedures used • Congenital syphilis to detect a group of viruses and other organisms that • Spirochete is transmitted from mother to fetus cause congenital disease • Difficult to detect serologically because when using • Toxoplasma a test that detects an IgG antibody, it is not known if • Other (viruses) CHAPTER 6 Immunology and Serology 189

• Rubella Rubella • Cytomegalovirus (CMV) • Togaviridae family • Herpes simplex virus • Also known as German measles Herpesviridae Family of Viruses • Causes systemic rash in children Epstein-Barr Virus • Severe complications if congenital • Most common infection is infectious mononucleosis (IM) • Vaccination to rubella is important in reducing the • Infects B cells that produce different types of antibodies number of infants born with congenital rubella • Virus-specific antibodies • Antibody titers are performed to determine immunity ○ Anti-VCA (viral capsid antigen) • Titers are compared to a standard provided by the ○ Anti–EA-D (early antigen diffuse) manufacture of the test kit • Both IgM anti–EA-D and anti-VCA indicates acute Rubeola infection • Not to be confused with rubella • IgG anti-VCA may persist for life, so may represent • Also known as measles a past infection • Caused by a virus in the Paramyxoviridae family • Anti-EBNA (EBV nuclear antigens) • Diagnosis is made typically by EIA for IgM antibody ○ Anti-EBNA may indicate the convalescent phase or rise in titer in subsequent specimens from the same of the infection patient • Heterophile antibodies West Nile Virus • In approximately 90% of patients with infectious • A flavivirus mononucleosis • Causes respiratory illness with possible encephalitis • React with similar antigens from unrelated species and fatality • Infectious mononucleosis heterophile antibodies • Fastest diagnosis is through serologic testing for IgM can react with sheep, bovine, and horse RBCs or IgG in the cerebrospinal fluid, if nervous system • Testing for heterophile antibodies is a screening test involvement is suspected • Should test for specific viral antibodies if patient has Hepatitis clinical symptoms and negative screening test • Hepatitis A (HAV) • Autoantibodies can be produced • Picornaviridae family Cytomegalovirus (CMV) • Contracted through the fecal-oral route • Population at risk • Is limiting • Transplant patients ○ Does not cause reinfection • Immunocompromised ○ Does not progress to a chronic stage • Newborns • Diagnosis is made by EIA for antibody • Up to 80% of people and infants with CMV are • IgM anti-HAV indicates initial infection asymptomatic • HAV antigen testing performed on fecal specimens • Can be diagnosed with viral culture, PCR, and • Testing for IgG anti-HAV: Recovery or immunity to antibody tests for IgM or IgG the virus • Presence of IgM indicates new or reactivation of • Hepatitis E (HEV) infection • Hepeviridae family Herpes Simplex 1 and 2 • Transmitted through fecal-oral route • Cause sores on and around the mouth and genital areas • Typically limiting, with very few cases becoming • Can be passed congenitally to infant severe • If untreated, progresses to disseminated disease • Diagnosis is by serologic testing for anti-HEV • Can be fatal for infant • Hepatitis B (HBV) • Serology: Currently only method approved by U.S. • Hepadnaviridae virus family Food and Drug Administration for diagnosing herpes • Transmitted parenterally (through contact with simplex virus infections blood and body fluids) Varicella-Zoster • Diagnosis by serologic testing for the HBV antigen • Initial disease is chickenpox or antibody markers • Causes a rash that blisters and forms scabs in recovery • Hepatitis B surface antigen (HBsAg): First marker • Severe cases can lead to varicella-zoster pneumonia to appear • After initial infection, virus lies dormant • Hepatitis E antigen (HBeAg): Also positive in the • Possible reactivation years later as shingles early stages • Diagnosis is made by testing for antigen (virus) by • Both HBsAg and HBeAg immunofluorescence ○ Present during the chronic/carrier state of the • Antibody testing: Rise in antibody titer using two disease specimens weeks apart is diagnostic ○ Patients with chronic HBV may be asymptomatic 190 CHAPTER 6 Immunology and Serology

TABLE 6-4 Hepatitis B Serology • Intracellular virus uses host cell machinery to replicate • Host cells are CD4+ T-helper lymphocytes Hepatitis B Positive or • Serologic testing includes testing for anti-HIV, HIV Infection Stage Marker Negative antigen, and viral nucleic acid Acute HBsAG Positive • Enzyme-linked immunosorbent assay (ELISA) used Acute Anti-HBc, IgM Positive to detect anti-HIV Chronic Anti-HBc, Total (IgM Positive ○ Positive samples are tested in triplicate and IgG) ○ If two of three samples are reactive with ELISA, Chronic Anti-HBs Negative (no confirmatory testing is required recovery) • Confirmatory testing with Western blot Chronic HBeAg Positive ○ Positive: Two of three HIV protein bands (p24, Recovery HBeAg Negative gp41, gp120/gp160) are present Recovery Anti-HBs Positive ○ Indeterminate: Present protein bands do not After vaccination Anti-HBs Positive meet the standard for positive After vaccination Anti-HBc Negative § Carrier HBsAG Positive Specimens should be recollected at a later date Carrier Anti-HBc Positive (IgG) and retested § These patients could be in the early stages of HBc, Hepatitis B core (antigen); HBs, hepatitis B surface (antigen); HBeAg, the disease hepatitis E antigen; HBsAg, hepatitis B surface antigen; Ig (G, M), • CD4 cell counts are measured frequently for effective- immunoglobulin. ness of treatment • Molecular testing: Recent and quantitative tests to ○ Presence of both indicates a highly infectious determine viral load state and viral replication • Anti-HBc, antibody to the viral core AUTOIMMUNE DISEASES IN ○ First antibody to be produced and detected IMMUNOLOGY ○ IgM anti-HBc indicates a recent acute infection ○ Total anti-HBc, IgM, and IgG indicates a past or • Autoimmunity current infection • Body produces antibody to self-components, such as ○ IgM levels of anti-HBc may decrease, but IgG proteins, hormones, and cellular components such anti-HBc remains positive for life as DNA • Anti-HBe, antibodies to HBeAg • Systemic ○ Produced after the disappearance of antibodies • Organ-specific ○ Indicates beginning of recovery • Anti-HBs, antibodies to HBsAg Systemic Lupus Erythematosus ○ Appear after the disappearance of HBsAg ○ An indicator of recovery and immunity to HBV • Immune complex autoimmune disease ○ Anti-HBs is the antibody produced after vaccina- • Autoantibodies produced to intracellular components tion (Table 6-4) of dying cells • Hepatitis D (HDV) • Cellular DNA • Also known as the d virus • RNA • Is transmitted parenterally • Other intracellular constituents • Only present in coexistence with HBV • Immune complexes are deposited in joints and major • Identified serologically by testing for anti-HDV or organs RNA testing • Complement deficiency is also seen • Hepatitis C (HCV) • Complement is continuously activated and used to • Flaviviridae family help clear these immune complexes • Transmitted through contact with contaminated • Autoantibodies formed in response to cellular blood and body fluids breakdown • Diagnosis: Detection of anti-HCV antibodies fol- • Anti-dsDNA: Anti–double-stranded DNA lowed by a confirmatory test for HCV RNA • Anti-ssDNA: Anti–single-stranded DNA Human Immune Deficiency Virus • Anti-ENA: Anti–extractable nuclear antigen • Retroviridae family • Antihistones • Transmitted • Anti-DNP (DNA coupled with histone) • Contact with contaminated blood/body fluids • Anti-Sm: Anti-Smith antibody • Sexual contact • Anti-dsDNA is most specific for systemic lupus • Mother to infant erythematosus (SLE) CHAPTER 6 Immunology and Serology 191

• Anti-Sm is seen only in SLE, but only a small percent- Grave’s Disease age of patients are positive for this autoantibody • Autoantibody to the thyroid-stimulating hormone • The other autoantibodies, as well as anti–SS-A and (TSH) receptor on the thyroid anti–SS-B can be found in other autoimmune • Hyperthyroid diseases Type I Diabetes Mellitus • Antinuclear antibody (ANA) test • Autoantibodies to islet cells of the pancreas • Characteristic laboratory test for these SLE • Autoantibodies to the enzyme glutamic acid autoantibodies decarboxylase • IFA staining of cells Other Autoimmune Diseases • Specific staining patterns are characteristic of • Multiple sclerosis certain diseases • Myasthenia gravis ○ Homogenous or diffuse staining • Goodpasture’s syndrome § Characteristic of SLE • Celiac disease § Pattern is staining of the whole nucleus § Indicates presence of anti-dsDNA, anti– dinitrophenyl antibody (anti-DNP), and IMMUNOPROLIFERATIVE DISEASES antihistones § Peripheral or rim pattern Leukemia ○ Presence of anti-dsDNA ○ Suggestive of SLE • Malignancy arises in the bone marrow or § Speckled pattern peripheral blood ○ Anti-Sm, anti-ENA, anti–SS-A, and anti–SS-B • Classification ○ Associated diseases are SLE, Sjo¨ gren’s syndrome, • Acute and other mixed connective tissue disease • Chronic § Nucleolar • Myeloid ○ Staining of the nucleolus • Lymphoid ○ Associated with anti–nucleolar RNA in SLE and scleroderma Lymphoma

• Malignancy is in the lymphoid tissue Rheumatoid Arthritis • Classification • Hodgkin’s: Classic Reed-Sternberg cells • Systemic autoimmune disease • Non-Hodgkin’s: Cancerous cells are mostly B cell • Immune complexes deposit in the joints, activate complement and continuous inflammation • Characteristic rheumatoid factor (RF) • IgM autoantibody produced Plasma Cell Dyscrasias • Autoantibody against the FC portion of IgG Multiple Myeloma • The majority of patients with rheumatoid arthritis • Malignant plasma cells produce monoclonal (RA) are positive for RF immunoglobulin • RF factor is not specific for RA • Serum protein electrophoresis shows increase in g ○ RF factor can be positive in other diseases region with monoclonal spike (M spike) • Agglutination tests for RF are indicative of RA • Monoclonal immunoglobulin is commonly IgG but • Anti–cyclic citrullinated peptides (anti-CCP) autoanti- can be IgA bodies are also assayed in RA • Bence Jones protein in urine: Free immunoglobulin • Present in the majority of patients with RA light chains • Rarely seen in patients without RA • Testing for light chains in serum may be more sensitive • Monitor RA • Light chains are rapidly absorbed by kidneys for • C-reactive protein (CRP) secretion • Complement levels • Urine levels may be undetectable before serum levels • Both are markers of inflammation • Free light chains in disease • Produced during maturation of B cells and Organ-Specific Autoimmune Diseases plasma cells • B cell tumors, diseases, and inflammatory responses Hashimoto’s Thyroiditis yield light chains in serum • Autoantibodies to thyroglobulin • Aids in diagnosis and disease monitoring/ • Results in hypothyroidism progression 192 CHAPTER 6 Immunology and Serology

Waldenstro¨m’s Macroglobulinemia • Associated with additional bone, facial, and cardiac • Malignancy of IgM-producing lymphocytes abnormalities • Serum protein electrophoresis indicates elevated levels of g region Severe Combined Immunodeficiency • Concentration of IgM antibody is confirmed using immunofixation • Several genetic causes Monoclonal Gammopathy of Unknown Significance • Autosomal recessive • Disorder with a monoclonal immunoglobulin in serum • X-linked • No clinical manifestations of myeloma or • Variety of molecular defects resulting in enzyme macroglobulinemia deficiencies • Small percent of patients progress to multiple mye- • Both T-cell and B-cell development is affected loma or Waldenstro¨ m’s macroglobulinemia • Identified in infancy • Patients are very susceptible to infections from all IMMUNODEFICIENT DISEASES organisms • Bone marrow transplantation is the traditional Transient Hypogammaglobulinemia of therapy Infancy • Gene therapy has been used

• Condition occurs during the newborn’s first year Chronic Granulomatous Disease • After circulating passive maternal antibodies disappear • Some infants’ immunoglobulins are not produced • Inherited disease-effecting neutrophil phagocytic early enough function • Low levels of immunoglobulin persist • Defective enzyme nicotinamide adenine dinucleotide • May acquire infections phosphate (NADPH) oxidase • Test low for IgG antibodies initially • Enzyme necessary for production of cellular hydro- • Later testing shows increased levels of immunoglobulin gen peroxide • Hydrogen peroxide essential for the oxidative burst Common Variable Immunodeficiency and microbicidal effect on catalase-positive organisms • Onset in early adulthood • No enzyme results in no production of hydrogen • Can be either congenital or acquired peroxide, with little to no oxidative burst • Usually identified after recurrent severe infections • Traditional test is the nitroblue tetrazolium test • B-cell levels appear normal but do mature to • Measures the oxidative reduction capability plasma cells • Produces blue color if positive • No to low immunoglobulin production • If no enzyme, test result is negative or no color • Common variable immunodeficiency remains for • Flow cytometry can now be used to identify neutro- life, with increased risk for other immunologic phils that lack the capability of producing the diseases oxidative burst

X-Linked Agammaglobulinemia (Bruton’s Leukocyte Adhesion Deficiency Agammaglobulinemia) • Genetic defect • X-linked inherited condition affecting males • Results in a defective molecule (b-integrin) • Early diagnosis as a result of recurrent infections as • Necessary for the adherence of leukocytes to infants endothelium • Genetically linked to the lack of Bruton’s tyrosine • Leukocytes/phagocytes cannot move through mem- kinase enzyme necessary for B-cell maturation branes into extravascular spaces • B cells are not detectable, but T cells have been dem- • Prolonged wound healing and skin and respiratory onstrated to be normal infections

DiGeorge’s Syndrome Complement Deficiencies

• Genetic deletion resulting in abnormal development of • MAC or C5-9 deficiencies are associated with recur- the thymus rent Neisseria infections. • T- cell function is normal • Early complement-component deficiencies • T-cell levels are low to nondetectable • C1qrs, C2, and C4, are associated with a lupus-like • Results in recurrent infections syndrome CHAPTER 6 Immunology and Serology 193

• Immune complexes fail to be cleared and are depos- • Precipitate out of the serum ited in joints and tissue • Deposit in tissue that mediates the involvement of • C1-Inhibitor (C1-INH) deficiency complement • Hereditary angioedema • Examples • Characterized by swelling and rash • Serum sickness (systemic) • Arthus reaction (localized) HYPERSENSITIVITY • Laboratory testing • Testing for specific disease Type 1 • Certain autoimmune disease reactions are considered as type III • Immediate hypersensitivity reactions ○ SLE • Also known as anaphylactic reactions ○ RA • Immune mediator is IgE • Cellular involvement includes basophils and mast cells Type IV • Production of IgE that binds to basophils and mast cells • Cross-linkage occurs between two or more IgE anti- • Delayed hypersensitivity bodies and the basophils and/or mast cells • Involves T-helper cells and cytotoxic T cells • Chemical meditators are released into the blood- • Symptoms appear several days to weeks after stream, causing the clinical symptoms exposure • Examples of type I hypersensitivity • Cytotoxic T cells and cytokines produced by T-helper • Urticaria cells • Hay fever • Cause inflammation • Asthma • Tissue damage at the site of allergen contact • Anaphylactic reactions to bee stings, venom, peanut • Examples allergies • Contact dermatitis • Direct inoculation of small amounts of allergen subcu- ○ Jewelry and latex taneously used to determine an individual’s sensitivity ○ Poison ivy to certain allergens ○ Farmer’s lung • Laboratory testing ○ Skin test for Mycobacterium tuberculosis • Testing for total IgE by the radioimmunosorbent test § Exposure to M. tuberculosis involves a T-cell– • Allergen-specific IgE by the radioallergosorbent test mediated response • Newer methods use the chemiluminescence format to test for IgE TUMOR IMMUNOLOGY

Type II • Tumor immunity: Evidence that tumors are eradicated • Tumor-infiltrating lymphocytes • Cytotoxic reactions mediated by IgG and IgM and • NK cells via loss of MHC class I molecules on complement defective cells • Cells destroyed by • Antibodies • Phagocytosis • Cytokines • Direct cytotoxic effects of complement triggered by ○ Tumor necrosis factor (TNF) is toxic to some tumors antibody coating of cells • Evasion of host immunity by tumors • Macrophages, neutrophils, and NK cells contribute • Tumor cells do not produce warning signals for to cell damage innate system • Examples ○ Failure of T-cell responses • Transfusion reactions • Certain tumor cells have decreased MHC class I • Hemolytic disease of the fetus and newborn molecules • Hemolytic anemia • Rapidly dividing tumor cells can acquire and share • Goodpasture’s syndrome antigens with normal tissue • Testing • Tumor antigens/markers • DAT • Tumor-specific antigens are produced by tumor ○ Detects in vivo binding of antibody or comple- • Tumor-associated antigens are surface molecules of ment to RBCs virally infected cells • Used in laboratory testing Type III ○ Aid diagnosis or staging of cancer ○ Monitor recurrence • Immune complex destruction ○ Help determine prognosis and treatment • Soluble antigen by IgG or IgM and complement ○ Screening in normal population (Table 6-5) 194 CHAPTER 6 Immunology and Serology

○ Selected Tumor Markers and Chronic TABLE 6-5 Associated Cancers § Months after transplant § Cell mediated along with cytokine production Tumor Markers Disease Association that enhances destruction of tissue and CEA Colorectal cancer transplant (carcinoembryonic antigen) • Graft-versus-host disease b-2 Microglobulin Multiple myeloma • Complication in bone marrow and stem cell CA125 Ovarian cancer transplants (carbohydrate antigen 125) • T cells in transplanted product recognize host cells CA15-3 Breast cancer as foreign CA19-9 Pancreas cancer • Severity is related to HLA match of donor HER2/neu Breast cancer • Graft-versus-leukemia effect PSA Prostate cancer • Benefit of T cells in transplant: Graft-versus-leukemia (prostate-specific antigen) effect ER/PR Breast cancer • (estrogen receptor/progesterone Promote cellular engraftment receptor) • Improve immune responses AFP Liver and testicular • Recipients of transplants are (a-fetoprotein) cancers • HLA typed • HLA matched with a donor • Cross-matched to rule out preformed antibodies to TRANSPLANTATION IMMUNOLOGY HLA from donor

Major Histocompatibility Complex CERTIFICATION PREPARATION QUESTIONS

• Genes on short arm of chromosome 6 For answers and rationales, please see Appendix A. • Code for proteins called human leukocyte antigens 1. Natural barriers of the immune system include all (HLAs) except which of the following? • Class I: HLA-A, HLA-B, HLA-C a. pH of secretions ○ At the same loci and are inherited together b. Coughing • Class II: HLA-DR, HLA-DQ, HLA-DP c. Hair follicles • Mendelian inheritance of haplotypes from each d. Intestinal bacteria parent 2. The fundamental difference between primary and • These proteins on cell surfaces have destructive secondary organs of the lymphatic system is: effect if transplants occur against HLAs a. Antibody production occurs only in the primary ○ HLA proteins regulate immune response of T cells lymph organs ○ Will process and present unmatched HLA as b. Complement production occurs only in the pri- foreign mary lymph organs • Transplants c. Maturation of lymphocytes occurs in secon- • Solid organ dary organs, and activation occurs in primary organs ○ Must be ABO group compatible d. Maturation of lymphocytes occurs in primary § Stem cell organs, and activation occurs in secondary organs § Bone marrow 3. Toll-like receptors act in which way? § Autograft: Recipient receives own tissue a. Enhance recognition of bacteria by phagocytic cells § Syngeneic graft: Recipient receives transplant b. Activate B cells to produce antibody from identical twin c. Activate helper T cells § Allograft: Transplant between individuals of d. Aid in processing antigen in the form of an MHC same species molecule § Xenograft: Transplant between individuals of 4. Neutrophils and monocytes have receptors for which different species part of the immunoglobulin molecule? • Rejection a. Fc • Transplanted cells are recognized as foreign b. Fab ○ Hyperacute rejection c. Hinge region § Minutes to hours after transplant d. Variable region § Transplants across ABO group barrier 5. One B-cell marker of early-stage B-cell development § Mediated by ABO blood group antibodies is _____, whereas ____ is a marker for later stages of ○ Acute B-cell development. § Days to weeks after transplant a. CD20; CD10 § Cytotoxic T-cell–mediated rejection b. CD21; CD10 CHAPTER 6 Immunology and Serology 195

c. CD10; CD20 15. A patient with a viral infection to the ABC virus is d. CD19; CD10 found to have a high antibody titer to the ABC virus’s 6. Adouble-positiveT cellwouldexpresswhichmarkers? RNA, or anti-ABCr. Which of the following is true? a. CD4+CD8+CD3+ a. MHC class I molecules presented antigen to b. CD4CD8+CD3+ CD4+ T cells c. CD4CD8CD3 b. MHC class II molecules presented antigen to d. CD4+CD8-CD3+ CD8+ T cells 7. Which cell is considered to be a bridge between the c. MHC class I molecules presented antigen to innate and adaptive immune systems? CD8+ T cells a. NK cell d. MHC class II molecules presented antigen to b. Mast cell CD4+ T cells c. Monocyte-macrophage 16. What is the main difference between agglutination d. T cell and precipitation reactions? 8. ______are involved in cell-mediated immunity, a. Agglutination occurs between a soluble antigen whereas ______are involved in humoral and antibody immunity. b. Agglutination occurs when the antigen is a. T cells; B cells particulate b. T cells; antibodies c. Precipitation occurs when the antigen is c. B cells; T cells particulate d. A and B d. Precipitation occurs when both antigen and anti- 9. Antigens that make very good immunogens include body are particulate which of the following? 17. Postzone causes false-negative reactions in antibody a. Carbohydrates titers as a result of which of the following? b. Proteins a. Too much diluent added to test c. Both a and b b. Excess antibody in test d. Neither a or b c. Excess antigen in test 10. The function of the complement system include(s) d. Incorrect diluent added to test which of the following? 18. Antibodies produced against two or more epitopes of a. Clearance of cellular debris specific antigen are considered ______. b. Chemotaxis a. Monoclonal c. Lysis of bacteria b. Pleomorphic d. All of the above c. Dimorphic 11. When C3 is cleaved by C3 convertase, what is the d. Polyclonal result? 19. In the radial immunodiffusion test, the gel contains a. C3a is released which of the following? b. C3b is used as an opsonin a. The antigen to be tested c. C3b is combined with other complement proteins b. Antibody to form C5 convertase c. Patient sample d. All of the above d. Noneoftheabove;thegelisthemediumtowhichthe 12. Characteristics of cytokines include which of the antibodyandantigenareappliedinequalproportion following? 20. Which statement is true regarding the radial immu- a. They can have a pleomorphic effect nodiffusion test? b. Cytokines are redundant a. The area of the precipitin ring is directly propor- c. Cytokines enhance cellular differentiation of tional to the concentration of antigen in the lymphocytes sample d. All of the above b. The area of the precipitin ring is directly propor- 13. Immunoglobulin idiotypes are antibodies with varia- tional to the concentration of antibody in the sample tions in the domains of which of the following? c. The area of the precipitin ring is directly propor- 1 2 a. CH and CH tional to the concentration antibody and the anti- b. VH and VL gen in the sample c. VH and CL d. The area of the precipitin ring indicates a partial 1 2 3 d. CH ,CH , and CH identity to the antibody in the sample 14. Mannose-binding lectin is similar to which compo- 21. The indirect antiglobulin test is for ______, nent of the classical pathway? whereas the direct antiglobulin test is for______. a. C3 a. Serum antigen; bound antigen b. C2 b. Serum antigen; bound antibody c. C1q c. Serum antibody; bound antigen d. C5a d. Serum antibody; bound antibody 196 CHAPTER 6 Immunology and Serology

22. In an indirect immunofluorescent antibody test for 29. A patient has the following hepatitis B serology: CMV antibodies, the conjugated antibody used for HBsAg: Negative visualizing is: Anti-HBc: Positive a. Antihumanglobulin conjugated to a fluorescent dye Anti-HBS: Positive b. Anti-CMVantibody conjugated to a fluorescent dye These results are consistent with which of the c. CMV virus conjugated to a fluorescent dye following? d. Antihuman globulin conjugated to an enzyme a. Acute hepatitis B 23. What is the difference between nephlometry and b. Chronic hepatitis B turbidimetry? c. Recovery from hepatitis B a. There is no difference between the two assays, d. Acute hepatitis A only in name 30. The HLA genes are inherited as: b. Nephlometry is a newer example of turbidimetry a. Diplotypes: Two diplotypes from each parent c. Nephlometry measures light transmitted through b. Haplotypes: One haplotype from each parent a solution, and turbidimetry measures light scat- c. HLAs are not inherited, instead are proteins tered in a solution absorbed onto cells d. Nephlometry measures light scattered in a solu- d. Only the HLA-A antigen is an inheritable trait tion, and turbidimetry measures light transmitted 31. Agglutination and precipitation that is visible through a solution depends on antigen–antibody ratios ______. 24. In an Ouchterlony immunodiffusion, the line of pre- a. With antigen in excess cipitation between the antibody and the antigen b. With antibody in excess wells forms an X. This reaction would be described c. That are equivalent as which of the following? d. All of the above 32. a. Nonidentity Which of the following cell types is implicated in b. Partial identity immediate hypersensitivity? c. Identity a. Neutrophil b. Mast cell 25. An initial titer of 4 followed by a subsequent titer c. Macrophage of 16 for the same patient, drawn 2 weeks later, is d. Monocyte indicative of which of the following? 33. Anti-dsDNA antibodies are associated with which of a. Infection the following? b. Convalescence a. Syphilis c. Past exposure b. CMV infection d. No exposure c. Systemic lupus erythematosus 26. A deficiency of T cells can result in which of the d. Hemolytic anemia following? 34. Rheumatoid factor is typically an IgM autoantibody a. Low levels of complement with specificity for which of the following? b. Dysfunctional macrophages a. SS-B c. Fewer B cells maturing to plasma cells b. Double-stranded DNA d. Contact dermatitis c. Ribonucleoprotein 27. A 2-week-old baby is seen for a possible infection with d. Fc portion of IgG CMV. Which of the following statements is false? 35. All of the following are autoimmune diseases except: a. A positive anti-CMV result from baby’s specimen a. Rheumatoid arthritis is inconclusive b. Rh disease of the fetus and newborn b. An initial titer of anti-CMV IgG would need to be c. Grave’s disease established d. Myasthenia gravis c. A positive result for anti-CMV IgM would indi- 36. In Grave’s disease, one of the main autoantibodies cate infection is: d. All are false statements a. Anti-CCP 28. What is the basic difference between the RPR and b. Antibody to islet cells of pancreas VDRL tests? c. Antibody to thyroid-stimulating hormone a. The RPR detects antigen, whereas the VDRL receptor detects antibody d. Anti-dsDNA b. The RPR test is read macroscopically, whereas the 37. An autoantibody found in patients with Hashimoto’s VDRL is read microscopically thyroiditis reacts with which of the following? c. The RPR test is a treponemal test, whereas the a. TSH receptor VDRL is nontreponemal b. Islet cells d. There is no difference because they are both spe- c. CRP cific tests for syphilis d. Thyroglobulin CHAPTER 6 Immunology and Serology 197

38. Skin testing for exposure to tuberculosis is an exam- 45. Severe combined immunodeficiency is characterized ple of which type of hypersensitivity? by which of the following? a. Type I a. Diagnosed in infancy b. Type II b. Shortened life span c. Type III c. No antibody production d. Type IV d. All of the above 39. Which of the following is a test for specific trepone- 46. A 3-year-old boy is seen by his physican because of mal antibody? many recent bacterial infections. Flow cytometery a. VDRL indicates normal levels of Tand B cells. The nitroblue b. RPR tetrazolium test for oxidative reduction is negative. c. FTA-ABS The most likely cause is: d. All of the above a. Wegener’s syndrome 40. Serum tested positive for HBsAg and anti-HBc IgM. b. Chronic granulomatous disease The patient most likely has which of the following? c. Bruton’s agammaglobulinemia a. Acute hepatitis C d. Diabetes mellitus b. Chronic hepatitis B 47. A 25-year-old man was seen by his physican for c. Acute hepatitis B recurrent infections. Immunoelectrophoresis d. Acute hepatitis A revealed hypogammaglobulinemia. This man most 41. The main difference between leukemias and lympho- likely has which of the following? mas is which of the following? a. Bruton’s agammaglobulinemia a. Leukemias are malignancies of cells in the bone b. Common variable immunodeficiency marrow c. X-linked agammaglobulinemia b. Lymphomas are maligniancies of cells in the bone d. DiGeorge’s syndrome marrow 48. In which disease would you expect to see an IgM c. Lymphomas are classified as either acute or spike on electrophoresis? chronic a. Transient hypogammaglobulinemia of infancy d. Leukemias are malignancies in lymphoid tissue b. Wiskott-Aldrich syndrome 42. A 1-year-old boy is seen for having many recurrent c. Leukocyte adhesion disease infections with Streptococcus pneumoniae. Labora- d. Waldenstro¨ m’s macroglobulinemia tory tests revealed a normal quantity of T cells, but 49. A person has an infected bug bite with pain, swelling, no B cells and no immunglobulins were seen on elec- and redness. What is the cause of these physical trophoresis. Which of the following would most symptoms of inflammation? likely be the cause? a. Production of antibody a. Chronic granulotomatous disease b. Secondary immune response b. Bruton’s agammaglobulinemia c. Increased blood flow and neutrophils to site c. DiGeorge’s syndrome d. Activation of NK cells d. Wiskott-Aldrich syndrome 50. The type of graft rejection that occurs within minutes 43. A patient with hereditary angiodema has which of of a tissue transplant is ______. the following deficiencies? a. Acute a. C5-9 b. Chronic b. Phagocytic cell function c. Hyperacute c. Mature B cells d. Accelerated d. C1 Inhibitor 44. A radiograph of a 1-year-old boy indicates the lack of a thymus. Complete blood count and flow cytometry confirm a below-normal lymphocyte count and a BIBLIOGRAPHY lack of T cells. Which of the following would most likely be the cause? Abbas AK, Lichtman AH: Basic immunology: functions and disorders of the immune system, ed 3, Philadelphia, 2011, Saunders. a. DiGeorge’s syndrome Brebner JA, Stockley RA: Polyclonal free light chains: a biomarker b. Wiskott-Aldrich syndrome of inflammatory disease or treatment target? Retrieved June c. Bare lymphocyte syndrome 2013 from http://f1000.com/prime/reports/m/5/4/. d. Bruton’s agammaglobulinemia Coico R, Sunshine G: Immunology: a short course, ed 6, Hoboken, NJ, 2009, John Wiley & Sons. Male D, Brostoff J, Roth DB, Roitt I: Immunology, ed 8, Philadelphia, 2013, Saunders. Turgeon ML: Immunology and serology in laboratory medicine, ed 4, St. Louis, 2009, Mosby. 198 CHAPTER 6 Immunology and Serology

SELF-ASSESSMENT

Content Area: ______

Score on Practice Questions: ______

List the specific topics covered in the missed questions:

List the specific topics covered in the correct questions: CHAPTER 6 Immunology and Serology 199

NOTES CHAPTER 7 Immunohematology and Blood Transfusion Medicine

Brenda C. Barnes and Elizabeth G. Hertenstein

BLOOD GROUP ANTIGENS AND ○ O gene does not encode a functional enzyme ANTIBODIES (silent allele—no detectable gene product) • H and Se genes • Red blood cells (RBCs) contain surface markers or • Hh and Se (secretor) are on chromosome 19 and are antigens on their surface or as part of their membranes closely linked • Surface markers are identified as antigens capable of • Each locus has two recognized alleles, one allele is initiating an immune response, which can affect an amorph RBC compatibility ○ H: Produces a glycosyltransferase that acts on • The antigens are either sugars or proteins type 2 chains—H antigen on RBCs ○ h: Amorph allele, does not express a detectable ABO Blood Group product—that is, no glycosyltransferase is produced See (Box 7-1) ○ Se: Produces a glycosyltransferase that acts on • Described by Karl Landsteiner in 1900 type 1 chains—H antigen in secretions • Most important blood group system: Individuals pos- ○ se: Amorph allele sess antibodies against antigens they lack • Bombay phenotypes • If A or B antigen is not present, person will make • Genotype hh known as Bombay phenotype antibody(ies) against the missing antigen(s) • hh: Very rare, does not make transferase to form the • This complementary relationship permits ABO test- H antigen ing of patient sera and RBCs • Characteristics • ABO antibodies are naturally occurring, stimulated ○ Bombay RBCs fail to react with anti-A, anti-B, or by nature anti-H lectin • Production initiated at birth, but titer low until 3 to ○ Bombay serum contains anti-A, anti-B, anti-A,B, 6 months of age and Anti-H (potent, reacts strongly at 37 C) • Genetics ○ Only blood from other Bombay phenotypes can • Antigens in this group exhibit autosomal codomi- be transfused nant inheritance • Para-Bombay phenotype ○ One locus on chromosome 9 occupied by one of • Genotype: hh Se three alleles: A, B, O • Normal amount of H antigen is detectable in saliva ○ Each person has a pair of chromosomes that because these people are secretors carry one allele • May produce a weak anti-H ○ Group O: Silent allele (gene product not • Subgroups detectable) • A subgroups • Genes at three separate loci control the presence ○ 80% A1 and location of the A, B, and H antigens: ABO, ○ 20% A2 or weaker Hh, and Se (Figure 7-1) ○ Inheritance of A2 gene: Small amount of H anti- • A and B genes encode glycosyltransferases that pro- gen conversion duce A and B antigens ○ Immunodominant sugars same ○ Genes do not directly encode for the antigen ○ Differentiation of A1 and A2 subgroups based on ○ Genes encode for the enzyme that transfers the reactivity with anti-A1 (lectin [Dolichos biflorus] immunodominant sugar that confers the specific- or human-based) ity (see Figure 7-1) § A1 cells react with anti-A1

200 CHAPTER 7 Immunohematology and Blood Transfusion Medicine 201

• Quantity of H Substance Found on Red Routine ABO testing (Table 7-1) BOX 7-1 Blood Cells by Blood Group • Forward typing or RBC typing ○ Anti-A and anti-B ○ H Substance Reagents used are generally monoclonal Large Amount Least Amount antibodies O>A2B>A2B>A1>A1B § Designed to give strong reactions § Should see at least a 3+ or greater reaction in the front type when using monoclonal reagents § A2 cells do not react with anti-A1 • Reverse typing or serum typing • Weak A subgroups ○ A1 cells and B cells: Reagent cells used for ABO ○ Show weaker reactivity than A2 typing are Rh negative ○ 1% population ○ Expect to see a 2+ reaction: Anything weaker ○ A3: Mixed-field pattern of agglutination with could be indicative of a serum problem anti-A • Both are required in patients and donors ○ Ax: Stronger reaction with anti-A,B than anti-A ○ Serve as a check for each other ○ Weaker subgroups detected only by elution and • Nonroutine reagents adsorption of anti-A ○ Anti-A,B • B subgroups § Used to aid in classification of subgroups ○ Very rare § Confirm group O units (retype) ○ Little consistency in description ○ A2 cells ○ Usually recognized by variations in strength of § Discrepancy resolution reaction with anti-B and anti-A,B ○ Lectins • Antibodies to A and B antigens § Anti-A1: D. biflorus • People typed as A and B generally produce IgM § Anti-H: Ulex europaeus class ABO antibodies • ABO discrepancy resolution • Small quantities of IgG are present • Causes of discrepancies are classified into four • People typed as O produce IgG class antibodies— groups anti-A,B • Weak or missing antigen reactivity: Cell grouping ○ Readily crosses the placenta tests

H antigen Protein Gal R or Gal GlcNAc Lipid Gene product: 1→ 4 1→ 3 L-Fucosyltransferase 1→ 2 Immunodominant sugar: L-Fucose Fuc

A antigen Protein Gal R or Gene product: Gal GlcNAc Lipid → → N-Acetylgalactosaminyltransferase GalNAc 1 4 1 3 1→ 3 Immunodominant sugar: → 1 2 N-Acetylgalactosamine

Fuc

B antigen Protein Gal R or Gene product: Lipid Gal GlcNAc D-Galactosyltransferase → → Gal 1 4 1 3 1→ 3 Immunodominant sugar: 1→ 2 D-Galactose

Fuc

FIGURE 7-1 Biochemical structures of the H, A, and B antigens. Gal, D-Galactose; GlcNAc, N-acetylglucosamine; Fuc, L-fucose; GalNAc, N-acetylgalactosamine. (From Funk MK, Grossman BJ, Hillyer CD, et al, editors: Technical manual, ed 18, Bethesda, MD, 2014, AABB, p 293.) 202 CHAPTER 7 Immunohematology and Blood Transfusion Medicine

§ TABLE 7-1 ABO Typing Interpretation Test RBCs with human anti-B acidified to pH 6.0 Forward Type ○ Positive direct antiglobulin test (DAT) (Patient Red Reverse Type § Strongly reactive DAT cells can spontane- Blood Cells) (Patient Serum) ously agglutinate with cell grouping reagents Anti-A Anti-B A Cells B Cells Interpretation § Most often seen with Rh typing reagents 1 § 00+ + O Can occur with ABO reagents if coating +00 + A antibody is cold-reactive § 0++ 0 B Resolution ++0 0 AB § Wash cells with 37 C saline § Incubate patient cell suspension at 37 C and wash with warm saline § Or can elute antibodies from RBCs with chlo- ○ Subgroups of A and B roquine diphosphate or dithiothreitol (DTT) § A3 subgroup ○ Contaminated cord blood samples § Mixed-field pattern with anti-A from § Wharton’s jelly is main cause group O or B donors § Resolution § Ax subgroup § Wash with saline 3 or 4 times, retest § No agglutination with human anti-A from § Request heel-stick sample group B donors ○ Unwashed cell suspensions § Agglutination with anti-A,B from group O § Washing can dissipate problems caused by donors § Patient antibodies to reagent components § May react with monoclonal anti-A § Rouleaux formation § Ael subgroup • Weak or missing antibody reactivity: Serum group- § Not agglutinated by anti-A or anti-A,B of ing tests any origin ○ Low antibody levels (newborns or older patients) § A antigen demonstrable only by adsorption ○ Missing antibodies (immunocompromised and elution studies patients) ○ B subgroups: Rare ○ Chimeras: Persistent chimeras develop a toler- ○ Cis-AB phenotype: Rare chromosome ance to both cell populations—check patient ○ Newborns: Lower number of A and/or B history! antigen sites ○ Subgroups: Cells from A subgroup patients often ○ Leukemia: Weak expression of A and/orB antigens typed as group O ○ Mixed-cell populations and chimeras: Review § Titer of anti-A is usually higher than that of transfusion and transplant history anti-B in most group O individuals ○ Transfusions are the most common cause of § Weak reactivity with A1 cells in group O per- chimerism son should be investigated ○ Excessive blood group substance: Can neutralize § Same is true for group B people reagents § May be due to anti-A1 or other alloantibodies • Extra antigen reactivity: Cell grouping tests and not by anti-A ○ Acquired B § Testing panel of A1,A2, and O cells can help § Patient types as AB, but serum contains anti-B determine if a discrepancy exists § Transient condition associated with disorders • Extra antibody reactivity: Serum grouping tests of gastrointestinal tract ○ Rouleaux § Certain clones used to make monoclonal anti-B § Resolution is generally to try saline replace- cause strong reactions with acquired B cells ment technique § Strength of reactivity weakened with reduced ○ Cold-reactive antibodies (autoantibody or reagent pH alloantibody) § Resolution § Mini–cold panel used to aid in resolution § Review patient’s clinical history and histor- § Testing with A2, O, and autologous cells, in ical blood type addition to tests with A1 and B cells can help § Associated with colonic bacterial explain serum discrepancies infections ○ Passively acquired antibodies: Check patient § Test autocontrol or other acquired B cell transfusion history! ○ Will be negative if patient has acquired B • Suggested resolution process for ABO serologic § Test cells with monoclonal anti-B reagent that problems does not detect acquired B ○ Repeat testing on same sample CHAPTER 7 Immunohematology and Blood Transfusion Medicine 203

○ Wash patient cells ○ Nonglycosylated, transmembrane, integral part ○ Obtain patient information of RBC membrane (Figure 7-3) § Diagnosis • D antigen § Historical blood group • Most clinically significant non-ABO antigen § History: Transfusions, transplants, medica- • Highly antigenic tions • Described as a “mosaic” ○ Review results with group O RBCs and ○ Several subparts make up the complete antigen autocontrol ○ D+individuals usually have all parts § Alloantibodies or autoantibodies ○ Dindividuals have no parts of the antigen ○ Obtain new sample if contamination is suspected • Weak D antigen ○ Weakened expression of D antigen Rh Blood System ○ D antigen reactive only at antihuman globulin (AHG) • Genetics ○ Results from an atypical inheritance at D locus • Two closely linked genes encode Rh antigens § Partial D (mosaic) ○ RHD: Determines D expression (D or non-D) § Position effect (D gene inherited trans to ○ RHCE: Determines C, c, E, e antigens (alleles: ce, C gene) Ce, cE, CE) § Genetic (variant Ro gene in blacks) • Over 50 antigens define system ○ Testing for weak D • D, C, E, c, e: Most typically discussed (Figure 7-2) § Performed at AHG phase, must include a con- • Biochemistry trol (must be negative for test to be valid)

Current Rh genetic theory: 2 loci Fisher-Race genetic theory: 3 loci Wiener genetic theory: 1 locus

RHD D or d Alleles: shorthand R0, R1, R2, Rz, Allele: D r, r , r , r y Antigens: D-positive or C, c, or Cw D-negative Antigenic specificities: RHCE Dce, DCe, DcE, DCE, E or e Alleles: RHCE, RHCe, RHcE, dce, dCe, dcE, dCE RHce Antigens: CE, Ce, cE, ce

FIGURE 7-2 Comparison of Rh genetic theories. Comparison of three Rh genetic theories that have influenced the nomenclature of the Rh blood group system. Modern molecular techniques have established that the Rh blood group system antigens are determined by two genetic loci. (From Blaney K: Basic & applied concepts of blood banking and transfusion practices, ed 3, St. Louis, 2012, Mosby.)

103 Cc 226 Ee Cell Exterior Amino acid chain

Lipid bilayer

NH2 COOH Cell Interior

Antigen Amino acid Number

C Serine 103 Cc c Proline 103 Cc E Proline 226 Ee e Alanine 226 Ee

FIGURE 7-3 Model of the Rh polypeptide. Model of the differences in the amino acid sequence for the antigens produced by the RHCE gene. The basic structure is similar. Differences in the amino acid at the residue number indicated determine the serologic typing to be C or c, E, or e. (From Blaney K: Basic & applied concepts of blood banking and transfusion practices, ed 3, St. Louis, 2012, Mosby). 204 CHAPTER 7 Immunohematology and Blood Transfusion Medicine

§ Donated units of blood and blood products must • Duffy antigens do not store well (elute) have weak D type determined before labeling ○ Destroyed by common enzymes § Transfusion recipients: Typing for weak D not ○ Only moderate immunogens required ○ Antibodies § Obstetric patients: D and weak D type deter- § IgG class mined in first trimester § May cause HTRs, HDFN is uncommon § Infants born to D-negative mothers should be § Nonreactive in enzyme tests (antigens are tested for D antigen including weak D degraded by enzymes) • Rh antibodies § May show dosage • Clinically significant (can cause hemolytic disease ○ Miscellaneous of the fetus and newborn [HDFN] and hemolytic § 68% of blacks are Fy(ab) transfusion reaction [HTR]) § Whites are most commonly Fy(a+b+) • Immunoglobulin G (IgG), subclass 1 or 3 • Ii • Produced after exposure to foreign RBCs • I: High-frequency antigen expressed on adult RBCs • Very immunogenic, especially D ○ Exists on precursor A, B, and H chains • Order of immunogenicity: D>c>E>C>e ○ Associated with branched chains • Most react at 37 C and AHG • i: Expressed on newborn or cord cells • Enhanced by enzymes or high-protein additives ○ Not antithetical to I • Usually do not bind complement ○ Expressed on linear precursor chains • May show dosage with a homozygous expression of ○ i matures to be I as precursor chains become antigen more branched • May occur in concert • Antibodies ○ Anti-c and anti-E § Anti-I is a common IgM autoantibody ○ Anti-D and anti-G (anti-G reacts with all § Enhanced by enzymes D-positive and C-positive RBCs) § May mask clinically significant alloantibodies • Persist for years and remain detectable • Kell • Rh (D) typing • K and k: Codominant alleles • Routine testing is for D antigen only • Antigens destroyed by sulfhydryl reagents • Most testing now performed with licensed mono- (2-mercaptoethanol [2-ME], DTT, s-[2-aminoethyl] clonal/polyclonal blend reagents isothiuronium bromide [AET]) • Rh control required if high-protein reagents used • Antibodies • Unusual phenotypes § Usually IgG, reactive at AHG • Rh null § Some examples do not react well in a low ionic ○ Individuals whose RBCs lack all Rh antigens strength saline (LISS) environment ○ Rh phenotype is / § Can show dosage ○ Two mechanisms § Can cause HTRs and HDFN ¼ ¼ § Amorph gene (r r ) at Rh locus • Miscellaneous 0 0 § Suppressor gene (X rXr) at regulator locus ○ Ko cells lack all antigens of the Kell system ○ Form anti-Rh 29 (total Rh) ○ McLeod phenotype: Depressed Kell system • Rh deletions antigens ○ Rh complexes lacking alleles at Ee or Cc locus § Inherited as X-linked trait ○ D––/D–– individuals § Persons have poorly defined abnormality of § Lack all C and c antigens neuromuscular system § Lack all E and e antigens § Associated with chronic granulomatous disease § Lack many high-incidence Rh antigens • Kidd a b § Form anti-Rh 17 (anti-Hro) if exposed to • Jk and Jk : Codominant alleles normal RBCs • Antigens located on RBC urea transporter ○ D–– RBCs have greatest amount of D antigen • Antibodies (because all Rh material is converted to ○ IgG, reactive at AHG D antigen) ○ Often show dosage ○ Enhanced by enzyme-treated cells Other Blood Group Systems ○ May bind complement ○ Do not store well, reactivity can quickly decline • Duffy in vitro • Fya and Fyb: Codominant alleles ○ Can cause HDFN, but usually mild • Most important antigens in Duffy system ○ HTRs and delayed HTRs because antibody titers • Antigens well developed at birth can rise and fall quickly in patients CHAPTER 7 Immunohematology and Blood Transfusion Medicine 205

• Lewis • MNS • Lea and Leb: Note that these are not alleles ○ Complex system of over 40 antigens: M, N, S, s, • Antigens are not intrinsic to RBCs and U are most important • Only expressed on type I precursor chains, adsorbed § M/N (glycophorin A [GYA]) and S/s (glyco- onto RBCs phorin B [GYB]) are both codominant ○ Oligosaccharides intrinsic to RBC membrane are alleles all type II § U expressed on GYB close to RBC membrane • Not found on cord cells because Lewis antigens are ○ Antibodies poorly developed at birth § Anti-M • Le gene fucosyltranferase attaches fucose in § Saline reactive (RT or lower) a(1!4) linkage to the subterminal GlcNAc § Often found in sera of persons with no his- ○ Lea antigen tory of exposure to RBCs • Se gene fucosyltranferase attaches fucose in § IgG or IgM a(1!2) linkage to the terminal Gal □ IgG is rarely clinically significant ○ Leb antigen § Usually do not bind complement • Le and Se gene interaction § Do not react with enzyme-treated cells ○ Le without Se § Acidic test system may aid detection § Lea on RBCs and in saliva § Demonstrates dosage ○ Le and Se § Common in children and burn patients § Leb on RBCs, Lea and Leb in saliva § Rarely causes HTR or HDFN ○ lele, secretor status irrelevant § Anti-N § Le(ab) on RBCs and in saliva § Rarely encountered • sese § IgM cold-reacting antibody ○ No H in secretions (Figure 7-4) § Usually not clinically significant • Lewis antibodies § Anti-S and anti-s ○ Almost always IgM: Do not cross placenta § Usually clinically significant IgG anti- ○ Not associated with HDFN bodies reactive at 37 C and at AHG phase ○ May bind complement § May/may not react with enzyme- ○ Most often room temperature (RT) reactive treated cells ○ May be seen at 37 C, but at decreased strength § May bind complement ○ Rarely seen at AHG phase § May cause HTR and HDFN ○ Neutralization techniques may be helpful to con- § Anti-U firm presence of Lewis antibody or determine § U is a high-incidence antigen presence of other underlying antibodies § Suspect anti-U in patients who are black and Ss, whose serum shows an antibody reacting to a high-incidence antigen • P blood group ○ Contains only P1 antigen ○ Globoside collection: P, Pk, LKE Lewis Antigens ○ Antigens are “built” on glycolipids, analogous to development of A, B, and H antigens ○ Phenotypes GAL GlcNAc GAL * ○ P1 § RBCs have P1 and P antigens § a Analogous to A1 subgroup •Le antigen ○ Fuc P2 •Le without Se § RBCs cells have P antigen § Analogous to A2 subgroup ○ Antibodies § Anti-P1 * GAL GlcNAc GAL § Formed by P2 individuals § Naturally occurring § •Leb antigen Optimal reactivity is at 4 C Fuc Fuc § Almost always IgM •Le and Se § Can be neutralized with commercially available P1 substance § Anti-P (usually found as an autoantibody) FIGURE 7-4 Lewis antigens. § Cold reactive IgG autoantibody 206 CHAPTER 7 Immunohematology and Blood Transfusion Medicine

§ Associated with paroxysmal cold ○ Fibrin clots formed with serum may be present in hemoglobinuria incompletely clotted specimens, may cause false- § Biphasic antibody positive reaction in gel testing § Attaches to RBC when cold ○ Clotting may be incomplete in certain patients— § Lysis occurs as RBC warms for example, patients being treated with heparin § Donath-Landsteiner test • Antibody screens and identification are performed to detect antibodies in ANTIBODY SCREEN AND • Patients requiring transfusion IDENTIFICATION • Obstetric patients • Patients with suspected transfusion reactions • Acceptable sample to use for transfusion testing • Blood and plasma donors • Patients who have blood drawn for transfusion ser- • Must include 37 C incubation and use of an vice requests must be properly identified by name antiglobulin test and unique identification number • Reagents • There must be a record of phlebotomist or person • Screening cells who drew the specimen ○ Group O RBCs that provide specific antigens • Date specimen was drawn must be on ○ Usually distributed in two-vial or three-vial sets specimen label (Table 7-2) • Specimen must not be hemolyzed • Enhancement media increases sensitivity of a test • Plasma and serum specimens are appropriate to use; system (Table 7-3) often plasma is preferred • AHG reagents

TABLE 7-2 Representative Antibody Screen Antigen Sheet (Three-Vial Set)

DCcEeKkFya Fyb Jka Jkb MNSs ++00+0 ++ 0 + 0 + +0+ +0 ++0+++ + + + + 0 +0 0 0 +0+0 +0 + 0 + 0 +0+

NOTE: Only major antigen systems are displayed.

TABLE 7-3 Comparison of Potentiators and Methods

Potentiator Use Limitations Low-ionic-strength saline Sensitive, economical, and allows for shorter Enhances cold autoantibodies (LISS) incubation time Some weak anti-K antibodies may be missed Equal parts of plasma/serum and LISS is important Bovine serum albumin Affects second stage of agglutination Needs longer incubation (BSA) Does not enhance warm autoantibodies Not sensitive for most antibodies except in Rh blood group system Polyethylene glycol (PEG) Shows increased sensitivity Enhances warm autoantibodies Recommend using anti-IgG AHG, not anti-IgG, -C3d AHG No 37 C readings May require extra wash Enzymes: ficin, papain Eliminates reactivity of Fya,Fyb, M, and N antigens; Enhances cold and warm autoantibodies S and s antigens are variable Should not be used as the only method Enhances Rh, JK, LE, P1 antibodies Gel technology Avoids cold reactive antibodies Enhances warm autoantibodies Shows increased sensitivity Weak anti-K may be missed owing to LISS-suspended red Can be automated cells Solid phase (SPRCA) Avoids cold reactive antibodies Enhances warm autoantibodies Shows increased sensitivity Weak anti-K may be missed owing to LISS potentiators Can be automated Manual method may be difficult to read From Blaney K, Howard P: Basic & applied concepts of blood banking and transfusion practices, ed 3, St. Louis, 2012, Mosby. AHG, Antihuman globulin. CHAPTER 7 Immunohematology and Blood Transfusion Medicine 207

○ Used to detect clinically significant antibodies • Screen will not detect antibodies to low-frequency ○ Must contain anti-IgG when used for antibody antigens that are not present on any of the detection and compatibility testing screening cells ○ Polyspecific AHG • Factors affecting sensitivity § Contains both anti-IgG and anti-complement • Cell-to-serum ratio (usually anti-C3d or anti-C3b) ○ Antibody in excess: False-negative because of § Used primarily in DAT testing prozone ○ Monospecific AHG ○ Antigen in excess: False-negative because of § Used in differential DAT testing, antibody postzone detection, and identification ○ Increasing the amount of serum in the test system § Directed against one type of globulin can help increase sensitivity when working with § Monospecific anti-IgG weak antibodies, but potentiators usually cannot § Monospecific anti-complement (anti-C3d be used (check manufacturer’s instructions) and/or anti-C3b) • pH ○ Coombs control cells ○ Most antibodies react best at neutral pH of 6.8 § Use required for control of negative AHG tests to 7.2 § Must react when added to negative AHG tests • Temperature or test must be repeated ○ Clinically significant antibodies generally react § Verifies that adequate washing was performed at 37 C or at AHG phase § Also verifies that AHG was added and work- ○ Immediate spin and RT phases are often omitted ing properly to limit detection of insignificant cold antibodies • Indirect antiglobulin test method (NOTE: This is the • Length of incubation method for tube testing; solid phase and gel testing ○ Too little contact time, not enough cells are procedures will differ) sensitized • Label tubes ○ Incubation time too long, bound antibody may • Add 2 drops patient serum to each tube dissociate • Add 1 drop reagent screen cells to each tube ○ Time depends on media used: Follow manufac- • Centrifuge and observe for hemolysis and turer’s instructions agglutination • Grading agglutination reactions (tubes) (Box 7-2) ○ Some institutions refer to this phase as • Reactions are graded using a qualitative standard immediate spin scale • Grade and record results (some institutions may ○ Gives information on omit this step) § Strength of reaction • Add 2 drops of enhancement reagent to tubes § Amount of antibody available to form anti- (if used) gen–antibody complexes • Incubate 15 to 20 minutes at 37 C ○ 4+ reaction • Centrifuge and observe for hemolysis and § Solid agglutinate, clear supernatant background agglutination § No free RBCs detected • Grade and record results (some institutions may ○ 3+ reaction omit this step) § RBC button breaks into several large aggluti- • Wash four times nates, clear supernatant background • Decant last wash to dry cell button ○ 2+ reaction • Add 2 drops AHG § RBC button breaks into many medium-sized • Spin, read, and record results agglutinates, clear supernatant background, ○ Negative: Add 1 drop of Coombs control cells, no free RBCs spin, and read; must be positive ○ 1+ reaction ○ Positive at any phase: Investigate § RBC breaks into numerous medium-sized and • Negative antibody screen small agglutinates, background is turbid with • Patient is eligible for immediate spin or electronic many RBCs crossmatches, if no history of clinically significant antibodies ○ History of antibodies, must do indirect antiglobulin test (IAT) crossmatch BOX 7-2 Grading Agglutination Reactions • Limitations of antibody screen It is important for reaction grading to be standardized to maintain • Antibody screen will not detect antibodies when the consistency among different technologists. Each institution will have titer has dropped below the sensitivity level of the policies in place describing their reaction grading procedure. screening method being used 208 CHAPTER 7 Immunohematology and Blood Transfusion Medicine

4ϩ Red cell button is a solid agglutinate; clear background

3ϩ Several large agglutinates; clear background

2ϩ Many medium-sized agglutinates; clear background

ϩ Medium- and small-sized agglutinates; background is turbid 1 with many free red cells

No agglutinated red cells are visible; red cells are observed 0 flowing off the red cell button during the process of grading

FIGURE 7-5 Grading antigen–antibody reactions. Consistency in grading reactions allows for correct interpretation of results in the immunohematology laboratory. (From Immucor, Inc.)

○ Negative § Intravenous immunoglobulin, Rh immuno- § No agglutinated RBCs are visible, RBCs are globulin (RhIG): Passive antibody transfer observed flowing off the cell button during ○ Diagnosis, age, race the process of grading (Figure 7-5) § Offer additional clues to nature of antibody • Antibody identification problem • Clinically significant antibody: Some define as an • Antibody panel antibody that reacts in vitro at 37 C and/or by ○ Group O RBCs IAT, but this is not always true ○ Available in sets ranging from 10 to 20 vials • Better characterized as an antibody that shortens ○ Thought of as extended antibody screens the survival of transfused RBCs or has been associ- (Table 7-4) ated with HDFN ○ Best to test using same enhancement media used ○ Rely on published data to determine clinical in the screen significance ○ Panel antigen profiles are lot-specific—make ○ If data are scarce or unavailable, respect the anti- sure you have the correct one bodies that react at 37 C and/or on IAT ○ Grade consistently using laboratory guidelines • Interpretation of the positive antibody screen can ○ Autocontrol (Figure 7-6) provide initial clues § Patient’s RBCs tested against patient’s serum • Initial considerations in the same manner as the antibody panel ○ Phase of reactivity § Optional to perform ○ Possible warm or cold autoantibody or § Not normally done with screen alloantibody? § Most workers test autocontrol (AC) with an ○ True agglutination or rouleaux? antibody panel ○ Does patient have previously identified antibodies? § Helps determine whether alloantibody or • Patient history: It is helpful to find out the following autoantibody specificity exists before the antibody identification process • Panel interpretation ○ Transfusion history ○ Phase of reactivity § Recent transfusions may indicate recent anti- § IgM reacts best at low temperatures (immedi- body stimulation ate spin phase) § Antigen typing considerations § IgG reacts best at AHG phase ○ Pregnancy history § Reactions at more than one phase may indi- § Recent antibody stimulation cate a combination of IgM and IgG antibodies ○ Recent drug therapy (Table 7-5) CHAPTER 7 Immunohematology and Blood Transfusion Medicine 209

AC pos at AHG No Suspect phase? alloantibody

Ye s No Suspect in vitro Positive? phenomenon Perform DAT Ye s

Ye s Patient transfused?

Elution No No further work required Ye s Evidence of No Hemolysis?

FIGURE 7-6 Suggested flowchart for interpretation of positive autocontrol.

TABLE 7-4 Representative Antibody Panel Antigen Sheet (11-vial set)

Cell No. D C c E E K k Fya Fyb Jka Jkb MNSs 1 ++0++0 ++ 0 + + + +0+ 2 ++00+++0 + 0 + + 00+ 3 +0 ++00 +0 + 0 + 0 +0+ 4 +0+0+0+0 0 + 0 + +00 5 0++0+0+0 + + + 0 +0+ 6 0 0 +++0 ++ 0 + + + 0 0+ 700+0+++0+++++++ 8 00+0+0++ 0 + 0 + +0+ 9 ++00+++0 + + + + ++0 10 ++0++0 ++ 0 + + + 0 ++ 11++00++++ 0 0 + 0 +0+ AC

NOTE: Only major antigen systems are displayed.

TABLE 7-5 Antibodies and Phase of Reactivity

Phase Room Temperature 37 C AHG a b a b a Antibodies Cold autoabs (I, H, IH), M, N, P1,Le,Le,Lu Potent cold autoabs, D, E, K Rh, K, Duffy, Kidd, S, s, Lu ,Xg Immunoglobulin class IgM IgG IgG Clinically significant No Yes Yes

§ Reaction strength § Suspect delayed transfusion reaction if § Clue to number of antibodies present patient recently has received a transfusion § Varying strengths suggest more than one § DAT should be performed antibody or dosage § Exclusion or ruling out – Provisional step only § Autocontrol (see Figure 7-6) § Panel cells that give negative reactions in all § If negative, suspect alloantibody phases can be used for exclusion § If positive, suspect autoantibody § Begin with the first nonreactive cell 210 CHAPTER 7 Immunohematology and Blood Transfusion Medicine

§ Examples of Substances for Clues that indicate multiple antibodies Neutralization for Certain □ Observed pattern does not fit that of a TABLE 7-6 Antibodies single antibody § Determine whether pattern fits com- Antibodies Neutralization Substances bined specificities □ Reactivity is present at different test Anti-P1 Hydatid cyst fluid, pigeon droppings, turtledoves’ egg white, commercial P1 phases substance § Evaluate each phase separately Anti-Lewis Plasma or serum, commercial Lewis substance □ Unexpected reactions occur when trying Anti-Chido, Anti- Plasma or serum to confirm the specificity of a suspected Rogers single antibody a Anti-Sd Urine § Test selected cells □ No discernible pattern § Perform exclusions to eliminate some specificities § Look across the panel and place a line § Test cells with strong antigen expres- through the antigen specificity that is posi- sion or increase sensitivity of the test tive (+) on the panel system § Continue with each nonreactive cell § Phenotype the patient § Provisional step only § Use of enzymes can help rule in or out § Matching the pattern • Finding compatible blood § Look at the reactions that are positive and ○ Phenotype frequency match the pattern § Can help determine number of units compat- § Single antibody will match one of the anti- ible for a patient with antibodies gen columns § How many units are compatible with a § If specificities remain that have not been patient with anti-Jka? excluded, additional testing is required § 77% of random population is Jk(a+) § Probability or “rule of three” § Combined phenotype calculations § Used to ensure pattern of reactivity is not § Can estimate number of units that will have due to chance alone to be tested to find a certain number of § Conclusive evidence derived by testing antigen-negative units enough antigen-positive and antigen- ○ Example negative cells § Patient has anti-c, anti-K, anti-Jka § Many laboratories require a probability (p) § How many units must be tested to find four of value of .05 or less the appropriate phenotype? □ Means a 5% chance the observed pat- § C negative: 20% tern happened by chance alone or you § K negative: 91% are correct 95% of the time § Jk(a) negative: 23% § Standard approach has been to require three § Multiply the individual phenotypes to get the antigen-positive cells that react and three combined phenotype antigen-negative cells that fail to react § 0.200.910.23¼0.04 or 4% of individ- □ Required for each antibody specificity uals will be cKJk(a) □ Institution’s approach may differ,but this § 100 units will have to be tested to find 4 units is the current academic recommenda- • Hardy-Weinburg law tion—answer test questions accordingly • Mathematical formula used to explain persistence § Phenotype the patient of recessive alleles in a population § Individuals cannot make alloantibodies to • Based on the following assumptions antigens they possess ○ Population must be large, and mating must occur § Test patient RBCs to ensure they are nega- at random tive for the antigen corresponding to the ○ Mutations must not occur identified antibody ○ There must be no migration, differential fertility, □ Testing complications include a positive or mortality of the genotype patent DAT or recent transfusion • Equation for a two-allele system § Multiple antibodies ○ p2 +2pq+q2 ¼1 § Serum with two or more alloantibodies ○ p+q¼1 may make interpretation of test results • Blood bank example difficult ○ Kidd blood group system (Jka and Jkb) CHAPTER 7 Immunohematology and Blood Transfusion Medicine 211

§ p¼frequency of Jka allele ○ Autoimmune hemolytic anemia § q¼frequency of Jkb allele § Patient autoantibody § p2 ¼frequency of JkaJka genotype ○ Hemolytic disease of the newborn § 2pq¼frequency of JkaJkb genotype § Maternal antibody § q2 ¼frequency of JkbJkb genotype ○ Drug-related mechanism ○ 77% of population expresses Jka antigen § Drug/antidrug complex § p2 +2pq¼frequency of persons who are Jk ○ Transfusion reactions (a+) and carry the allele Jka § Recipient antibody against donor cells § q2 ¼1(p2 +2pq)¼frequency of persons who • Elution is a technique used to dissociate IgG anti- are Jk(a) bodies from sensitized RBCs § q2 ¼p1ffiffiffiffiffiffiffiffiffiffi0.77¼0.23 • The recovered antibody is called an eluate and can § q¼ 0:23 be tested like serum to determine antibody § q¼0.48 (allele frequency of Jkb) specificity ○ The sum of frequencies of both alleles must equal • Various methods 1.00 ○ Temperature variation § p+q¼1.00 § Heat elution § p¼1q § Freeze–thaw elution § p¼10.48 ○ pH manipulation § p¼0.52 (allele frequency of Jka) § Acid elution is most commonly performed ○ Number of Jk(b+) persons can be calculated § Acidic solution decreases pH and disrupts § 2pq+q2 ¼frequency of Jk(b+) antigen–antibody bond, releasing antibody § ¼2 (0.520.48)+(0.48)2 into acid solution (eluate), with buffer § ¼0.73 added at end of procedure to raise pH ○ Chemical CROSSMATCH AND SPECIAL TESTS • Adsorption is the process of removing antibody from serum • Crossmatch: Performed before transfusion of donor ○ Patient serum is incubated with appropriate products containing RBCs RBCs under optimal conditions depending on • Immediate spin crossmatch the antibody to be removed ○ Patient has no clinically significant antibody(ies) § Warm: 37 C detected and no history of antibody(ies) § Cold: 4 C ○ Checks for ABO compatibility • Most commonly used to remove autoantibodies ○ Procedure from serum § Mix patient serum with donor cells • Autoadsorption § Spin and read ○ Limited by transfusion history and amount of • Antiglobulin crossmatch sample ○ Patient has currently reactive antibody(ies) or ○ Patients transfused within previous 3 months do history of an antibody(ies) that may or may not qualify not be demonstrating ○ Severely anemic patients may not have enough ○ Procedure RBCs to carry out procedure § Same as immediate spin ○ Cells are usually treated with enzyme to remove § Continues to include 37 C incubation bound autoantibody § Concludes with AHG phase (review IAT § Examples: DTT, chloroquine diphosphate procedure) (CDP), ethylenediaminetetraacetic acid § Enhancement media can be used (EDTA)-glycine-hydrogen chloride (HCL) § Recommended to use same methodology as ○ Alloadsorption used for the antibody screen/identification § Cells phenotypically similar to those of • Electronic crossmatch patient are used ○ Computer is used to make the final check of ABO § Enzyme treatment removes some antigens to compatibility aid in matching and enhances antibody uptake ○ Specific eligibility requirements apply • Titration studies are performed to measure the titer ○ Patient cannot have history of or any currently of an antibody (quantify amount of antibody reacting unexpected antibodies present) • Special tests ○ Twofold serial dilutions of serum are tested • DAT: One-step test to detect RBCs sensitized with against antigen-positive RBCs antibody or complement in vivo ○ Reciprocal of the highest dilution showing visi- • Used to diagnose certain clinical events ble agglutination is the antibody titer 212 CHAPTER 7 Immunohematology and Blood Transfusion Medicine

○ Most often used to monitor quantity of antibody BOX 7-3 Kleihauer-Betke Calculation Example in a woman’s serum during pregnancy ○ Considered significant: Fourfold increase Example: (i.e., 4 to 16) • Kleihauer-Betke reported as 1.3% • Prewarm technique is a procedure in which RBCs • (1.3/100)5000 mL* ¼ 65 mL of fetal blood and serum (or plasma) are prewarmed separately • 65/(30 mL/dose)¼2.2 doses of Rh immunoglobulin required to 37 C before they are combined *Mother’s arbitrarily assigned blood volume. ○ May be used when cold autoantibodies are present that may mask the presence of clinically • Principle: In slides subjected to an acid elution step, significant antibodies adult Hgb dissolves out of cells, whereas HbF, ○ Should be used with caution which is acid resistant, remains intracellular and § Reactivity of potentially significant antibodies is stained can be diminished • Procedure § Weak antibodies can be missed ○ Maternal blood smear treated with acid • Neutralization uses soluble antigen to inhibit reac- ○ Stained with counterstain tivity of certain antibodies ○ 2000 cells counted under microscope ○ Soluble antigen added to serum sample ○ Percent of fetal cells calculated ○ Mixture incubated at room temperature to neu- • Limitations tralize (by occupying antigen-binding sites) ○ Precision and accuracy may be poor ○ Neutralized serum tested against RBCs (panel ○ Slide quality cells) ○ Area of slide counted ○ Must have control (saline), it must be reactive to ○ Judgment of technologist ensure neutralization occurred and reactivity was • Calculation not eliminated because of dilution (see Table 7-6) ○ 300 mg dose of RhIG will protect against up to • Rosette test (Figure 7-7) 15 mL of D-positive RBCs (approximately ○ Principle: Demonstrates small number of D- 30 mL of fetal whole blood) positive cells in D-negative cell suspension ○ Number of doses of RhIG required is determined § Cell suspension is incubated with human ori- by dividing the estimated volume of fetal blood gin anti-D present by 30 (Box 7-3) § Some kits use chemically modified anti-D • Safety margin § Antibody attaches to sites on D-positive cells, ○ When number to the right of the decimal point is if present less than 5, round down and add 1 dose of RhIG § Indicator D-positive cells are added § 2.2¼3 doses § If antibody is present, indicator cells bind to ○ When number to the right of the decimal point is the antibody attached to the D-positive cells 5 or greater, round up to the next number and in suspension add on dose of RhIG § Visible agglutinates (rosettes) are formed § 2.8¼4 doses ○ Limitations § Weak D-positive cells do not react as strongly BLOOD DONATION, TRANSFUSION as normal D-positive cells THERAPY, TRANSFUSION REACTIONS, § If a newborn is weakly D-positive, must quan- AND HEMOLYTIC DISEASE OF THE FETUS tify fetal maternal hemorrhage with the AND NEWBORN Kleihauer-Betke stain • Kleihauer-Betke stain identifies fetal hemoglobin F Blood Donation (Hgb F) • Starts the process of providing safe and adequate blood D+ cell - suspension products for patients who need transfusion therapy support • Donor requirements • Donor screening: Regulated by the U.S. Food and Drug Administration (FDA) ○ Registration: Donor must be fully identified Indicator cells according to specific requirements ○ Health history: Performed for the protection of This method will detect a 10 mL FMH. the donor and recipient This is considered sensitive enough for a screening test. ○ Helps identify situations in which prospective FIGURE 7-7 Principle of the rosette test. donor would be deferred CHAPTER 7 Immunohematology and Blood Transfusion Medicine 213

○ Permanent deferral: Prospective donors are ○ Anti-HBc ineligible to donate for a recipient other than ○ HCV NAT themselves ○ Anti-HIV-1/2 ○ Indefinite deferral: Prospective donors are ○ HIV NAT unable to donate for a recipient other than them- ○ Anti-HTLV-I/II selves for an unspecified period; may become eli- ○ Syphilis: Rapid plasma reagin or gible if current requirements change hemagglutination ○ Temporary deferral: Prospective donors are ○ West Nile virus nucleic acid testing (NAT) unable to donate for a limited period ○ IgG antibody to Trypanosoma cruzi (Chagas’ ○ Physical examination: Performed for the protec- disease) tion of the donor (Table 7-7) • Eligibility (per FDA guidelines as reported in the Transfusion Therapy AABB Technical Manual) • ○ Whole blood: 8 weeks RBCs • Contain Hgb ○ Two-unit RBC collection: 16 weeks • Serve as primary mechanism for tissue O transport ○ Infrequent plasmapheresis: 4 weeks 2 • Provide increased O carrying capacity and ○ Plasmapheresis, plateletpheresis, or leukapher- 2 increased RBC mass esis: More than 2 days • Indications: • Donation types ○ Treatment of anemia in normovolemic patients • Allogeneic: Intended for patient use ○ Based on clinical status (Table 7-8) • Autologous: Intended for donor use • ○ Screening procedures focus Platelets • Essential to formation of primary hemostatic plug § Medications • Provide surface for fibrin formation § Associated medical illnesses • Decreased numbers result from § Cardiovascular risk factors ○ Decreased platelet production § Presence of bacteremia ○ Increased destruction ○ Unused units cannot be “crossed over” into allogeneic inventory • Directed: Intended for target patient from selected ABO Compatibility for Whole Blood, donors Red Blood Cells, and Plasma TABLE 7-8 Transfusions ○ Subjected to same selection and testing criteria as allogeneic donors Recipient Donor § Units can be “crossed over” § 56-day waiting period may not apply ABO Whole Red Blood Phenotype Blood Cells Plasma • Volume collected (whole blood donation) should not exceed 10.5 mL/kg A A A, O A, AB • Donor testing: FDA required tests B B B, O B, AB ○ ABO and D phenotype AB AB AB, A, B, O AB OOOO,A,B,AB ○ Antibody screen ○ HBsAg From Blaney K, Howard P: Basic & applied concepts of blood banking and ○ Anti-HCV transfusion practices, ed 3, St. Louis, 2012, Mosby.

TABLE 7-7 Physical Examination Criteria

Acceptable Limit Criteria Allogeneic Autologous Age Applicable state law or 16 Determined by medical director Blood pressure Systolic, 180 mm Hg Determined by medical director Diastolic, 100 mm Hg Pulse 50-100 beats/min without pathologic irregularity, <50 beats/min Determined by medical director acceptable if an otherwise healthy athlete Temperature 37.5 C (99.5 F) if measured orally, or equivalent if Deferral for conditions presenting risk measured by another method for bacteremia Hemoglobin/ 12.5 g/dL or a hematocrit value of 38% 11 g/dL or a hematocrit value of hematocrit 33%

Data from information from the AABB Technical manual, ed 17, Bethesda, Md, 2011, American Association of Blood Banks. 214 CHAPTER 7 Immunohematology and Blood Transfusion Medicine

• Platelet components ○ Used for first-trimester miscarriages or abortions ○ Random donor platelets ○ Rarely used § Prepared from a unit of whole blood § Fear of miscalculating length of pregnancy § Suspended in 40 to 70 mL plasma § Cost § Contain 5.51010 platelets • Special component considerations ○ Plateletpheresis • Leukocyte reduction § Collected by apheresis from a single donor ○ Less than 5106 residual white blood cells § Contains 31011 or more platelets (WBCs) § Comparable to 4 to 8 platelets units § Cells, plateletpheresis, and pooled platelets § Volume of 100 to 500 mL (Table 7-9) ○ Less than 8.3105 residual WBCs • Fresh frozen plasma (FFP) § Platelets, leukocytes reduced (prepared from • Contains all clotting factors, including labile factors whole blood) V and VIII ○ May reduce risk for • Only approved component for clinically significant § Febrile nonhemolytic reactions deficiency of factors II, V, X, and XI § Cytomegalovirus (CMV) transmission (often • Indications considered “CMV safe” [not the same as ○ Used in bleeding patients with multiple coagula- CMV negative]) tion factor deficiencies § Human leukocyte antigen (HLA) alloimmuni- ○ Liver disease zation • Warfarin treatment • CMV negative • Dilutional and consumption coagulopathy ○ Collected from seronegative donors • Cryoprecipitated AHF • Washed • Cold-insoluble portion of FFP thawed at 1 to 6 C ○ RBCs • Contains § Removes 99% of plasma proteins, electro- ○ 80 international units factor VIII lytes, and antibodies ○ 150 mg fibrinogen (AABB quality control § Can lose up to 20% of unit requirement) § Expiration is 24 hours ○ On average, each unit contains approximately ○ Platelets 250 mg fibrinogen § Expiration is 4 hrs after washing ○ 20% to 30% of factor XIII of original unit ○ Irradiation ○ 40% to 70% von Willebrand factor (vWF) of § Only acceptable method to prevent graft- original unit versus-host disease (GVHD) • Indications § Ordered for immunocompromised patients ○ Second-line therapy receiving components that contain viable § von Willebrand’s disease lymphocytes § Hemophilia A § RBCs, platelets, granulocytes, nonfrozen § Control bleeding associated with fibrinogen plasma deficiency § Prevents proliferation of transfused lympho- § Factor XIII deficiency cytes • RhIG § Some damage is done to RBCs, and viability is • IgG anti-D derived from pools of human plasma affected ○ Full 300-mg dose counteracts 15 mL of § Expiration is original expiration of unit or D-positive RBCs 28 days, whichever comes first ○ Equals 30 mL of fetal whole blood • Neonatal considerations • 50-mg dose counteracts 2.5 mL of D-positive RBCs • Washed or fresh cells often indicated ○ Difficulty in metabolizing citrate and potassium (washed) TABLE 7-9 Platelet ABO and Rh Compatibility ○ Maximize level of 2,3-diphosphoglycerate Recipient Donor (fresh) • CMV negative ABOABRh+RH ○ Infection risk to preterm infants born to seroneg- A üü ative mothers B üü ○ Leukocyte reduction is also recommended O üüüü • Irradiation AB ü ○ Recommended to prevent possible transfusion- Rh positive üü Rh negative ü associated GVHD (TA-GVHD) in § Intrauterine transfusions CHAPTER 7 Immunohematology and Blood Transfusion Medicine 215

§ Exchange transfusions ○ Crossmatch is not necessary as long as initial § Transfusion to a premature (<1200 g) antibody screen from heel stick or maternal spec- neonate imen is negative ○ Transfusions in a full-term newborn infant do ○ Infants do not form antibodies during first not require routine irradiation 4 months of life • Hemoglobin S (Hgb S) ○ Antigen-negative blood must be provided if ○ Infants who are hypoxic or acidotic should mother or infant possesses an RBC alloantibody receive blood tested and negative for Hgb S ○ ABO identical or ABO compatible D negative or ○ Hgb S–negative units are needed for exchange same type as infant can be transfused during first transfusions 4 months • Intrauterine transfusion ○ Group O RBCs are most generally used for intra- ○ Hematocrit (Hct) of RBCs should be greater than uterine and neonatal transfusions 70% because ○ Rh-negative blood used for fetuses and neonates § Small volume transfused when blood type is unknown or Rh negative § Need to correct severe anemia (Table 7-10) • Testing considerations • Issuing of components

TABLE 7-10 Component Information

Component Storage Expiration Whole blood 1-6 C ACD/CPD/CP2D: 21 days If intended for room temperature components, store CPDA-1: 35 days at 1-6 C within 8 hr of collection Whole blood irradiated 1-6 C Original outdate or 28 days from date of irradiation, whichever is earlier Red blood cells (RBCs) 1-6 C ACD/CPD/CP2D: 21 days CPDA-1: 35 days Open system: 24 hr Additive solutions: 42 days Washed RBCs 1-6 C24hr RBCs leukocytes, reduced 1-6 C ACD/CPD/CP2D: 21 days CPDA-1: 35 days Open system: 24 hr Additive solutions: 42 days Washed and rejuvenated RBCs 1-6 C24hr RBCs irradiated 1-6 C Original outdate or 28 days from date of irradiation, whichever is earlier RBCs frozen, 40% glycerol 65 C10yr RBCs frozen, 20% glycerol 120 C10yr Deglycerolized RBCs 1-6 C24hr Platelets 20-24 C with continuous gentle agitation 24 hr to 7 days, depending on collection system* Pooled platelets (or open 20-24 C with continuous gentle agitation 4 hr, unless otherwise specified system) Platelets leukocytes, reduced 20-24 C with continuous gentle agitation Open system: 4 hr* Closed system: No change from original expiration date* Platelets irradiated 20-24 C with continuous gentle agitation Open system: 4 hr Closed system: No change from original expiration date* Granulocytes 20-2 C without agitation 24 hr Fresh frozen plasma 18 Cor65 C12mo(18 C) 7yr(65 C, with approval of FDA) Fresh frozen plasma (after 1-6 C24hr thawing) Cryoprecipitated AHF 18 C12mo Cryoprecipitated AHF (after 20-24 C 4 hr if open system or pooled, 6 hr if single unit thawing) or pooled

*Note: Maximum time without agitation is 24 hr. Data from information from the AABB Technical Manual, ed 17, Bethesda, Md, 2011, American Association of Blood Banks. 216 CHAPTER 7 Immunohematology and Blood Transfusion Medicine

• Systems should be in place to ensure the correct • Laboratory investigation of suspected transfusion blood component is being issued to the correct reaction patient • Clerical check of component bag, label, paperwork, • When component is issued to an individual (as and patient sample opposed to being sent to a location—e.g., surgical • Repeat ABO testing on posttransfusion sample suite), a second check of information reviewed • Visual check of pretransfusion and postransfusion should occur and be documented sample to look for hemolysis • Electronic or manual processes should be in place to • Perform DAT on posttransfusion sample check • Report findings to blood bank supervisor or medi- ○ Component’s unique blood number cal director ○ Recipient’s name and another patient identifier • Acute HTR ○ Blood type ○ Incompatible RBCs transfused to recipient with ○ Expiration date preformed antibody ○ Crossmatch status ○ Misidentification most common cause of ABO ○ Other serologic information (e.g., CMV negative) incompatibility ○ Documentation of status of visual inspection ○ Occurs within minutes of start of infusion ○ Time and date of issue ○ Intravascular reaction ○ Person (or location) to whom it was issued § IgM or complement-fixing IgG § Most severe reactions associated with ABO Transfusion Reactions incompatible transfusions § Hemoglobinemia, hemoglobinuria • A transfusion reaction is any adverse effect of transfu- § Decreased Hct, decreased haptoglobin, sion therapy that occurs during or after administration increased lactate dehydrogenase, increased of a blood component plasma hemoglobin • Transfusion of any blood component can result in a § Increased serum bilirubin 6 to 12 hours later transfusion reaction § Severe clinical symptoms: Shock, hypoten- • Signs and symptoms sion, bronchospasm, disseminated intravascu- • Fever lar coagulation (DIC) ○ Generally defined as 1 C or greater rise in tem- § Complement fragments, anaphylatoxins perature above 37 C C3a and C5a • Chills with or without rigors § Renal ischemia, tubular necrosis, acute • Respiratory distress, including wheezing, coughing, renal failure and dyspnea § Activation of coagulation cascade, DIC • Hypertension or hypotension § Cytokines interleukin–1b (IL-1b), IL-6, • Abdominal, chest, flank, or back pain IL-8, TNF-a • Pain at the infusion site § Extravascular reaction • Skin manifestations, including urticaria, rash, flush- § Complement activation incomplete, extra- ing, pruritus, and localized edema vascular clearance • Jaundice or hemoglobinuria □ Typical with non-ABO antibodies • Nausea/vomiting § Milder clinical symptoms • Abnormal bleeding □ Fever, new positive DAT, falling Hct • Oliguria/anuria with no overt signs of bleeding • Clinical evaluation of suspected transfusion reaction □ Hemoglobinemia, hemoglobinuria rarely • Patient-focused steps seen ○ Stop transfusion and maintain intravenous □ Non–immune-mediated hemolysis access with normal saline ○ “Abused” unit ○ Perform clerical recheck § Improper storage/shipping temperatures, ○ Contact treating physician immediately for mishandling instructions for patient care § Malfunctioning blood warmers, micro- • Component-focused steps wave ovens, hot water baths, inadvertent ○ Contact transfusion service for directions for freezing investigation ○ Mechanical hemolysis ○ Obtain instructions for returning any remaining § Roller pumps, pressure infusion pumps, component, associated intravenous fluid bags, small-bore needles and tubing ○ Osmotic hemolysis ○ Determine appropriate samples to send to § Addition of drugs or hypotonic solutions laboratory § Inadequate deglycerolization of frozen RBCs CHAPTER 7 Immunohematology and Blood Transfusion Medicine 217

○ Bacterial growth in blood units ○ Pathophysiology ○ Intrinsic RBC defect such as glucose-6- § Associated with infusion of antibodies to leu- phosphate dehydrogenase (G6PD) deficiency in kocyte antigens and infusion of biologic patient or donor response modifiers (BRMs) • Transfusion-associated sepsis § Either may initiate cellular activation and ○ Fever, shock, hemoglobinuria, DIC, abdominal damage of basement membrane cramps, diarrhea, vomiting § Pulmonary edema occurs secondary to leak- ○ Mortality rates vary by component age of protein-rich fluid into alveolar space ○ More likely to affect products stored at room § HLA antibodies have been implicated in temperature (platelets) some cases ○ Laboratory investigation: Rule out hemolytic § BRMs can accumulate during storage reaction, Gram stain and culture of unit and • TACO recipient, visual inspection of blood bag ○ Massive transfusion or single unit • Febrile nonhemolytic reactions § Volume overload or rapid infusion ○ Temperature increase 1 C(2 F) or more asso- ○ Young children and elderly at most risk ciated with transfusion (during or delayed until ○ Dyspnea, cough, cyanosis, severe headache, after transfusion) peripheral edema, systolic hypertension, conges- ○ Incidence: 0.1% to 1% with universal leukocyte tive heart failure reduction § Must differentiate from TRALI ○ Usually benign, may be accompanied by chills, • Complications of massive transfusion rigors, and/or discomfort ○ Citrate toxicity ○ Causes ○ Hyperkalemia and hypokalemia § Interaction between preformed antibody in ○ Hemostatic abnormalities recipient’s plasma and antigen on donor lym- ○ Air embolism phocytes, granulocytes, or platelets (HLA ○ Hypothermia antibodies are most notable) • Delayed immunologic response: Alloimmunization § Cytokine release in the recipient in response to ○ Immune response to foreign antigens on RBC, or antigen–antibody reactions may increase WBC and platelets (HLA) severity of reaction ○ Primary versus secondary response § Any unexplained, transfusion-associated rise ○ Approximately 1% to 1.6% of RBC transfusions in temperature deserves prompt attention are associated with antibody formation § Rule out other serious causes (acute HTR, • TA-GVHD sepsis) ○ Immunologic transfusion complication • Allergic reaction ○ Donor lymphocytes proliferate and attack ○ Hypersensitivity reaction (IgE-mediated allergic recipient reaction) ○ Greater than 90% mortality rate ○ Triggered by exposure to soluble substance in ○ Clinical: Fever, skin rash, hepatitis, enterocolitis, donor plasma to which recipient is sensitized pancytopenia, and immunodeficiency ○ Ranges from rash and/or urticaria (hives) and ○ Symptoms usually appear within 8 to 10 days of itching to an anaphylactoid reaction transfusion ○ Usually not accompanied by fever ○ Three requirements for GVHD to develop • Transfusion-related acute lung injury (TRALI) § Expressed HLA antigens different between ○ May occur as frequently as 1 in 5000 donor and recipient transfusions § Graft must contain immunocompetent cells ○ Signs and symptoms § Host must be incapable of rejecting immuno- § Fever, chills, dyspnea, cyanosis, hypotension, competent cells new onset of bilateral pulmonary edema ○ Irradiation § Symptoms arise within 6 hours of transfusion § Irradiation of cellular components recom- § Most cases evident within 1 to 2 hours after mended transfusion § Patients identified at risk for TA-GVHD ○ All plasma-containing components have been § Transfusions of cellular components between implicated blood relatives ○ Must distinguish TRALI reaction from § Transfusion of HLA-selected products § Anaphylactic reaction • Posttransfusion purpura § Transfusion-associated circulatory overload ○ Uncommon, usually in women (TACO) ○ Abrupt onset of severe thrombocytopenia § Transfusion-related sepsis (<10,000/mL), 1 to 24 days after blood 218 CHAPTER 7 Immunohematology and Blood Transfusion Medicine

transfusion in a previously pregnant or trans- • Rh HDFN fused patient ○ Anti-D alone or in combination with Anti-C or ○ 70% of posttransfusion purpura cases associated anti-E with antibodies against HPA-1a (PlA1)Ag • “Other” HDFN ○ Usually self-limited with full recovery, although ○ Other antigens in Rh system some patients can die from intracranial bleeding ○ Antigens in other systems • Iron overload • ABO HDFN ○ One RBC unit contains approximately ○ Can occur in any pregnancy 250 mg iron ○ Group A or B infants born to group O mothers ○ Chronically transfused patients at risk § Humans can make IgG anti-A,B ○ Iron deposits interfere with heart, liver, and § ABO IgG antibodies occur without history of endocrine glands, causing cardiomyopathy, prior exposure (Table 7-11) arrhythmias, and hepatic and pancreatic failure • Serologic studies ○ Threshold for clinical damage: Lifetime expo- • Early pregnancy sure to more than 50 units of RBCs in a non- ○ Performance of ABO/Rh, weak D if D negative bleeding person ○ Antibody screen, identify if antibodies detected ○ Treatment: Iron-chelating agents, “fresh” blood • 28 weeks • Transfusion fatalities ○ Rh-negative women with initial negative anti- ○ Fatalities resulting directly from the effects of body screen transfusion must be reported to the FDA (Center § Repeat antibody screen for Biologics Evaluation and Research) within § Administer RhIG 24 hours and by written report within 7 days ○ Positive antibody screen ○ Most deaths are unrelated to transfusion, but if § Identify antibody there is any suggestion that transfusion contrib- § Presence of antibody does not mean HDFN uted, initiate investigation into case will occur § Not all antibodies are risk to fetus § Anti-Lea, anti-I Hemolytic Disease of the Fetus and □ Baby may lack corresponding antigen Newborn □ Fetal involvement may be predicted by typing father’s RBC antigens • Fetal RBCs are coated with maternal alloantibody § Maternal antibody titer • Directed against antigen inherited from the father that § Titration studies can aid in treatment is absent from the mother decisions • IgG-coated RBCs are destroyed § Establish baseline in first trimester • Before and after birth § Repeat at intervals determined by clinician • Severity ranges from intrauterine death to asymptom- § Usually not repeated until 16 to 18 weeks atic (serologic detection only) § Use is controversial • Three prerequisites for HDFN § No established critical titers for antibodies • Mother lacks antigen (exposed through pregnancy other than anti-D or transfusion) § Represents a noninvasive means to monitor • Fetus possesses antigen, inherited from father pregnancy • Mother has made an IgG antibody ○ Sensitization depends on § Recognition of foreign antigen ABO Versus Rh Hemolytic Disease § Responder TABLE 7-11 of the Fetus and Newborn § Antigen is immunologic § Amount of fetal-maternal bleed ABO HDFN Rh HDFN § ABO compatibility Most common Not caused by Rh immunoglobulin • Complications of HDFN include rising levels of Cannot be diagnosed Followed with titers unconjugated bilirubin, which is the biggest risk Can affect first child Immune exposure (second child) ○ Decision to perform exchange transfusion driven Weak-to-negative DAT Very strong DAT by bilirubin levels Occurs in group type O Can affect any Rh-negative mother • Central nervous system damage caused by mothers ○ Prematurity Slight rise in bilirubin (treat High rise in bilirubin (may need to with phototherapy) perform exchange transfusion) ○ Acidosis ○ Hypoxia DAT, Direct antiglobulin test; HDFN, hemolytic disease of the fetus and ○ Hypoalbuminemia newborn. CHAPTER 7 Immunohematology and Blood Transfusion Medicine 219

CERTIFICATION PREPARATION QUESTIONS c. AHG d. LISS For answers and rationales, please see Appendix A. 10. Which is true of antibodies to Kidd blood group sys- 1. Which immunodominant sugar confers A antigen tem antigens? specificity? a. They are enhanced by enzymes a. D-Galactose b. Titers can quickly drop in patients b. L-Fucose c. Both A and B c. N-Acetylgalactosamine d. None of the above d. Both A and C 11. Which of the following is true of the Lewis system? 2. If a patient has an A2 ABO type, which of the follow- a. Lewis antigens are found on type II precursor cells ing statements is true? b. Lewis antigens are well developed at birth a. The patient’s red cells will react with anti-A1 lectin c. Antibodies to Lewis antigens always cause HTRs b. The patient’s serum will react with A2 cells d. Antibodies to Lewis antigens rarely cross the c. The patient’s red cells will react with anti-A2 lectin placenta 12. d. The patient’s serum will react with A1 cells if Which of the following is true of antibodies to MNS anti-A1 is present blood group system antigens? 3. Which genotype confers the Bombay blood type? a. Anti-U is directed at a high-incidence antigen a. Hh b. Anti-N is commonly found b. hh c. Anti-M is always clinically significant c. Sese d. Anti-S is reactive with enzyme-treated cells d. Lele 13. Which of the following antibodies is classified as 4. Which genes encode for Rh antigens? “biphasic” and an autoantibody? a. RHDCE a. Anti-B b. RHD b. Anti-P c. RHCE c. Anti-H d. Both b and c d. Anti-Lea 5. Testing for the D antigen was conducted at the IAT 14. You have performed an antibody screen using the phase. A control was included in the testing. Both tube method. All three screening cells tested negative. the patient’s red cells and the control tube reacted The Coombs check cells in all three tubes are also at 4+. How would you interpret this test? nonreactive. What should you do? a. The test is invalid because the control tube was a. Respin the tubes and reread them positive b. Report the antibody screen as negative b. The patient is D positive c. Repeat the antibody screen c. The patient is D negative d. Perform an antibody identification panel d. The test should be repeated and the control tube 15. An antibody panel has six 2+ reactive cells at AHG omitted phase. Panel testing using enzyme-treated cells 6. Of the red cells listed, which has the most D antigen showed no reactivity. Which is the most likely anti- present? body that is present? a. Rh null a. Anti-Fya b. D positive b. Anti-e c. dce/dce c. Anti-k d. D d. Anti-Lua 7. Which is true of the Duffy blood group system? 16. A patient has a currently nonreactive antibody screen a. Antigens are resistant to enzyme treatment but has a history of anti-Jka in the patient file. Which b. Antibodies never show dosage type of crossmatch must be performed on this patient? c. Fya and Fyb are codominant alleles a. Immediate spin crossmatch d. The majority of whites are Fy(ab) b. IAT crossmatch 8. Which antibody is typically considered to be an auto- c. Electronic crossmatch antibody if found in the serum of an adult? d. Both a and c a. Anti-K 17. A recently transfused patient has a 3+ reactive DAT b. Anti-I with anti-IgG. Which procedure should be used to c. Anti-D identify the specificity of the IgG antibody attached d. Anti-Fya to the red cells? 9. Which reagent destroys all of the Kell blood group a. Adsorption system antigens? b. Neutralization a. DTT c. Titration b. Chloroquine diphosphate d. Elution 220 CHAPTER 7 Immunohematology and Blood Transfusion Medicine

18. An O-negative mother gave birth to an O-positive a. Autosomal recessive inheritance baby. Her rosette test was positive. Which of the fol- b. X-linked dominant inheritance lowing is true? c. Y-linked recessive inheritance a. The test is invalid because of the mother’s d. Autosomal codominant inheritance ABO type 25. What blood type is not possible for the offspring of b. A Kleihauer-Betke test should be performed to AO and BO parents? quantify the fetal maternal hemorrhage a. AB c. The mother should be given a 300-mg dose b. A or B of RhIG c. O d. A weak D test should be performed on the baby d. All are possible 19. In which of the following settings are platelet trans- 26. How many molecules of IgM are needed to fix fusions not indicated? complement? a. Thrombotic thrombocytopenic purpura a. 1 b. Immune thrombocytopenic purpura with severe b. 2 intracranial hemorrhage c. 3 c. Massive transfusion d. 4 d. Vascular catheter placement, platelet count 27. For lysis of red blood cells to occur after antigen– 24,000/mL antibody reaction, which compound is required? e. Brain biopsy, platelet count 62,000/mL a. Albumin 20. An obstetric patient presents to the hospital with b. Glucose-6-phosphate dehydrogenase (G6PD) marked vaginal bleeding and severe lower abdominal c. Complement pain. During placement of an intravenous catheter, d. Antihuman globulin (AHG) she was noted to have marked oozing. She is diag- 28. An end-point of tube testing other than agglutination nosed with disseminated intravascular coagulation that must also be considered a positive reaction is as a complication of her primary problem. She is called: given cryoprecipitate and fresh frozen plasma before a. Clumping going to the operating room. What element of cryo- b. Mixed field precipitate is important in treating this patient? c. Hemolysis a. Factor I d. Microscopic b. Factor II 29. Mixed-field (mf) agglutination can be observed c. Factor VIII:c in the: d. Factor VIII:vWF a. DAT on a person undergoing delayed hemolytic e. Factor XIII transfusion reaction 21. A patient’s ABO blood type is determined by which b. IAT result of a patient who has anti-Lea of the following? c. DAT on a patient on high doses of penicillin a. Genetic inheritance and environmental factors d. Typing result with anti-A of a patient who is A2 b. Genetic inheritance subgroup c. Environmental factors 30. In which situation(s) may the ABO serum grouping d. Immune function not be valid? e. Maternal blood type a. The patient has hypogammaglobulinemia 22. A trauma patient with type AB is seen at a rural hos- b. IgM antibodies are present pital. The hospital only has 3 units of type AB RBCs. c. Cold autoantibodies are present What blood type of RBCs can the patient receive as d. All of the above an alternative? 31. If you knew the DAT is positive, what would you a. Type O expect the Rh control to be when doing a weak D test b. Type B through AHG? c. Type A a. Negative d. None of the above b. Positive e. All of the above c. Mixed field 23. A genetic state in which no detectable trait exists is d. Hemolysis at 37 C would be seen called: 32. How can IgG antibodies be removed from red cells? a. Recessive a. Elution b. Dominant b. Adsorption c. Incomplete dominance c. Prewarming d. Amorph d. Neutralization 24. Most blood group antigens are expressed as a result 33. Testing needs to be done with an antiserum that is of which of the following? rarely used. The appropriate steps to take in using CHAPTER 7 Immunohematology and Blood Transfusion Medicine 221

this antiserum include following the manufacturer’s c. 10 g/dL procedure and: d. 11 g/dL a. Performing a cell panel to be sure that the antise- 41. An IgA-deficient patient with clinically significant rum is performing correctly anti-IgA requires which of the following? b. Performing the testing on screen cells a. Leukocyte-reduced fresh frozen plasma c. Testing in duplicate to ensure the repeatability of b. CMV-seronegative RBCs the results c. Irradiated RBCs and platelets d. Testing a cell that is negative for the antigen and d. Washed RBCs one that is heterozygous for the antigen 42. Anti-H will react weakest with blood from a person 34. Based on the following antigram, which cell is het- with ______. erozygous for M? a. Group O b. Group A1 a b a b a b DCEcEMNSsP1 Le Le KkFy Fy Jk Jk c. Group A2 10 +0+++ ++00 + 0 0+0 + 0 + d. Group A2B 2++00+0 +0++ 0 + 0++ + + 0 43. Which of the following antibodies do not match the 30 0 ++++ 0 +++ 0 0 +++ 0 + + others in terms of optimal reactive temperature? a. Anti-Fya a. Cell 1 b. Anti-M b. Cell 2 c. Anti-K c. Cell 3 d. Anti-S d. None of the above 44. What antibody can an R1r patient make if transfused 35. Which antibody can be neutralized with a specific with R2R2 blood? reagent? a. Anti-D a. Anti-D b. Anti-C b. Anti-Jka c. Anti-E c. Anti-M d. Anti-c d. Anti-Lea e. Anti-e 36. Group O red blood cells are used as a source of com- 45. What is the probability of finding blood negative for mercial screening cells because: the Jka and Fya antigens (23% of population is Jk a. Anti-A is detected using group O cells [a] and 34% of population is Fy[a])? b. Anti-D reacts with most group O cells a. 5.1% c. Weak subgroups of A react with group O cells b. 51% d. ABO antibodies do not react with group O cells c. 7.8% 37. The use of EDTA samples for the direct antiglobulin d. 78% test prevents activation of the classical complement 46. If the following patient’s RBCs were tested against pathway by: anti-H lectin and did not react, this person would a. Causing rapid decay of complement proteins be identified as a(an): b. Chelating Mg2+ ions, preventing assembly of C6 c. Chelating Ca2+ ions, preventing assembly of C1 Serum Antibody Screening d. Preventing chemotaxis 38. Cell typing typing results Check (Coombs control) cells are: A1 B a. Added to every negative antiglobulin test Anti-A Anti-B cells cells SCI SCII Autocontrol b. Added to negative direct antiglobulin tests only 0 0 4+ 4+ IS 4+ 4+ 0 c. Used to confirm a positive Coombs’ reaction 37C LISS 4+ 4+ 0 d. Coated with both IgM and C3d AHG 4+ 4+ 0 39. What type(s) of red cells is(are) acceptable to trans- Check cell 2+ fuse to an AB-negative patient? a. A negative, B negative, AB negative, O negative a. Acquired B b. O negative only b. Secretor c. AB negative only c. Oh phenotype d. AB negative, A negative, B negative only d. Subgroup of A 40. A nonbleeding adult of average height and weight 47. If a person has the genetic makeup Hh, AO, LeLe, with chronic anemia is transfused with 2 units of sese, what substance will be found in the secretions? red blood cells. The pretransfusion Hgb is 7.0 g/dL. a. A substance What is the expected posttransfusion Hgb? b. H Substance a. 8 g/dL c. Lea substance b. 9 g/dL d. Leb substance 222 CHAPTER 7 Immunohematology and Blood Transfusion Medicine

48. Before A and B antigens can be expressed, the precur- c. Anti-Coa and anti-Cob sor substance must have the terminal sugar d. Anti-Doa and anti-Jsb ______. 57. Donors who have received RBC transfusion within a. d-Galactose the last 12 months are deferred because: b. N-Acetylgalactosamine a. Blood could transmit hepatitis or HIV c. Glucose b. Donor red cell hemoglobin level may be too low d. L-Fucose c. Donor health would prohibit the donation 49. A white female’s RBCs gave the following process reactions: D+, C+, E-, c+, e+. The most probable d. There will be two cell populations in this Rh genotype is: donor a. DCe/Dce 58. Autologous presurgical donations are not allowed b. DCe/dce for which of the following patients? c. DCe/DcE a. Weigh less than 100 lb d. Dce/dCe b. Under the age of 14 50. If a D-positive person makes anti-D, this person is c. With hemoglobin of 13 g/dL most likely: d. With bacteremia a. Partial D 59. Which of the following viruses resides exclusively in b. D negative leukocytes? c. Weak D as position effect a. HCV d. Weak D because of transmissible genes b. HBV 51. A serum containing anti-k is not frequently encoun- c. CMV tered because of which of the following? d. HIV a. People who lack the k antigen are rare 60. Which product is least likely to transmit hepatitis? b. People who possess the k antigen are rare a. Cryoprecipitate c. The k antigen is not a good immunogen b. Plasma protein fraction d. Kell-null people are rare c. RBC 52. A characteristic of the Xga antigen is that the Xga d. Platelets antigen: 61. In preparing platelets from a unit of whole blood, the a. Has a higher frequency in women than in men correct order of centrifugation is: b. Has a higher frequency in men than in women a. Hard spin followed by a hard spin c. Is enhanced by enzymes b. Light spin followed by a light spin d. Is usually a saline reacting antibody c. Light spin followed by a hard spin 53. Which of the following is a characteristic of the Kidd d. Hard spin followed by a light spin system antibodies? 62. Which antibody could cause hemolytic disease of the a. The antibodies are usually IgM fetus and newborn? b. The corresponding antigens are destroyed by a. Anti-I enzymes b. Anti-K c. The antibodies are usually strong and stable dur- c. Anti-Lea ing storage d. Anti-N d. The antibodies are often implicated in delayed 63. A group A, D-negative obstetric patient with hemolytic transfusion reactions anti-D (titer 256) is carrying a fetus who needs an 54. Anti-E will react with which of the following cells? intrauterine transfusion. The blood needed a. RoRo should be: b. R1R1 a. Group A, D-negative RBC c. R2R2 b. Group A, D-negative whole blood d. rr c. Group O, D-negative RBC 55. Which statement is not true concerning anti-Fya and d. Group O, D-negative whole blood anti-Fyb? 64. Which of the following mothers should receive a. Are clinically significant RhIG? b. React well with enzyme-treated panel cells a. A-negative mother; O-negative baby; no prenatal c. Cause hemolytic transfusion reactions care, anti-D in mother d. Cause a generally mild hemolytic disease of the b. AB-negative mother; B-positive baby; anti-D newborn in mother 56. Which of the following antibodies can be neutralized c. O-negative mother; A-positive baby; no anti-D with pooled human plasma? in mother a. Anti-Hy and anti-Ge:1 d. A-positive mother; A-positive baby; no anti-D b. Anti-Cha and anti-Rga in mother CHAPTER 7 Immunohematology and Blood Transfusion Medicine 223

65. How many doses of RhIG are indicated for a 73. How would you interpret the following reactions? Kleihauer-Betke reading of 0.6%? Forward Type Reverse Type

a. 1 Anti-A Anti-B A1 Cells B Cells b. 2 004+4+ c. 3 d. 4 a. Blood type A 66. What should be done first if a mother types as O and b. Blood type O the baby types as AB? c. Blood type B a. Report the results with no further testing d. Blood type AB b. Try to get a sample from the father 74. Noting these reactions, if they patient needed blood c. Recheck all labels, get new samples, if necessary, now, what type of blood should be transfused? and retest Forward Type Reverse Type d. Retype using all new reagents Anti-A Anti-B A Cells B Cells 67. 1 A newborn has a positive DAT. What is the best pro- 4+ 0 1+ 4+ cedure to determine the antibody causing a positive DAT in this newborn? a. An antibody titer on the mother’s serum a. Blood type A b. An antibody panel on the mother’s serum b. Blood type O c. An antibody panel performed on the eluate of the c. Blood type A2 mother’s cells d. Blood type A 75. d. An antibody panel performed on the eluate of the Blood group antibodies made by type A and type B baby’s cells people are predominantly which class? 68. Which of the following is(are) an example(s) of a a. IgE record-keeping error? b. IgA a. Use of correction fluid or tape c. IgG b. Using pencil d. IgM 76. c. Documentation after the fact Based on these reactions, what should be the next step? d. All of the above Forward Type Reverse Type 69. Which of the antigens below is considered low Anti-A Anti-B A1 Cells B Cells incidence? 4+ 0 1+ 4+ a. Fya b. S a. Test the serum with A2 cells c. C b. Report the patient as type A a d. Kp c. Test the cells with anti-A1 lectin 70. Which of the antigens below is considered high d. Both a and c incidence? e. Request a new specimen a. Fyb 77. A “directed donor” unit of blood is defined as a unit b. Vel of blood from a person who gives blood for: c. E a. Relief of polycythemia or other blood disorder d. S b. His or her specific use only 71. In performing tube testing, you see many medium- c. First-degree blood relative sized agglutinates in a clear background. How would d. Another person he or she has specified you grade this reaction? 78. Before the patient can receive a directed donation a. 2+ unit, the patient requires which of the following tests b. 1+ to be completed? c. 4+ a. Type and screen only d. 3+ b. Type and screen and compatibility testing 72. Of the following, which genotypes would result in c. Retype of patient and donor unit the B phenotype? d. No additional testing is required a. BB 79. An 18-year-old female with a hematocrit of 38%, b. AB temperature of 37 C, and blood pressure of 175/ c. BO 90 mm Hg presents for whole blood donation. Based d. a and b on this information, would you accept, permanently e. a and c defer (PD), or temporarily defer (TD) the donor? 224 CHAPTER 7 Immunohematology and Blood Transfusion Medicine

a. Accept c. Cryoprecipitate at 1 to 6 C, then pooling the b. TD, blood pressure is too high for a person of thawed cryoprecipitate in batches of 10 units, her age then quickly refreezing c. TD, temperature is too high d. Cryoprecipitate at room temperature, then centri- d. PD, for all values listed fugation in the cold to concentrate the cryopreci- 80. A 63-year-old man with a hemoglobin value of pitate to the bottom before adding more plasma 130 g/dL and pulse of 80 beats/min, who received to reconstitute human pituitary growth hormone (PGH) when he 86. Platelets must be kept in constant motion for which was 10 years old, presents for whole blood donation. of the following reasons? Based on this information, would you accept, perma- a. Maintain the pH so the platelets will be alive nently defer (PD,) or temporarily defer (TD) the donor? before transfusion a. Accept the donor b. Keep the platelets in suspension and prevent b. TD, because of the human PGH clumping of the platelets c. PD, because of the human PGH c. Mimic what is going on in the blood vessels d. PD, because of the high hemoglobin value d. Preserve the coagulation factors and platelet 81. A 38-year-old female weighing 153 lb, who received viability the rubella vaccine 2 months previously, presents to 87. After thawing and pooling cryoprecipitate for trans- donate whole blood. She also received 2 units of fusion to a patient, the product should be stored at: packed cells after the delivery of her eighth child a. Room temperature 8 weeks ago. Based on this information, would you b. 1 to 6 C accept, permanently defer (PD), or temporarily defer c. 37 C (TD) the donor? d. 0 C a. Accept the donor 88. Fresh frozen plasma must be thawed at which b. TD because of the packed cells 8 weeks ago temperature? c. PD because of receiving blood products a. 1 to 6 C d. TD because of the rubella vaccine b. Room temperature 82. A 22-year-oldfemale witha cousin with AIDSwho had c. 37 C taken aspirin the day before and with needle marks on d. 40 C or higher both arms presents to donate whole blood. Based on 89. Frozen red blood cells are prepared for transfusion this information, would you accept, permanently defer by thawing at: (PD), or temporarily defer (TD) the donor? a. Room temperature and then washing with saline a. PD, needle marks on both arms b. 37 C in a water bath and then washing with dif- b. TD, needle marks on both arms ferent concentrations of saline c. PD, cousin with AIDS c. 37 C control incubator and then mixing well d. TD, because of the aspirin before transfusion 83. Each unit of blood must be tested for all of the fol- d. 1 to 6 C for 2 days and then washing with dif- lowing except: ferent concentrations of dextrose a. Anti-HIV 1/2 90. Which is the most likely reason frozen deglycerolized b. HBsAg red blood cells would be used? c. Anti-HCV a. A patient with antibodies to a high-frequency d. Antigen to HCV antigen 84. The principle of the HBsAg test is to detect which of b. Pregnant women requiring intrauterine the following? transfusions a. Antigen in patient’s plasma c. Emergency transfusion situations b. Antigen on the patient’s RBCs d. Group AB Rh-negative patients c. Antibody in patient’s serum 91. One indication for transfusion of thawed/pooled d. Antigen and antibody in patient’s serum cryoprecipitate would be replacement of which of 85. Cryoprecipitate is prepared by first thawing: the following? a. Fresh frozen plasma at 1 to 6 C, and then doing a. Factor X in hemophiliacs a cold centrifugation to pack the cryoprecipitate b. Factor VIII in massively transfused patients to the bottom so the plasma may be removed c. Fibrinogen b. Fresh frozen plasma at room temperature, then d. Volume placing in the freezer for 2 hours, then centrifug- 92. A contraindication for transfusing red blood cells to ing and removing the cryoprecipitate a patient is if the patient: CHAPTER 7 Immunohematology and Blood Transfusion Medicine 225

a. Is massively bleeding 96. Platelets made from a single whole blood donation b. Has well-compensated anemia should contain which of the following? c. Has bone marrow failure a. 31011 platelets in 90% of samples d. Has decreased red blood cell survival b. 3.3109 platelets in 75% of samples 93. Concerning the component and the required quality c. 5.51010 platelets in 90% of samples control results, which of the following is a true d. 101010 platelets in 75% of samples statement? 97. Frozen red blood cells must be stored at a. FFP must have 80 international units of fibrino- ______. gen in 7 units tested a. 180 C or less b. Cryoprecipitate must have 80 international units b. 18 C or less of factor VIII c. 32 C or less c. Leukocyte-reduced red blood cells must have d. 65 C or less fewer than 3.31011 WBCs in each unit 98. The temperature for incubation of the indirect anti- d. Platelets must have no red blood cells globulin test (IAT) should be ______. 94. Fresh frozen plasma must be stored at: a. 24 C a. Colder than 18 C for no longer than 1 year b. 6 C from donation c. 37 C b. Colder than 38 C for no longer than 1 year d. 37 10 C from donation 99. The temperature of a blood refrigerator without a c. Exactly 18 C for no longer than 1 year from continuous recording device should be recorded: donation a. Daily d. 18 Cto38 C for up to 10 years from b. Every 4 hours donation c. Once every 24 hours 95. The storage temperature for packed red blood cells is d. Every 30 minutes ______. 100. When should quality control be performed on rou- a. 1 to 10 C tine blood typing reagents? b. 1 to 4 C a. At the beginning of each shift c. 1 to 6 C b. Once daily d. 20 to 25 F c. Weekly d. Only when opening a new vial 226 CHAPTER 7 Immunohematology and Blood Transfusion Medicine

SELF-ASSESSMENT

Content Area: ______

Score on Practice Questions: ______

List the specific topics covered in the missed questions:

List the specific topics covered in the correct questions: CHAPTER 7 Immunohematology and Blood Transfusion Medicine 227

NOTES CHAPTER 8 Clinical Chemistry

Janelle M. Chiasera

LIPIDS AND LIPOPROTEINS • Intermediate density lipoprotein{: A lipolytic product of VLDL catabolism; taken up into the liver cell Lipids or converted to LDL • Lp(a): An LDL-like molecule with one Apo(a) • Essential components, required for many body functions linked to the Apo B-100, high level of homology • Functions: Hormone precursors, hormones, cell mem- with plasminogen, increased concentrations associ- brane structure and function, fuel, energy storage, aid ated with increased risk for coronary heart in nerve conduction, aid in digestion disease (CHD) • Analysis commonly used to assess risk for cardiovascular disease • Major lipids analyzed in the clinical laboratory include Apoproteins total cholesterol and triglycerides • Functional and structural protein components of the • Total cholesterol and triglycerides most commonly lipoprotein molecule measured using enzymatic assays (Table 8-1) • Each lipoprotein molecule contains one or more apoproteins Lipoproteins • Three main functions • Activate enzymes to aid in lipid metabolism • Spherical lipid and protein complex (Figure 8-1), lipid • Maintain structural integrity of the lipoprotein core (triglycerides and cholesterol esters) and an outer molecule shell of phospholipids, protein, and free cholesterol • Enhance cellular uptake of lipoproteins (apoproteins • The smaller the diameter of the molecule, the more are recognized by cell surface receptors) (see dense the molecule Table 8-4 for specific apoprotein function by class) • Each lipoprotein molecule has a different physical and Specimen Considerations chemical makeup; lipoproteins may be separated and • Serum or plasma after 12- to 16-hour fast (fast measured based on one or more of these characteristics required for triglyceride analysis) (Table 8-2) • Ethylenediaminetetraacetic acid (EDTA) (1 mg/1 mL • High-density lipoprotein (HDL) and low-density blood) plasma preferred for analysis of lipoproteins lipoprotein (LDL) are the only lipoproteins currently (EDTA preserves lipoproteins); separate plasma within routinely measured in the clinical laboratory (see 2 hours Table 8-3 for a list of methods used to measure HDL • National Cholesterol Education Program recommends and LDL) multiplying plasma concentrations by 1.03 to convert • Functions: Transport lipids through circulatory sys- to serum values tem, facilitate lipid metabolism • No alcohol 72 hours before blood collection (alcohol • Five major lipoproteins: Chylomicrons, very-low- transiently increases triglycerides) density lipoprotein (VLDL), LDL, and HDL • Patient should be seated during collection • Additional lipoproteins identified include chylomicron • Avoid citrate, fluoride, oxalate anticoagulants (can remnant, intermediate density lipoprotein (IDL), and cause dilution of plasma components) (Box 8-1) Lp(a) Pathology • Chylomicron remnant*: A lipolytic product of chy- • Abnormalities in lipids and lipoproteins are referred lomicron catabolism, rapidly taken into the liver to as hyperlipidemias and hyperlipoproteinemias, cell by specific receptors respectively • Elevated levels of lipids and lipoproteins are associated *Remnant lipoproteins have been shown to be predictive of coro- with risk for coronary heart disease, specifically,increased nary heart disease risk. total cholesterol, increased LDL, decreased HDL

228 CHAPTER 8 Clinical Chemistry 229

TABLE 8-1 Methods to Measure Total Cholesterol and Triglyceride

Analyte Method Principle Comments Total cholesterol Enzymatic, Reaction Steps Most end-point 1. CholesterolEsters Cholesterol esterase! Cholesterol + Fatty acids common

2. Cholesterol+O2 cholesterol oxidase!cholest-4-en-3-one +H2O2 method

Detection: peroxidase A. H2O2 + 4-aminophenazoneðor other dyeÞ ! oxidized dyeðAmax, 500nmÞ +H2O2 B. Monitor O2 consumption with an O2 electrode CDC-modified Cholesterol extracted with zeolite, esters chemically hydrolyzed (saponification), and total Reference Abell reaction cholesterol measured by Liebermann-Burchard (L-B) reaction method Triglycerides* Enzymatic Common Steps to All Enzymatic Assays Below: Most Lipase, Protease 1. Triglycerides ! Glycerol + 3 Fatty acids common Glycerol Kinase 2. Glycerol + ATP ! Glycerol-3 phosphate + ADP methods Pyruvate Kinase (NADH Consumption): Pyruvate Kinase 3. ADP + Phosphoenolpyruvate ! ATP + Pyruvate Lactate Dehydrogenase 4. Pyruvate + NADH + H + ! Lactate + NAD + Glycerol Phosphate Dehydrogenase (Formazan Colorimetric): Glycerol Phosphate Dehydrogenase 3. Glycerol-3-phosphate + NAD + ! Dihydroxyacetone phosphate + NADH + H + Diaphorase 4. NADH + Oxidized tetrazolium ! Reduced tetrazolium Glycerol Dehydrogenase: Glycerol Dehydrogenase 3. Glycerol + NAD + ! Dihydroxyacetone + NADH + H + Diaphorase 4. NADH + H + + Resazurin ! Resorufin + NAD +

CDC, Centers for Disease Control and Prevention. *National Cholesterol Education Program recommends all laboratories offer glycerol blanking.

Apolipoprotein • Tangier disease: Autosomal dominant lipoprotein disor- Unesterified cholesterol Cholesteryl der resulting from catabolism of HDL. Characterized by ester extremely reduced HDL concentrations and accumulation of cholesterol esters in body tissues, causing orange Phospholipid Triglycerides tonsils, splenomegaly, and peripheral neuropathy

Carbohydrates

• Aldoses or ketoses, nomenclature based on configura- tion (D or L); D sugars represent majority found in the body • Three main classes of carbohydrates • Monosaccharides ○ Simple sugars ○ Glucose ○ Fructose • Disaccharides Lipoprotein particle. FIGURE 8-1 ○ Two monosaccharides linked by a glycosidic bond § Maltose (glucose and glucose) § Lactose (glucose and galactose) § Sucrose (glucose and fructose) • Pathologic processes are classified based on lipid levels, • Polysaccharides lipoprotein pattern, and clinical and biochemical phe- ○ Two monosaccharides linked by glycosidic bond notype, with most very rare in occurrence (Table 8-5) ○ Starch and glycogen 230 CHAPTER 8 Clinical Chemistry

TABLE 8-2 Lipoprotein Characteristics

Lipoprotein Density Electrophoretic Protein Content (Diameter (nm) (g/mL) Major Lipids (%) Mobility (%) Chylomicrons <0.95 90 TGex Origin 1-2 (>70) VLDL 0.95-1.006 65 TG (end), 15 chol Pre-b 8-10 (26-70) LDL 1.019- 50 chol b 20 (19-23) 1.063 HDL 1.063-1.21 20 chol, 25 phos a 50-55 (4-10)

Chol, Cholesterol esters; HDL, high-density lipoproteins; LDL, low-density lipoproteins; phos, phospholipids; TGend, endogenous triglycerides; TGex, exogenous triglycerides; VLDL, very-low-density lipoproteins.

TABLE 8-3 Methods to Measure High-Density and Low-Density Lipoproteins

Analyte Method Description Comments HDL Ultracentrifugation* Sample adjusted to a density of 1.063 (potassium bromide) and centrifuged Specialized laboratories only at high speed for 24 hr. Sample separates based on density (see Table 8-2) Precipitation* 1. Precipitation of Apo B containing lipoproteins using polyanion-divalent Commonly used method cation solutions. HDL quantitated in the supernatant after precipitation and centrifugation 2. Precipitation of Apo B containing lipoproteins using dextran sulfate and iron; no centrifugation needed

Electrophoresis Separation based on size and charge; mobility equal to a1-globulins; Frequently used agarose gel Homogeneous Immunologic assay involving blockage of non-HDL lipoproteins; HDL free to Commonly used method (direct) react with reagent enzymes LDL Friedewald formula LDL-C¼cholesterol (VLDL-C+HDL-C) Commonly used; not reliable TG/5 approximates VLDL-C estimation when TG >400 mg/dL b Quantification Uses ultracentrifugation (to separate VLDL and chylo) and precipitation (to Research only remove HDL), LDL calculated as density >1.006 HDL Homogeneous Uses detergents or other chemicals to block or solubilize lipoprotein classes Commonly used method (direct) to allow for quantitation of LDL

HDL, high-density lipoprotein; LDL, low-density lipoprotein; TG, triglycerides; VLDL, very-low-density lipoprotein. *Centers for Disease Control and Prevention reference method involves ultracentrifugation (to remove VLDL), heparin manganese precipitation from 1.006 infranatant (to remove LDL), and analysis of supernatant by Abell-Kendall method.

Apoprotein Classes TABLE 8-4 Function and Carriers

Apoprotein Class Lipoprotein Carrier Function AAIHDL, chylomicrons Cofactor for LCAT AII HDL Unknown AIV HDL, chylomicrons Activates LCAT B48Chylomicrons Secretion of TG from intestines 100 VLDL, LDL LDL receptor binding CIChylomicrons, VLDL, HDL Activates LCAT II Chylomicrons, VLDL, HDL Activates LpL III Chylomicrons, VLDL, HDL Inhibits activation of LpL by C-II E Chylomicrons, VLDL, HDL Facilitate uptake of remnant particles (chylomicrons and IDL)

HDL, High-density lipoproteins; LCAT, lecithin cholesterol acyltranferase; LDL, low-density lipoproteins; LpL, lipoprotein lipase; TG, triglycerides; VLDL, very-low- density lipoproteins. CHAPTER 8 Clinical Chemistry 231

• Recommended Ranges Measurement done to diagnose hyperglycemia (diabetes mellitus) or hypoglycemia National Cholesterol Education Program: • Hemoglobin A1c is used as a measure of long-term gly- Adult Treatment Panel Recommended cemic control (2- to 3-month period). A1c is used to BOX 8-1 Ranges (Based on Serum Samples) diagnose and monitor treatment of diabetes. The Total cholesterol: <200 mg/dL American Diabetes Association (ADA) recommends Triglycerides: <150 mg/dL that A1c measurements be performed at least twice HDL: >40 mg/dL; >60 mg/dL protective (subtract a risk factor) per year in those with stable glycemic control and LDL: <100 mg/dL optimal or: quarterly in those with unstable glycemic control. <100 mg/dL with CHD or CHD risk equivalent The ADA recommends an A1c goal of approximately <130 mg/dL with two or more risk factors 7% in those with diabetes to reduce the complications <160 mg/dL with 0 to 1 risk factor associated with diabetes (microvascular, macrovascu- Risk factors: Cigarette smoking, hypertension (blood pressure >140/90 mm Hg or on hypertensive medication), HDL <40 mg/dL, lar, and neuropathic) family history of premature CHD, age (men 45 years, women • Hemoglobin A1c is made through the three-step 55 years). glycation process ○ Glucose reversibly binds to the amino terminal CHD, Coronary heart disease; HDL, high-density lipoprotein; LDL, low-density end of hemoglobin to form a labile aldimine lipoprotein. ○ The aldimine undergoes irreversible rearrangement (Amadori arrangement) to form a stable • Serve as major source of energy for the body; glucose is ketoamine the only monosaccharide used by the body for energy; ○ Glucose undergoes a conformational change to a all sugars must be digested to this monosaccharide cyclic structure for use Measurement • Glucose regulation involves primarily insulin • Three enzymatic methods are available: Hexokinase, (decreased glucose) and glucagon (increased glucose), glucose oxidase, and glucose dehydrogenase both produced from the pancreas, acting in conjunc- (Table 8-6) tion. To a lesser extent, epinephrine, cortisol, and Specimen Considerations growth hormone (all increase glucose) contribute to • Glucose may be analyzed on a variety of samples, glucose regulation; insulin is the only hormone that including acts to decrease glucose concentrations • Serum • Carbohydrate metabolism terminology • Plasma (5% lower than serum) • Glycolysis: Process that converts glucose to pyru- • Whole blood (15% lower than plasma) vate for energy purposes • Capillary (2 to 5 mg/dL higher than venous col- • Gluconeogenesis: Formation of glucose from non- lected samples) carbohydrate sources (amino acids, lactate, glycerol • Urine [lipids]) • Other fluids (cerebrospinal fluid [CSF], ascetic, • Glycogenolysis: Breakdown of glycogen to form pleural, peritoneal, and drainage fluids) collect in glucose tubes containing fluoride/oxalate preservative; ana- • Glycogenesis: Conversion of glucose to glycogen for lyze immediately after collection, centrifuge if storage purposes not clear

TABLE 8-5 Primary Hyperlipoproteinemias

Fredrickson Name Phenotype Increased fraction(s) Plasma Appearance Defect Hyperchylomicronemia I TG, chylomicrons Cream layer over clear plasma LPL or Apo-CII deficiency Hyperbetalipoproteinemia IIA IIA: LDL, TC IIA: Clear LDL receptor mutation IIB IIB: LDL, TG, VLDL IIB: Turbid Decreased LDL clearance Dysbetalipoproteinemia III TC, TG, LDL (IDL) Turbid Defective ApoE Hyperprebetalipoproteinemia IV VLDL, TG Turbid Mixed hyperlipoproteinemia V Chylomicrons, VLDL, TC, Cream layer over turbid plasma TG

HDL, High-density lipoproteins; LCAT, lecithin cholesterol acyltransferase; LDL, low density lipoproteins; LpL, lipoprotein lipase; TG, triglycerides; VLDL, very low density lipoproteins. 232 CHAPTER 8 Clinical Chemistry

TABLE 8-6 Enzymatic Methods to Measure Glucose

Method Principle Hexokinase Hexokinase Glucose + ATP ! Glucose-6-phosphate + ADP Glucose-6-phosphate + NAD + G6PD ! 6-Phosphogluconate + NADH + H + Glucose oxidase GlucoseOxidase Glucose + O2 ! Gluconic acid + H2O2 Peroxidase o-Dianisidine + H2O2 ! Oxidized o-dianisidine + H2O Glucose dehydrogenase Glucose Dehydrogenase Glucose + NAD + ! D-Glucono-d-lactone + NADH + H +

Recommended Ranges Screening Oral Glucose Tolerance Test for the Diagnosis of Gestational American Diabetes Association TABLE 8-8 Diabetes Mellitus BOX 8-2 Recommended Range for Glucose Gestational Diabetes Screening: Glucose Challenge Test Glucose <100 mg/dL Sample drawn 1 hr after a 50-g glucose drink Glucose Level Interpretation • Separate immediately after collection to avoid glycolysis; glucose in whole blood at room temperature <140* mg/dL Normal screen undergoes glycolysis at a rate of 5% to 7% per hour. 140*-199 mg/dL Abnormal screen; perform diagnostic oral glucose tolerance test Collect in sodium fluoride if cannot be separated immediately (Box 8-2) *Some use a cutoff of >130 mg/dL because that identifies 90% of women • ADA criteria for the diagnosis of diabetes mellitus are with gestational diabetes mellitus in contrast to 80% identified using the found in Table 8-9 threshold of >140 mg/dL. • American Diabetes Association Criteria for the Diag- nosis of Gestational Diabetes. American Diabetes Association • Perform in those with high-risk characteristics: Diagnostic Criteria for Diabetes Marked obesity, history of gestational diabetes mel- TABLE 8-9 Mellitus litus (GDM), glycosuria, or a strong family history. Criteria Comments Initial screening at first prenatal visit; retest at 24 to 28 weeks of gestation if negative 1. Hemoglobin A1c Using a method traceable to the • Fasting plasma glucose (FPG) greater than 126 mg/ 6.5% National Glycohemoglobin Standardization Program dL or random plasma glucose (RPG) greater than or 2. Fasting plasma 200 mg/dL meets the diagnosis of GDM; if not diag- glucose nostic (in women with high-risk characteristics), 126 mg/dL perform either of the following or ○ One-step approach 3. Random plasma With symptoms of hyperglycemia § Perform a diagnostic oral glucose tolerance test glucose (OGTT) without prior screening (Table 8-8) 200 mg/dL ○ Two-step approach or 4. 2-Hr plasma glucose During an oral glucose tolerance test as Oral Glucose Tolerance Test in 200 mg/dL described by the World Health Diagnosis of Gestational Diabetes Organization with a 75-g glucose load Samples Drawn After 100-g* NOTE: In the absence of symptoms of hyperglycemia, 1-3 should be confirmed TABLE 8-7 Glucose Drink by repeat testing.

Time of Sample Collection Target Level § Perform a screening OGTT, if the screening Fasting (before glucose load) 95 mg/dL OGTT is abnormal (Table 8-9) 1 Hr after glucose load 180 mg/dL § Perform the diagnostic OGTT (see Table 8-8) 2 Hr after glucose load 155 mg/dL Pathologic Processes 3 Hr after glucose load{ 140 mg/dL Hypoglycemia • Defined as glucose value of 45 mg/dL or less, life- Interpretation: If two or more values meet or exceed the target level, gestational diabetes mellitus is diagnosed. threatening condition *A 75-g glucose load may be used, although this method is not as well • Diagnosed based on Whipple triad: (1) glucose 45 mg/dL validated as the 100-g oral glucose tolerance test. or less, (2) symptoms of hypoglycemia, (3) resolution {The 3-hr sample is not drawn if 75 g is used. of symptoms after glucose administration CHAPTER 8 Clinical Chemistry 233

• Two types • Types of DM • Reactive “fictitious” hypoglycemia: Caused by • Type 1 DM (T1DM) (5%-10% of all DM cases) insulin injections ○ Cause: Absolute deficiency of insulin secretion • Spontaneous: Caused by excessive insulin secretion • Type 2 DM (T2DM) (90%-95% of all DM cases) (insulin-producing tumor) ○ Cause: Combination of insulin resistance and • C-peptide and insulin levels are helpful in differentiat- inadequate compensatory secretion of insulin ing the types • Gestational DM (GDM) • Fictitious: See increased insulin, low or undetect- ○ Any degree of glucose intolerance with onset or able C-peptide first recognition during pregnancy • Spontaneous: See increased insulin, increased • Other specific types C-peptide ○ Caused by genetic abnormalities, diseases, endo- • Unexplained hypoglycemia in children (<5 years of crinopathies, infections, drugs, etc. age) may indicate galactosemia (inborn error of glu- Markers of Glucose Control: Analytes Used to Assess cose metabolism), Clinitest can aid in the diagnosis Glucose Control • Galactosemia suggested when urine glucose • Glucose: Glucose level at the time of draw (dipstick) is negative and Clinitest positive • Hemoglobin A1c (HbA1c) also known as glycated • Hypoglycemia in children with liver enlargement is hemoglobin. Measure of glucose control over the pre- suggestive of one of 14 glycogen storage diseases, vious of 2 to 3 months (life span of a red cell), repre- von Gierke’s disease. This is an inherited disease sents glucose attached to the N-terminal valine of resulting from a deficiency in glucose-6-phosphatase the hemoglobin molecule (enzyme responsible for breaking down glycogen). • A very strong association between HbA1c and esti- • Clinically significant glycogen storage diseases mated average glucose • Glycogen storage disease IA (von Gierke’s disease): • The ADA and the American Society for Clinical Deficiency in glucose-6-phosphatase Chemistry recommend estimated average glucose • Glycogen storage disease II (Pompe’s disease): (eAG) be reported along with every HbA1c result Deficiency in acid maltase • eAG: Calculated average glucose that corresponds to a • Glycogen storage disease III (Cori’s disease): measured HbA1c result. Deficiency in debranching enzyme eAG mg=dL ¼ 28:7 A1c 46:7 • Glycogen storage disease V (McArdle’s disease): Deficiency in myophosphorylase • Fructosamine: Refers to glycated albumin. A measure • Glycogen storage disease VII (Tarui’s disease): of glycemic control over a 2- to 3-week period Deficiency in phosphofructokinase Hyperglycemia: Diabetes Mellitus Blood Gases • Defined as a group of metabolic diseases represented by hyperglycemia and caused by • Includes pH, pCO2 pO2, HCO3-, base excess, O2 • Defect in insulin secretion saturation • Defect in insulin action • pH, pCO2,pO2 are measured, all others are calculated • Defect in both insulin secretion and action parameters • Symptoms of hyperglycemia include polyuria, poly- • Normal acid-base status refers to a pH of 7.40 and a dipsia, weight loss with polyphagia, blurred vision ratio of bicarbonate to carbonic acid of 20:1 • Long-term complications include retinopathy, neurop- • pH athy, nephropathy, and atherosclerotic cardiovascular • Index of acidity or alkalinity of blood disease • pH¼log [H+]; inverse, nonlinear relationship to • Diagnostic criteria (see Table 8-9) [H+]; unitless; increase (H) ¼ decrease pH • Intermediate group ○ Decrease in 1 pH unit results in a 10-fold increase • The ADA also defined an intermediate group in [H+] activity located between the normal and diagnostic range, • Decreased pH (<7.35) indicates acidemia, referred to as increased risk for diabetes increased pH (>7.45) indicates alkalemia • Defined as follows • pH is regulated within a very narrow limit, optimal ○ FPG 100 to 125 mg/dL: Impaired fasting glucose pH¼7.40, which corresponds to an [H+]of ○ 2-hour PG in the 75-g OGTT 140 to 199 mg/dL: 40 nmol/L Impaired glucose tolerance • pCO2 is the respiratory component ○ A1c 5.7 to 6.4%: At risk for developing diabetes • pCO2 refers to the pressure of carbon dioxide dis- mellitus (DM) solved in the blood ○ NOTE: This intermediate group is not a clinical • A normal pCO2 refers to the ability of the body to entity, but rather serves as a risk factor for the remove CO2 at a rate equal to the cellular produc- development of DM and cardiovascular disease tion of CO2 234 CHAPTER 8 Clinical Chemistry

• The body rids itself of CO2 via two mechanisms: • Arterial, venous, and capillary samples acceptable The lungs and conversion of CO2 to carbonic acid • Samples are stable for 15 minutes at room tempera- - + that dissociates to HCO3 and H (in the presence ture, 45 minutes if stored on crushed ice of carbonic anhydrase) • Avoid pneumatic tube transport unless a pressure- • Increased pCO2 indicates respiratory acidosis (see sealed container is used Table 8-11 for causes of acid-base disturbances) • Anaerobic conditions are essential during collection; • Decreased pCO2 indicates respiratory alkalosis (see room air has a pCO2 of 0 and a pO2 of approximately Table 8-11 for causes of acid-base disturbances) 150 mm Hg, which may affect sample values if they • Bicarbonate is the metabolic component are uncapped or contain air bubbles • Calculated parameter, calculated from pH and pCO2 • Samples exposed to room air (uncapped or air bub- using the Henderson-Hasselbalch (HH) equation bles) may result in false elevations in pO2 and false ○ HH equation decrease in pCO2 as a result of equilibration with

0 room air pH ¼ pK + log HCO =H CO 3 2 3 • Collect in syringe with dry heparin anticoagulant; H CO ¼ pCO ðÞ¼ 0:0306 2 3 a 2 a glass syringe preferred stored on ice; if collected in Optimal HCO3 =H2CO3 ¼ 20:1 plastic syringe, store at room temperature and assay within 15 minutes • An indication of the buffering capacity of the blood Measurement • Increased bicarbonate indicates metabolic alkalosis • pH, pCO2, and pO2 are measured using an electro- (see Table 8-11 for causes of acid-base disturbances) chemical cell (galvanic cell) that contains two half cells • Decreased bicarbonate indicates metabolic acidosis (reference and indictor) connected by a salt bridge (see Table 8-11 for causes of acid-base disturbances) • Reference electrodes include standard hydrogen • pO2 (SHE), saturated calomel (SCE), and silver/silver • Is a measure of the pressure of dissolved O2 in chloride (Ag/AgCL) electrodes. Most commonly the blood used reference electrode is the silver/silver chloride • Value is related to the ability of the lungs to electrode oxygenate blood ○ Saturated with KCL (SCE and Ag/AgCL) or HCL • Decreased pO2 may be related to decreased pulmo- (SHE) so the electrode has a constant, fixed nary ventilation, impaired gas exchange, altered potential blood flow within the heart or lungs • Indicator electrodes include glass, liquid, solid- Physiologic Buffers state, gas sensing, and enzyme • Body’s first line of defense in all body fluids against ○ Potential of this electrode varies depending on changes in pH the analyte in the patient sample • All buffer systems are intercellular • pH and pCO are measured using potentiometry (cur- • 2 The body contains four buffer systems rent is maintained at zero and voltage is measured) and • Bicarbonate: Carbonic acid buffer system pO is measured using amperometry (voltage is con- ○ 2 Most physiologically important buffer system, stant and current is measured) works in conjunction with the hemoglobin buffer • pH is measured using a pH-sensitive glass indicator system to maintain normal blood gas status electrode with an Ag/AgCl reference electrode ○ Is of primary importance in acid-base regulation • pCO2 is measured using a modified pH electrode because CO2 (a volatile acidic gas) is produced covered by a silicone rubber membrane permeable from energy metabolism to gas. The pH electrode is modified by the addition ○ Combats the production of large amounts of acid of an aqueous layer of bicarbonate solution on the (H2CO3) according to the following equation inside surface of the membrane; the bicarbonate Carbonic Anhydrase buffer reacts with the CO2 in the patient sample, H CO ! H O+CO 2 3 2 2 producing an H+ ion that is detected by the internal • Hemoglobin buffer system pH meter ○ Reduced hemoglobin has an increased affinity • pO2 is measured using a cell containing a platinum + for H rather than oxygenated hemoglobin cathode, an Ag/AgCL reference electrode, a phos- ○ Buffers by transporting acid from the tissues to phatase buffer (in contact with both electrodes), the lungs for removal and a polypropylene membrane. Voltage is applied • Plasma protein buffer system and phosphate buffer to the cathode (0.6 V) and pO2 crosses the mem- system brane and reduces, causing four electrons of current ○ Of minor importance to flow for every O2 molecule Sample Considerations • Recommended ranges for blood gases are found in • Sample is highly susceptible to improper collection and Table 8-10 handling • Acid-base disorders are presented in Table 8-11 CHAPTER 8 Clinical Chemistry 235

Total Body Water, Electrolytes, and moving into the cell), largely determines osmolality Minerals of extracellular fluid • Potassium: Major cation of intracellular fluid, Anion Gap maintained by Na+/K+ ATPase pump (exchanges • Accounts for approximately 65% and 55% of total three sodium ions out of cell for two potassium ions body mass in males and females, respectively moving into the cell), involved with neuromuscular • Divided into two main categories: Intracellular water excitability and contraction of the heart (ICW) and extracellular water (ECW) (Table 8-12) • Chloride: Major extracellular anion, exact function • The composition of interstitial fluid is similar to that of not well understood plasma, except that plasma contains large protein • Bicarbonate: Second most abundant ion in extracel- molecules lular fluid, major component of the blood buffering • Regulation of fluid volume depends on plasma osmo- system lality and blood volume • Movement of fluid between compartments is a result • The plasma component of ECW is the only compart- of osmotic pressure and hydrostatic and oncotic pres- ment directly measurable sure differences • Electrolytes include bicarbonate, sodium, potassium, • Osmotic pressure is the most important factor mov- chloride, osmolality, and anion gap ing water (from lower concentrations to higher con- • Sodium: Major cation of extracellular fluid, main- + + centrations) between the intracellular space and tained by Na /K ATPase pump (exchanges three plasma; caused by the difference in concentrations sodium ions out of cell for two potassium ions of solutions on either side of a membrane • Hydrostatic pressure drives fluid out of the vessels into the surrounding tissue Recommended Ranges for Blood TABLE 8-10 Gases Analytes Osmotic Pressure, Osmolality, and Volume Analyte Range Units Regulation pH 7.35-7.45 No units • Osmotic pressure determines water distribution pCO2 35-45 mm Hg among body water compartments; force that moves pO2 80-100 mm Hg water from dilute to concentrated solutions HCO 22-28 mmol/L 3 • Osmolality is a measure of solute concentration of a Base excess 2-+2 mmol/L solution; a measure dependent on the number (not size O saturation 95 % 2 and charge) of particles in solution

TABLE 8-11 Acid-Base Disorders, Cause, and Laboratory Values

Associated Disorder Data Uncompensated Compensated* Cause

Respiratory Decreased pH N HCO3 Increased HCO3 Obstructive lung disease, acidosis Increased (1 mmol/L for every acute airway obstruction,

pCO2 10–mm Hg increase circulatory failure, impaired respiratory system { in pCO2) Metabolic Decreased pH N pCO2 Decreased pCO2 Ketoacidosis, hypoxic acidosis, renal failure acidosis Decreased (1-1.3 mm Hg for every

HCO3 1-mmol/L decrease in HCO3) Respiratory Increased pH N HCO3 Decreased HCO3 Hypoxia-induced hyperventilation, pulmonary embolism, alkalosis Decreased (1-2 mmol/L for every pulmonary edema, anxiety, overventilation, drugs,

pCO2 10–mm Hg decrease CNS disorders { in pCO2) Metabolic Increased pH N pCO2 Increased pCO2 Hypokalemia, excess administration of HCO3, vomiting, alkalosis Increased (0.6-0.7 mm Hg for every GI suction, corticosteroid excess

HCO3 1-mmol/L increase in HCO3)

↑, Increased; #, decreased; CNS, central nervous system; GI, gastrointestinal; N, normal. *The terms uncompensated and compensated refer to whether the body has made changes to restore the pH to normal (20:1 ratio). Uncompensated means the body has not yet attempted to restore the pH to normal; compensated refers to the fact that the body has made changes to restore the pH to normal. { Refers to compensation for acute situations only. Chronic situations will increase or decrease HCO3 by 3-5 mmol/L for every 10–mm Hg change in pCO2 236 CHAPTER 8 Clinical Chemistry

TABLE 8-12 Total Body Water Compartments and Electrolytes

Percent of Total Body Compartment Components Water (%) Major Cations Major Anions

Intracellular 60 K, Mg PO4 Extracellular Plasma 10 Na, K, Ca Cl, HCO3 Interstitial fluid 30 Na, K, Ca Cl, HCO3

Major Cations and Anions in Recommended Ranges for Extracellular and Intracellular Fluid TABLE 8-14 Electrolytes TABLE 8-13 Compartments Analyte Range Units Primary Cations Primary Anions Sodium 135-145 mmol/L Sodium (Na+) Chloride (Cl) Potassium 3.5-5.0 mmol/L + Potassium (K ) Bicarbonate (HCO3 ) Chloride 98-107 mmol/L + Calcium (Ca ) Phosphate (HPO4) Total carbon dioxide 21-28 mmol/L + 2 Magnesium (Mg ) Sulfate (SO4 ) Osmolality (plasma) 275-295 mOsm/kg Anion gap 8-16 (without potassium) mmol/L 12-20 (with potassium) • Osmolality formula

OsmolalitycalculatedðÞ¼mOsm=kg 2NaðÞ+ ðÞBlood urea nitrogen½ mg=dL 2:8 +ðÞ Glucose½ mg=dL =18 • • The measured osmolality and calculated osmolality Electrolytes are molecules capable of carrying a charge may be used to calculate the osmolal gap. A difference and are characterized as anions or cations • between the calculated and measured osmolality may Anions carry a negative charge • be due to the following Cations carry a positive charge • • Presence of other osmotically active substances Electrolytes are essential components in numerous (other than sodium, glucose, urea) processes • • Metabolic acidosis caused by nonelectrolytes (lactic Volume and osmotic regulation: Sodium, chloride, acid, ketoacids, alcohol, ethylene glycol, organic acids) and potassium • • Osmolal gap formula Heart contractility: Potassium and magnesium • Enzyme activation: Magnesium Osmolal gap ¼ Measured osmolality Calculated osmolality • Acid-base balance: Bicarbonate, potassium, and • Regulation of osmolality and volume depends mainly chloride on thirst and to a lesser extent antidiuretic hormone • Blood coagulation: Calcium and magnesium (ADH), renin-angiotensin-aldosterone (RAA) system, • Sodium is the major cation in extracellular fluid, and natriuretic peptides potassium is the major cation in intercellular fluid + + • Thirst, stimulated by increased osmolality and • The Na /-K ATPase pump maintains the distribution decreased volume, acts to increase water intake of sodium inside the cell and potassium outside the cell and decrease osmolality by exchanging three sodium ions moving out of cells • ADH, stimulated by increased osmolality and for two potassium ions moving into the cell decreased volume, results in the reabsorption of • The kidneys maintain or excrete sodium based on water by the kidneys and decreased osmolality osmolality and blood volume • RAA system, stimulated by decreased volume and • Reference ranges for electrolytes are found in decreased sodium, increases blood pressure, serum Table 8-14 sodium, urine potassium, and urine hydrogen and Pathologic Processes decreases serum potassium and hydrogen Sodium • Natriuretic peptides, stimulated by increased car- • Hyponatremia diac volume, increase urine sodium and decrease • Defined as plasma sodium less than 135 mmol/L; blood volume and aldosterone weakness and confusion seen at values less than 20 mmol/L; paralysis and severe mental impair- Electrolytes ment seen at values less than 110 mmol/L • Hyponatremia should be confirmed by decreased • The term electrolytes refer to the majority of osmoti- plasma osmolality; both urine and plasma osmolal- cally active ions (cations and anions) in the body ity are helpful in differentiating between causes of • Primary cations and anions are listed in Table 8-13 hyponatremia CHAPTER 8 Clinical Chemistry 237

○ Causes of Hypernatremia and Urine osmolality 300 to 800 mOsm/kg: Impaired TABLE 8-15 Hyponatremia ADH release, diuretics, osmotic diuresis ○ Urine osmolality less than 300 mOsm/kg: Volume Diabetes insipidus Pathology Status Cause Hyponatremia Hypovolemia Sodium loss in excess of water Potassium Thiazide diuretics GI factors, burns, sweat Hypokalemia Potassium depletion • Defined as potassium less than 3 mmol/L Hyponatremia Normovolemia Water balance issues • Poses a serious concern in all people; characterized SAIDH by muscle weakness, irritability, paralysis, cardiac Hyperlipidemia (artifactual) abnormalities Adrenal insufficiency • Potassium less than 2.5 mmol/L induces cardiac Altered osmolality arrhythmias, including premature atrial and ven- regulation tricular beats and ventricular tachycardia and Hyponatremia Hypervolemia Movement of fluid from intravascular to interstitial fibrillation fluid • Potassium less than 3.5 mmol/L before surgery is asso- CHF ciated with increased incidence of arrhythmias requir- Hepatic cirrhosis ing cardiopulmonary resuscitation Advanced renal failure • Causes include Excess water intake • GI or urinary loss Nephrotic syndrome ○ Vomiting, diarrhea, gastric suction Hypernatremia Renal loss ○ Kidney disorders, including tubular acidosis GI loss ○ Excess aldosterone Sweating ○ Hypomagnesemia Inadequate thirst mechanism • Increased cellular uptake Those without access to water ○ Ingestion of excessive salt Alkalemia increases cellular uptake of potassium ○ Insulin increases uptake of potassium into CHF, Congestive heart failure; GI, gastrointestinal; SIADH, syndrome of muscle and liver cells inappropriate antidiuretic hormone. • Reduced dietary intake Hyperkalemia • Pathogenesis of hyponatremia is associated with • Defined as potassium greater than 5 mmol/L volume status (hypovolemic, normovolemic, and • Potassium 6 to 7 mmol/L associated with altered hypervolemic) (Table 8-15) electrocardiogram • Diagnosis of hyponatremia includes decreased • Potassium greater than 8 mmol/L associated with plasma sodium, decreased plasma osmolality, and muscle weakness differentiation of cause by urine sodium • Potassium greater than 10 mmol/L associated with ○ Urine sodium less than 15 mmol/L: Hypovole- cardiac arrest mia with fluid replacement (diarrhea, vomiting) • Poses a serious concern in all people; characterized by ○ Urine sodium greater than 20 mmol/L: Syn- muscle weakness, cardiac arrhythmias, and cardiac drome of inappropriate secretion of antidiuretic arrest hormone, thiazides, aldosterone deficiency, renal • Often multiple underlying conditions exist failure with excess water • Causes • Hypernatremia • Excess intake (oral or intravenous) • Defined as plasma sodium greater than 150 mmol/L ○ Likely seen with renal impairment • Rarely occurs in those with a normal thirst response • Cellular loss • Results from excess water loss relative to sodium ○ Processes that promote the release of potassium levels, including renal loss, gastrointestinal (GI) loss from cells (trauma, severe tissue hypoxia, mas- and sweating, fever, burns, and exposure to heat sive hemolysis, metabolic acidosis, administra- • Measurement of osmolality is used to differentiate tion of cytotoxic agents, hypothermia) the causes of hypernatremia • Decreased renal excretion • Diagnosis of hypernatremia includes increased ○ Glomerular filtration impairment plasma sodium and differentiation of cause by urine ○ Tubular function impairment osmolality ○ Conditions that reduce aldosterone production ○ Urine osmolality greater than mOsm/kg: GI loss, • Factitious (artifactual) diminished thirst, excessive intravenous or oral ○ Prolonged tourniquet use during collection intake of sodium ○ Excessive clenching of fist during venipuncture 238 CHAPTER 8 Clinical Chemistry

+ + ○ Storage of blood on ice; whole blood samples for AG ¼ ðÞNa +K ðÞCl + HCO3 ; normal range 10 to 20 mmol=L + potassium analysis should be collected and AG ¼ ðÞNa ðÞCl + HCO3 ; normal range 8 to 16 mmol=L stored at room temperature ○ Potassium is frequently omitted because its con- ○ Hemolysis tribution is so small and it is found elevated as a result of hemolysis Chloride • Clinical utility: Acid-base disturbances, laboratory error (quality control), assessment of increases in Hyperchloremia (Chloride >107 mmol/L) unmeasured cations and anions • Frequently follows hypernatremia • Causes: GI loss, renal tubular acidosis, mineralocorti- Bone and Minerals coid deficiency • Compensated respiratory alkalosis Bone • Often indicates an acidotic process • Two main functions of bone: Support and mineral Hypochloremia (Chloride <98 mmol/L) homeostasis • Causes: Excessive loss (GI loss), mineralocorticoid • Bone contains 99% of calcium, 85% of phosphorus, excess, salt-losing renal disease and 55% of magnesium • Often indicates an alkalotic process • Classification of bone: Long bones (i.e., limbs) or flat bones (i.e., skull) • Three main cell types in bone Bicarbonate • Osteoblasts: Synthesize bone, very rich in alkaline Increased Bicarbonate phosphatase (ALP), contain receptors for parathy- • Causes: Severe vomiting, hypokalemia, excessive roid hormone (PTH), calcitriol, and estrogen • Osteocytes: Synthesize small amount of matrix for alkali intake Decreased Bicarbonate bone integrity, capable of bone resorption when mineral concentrations are altered • Causes: Hypoxia, ketoacidosis, diarrhea • Osteoclasts: Demineralize and digest bone • Strength of bone determined by bone density (peak Anion Gap (AG) bone mass and amount of bone loss) and bone quality (structure, turnover, damage, and mineralization • The difference between the sum of routinely measured Bone Remodeling cations and anions (Table 8-16) • Remodeling of bone involves a coupling process of • Anion gap formulas bone resorption and formation • 10% of bone mass participates in the remodeling process Causes of Increased and Decreased • The coupled processes differ according to age; as peo- TABLE 8-16 Anion Gap ple age, they are predisposed to overall net bone loss (Table 8-17) Anion • Hormonal regulation of remodeling is controlled by Gap Cause Example PTH, calcitonin, vitamin D (systemic regulators), Increased Decreased Uremia and prostaglandin and growth factors (e.g., insulin like unmeasured growth factor) (local regulators) (see Table 8-18 for cations role of PTH, calcitonin, and vitamin D on calcium Increased Lactic acidosis, ketoacidosis, unmeasured ingestion of toxic and phosphorus anions substances (methanol, ethanol, ethylene glycol) Minerals Laboratory error Overestimation of sodium, underestimation of chloride • Include calcium, magnesium, and phosphorus or bicarbonate • Found predominantly in bone; bone contains 99% of Decreased Increased Calcium, magnesium calcium, 85% of phosphorus, 55% of magnesium unmeasured cations Decreased Hypoalbuminemia TABLE 8-17 Effect of Age on Bone Homeostasis unmeasured anions Age Group Net Effect Laboratory error Underestimation of sodium, Infancy and adolescents Bone formation>bone resorption overestimation of chloride Young adults Bone formation¼bone resorption or bicarbonate Older adults Bone formation

Effect of Hormones on Mineral Specimen Considerations for TABLE 8-18 Homeostasis Calcium, Magnesium, and TABLE 8-20 Phosphorus Hormone Effect Net Effect Analyte Specimen Comments Parathyroid Bone: Activates osteoclasts, Increased hormone mobilizes calcium and calcium Calcium Serum or plasma pH will alter calcium phosphorus Decreased (heparin only) levels (every 0.1 Kidney: Calcium reabsorption, phosphorus Separate as quickly as decrease in pH will phosphorus excretion Activation of possible to avoid increase the ionized Kidney: Stimulate 1- vitamin D uptake of calcium by calcium (IoCa) by a-hydroxylase activity cells 0.16 mg/dL (activates vitamin D) No hemolysis Calcitonin Activates osteoblasts, retention Decreased Magnesium Serum or plasma of calcium in bone calcium (heparin only) Decreased Separate as quickly as phosphorus possible to avoid Vitamin D Intestines: Reabsorption of Increased elution of magnesium calcium and phosphorus calcium from cells) Kidneys: Reabsorption of calcium Increased No hemolysis and phosphorus phosphorus Phosphorus Serum or plasma (heparin only) Separate as quickly as possible

TABLE 8-19 Recommended Ranges for Minerals • Most commonly used methods to measure calcium, magnesium, and phosphorus are found in Analyte Range Units Table 8-22 Calcium 8.5-10.5 mg/dL Ionized calcium 4.48-4.92 mg/dL Magnesium 1.5-2.5 mEq/L Proteins Phosphorus 2.8-4.0 mg/dL • PTH 10-65 pg/mL Serve a key role in transport, synthesis, storage, and Vitamin D clearance of substances. • May be classified by their function (Table 8-23) PTH, Parathyroid hormone. • Comprise 50% to 70% of dry weight of cells • Are classified as positive and negative acute phase reactants (see Table 8-23) • Metabolism depends on mineral availability and the • Positive acute phase reactants: Serum proteins that interaction of bone, kidney, and GI tract with PTH, increase in response to inflammation calcitonin, and vitamin D • Negative acute phase proteins: Serum proteins that • Calcium decrease in response to inflammation • Three forms present in plasma: 45% ionized cal- • Complex macromolecules composed of amino acids cium (biologically active form), 45% protein linked by peptide bonds in a head to tail fashion bound, and 10% complexed with anions • Measured clinically in blood, urine, CSF, amniotic • Controlled by action of PTH and vitamin D on bone fluid, saliva, feces, peritoneal fluid, and pleural fluid and kidney and intestines • May be separated by electrophoresis into the following • Has widespread functions, including formation of regions: Albumin, a1, a2, b, and g (see Table 8-24 for bone, coagulation, neurologic and neuromuscular list of globulins in each category and the function) function, and nonspecific binding • Phosphorus Proteins and Their Function • Functions in energy metabolism, nucleic acid metabolism, bone formation, cell signaling, and • Synthesized in the liver except for the immunoglobu- acid-base homeostasis lins, which are synthesized by plasma cells • Regulated secondary to calcium • Metabolism originates in the GI tract by proteolytic • Recommended ranges for minerals are found in enzymes, absorbed through the jejunum, and trans- Table 8-19 ported through portal circulation to amino acid pools • Specimen considerations are found in Table 8-20 • May be classified by their function (see Table 8-23) • Calcium, magnesium, and phosphorus status, • Are amphoteric in nature, therefore contain two ioniz- symptoms, and causes are presented in Table 8-21 able sites (proton-donating and proton-accepting sites) TABLE 8-21 Calcium Magnesium and Phosphorus Status, Symptoms, and Causes

Analyte Status Symptoms Causes Calcium Hypocalcemia Hyperreflexia Hypoparathyroidism Tetany Vitamin D deficiency Muscle pain Pseudohypoparathyroidism (PTH resistance) Cramps Calcitonin Paresthesias Magnesium deficiency Seizures Dietary deficiency Heart arrhythmias Phosphate infusion Calcium Hypercalcemia Fatigue/malaise Hyperparathyroidism Weakness Tumors/malignancies Depression Endocrine disturbances (thyrotoxicosis and Addison’s disease) Apathy Thiazide diuretics Nephrolithiasis Phosphorus Hyperphosphatemia Increased intake Increase GI absorption Cell injury/lysis Decreased renal excretion Phosphorus Hypophosphatemia Decreased intake Vitamin D deficiency Redistribution into the cell (i.e., respiratory alkalosis, insulin administration, epinephrine injection, healing bone) Excessive loss (renal disorders, proximal convoluted tubule injury Increased GFR Magnesium Hypermagnesemia Iatrogenic Increased intake Increased GI absorption Cellular release Magnesium Hypomagnesemia Malabsorption\malnutrition Diuretics Endocrine disorders (hyperaldosteronism, hyperthyroidism)

GFR, Glomerular filtration rate; GI, gastrointestinal; PTH, parathyroid hormone.

TABLE 8-22 Most Commonly Used Methods to Measure Calcium, Magnesium, and Phosphorus

Method Principle Comments Calcium Calcium+OCPC red complex ! Colorimetric using OCPC or arsenazo III Ionized calcium Calcium-specific ion selective electrode Potentiometry Magnesium Magnesium+Calmagite ! Most current methods use metallochromic indicators or dyes Colored product that change color as they bind magnesium (calmagite, methylthymol blue, formazan dye, arsenazo) Phosphorus Phosphate ions+Ammonium molybdate!Phosphomolybdate Detection steps: 1. Read phosphomolybdate at 340 nm 2. Reduce phosphomolybdate to molybdenum blue and read at 600-700 nm

OCPC, Orthocresolphthalein complexone.

TABLE 8-23 Classification of Proteins by Function

Function Description Example(s) Immune defense Immunoglobulins used for the elimination of foreign antigens IgA, IgG, IgM, IgD, and IgE Acute phase reactants Proteins associated with inflammation Negative APR: Albumin, prealbumin, and transferrin (APR) Positive APR: a1, a2, b, d globulins Transport Proteins used to bind and transport substances Prealbumin, albumin, ceruloplasmin, a2- macroglobulin, transferrin Coagulation Proteins aid clot formation and work with complement Complement CHAPTER 8 Clinical Chemistry 241

TABLE 8-24 Serum Protein Electrophoresis Regions With Associated Proteins and Their Function

Region Relative (%) Proteins Function Albumin 60 Albumin Transport, oncotic pressure, amino acid reserve

a1 31.a1-Antitrypsin 1. Binding, inactivation of trypsin-like enzymes 2. a1-Acid glycoprotein 2. Binds and inactivates progestin and neutral or cationic drugs 3. a1-Antichymotrypsin 3. Inactivation of chymotrypsin-like enzymes a2 10 1. Ceruloplasmin 1. Copper oxidase, oxidation of iron and incorporation into transferrin 2. a2-Macroglobulin 2. Inhibitor of proteolytic enzymes, transport of cytokines and growth factors 3. Haptoglobin 3. Binds and transports free hemoglobin b 11 1. Transferrin 1. Transports iron to tissues 2. Complement (C3 and C4) 2. Essential factors in complement pathways g 16 1. Immunoglobulins 1. Protection against foreign antigens 2. C-reactive protein 2. Nonspecific acute phase protein

• Can use total protein and albumin to calculate an A/G blood cell (RBC) count, hemoglobin, and hemat- ratio ocrit with an increased reticulocyte count ○ Globulin ¼ total proteinðÞ g=dL AlbuminðÞ g=dL Ceruloplasmin: Positive acute phase protein, functions to bind serum copper (90% bound A=G Ratio ¼ AlbuminðÞ g=dL =GlobulinðÞ g=dL by ceruloplasmin), used clinically to aid in the diagnosis of Wilson’s disease (see decreased total • High A/G suggests underproduction of globulins serum copper and ceruloplasmin with increased found in some leukemias and genetic deficiencies urine copper) • Low A/G suggests overproduction of globulins ○ Other includes a2-macroglobulin (multiple myeloma or autoimmune disease) or • b Region underproduction of albumin (liver disease or ○ Transferrin: Negative acute phase protein; func- nephrotic syndrome) tions in iron transport to the tissues; used clinically to investigate causes of anemia, gauge Normal A=G ¼ 1:0to1:8 iron metabolism, and determine iron-carrying capacity in the blood • Prealbumin: Negative acute phase protein, transport ○ Others include hemopexin, b -microglobulin, protein for thyroxine and triiodothyronine sensitive, 2 and complement marker of poor nutritional status, increased concentra- • g Region tions seen with steroid use and in alcoholics ○ C-reactive protein (CRP): Positive acute phase • Albumin: Negative acute phase protein, nonspecific protein, marker of inflammation, but more binder of many substances, major function is to main- recently high sensitivity CRP tain colloid osmotic pressure § Used to determine risk for development of car- • Globulins: Includes a , a , b, and d fractions 1 2 diovascular disease • a Region 1 ○ Immunoglobulins: Includes IgG, IgA, IgM, IgD, ○ a -Antitrypsin: Positive acute phase protein; 1 and IgE major component of the a region (90%); func- 1 • Separated by electrophoresis from anode to cathode tions to inhibit protease neutrophil elastase; defi- (Figure 8-2) ciency is associated with a mutation in the • Common abnormal electrophoretic patterns SERPINA1 gene causing a -antitrypsin defi- 1 • b-g Bridge: Fusion of the b and g bands caused by ciency. a -Antitrypsin is associated with emphy- 1 fast moving g-globulins; most common cause is cir- sema and sometimes cirrhosis rhosis, but this also may be seen in rheumatoid ○ Others include a -fetoprotein, a -glycoprotein, 1 1 arthritis and respiratory and skin infections and a1-antichymotrypsin • a2 Region ○ Haptoglobin: Positive acute phase protein; serves − to bind free hemoglobin in the blood for clear- + ance; used clinically to detect and evaluate hemo- α α lytic anemia. Laboratory data in hemolytic Anode Prealbumin Albumin 1 2 βδCathode anemia includes decreased haptoglobin, red FIGURE 8-2 Electrophoresis from anode to cathode. 242 CHAPTER 8 Clinical Chemistry

TABLE 8-25 Methods to Measure Total Protein and Albumin

Method Principle Comments Albumin Albumin+BCG!Color change Dye binding methods using BCG or BCP Total protein Protein + Cupric divalant ions Alkaline! Violet chelate Biuret reaction based on the presence of peptide bonds Serum protein Electrophoresis at pH 8.6 on cellulose acetate or agarose gel Uses barbital buffer at pH 8.6, conferring a net negative charge electrophoresis resulting in an electrophoresis pattern as follows: on proteins; proteins migrate from cathode to anode

Anode prealbumin albumin a1, a2, b, d cathode

BCG, Bromocresol green; BCP, bromocresol purple.

• Acute phase reaction: Increased bands in the a1, a2, TABLE 8-26 Recommended Ranges for Proteins and b regions with a decreased albumin band; seen with infection, tumor growth, hepatitis, surgery, Analyte Range Units trauma, burns Albumin 3.5-5.0 g/dL • Nephrotic syndrome pattern: Decrease in albumin a1 0.1-0.3 g/dL and g bands, with an increase in a -globulins 2 a2 0.6-1.0 g/dL • M-spike: Diffuse increase in the g-globulin band, b 0.7-1.1 g/dL found in polyclonal gammopathy g 0.8-1.6 g/dL Total protein 6.5-8.3 g/dL Specimen Considerations • Albumin: Serum or plasma (heparin), marked lipemia, may interfere with the bromocresol green assay Jaundice • Protein: Serum or plasma, plasma values will be 2 to • Condition characterized by yellow discoloration of 4 g/L higher than serum because of the presence of skin, sclera, and mucous membranes fibrinogen • Most commonly caused by increased bilirubin, not clin- • Methods to measure total protein and albumin are ically seen until bilirubin values exceed 3 to 5 mg/dL found in Table 8-25 • Total bilirubin is composed of three fractions • Recommended ranges for proteins are found in ○ Conjugated bilirubin (soluble and excretable) Table 8-26 ○ Unconjugated (water-insoluble and nonexcre- table) Liver ○ d Bilirubin (bilirubin covalently bound to albumin) • Largest internal organ, weighing 1200 to 1600 g • Elevations in unconjugated bilirubin pose great risk • Extremely vascular organ, receives 1500 mL blood/ for development of kernicterus, especially in infants min from two major vessels—the hepatic artery and • Jaundice may be divided into three categories the portal vein • Prehepatic • Performs many diverse functions, including metabo- ○ Occurs when increased amounts of bilirubin are lism (carbohydrate, amino acid, lipid, and bilirubin), brought to the liver cell, most commonly result- detoxification (serves as a barrier between potentially ing from increased RBC destruction harmful substances absorbed in the GI tract and sys- § Causes temic circulation), elimination (through the produc- § Hemolytic anemia tion of bile), and storage (provides a source of § Exposure to chemicals energy during fasting states) § Some cancers • Functional unit of the liver is the lobule § Autoimmune hemolytic anemia • Lobules contain two cell types § Transfusion reaction ○ Hepatocytes § Hemolytic disease of the newborn § Responsible for metabolic function of the § Congestive heart failure liver • Hepatic ○ Kupffer cells ○ Occurs from direct damage to the liver cell § Line the vascular spaces and are phagocytic § Causes macrophages capable of ingesting bacteria § Gilbert’s disease and other foreign material § Crigler-Najjar syndrome • The liver is the only organ involved in bilirubin metab- § Dubin-Johnson syndrome olism, because bilirubin is formed from the breakdown § Rotor’s syndrome of old RBCs (Figure 8-3) § Cirrhosis CHAPTER 8 Clinical Chemistry 243

Red Blood Cell Destruction

Heme

Hemoglobin oxygenase

Biliverdin reductase

Urobilinogen Unconjugated Bilirubin (insoluble)

80% 20% Bound by albumin and transported to the liver

Fecal Extrahepatic Liver Excretion Circulation Unconjugated Bilirubin (50-250 mg) UDP Glucoronyl Transferase

Conjugated Bilirubin

Renal excretion (1-4 mg) Conjugated Bilirubin (soluble and excretable)

Secreted from liver to the intestines

Intestines Conjugated Bilirubin

GI Bacteria

Urobilinogen

FIGURE 8-3 Bilirubin metabolism. GI, Gastrointestinal.

§ Viral hepatitis ○ Urine urobilinogen: Increased § Alcoholic liver disease • Hepatic § Drug-induced liver disease ○ Total bilirubin: Increased § Hepatocellular carcinoma ○ Conjugated bilirubin: Increased § Neonatal physiologic jaundice ○ Unconjugated bilirubin: Normal or increased • Posthepatic ○ Urine urobilinogen: Normal or increased ○ Occurs from the blockage of the flow of bile from • Posthepatic the liver ○ Total bilirubin: Increased § Causes ○ Conjugated bilirubin: Increased § Bile duct stones ○ Unconjugated bilirubin: increased § Gallbladder stones ○ Urine urobilinogen: Decreased § Cancer of the bile ducts Description of Disorders of Bilirubin Metabolism § Bile duct stenosis Gilbert’s Disease • Typical laboratory findings in prehepatic, hepatic, and • Inherited unconjugated hyperbilirubinemia posthepatic jaundice • Mild, benign condition, bilirubin only slightly elevated • Prehepatic (usually <3 mg/dL) ○ Total bilirubin: Normal to increased • Affects 2% to 12% of population (male gender bias) ○ Conjugated bilirubin: Normal to increased • Caused by defective transport of bilirubin through the ○ Unconjugated bilirubin: Increased liver cell membrane 244 CHAPTER 8 Clinical Chemistry

• Activity of uridine diphosphate (UDP)–glucuronyl condition resulting in the infiltration of fat into transferase is 20% to 50% the liver; condition is reversible, with cessation • Requires no treatment of alcohol resulting in complete recovery; very Crigler-Najjar Syndrome few laboratory abnormalities seen • Rare, inherited condition ○ Alcoholic hepatitis: Occurs with moderate alco- • Caused by decreased or absent UDP–glucuronyl hol consumption over a longer period; presents transferase with a variety of symptoms, including hepato- • Classified as type 1 (no UDP–glucuronyl transferase, megaly, vomiting, jaundice, abdominal pain. bad prognosis) and type 2 (decreased UDP–glucuronyl Laboratory results reflect liver damage and transferase) include increased aspartate aminotransferase Dubin-Johnson Syndrome (AST), alanine aminotransferase (ALT), g- • Chronic, benign condition producing obstructive liver glutamyl transferase (GGT), ALP, and bilirubin disease increased acute phase reactants • Results in the ineffective removal of conjugated ○ Alcoholic cirrhosis: Occurs with heavy alco- bilirubin from the hepatocyte hol consumption over an extended period; • Causes conjugated hyperbilirubinemia results in the most severe damage to the • Requires no treatment liver that is irreversible; symptoms include Rotor’s Syndrome weight loss, weakness, hepatomegaly, spleno- • Similar to Dubin-Johnson syndrome megaly, jaundice, ascites, fever, malnutrition, • Benign conjugated hyperbilirubinemia and edema. Laboratory findings include ele- • Caused by a reduction in the concentration or activity vated AST and ALT, decreased albumin, of the intracellular binding protein ligandin prolonged prothrombin time, and elevations Other Liver Disorders in ALP and bilirubin associated with the cholestatic form Reye’s Syndrome Specimen Collection and Handling • Acute and often fatal childhood condition (5-15 years) • Bilirubin: Serum or plasma acceptable, protect from • Hypothesized to be caused by a virus that improves light (bilirubin will deteriorate 30% to 50%/hr when within 2 and 4 days and then is followed by the abrupt exposed to light), no hemolysis, fasting sample onset of vomiting and diarrhea that may progress to preferred coma, respiratory arrest, and often death • Methods • Hallmark signs include encephalopathy and fatty • Most commonly used methods to measure bilirubin degeneration of the liver are modifications of the diazotized sulfanilic acid • Aspirin has been found to be associated with Reye’s method (diazo method) described by Jendrassik syndrome and Grof in 1938 (Table 8-27) Alcoholic Liver Disease • Recommended ranges for bilirubin are found in • Relatively common condition resulting from long- Table 8-28 term consumption of alcohol • Disease is caused by the hepatotoxic effects of acetaldehyde in high concentrations General Endocrinology • Acetaldehyde is formed from the catabolism of alcohol in the presence of alcohol dehydrogenase • Hormone: A chemical substance produced by an organ • Three associated stages to result in an effect on a target organ ○ Alcoholic fatty liver: Occurs with moderate alco- • Hormones are classified into three groups: Protein, ste- hol consumption for 6 to 12 months; reversible roid, and aromatic amino acid derivatives

TABLE 8-27 Commonly Used Methods to Measure Bilirubin

Method Principle Comments Total Bilirubin+Diazotized sulfanilic acid+ Classic diazo reaction described by Ehrlich; accelerator is caffeine and sodium bilirubin Accelerator!Azodipyroles (reddish-purple) benzoate; all fractions of bilirubin react with this method Direct Bilirubin+Diazotized sulfanilic acid ! Classic diazo reaction minus the accelerator; only conjugated and d bilirubin bilirubin Azodipyroles (reddish-purple) react in this method Indirect Indirect bilirubin¼Total bilirubinDirect bilirubin Calculation bilirubin CHAPTER 8 Clinical Chemistry 245

• Protein hormones are produced by the hypothala- • The hypothalamus (located in the brain) synthesizes mus, pituitary gland, and other target glands and and secretes releasing or inhibiting factors to turn on circulate in a free form (unbound) or shut off the HPT axis; the central nervous system • Steroid hormones are all derived from cholesterol, and concentration of target gland hormones regulate majority circulate bound to carrier plasma proteins the release of these hormones • Aromatic amino acid derived hormones are synthe- • The pituitary gland (found at the base of the skull) pro- sized from tyrosine duces hormones (tropic and nontropic) that act • Hormone concentrations in the body are tightly directly on a target organ to produce hormones controlled by feedback control of the hypothalamic- • Hypothalamus and pituitary hormones are synthe- pituitary-target organ axis (HPT axis) (Figure 8-4) sized and released in a minute-to-minute pulsatile fash- • Two types of feedback exist ion; pituitary hormones also exhibit circadian rhythm • Positive • Transport proteins (found in circulation) carry steroids ○ Positive feedback begins when the hypothalamus from the organ of synthesis to the target organ or receives input to produce a releasing factor that tissue (see Table 8-29 for a list of common transport acts on the pituitary gland. The pituitary proteins) responds by releasing tropic hormones that act • See Table 8-30 for a list of hypothalamus and pituitary on a specific target gland to promote hormone hormones, their associated target glands, and hor- synthesis and release mones produced • Negative • Disorders occur as a result of hyperfunction or hypo- ○ Negative feedback: Hormones that are synthe- function of an endocrine gland; classified as primary, sized and released from target glands feed back secondary, or tertiary depending on the site of the to the pituitary gland and hypothalamus to stop defect further production of releasing and tropic • Primary disorders refer to a defect in the hormones target gland

TABLE 8-28 Recommended Ranges for Bilirubin Hormone Transport Proteins and Analyte Range Units TABLE 8-29 Associated Hormones Total bilirubin 0.2-1.0 mg/dL Protein Hormone(s) Conjugated bilirubin 0.0-0.2 mg/dL Cortisol-binding globulin Cortisol Sex hormone–binding globulin Estradiol, testosterone CNS Input Thyroid-binding globulin T3,T4 Cerebral Input Thyroxine-binding prealbumin T (+) 4 (–) Albumin All hormones (+) (–) , Triiodothyronine; , thyroxine. Hypothalamus T3 T4

Major Hypothalamus and Pituitary Releasing factors (+) Hormones: Their Target Organs Inhlbitory factors (–) TABLE 8-30 and Hormones

Hypothalamus Pituitary Target Pituitary Hormones Hormones Gland Hormones CRH ACTH Adrenal Glucocorticoids gland Mineralocorticoids Trophic hormones (+) Catecholamines

TRH TSH Thyroid T3 and T4 gland Endo Gland GnRH LH/FSH Ovaries/ Sex steroids testes GHRH GH Bone Insulin-like growth factor Secretory hormone ACTH, Adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; FSH, follicle-stimulating hormone; GH, growth hormone; GnRH, Target tissue gonadotropin-releasing hormone; LH, luteinizing hormone; TRH, thyrotropin- FIGURE 8-4 Hormone feedback control system. CNS, Central releasing hormone; TSH, thyroid-stimulating hormone; T3, triiodothyronine; nervous system; Endo gland, endocrine gland. T4, tetraiodothyronine or thyroxine. 246 CHAPTER 8 Clinical Chemistry

(Peroxidase Colloid Analyte Range Units transaminase) sTSH 0.4-4.2 mU/mL fT 0.8-2.7 ng/dL 4 (Peroxidase) fT 1.4-4.4 pg/mL (b) 3 (d) Colloid THBR 0.72-1.25 H2O2 drops Cell fT3, Free triiodothyronine; fT4, free thyronine; sTSH, serum thyroid- Protease stimulating hormone; THBR, thyroid hormone–binding ratio. (e) THYROGLOBULIN peptidase SYNTHESIS

BOX 8-3 Function of Thyroid Hormones (f) MIT DIT Deiodinase Fetal growth and development T3 T4 Sexual maturation (a) Central nervous system development Capillary Caloric and metabolic activity T3 T4 Stimulate protein synthesis FIGURE 8-5 Thyroid hormone synthesis. DIT, diiodotyrosine; Stimulate carbohydrate and lipid metabolism MIT, moniodotyrosine; T3, triiodothyronine; T4, thyroxine. Influence calcium and phosphorous metabolism Increased blood flow, cardiac output, and heart rate

• Coupling of iodinated tyrosyl residues to • Secondary disorders refer to a defect in the thyroglobulin (Tg) pituitary gland • Release of iodothyronines through the cleavage of • Tertiary disorders refer to a defect in the follicular Tg by proteases (Figure 8-5) hypothalamus • The synthesis of thyroid hormones requires iodine, • Recommended ranges for thyroid testing can be which is ingested in the form of iodide; the transport found in Table 8-31 of iodide to the follicle is the rate-limiting step in the synthesis of thyroid hormones Thyroid Function, Hormones, and Disorders • Approximately 80% of secreted hormone is T4 and 20% is T3; most of the physiologically available T3 • The thyroid gland is a small bilobed organ located in is produced from the deiodination of T4; deiodination the lower front of the neck of the b ring of T4 produces T3; deiodination of the a • The thyroid gland contains two main cell types: Follic- ring of T4 produces reverse T3 (rT3), a biologically ular and parafollicular inactive compound • Follicular cells produce thyroid hormones (triiodo- • T3 and rT3 are produced in approximately equal amounts thyronine [T3] and thyroxine [T4]), which are • T3 and T4 circulate bound to carrier proteins stored in the colloid of the follicle cell (99% bound) • Parafollicular cells produce calcitonin and are • Disorders of the thyroid gland are divided into three referred to as C-cells main groups • The production and secretion of thyroid hormones (T3 • Hyperthyroidism (overproduction of thyroid and T4) is regulated by the anterior pituitary hormone, hormones) thyroid-stimulating hormone (TSH), also known as ○ Caused by autoantibodies to TSH receptors thyrotropin (Graves’ disease), thyroidal nodules (toxic nodu- • Production and secretion of TSH are regulated by lar goiters), thyroidal TSH receptor sensitivity to thyrotropin-releasing hormone (TRH) produced by human chorionic gonadotropin (seen in the hypothalamus pregnancy) • TRH and TSH secretion is turned on and off depend- ○ Most common of all causes is Graves’ disease ing on thyroid hormone levels by positive and negative ○ Occurs most commonly in females control of the HPT axis ○ Manifests with characteristic symptoms, includ- • Thyroid hormones are responsible for multiple physi- ing goiter and exophthalmos ologic processes (Box 8-3) ○ Anti–thyroid peroxidase and TSH receptor anti- • Thyroid hormone production involves four steps bodies are present in up to 95% of cases • Iodide trapping by the thyroid gland ○ May manifest as subclinical or overt (see • Incorporation of iodide into tyrosine using Table 8-32 for laboratory findings in thyroid peroxidase disorders) CHAPTER 8 Clinical Chemistry 247

TABLE 8-32 Laboratory Findings in Thyroid Disorders

Disorder TSH fT4 T3 Antibodies Comment Subclinical hypothyroidism Increased N N +/ Symptoms generally not present Overt hypothyroidism Increased Decreased Decreased Often present Subclinical hyperthyroidism Decreased N N +/ Overt hyperthyroidism Decreased Increased Increased Often present

Euthyroid sick syndrome Increased or decreased N Decreased Not present Increased rT3

N, Normal; rT3, reversed triiodothyronine.

○ Symptoms include nervousness, tremor palpita- unavoidable, FT4 is the most reliable indicator of thy- tions, fatigue, weakness, weight loss, heat intol- roid status in sick individuals erance, menstrual change, neck mass, muscle • A battery of tests is available to assess thyroid func- weakness, and exophthalmos (prominence of tion, including TSH, fT4, total thyroxine (TT4), free the eyes) triiodothyronine (fT3), total triiodothyronine • Hypothyroidism (underproduction of thyroid (TT3), index methods (thyroid hormone–binding hormones) ratio [THBR] or free thyroxine factor [T-7]), Tg, ○ Primary hypothyroidism may be caused by insuf- thyroid antibodies, and thyroxine binding globulin ficient iodine or autoimmune destruction of the (TBG) measurements thyroid gland (Hashimoto’s thyroiditis) • Free hormone levels (fT4 and fT3) are better indica- ○ Most common primary cause is Hashimoto’s tors of thyroid status because they are independent thyroiditis of binding protein concentrations ○ Secondary and tertiary hypothyroidism are • The ATA recommends using TSH and fT4 for diag- caused by pituitary and hypothalamus dysfunc- nostic purposes; if necessary, test for fT3 also may tion, respectively be ordered ○ May present as subclinical or overt (see • A log linear relationship exists between TSH and Table 8-32) fT4 such that a doubling of thyroid hormones ○ Symptoms include cold intolerance, dyspnea, results in a 100-fold inverse change in TSH; there- weight gain, cognitive dysfunction, constipation, fore, TSH is the most sensitive indicator of thyroid dry skin, hoarseness, edema, myalgia, depres- status sion, menorrhagia • TSH • Euthyroid sick syndrome (abnormal thyroid hor- ○ Assays are labeled as “generation” assays, which mones in the absence of thyroid disease) refers to the sensitivity of the method; first- ○ Refers to abnormalities in thyroid hormone con- generation methods refer to assays with analyti- centrations in the absence of thyroid disease cal sensitivities of 1.0 mIU/L, second-generation ○ Common in hospitalized patients assays have a sensitivity of 0.1 mIU/L, third- ○ Characterized by decreased conversion of T4 to generation assays have a sensitivity of 0.01 T3 with an increase in rT3; TSH may be low mIU/L, etc. and the response to TRH blunted ○ Most current assays for TSH employ a two-site ○ There is no benefit to treating this with thyroid (sandwich) heterogeneous immunoassay that hormone replacement uses a capture antibody directed toward the a • Disorders may be further characterized as the follow- subunit and a second antibody (labeled) directed ing depending on the site of the defect toward the b subunit of the TSH molecule. The • Primary: Disease originates in the thyroid gland second antibody is commonly labeled with per- • Secondary: Disease originates in the pituitary gland oxidase, ALP, and/or chemiluminescent or fluo- • Tertiary: Disease originates in the hypothalamus rescent labels • American Thyroid Council (ATC) published recom- • fT4 mendations for thyroid disease screening; high-risk ○ Reference method involves dialysis of sample to persons should be screened (high risk includes which a known tracer amount of iodinated T4 elderly, neonates, postpartum females, those with a has been added family history of autoimmune disease or thyroid § Sample is assayed for TT4 and fT4 and calcu- disease) lated using the formula • Diagnosis of hyperthyroidism and hypothyroidism % fT4 ¼ TT4 % Tracer T4ðÞ dialyzed should include TSH and free thyroxine (fT ) 4 ○ • The ATC recommends delaying thyroid testing in Current method includes measurement of fT4 those who are sick until the illness subsides. If using immunometric assays 248 CHAPTER 8 Clinical Chemistry

• THBR syndrome), hyperaldosteronism (Conn’s syndrome), ○ Used to estimate the number of unoccupied thy- and congenital adrenal hyperplasia roid hormone binding sites • Cushing’s syndrome (hypercortisolism) ○ Represents the percent uptake of patient sample ○ General term used to describe any condition relative to the percent uptake of a euthyroid per- resulting from increased cortisol son; formula ○ A condition referred to as pseudo-Cushing’s syn- THBR ¼ % UptakeðÞ patient serum drome may exist with chronic alcoholism and/or % UptakeðÞ reference serum a high level of cortisol-binding globulin associated with pregnancy or the use of contraception, ○ A THBR of 1.0 indicates that the patient uptake which can be confused with Cushing’s syndrome and the reference sample uptake are the same ○ Causes include iatrogenic and noniatrogenic; ○ More accurately reflects thyroid status in those noniatrogenic causes include pituitary tumors with abnormal binding protein concentrations (60% of all cases), ectopic ACTH (20% of all ○ THBR is directly proportional to the free hor- cases), and adrenal adenoma and adrenal carci- mone fraction noma (combined 20% of all cases) Sample Collection and Handling ○ Cushing’s syndrome is divided into two broad • Serum or plasma (some methods require serum only) categories: ACTH dependent (Cushing’s disease) • Preferable free from hemolysis and lipemia and ACTH independent (Cushing’s syndrome) • Newborn screening is whole blood collected by heel § Cushing’s disease is caused by an ACTH- puncture within 48 to 72 hours after birth producing pituitary tumor; the feedback con- • Sample should be stored at 2 to 8 C if not analyzed trol system is nonfunctional. Condition is within 24 hours characterized by § Increased cortisol and ACTH Adrenal Cortex and Medulla § Increased glucose, sodium, and aldosterone § NOTE: Cortisol in very high levels can have • Adrenal glands are located at the upper pole of each mineralocorticoid activity kidney § Cushing’s syndrome is caused by adrenal ade- • Adrenal cortex constitutes 90% of total gland noma that produces excess cortisol; the feed- volume, and the medulla constitutes 10% of total back control system is functional. Condition gland volume is characterized by • Adrenal cortex secretes corticosteroids, including glu- § Increased cortisol and decreased ACTH cocorticoids (cortisol), mineralocorticoids (aldoste- § Increased glucose, sodium, and aldosterone rone), and sex steroids (androgens and estrogens) ○ Clinical features of Cushing’s syndrome are listed • Adrenal medulla secretes catecholamines (epineph- in Box 8-4 rine, norepinephrine, dopamine) • Laboratory diagnosis of Cushing’s syndrome • All hormones are steroid hormones that are produced • Includes two phases of testing from cholesterol ○ Screening tests: Used to detect hypercortisolism Adrenal Cortex Hormones § Overnight dexamethasone test (1 mg dose at • Glucocorticoids: Cortisol 11 PM); at 8 AM plasma cortisol levels are • Regulated by HPA axis (adrenocorticotropic hor- collected mone [ACTH] at the pituitary; corticotropin- § Cortisol 5 mg/dL or less: Normal releasing hormone [CRH] at the hypothalamus) • Mineralocorticoids: Aldosterone • Regulated by the RAA system and to a lesser extent Clinical Feature of Adrenal ACTH (pituitary) and potassium levels BOX 8-4 Hypercortisolism • Androgens: Dehydroepiandrosterone sulfate (DHEA-S), dehydroepiandrosterone (DHEA), and androstenedione Truncal obesity Adrenal Medulla Hormones Hypertension • Catecholamines Glucose intolerance • Epinephrine Plethoric facies • Norepinephrine Skin atrophy • Dopamine Muscle weakness Adrenal Cortex Disorders Menstrual and gonadal dysfunction • Diseases of this organ are classified as resulting from Acne Hirsutism hypofunction or hyperfunction of the adrenal cortex Osteoporosis • Adrenal gland hyperfunction may result in three broad Psychiatric problems categories of disorders: Hypercortisolism (Cushing’s CHAPTER 8 Clinical Chemistry 249

§ Cortisol 10 mg/dL or greater: increased aldosterone are considered suspect Hypercortisolism for hyperaldosteronism. The positive screen test § Urine free cortisol test, involves a 24-hour must be confirmed using an aldosterone urine collection suppression test § Urine cortisol less than 50 mg/24 hours: ○ Confirmation of hyperaldosteronism can be Normal accomplished using one of three tests § Cortisol at midnight (when cortisol levels are § Oral salt load test at their lowest); a plasma sample or more § Intravenous saline infusion test commonly a salivary sample is collected at § Fludrocortisone suppression test midnight • Once the diagnosis is made, differentiation between § Cortisol less than 5 mg/dL: Normal aldosterone-producing adrenal adenoma and IHA § Cortisol 5 mg/dL or greater: Hypercortisolism is required to ensure proper treatment • Differentiating tests: Used to differentiate the ○ Bilateral adrenal venous sampling coupled with causes of hypercortisolism simultaneous determination of ACTH- ○ Plasma ACTH stimulated cortisol from both glands may be used § Less than 2 pmol/L: Cushing’s syndrome • Congenital adrenal hyperplasia § Approximately 11 pmol/L: Cushing’s disease ○ Describes a group of inborn errors of metabolism § 50 pmol/L¼Ectopic sources caused by enzyme deficiencies in the biosynthesis ○ Bilateral petrosal sinus sampling after CRH (100 of cortisol and aldosterone mg) administration. Three venous catheters are § Can cause adrenal insufficiency and excess placed: one each in the left and right inferior androgen synthesis petrosal sinus sampling (IPSS) that drain the § Can cause females at birth to appear pituitary gland and one in the inferior vena cava masculinized (IVC). Baseline samples are collected, CRH is ○ At least six defects are known to exist. Most administered, and collections are taken at 2, 5, common defect is 21-hydroxylase deficiency and 10 minutes. The ratio of IPS to IVC is inter- (partial), which occurs in 95% of all congenital preted as follows adrenal hyperplasia cases § IPSS:IVC greater than 2: Cushing’s disease ○ 21-Hydroxylase deficiency is inherited as an § IPSS:IVC less than 1.4: Ectopic cause autosomal recessive disorder affecting 1 in • Conn’s syndrome (hyperaldosteronism) 10,000 births. Much higher frequencies may be ○ Results from an excess of mineralocorticoids, seen in Ashkenazi Jews (1 in 30) mainly aldosterone. Aldosterone in excess will ○ Diagnosis involves demonstration of low corti- cause reabsorption of sodium (water and chlo- sol, elevated 17-hydroxyprogesterone, low ride secondarily) and excretion of potassium aldosterone and hydrogen • Adrenal gland hypofunction results in adrenal ○ Characterized by hypertension, hypokalemia, insufficiency and is caused by and alkalosis as a result of the action of ○ Primary adrenal disease (Addison’s disease) aldosterone ○ Secondary adrenal insufficiency (decreased levels ○ Caused mainly by two disorders: Aldosterone- of ACTH or CRH) producing adrenal adenoma (Conn’s syndrome) ○ Long-term suppression of the HPT axis by exog- or idiopathic hyperaldosteronism (IHA); differ- enous administration of glucocorticoids entiation is critical, because 70% of the cases • A relatively rare disorder (prevalence of 1 in of Conn’s syndrome are medically curable 50,000) ○ Conn’s syndrome is the most common cause of • Most common cause is autoimmune adrenalitis and hyperaldosteronism; condition is characterized is commonly associated with other autoimmune by disorders § Increased aldosterone and decreased renin • This disorder develops slowly with the gradual loss ○ Idiopathic adrenal hyperplasia characterized by of cortisol and increased ACTH. The melanocyte- § Increased aldosterone and renin stimulating property of ACTH is what results in • Diagnosis of primary hyperaldosteronism involves the clinical feature of hyperpigmentation a two-step approach • Symptoms appear after approximately 90% of the ○ Screening for hyperaldosteronism adrenal gland is destroyed ○ Confirmation of hyperaldosteronism using one • Generally the disorder is considered in people with of three aldosterone suppression tests uncontrollable hypertension and unexplained ○ Screening involves a morning (between 8 AM and hypokalemia with increased urinary potassium 10 AM) plasma aldosterone–to–plasma renin (>30 mmol/day). Clinical features of Addison’s dis- ratio. Ratios above 25 in the presence of ease are listed in Box 8-5. 250 CHAPTER 8 Clinical Chemistry

Clinical Features of Primary Adrenal Clinical Features of BOX 8-5 Insufficiency BOX 8-6 Pheochromocytoma

Muscle weakness Hypertension Fatigue Headache Weight loss Palpitations Pigmentation Pallor Anorexia Dyspnea Fever Nausea Dehydration Anxiety attacks Nausea General weakness Hypotension Abdominal pain

Common Enzymes and Reactions TABLE 8-33 Catalyzed • Diagnosis of Addison’s disease may be made one of two ways Enzyme Reaction Catalyzed Comments ○ 0 Symptoms alone AST L-Aspartate+ -Ketoglutarate P-5 -P required for § a Symptoms alone are enough to diagnose this $ Oxaloacetate+ L- full catalytic condition in most cases Glutamate activity 0 § Symptoms include ALT L-Alanine+a-Ketoglutarate P-5 -P required for § Hyponatremia $ L-Glutamate+Pyruvate full catalytic § Hyperkalemia activity § Acidosis ALP p-Nitrophenyl phosphate $ Magnesium required § Hypovolemia p-Nitrophenol+HPO4 for full catalytic § Hypotension activity ○ LD L-Lactate+NAD $ Pyruvate pH 8.3-8.9 favors Plasma aldosterone and renin results + § +NADH+H forward reaction An elevated plasma renin result and decreased pH 7.1-7.4 favors plasma aldosterone result are diagnostic for reverse reaction Addison’s disease CK Creatine+ATP $ Creatine Magnesium required Adrenal Medulla Disorders phosphate+ADP for full catalytic • Disorders of the adrenal medulla are divided into cat- activity echolamine excess and catecholamine deficiency Alkaline phosphatase; alanine aminotransferase; creatine • Catecholamine excess results as a consequence of ALP, ALT, CK, kinase; LD, lactate dehydrogenase. adrenal medullary chromaffin cell tumors; the most important tumor clinically is pheochromocytoma ○ Although rare in occurrence (0.2% occurrence) and mostly benign, accurate and timely Enzymology diagnosis is essential because pheochromocytoma will cause severe cardiovascular compli- • Enzymes play an integral role in many cellular cations and death if not identified and properly processes treated • Thousands have been identified; the clinical labora- ○ 10% to 15% may be malignant tory routinely measures fewer than 15 for diagnostic ○ Pheochromocytoma is divided into two types: purposes Multiple endocrine neoplasia (MEN) 1 and • Enzymes are proteins with catalytic activity; they MEN 2 accelerate the rate at which a chemical reaction takes ○ Clinical features are presented in Box 8-6 place without themselves being consumed in the pro- ○ Vanillylmandelic acid (VMA), a metabolite cess (see Table 8-33); different forms of enzymes exist of epinephrine and norepinephrine, is used to that are referred to as isoenzymes aid in the detection of excess catecholamine • Lactate dehydrogenase is a tetramer containing production caused by neuroblastomas, pheo- two subunits, heart and muscle, that form five chromocytomas, and other neuroendocrine isoenzymes: LD-1 (HHHH), LD-2 (HHHM), LD- tumors 3 (HHMM), LD-4 (HMMM), and LD-5 § NOTE: VMA is subject to many interferences; (MMMM); historically the LD-1, LD-2 flipped therefore an increased VMA is not diagnostic ratio was used in the diagnosis of acute myocardial of the previously mentioned disorders infarction CHAPTER 8 Clinical Chemistry 251

Rise and Fall Pattern of Creatine Zero order kinetics Kinase–Myocardial Bound and Troponins After Acute Myocardial TABLE 8-34 Infarction First Rise (hr After Peak (hr After Back to order kinetics

Analyte AMI) AMI) Normal Reaction Rate CK-MB 4-6 12-24 72 hr after AMI Troponin I 4-6 12-24 3-7 days Substrate Troponin T 4-6 12-24 7-10 days FIGURE 8-6 Zero order kinetics. AMI, Acute myocardial infarction; CK-MB, creatine kinase–myocardial bound.

• Creatine kinase is a dimer containing two subunits, Test Procedures muscle and brain, that form three isoenzymes: CK- • Enzymes are measured using activity or mass assays BB (brain type), CK-MB (myocardial bound), and • Activity assays measure the catalytic effect of an CK-MM (muscle type); CK-MB is used in the enzyme on a substrate; activity is measured at optimal diagnosis of acute myocardial infarction temperatureand pH for enzymes of interest; substrate (AMI) (CK-MB >6% of total CK is diagnostic for is added in excess so reaction rate depends only on the AMI); additionally, CK-MB has a typical rise amountofenzymeinthereaction(zeroorderkinetics) and fall pattern after an AMI that is used in • Activity assays are also referred to as kinetic assays; conjunction with troponin levels to diagnose AMI kinetic assays measure enzyme activity over a (Table 8-34) specified period ○ According to the Joint European Society • Activity assays report activity as an international for Cardiology/American Heart Association unit (IU) (AHA) Science Advisory and the Coordinating ○ The IU is defined as the amount of enzyme Committee/AHA/World Heart Federation Task needed to convert 1 mmol of substrate per minute Force for the redefinition of myocardial infarc- using standardized conditions tion, the diagnosis of acute myocardial infarc- ○ Enzyme concentrations (international units per tion, is based on meeting two of the following liter) are calculated using the formula three criteria 6 IU=L ¼ DA=E L 10 1=T TV=SV § Clinical symptoms of ischemia ¼ § Electrocardiographic changes where DA Average change in absorbance per § Rise and fall of highly sensitive biochemical minute ¼ markers (preferably troponins) E Molar absorptivity ¼ ○ Additionally, the European Society of Cardiol- T Time ¼ ogy and the American College of Cardiology TV Total volume ¼ consensus report recommended that samples SV Sample volume 6 ¼ for the determination of AMI be collected as 10 Conversion factor from moles to follows micromoles ¼ § Admission L Light path § 6 to 9 hours after admission • Mass assays that measure either protein mass or § 12 to 14 hours after admission (if the earlier enzyme concentrations have been developed samples were negative) and are in use clinically for determining isoenzymes • Enzymes combine with substrate to form enzyme/sub- such as CK-MB, LD-1, and the bone fraction of ALP strate complexes that dissociate, releasing enzyme and • Diagnostic utility is related to their tissue distribution product according to the following Michaelis-Menten (Table 8-35) theory • Recommended ranges for commonly measured enzymes can be found in Table 8-36 E+S$ ES $ E+P Sample Collection and Handling

• Rates of the forward and reverse reaction¼k1 and k2 • Serum or plasma (heparin) • Enzyme assays in chemistry are designed for zero order • Calcium and magnesium chelating anticoagulants must kinetics in which the rate of reaction is proportional to be avoided because calcium and/or magnesium are enzyme present (Figure 8-6) required for full catalytic activity of certain enzymes 252 CHAPTER 8 Clinical Chemistry

Enzyme Distribution and Clinical b. Covertcholesterolestersto cholesterolfor detection TABLE 8-35 Significance c. Precipitate all Apo B and Apo A containing lipoproteins Clinical d. Precipitate all Apo B containing proteins Enzyme Tissue Distribution Significance 3. Which of the following lipoproteins is the smallest of Alkaline phosphatase Placenta, intestinal Hepatobiliary all the lipoproteins and is composed of 50% protein? mucosa, kidney, damage a. HDL bone, liver b. Chylomicrons Alanine Liver, kidney Hepatocellular c. LDL aminotransferase damage d. Triglycerides Aspartate Heart, liver, skeletal Hepatocellular 4. Which of the following would be most adversely aminotransferase muscle, kidney, damage affected by a nonfasting sample? pancreas a. HDL g-Glutamyltransferase Kidney, biliary tract of Hepatobiliary liver damage b. LDL Lactate Brain, heart, Nonspecific c. Cholesterol dehydrogenase erythrocytes, kidney, d. Triglycerides lung, skeletal 5. Which of the following apoproteins is responsible for muscle, liver, receptor binding for IDL and the chylomicron rem- pancreas, stomach nant produced in fat transport? Creatine kinase Skeletal muscle, Myocardial a. Apo A1 myocardium, brain, damage b. Apo C colon, stomach, and skeletal c. Apo E urinary bladder muscle d. Apo B injury 6. Which of the following enzymes is found bound to Amylase Pancreas, salivary Pancreatitis glands, fallopian HDL and LDL in blood plasma and acts to convert tube free cholesterol into cholesteryl esters? Lipase Pancreas Pancreatitis a. Cholesterol esterase b. Cholesterol oxidase c. Lecithin-cholesterol acyltransferase Recommended Ranges for d. Lipase TABLE 8-36 Commonly Measured Enzymes 7. Which of the following blood samples would serve best to assay lipoproteins because this anticoagulant Enzyme Recommended Range Units acts to preserve lipoproteins? Alkaline phosphatase 30-90 Units/L a. EDTA plasma sample Alanine 6-37 Units/L b. Heparin plasma sample aminotransferase c. Citrate plasma sample Aspartate 5-30 Units/L d. Fluoride plasma sample aminotransferase 8. Exogenous triglycerides are transported in the g-Glutamyltransferase 6-45 Units/L plasma in which of the following forms? Lactate dehydrogenase (L $ P) 100-225 Units/L a. VLDL (P $ L) 80-280 b. Chylomicrons Creatine kinase 15-160 Units/L Amylase 60-180 Units/L c. LDL Lipase 10-200 Units/L d. Cholesteryl esters 9. A patient presents to his physician for a lipid profile. The following results are received: HDL¼50 mg/dL CERTIFICATION PREPARATION QUESTIONS Total cholesterol¼300 mg/dL Triglycerides¼200 mg/dL For answers and rationales, please see Appendix A. The calculated LDL cholesterol is: 1. Which of the following is considered a lipid? a. 200 a. Chylomicrons b. 210 b. LDL c. 290 c. Cholesterol d. 350 d. HDL 10. According to the National Cholesterol Education 2. In the laboratory procedure for the quantification of Program, which lipid or lipoprotein class is more HDL, the purpose of the dextran sulfate is to: important for therapeutic decision making (diet a. Precipitate all Apo A1 containing lipoproteins and medication decisions)? CHAPTER 8 Clinical Chemistry 253

a. Chylomicrons a. Glucose b. LDL b. Galactose and glucose c. HDL c. Maltose and glucose d. Cholesterol d. Fructose and glucose 11. Which of the following mechanisms accounts for 18. Laboratory tests are performed for a postmeno- the elevated plasma level of b-lipoproteins seen in pausal, 57-year-old woman as part of an annual hyperbetalipoproteinemia (Fredrickson’s type II physical examination. The patient’s random serum lipoproteinemia)? glucose is 220 mg/dL, and the glycated hemoglobin a. Elevated insulin found in these patients (HbA1c) is 11%. Based on this information, this b. Apo B-100 receptor defect patient would mostly likely be classified as: c. Apo C-II–activated lipase deficiency a. Normal d. LCAT deficiency b. Impaired 12. Which enzyme is common to all enzymatic methods c. Having type 1 diabetes for triglyceride measurement? d. Having type 2 diabetes a. Glycerol phosphate oxidase 19. Which of the biochemical processes below is pro- b. Glycerol phosphate dehydrogenase moted by insulin? c. Pyruvate kinase a. Glycogenolysis d. Glycerol kinase b. Gluconeogenesis 13. A patient sample is assayed for fasting triglycerides c. Esterification of cholesterol and a triglyceride value of 1036 mg/dL. This value d. Uptake of glucose by the cells is of immediate concern because of its association 20. Laboratory results for a patient with type 2 diabetes with which of the following conditions? are as follows: a. Coronary heart disease Analyte Result b. Diabetes Glucose 128 mg/dL c. Pancreatitis Total cholesterol 195 mg/dL d. Gout HDL 45 mg/dL 14. Which of the following apoproteins is inversely LDL 105 mg/dL related to risk for coronary heart disease and is a sur- BUN 38 mg/dL rogate marker for HDL? Creatinine 2.1 mg/dL Microalbuminuria 54 g/Ml a. Apo A-I m AST 28 U/L b. Apo B ALT 38 U/L c. Apo B100 d. APO E Which of the following statements is correct regard- 15. What is the most appropriate fasting procedure when ing this patient? a lipid study of triglycerides, total cholesterol, HDL, a. Patientisatincreasedriskfor cardiovascular disease and LDL tests are ordered? b. Patient is at increased risk for diabetic nephropathy a. 8 hours, nothing but water allowed c. Patient is at increased risk for liver failure b. 10 hours, water, smoking, coffee, tea (no sugar or d. Patient is at risk for hypoglycemia 21. cream) allowed At what serum glucose concentration would glucose c. 12 hours, nothing but water allowed begin to appear in the urine? d. 16 hours, water, smoking, coffee, tea (no sugar or a. 50 mg/dL cream) allowed b. 75 mg/dL 16. John Smithers (21 years of age) is in to see his phy- c. 100 mg/dL sician for a pre-college physical and checkup. John d. 170 mg/dL has always been extremely healthy. The following 22. Which of the following laboratory tests is the best laboratory results are received: marker to detect patients with diabetes who are at risk for developing diabetic nephropathy? A ¼0.679 A ¼0.650 Standard Control a. Creatinine ASmithers ¼0.729 CStandard ¼200 mg/dL Control range 190-195 mg/dL b. BUN c. Microalbuminuria test John’s cholesterol concentration is approximately: d. Glucose a. 186 mg/dL 23. A 68-year-old obese woman visits her doctor report- b. 199 mg/dL ing increased urination (especially at night), c. 209 mg/dL increased thirst, and increased appetite. Her glucose d. 215 mg/dL on examination was 210 mg/dL (fasting). Which of 17. Sucrose is considered a disaccharide that on hydroly- the following statements best fits with the given sis yields which of the following sugars? information above? 254 CHAPTER 8 Clinical Chemistry

a. The patient most likely has type 1 diabetes mellitus 30. The kidney is responsible for acid-base balance b. The patient would show a positive glucose in through the removal of H ions via four major mech- her urine anisms. Which of the following describes one of c. The patient would have a decreased glycated those mechanisms? hemoglobin a. Reabsorption of H ions in the proximal convo- d. Additional testing of this patient should include luted tubule assessment of hypoglycemia b. Reaction of H ions with Na in the descending 24. Which of the following hemoglobin A1c results rep- loop of Henle resents an impaired state according to the American c. Reaction of H ions with filtered bicarbonate ions Diabetes Association? d. Reaction of H ions with ADH in the a. 4.5% collecting ducts b. 5.5% 31. Given the data below, the calculated creatinine clear- c. 6.0% ance corrected for body surface area approximates d. 6.5% ______. 25. A plasma glucose result is 100 mg/dL. The corresponding glucose in whole blood would approximate: Analyte Result Serum creatinine 1.2 mg/dL a. 58 mg/dL Urine creatinine 120 mg/dL b. 87 mg/dL Urine volume 1.75 L/day c. 98 mg/dL Surface area 1.80 m2 d. 114 mg/dL 26. Which of the following methods is virtually specific a. 16 mL/min for glucose and employs G6PD as a second coupling b. 115 mL/min step requiring magnesium? c. 126 mL/min a. Hexokinase d. 210 mL/min b. Glucose oxidase 32. Which formula is most accurate in predicting plasma c. Glucose dehydrogenase osmolality? d. Pyruvate kinase a. Na+2(Cl)+BUN+Glucose 27. A 62-year-old patient presents to the physician with b. 2(Na)+2 (Cl)+Glucose+BUN report of increased thirst and increased urination, c. 2(Na)+Glucose/18+BUN/2.8 particularly at night. The physician requests a series d. 2(BUN)+Glucose/18+Cl/2.8 of tests over the next few days. The following data 33. Which of the following statements regarding serum are received: urea is true? Analyte Result a. Levels are independent of diet Random glucose 186 mg/dL b. High BUN levels can result from necrotic liver Fasting glucose 114 mg/dL disease 2-Hour OGTT 153 mg/dL c.BUNiselevatedinprerenalaswellasrenal HbA1c 5.9% failure Which of the following conclusions may be made d. BUN rises earlier and quicker than creatinine in regarding these data? renal damage a. Data represents normal glucose status 34. Osmolality can be defined as a measure of the con- b. Data represents an impaired glucose status centration of a solution based on: c. Data represents the presence of an insulinoma a. The number of particles present d. Data represents the diagnosis of diabetes b. The number and size of particles present 28. Which of the following renal conditions is associated c. The density of particles present with a recent group A b-hemolytic streptococcus d. The isoelectric point of a particle infection? 35. An increased osmole gap is most commonly seen in a. Kidney obstruction which of the following? b. Acute renal failure a. Type 2 diabetes c. Uremic syndrome b. Pancreatitis d. Acute glomerulonephritis c. Presence of toxins such as ethanol and ethylene 29. The red complex developed in the Jaffe method to glycol determine creatinine measurements is a result of the d. Liver failure complexing of creatinine with which of the following? 36. A patient with type 2 diabetes is in for a routine a. Alkaline picrate examination with the physician. A series of labora- b. Diacetyl monoxide tory tests are performed, including calculation of c. Sulfuric acid an eGFR. The patient’s calculated eGFR is 64 mL/ d. Sodium hydroxide min. This result is most indicative of: CHAPTER 8 Clinical Chemistry 255

a. A normal state 44. Which of the following conditions is caused by defi- b. Abnormal glucose control cient secretion of bilirubin into the bile canaliculi? c. Mild kidney damage a. Gilbert’s disease d. Kidney failure b. Physiologic jaundice of the newborn 37. A healthy 28-year-old female sees her physician for a c. Dubin-Johnson syndrome routine examination and receives a “relatively” clean d. Hemolytic jaundice bill of health except for the results below. 45. Which of the following enzymes is responsible for the Total bilirubin 2.8 mg/dL conjugation of bilirubin? Direct bilirubin 0.1 mg/dL a. Biliverdin reductase Indirect bilirubin 2.7 mg/dL b. Peroxidase These results most likely indicate which of the c. UDP–glucuronyl transferase following? d. b-Glucuronidase a. Normal bilirubin metabolism 46. Which of the following analytes is the best indicator b. Extrahepatic obstruction of hepatobiliary damage? c. Dubin-Johnson syndrome a. AST d. Gilbert’s disease b. ALT 38. Which of the following is measured using glutamate c. ALP dehydrogenase and is a measure of advanced stages, d. Bilirubin poor prognosis, and coma in liver disease? 47. Which of the following fractions of bilirubin in high a. Total bilirubin concentrations is associated with kernicterus in b. Ammonia newborns? c. Unconjugated bilirubin a. Delta bilirubin d. Urea b. Unconjugated bilirubin 39. In which of the following disease states would you c. Conjugated bilirubin see an elevation in total bilirubin and conjugated bil- d. Unconjugated and delta bilirubin irubin only? 48. The characteristic laboratory finding in alcoholic cir- a. Biliary obstruction rhosis includes: b. Hemolysis a. Moderate elevations in AST and ALT, normal c. Neonatal jaundice GGT, and normal ALP d. Hepatitis b. Slight elevations in AST and ALT, marked eleva- 40. In which of the following conditions does no activity tions in ALP, normal GGT of glucuronyl transferase result in increased unconju- c. Slight elevations in AST, ALT, and GGT and gated bilirubin and kernicterus in neonates and even- marked elevations in 50 nucleotidase tual death within 18 months? d. Slight elevations in AST and ALT (AST>ALT), a. Gilbert’s disease marked elevations in GGT, slight elevations b. Dubin-Johnson syndrome in ALP c. Crigler-Najjar syndrome 49. Which of the following liver conditions shows an d. Intravascular hemolysis increase in both conjugated bilirubin and ALP, man- 41. As a reduction product of bilirubin catabolism, this ifests with antimitochondrial antibodies, and shows compound is partially reabsorbed from the intestines a characteristic lipoprotein X on electrophoresis? through the portal circulation for reexcretion by the a. Hemochromatosis liver. What is this compound? b. Primary biliary cirrhosis a. Urobilinogen c. Alcoholic fatty liver b. Azobilirubin d. Hepatic tumors c. Biliverdin 50. Which set of results is consistent with uncompen- d. Urobilin sated metabolic acidosis? 42. In the liver, bilirubin is conjugated in the presence of a. pH 7.25, HCO3 15 mmol/L, PCO2 37 mm Hg which of the following? b. pH 7.30, HCO3 16 mmol/L, PCO2 28 mm Hg a. b-Glucuronidase c. pH 7.45, HCO3 22 mmol/L, PCO2 40 mm Hg b. Bilirubin oxidase d. pH 7.40, HCO3 25 mmol/L, PCO2 40 mm Hg c. Uridine diphosphate (UDP)–glucuronyltransferase 51. A patient with emphysema who has fluid accu- d. Peroxidase mulation in the alveolar sacs (causing decreased 43. Hepatocellular damage may be best assessed by ventilation) is likely to be in which of the following which of the following parameters? acid-base clinical states? a. Serum AST and ALT levels a. Respiratory alkalosis b. GGT and ALP b. Respiratory acidosis c. Bilirubin, GGT, and ALP c. Metabolic acidosis d. Ammonia and urea d. Metabolic alkalosis 256 CHAPTER 8 Clinical Chemistry

52. Which of the following buffer systems is the 60. Which of the following values would be seen in most important physiologic buffer system in the uncompensated metabolic acidosis? body? a. pH 7.38 a. Hemoglobin b. pCO2 52 mm Hg b. Protein c. HCO3 15 mmol/L c. Phosphate d. pH 7.53 d. Bicarbonate/carbonic acid 61. Which of the following blood gas disorders is 53. To maintain electrical neutrality in the red blood cell, most commonly associated with an abnormal anion bicarbonate leaves the red blood cell and enters the gap? plasma through an exchange mechanism with which a. Metabolic acidosis of the following? b. Metabolic alkalosis a. TCO2 c. Respiratory acidosis b. Sodium d. Respiratory alkalosis c. Chloride 62. Which of the following statements best describes the d. Phosphate predominant feedback system associated with 54. Increased PCO2 in a patient most commonly results endocrinology? in which of the following primary acid-base a. Decreased levels of circulating hormones directly abnormalities? result in the production of hormone from the a. Respiratory acidosis target organ b. Metabolic acidosis b. Increased circulating levels of hormones directly c. Respiratory alkalosis result in the production of releasing factor from d. Metabolic alkalosis the hypothalamus 55. Which of the following changes will occur with a c. Increased circulating levels of hormones directly blood gas sample exposed to room air? result in the production of inhibiting factor from a. pH increased the hypothalamus b. pCOO2 increased d. Normal levels of circulating hormones directly c. pO2 decreased result in the production of hormone from the d. Ionized calcium increased target organ 56. Which of the following is the correct collection and 63. The following laboratory results are from a 54-year- handling for the analysis of blood gases? old woman complaining of weight gain, intolerance a. Plastic syringe, dry heparin, store on ice, assay to heat, fatigue, and not being able to stay awake. within 1 hour Analyte Result b. Glass syringe, liquid heparin, store on ice, assay Na 140 mmol/L within 15 minutes K 4.0 mmol/L c. Glass syringe, no additive, store on ice, assay Glucose 75 mg/dL within 15 minutes Aldosterone 8 ng/dL Ionized Ca 4.8 mg/dL d. Plastic syringe, dry heparin, store at room temper- Mg 2.0 mEq/L ature, assay within 15 minutes Phos 3.0 mg/dL 57. What is the blood pH when the partial pressure of TSH 7.2 mU/mL carbon dioxide (pCO2) is 45 mm Hg and the bicar- FT4 1.0 ng/dL bonate is 28 mmol/L? Cortisol 10 mg/dL a. 7.00 Which of the following conditions best fits with the b. 7.11 history and data? c. 7.33 a. Hyperthyroidism d. 7.41 b. Cushing’s syndrome 58. What is the normal ratio of bicarbonate to dissolved c. Hyperaldosteronism carbon dioxide in arterial blood? d. Hypothyroidism a. 1:10 64. A 42-year-old woman presents to her physician with b. 10:1 truncal obesity, bruising, hypertension, hyperglyce- c. 20:1 mia, and increased facial hair. The physician suspects d. 1:20 an endocrine disturbance. Significant test results are 59. Which of the following sets of blood gas data is as follows: considered normal? Analyte Result a. pH 7.33, HCO3 18 mmol/L, pCO2 32 mm Hg TSH 3.0 mU/mL b. pH 7.30, HCO3 16 mmol/L, pCO2 28 mm Hg FT4 1.0 ng/dL c. pH 7.45, HCO3 22 mmol/L, pCO2 40 mm Hg Glucose 90 mg/dL d. pH 7.40, HCO3 25 mmol/L, pCO2 40 mm Hg Serum cortisol (8 AM)45mg/dL CHAPTER 8 Clinical Chemistry 257

Plasma ACTH (8 AM) 152 pg/mL Plasma ACTH Decreased Urine free cortisol Increased Dexamethasone Overnight: No Dexamethasone suppression tests suppression test suppression Overnight 300 nmol/L High dose: No High dose >50% suppression suppression What is the most probable condition? a. Pituitary tumor What is the most probable condition? b. Addison’s disease a. Addison’s disease c. Adrenal adenoma b. Cushing’s disease d. Ectopic ACTH production c. Conn’s syndrome 65. Hypothyroidism is best characterized by which of the d. Cushing’s syndrome 71. following sets of test results? Trophic hormones are produced by the ______, and releasing factors are produced by the ______. a. TSH 0.2 mU/mL, FT3 8.9 pg/mL, FT4 4.5 ng/dL a. Hypothalamus; pituitary b. TSH 8.5 mU/mL, FT3 1.0 pg/mL, FT4 0.5 ng/dL b. Pituitary; hypothalamus c. TSH 0.1 mU/mL, FT3 1.1 pg/mL, FT4 0.8 ng/dL c. Specific endocrine glands; hypothalamus d. TSH 3.9 mU/mL, FT3 3.0 pg/mL, FT4 1.0 ng/dL 66. The release of thyroid-releasing hormone (TRH) d. Pituitary; target gland 72. would result in which of the following actions from When free thyroxine cannot be measured directly, the HPT axis? the free thyroxine index (FT4I) may be calculated a. Decreased release of thyroid-stimulating hor- by using which measured laboratory data? mone from the pituitary gland a. TSH and T3 resin uptake b. Increased release of thyroid-stimulating hormone b. T4 and T3 resin uptake from the thyroid gland c. TSH and T4 c. Increased release of thyroid hormones from the d. T3 and T3 resin uptake 73. thyroid glands The most commonly used challenge test to assist in d. Increased release of thyroid hormones from the evaluating a potential growth hormone deficiency pituitary gland is the: a. Insulin challenge test 67. A serum thyroid panel reveals an increase in total T4, b. Dexamethasone suppression test normal TSH, and a normal fT4. What is the most likely cause of these results? c. Oral glucose tolerance test a. Increased thyroxine-binding protein d. Captopril suppression test 74. b. Secondary hyperthyroidism Symptoms of primary adrenal insufficiency c. Subclinical hypothyroidism (Addison’s disease) include which of the following? d. Subclinical hyperthyroidism a. Hypercortisolism 68. Thyroid hormones are derived from which of the b. Hypokalemia following? c. Hypertension a. Histidine d. Acidosis 75. b. Cholesterol A TRH stimulation test is performed, and a flat c. Tyrosine response is received from this test procedure. This d. Phenylalanine most likely indicates: 69. In patients with developing subclinical hyperthyroid- a. Secondary hypothyroidism b. Tertiary hypothyroidism ism, TSH levels will likely be ______, and fT4 will likely be ______. c. Primary hypothyroidism a. Decreased, increased d. Secondary hyperthyroidism 76. b. Increased, decreased The first step in the synthesis of thyroid hormones is: c. Decreased, normal a. Iodide trapping d. Increased, normal b. Binding of thyroglobulin 70. A 30-year-old woman is admitted to the hospital. c. Oxidation of iodine She has truncal obesity, buffalo humpback, moon d. Oxidation of TG molecule 77. face, purple striae, hypertension, hyperglycemia, Which of the following conditions is a result of cat- increased facial hair and amenorrhea. The physi- echolamine excess, includes two classifications cian orders endocrine testing. The results are as fol- (MEN 1 and MEN 2), and may result in death from lows: severe cardiovascular complications? a. Cushing’s syndrome Analyte Result b. Conn’s syndrome Urine free cortisol Increased c. Addison’s disease Serum cortisol (8 AM) Increased d. Pheochromocytoma 258 CHAPTER 8 Clinical Chemistry

78. The main estrogen produced by the ovaries and used 86. Kinetic enzymatic assays are best performed during to evaluate ovarian function is: which phase of an enzymatic reaction? a. Estriol a. Linear phase b. Estradiol b. Lag phase c. Epiestriol c. Plateau phase d. Estrogen d. Any phase as long as temperature and pH are 79. The Michaelis-Menten theory states which of the constant following? 87. A nurse calls the laboratory technologist on duty ask- a. E+S+I!ES+EI+ESI!E+P ing about blood collection for the analysis of b. E+S $ ES!E+P enzymes (AST, ALP, ALT, GGT, CK). Which of the c. E+S+I!ES+EI!E+P following tubes would you suggest the technologist d. E $ ES!E+P collect? 80. Which of the following enzymes is the best indicator a. Red top of pancreatic function? b. EDTA a. AST c. Oxalate b. ALT d. Fluoride c. GGT 88. Which of the following enzymes catalyzes the con- d. Lipase version of starch to glucose and maltose? 81. Which of the following enzymes catalyzes the con- a. Lipase version of p-nitrophenyl phosphate to a colored p- b. Amylase nitrophenol product? c. ALT a. AST d. GGT b. ALT 89. Hyperparathyroidism is most consistently associated c. ALP with which of the following? d. GGT a. Hypocalcemia 82. One international unit of enzyme activity is the b. Hypercalciuria amount of enzyme that under specified reaction con- c. Hypophosphatemia ditions of substrate concentration, pH, and tempera- d. Metabolic alkalosis ture, causes usage of substrate at the rate of: 90. What percentage of serum calcium is in the ionized a. 1 millimole/min form? b. 1 micromole/min a. 30% c. 1 nanomole/min b. 50% d. 1 picomole/min c. 60% 83. A physician calls to request a CK test on a d. 80% sample already in the laboratory for coagulation stud- 91. Which of the following best describes the action of ies. The sample is 1 hour old and has been stored parathyroid hormone? at 4 c. The plasma shows very slight hemolysis. a. PTH increases calcium and phosphorus reabsorp- What is the best course of action and the reason for it? tion in the kidney a. Perform the CK assay because no interferent is b. PTH decreases calcium and phosphorus release present from bone b. Reject the sample because it is slightly hemolyzed c. PTH decreases calcium and increases phosphorus c. Reject the sample because it has been stored too long reabsorption in the liver d. Reject the sample because the citrate will interfere d. PTH increases calcium reabsorption and 84. Which of the following statements regarding CK is decreases phosphorus reabsorption in the kidney true? 92. Which of the following is most likely to produce an a. Levels are unaffected by strenuous exercise elevated plasma potassium result? b. Levels are unaffected by repeated intramuscular a. Hypoparathyroidism injections b. Cushing’s syndrome c. Highest levels are seen in Duchenne’s muscular c. Diarrhea dystrophy d. Hemolysis d. The enzyme is highly specific for heart injury 93. Which of the following hormones involved in cal- 85. Which of the following conditions can “physiologi- cium regulation acts by decreasing both calcium cally” elevate serum alkaline phosphatase? and phosphorous? a. Hyperparathyroidism a. PTH b. Diabetes b. Calcitonin c. Third-trimester pregnancy c. Vitamin D d. Nephrotic syndrome d. Cortisol CHAPTER 8 Clinical Chemistry 259

94. Which of the following electrolytes is the chief 98. Psuedohyperkalemia is most commonly a result of plasma cation whose main function is maintaining which of the following? osmotic pressure? a. Metabolic acidosis a. Chloride b. Hemolysis b. Potassium c. Hyperaldosteronism c. Sodium d. Hyperparathyroidism d. Bicarbonate 99. The following results were seen on a blood sample: 95. Which of the following conditions is associated with Analyte Result hypernatremia? Na+ ¼140 mEq/L K+ ¼15.0 mEq/L a. Diabetes insipidus Cl ¼105 mEq/L HCO3 ¼22 mmol/L b. Hypoaldosteronism The technologist should do which of the following? c. Diarrhea a. Report the results d. Acidemia b. Repeat and check the chloride result 96. Which of the following conditions will elevate ion- c. Repeat and check the Na+ result ized calcium? d. Check the sample for hemolysis a. Diabetes mellitus 100. The major intracellular cation is which of the b. Hyperlipidemia following? c. Acidosis a. Potassium d. Alkalosis b. Sodium 97. The anion gap is useful (among other things) as an c. Chloride inexpensive measure of quality control for which d. Bicarbonate of the following analytes? a. Blood gas analyses b. Sodium, potassium, chloride, and total carbon dioxide c. Calcium, phosphorus, and magnesium d. AST, ALT, GGT, and ALP 260 CHAPTER 8 Clinical Chemistry

SELF-ASSESSMENT

Content Area: ______

Score on Practice Questions: ______

List the specific topics covered in the missed questions:

List the specific topics covered in the correct questions: CHAPTER 8 Clinical Chemistry 261

NOTES CHAPTER 9 Molecular Diagnostics

Gideon H. Labiner

MOLECULAR BIOLOGY BASICS ○ Histone: Protein involved in the organization of nuclear DNA Chromosomes ○ Double helix: Sugar-phosphate backbone with base pairs oriented in the core • Genetic material is contained on chromosomes Nucleic Acid Structure • Maternal and paternal inheritance of genetic • Molecular composition of DNA and RNA (Figure 9-2) material • Base ○ Haploid: A single copy is 23 chromosomes § Purines: Double carbon–nitrogen rings ○ Diploid: Two copies of each chromosome, 46 § Adenine chromosomes § Guanine • Parts of a chromosome § Pyrimidines: Single carbon–nitrogen ring ○ Telomeres: Area at the end of a chromosome § Cytosine ○ Centromere: Area in the middle of the chromo- § Thymine some where the chromatids (“arms”) meet § Uracil: Replaces thymine as base in RNA § Metacentric ○ Sugar: Pentose or five-carbon ring § Submetacentric § Deoxyribose in DNA § Acrocentric § Ribose in RNA ○ Heterochromatin: Tightly packed DNA ○ Phosphate bonds: Monophosphate, diphosphate, ○ Euchromatin: Looser packaging of DNA, which triphosphate could indicate that a gene is actively being ○ Nucleotide: Base+Sugar+Phosphate transcribed § Phosphodiester bond links the 50 carbon of • Chromosome structure one nucleotide to the 30 carbon of another ○ Primary structure: Sequence of nucleic acids § Nucleotides always join in a 50 to 30 orientation ○ Secondary structure: Folding of sequence based § This bonding makes the alternating sugar on hydrogen bonding and phosphate backbone of DNA ○ Tertiary structure: Three-dimensional shape dis- § Nucleoside: Base+Sugar playing the turns of the helix and major and ○ Base pairing: Joining of a purine with a pyrimi- minor groove dine by hydrogen bonding § B-DNA: Right-handed helix and most § Adenine binds with thymine: Two common form hydrogen bonds § A-DNA: Right-handed and similar to B form § Guanine binds with cytosine: Three hydrogen § Z-DNA: Left-handed DNA bonds • Organization of DNA (Figure 9-1) ○ Orientation of molecules ○ Chromatin: Nuclear DNA strand and its associ- § Forms right-handed double helix ated structural proteins § 10 bases per turn § Arranged and organized in a hierarchical fash- § One helical turn¼3.4 nm ion in which the degree of its condensation § Polymers of sugar and phosphates run in increases with higher levels of structural opposite direction organization § Antiparallel ○ Solenoid: Supercoiled chromatin fibers § Complementary strands join together to ○ Nucleosome: Eight histone proteins with a form double-stranded DNA (dsDNA) strand of DNA (170 base pairs long) wrapped § RNA has a hydroxyl group at the 20 carbon around them, giving a “beads-on-a-string” § RNA is usually found in the single-stranded appearance form

262 CHAPTER 9 Molecular Diagnostics 263

DNA double helix

Histone DNA Nucleosomes

200 bp of DNA Solenoid

Chromatin

Chromatin loop contains approximately 100,000 bp of DNA

Telomere

Centromere Chromatid

FIGURE 9-1 Structural organization of human chromosomal DNA. bp, Base pairs. (From Jorde B, Carey J, Bamshad M: Medical genetics,ed4, Philadelphia, 2009, Mosby.)

Nucleic Acid Physiology and Function ○ DNA polymerases: Enzymes that synthesize • Central dogma DNA with proofreading and exonuclease • DNA replicates itself to produce DNA capabilities • DNA is transcribed to make RNA ○ Okazaki fragments • RNA is translated to produce protein § Leading strand: Continuous synthesis of • Replication: Generating or producing new DNA from daughter strand parent DNA § Lagging strand: Discontinues synthesis of • Both strands of parent DNA act as the template for daughter strand synthesizing daughter stands ○ DNA ligase: Digests primer and joins Okazaki ○ Process is semiconservative fragments ○ New dsDNA will be made up of one parent and • Transcription one daughter strand • Genes are located within DNA • Replication forks are produced through the ○ Allele: A form of a gene that is variable unwinding enzymes (helicase and topoisomerase) § Two alleles make up a genotype ○ Primase: Enzyme that synthesizes a short RNA to • Locus: Location on the chromosome where the gene/ prime DNA synthesis alleles are located, plural is loci 264 CHAPTER 9 Molecular Diagnostics

3؅ 5؅ ○ Exons: Coding regions H H ○ Introns: Noncoding regions, sometimes referred CH to as junk DNA Thymine CCH N C N C Cytosine ○ Promoter: Regulatory region that influences the C C C C initiation and rate of transcription O O N O NNH • Messenger RNA (mRNA) is the ultimate product of H H H HH transcription Hydrogen H N NNNO bond ○ Chain termination: When RNA polymerase CCHH C C Adenine Guanine reaches a termination sequence NNCC § pre-mRNA and heterogeneous nuclear CCN N (hnRNA) are the sequences before processing NN § Introns are removed by splicing C C HH § Spliceosomes are formed from small (5؅ 3؅ nuclear ribonucleic particles (snRNPs § Exons are placed next to one another § Capping: Guanine added at 50 end Sugar-phosphate backbone § Polyadenylation: Poly A tail added to the 30 end FIGURE 9-2 Chemical structure of the four bases, showing ○ hydrogen bonds between base pairs. Three hydrogen bonds are The mature mRNA can now leave the nucleus formed between cytosine–guanine pairs, and two bonds are and enter the cytoplasm formed between adenine–thymine pairs (From Jorde B, Carey J, • Translation Bamshad M: Medical genetics, ed 4, Philadelphia, 2009, Mosby.) • Transfer RNA (tRNA) and ribosomal RNA (rRNA) form the site for protein synthesis • Genes are composed of nucleic acids arranged into • Genes contain information needed for the produc- codons tion of RNA and proteins • Codons are 3 nucleic acids that together code for 1 ○ Also needed for cellular maintenance amino acid ○ Transcription is similar to replication ○ 64 possible codons for 20 amino acids § DNA is the source and produces single- § Redundancy protects against possible stranded RNA (ssRNA) from dsDNA mutations § The process uses the enzyme RNA polymerase • Amino acids are the building blocks for protein • Only specific genes are transcribed at any given time • 20 amino acids are involved in protein synthesis • Genes contain both coding and noncoding sections (Table 9-1)

TABLE 9-1 The Genetic Code: Translation of Messenger RNA to Amino Acids During Protein Synthesis

Nucleotide Position in the Codon First Second Third UCA G UU Phenylalanine Phenylalanine Leucine Leucine C Serine Serine Serine Serine A Tyrosine Tyrosine Stop Stop G Cysteine Cysteine Selenocysteine* Tryptophan CU Leucine Leucine Leucine Leucine C Proline Proline Proline Proline A Histidine Histidine Glutamine Glutamine G Arginine Arginine Arginine Arginine AU Isoleucine Isoleucine Isoleucine Methionine C Threonine Threonine Threonine Threonine A Asparagine Asparagine Lysine Lysine G Serine Serine Arginine Arginine GU Valine Valine Valine Valine C Alanine Alanine Alanine Alanine A Aspartic acid Aspartic acid Glutamic acid Glutamic acid G Glycine Glycine Glycine Glycine

*The codon UGA can code for either selenocysteine or stop. From Bruns D, Ashwood E, Burtis, C: Fundamentals of molecular diagnostics, St. Louis, Saunders, 2007. CHAPTER 9 Molecular Diagnostics 265

○ One start codon: Acts as initiation point for ○ Reverse transcriptase reading § Synthesizes a DNA strand from either DNA § AUG or RNA ○ Three stop codons: Terminates the elongation of § Found in retroviruses the polypeptide chain ○ Kinase: Transfers phosphate groups § UAA ○ Phosphatase: Removes phosphate groups § UAG ○ Methylase: Adds methyl groups to nitrogen bases § UGA ○ Deaminase: Remove amino groups from • tRNA and anticodon nitrogen bases ○ tRNA carries amino acids specified by the codon in the mRNA NUCLEIC ACID ISOLATION • Wobble principle: Allows for loose-fitting match between the tRNA anticodons and the mRNA • DNA and RNA isolation codons • The process of separating nucleic acid material • Epigenetics from its surroundings • Alteration of gene function without changing DNA ○ Surrounding can include tissues, debris, cells, sequence proteins, lipids, or carbohydrates ○ DNA methylation • Cell lysis, extraction, and purification are part of ○ Genomic imprinting the isolation process ○ Histone modification • DNA is fairly stable over a wide range of tempera- • Mitochondrial DNA tures; hydrolysis poses a major threat to nucleic • Circular DNA that is 16,569 base pairs acids remaining intact • Maternal inheritance pattern • RNA is easily degraded by environmental RNase • Number of mitochondria per cell is variable, some enzymes cells have thousands • Considerations for choosing an isolation method ○ Specimen type (Table 9-2) ○ Amount of sample and desired yield GENOMES AND NUCLEIC ACID ○ Purity and size of isolate ALTERATIONS ○ Ease of operation and throughput ○ Costs and hazards • Human genome • Extraction methods • 3.2 billion base pairs • Liquid phase • 23 chromosome pairs (46 chromosomes) ○ Used for large sample volumes • Approximately 30,000 genes ○ Phenol-chloroform is a biphasic organic extrac- ○ Sequence variation tion method § 99.9% of the genome between random indi- § Hydrophobic portion: Lipids and debris on viduals is identical the bottom § Intron sequences are often simple repeat § Hydrophilic portion: Aqueous phase contains sequences the DNA on top • Microsatellites also called short tandem repeats (STRs) ○ Inorganic method substitutes the harsh chemi- ○ Repeat units are two to six base pairs in length cals of organic extraction with high salt condi- • Minisatellites also called variable number of tan- tions at a low pH dem repeats (VNTRs) § The DNA is collected from the upper phase ○ Repeat units are 40 to 500 base pairs in length and then precipitated with isopropyl alcohol • A polymorphism is a sequence variation occurring in • Solids phase more than 1% of a population ○ More commonly used because of ease of use, • Single nucleotide polymorphism (SNP) fewer safety concerns, ability for high through- • Nucleic acid enzymes put, and automation • Used in molecular laboratory procedures to manip- § Column filter ulate nucleic acids § Magnetic beads ○ Polymerase: Catalyzes the extension of nucleo- • General steps of extraction tides in the presence of a template strand ○ Cell lysis step to disrupt membranes ○ Ligase: Catalyzes the linkage between two nucle- ○ Salt preparation to remove proteins otides by a phosphodiester bond ○ DNA precipitated by alcohol ○ Nuclease: Cleaves nucleic acids • Assessment of nucleic acid yield and quality § Endonuclease: Acts on internal bonds • Ultraviolet (UV) light is absorbed by DNA and § Exonuclease: Acts on external bonds RNA at a wavelength of 260 nm 266 CHAPTER 9 Molecular Diagnostics

TABLE 9-2 Specimen Collection and Storage

Storage Temperature ( C) Specimen Collection and Transport (Short/Long) Considerations Whole blood or Lavender or yellow-top tube for whole 4/-70 Avoid heparin tube, remove red blood cells before bone marrow blood storage to avoid hemolysis Tissue Freeze solid tissues or on ice 4/-70 May be paraffin-embedded Buccal swabs Rinse or swab oral cavity, collect in 4/-20 Less invasive collection procedure buffer or transport medium Microorganism Special collection systems for various 4/-70 Viral RNA should be stored at 24 C, avoid target organisms contamination that may result in false-positive findings Forensic: Blood, Evidence labeled, air dry blood-stained 4/-70 Chain of custody for evidence, avoid heat and hair, nails, clothes, separate paper bags for 24 C for blood stored on contamination, aliquot to avoid repeated secretions, etc. each item special filter paper heating and thawing

• Yield can be measured with a spectrophotometer phosphate backbone of the dsDNA making frag- ○ 50 mg/L of dsDNA has an absorbance of 1.0 at ments with 260 nm § Blunt ends ○ RNA at an absorbance of 1.0 at 260 nm equals § Stagger or sticky ends (50 or 30) 40 mg/L • Changes in DNA may result in loss or gain of a • Quality and purity can be measured by looking at cleavage site the absorbance ratio A260/A280 • Result will be a change in fragment size that can be ○ 280 nm is the absorbance of proteins detected by electrophoresis ○ If the ratio is less than 1.6, contamination with ○ Fragments are compared to a known protein is present DNA ladder ○ If the ratio is 1.6 to 1.9, minimal contamination ○ Tracking dye and a density agent, such as glyc- may be present in the extract and the DNA is erol, are added to the DNA sample not pure • Southern blotting (Figure 9-3) ○ Extracts from 1.8 to 2.0 are typically acceptable • After digestion of DNA by a restriction enzyme and for clinical specimens separation by electrophoresis, Southern blotting is ○ A ratio of 2.0 indicates a pure DNA extract the act of transferring the resulting fragments to a solid support medium NUCLEIC ACID TECHNIQUES ○ Nylon or nitrocellulose membrane/filter is medium of choice Electrophoresis ○ DNA can be fragmented using an acid treatment during transfer as part of depurination • Both DNA and RNA carry a negative charge and will ○ Alkaline denaturation is used to form ssDNA migrate toward the positive-charge electrode (anode) ○ Neutralization • Gels act like a sieve for nucleic acid molecules • Transfer (blotting) methods • Slab gel (vertical or horizontal) or using gel polymer ○ Capillary action inside a capillary ○ Vacuum transfer • Agarose gel: Larger fragments (20 bp - 10 Mb) • Immobilization on nitrocellulose membrane • Polyacrylamide gel: Smaller sequences (1 bp - 2 kb) through UV cross-linkage ○ Neurotoxin ○ Single-stranded probes are incubated with the ○ Used for sequencing membranes and hybridize to complementary tar- • Based on molecular weight of the fragment get regions • Smaller molecules move faster § Probes contain a label for detection • Molecules may form secondary structures or § Wash excess probe under appropriate strin- heteroplexes gency conditions to discourage nonspecific • Restriction fragment length polymorphism (RFLP) binding to nontarget nucleic acid • Using restriction enzymes to cut dsDNA at specific § Formamide concentration cleavage sites § Temperature ○ Restriction enzymes are derived from bacteria ○ Fragments are visualized by and read nucleic acid sequences and cut the § Autoradiography film exposed to membrane CHAPTER 9 Molecular Diagnostics 267

Restriction enzyme

Electrophoresis on agarose gel

Denaturation with alkali

Blotting

Dry paper towels Cellulose nitrate filter Gel containing denaturated DNA

Filter paper Buffer

Hybridization with 32P DNA probe Denature X X X X

X Expose to X-ray film

Autoradiograph X showing bands(s)

FIGURE 9-3 The Southern blotting procedure. 268 CHAPTER 9 Molecular Diagnostics

Genomic DNA to Long products to Short products to long products short products short products

Denaturation 94° C

Annealing 55° C

Extension 72° C

FIGURE 9-4 Schematic diagram of polymerase chain reaction. (From Bruns D, Ashwood E, Burtis C: Fundamentals of molecular diagnostics, St. Louis, Saunders, 2007.)

§ Ethidium bromide (EtBr): Intercalates into § When half of the primers are bound (double DNA and fluoresces under UV light stranded) and half are unbound (single stranded) § Chemiluminescence signal § Relates to the optimal temperature needed for • Pulsed-field gel electrophoresis (PFGE) annealing step • Used for very large pieces of DNA • Magnesium chloride: Cofactor in reaction • Current applied in alternating orientations • Nucleotide bases • Used for epidemiologic studies for infectious diseases ○ Deoxynucleotide triphosphates: dNTP • Northern blotting ○ dATP, dCTP, dGTP, and dTTP • Technique similar to Southern blotting, but the • Buffer for optimal pH target is RNA • Polymerase adds dNTPs • Electrophoresis under denaturing conditions ○ The PCR reaction uses a thermocycler, which • Used for looking at mRNA transcripts allows for the rapid change in temperature • Western blotting needed for each step • Technique similar to Southern blotting, but the • Thermostable Taq polymerase withstands the extreme target is proteins temperature fluctuations necessary to complete the • Probes are antibodies three steps of a PCR cycle • Derived from Thermus aquaticus, which lives in hot Amplification Techniques springs • Steps in PCR (Table 9-4) Polymerase Chain Reaction. See Figure 9-4. ○ Denaturation: dsDNA is heated and separated • Target amplification: Known nucleic acid sequence of in ssDNA interest targeted for enzymatic replication in an ○ Annealing: Cooler temperature allows for amplified manner primers to bind to target sequence • Polymerase chain reaction (PCR) uses a master mix of ○ Extension: Temperature optimal for polymerase all components needed for the reaction (Tables 9-3) activity • Template DNA containing target sequence • Traditional PCR also is referred to as end-point PCR ○ Purified dsDNA Polymerase Chain Reaction Kinetics • Forward and reverse primers • Allows for exponential expansion of products ○ Oligodeoxynucleotides (20-30 base pairs) • Typical PCR run has 25 to 30 cycles, making millions ○ Complementary to the opposing strands flanking of copies of the target sequence the sequences of interest • Amplified product is known as an amplicon ○ Melting temperature (Tm) ○ Clones or replicas of target sequence CHAPTER 9 Molecular Diagnostics 269

Polymerase Chain Reaction • SYBR green I: Specific dye for dsDNA TABLE 9-3 Components ○ Lets off signal when DNA is denatured ○ Safer to use than EtBr Components Function • Probes-specific detection Template DNA Includes target sequence • Hybridization probes Primers 20-30 base pairs long, flank target sequence ○ Fluorescence resonance energy transfer (FRET) at 50 ends § Uses two probes: Acceptor probe and Magnesium Enzyme cofactor donor probe chloride § Energy is transferred from donor to acceptor, dNTP Nucleotides being added (A, G, T, C) and a fluorescent signal is released Buffer Maintains pH of reaction • Hydrolysis probes 0 Polymerase Allows for addition of dNTPs at 5 end during ○ Reporter and quenchers extension ○ TaqMan is an example that uses the 50 exonuclease activity of Taq polymerase for signal generation • TABLE 9-4 Polymerase Chain Reaction Cycles Molecular beacons and Scorpion probes • Melting curve analysis Steps Temperature ( C) • Separation of strands based on temperature ○ Denaturation 94 (30-60 sec) For short strands this can be estimated by

Annealing 55 (30-90 sec) 2ATðÞ+4GCðÞ¼Tm Extension 72 (30-90 sec) ○ More complicated for longer strands, must account for § Salt concentration ○ At some point in the PCR run, the production of § GC versus AT binding (GC has more H bonds) amplicons plateaus § Total length of fragment • Amount of product is related to the efficiency of the • Allows for genotyping cycles • SNP selection Variations on Polymerase Chain Reaction Polymerase Chain Reaction Controls and Reverse-Transcriptase Polymerase Chain Reaction Contamination Issues • Technique used if the template of interest is RNA • Controls in PCR must monitor for • Uses RNA viral polymerase reverse transcriptase • False positive findings resulting from carryover or • Makes RNA/DNA complex other contamination • Then replaces the RNA with DNA • False negative findings resulting from presence of ○ The resulting strand is referred to as cDNA or inhibitors and/or error in master-mix components complementary DNA • Purpose for controls ○ Used to measure or detect various types of RNA • Positive control: Contains sequence of interest ○ Does not include the intron sequences of a gene • Negative control: Does not contain sequence of Multiplex Polymerase Chain Reaction interest • The use of multiple primer pairs in one reaction tube • Reagent blank control: All reactants except DNA • Allows for detection of multiple targets • Amplification control • Internal amplification controls ○ Amplification of a different target region • Multiple pathogens ○ Tests for inhibition of PCR • Multiple polymorphism in identity testing § If amplification control is negative, corrective • Multiplex ligation-dependent probe amplification action is to repurify DNA extract • Method that uses multiple probes for areas of inter- ○ Differentiates true negative from false negative est and then amplifies with a single primer § Sensitivity control: Defines the lower limits of Real-Time Polymerase Chain Reaction testing • Also referred to as quantitative PCR (qPCR) because • Primer-dimers data, by way of florescence signaling, are collected • Result of unintended binding of primers to one another during nucleic acid amplification • Distinguished from true target by molecular weight • Amplification and fluorescence monitoring takes place or Tm in same reaction tube, minimizing risk for • Cleaning and decontamination contamination • Uracil-N-glycosylase: Degrades carryover amplicons • Real-time PCR dyes • UV light • EtBr • 10% bleach 270 CHAPTER 9 Molecular Diagnostics

• Laboratory design Additional Molecular Technologies • Separate preamplification and postamplification Microarrays: DNA Hybridization Arrays steps • • Dedicated equipment: Pipettes Also known as a DNA chip • • Prealiquoted reagents Solid support: Silicon, plastic, or glass • • Negative airflow and unidirectional processing Oligonucleotide probes are added by photolithogra- phy or printing to specific locations to form the array • Thousands of probes are contained on the support chip Alternative Amplification Techniques • Applications • Bacterial identification Ligase Chain Reaction • SNP and mutation detection • Uses four primer/probes and DNA ligase • Gene expression • When the oligonucleotide primer/probes bind to target • DNA sequencing region separated by a few base pairs, the ligase will join • Comparative genome hybridization the probes together Mass Spectrometry Transcription-Based Amplification • Matrix-assisted laser-desorption ionization time-of- • Target is dsDNA or ssRNA and is used to flight (MALDI-TOF) synthesize cDNA • No labeling needed • Isothermal reaction conditions, do not need • Genotype derived from differing mass of alleles thermocycler received by a detector • Uses reverse transcriptase, RNase H, RNA polymerase, and primers • End result is ssRNA amplification Nucleic Acid Sequencing • Examples of transcription-based amplification ○ Transcription-mediated amplification (TMA) Maxam-Gilbert Chemical Sequencing ○ Nucleic acid sequence–based assay (NASBA) • Strong reducing agents used to break nucleotide frag- Strand Displacement Amplification ments in predictable places • Isothermal application reaction that takes place in two • Dimethyl sulfate stages • Formic acid • Stage one: Target generation • Hydrazine and salt • Stage two: Probe amplification • Electrophoresis run on polyacrylamide gel • Uses a modified deoxynucleotide • Method is not amenable to automation and uses haz- • During the extension step the modified deoxynucleo- ardous chemicals tide is nicked by a restriction enzyme Sanger Sequencing • The act of nicking and extension allows for amplifica- • Uses four dideoxynucleotide (ddNTP) base analogs tion of the probes • Results in chain termination as ddNTPs are incorporated randomly among the other dNTPs • Dideoxynucleotide lacks 30 hydroxyl group (OH) Signal Amplification • Separated by polyacrylamide gel electrophoresis or capillary electrophoresis Branched-Chain DNA (bDNA) • Automated methods use fluorescent labeling • Capture probes on a microtiter well plate used to • Dye primer hybridize target • Dye terminator • Extenders Pyrosequencing • Preamplifiers • Uses four enzymes: DNA polymerase, adenosine tri- • Amplifier probes phosphate (ATP) sulfurylase, luciferase, and apyrase Hybrid Capture • Uses two substrates: Adenosine 50 phosphosulfate and • RNA probes luciferin • Make DNA-RNA hybrids • Pyrophosphate (PPi) is released, light is produced that • Sandwich assay corresponds to the dNTP added Cleavage-Based Amplification Next-Generation Sequencing • Invader system: Cuts overlapping regions of • Instruments with the capacity to sequence large vol- nucleotides umes of samples • Use the enzyme cleavase • Whole-genome sequencing for affordable cost • FRET probe (<$1000) • Reporter molecule • Used for personalized medicine CHAPTER 9 Molecular Diagnostics 271

Detection Techniques • Mutations detected by many platforms, including ○ PCR-RFLP • Labeled probes ○ Single-strand confirmation polymorphism • Radioactive (SSCP) ○ 32 Phosphorus atoms ( P) ○ Invader (cleavage-based assay) • Nonradioactive ○ Microarray ○ Digoxygenin Hereditary Hemochromatosis ○ Biotin • Excess iron absorption ○ Streptavidin and alkaline phosphatase • Treatment is via therapeutic phlebotomy ○ Fluorescent labels • Several common base substitutions in HFE gene have been found INHERITED DISEASES • G to A at amino acid 282 (C282Y) • C to G in exon 63 (H63D) Diseases With Mendelian Inheritance • A to T in codon 65 (S65C) • Individuals with mutations may be asymptomatic • Inheritance pedigree that follows a dominant and because of incomplete penetrance recessive pattern Autosomal Dominant Diseases • Punnett square (Figure 9-5) • Presence of one mutated gene is enough to cause ○ Recurrence risk disease • Genes carrying the mutation are on autosomes Factor V Leiden (chromosomes 1-22) • Hereditary hypercoagulability • Typically equal frequency for being affected • Factor V gene is on chromosome 1 (1q23) • Heterozygous: One normal gene (wild-type) and • Leiden mutation is 1691 A!G (R506Q) one abnormal gene (mutation) • Oral contraceptives increase risk for thrombosis • Homozygous: Inherited two genes with the same • Mutations detected by many platforms, including mutation • Invader Autosomal Recessive Diseases • PCR followed by electrophoresis Cystic Fibrosis • Real-time PCR using melting curve analysis • Gene on chromosome 7 Huntington’s Disease • Codes for the cystic fibrosis (CF) transmembrane • Late-onset neurodegenerative disorder conductance regulator protein (CFTR) • Gene for Huntington’s disease is huntingtin and is on • Over 1300 mutations have been identified chromosome 4 • Most common mutation is delta-F508, which is a • Trinucleotide repeat three–base pair deletion • Expansion of CAG • Phenotypic expression will vary based on • Normal repeat range from 10 to 27 copies mutation • Repeats of 28 to 35 “mutable” • Sweat chloride test performed as part of diagnosis • Repeats of 36 to 39 reduced penetrance • Repeatsof40orgreaterareassociatedwith disease Carrier parent • Anticipation: Higher number of repeats in offspring with earlier onset • Ethical issues surround testing for Huntington A a disease • PCR with fluorescent primers to determine the exact repeat number X-Linked Diseases A AA Aa • X-linked recessive females are carriers and usually not affected • Males will receive the mutated gene only from their mother and will be affected • Carrier parent Hemizygous a Aa aa • Many cases are due to new mutations Hemophilia A • Deficiency of coagulation factor VIII (FVIII) FIGURE 9-5 Punnett square illustrating the mating of two heterozygous carriers of an autosomal recessive gene. The genotype • X-linked recessive of the affected offspring is shaded. (From Jorde B, Carey J, • Mostly male cases, but female cases have been Bamshad M: Medical genetics, ed 4, Philadelphia, 2009, Mosby.) reported 272 CHAPTER 9 Molecular Diagnostics

• Female cases related to skewed X-inactivation of • Deletion of allele during egg and sperm production mutated FVIII gene (gametogenesis) • Female cases could be due to two mutated • Different phenotypic presentation depending on FVIII genes maternal or paternal deletion inheritance • PCR assay for detection of inversion mutation • Imprinting on chromosome, 15 del(q11q13) • Sequence the FVIII gene for mutation if inversion ○ Prader-Willi syndrome: Paternal inheritance assay is negative § Learning disability Duchenne’s Muscular Dystrophy § Short stature • X-linked recessive § Behavioral issues • Mostly male cases, but female cases have been ○ Angelman’s syndrome: Maternal inheritance reported § Learning disability • Largest gene in the human genome with a length of 2.2 § Attacks of laughter megabases § Absences of speech • Dystrophin is the protein product ○ Loss of gene expression caused by several • Most common neuromuscular disorder mechanisms • Progressive myopathic weakness • Mutations detected by many platforms, including • Elevated serum creatine kinase • Cytogenetic detection • Diagnosis can be made through immunohistochem- ○ Karyotyping: For chromosome rearrangement ical studies ○ Fluorescence in situ hybridization (FISH): For • Multiplex PCR for prenatal testing deletion detection Fragile X Syndrome • Methylation-specific PCR (mPCR) • X-linked dominant disorder with reduced penetrance • PCR for STRs for uniparental disomy • Penetrance increases in subsequent generations • Sequencing • Common inherited form of learning disability Complex Diseases • Name derived from cytogenetic abnormality of a • Mulitfactoral inheritance patterns breakpoint or fragile spot within the telomere of a • One or more genes with one or more environmental metaphase X chromosome factors • Trinucleotide repeat expansion of CGG • Difficult-to-study combined effects of environmen- • FMR-1 gene tal and genetic factors • Normal range alleles contain 5 to 45 repeats ○ Twin studies are often used • Premutation 55 to 200 repeats § Greater concordance of disease among mono- • Mutation with more than 200 repeats zygotic twins who share all genes than dizy- • Southern blotting for genotype analysis gotic twins who share half their genes • PCR with capillary electrophoresis for repeat number § Less than 100% concordance in monozygotic twins indicates environmental contributors Diseases With Non-Mendelian Inheritance Inherited Breast Cancer • Familial breast cancer accounts for only 5% to 10% of Mitochondrial DNA Diseases all breast carcinomas • Mitochondria generate energy by producing ATP • Mutations in two major breast cancer genes predis- through oxidative phosphorylation pose individuals to breast and ovarian cancer • Mitochondria contain their own circular DNA mole- • BRCA1 gene also associated with increased risk for cule, mtDNA prostate and colon cancer • 16,569 base pairs containing 37 genes • BRCA2 gene also associated with increased risk for • Heteroplasmy: Normal and mutated mtDNA cop- pancreatic cancer ies can coexist in a cell ○ Mutations in BRCA1 and BRCA2 are inherited • Two types of mtDNA mutations in an autosomal dominant manner ○ Those that affect mitochondria protein synthesis § Inheriting gene does not mean that an individ- § tRNA genes ual will develop cancer § rRNA genes § Cannot tell what type of cancer might develop ○ Mutations with the protein coding changes § Cannot tell at what age onset of cancer • Acquired mtDNA deletions are part of the aging might occur process § Men who carry the genes have greater risk for • Sequencing of mtDNA is ideal developing cancer • PCR and Southern blotting ○ BRCA1 and BRCA2 are tumor-suppressor genes Imprinting • DNA sequencing for mutations on BRCA1 and • Histone or DNA modification BRCA2 • Results in transcriptional silencing • Genetic counseling for individuals before testing CHAPTER 9 Molecular Diagnostics 273

Reporting of Test Results • Amelogenin gene: Codes for tooth enamel and is used to identify the presence of X chromosomes • Result should be written so easily understood and or an X and a Y chromosome interpreted in an accurate manner • The Y chromosome has polymorphic loci that are • Along with typical information found on a laboratory used forensically to differentiate the male fraction report (name, birth date, laboratory name), a DNA (sperm) from the female fraction (epithelial cells) test for an inherited disease should include from rape kit evidence • Clinical history and reason for referral • Y-chromosome DNA can be used for ancestry • Explanation of the methodology used studies • Description of patient’s results ○ Fathers and sons will have the same Y • Diagnostic accuracy chromosome STRs • Clinical significance of results • mtDNA can be used forensically • Statement from or meeting with genetic counselors ○ When nuclear DNA is not available ○ Implications of results ○ When nuclear DNA is highly degraded ○ Potential risk for disease for family members ○ When only distant relatives are available for ref- • Statement regarding whether the test is approved by erence sample the U.S. Food and Drug Administration • SNP: Locus for which a single base pair varies in a population ○ Microarray IDENTITY ASSESSMENT ○ Large number must be obtained for significant discriminatory value Forensic DNA Typing Statistical Interpretation • Loci used in DNA typing exhibit Hardy-Weinberg • Genetic variation useful in identity testing equilibrium • Genetic locus variations in a population are called • p2 +2pq+q2 ¼1.0 alleles • In a population containing the genotypes AA, Aa, • A locus is polymorphic if the least common allele is and aa present in greater than 1% of the population ○ p is the frequency of A • Typically introns (noncoding regions) are used for ○ q is the frequency of a identity testing ○ frequency of AA is p2 ○ VNTR or minisatellite ○ frequency of Aa is 2pq ○ STR or microsatellite • Ideal for identity testing because ○ Thousands are scattered throughout the genome Combined DNA Index System ○ Automated fluorescence analysis ○ A specific allele can be identified against a • DNA evidence from crime scene is added to Combined known ladder of alleles DNA Index System (CODIS) database ○ STRs are typically transmitted in mendelian • Convicted offender database fashion • Missing person database ○ The greater the number of alleles present in a • 13 core STR loci locus, the more informative the identification ○ A “hit” is a match between offender and DNA of a specific allelic combination will be left at crime scene ○ Extensive information is available about allele ○ Ethical issues surround how these databases can frequency in many populations be searched Forensic Applications • Exclusion results are often found during DNA identity testing Human Leukocyte Antigen Typing • One or more alleles different from known sample needed for exclusion • Genetic features of human leukocyte antigen (HLA) • Suspect did not commit the crime typing genes • Alleged man did not father the child • Alleles determine the tissue compatibility of trans- • Likelihood of inclusion of tested individuals planted grafts from another donor (allograft) • Discriminatory power: Ability to distinguish an • HLA genes code for highly polymorphic surface individual from the rest of a population molecules which serve as strong alloantigens ○ Based on allelic frequency over several loci • HLA genes are located in the major histocompati- • ABO system has a poorer discriminatory factor bility complex (MHC) than DNA identity testing using RFLP or STRs ○ Chromosome 6 274 CHAPTER 9 Molecular Diagnostics

○ HLA class I Specific Pathogens § A, B, and C • ○ HLA class II Chlamydia trachomatis and Neisseria gonorrhoeae • § DR, DQ, and DP Usually run together on multiplex assays • ○ HLA genes are codominantly expressed Many platforms for detection, including ○ § Variability exists among both racial and eth- Hybrid Capture ○ nic groups PCR ○ • Applications of HLA typing Strand displacement amplification (SDA) ○ • Transplantation Transcription-mediated amplification (TMA) • ○ Matching donor and recipient HLA alleles Group B streptococcal disease • improve long-term organ graft survival Important in perinatal testing to decrease infection ○ Hematopoietic cell transplantation (HCT) also of neonate • has the risk for developing acute or chronic Real-time PCR • graft-versus-host disease (GVHD) Mycobacterium tuberculosis • • DNA-based HLA allele identification Standard growth methods take 6 to 8 weeks • ○ DNA sequencing Molecular testing is rapid and allows for prompt ○ Allele-specific PCR treatment ○ ○ Nomenclature for HLA alleles is frequently TMA of ribosomal RNA ○ updated because of new discoveries PCR for 16S ribosomal RNA gene • • Hematopoietic cell engraftment (HCT) analysis Detection of antimicrobial resistance is possible • • May display chimerism, which is a mosaicism con- Drawback to nucleic acid detection of M. tubercu- sisting of the coexistence of cells derived from the losis nonviable organisms ○ recipient and the donor Should not test individuals who have received • Methods for performing engraftment analysis antituberculosis medication for infection • ○ Obtain sample from recipient before transplant Methicillin-resistant Staphylococcus aureus (MRSA) • for native genotype mecA gene is responsible for resistance ○ ○ Posttransplantation STR loci are informative Gene allows for drug resistance • (nonidentical genotype) and a calculation is per- Tests using PCR and real-time PCR • formed from this data Vancomycin-resistant enterococcus (VRE) • vanA, vanB, and vanC genes are responsible for resistance MOLECULAR METHODS IN INFECTIOUS • Tests using PCR and real-time PCR DISEASES

Indications for Molecular Testing Molecular Virology • Viral genomes • Molecular methods are faster than microbial culture • Less complex then bacterial and human methods or are slow growing • Range from 5000 to 250,000 base pairs • Can detect pathogens that might not culture by tradi- • Variable in structure tional methods ○ DNA or RNA • Provides genetic information that can be used for ○ ssDNA or dsDNA antimicrobial drugs ○ Linear or circular • Used to monitor therapy or provide prognosis • Sequence variations are common • Trace outbreak of infection ○ Single base changes • Provides greater sensitivity and specificity ○ Insertions • Capable of high throughput ○ Deletions Molecular Bacteriology • Viral-load testing • Test to quantify amount of viral nucleic acid in • Bacterial genomes host • Less complex than human genomes ○ Used to predict time and course of disease • Usually circular DNA ○ When to initiate antiviral therapy • Approximately 4.5 million base pairs ○ Monitor response to therapy • Plasmids: Additional genetic material carried by • Test platforms include bacteria ○ RT-PCR ○ dsDNA and circular ○ Branch DNA testing ○ Codes for antibiotic resistance and pathogenicity ○ Nucleic acid sequence–based amplification ○ Used for identification purposes (NASBA) CHAPTER 9 Molecular Diagnostics 275

• Human immune deficiency virus (HIV) type 1 MOLECULAR GENETICS IN HUMAN • RNA retrovirus CANCERS ○ Uses reverse transcriptase to enter into host DNA ○ gag and pol genes are targets for identification Oncogenes and Tumor Suppressor Genes • CD4 count and viral load are factors in therapy and progression to acquired immunodeficiency • Oncogenes syndrome • Proto-oncogene is a normal gene that codes for pro- ○ Goal of therapy is to obtain viral loads below teins that regulate cell growth and differentiation 50 copies/mL • When the proto-oncogene has been altered by • Enzyme-linked immunosorbent assay for screening mutations that affect gene function it becomes an test followed by Western blot for confirmation oncogene • Resistance testing • Oncogene proteins also function on the stages of ○ Sequencing for nucleotide changes, which might cell division decrease effectiveness of antiviral therapy ○ Growth factor signaling pathways ○ Reverse transcriptase has no proofreading capa- ○ Promote the transformation of cells into bilities, so errors are common cancer cells ○ Phenotypic resistance measures viral replication • Tumor suppressor genes when combined with an antiviral drug • Genes that suppress oncogenesis by suppressing • Hepatitis C virus malignant phenotypes • RNA virus • Cancers can develop when there is loss of function • Test platforms include of the gene ○ RT-PCR • Involved in the regulation of progression through ○ TMA the cell cycle or the regulation of DNA repair ○ bDNA ○ Real-time PCR Molecular Diagnosis of Hematopoietic • Cytomegalovirus (CMV) Neoplasms • dsDNA virus in the herpesvirus family • Test platforms include Leukemias Versus Lymphomas ○ Hybrid Capture • Lineage and stage of differentiation ○ NASBA • Acute and chronic forms • Human papillomavirus (HPV) Clonality of Lymphomas • dsDNA • Important step in detecting malignant from benign • Test platforms include proliferation of lymphocytes ○ Hybrid Capture • B lymphocytes: Are able to rearrange segments of ○ Real-time PCR the DNA that code for immunoglobulins • Herpes simplex virus • T lymphocytes: Are able to rearrange segments of • dsDNA virus in the herpesvirus family the gene that codes for T-cell receptors (TCRs) • Test platforms include • Some malignant lymphomas and leukemias demon- ○ PCR strate both immunoglobulin and TCR ○ Real-time PCR rearrangements • Enteroviruses • Assays to determine clonality • Group of RNA viruses • Southern blotting of antigen-receptor gene • Test platforms include rearrangements ○ RT-PCR ○ Positive for clonality when one or two distinct ○ NASBA non-germline bands appear Bioterrorism Agents ○ SNPs could lead to false identification of monoclonality • The key elements of a biologic weapon are difficulty of • PCR analysis of antigen-receptor gene detection, ease of dissemination, and ability to cause rearrangements severe illness or death ○ Amplification of DNA across the V(D)J • Molecular testing is well suited for the rapid detection junctions of bioterrorism organisms ○ Evaluation of products by electrophoresis • Bacillus anthracis ○ Monoclonal populations yield one or two bands • Yersinia pestis or peaks • Francisella tularensis ○ For polyclonal mixture each cell carries a unique • Brucella abortus gene rearrangement and would appear as a • Variola virus (smallpox) smear on a gel 276 CHAPTER 9 Molecular Diagnostics

Hematopoietic Malignancies • Drugs designed to inhibit binding to the EGFR Follicular Lymphoma when overexpressed • t(14:18) translocation brings the gene for BCL-2 from ○ Some mutations to EGFR respond to drugs better chromosome 18 and the gene for the immunoglobulin ○ Sequencing the EGFR gene for these mutations heavy chain (IgH) from chromosome 14 can support optimal treatment • FISH analysis can detect via DNA probes • K-ras (Kirsten rat sarcoma viral oncogene) • Fluorescent red dye probes for the IgH locus • This protooncogene family is the most commonly • Fluorescent green probes for the BCL-2 gene found mutation in human cancers • When the translocation is present, the signal • In G-protein family appears as yellow fluorescence ○ Act as components for energy production Chronic Myeloid Leukemia ○ Part of pathway to communicate growth signals • Translocation of chromosomes 9 and 22 from cell membrane to nucleus • Philadelphia chromosome ○ When mutation is present, remains in an • Diagnostic hallmark for chronic myeloid active state leukemia (CML) • Detection can be through DNA sequencing, pyrose- • BCR-ABL gene fusion encodes for a chimeric pro- quencing, or PCR tein with constitutively activated tyrosine kinase activity • 210-kDa protein (p210BCR/ABL) MUTATIONS AND CYTOGENETIC • t(9:22) may be seen in acute lymphocytic leukemia ANALYSIS (ALL) with poor prognosis • Cytogenetics analysis • Detection can be done using • Karyotyping • Conventional cytogenetics ○ Metaphase chromosomes ○ Minimal residual disease detection ○ Arrest mitosis at metaphase with colcemid • FISH • FISH • RT-PCR • Microarray • Real-time PCR to quantify BCR-ABL transcripts ○ Comparative genome hybridization (CGH) ○ Tyrosine kinase inhibitor imatinib mesylate • Mutation classifications (Gleevec) is used very successfully for treatment • A polymorphism is a genotypic change found in of CML more than 1% of the population Myeloproliferative Disorders (non–BCR-ABL) • A mutation or variant is a genotypic change found • Jak2 mutation: Janus kinase 2, a protein tyrosine in less than 1% of the population kinase ○ Types of point mutations • Polycythemia vera § Silent • Essential thrombocythemia § Conservative • Idiopathic myelofibrosis § Missense • Specific change is V617F can be detected by: § Nonsense • Real-time PCR with melting curve analysis § Frameshift • PCR ○ Several platforms can be used to identify point • DNA sequencing mutations Molecular Genetics of Solid Tumors § SSCP • HER2/neu (human epidermal growth factor 2) § Allele-specific oligomer hybridization • Transmembrane protein with tyrosine kinase § Real-time PCR with melting curves activity § Sequence-specific primer–PCR • HER2/neu is overexpressed in some forms of breast § MALDI-TOF cancer • Aneuploidy: Loss or gain of a chromosome ○ Its presence is an indication for the use of trastu- • Chromosome abnormalities zumab (Herceptin), an anti- HER2/neu ○ Translocation antibody drug ○ Deletion ○ Overexpressed products resulting from the ○ Inversion HER2/neu gene can be detected by ○ Insertion immunohistochemistry ○ FISH can detect increase in DNA copy number PHARMACOGENETICS • Epidermal growth factor receptor (EGFR) • Receptor on cell membrane that when activated ini- • Study of genetic variations in drug response using data tiates proliferation from many assayed across a genome CHAPTER 9 Molecular Diagnostics 277

• Approach to personalized medicine 6. Transfer RNA (tRNA): • Clinical application of pharmacogenetic testing a. Is a gene silencing RNA used in cancer research ○ Polymorphism that can inhibit or induce drug b. Is a macromolecular complex delivered from the metabolism nucleus by ribosomal RNA ○ Cytochrome P450 polymorphisms c. Contains the codon sequence that synthesizes an ○ Drug metabolism enzymes amino acid ○ Test can predict response or effectiveness of d. Contains the anticodon region that binds to certain drugs mRNA codon in the ribosome ○ Test platforms 7. Which one of the following statements concerning § PCR mitochondrial DNA (mtDNA) is incorrect? § Microarray a. Pseudogenes are small pieces of nuclear DNA that § Mass spectrometry share significant homology with mtDNA b. mtDNA is circular and contains approximately CERTIFICATION PREPARATION QUESTIONS 16,500 base pairs c. mtDNA is inherited from the mother because For answers and rationales, please see Appendix A. only ova contain mitochondria 1. During replication the “parent” strand of DNA d. Follows mendelian inheritance patterns serves which purpose? 8. The enzyme ligase joins the Okazaki fragments of the a. It is completely excised by exonuclease enzymes ______. when replication of the strand is complete a. Template strand b. It has a sequence that is complementary to the b. Lagging strand daughter strand that is being replicated c. Leading strand c. It is also referred to as the lagging strand d. Primer fragments d. It will be copied by a DNA polymerase to form 9. How does ribonucleic acid (RNA) differ from deox- two new daughter DNA strands yribonucleic acid (DNA)? 2. Which of the following are forms of nucleic acids? a. RNA has a uracil and DNA has a thymine a. Nucleosides b. RNA does not contain nucleotides and b. DNA or RNA DNA does c. Base pairs c. DNA resides in the cytoplasm of the cell and RNA d. Trinucleotide sequences is in the nucleus 3. The central dogma speaks to the function of the d. DNA has a messenger form and RNA does not molecular components of DNA and states that: 10. The chromosomes in a eukaryotic cell: a. The main function of genes is to store and trans- a. Are in their most compact state and appear as mit genetic information chromatin arms joined at the center during the b. There are specific nucleotide triplets that code for cell division stage called metaphase specific amino acids b. Contain genomic regions that are rich in genes, c. Genes are perpetuated as sequences of nucleic less compactly organized, and termed acid, but function by being expressed in the form heterochromatin of protein c. Contain two specialized regions of euchromatin, d. All sequences of DNA in the human genome will telomeres, and centromeres result in the production of RNAs d. Are highly ordered structures of a single RNA 4. An exon is defined as: molecule, compacted many times with the aid a. A region of DNA present in a mature strand of of structural RNA-binding proteins mRNA and can be translated into protein 11. Which of the following is a description of restriction b. A region of DNA that is recognized by RNA poly- endonucleases? merase to start transcription a. A family of bacteria from which endonuclease c. A region of DNA that is transcribed then removed that cuts DNA into fragments is produced from mRNA by excision and is not translated into b. Only able to digest specific genes on specific chro- protein mosomes with their endonuclease action d. A region of DNA comprising three base pairs that c. Enzymes that specifically degrade DNA in nucleic signal for termination of replication acid mixtures when plasmids are present 5. During the replication process the addition of bases d. Enzymes produced by bacteria to prevent inva- occurs: sion and replication of foreign DNA in their bac- a. At the telomeric end of a DNA strand terial genome b. In the 30 to 50 direction 12. Which enzyme catalyzes DNA replication? c. In the 50 to 30 direction a. Endonuclease d. In both the 50 to 30 and 30 to 50 directions b. Ligase 278 CHAPTER 9 Molecular Diagnostics

c. Polymerase b. Denature, anneal, and extend d. Reverse transcriptase c. Extend, anneal, and denature 13. Which of the following is a block of specific sequence d. Extend, denature, and anneal variants that are inherited together? 21. When RNA is to be used in a PCR amplification a. Allele procedure, what is the first step that must be b. Haplotype performed? c. Locus a. RNA cannot be used in a PCR reaction because it d. Polymorphism will disintegrate during the denaturation step 14. Messenger RNA (mRNA) is produced in the b. RNA must be denatured to form single strands to ______. allow for the annealing of primers a. Golgi c. A reverse-transcription procedure must be per- b. Mitochondria formed to form cDNA c. Nucleus d. RNA must first be treated with RNases to remove d. Ribosomes interfering substances from the target 15. In the organic liquid phase (phenol-chloroform) 22. In the PCR, a ______initiates extension of DNA extraction procedure, proteins are precipitated the sequence of interest by allowing Taq polymerase out of solution: to begin adding nucleotides to single-stranded DNA. a. In the aqueous phase a. Probe b. As a pellet on the filter b. Ligase c. In the organic phase c. Promoter d. On the silica-based gel d. Primer 16. The purity of an extract of nucleic acid can be deter- 23. When performing a PCR procedure, which is the mined by which of the following? most important control to run to check for the pres- a. Measuring absorbances at 260 nm and 280 nm ence of amplicons? and dividing A260 by A280 a. Blank control b. Measuring the bands on an agarose gel b. dTTP control c. Measuring absorbances at 260 nm and 280 nm c. Control and subtracting A280 from A260 and then multi- d. Oligo dT control plying the result by a dilution factor 24. The process of transferring the digested DNA out of a d. Multiplying the concentration of the nucleic acid gel after electrophoresis and onto a nylon membrane by the dilution factor is referred to as: 17. Solid-phase DNA extraction procedures are more a. Hybridization blotting commonly used in a clinical laboratory because: b. Northern blotting a. They can be coupled with a gas chromatograph c. Southern blotting linked to a mass spectrometer d. Western blotting b. They perform best when a large volume of sample 25. In a real-time PCR assay, which probe type is used in is submitted for DNA extraction conjunction with fluorescence resonance energy c. They are adaptable to automation transfer (FRET) on the formation of a duplex? d. They involve organic solvents that are easily a. Hybridization found in a laboratory b. Hydrolysis 18. The rate of DNA migration in a gel electrophoresis c. Hexamere depends primarily on the ______. d. Primer a. Buffer temperature 26. The increase in quantifiable signal observed early in a b. Pore size of the gel real-time PCR run depends on which of the c. Shape of the DNA molecule following? d. Size of the genomic DNA a. The wavelength of the fluorescent dye in the 19. Signal amplification differs from target amplification reaction when designing protocols for identification of b. The number of cycles in the run nucleic acids. Which of the following is an example c. The amount of fluorescent quencher of a signal amplification technique? d. The initial amount of target DNA a. Branched-chain DNA detection 27. Which of the following practices should be employed b. Ligase chain reaction to prevent contamination of patient samples and c. Polymerase chain reaction reagents with amplicons? d. Reverse-transcriptase PCR a. Use bleach solution for cleaning work area 20. Identify the correct sequence of events for a polymer- b. Use UV lights in hooded work area ase chain reaction (PCR) cycle. c. Maintain closed analytical systems a. Anneal, extend, and denature d. All of the above CHAPTER 9 Molecular Diagnostics 279

28. Which of the following statements is true regarding 36. In a chain-termination DNA sequencing reaction, agarose gel electrophoresis? which one of the following components are tagged a. Nucleic acids are separated in an electrical field with a fluorescent dye? because of their net positive charge a. Dideoxynuclotide triphosphates (ddNTP) b. Larger nucleic acid molecules are able to migrate b. Deoxynucleotide triphosphates (dNTP) through the agarose gel faster than smaller c. Pyrophosphates (PPi) molecules d. Capillary probe (CaP) c. Nucleic acids are separated in agarose by shape, 37. Pyrosequencing sequence analysis and quantification charge, and size depends on the release of ______in a quantity d. Agarose is a dye that binds in double- equal to that of an incorporated dNTP. stranded DNA a. Apyrase 29. PCR requires all of the following components except: b. Luciferase a. Deoxynucleotide triphosphates c. Nucleotides b. DNA endonuclease d. Pyrophosphate c. DNA polymerase 38. In which of the following inheritance patterns are d. Oligonucleotides (primers) homozygous alleles necessary to express the disease 30. What is the purpose of the primer extension? phenotype? a. To cut the native DNA into small pieces with a a. Autosomal dominant restriction enzyme b. X-linked dominant b. To hybridize the oligonucleotide primers to the c. Autosomal recessive single-stranded DNA pieces d. Trinucleotide repeats c. To produce PCR amplicons 39. The differential activation of genes depending on d. To activate the DNA polymerase to form hybrids the parent from which they are inherited is referred with the oligonucleotide primers to as: 31. The most common nucleic acid stain used after sep- a. Allelic heterogeneity aration by agarose gel electrophoresis is: b. Imprinting a. Bromothymol blue c. Mosaicism b. Bromocresol green d. Pleiotropy c. Ethidium bromide 40. What is the preferred specimen type for molecular d. Phenolphthalein studies for the diagnosis of inherited mutations? 32. This method is based on the microscopic grouping a. RNA extracted from peripheral mature red cells of probe DNA molecules attached to a solid sup- b. RNA extracted from fresh serum port mechanism such as glass, silicon, or plastic c. DNA extracted from peripheral blood white cells chips. d. DNA extracted from peripheral blood mature a. DNA microarray red cells b. Multilocus enzymes electrophoresis 41. A type of polymorphism that consists of a series of c. Restriction fragment length polymorphism (RFLP) trinucleotide repeats that can be two to seven base d. Polymerase chain reaction (PCR) pairs in length and is known as a microsatellite 33. The field of proteomics studies which of the sequence is also referred to as a: following? a. Restriction fragment length polymorphism a. Proteins on a cellular level (RFLP) b. Serum proteins b. Variable number tandem repeat (VNTR) c. Proteins in genes c. Restriction endonuclease d. The human genome d. Short tandem repeat (STR) 34. Which of the following is not a factor that influences 42. The site of a particular nucleotide sequence on a hybridization reactions? chromosome is referred to as a(n): a. Degree of complementarity between the probe a. Allele and target nucleic acid b. Locus b. pH c. Polymorphism c. Size of the target’s genome d. Genotype d. Temperature 43. The HLA A, B, and C molecules are encoded for by 35. Which of the following is false about primers? which class gene(s)s within the major histocompati- a. Primers should be at least 100 nucleotides long bility complex (MHC)? b. Primers are typically 15 to 30 nucleotides long a. Class I c. Primers should have a GC percentage of 40% b. Class II to 60% c. Class III d. Primers should anneal to a specific target d. All groups in MHC class 280 CHAPTER 9 Molecular Diagnostics

44. The detection of the DNA from cytomegalovirus 48. The technique that uses fluorescent DNA probes to (CMV) and human papillomavirus (HPV) is typically detect chromosomal abnormalities within cells in performed using the Hybrid Capture assay. What cytogenetic studies is referred to as: type of assay is Hybrid Capture? a. Fluorescence in situ hybridization (FISH) a. Target amplification assay b. Karyotype in situ hybridization (KISH) b. Signal amplification assay c. Fluorescence in situ PCR c. Reverse transcriptase assay d. Microarray d. Viral load assay 49. A tumor-suppressor gene performs which of the fol- 45. The major advantage for using nucleic acid tech- lowing task(s)? niques for the identification of infectious disease is: a. Codes for normal growth-promoting proteins a. The lower cost involved with molecular analysis b. Codes for proteins that control cell division b. The high specificity for identification of an c. Repairs nucleotide mismatches in the DNA strand organism d. All of the above c. The lack of false negatives 50. Which term describes a normally occurring gene that d. The ability to distinguish normal flora from when altered is often associated with cancers? disease-causing organisms a. Oncogene 46. Which of the following genes is not found in b. Proto-oncogene retroviruses? c. Meta-oncogene a. Gag d. Post-oncogene b. Pol c. Env BIBLIOGRAPHY d. Onc 47. In chronic myeloid leukemia (CML) the fusion of Bruns D, Ashwood E, Burtis C: Fundamentals of molecular diagnos- chromosomes 9 and 22 produces a hybrid gene, tics, St. Louis, 2007, Saunders. BCR-ABL. This chromosome is referred to as the: Buckingham L: Molecular diagnostics: fundamentals, methods, and clinical applications, ed 2, Philadelphia, 2011, F. A. Davis. a. Cincinnati chromosome Mahon C, Lehman D, Manuselis G: Textbook of diagnostic micro- b. Legionnaires chromosome biology, ed 4, St. Louis, 2011, Saunders. c. Myeloprolifererative chromosome Rodak B, Fritsma G, Keohane E: Hematology: clinical principles d. Philadelphia chromosome and applications, ed 4, St. Louis, 2012, Elsevier. Turnpenny PE, Ellard S: Emery’s elements of medical genetics, ed 14, Philadelphia, 2012, Churchill Livingston. CHAPTER 9 Molecular Diagnostics 281

SELF-ASSESSMENT

Content Area: ______

Score on Practice Questions: ______

List the specific topics covered in the missed questions:

List the specific topics covered in the correct questions: 282 CHAPTER 9 Molecular Diagnostics

NOTES CHAPTER 10 Laboratory Operations

Paul R. Labbe and Linda J. Graeter

ORGANIZATIONAL MANAGEMENT Management Styles STRUCTURE FOR THE CLINICAL • Management by objective (MBO): Targets organiza- LABORATORY tional and employee performance by aligning goals and objectives throughout the organization, including • Many types of management organizational charts, timelines, tracking, and feedback in the process depending on whether it is in an academic medical cen- • Continuous quality improvement (CQI): Analytical ter, a large hospital network system, an independent decision-making tool that determines when a process laboratory, research laboratory, or a physician office is working predictably and when it is not, and identi- laboratory organization. General laboratory manage- fying the variation to lessen or eliminate it, using pro- ment structures include cess control charts (Figure 10-1) • Laboratory director: Strategic oversight of labora- • Total quality management (TQM): Management tory services approach to long-term success through customer satis- • Laboratory manager: Daily workflow of the faction, incorporating all members of an organization laboratory team participating in improving process, products, services, • Technical managers: Specialists in ensuring total and the culture in which they work service quality and accurate testing Motivational Theories and Styles • Logistical supervisors: Specialists in preanalytical • Maslow’s Hierarchy of Needs: Psychology theory and postanalytical collection to reporting and bill- proposed by Abraham Maslow in 1943 in which all ing of laboratory testing individuals focus on the fundamental needs and once • Medical laboratory scientists and technicians: Daily those are fulfilled will progress to higher needs testing production and evaluation of results (Figure 10-2) • Laboratory assistants: Support services to the labora- • Frederick Herzberg’s Motivator-Hygiene Theory: The- tory workflow (pretesting, testing, and posttesting) ory of motivation in which employees base their satisfaction or dissatisfaction with work on hygiene factors (company policies, wages, job security) and motivator LEADERSHIP factors (status, advancement opportunity, recognition, personal achievement) Management and Motivation • McGregor’s Theory X and Y: Theoretical assumptions proposed by Douglas McGregor in 1960 based on Leadership Styles (Kurt Lewin, 1939) behavior of individuals at work • Authoritative/autocratic: Leader informs employees • Theory X is that humans have an inherent dislike of what is to be done and how it is to be performed— work and will avoid it if they can—therefore they closed system need to be “controlled” (boss-centered leadership) • Participative/democratic: Includes one or more • Theory Y is that the expenditure of physical employees in the decision-making process, with the and mental effort in work is as natural as in leader maintaining the final decision-making play or rest—therefore, if the job is satisfying, authority—open system the employee will be self-motivated (employee- • Delegative: Leader confers the decision-making ability centered leadership) to the employees, with the leader still responsible for • Tannenbaum Schmidt Theory: Continuum of leader- the decisions made by the employees—free reign or ship proposed in 1958 by Robert Tannenbaum and laissez faire system Warren Schmidt and later updated in 1973 suggests • Combination: All three styles are used, depending on a manager uses a broad range of leadership styles the issues involved—generally an indication of a based on the prevailing circumstances in the current good leader environment

283 284 CHAPTER 10 Laboratory Operations

Self Actualization

Self-esteem

Love

Security and Safety

Physiological

FIGURE 10-2 Maslow’s hierarchy of human needs. (From Fuller J: Surgical technology: principles and practice, ed 6, St. Louis, 2012, Saunders.)

suggesting a human relations orientation in contrast to a low LPC score indicating a task orientation • Myers-Briggs Type Indicator (MBTI): Uses a theory of psychological types to make insights of type theory applicable to individuals and groups. A grid displays the 16 personality types based on your indicators: Extraversion(E)/Introversion(I), Sensing (S)/Intuition(N), Thinking(T)/Feeling(F), and Judg- ing(J)/Perceiving(P) and is determined using the MBTI instrument

PROFESSIONALISM FIGURE 10-1 Conceptual basis of control charts. A, Frequency distributions of control observations for different error conditions. • B, Display of control values representing those distributions when Profession: Principle, vocation, employment, or call- concentration is plotted versus time on a control chart. (From ing that requires specialized knowledge and intensive Burtis C, Ashwood E, Bruns D: Tietz fundamentals of clinical academic preparation chemistry, ed 6, St. Louis, 2008, Saunders.) • Professional: Member of a vocation who has obtained a degree in a highly specialized field, commonly engaged in creative and intellectually challenging work • Blake Mouton: Robert Blake and Jane Mouton in • Professionalism: Practice of an activity using the con- 1964 plotted leadership behavior on a grid based on duct, aims, and qualities based on a code of ethics for concern for people versus concern for production, with that profession a scale of 1 to 9 based on the level of concern. Snap- • Code of ethics: American Society for Clinical Labora- shots of five styles of leadership include impoverished, tory Science sets forth the principles and standards by country club, politician, authoritarian, and team which clinical laboratory professionals practice their • Hershey Blanchard: Ken Blanchard and Paul Hershey profession, based on conceived a leadership theory in the mid-1970s based • Duty to patient on situational models; there is no single “best” style of • Duty to colleagues and the profession leadership, but is more dependent on the task, knowl- • Duty to society edge, and group dynamics for each situation • Fiedler Theory: Developed by Fred Fiedler, this leader- COMMUNICATION ship theory is based on two factors of situational con- tingency: leadership style and situation control. An • Effective laboratory operation depends on individual index designated the least-preferred coworker (LPC) specialists working as an effective team within the scale is a rating used by leaders, with a high score health care system of professionals CHAPTER 10 Laboratory Operations 285

• Communication is a key to an effective laboratory CLIA-approved accrediting organizations. Cur- • Verbal communication: Using sounds, words, rently more than 200,000 laboratories are speech, and language to interact between individ- registered under CLIA, approximately 50% of uals and to groups which are physician office laboratories • Nonverbal communication: Using touch, body lan- • CLIA-approved providers: 2013 current listing of guage, timeliness, and personal space to convey approved accreditation organizations for issuing a interaction and listening attentiveness with others CLIA accreditation certificate: American Associa- • Paraverbal communication: Messages transmitted tion of Blood Banks, American Osteopathic Asso- through the tone, pitch, and pacing of the voice ciation, American Society for Histocompatibility • Informative communication: Using data, instruc- and Immunogenetics, College of American tions, and knowledge to convey information to Pathologists, COLA, Joint Commission http:// others www.cms.gov/MLNProducts/downloads/ • Communication styles can be affective (emotional), CLIABrochure.pdf imaginative (perceived image), persuasive (inducing • Family Medical Leave Act (FMLA): Supplies any or commonly accepted), and ritualistic (cultural or employee who has been on the job for over 1 year routinely established) up to 12 weeks of unpaid, job-protected leave • Active listening requires the listener to be attentive to each year the communicator and is a structured way of respond- • Fair Labor Standards Act (FLSA): In 1938 it set ing to others. Suspending one’s own frame of refer- guidelines on pay practices in government, busi- ence, using good judgment, and avoiding other nesses, and health care and defined overtime pay activities will focus the listener on evaluation of what practices. The Equal Pay Act of 1963 is a section the communicator is conveying. Roadblocks to active of the FLSA that affirms equal pay for equal work listening are performing other tasks and multitasking • Food and Drug Administration (FDA): Government during the communication process agency that regulates clinical laboratories through approval of laboratory test kits and medical devices REGULATORY and provides clarification on how workload should be calculated when using current FDA-approved • U.S. clinical laboratories are governed by the laws and semiautomated gynecologic cytology screening regulatory guidelines of the following federal and state devices agencies and programs • Centers for Disease Control and Prevention (CDC): • Department of Health and Human Services Government laboratory working on the Battelle Pro- (DHHS): Oversight federal government agency that ject to identify best practices in laboratory medicine regulates clinical laboratories based on the federal • Department of Transportation (DOT): Provides the laws passed by the U.S. Congress regulations for shipment of biological and hazard- • Centers for Medicare & Medicaid Services (CMS): ous materials, including clinical laboratory Within the DHHS, regulates all laboratory testing specimens (except research) performed on humans in the • Environmental Protection Agency (EPA): Regulates United States through the Clinical Laboratory and oversees the disposal of contaminated wastes Improvement Amendments (CLIA) from the clinical laboratory • CLIA Program: Program administered through the • Health Insurance Portability and Accountability federal and state agencies to ensure high-quality Act (HIPAA): In 1996 identified rules to protect laboratory testing individuals’ identifiable health information, the • CLIA 88: Law established by the U.S. Congress to rights granted to that individual, enforcement activ- create uniform federal standards for regulating clin- ities, and methods to monitor and file a complaint ical laboratories; also extended government over- • Health Information Technology for Economic and sight to all testing facilities, including physician Clinical Health Act (HITECH): In 2009 defined office laboratories. The government agencies regulations requiring all health providers to notify responsible for overseeing the laboratory standards individuals when their health information has been are CMS, Centers for Disease Control and Preven- breached; is monitored by the Office for Civil tion, and Food and Drug Administration Rights • CLIA Certificates: CLIA 88 requires clinical • National Labor Relations Board (NLRB): Federal laboratories to obtain one of four certificates to agency to safeguard employee rights and determine perform laboratory testing: Certificate of Waiver, whether they have the right to form a union; the lab- Certificate of Provider Performed Microscopy, oratory is recognized as a bargaining unit by Certificate of Compliance, or Certificate of Accred- the NLRB itation. CMS issues the first three certificates, and • National Institute for Occupational Safety and the Certificate of Accreditation is obtained through Health (NIOSH): Division of the CDC responsible 286 CHAPTER 10 Laboratory Operations

for conducting research and making recommenda- laboratories for the next 6 years to help laboratories tions for the prevention of work-related illnesses meet CLIA requirements. The increase in oversight and injuries by CMS was driven by a government investigation • Occupational Safety and Health Administration in 2006 into how some highly publicized laboratory (OSHA): Main federal agency charged with errors had occurred and could have been prevented enforcement of safety and health legislation • The Joint Commission (TJC) accredits more organi- • Office of Inspector General (OIG): Monitors the zations than any other agency in health care. It business aspects of the clinical laboratory and estab- accreditsmorethan18,000healthcareorganizations, lishes guidelines to prevent fraud and abuse from including approximately 3000 clinical laboratories unethical and improper reimbursement for labora- • Individual state health departments issue the other tory services under government programs such as three CLIA certificates as a governmental arm of CMS Medicare and Medicaid • Other certification and accreditation agencies for specific health or specialty laboratories • Det Norske Veritas (DNV): Recently instituted ACCREDITATION accreditation organization within the United States for accreditation of hospitals using the Interna- • All U.S. clinical laboratories that apply for a Certifi- tional Standards Organization (ISO) 9001 certifica- cate of Accreditation must be certified by one of the tion. If a hospital uses DNV for accreditation, it seven deemed status accreditation organizations desig- then partners with one of the other deemed organi- nated on the CMS website zations for accreditation of the hospital laboratory • American Association of Blood Banks (AABB): • Substance Abuse and Mental Health Services Accreditation is granted for collection, processing, Administration (SAMHSA): Offers a certification testing, distribution, and administration of blood required by all laboratories that are performing fed- and blood components; hematopoietic progenitor eral workplace drug testing programs cell activities; cord blood activities; perioperative • National Accrediting Agency for Clinical Labora- activities; relationship testing activities; immunohe- tory Sciences (NAACLS): International agency for matology reference laboratories; and Specialist in accreditation and approval of educational pro- Blood Bank schools grams in the clinical laboratory sciences and related • American Association for Laboratory Accredita- health professions tion: provides acceditation services and training • American Osteopathic Association (AOA): The AOA Healthcare Facilities Accreditation Program HUMAN RESOURCES (HFAP) is authorized by the CMS to survey hospitals, clinical laboratories, and other health care • Job position: Determination of a job position is done facilities for compliance with CMS standards through an evaluation instrument or job position anal- • American Society for Histocompatibility and ysis checklist Immunogenetics (ASHI): Accreditation program • The job analysis is a review of the job to be done and to evaluate laboratory personnel, procedures, and the work to be performed. Once this is completed a facilities to determine if they are in compliance with job description, performance analysis, training published standards of ASHI and standards of orga- assessment, and promotional criteria may be set nizations by which ASHI is deemed: HCS/BM • Job advertisement: Once a vacancy in a job position Transplantation—Related and Unrelated Donor; occurs,ajobadvertisementisplacedtoattractapplicants Solid Organ Transplantation—Deceased and Live and list the role of the job, experience required, purpose, Donor; Parentage Testing; Histocompatibility Test- location, general schedule, and responsibilities. The ing; and Transfusion Support advertisement should be short and to the point, using ter- • College of American Pathologists (CAP): Labora- minology that will attract the professional laboratory tory Accreditation Program meets the needs of a personnel desired for the position variety of laboratory settings from complex univer- • Job description: Written statements that describe the sity medical centers to physician office laboratories. duties, responsibilities, outcomes expected, qualifica- The program also covers a complete array of disci- tions needed, and reporting responsibilities to co- plines and testing procedures. Because of its com- workers and supervisor prehensive nature, CAP accreditation can help • Full time equivalent (FTE): Defined by the Govern- achieve a consistently high level of service through- ment Accountability Office (GAO) as the number of out an institution or health care system hours worked divided by the compensable hours in a • COLA: First organization to be renewed since work year as defined by law increased government scrutiny of survey organiza- • 2080 hours/year equates to 1 FTE tions and was given permission to accredit • 1040 hours/year equates to 0.5 FTE CHAPTER 10 Laboratory Operations 287

• Interview and selection process: An interview is con- • Good customer service is the best public relations ducted to determine the best candidate for the position and positive advertising strategy • A standard set of questions should be used with all • Successfully handling a dissatisfied customer will candidates that are job-related and review objective 95% of the time ensure that this customer will criteria based on the job description. Not all candi- use your services again, because you have attempted dates need to be interviewed for the job they apply to “right the wrong” to, but standard cutoff criteria should be applied to • Legal considerations guide the policies and procedures all candidates to avoid bias and discrimination of the laboratory in relation to human resources and • Employee orientation and training: A checklist should the appropriate practices of laboratory testing be used to ensure all pertinent issues are reviewed with • Refer to the accreditation and certification section the new employee to ensure complete understanding of of this chapter, along with the definitions of the var- the responsibilities of the position ious governmental institutions and regulations, • This review should cover administrative policies, such as the National Labor Relations Board, Fair the general layout of the work environment, organi- Labor Standards Act, Equal Pay Act, and Family zational policies, department and job duties, and an Medical Leave Act identified mentor to assist in the new employee training FINANCIAL ASPECTS OF LABORATORY • Many organizations have a probationary period for the new training to take place to determine if the • Budget preparation: A template for forecasting reve- employee is a good fit for the job and the job is a nue and expenses for an organization good fit for the employee • A budget involves four processes: Development of • Employee evaluation/annual review: Assessment and goals, forecasting of revenue using budget assump- review of a worker’s job performance tions, forecasting of expenses, and ongoing moni- • Employees are evaluated on a regular basis (often toring of both revenue and expenses once per year) • Capital versus operational expense: Capital expense is • Reminds workers what is expected of them in the money used for physical assets such as acquiring or workplace and provides employers with informa- upgrading facilities or equipment and is often depreci- tion to use when making employment decisions, ated over a 5-plus year period. Operating expense is such as promotions, pay raises, and layoffs money used for day-to-day operations, such as • Employee corrective action and discipline: Used to redi- employee wages, taxes, and the general costs of doing rect the poor or inappropriate performance of a labora- business tory employee through specific goals and retraining • Justification for both capital and operating costs often • Step-wise action plan documented through a set of use return on investment process thinking targeted objectives and a timeline for completion • Determine if the expenditure is essential, necessary, • Team building: Uses a skill set to bring members of dif- or desired; will reduce day-to-day costs over a ferent backgrounds and skill sets together for a com- period; or will add additional revenue mon purpose • Classification of expenses: Fixed versus variable and • Within the laboratory, among phlebotomists, direct versus indirect aides, technicians, technologists, specialists, and • A fixed cost is a routine charge that does not change pathologists with test production (rent of facility space, taxes, etc.) • Outside the laboratory, incorporating the health • A variable cost changes based on fluctuations in test care team of physicians, nurses, and ancillary health volume (reagent usage) and hours worked (regular care team members and overtime costs) • Conflict management: Uses strategies to improve the • A direct expense is related to all components asso- positive aspects of conflict and decrease the negative ciated with performing the test (laboratory equip- aspects of conflict, for improving the group outcome ment, reagents, service contract, quality control) and improving learning and performance of the group • Indirect expense is related to all components consid- • Conflict is an expected process within a group and ered overhead or administrative, such as marketing/ when managed properly may increase the perfor- sales, insurance, etc. mance outcome of the group • A laboratory could have fixed indirect and fixed • Five approaches to conflict management: Avoiding, direct costs and the same with variable classifications accommodating, compromising, forcing, and • Cost allocations: Cost/billable is calculated using the collaborating total direct costs of performing a test as the numerator • Customer service applies to internal and external and the revenue generated sample as the denominator customers • Cost/billable is used in many reagent rental agree- • The three Rs of customer service: Results, Relation- ments in which the instrument is placed in the ship, and Resource laboratory and an agreed-on cost per billable test 288 CHAPTER 10 Laboratory Operations

(does not count quality control, standards, or patient • Laboratory Management Index Program (LMIP): repeat samples tested) is calculated for the laboratory CAP service that accounts for workload recording to pay the instrument vendor on an ongoing basis via for labor per billable test plus the expenses for non- their operating budget instead of a capital expense billable tests (quality control) for buying the instrument outright • Test/FTE is often used today to benchmark with other • A cost/test may be different from a cost/billable laboratories using a standardized test terminology and because this ratio includes all tests performed on the FTE, which equals 40 hours/week or 2080 hours/ the instrument, including quality control and year standards • Scheduling to maximize efficiency, productivity, and • Reimbursement: Fee for service—before the 1980s all customer service is based on the workload units, bud- laboratory testing was reimbursed at the fee charged get, test menu and volume, education and experience for the service level of employee, and complexity of the work • Diagnostic related groups (DRGs): Established in • By assigning a standard unit (estimate of time) to 1982 to determine the level of payment for the ser- each billable test allows you to calculate the number vice provided—laboratory services were combined of FTEs needed for scheduling and the approximate with other ancillary services for the inpatient popu- costs to budget lation of hospitals ○ Standard unit measured as 0.25 (time to perform • Resource-Based Relative Value Scale: AMA coding one test) system to reimburse physicians for outpatient work 0:25 12, 000 tests over a 4-week period ¼ 3000 hours needed or Part B of Medicare reimbursement—based on 3000 hours=160 hoursðÞ¼ 4 weeks 18:75 FTEs relative value units of (1) work, (2) practice • expense, (3) malpractice expense Staff mix is determined by the work complexity, the • Ambulatory Payment Categories (APCs): Are hours of operation (days only or 24 hours), and the assigned to outpatient and emergency room proce- resources available for checks/balances and supervision • dures and tests and determine the outpatient pro- Experience, training, and maturity within the prospective payment system fession will aid in determining aide/technician/tech- • Current Procedural Terminology (CPT): A regis- nologist mix per shift in the laboratory setting • tered product of the AMA to standardize the Pool/as-needed (PRN)/contract staff are often used to language of various medical procedures fill temporary vacancies in the laboratory workforce • • International Classification of Diseases 9 (ICD-9). Pool staff may be employees who work only as Procedure-based classifications assigned to all tests needed and when called by the laboratory • within the laboratory PRN: Acronym for Latin term pro re nata, or • Healthcare Common Procedure Coding System “as needed” • (HCPCS): Standardized identification system to Staff also may be acquired through temporary staff- ensure claims are processed in a consistent manner ing agencies and are contracted for a period to fulfill for reimbursement (originally HCFA for the earlier specific job duties but are not employees of the name of the CMS Health Care Financing organization Administration) • Since 1984 and the enactment of the Deficit Reduc- SAFETY tion Act, laboratories must bill Medicare directly based on a universal laboratory fee schedule that • OSHA mandates personal protective equipment and is capped, and the Act eliminated any copay by ergonomic work stations be supplied to all employees the patients under Medicare versus other insurances within the laboratory • A safety manual, training, and safety officer are crit- WORKLOAD AND PRODUCTIVITY ical to ensure a safe work environment within the laboratory, with appropriate documentation and • CAP workload is a measurement that is over 25 years counseling for employees who improperly skip old; it is a productivity measurement based on the time safety protocols when performing their job to perform a test, the specimen processing involved, • Safety “right to know” addresses hazards and provides and the reporting time aspects of completing a report guidelines for employees to understand the general lab- • Hospitals often use an adjusted patient day–to-FTE oratory information on safety, including occupational ratio to determine productivity for the laboratory. exposure to blood-borne pathogens and hazardous However, the inpatient-to-outpatient testing ratio chemicals and the protection devices and communica- along with revenue received rather than number tions necessary to ensure a safe work environment of tests performed could skew the data • The mandate of right to know lists the hazardous • Billable tests/FTE could be used but does not take into substances with which the employees work, what account the variance in the test menu complexity protection is available, training and education, CHAPTER 10 Laboratory Operations 289

and the ability of employees to make a written series of statistical calculations to determine whether request for further information on safety for variations in quality control are random or systemic, their job giving guidance on acceptability and reportablity of • Blood-borne pathogens and chemical hygiene plan are accurate laboratory test values specific and targeted plans that describe the hazards, • Troubleshooting: Erroneous laboratory results are the education and training on those hazards, annual investigated through evaluation of the preanalytical, documentation of this training, protective equipment, analytical, and postanalytical steps and medical surveillance for exposures • Process improvement: Six Sigma, Lean, and TQM are • Hepatitis B vaccine must be offered to all employees proactive process improvement techniques and systems at no cost, because of the significant risk for infec- • Six Sigma improves laboratory processes by remov- tion of employees through needle sticks, cuts, or ing the causes of errors and minimizes variations splashes to mucous membranes (improving precision). Six Sigma is a statistical term • Safety program must be present in all laboratories to defined as 3.4 errors/million opportunities or deal with the multiple regulatory agencies’ guidelines 99.99966% free from defects (OSHA, EPA, DOT, NIOSH, etc.) and to ensure a safe • Lean, or Kaizen, is a method to remove non–value work environment added steps in any process within the laboratory, • A safety officer develops and maintains safety with the result of improving turnaround time or procedures, with ongoing updates through com- space usage efficiency munication and training and comprehensive docu- • Total Quality Management promotes a philosophy mentation of these processes that everyone involved in the laboratory workflow • All laboratory employees and students must play a has a responsibility for a quality product; this proactive role in their own safety and the safety of encompasses suppliers/vendors, the health care their co-workers team, management, and patients • Continuing education: Professional development and professionalism depend on ongoing education PROCEDURES throughout a 40-year career • CLIA 88 recommends and some state agencies and • Laboratory procedures are the standard written pro- employers require, continuing education on an cesses that all laboratory employees follow to function annual basis effectively in their roles • Professional Acknowledgment for Continuing Edu- • CAP requires procedures based on the guidelines cation (P.A.C.E.) credits are offered through ASCLS presented in the CLSI Laboratory Documents and are approved for laboratory-focused programs Development and Control • This guideline follows the common elements that should be in all procedures: Title, purpose/princi- EDUCATION ple, procedure instructions, references, author, approval signatures, safety information, reagents, Medical Laboratory Education supplies and equipment needed, limitations of the • Encompasses training new employees, current method, calculations, and expected values employees, students, other health care professionals, and the community; includes continuing education QUALITY programs • Overseen by the National Accrediting Agency for Clin- • Daily quality control: Process used to ensure that the ical Laboratory Sciences (NAACLS). NAACLS daily laboratory reports are generated from appropri- accredits educational programs in Cytogenetic Tech- ate patient specimens following standard procedures nology, Medical Laboratory Scientist, Medical Labo- and using metrics and monitors throughout the prea- ratory Technician, Diagnostic Molecular Scientist, nalytical, analytical, and postanalytical processes to Histotechnician, Histotechnologist, and Pathologists’ ensure accurate and precise results Assistant • Quality assurance: Systematic process that ensures a • NAACLS publishes and administers the Guide to quality laboratory service and result every time, all Accreditation and Accreditation Standards for each of the time. A quality assurance leader or team reviews program type the processes in place for a consistent standard accu- • The goals and responsibilities of laboratory educa- rate laboratory result and evaluates these processes tional training programs are guided by NAACLS to be proactive in process improvement techniques and the profession itself through its various profes- • Westgard’s quality management framework: James sional societies Westgard developed multiple-rule quality control for ○ Scope of practice: Defines the roles of and the ser- the laboratory known as the “Westgard Rules,” a vices provided by laboratory professionals 290 CHAPTER 10 Laboratory Operations

○ Body of knowledge: Within the scope of practice, • Cognitive LOs: Cognitive LOs are based on knowl- describes the knowledge, skills, and attitudes edge; the student must possess the knowledge needed that are required of laboratory professionals. to meet the objective Educational programs are obligated to provide • Level I: Recall of learned information; verbs include the content of its discipline’s body of knowledge define, describe, list to students • Level II: Applying the learned information; verbs ○ Curriculum: Defined course of studies; the total include calculate, discuss, explain content encompasses the various components of • Level III: Analyzing information, making decisions; the body of knowledge verbs include analyze, compare, distinguish • Cognitive LO examples Learning Domains ○ Define the term nosocomial ○ Discuss the advantages of using automatic • Learning is categorized into three domains: Cognitive, pipettors Psychomotor, and Affective. Each domain is further ○ Give a patient’s medical history and laboratory divided into levels of learning called taxonomy levels. results accurately Taxonomy levels within each domain begin at the • Psychomotor LOs: Psychomotor LOs are based on respective fundamental level and progress to the more skill; the student must be able to successfully utilize complex level. This provides a performance contin- learned motor skills to meet the objective. uum for the learner • Level I: Awareness of ability to perform a motor • Cognitive: Knowledge based on the following skill; verbs include prepare, label, set up • Recall: Remembering facts • Level II: Proficiency in performing the skill; verbs • Comprehension: Understanding of the material include operate, measure, perform • Application: Relating learned material to new • Level III: Ability to alter the procedure; verbs situations include revise, design, develop • Analysis: Breaking down a situation into interre- • Psychomotor LO examples lated components ○ Motor skills to meet the objective • Synthesis: Tying interrelated components together § Label the tubes for a serial dilution procedure in a useful way § Perform a manual platelet count within 20% • Evaluation: Judging the value of information, “crit- of the automated analyzer result ical thinking” § Design the procedure using the new auto- • Psychomotor: Performance based, includes coordina- mated instrument tion, thoroughness, and efficiency • Affective LOs: Affective LOs are behavior based; the • Observing student must exhibit appropriate and professional • Preparing behaviors and attitudes • Performing • Level I: Awareness of activity; verbs include comply, • Affective: Behavior and attitude based attend, obey • Listening and learning • Level II: Associating a value to an activity; verbs • Applying appropriate behavioral patterns include cooperate with, assist, share • Commitment to and a respect for life-long • Level III: Commitment to a set of values; verbs professionalism include defend, exhibit, influence Bloom’s Taxonomy • Affective LO examples ○ Complies with the class attendance policy • Developed by Dr. Benjamin Bloom, Bloom’s Taxon- ○ Assists classmates with maintaining an organized omy is a set of principles that outline the learning levels laboratory space within the cognitive domain. Bloom’s Taxonomy has ○ Exhibits a respectful demeanor at all times become an underlying guideline for the education community, regardless of the discipline Learning Objectives LEARNING ASSESSMENTS • Learning objectives (LOs) are statements that explicitly describe what the learner should know or be able • Learning assessments are measures of student perfor- to do after a period of instruction mance within the three learning domains. Assessments • LOs are also categorized as Cognitive, Psychomotor, can include examinations (tests) and quizzes, practical or Affective examinations, writing a paper, doing a presentation, • LOs are further categorized into levels I, II, and III and being observed by an instructor in class or in the from simple to complex laboratory • LOs must be measurable with respect to student • Examination questions are categorized into three tax- performance onomy levels: I, II, and III CHAPTER 10 Laboratory Operations 291

• Level I: Simple recall questions—examples • Cooperative learning: Students work in groups, dis- • What color is a positive spot indole test? cussing and learning material • What is the normal value for serum chloride? • Distance education: Students participate and learn • Level II: Using learned material to interpret and from a distance using online materials answer the question—examples Clinical (Bench) Teaching • A Gram stain of a pleural fluid specimen shows • For laboratory professionals, the first type of teaching gram-negative coccobacilli. Which of the follow- often encountered is training students or new ing is the organism most likely seen in the Gram employees “at the bench.” Teaching clinical skills stain? requires a basic understanding of the educational pro- • An anion gap was found to be 28 mmol/L. Which cess, including planning. The steps involved in clinical of the following is a possible cause of this result? training are • Level III: Using learned materials to solve a problem— • Students are provided with the didactic knowledge examples and psychomotor skills necessary to accurately • A patient was found to have circulating blasts. The understand and perform the procedure marrow blast count was 43%. No Auer rods were • Students observe the procedure step by step, dem- identified, and the peroxidase and nonspecific ester- onstrated by the instructor. This step includes ase were negative. Flow cytometry immunopheno- obtaining specific information about the procedure typing revealed the presence of CD 13, CD 33, from the Standard Operating Procedure and CD 34 in the abnormal cells. CD 41, CD 19, manual (SOP) and TdT were negative. Which of the following is • Students practice the procedure as the instructor the most likely cause of these findings? observes until the expected competency is attained. • The image shown is an auramine-rhodamine stain The instructor assesses performance and provides of an organism recovered from an infected finger feedback to the students wound that was slowly progressive in spite of top- • Students perform the procedure under the supervi- ical antibiotic treatment. The infection developed sion of an instructor after the patient cut his finger while cleaning his home aquarium. The organism grew optimally at 30 C on Middlebrook 7H11 medium and formed deep yellow pigment when exposed to light. The organism was negative for nitrates and heat-stable CERTIFICATION PREPARATION QUESTIONS catalase, but hydrolyzed Tween and produced urease and pryazinamidase. Which of the following is For answers and rationales, please see Appendix A. the most probable identification of this organism? 1. During the morning rush, your laboratory manager Learner Types comes into the laboratory and starts explaining a new policy regarding vacation requests. Word • The three fundamental types of learners are visual, spreads of the change throughout the day, and the auditory, and kinesthetic message has changed somewhat. Several in the labo- • Visual learners learn primarily from written mate- ratory are upset and complain to the laboratory man- rials and by sight ager. Which of the following actions is the most • Auditory learners learn primarily by listening and appropriate way to handle such a situation? talking a. Nothing should be changed, it was handled • Kinesthetic learners learn primarily by doing appropriately • Instructional materials and methods should be b. The manager should have posted the change on designed with consideration for all three learning types the bulletin board in the break room Teaching Methods c. The manager should have announced the policy • Lecture: Instructor presents material to a group of on each shift students d. The manager should have discussed and distrib- • Laboratory demonstration: Method, technique, or uted the policy at a laboratory meeting, or several instrument is demonstrated laboratory meetings, so that all employees heard • Discussion: Topics are interactively discussed in class the policy from the manager by the instructor and students 2. What is the most important role of the manager in • Role playing: Students assume roles in a given sce- charge? nario; helpful in demonstrating affective behaviors a. Independent decision making • Case study based: Students are provided with patient b. Communication case studies, typically used to facilitate critical think- c. Informal discussions ing in patient diagnoses d. None of the above • Problem-based learning: Students work in groups to 3. Your laboratory was just inspected by CAP. Your solve a problem inspector noted a phase I (lower level) citation that 292 CHAPTER 10 Laboratory Operations

the hematology laboratory space is quite small. What 14. Rewards such as a reserved parking spot or the abil- action should the laboratory take? ity to attend conferences may be used to recognize a. Submit a written plan to CAP outlining that the employee accomplishments. laboratory will reorganize the space to meet the a. True needs of the department b. False b. Immediately begin remodeling the laboratory, 15. Which of the following is a benefit of teams in the because this type of citation must be corrected workplace? within 30 days a. Sense of accomplishment c. Ignore the phase I citation, because it is only a b. Increased communication recommendation c. Relief for employees d. Reply to the CAP that space issue is not their d. All of the above concern 16. Which of the following is a category for justification 4. The role of NAACLS is to: of capital expenditures? a. Accredit laboratories a. Replacement b. Offer certification examinations b. Cost reduction c. Offer continuing education c. New equipment d. Accredit educational programs d. All of the above 5. COLA is similar to which of the following agencies? 17. Which of the following government regulations a. CAP apply standards for employers to apply for medical b. TJC leave of absence: c. AABB a. CAP d. ASCLS b. FMLA 6. Your laboratory is going to begin offering drug testing c. CLMA to local employers. While setting up the laboratory, d. CLSI you will likely work with which of the following agen- 18. You are the lead chemistry MLS. Your laboratory cies for accreditation of the laboratory? manager has asked you to evaluate two new methods a. COLA for cholesterol analysis. In your evaluation, you b. SAMHSA found that method A was very accurate and precise c. AABB and that method B was not very accurate and precise. d. CLSI However, the laboratory will make more money by Match the following three agencies with the correct application investing in method B. Which of the following deci- they oversee. sions would exhibit professionalism? 7. __ CMS a. Recommend method A to your laboratory man- 8. __ FDA ager. It is important that the laboratory produce 9. __ DOT the most accurate and precise results a. Regulates shipments of human specimens b. Recommend method B to your laboratory man- b. Approves new testing procedures and methods ager. It is important that the laboratory make as c. Regulate laboratory testing via CLIA much money as possible 10. Job descriptions can be viewed as a summary of the c. State that you are unable to make a recommenda- findings obtained from a job analysis. tion, because no difference in the methods a. True was noted b. False d. Recommend that you need more time to evaluate 11. Which of the following items should not be con- both methods tained within a job advertisement? 19. The three Rs of customer service include results, rela- a. Name of organization tionships, and reliability. b. Starting salary a. True c. Position title b. False d. Certification/licensure requirements 20. Legal personnel issues in the clinical laboratory 12. Which of the following qualifications is(are) neces- include all of the following except: sary to be an effective evaluator? a. Medical leaves a. Knowledge of job and work being done b. Reimbursement b. Proximity to the person being judged c. Termination procedures c. Time to conduct the review d. Interview questions d. All of the above 21. Which of the following regulates overtime pay? 13. There is no relationship between an evaluation a. NLRB instrument and the job description. b. FLSA a. True c. EPA b. False d. FMLA CHAPTER 10 Laboratory Operations 293

22. A hospital is reimbursed under the Medicare DRG 31. The Clinical Laboratory Improvement Act of 1988 program according to which of the following? applies to which of the following? a. Amount of service provided a. All clinical laboratories b. Costs incurred in providing care b. Hospital laboratories only c. Nature of the illness c. Laboratories engaged in interstate commerce d. Profit margin of the laboratory only 23. Which statement describes the appropriate schedul- d. Physician office laboratories only ing process? 32. What is the purpose of a compliance plan? a. Matching the people presently working in the lab- a. Reduce fraud and abuse billing practices oratory with current workload requirements b. Monitor employee compliance to safety practices b. The setting of long-term goals and objectives for c. Train employees on customer satisfaction the number and types of personnel needed to meet d. Ensure fairness in scheduling the labor requirements of the laboratory 33. Which organization publishes guidelines for writing c. Allow a democratic approach and have the staff technical procedures that are usually acceptable to schedule when they want to work agencies that govern laboratory operations, such as d. Placing all of the experienced personnel on CAP and COLA? one shift a. AABB 24. Which of the following is a goal of an interview? b. CLSI a. Establish social contacts c. OSHA b. Evaluate applicant’s skills and personality d. ASM c. Discuss the religious background of the 34. Which of the following arrangements offers the most applicant instrument service coverage and “piece of mind” for d. Discuss prior arrests of the applicant laboratory managers? 25. Annual budget assumptions are based on a. Pay as needed for time and materials ______. b. Establishing a service contract for key a. Estimated direct costs instruments b. Estimated indirect costs c. Fingers crossed that nothing breaks down c. Projected reimbursement and revenue d. Order new instruments when the warranty runs d. All of the above out on the old one 26. Ambulatory Payment Category (APC) codes are used 35. Given the following information, calculate the for reimbursement purposes for which of the needed FTEs. following? Standard unit: 0.5 a. Inpatients Number of billable tests in 14 days: 10,000 b. Medicare inpatients a. 62.5 c. Outpatient and emergency room patients b. 125 d. None of the above c. 250 27. According to most managed care plans, the type of d. None of the above reimbursement similar to federal systems is: 36. Productivity can be calculated by which of the a. Capitation following? b. Per diem a. Dividing actual hours worked by earned hours c. Per case and multiply by 100 d. Carve out b. Dividing earned hours by actual hours and multi- 28. Budgets should be reviewed ______to identify ply by 100 budget variances. c. Calculating the number of people on your staff a. Weekly and dividing by the number of shifts b. Monthly d. Comparing the number of radiology technicians c. Quarterly in comparison to laboratory technicians d. Annually 37. Legal institutional issues in the clinical laboratory 29. When calculating the costs per billable tests, the total include all of the following except: test volume in the laboratory is not needed. a. Confidentiality a. True b. Job announcements b. False c. Procedures 30. Which of the following agency(ies) developed the d. Chain of custody Medical Compliance Plan (MCP)? 38. PRN employees are useful in the scheduling process a. CAP because they help to decrease overtime and increase b. TJC flexibility. c. HHS OIG a. True d. NAACLS b. False 294 CHAPTER 10 Laboratory Operations

39. When using the 8 and 80 payroll regulation and 45. Which of the following methods would be most help- guidelines, are employees paid overtime when they ful during the interview to assess an applicant’s abil- work more than 8 hours in one day? ities to resolve problems in a mature manner? a. Yes, the employees would be paid overtime a. Read a scenario and ask the applicant how he or b. No, the employees would not be paid overtime she would react if in the situation because they must have worked more than b. Directly ask the applicant if he or she is mature 80 hours also c. Observe the applicant during the interview c. It depends on how the employees desire to be paid d. There is no way to assess these abilities during the d. The employees are not paid overtime regardless interview process of how many hours are worked 46. An educational learning objective must: 40. You are in charge of planning the schedule for the a. Be written in broad terms day shift. Your laboratory manager asks that you b. Not be measurable identify three ways to reduce the labor costs for that c. Be provided after the educational unit ends shift. Of the following, which is not a way to reduce d. Explain exactly what the student should be able the costs? to do a. Use PRN employees to reduce overtime costs 47. The cognitive learning domain includes which of the b. Switch to a 40-hour work week following? c. Schedule your most educated and trained a. Evaluation employees during first shift b. Demonstration d. Use a mix of highly educated employees and rel- c. Attitudes atively new employees d. Motor skills 41. As part of the Blood-borne Pathogens Plan, 48. Being described as a team player falls primarily employers must offer the hepatitis B vaccine to all within which learning domain? employees at the expense of the clinical laboratory a. Cognitive (or organization). b. Psychomotor a. True c. Affective b. False d. Level III 42. Which of the following components should not be 49. Which of the following learning objectives is appro- included in a written procedure? priately written? a. Test method a. The student will understand how to perform b. Cost per billable test urine microscopy c. Quality control procedures b. The student will be able to accurately list the steps d. Reagents and media needs in performing a venipuncture 43. Which of the following agencies has the inspection c. The student will be aware of professional authority for laboratory accreditation? behavior a. CAP d. The student will not make mistakes b. TJC 50. An examination question that includes a comprehen- c. State health departments sive list of laboratory tests from a given patient and d. All of the above requests the examinee to make a diagnosis is an 44. Which of the following questions can be legally example of a question that belongs to which taxo- asked during an interview? nomic level? a. What is your date of birth? a. Level I b. Where have you previously worked? b. Level II c. Do you have any dependents? c. Level III d. Do you have any long-term health problems? d. Level IV CHAPTER 10 Laboratory Operations 295

BIBLIOGRAPHY

American Association for Clinical Chemistry: Communicating the Jones S: Clinical laboratory pearls, ed 1, Philadelphia, Pa, 2000, laboratory’s value to health care: an interview with Rodney Lippincott Williams & Wilkins. Forsman, Clin Lab News: 12–13, 2004. Lewin K, Lippitt R: An experimental approach to the study of autoc- Cohen C, Cohen SL: Lab dynamics: management skills for scientist, racy and democracy: a preliminary note, Sociometry ed 1, New York, 2005, Cold Spring Harbor Laboratory Press. 1:292–300, 2003. Drucker P: The practice of management, ed 1, New York, 1993, de Kieviet W, Frank E, Stekel H: Essentials of clinical laboratory Harper Business. management in developing regions, 2008, Committee on Garcia L: Clinical laboratory management, ed 1, Washington, DC, Clinical Laboratory Management, Education and Management 2004, ASM Press. Division, International Federation of Clinical Chemistry. Harmening DM: Laboratory management: principles and processes, Hofstede G: “Cultures and Organizations: Software of the ed 2, St. Petersburg, Fla, 2007, D.H. Publishing Consulting. Mind,”Admin Sci Quart (Johnson Graduate School of Man- Hudson J: Principles of clinical laboratory management: a study agement, Cornell University) 38(1):132–134, March 1993, guide and workbook, ed 1, Upper Saddle River, NJ, 2003, Pren- JSTOR 2393257. tice Hall.

CLINICAL LABORATORY MANAGEMENT ASCLS: http://www.ascls.org/ WEBSITES NILA: http://www.aab.org AAB http://www.aab.org/ CMS: http://www.cms.hhs.gov/ AMT: http://www.amt1.com/ CLIA application: http://www.cms.gov/Medicare/CMS- Forms/CMS-Forms/Downloads/CMS116.pdf Continuing Education

Credentialing Agencies MTS: http://www.medtraining.org/ Body of Knowledge: http://www.clma.org/?page¼BOK_ CAP: http://www.cap.org/apps/cap.portal Logo ASCP: http://www.ascp.org/ CE organizer: http://ceorganizer.ascls.org/ TJC: http://www.jointcommission.org/ COLA: http://www.cola.org/ NAACLS: http://naacls.org/ Government and Legislative ASHI: http://www.ashi-hla.org/ Library of Congress: http://thomas.loc.gov/ AO: http://www.osteopathic.org/Pages/default.aspx House of Representatives: https://writerep.house.gov/ DNV: http://dnvaccreditation.com/pr/dnv/default.aspx writerep/welcome.shtml Senate: http://www.senate.gov/general/contact_ Laboratory Resource Sites information/senators_cfm.cfm SaveOurLabs: http://www.saveourlabs.com/ CLSI http://clsi.org/ A2LA: http://A2la.org POCT: http://www.pointofcare.net/ Quality Control Reference NIT: http://www.nih.gov/ Westgard Rules: http://www.westgard.com/ LOINC: /https://loinc.org/ SNOMED: http://www.ihtsdo.org/ Lab Tests Online: http://labtestsonline.org/ Human Resources Reference Labs Are Vital: http://www.labsarevital.com/ Performance reviews: http://performancesolutions.com/ Results for Life: http://www.labresultsforlife.org/

Professional Organizations and Associations

CLMA: http://clma.org/ AACC: http://aacc.org/AACC/ ASM: http://www.asm.org 296 CHAPTER 10 Laboratory Operations

SELF-ASSESSMENT

Content Area: ______

Score on Practice Questions: ______

List the specific topics covered in the missed questions:

List the specific topics covered in the correct questions: CHAPTER 10 Laboratory Operations 297

NOTES CHAPTER 11 Laboratory Calculations

Melanie J. Giusti and Mark W. Ireton

SIGNIFICANT FIGURES AND ROUNDING • If the left-most digit removed is less than 5, the final retained digit remains the same • Measured numbers include a certain amount of cer- • If the left-most digit to be removed is 5, the final tainty and uncertainty retained digit is increased if it is odd, or it stays • The uncertainty is in the digit furthest to the right, the same if it is even and it reflects the precision of the measuring instru- • Only measured numbers determine significant figures ment and the skill of the person using it • Exact, counted, or ideal numbers do not determine • The significant figures in a measured number include significant figures. For example, 1000 mg in 1 g is every digit except nonmeasured zeroes that only hold an exact amount the decimal place • Counted numbers, such as 5 nucleated RBCs on a • Table 11-1 shows examples of numbers and the num- blood smear, or the valence of a molecule, also do ber of significant figures in each one not affect significant figures • When making calculations, the final answer is rounded • When a practice problem asks to make a 1-L solu- off to reflect the value with the least certainty (the few- tion, that volume does not affect significant figures est significant figures) because it is a theoretical amount, with no indica- • For multiplication and division, the answer must have tion of how it would be measured the same number of significant figures as the starting • Numbers are rounded only after all calculations are value with the least number of significant figures complete. Rounding before calculations are com- • For example, multiplying 2.5 g/L by 0.5250 L plete introduces inaccuracies that can become exag- results in a product of 1.3125 g. However, this must gerated in further calculations be rounded to 1.3 g because 2.5 g/L has only two significant figures • For addition and subtraction, the answer must have the same number of decimal places as the starting EXPONENTIAL NOTATION value with the fewest number of decimal places • Very large and very small numbers are written in expo- • For example, the sum of 15.5 mL of solution plus nential notation—usually one to three digits to the left 25.92 mL of solution (measured with a more pre- of the decimal place and a multiplier based on 10x cise instrument) equals 41.42 mL. This number • When this exponential x is a positive integer, the must be rounded to 41.4 mL because 15.5 mL has number is multiplied by 10 x-number of times only one decimal place • When the exponential x is a negative integer, the • When rounding numbers number is divided by 10 x-number of times • If the left-most digit removed is greater than 5, • In other words, if the exponential form is written out round up the final retained digit by one in standard notation, the decimal point is moved x number of digits to the right when x is positive Example Numbers and Their and x number of digits to the left when x is negative TABLE 11-1 Significant Figures • Example ○ 2.5 102 ¼2.5 10 10: Measurement Significant Figures Move the decimal point two digits to the 101right¼250 10.2○ 2.5 10 2 ¼2.5 10 10: 10.0 3 Move the decimal point two digits to the 35 2 left¼0.025 0.35 2 • In mathematical operations involving exponential 0.0035 2 0.00350 3 notation, the significant digits and the exponential powers are handled separately

298 CHAPTER 11 Laboratory Calculations 299

• In multiplication, the exponents are added • Example • In division, the exponents are subtracted ○ What is 20 mg converted to micrograms? • Example § Answer: Knowing that a gram is equivalent to 103 mg, and that a gram is also equivalent to 3 104 5 105 106 mg, use the factor-label method to solve for micrograms ○ Answer: The product of the significant digits is g 106 mg 15. The sum of the exponents is 9. Therefore 20 mg ¼ 20 103 mg the answer is 15 109 or 1.5 1010 103 mg g • Example 4 103 2 105 TEMPERATURE CONVERSIONS • Temperatures are measured in degrees Celsius (C), ○ Answer: The quotient of the significant digits is degrees Fahrenheit (F), or Kelvins (K) 2. The exponent in the denominator is subtracted • In the medical laboratory, the Celsius scale is most from the exponent in the numerator. Therefore often used 2 the answer is 2 10 • Formulas used to convert between these scales • If it is necessary to add or subtract numbers in expo- • F¼9/5 C+32 nential notation, convert the exponential number to • C¼5/9 (F 32) standard notation before making calculations, then • K ¼ C+273 convert back to exponential notation when finished • To use any of these equations, insert the known values to solve for the unknown values UNITS OF MEASURE • Example ○ 99.2 F equals how many degrees Celsius? Metric System § Answer: Insert the known Fahrenheit value into the equation for Celsius, and solve • The metric system is used for measuring size, volume, and weight C ¼ 5=999ðÞ:2 32 • Very large and small amounts are modified by prefixes C ¼ 5=967ðÞ:2 based on factors of 10 C ¼ 37:3 • For example, “kilo” is equal to 1000 units or 1 3 10 , so there are 1000 meters per kilometer, or § Hint 3 10 kilometers per meter (Table 11-2) ○ If in doubt about correctly remember- • Knowing these values, it is possible to convert between ing the Celsius/Fahrenheit conversion units using the factor-label method formulas during an examination, it • Example is possible to double-check the correct- ○ How many grams are in 2 kg? ness of the equation by using the § Answer: Knowing that a kilogram is equiva- known freezing and boiling points of 3 3 lent to 10 g, multiply 2 kg by 10 g/kg and water: 0 Cor32 F and 100 Cor cancel the kilogram units 212 F, respectively ○ 103g Insert either the Celsius or Fahrenheit 2kg ¼ 2 103g or 2000g kg boiling point or freezing point of water into a formula, and if the answer is correct then so is the formula TABLE 11-2 Metric System

Prefix Factor FACTORS AND DILUTIONS 6 mega- (M) 1,000,000 or 1 10 Dilutions and Ratios kilo- (k) 1,000 or 1 103 1 deci- (d) 0.1 or 1 10 • A dilution can be expressed as a fraction of specimen 2 centi- (c) 0.01 or 1 10 volume divided by the sum of specimen volume and milli- (m) 0.001 or 1 103 diluent. The resulting fraction is the dilution (or dilu- micro- (m) 0.000001 or 1 106 nano- (n) 0.000000001 or 1 109 tion factor) 12 pico- (p) 0.000000000001 or 1 10 Specimen volume Specimen volume femto- (f) 0.000000000000001 or 1 1015 Dilution ¼ ¼ Specimen volume + diluent volume Total volume 300 CHAPTER 11 Laboratory Calculations

• Example ○ 10 mL of saline is added to 10 mL of blood. What is the dilution? § Answer: Divide the volume of blood (10 mL) by the total volume contained § This is a ½ dilution 10 mL 10 mL 1 Dilution ¼ ¼ ¼ 10 mL + 10 mL 20 mL 2 • Example ○ What is the dilution if 10 mL is diluted to 100 mL? ○ Answer: Divide the sample volume by the total volume 10 L 1 Dilution ¼ m ¼ 100 mL 10 • Example ○ How do you prepare a solution of 20 mL of serum that is diluted to 100 mL, and what is FIGURE 11-1 Example of a dilution series. the dilution? § Determine the volume of diluent by subtracting the specimen volume from the total volume 100 mL 20 mL ¼ 80 mL § Next divide the amount of sample (20 mL) by the total volume (100 mL), which equals 1=5 • If a 1-mL aliquot is taken from tube A and added to § Answer: Add 80 mL of diluent to the 20-mL tube B, this represents a 1=10 dilution in tube B sample, which makes a 1=5 dilution • If 1 mL of the dilution in tube B is added to tube C, • The concentration of a diluted solution is the product and 1 mL of the tube C dilution is added to tube D, of its original concentration times the dilution factor each of these represents a 1=10 dilution • Example • The dilution factors in each tube are ○ 20 mL of a 20-mg/dL reagent is diluted 1=5 ○ Tube A¼Undiluted (neat) ○ What is the final concentration? ○ Tube B¼1=10 § Answer: Multiply the initial concentration by ○ Tube C¼(1=10) (1=10)¼1=100 the dilution factor ○ Tube D¼(1=10) (1=10) (1=10)¼1=1000 • 20 mg 1 4mg The concentration of the solution in any selected tube ¼ is the product of the concentration of tube A times the dL 5 dL dilution factor of the selected tube • Dilutions written as fractions are always considered to be volume of sample per total volume • Ratios can be used to express part-per-part or part-per- CONVERSION AND CORRECTION whole, and are written with a colon between figures FACTORS • In a solution containing 1 mL of serum and 4 mL of saline (total volume 5 mL), the following dilution • Factor: A fraction that can be used to: and ratio relationships exist • Correct a variance in procedure • Dilution is 1/5 • Express a quantity in different terms • Ratio of serum to total volume is 1:5 • Combine calculations • Ratio of serum to saline is 1:4 • The most basic factor is a unit-conversion, which is the • Ratio of saline to total volume is 4:5 basis for the factor-label method • Ratio of total volume to serum is 5:1 • Example • Ratio of saline to serum is 4:1 ○ To convert a volume measured in milliliters to liters, multiply the original figure by a fraction with equivalent numerator and denominator Dilution Series and cancel out the corresponding units 1L • When a diluted solution is diluted again, the final dilu- 450 mL ¼ 0:45 L tion is the product of its dilution factors (Figure 11-1) 1000 mL • Tube A is an undiluted patient sample • To use a factor to correct for a variance in procedure, • Tubes B to D contain 9 mL diluent the equation is: CHAPTER 11 Laboratory Calculations 301

Volume that should be used ○ Measured value ¼ Corrected value Amount per 100 Available sample volume ○ In the clinical laboratory, percent solutions are • In the case of a dilution, the numerator in the previous expressed as grams per 100 mL fraction example would be volume of the diluted ○ Example specimen § 0.9% saline solution contains 0.9 g NaCl per • Example 100 mL water ○ A urine specimen is delivered to the laboratory for a sodium measurement. The procedure calls Molarity for 5.0 mL but the specimen is only 4.0 mL, and acquiring a new specimen is not possible. • Molarity (M) is the number of moles (mol) of solute The medical laboratory scientist at the bench per liter of solution dilutes the specimen with 1.0 mL of water to mol 5.0 mL. The analyzer measures a sodium concen- M ¼ L tration of 44 mEq/L. What is the corrected (actual) concentration of the patient sample? § Answer: To correct for the dilution, multiply the • Example ○ result by the diluted volume (how much should What is the molarity of a solution containing have been used) and divide by the available spec- 3 mol of glucose in 500 mL of water? § imen volume Answer: Use the equation previous and the factor-label method to solve for moles per sin- 5:0mL 44 mEq=L ¼ 55 mEq=L gle liter 4:0mL 3 mol 1000 mL 6 mol • When the dilution factor is known, the corrected M ¼ ¼ ¼ 6M 500 mL L L value can also be found by multiplying the measured result by the inverse of the dilution factor. In the pre- • Example ○ vious example, the dilution factor was 4=5, so the How many moles of glucose are in 400 mL of a measured value can be multiplied by 5=4 for the 5-M glucose solution? § corrected value Answer: Multiply molarity (which is converted from Moles to moles per liter) by volume to solve for moles UNITS OF CONCENTRATION AND 5 mol L PREPARATION OF SOLUTIONS 400 mL 5M¼ 400 mL ¼ 2 mol L 1000 mL Concentration as Parts per Parts and as Percent • Molarity and weight/volume are interchangeable using molecular weight (MW), also called molar mass, • Solution concentration can be expressed proportion- which is based on atomic mass and is equal to grams ally as: per mole • Weight per volume (w/v) g ○ MW ¼ Weight per volume concentrations are often mol expressed as grams per deciliter or milligrams g mol g MW M ¼ ¼ per deciliter mol L L ○ Example § 3.2 g glucose dissolved in 2 dL of water has a • Example concentration or 3.2 g/2 dL or 1.6 g/dL ○ How many moles are in 23.4 g of potassium ○ In a weight per volume solution, addition of sol- chloride? (MW of KCl¼74.5 g/mol) ute does not affect volume (assuming the solu- § Answer: Use the factor-label method to multi- tion is not saturated) ply mass by the inverse of MW to eliminate • Volume per volume (v/v) grams and solve for moles • Weight per weight (w/w) mol ○ The addition of solute adds to the mass of the sol- 23:4g ¼ 0:314 mol 74:5g ute and to the total solution but not to the mass of solvent ○ Example § NOTE: Grams per mole can be inverted § 1 g sodium chloride in 100 g H2O describes a to moles per gram when solving for solution that is 1 g/100 g or 0.01 g/g NaCl moles because proportionally 1 mol of • Number per volume (n/v) KCl represents the same amount as 74.5 g • Percent solution of KCl 302 CHAPTER 11 Laboratory Calculations

• Example of the substance (also known as formula weight) ○ How much water is needed to make a 0.5-M divided by its valence solution from 150 g of NaCl? (MW of • Calculate the number of equivalents in solution, NaCl¼58.5 g/mol) which is the weight of the solute in the sample § Answer: Knowing that 0.5 M equals 0.5 mol/ divided by the gram equivalent weight L, solve for volume by multiplying the weight • Solve for normality with the equation, N¼Eq/L of the sample by the inverse of the molarity by • Equation review the inverse of molecular weight (to isolate MW 1 mol liters in the numerator) Gram equivalent weight ¼ Valence L mol Grams in sample 150 g ¼ 5:1L EquivalentsðÞ¼ Eq 0:5 mol 58:5g Gram equivalent weight Eq N ¼ Molality L • Example • Molality (m) is the number of moles of solute per kilo- ○ What is the normality of a 1-L solution contain- gram of solvent ing 46.3 g Ca(OH)2? (MW of Ca(OH)2 ¼74.1 mol m ¼ g/mol) kg § Answer § • Remember that the denominator is the mass of solvent, Determine the valence to calculate gram equivalent weight. Ca(OH)2 dissociates not of the entire solution • Example: into two OH ions per molecule, so the ○ What is the molality of a solution containing valence is 2. Gram equivalent weight is 5.0 mol glucose and 4.0 kg water? MW 1 mol 74:1g § Gram equivalent weight ¼ ¼ ¼ 37:0g Answer: Insert the measurements into the Valence 2 molality formula and divide to determine § moles per 1 kg Determine the number of equivalents in the sample 5:0 mol 1:2 mol m ¼ ¼ Grams in sample 46:3g 4:0kg kg Eq ¼ ¼ ¼ 1:25 Gram equivalent weight 37:05 g

Normality § Finally, solve for the normality of this • Normality (N) is a term of concentration based on the solution ability of some substances to release hydrogen (H+)or Eq 1:25 Eq N ¼ ¼ hydroxide (OH ) ions in solution L 1L • In biological systems, the number of H+ or OH ions § The normality of this solution can be writ- that are released per molecule in solution is equivalent ten as 1.25 N or 1.25 Eq/L to valence, which is the bonding ability of a molecule • The simplest definition of normality is based on molarity and valence Osmolality N ¼ M Valence • One osmole is equivalent to a mole of substance multiplied by the number of osmotically active particles per mole the substance dissociates into in • Example, Ca(OH)2 dissociates in solution to release two OH ions per molecule, so the valence is 2. solution Therefore the normality of a 1-M solution of Ca • For example, glucose molecules do not dissociate into smaller particles in water, so 1 osmole of glucose (OH)2 is 2 N equals 1 mole of glucose N ¼ M Valence ¼ 1 2 ¼ 2 • NaCl dissociates into one Na+ and one Cl (two • For substances with a valence of 1, normality equals total particles) per mole, so 1 mole of NaCl equals molarity 2 osmoles NaCl • Normality is also defined as equivalents per liter and ○ Osmoles¼Number of moles number of osmot- can be determined given the weight of solute and the ically active particles per mole volume of solution using the following steps: • Osmolality measures concentration in terms of • Determine valence to calculate the gram equivalent osmoles per kilogram of solvent weight of the solute, which is the weight of 1 mole ○ Osmolality¼Osmoles per kilogram CHAPTER 11 Laboratory Calculations 303

• Osmolality is the preferred unit in the clinical labora- ○ How many milliequivalents of CO2 are con- tory for osmometry measurements of electrolytes, tained in the 10-mL collection tube? freezing point depression, and vapor pressure depres- § Answer: Assign values to set up the equation sion. Note that in the abbreviation for milliosmoles, § n1 ¼25 mEq the “O” is capitalized—mOsmol § v1 ¼1L¼1000 mL • Example § n2 ¼Unknown ○ What is the osmolality of 32.7 g NaCl dissolved § v2 ¼10 mL in 2.0 kg water? (MW of NaCl¼58.5 g/mol) ○ Insert these values into the equation and cross- ○ Answer: Multiply the weight of NaCl by the multiply to solve for the unknown number of inverse of its molecular weight to determine milliequivalents (n2) in the 10-mL tube: how many moles are present 25 mEq n1 mol ¼ 32:7g ¼ 0:55897 mol 1000 mL 10 mL 58:5g 25 mEq 10 mL ¼ n ○ NaCl dissociates into two osmotically active par- 1000 mL 1 ticles per molecule in solution 0:25 mEq ¼ n1 ○ Determine number of osmoles Osmoles ¼ Moles Active particles ¼ 0:55897 2 ¼ 1:1179 ○ Solve for osmolality Dilution Equations (Changing osmol 1:1179 osmol Concentrations) Osmolality ¼ ¼ ¼ 0:56 osmol=kg kg 2:0kg • When making a dilution, the volume of the original solution is less than the volume of the diluted solution • The amount of solute remains the same Proportionally Equivalent Solutions • Because volume (V) times concentration (C) equals amount of solute, and the amount of solute remains the • Calculations with two solutions whose concentrations same, the resulting equation can be used to solve for do not change are known as ratio and proportion any one missing factor in this type of dilution problem calculations V C ¼ V C • The general formula for ratio and proportion is 1 1 2 2 w w 1 ¼ 2 • Example v1 v2 ○ What volume of a 0.5-M glucose solution can be • Weight per volume (w/v) is most common, but weight made from 100 mL of a 3-M glucose stock per weight (w/w), volume per volume (v/v), number solution? per volume (n/v), and other combinations are also § Answer: Assign values for the equation ¼ used § V1 100 mL § ¼ • Example C1 3M § ¼ ○ If a 5.0-dL solution contains 4.5 mg of bilirubin, C2 0.5 M § ¼ how much bilirubin would there be in a 2.0-dL V2 Unknown aliquot of the solution? ○ Divide both sides of the equation ¼ § Answer: Assign values to set up the equation V1 C1 V2 C2 by C2 to isolate V2, then insert § w1 ¼4.5 mg the known quantities and solve for V2 § ¼ v1 5.0 dL V C 100 mL 3M § ¼ V ¼ 1 1 ¼ ¼ 600 mL w2 Unknown 2 C 0:5M § v ¼2.0 dL 2 2 ○ ○ Insert these values into the equation and cross- Note that in this problem there is no need to convert M to moles per liter because these units can- multiply to solve for the unknown mass (w2)in the 2.0-dL aliquot cel each other • 4:5mg w Example ¼ 2 ○ 5:0dL 2:0dL A 3.5-L solution was diluted with 1.5 L of water to create a 0.5-M solution with a total volume of 4:5mg 2:0dL ¼ w 5.0 L 5:0dL 2 ○ What was the concentration of the original 1:8mg¼ w2 solution? • Example § Answer: Assign values for the equation ○ The total carbon dioxide concentration of a § V1 ¼3.5 L serum sample is 25 mEq/L § C1 ¼Unknown 304 CHAPTER 11 Laboratory Calculations

§ ¼ L+H 2+8 V2 5.0 L M ¼ ¼ ¼ 5 2 2 § C2 ¼0.5 M ○ Divide both sides of the formula V1 C1 ¼V2 C2 by V1 to solve for C1, which represents the concentration of the original Measures of Variability solution • Range (R) • The difference between the highest value and the V2 C2 5:0L 0:5M C1 ¼ ¼ ¼ 0:7M lowest value in a sample set V1 3:5L R ¼ H L STATISTICS AND QUALITY ASSESSMENT • Not often used, but an easy calculation for quick assessment of the comparative precision of Measures of Center test runs ○ A larger range indicates decreased precision • Mean • Variance (SD2 or s2) and standard deviation (SD or s) • The mean (x) is the average of the individual values • First, determine the mean of the sample set (x or xi) in the sample set • Next, a data table is created • Calculated by dividing the sum (S) of all the individ- ○ Each x value is placed in the first column ual values by the number (n) of values in the set ○ The mean subtracted from each x value is in the X second column (x x) x ○ x ¼ The square of the difference between each x n and the mean is listed in the third column • Example [(x x)2] ○ What is the mean of the following data set: 2.5, ○ The sum of the values in the third column is 3.1, 4.3, 4.0, 5.2, 4.7, 4.8? inserted into the equation for variance § Answer: Divide the sum of the values by the X 2 total number of values ðÞx x Variance ¼ SD2 ¼ 28:6 n 1 ¼ ¼ 4:1 x 7 • Example • Median ○ For the data set {2.4, 2.6, 2.8, 3.3, 3.6, 3.9}, the • The number in the middle of an ordered data set number of values (n) is 6. The mean is ○ Place the data set in numerical order ○ If an odd number of values, select the number in X x 18:6 the middle x ¼ ¼ ¼ 3:1 ○ If an even number of values, the median is the n 6 average of the two middle numbers ○ • Example Create the data table as described previously ○ What is the median of the following data set: 1, using each x value and the mean 2 2, 3, 9, 10, 12, 14 x(x x)(x x) § Answer: The median is 9 2.4 0.7 0.49 • Example 2.6 0.5 0.25 2.8 0.3 0.09 ○ What is the median of the following data set: 2, 3.3 0.2 0.04 0, 2, 4,5,8 3.6 0.5 0.25 § Answer: The median is (2+4)/2¼3 3.9 0.8 0.64X • 2 Mode ðÞx x ¼ 1:76 • The most frequently found number(s) in a data set ○ • Example Solve for variance X ○ In the data set {2, 5, 5, 5, 7}, the mode is 5 2 ðÞx x 1:76 ○ In the data set {1, 4, 4, 4,8,9, 9, 9, 15, 20}, the SD2 ¼ ¼ ¼ 0:352 1 5 modes are 4 and 9 n ○ • Midrange (M) Standard variation is, by definition, the square • The average of the lowest (L) and highest (H) values root of variance in a data set sXffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 • Example pffiffiffiffiffiffiffi ðÞx x pffiffiffiffiffiffiffiffiffiffiffi Standard deviation ¼ SD ¼ 2 ¼ ¼ 0:352 ○ In the data set {2, 3, 5, 6, 8}, what is the midrange? SD n 1 § Answer ¼ 0:593 CHAPTER 11 Laboratory Calculations 305

Coefficient of Variation ○ Patient does not have the disease, but the test results are positive • To use standard deviation to compare the precision of • False negative (FN) one assay to the precision of another assay, the relative ○ Patient has the disease, but the test results are differences in the size of values between the different negative assays must be normalized • Clinical sensitivity • The standard deviation of an assay is divided by the • Total number of patients with the disease can be mean of its results defined as the sum of the true positives and false • The resulting quotient is the coefficient of variability negatives (CV), which is expressed as a percent • Proportion of true positive test results to the total SD patients with the disease CV ¼ 100% x True positives TP Sensitivity ¼ ¼ 100 • The assay with the lower CV value is the more precise Total diseased patients TP + FN % assay • The sensitivity describes the ability to detect the tar- • Example get analyte even in low concentrations ○ Using the figures from the earlier section on stan- • A highly sensitive test can detect disease even when dard deviation, the CV for that assay would be the disease marker is present in small amounts, thus 0:593 CV ¼ 100% ¼ 19:1% preventing false negatives 3:1 • A test with poor sensitivity has many false negatives ○ If another assay has a standard deviation of because the test often misses the marker for the dis- 0.235 and a mean of 2.12, its CV would be ease in specimens 0:235 • Clinical specificity CV ¼ 100 ¼ 11:1 2:12 % % • The total number of patients who do not have the disease is the sum of the true negatives and false ○ The second assay is more precise because its CV positives is lower • Proportion of the true negative test results to the total number of patients who do not have the disease Accuracy and Precision True negatives TN Specificity ¼ ¼ 100 Total patients without disease TN + FP % • Accuracy • Closeness of an individual result to the true value • Describes the ability to correctly detect only the • In terms of the multiple results in a test procedure, intended marker of disease, not similar analytes that accuracy can be judged in terms of measures of center may be present ○ Example: An assay whose mean and/or median • A highly specific test will have few false positives are close to the actual result would be considered because only the specific analyte of disease is accurate detected • Precision • A test with poor specificity will have many false • The reproducibility of a test procedure positives because it will incorrectly interpret benign • Can be evaluated in terms of the measures of analytes as markers of disease + variability • Positive predictive value (PPV or PV ) ○ Example: An assay with a low standard devia- • Indicates the degree of certainty that positive or tion or coefficient of variability can be consid- negative test results are correct ered a precise assay • Proportion of the patients with positive results who actually have disease • Calculated by dividing the true positives by all pos- Sensitivity, Specificity, and Predictive itive test results Values True positives TP PPV ¼ ¼ 100% • Four terms that correlate a patient’s disease state to the Total positives TP + FP test results • Indicates the certainty that a positive test result is • True positive (TP) correct ○ Patient has the disease and correctly tests positive • Negative predictive value (NPV or PV ) of a test is the • True negative (TN) proportion of the patients with negative results who ○ Patient does not have the disease and correctly are actually disease free tests negative • Calculated by dividing the true negatives by all neg- • False positive (FP) ative test results 306 CHAPTER 11 Laboratory Calculations

True negatives TN • NPV ¼ ¼ 100% The area under the curve represents the sum of all Total negatives TN + FN the frequencies of the numbers in the sample, and • Example the total sum is 1 ○ Determine the sensitivity, specificity, and predic- • The normal curve is useful in the clinical laboratory tive values in a new assay to detect influenza when the units on the x-axis are SD on either side of infection was developed and tested on 500 peo- the mean ple; 200 people tested positive for the flu. Of • In a normal curve these 200 positives, 185 were confirmed as being • 68% of all numbers will fall between 1 SD and +1 infected using the reference method. Of the SD of the mean remaining 300 people who tested negative, 6 • 95% will fall between 2 SD and +2 SD were later shown to be infected with influenza • 99% will fall between 3 SD and +3 SD § Answer • The values that fall within 2 SD of the mean are § First, define basic information considered normal and within the 95% confidence ○ Of the 200 total positive test results, 185 interval were shown to be true positive results, • The values outside 2 SD but within 3 SD of the with the remaining 15 patients with mean are questionable positive results actually having false pos- • The Levey-Jennings chart itive results • Horizontal graph of quality control data ○ Of the 300 negative test results, 294 were • X-axis of the normal curve is converted to the ver- confirmed as true negatives and the re- tical axis maining 6 were shown to be false negatives ○ Median flanked by 3 positive standard deviation ○ The total number of patients with dis- increments above and 3 negative standard devia- ease is the sum of the true positives tion increments below and false negatives: 191 ○ Data points for each day plotted horizontally ○ The total number of patients who were ○ Once the mean and standard deviations for an disease free is the sum of the true nega- assay are determined, the Levey-Jennings chart tive and false positive results: 309 provides an easy-to-read visual representation ○ True positive (TP)¼185 of quality control data generated over time ○ False positive (FP)¼15 • Westgard Multirule set ○ True negative (TN)¼294 • Monitor quality control performance is based on ○ False negative (FN)¼6 control rules § Next, use the previous equations to solve • Refer to Figure 11-2 for examples listed parenthet- for sensitivity, specificity, PPV, and NPV ically in the following list TP 185 ○ 1 : One control exceeds 2 SD of the mean sensitivity ¼ 100 ¼ 100 ¼ 96:8 2s TP + FN % 191 % % (day 1, level 1) TN 294 ○ 13s: One control exceeds 3 SD of the mean; specificity ¼ 100 ¼ 100 ¼ 95:1 TN + FP % 309 % % indicative of random error (day 2, level 1) ○ TP 185 22s: Two consecutive controls are either +2 SD or PPV ¼ 100 ¼ 100 ¼ 92:5 TP + FP % 200 % % 2 SD of the mean; indicative of systematic error TN 294 (day 4, levels 1 and 2; or day 4 and 5, level 1) NPV ¼ 100 ¼ 100 ¼ 98:0 ○ R : Combination of one control +2 SD of TN + FN % 300 % % 4s the mean and another 2 SD of the mean; indicative of random error (day 6, levels 1 and 2) ○ QUALITY CONTROL 41s: Four consecutive controls exceed either +1 SD or 1 SD of the mean; indicative of system- Normal Distribution atic error (days 7-10, level 1; or days 8-9, levels 1and2) • In any large sample of random data, if the data points ○ 10x: 10 consecutive controls falling on one side on the x-axis are plotted against frequency on the or the other of the mean; indicative of systematic y-axis, a bell-shaped curve will form, with its peak error (no example illustrated) coinciding on the x-axis with the mean of the sample • Whether violation of these rules constitutes a warn- • Normal (Gaussian) distribution ing or a rejection depends on individual laboratory • Curve is symmetrical around the vertical line that policies passes through the mean on the x-axis, and the curve • Some patterns in quality control results are defined approaches but never touches the x-axis—is consid- more qualitatively ered a normal, or Gaussian, distribution • Shift CHAPTER 11 Laboratory Calculations 307

Level 1 Control 2. How many liters are in 3 mL? a. 0.003 +3SD b. 0.000003 +2SD c. 0.0000003 d. 0.000000003 +1SD 3. How many micrograms are in 10 mg? Mean a. 100 −1SD b. 1000 −2SD c. 10000 −3SD d. 100000 4. How many grams are in 0.85 kg? 12345678910 a. 8.5 b. 85 Level 2 Control c. 850 d. 8500 +3SD 5. 25 C is equivalent to how many degrees Fahrenheit? +2SD a. 31.7 +1SD b. 45.9 c. 77 Mean d. 102.6 − 1SD 6. 80 F is equivalent to how many Kelvin? −2SD a. 285.4 −3SD b. 299.7 c. 353.0 12345678910 d. 359.4 FIGURE 11-2 Examples of Levey-Jennings chart used for 7. How would you prepare a 1=5 dilution of a urine evaluation of quality control data. sample? a. 1 part urine+3 parts diluent b. 1 part urine+4 parts diluent ○ Six or more control values fall on the same side of c. 1 part urine+5 parts diluent the mean d. 1 part urine+6 parts diluent ○ The distance from the mean does not matter 8. A 50-g/L solution was diluted 1:5. This diluted sam- ○ The problem does not necessarily grow ple was then diluted 1:10. What is the concentration steadily worse of the final solution? ○ A shift represents that a new mean has been a. 0.8 g/L established b. 1 g/L • Trend c. 2.9 g/L ○ Six or more consecutive control values get con- d. 3.3 g/L tinuously larger or smaller (i.e., a linear direction 9. How much diluent is needed to prepare 300 mL of of values) a 0.2-M working solution from a 0.8-M stock ○ Can move away from, toward, or across solution? the mean a. 75 mL ○ Represents a progressively worsening problem b. 225 mL • Dispersion c. 900 mL ○ Uneven distribution of control values outside 1 d. 1200 mL SD but within 3 SD of the mean 10. What is the molarity of a solution containing 2 mol ○ Increased dispersion represents loss of precision of sodium in 400 mL of water? without loss of accuracy a. 0.005 M b. 0.5 M CERTIFICATION PREPARATION QUESTIONS c. 5 M d. 50 M For answers and rationales, please see Appendix A. 11. How many moles of glucose are in 300 mL of a 2-M 1. How many liters are in 4 dL? glucose solution? a. 400 a. 0.6 b. 40 b. 6 c. 0.4 c. 60 d. 0.04 d. 600 308 CHAPTER 11 Laboratory Calculations

12. How many grams of NaCl are required to make 20. Using the data in Box 11-1, what is the mode of cho- 0.50 L of a 1.5-M NaCl solution? (MW of lesterol results? NaCl¼58.5 g/mol) a. 150 a. 4 b. 165 b. 19.5 c. 168 c. 44 d. 187 d. 175.5 21. Using the data in Box 11-1, what is the standard 13. What volume of diluent is in a 4-M solution contain- deviation of the cholesterol results? ing 125.6 g of KCl (MW¼74.5 g/mol)? a. 13.8 a. 0.42 L b. 15.1 b. 2.37 L c. 21.2 c. 6.74 L d. 26.1 d. 421 L 22. What is the midrange of the following data set: 10, 14. What is the normality of a 2-L solution of 54.2 g 4, 6, 7, 12, 9, 14? H2SO4 (MW¼98.0 g)? a. 7 a. 0.28 N b. 8.8 b. 0.56 N c. 9 c. 0.91 N d. 9.5 d. 1.11 N 23. Which of the following is correct when rounding 15. How many grams of NaCl (MW¼58.5 g/mol) are in 2.25 to one decimal place? a 0.67-osmol/kg solution that was made from 3.0 kg a. 2.2 of water? b. 2.3 a. 39.4 24. What is the sum of the following figures: b. 59.1 0.125+3.45+32.981? c. 102.2 a. 36.556 d. 236.4 b. 36.55 16. In the calculation of the mean, what does “n” represent? c. 36.56 a. The sum of the values d. 36.6 b. The number of values in the set 25. Which of the following represents the product of c. The average of the values (4 103)(6 102)? d. The middle number of the set a. 10 105 17. What is the mode a reflection of in a data set? b. 10 106 a. The average of the individual values in the data set c. 24 105 b. The most frequent number in the data set d. 24 106 c. The average of the lowest and highest numbers in 26. The closeness of a test value to the actual value the data set describes which of the following? d. The number in the middle of the date set a. Accuracy 18. Which of the following statistics is equivalent to the b. Precision square root of the variance? c. Reproducibility a. Coefficient of variation d. Reliability b. Standard deviation 27. Which of the Westgard rules is violated in the control c. Sensitivity data below? (Figure 11-3) d. Specificity a. 22s Use the data in Box 11-1 to answer questions 19 to 21. b. R4s c. 41S BOX 11-1 Cholesterol Results for 10 Patients d. 10x

180 150 150 +3SD 200 165 168 150 205 145 +2SD 170 +1SD Mean 19. Using the data in Box 11-1, what is the mean of the −1SD cholesterol results? −2SD a. 150 − b. 168 3SD c. 187 d. 189 FIGURE 11-3 Quality control data. CHAPTER 11 Laboratory Calculations 309

28. Which of the following Westgard rules is indicative BIBLIOGRAPHY of random error? a. 1 Bettelheim FA, March J: Introduction to organic & biochemistry, 3S ed 3, Fort Worth, Tex, 1998, Harcourt Brace College. b. 22s Burtis CA, Ashwood ER, Bruns DE: Tietz fundamentals of clinical c. 41s chemistry, ed 6, St. Louis, 2008, Saunders. d. 10x Campbell JB, Campbell JM: Laboratory mathematics: medical and 29. Which of the following pairs of concepts are cor- biological applications, ed 5, St. Louis, 1997, Mosby. rectly matched? Davidsohn I, Henry JB, editors: Todd-Sanford clinical diagnosis by laboratory methods, ed 14, Philadelphia, 1969, W.B. a. Sensitivity: prevent false negatives :: specificity: Saunders. prevent false positives Pauling L, Pauling P: Chemistry, San Francisco, 1975, W.H. b. Sensitivity: prevent false positives :: specificity: Freeman. prevent false negatives c. Sensitivity: precision :: specificity: accuracy d. Sensitivity: coefficient of variation :: specificity: standard deviation 30. Which of the following equations represents the clinical sensitivity of a test? TP a. TP + FN 100% TN b. TN + FP 100% TP c. TP + FP 100% TP d. TP + FP 100% 310 CHAPTER 11 Laboratory Calculations

SELF-ASSESSMENT

Content Area: ______

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List the specific topics covered in the missed questions:

List the specific topics covered in the correct questions: CHAPTER 11 Laboratory Calculations 311

NOTES APPENDIX A Answers and Rationales to Certification Preparation Questions

CHAPTER 1 5. d. Transduction refers to moving genetic informa- 1. a. Some bacteria form thick-walled structures tion from one prokaryote to another via a bacte- termed spores. These structures are formed during riophage or virus. a process that makes a copy of a chromosome and encloses it with a thick protein coat. This spore is 6. c. The Gram stain procedure involves four steps: resistant to heat, cold, drying, most chemicals, applying crystal violet—the initial purple dye that and boiling. Spores can remain viable for many enters the cell; applying Gram’s iodine—the mor- years in soil or dust. dent or chemical that binds to the crystal violet forming a complex; decolorizing—alcohol or ace- 2. c. Genus and species are italicized or underlined. tone/alcohol is used to remove the crystal violet/ Genus is capitalized; species is lowercase. iodine complex from gram-negative cells; and safranin counter stain—the red dye is used as a second stain. Gram-positive bacteria retained the 3. b. Bacterial cells generate stored energy in the form of initial crystal violet, the red counter stain blends ATP by one of two basic mechanisms: respiration with the violet, and gram-negative bacteria are or fermentation. Fermentation is metabolism in only stained with the safranin and appear red. the absence of oxygen, an anaerobic process. In fermentation, glucose is converted into pyruvate by 7. d. Although many different bacteria can be isolated either the Embdem-Meyerhof pathway or glycoly- from skin and soft tissue infections, the two most sis. Pyruvate can then enter several other cycles, common organisms are Staphylococcus aureus and the end-products vary depending on cycle and Streptococcus pyogenes. We should be aware entered. In aerobic respiration, glucose usage of these likely organisms as we read and report under aerobic conditions and pyruvate enters the gram-stained smear results. S. pneumoniae and Kreb’s cycle (TCA cycle). The end-products of res- N. gonorrhoeae are important pathogens but piration are carbon dioxide and water. The unique rarely associated with skin infections. Although metabolic pathways and end-products produced Pseudomonas can be associated with some unique by bacteria can be used to aid in the identification infections of the skin, they are uncommon. of particular genera or species. 8. d. The two major uses for gram-stained smears are to 4. d. Bacteria can acquire or exchange genetic material evaluate the quality of a specimen (inflammation with other bacteria through several basic mecha- versus normal flora contamination) and describe nisms: conjugation, transformation, or transduc- the types of bacteria—gram-positive versus gram- tion. Conjugation involves cell-to-cell contact, a negative. For sputum specimens, expectorated series of complex chemical signals between lower respiratory tract specimens, the following cells, and usually the formation of a bridge or criteria are often applied: (1) more than 25 epithe- sex pilus. Transformation is the uptake of free lial cells/low-power field¼saliva, significant num- DNA by a recipient cell. Transduction is the ber of normal flora bacteria. (2) Few epithelial cells, acquisition of DNA through the action of a bac- many white blood cells¼specimen more likely to teria phage. yield a pathogen. In adult patients, specimens with

312 APPENDIX A Answers and Rationales to Certification Preparation Questions 313

significant numbers of epithelial cells often are not It causes urinary tract infection in some popula- cultured and new samples are collected. tions, especially young, sexually active women. In this population, it is considered significant in 9. b. The atmosphere in which specimens are incubated urine cultures even if it is found in small numbers. can have a significant effect on the growth of potential pathogens. Many bacteria isolates will 15. d. Staphylococcus aureus is capable of producing a grow easily in routine air or with a slight increase large number of molecules, which may contribute in carbon dioxide, 3% to 5% CO2 (aerobes). to its ability to cause disease. Toxic shock syn- Other pathogens require special atmospheres for drome is a disease associated with the production optimal growth, including increased CO2, nitro- of a specific toxin, toxic shock syndrome toxin–1 gen, or decreased or complete absence of oxygen: (TSST-1), which belongs to the group of toxins Microaerophilic: 5% O2, 10% CO2, 85% N known as enterotoxins. Anaerobic: 85% N2, 10% H2,5%CO2 16. a. Several media can be used for selection of Staph- 10. d. Hektoen enteric (HE) contains meat peptones and ylococcus aureus from mixed cultures. PEA or yeast extract, bile salts (inhibit gram-positive CNA would select for gram-positive organisms organisms), lactose, sucrose, salacin (carbohy- but are not specific for S. aureus. Mannitol salt drate sources), pH indicators (bromophenol blue agar was developed specifically to select for and acid fuchsin), and ferric ammonium citrate Staphylococcus spp. and to differentiate between for the detection of H2S production. This medium S. aureus and other Staphylococcus spp., because is very selective for enteric pathogens and is differ- S. aureus ferments mannitol and other species ential for Salmonella and Shigella. Yellow-orange generally do not. More recently, chromogenic colonies¼lactose fermenter (E. coli), colorless/ agars have been developed that are more specific green colonies with unchanged medium¼non– for S. aureus and some have been designed to spe- lactose fermenter (Shigella, Providencia), and cifically detect methicillin-resistant S. aureus black colonies¼H2S production (Salmonella). 17. a. Although some bacterial pathogens can cause 11. c. The genus Staphylococcus represents one of the vomiting and diarrhea, the hallmark of the staph- most important groups of potentially pathogenic ylococcal enterotoxins is that because the toxins bacteria and most common normal flora organ- are preformed in the contaminated food, the isms. This genus has the general characteristics organism does not need to grow in the host and of gram-positive cocci in clusters and is catalase- therefore the action is very rapid. Most other positive, facultative anaerobic, ferments glucose, enteric pathogens need to proliferate before the and nonmotile. host experiences disease.

12. a. A unique pathogenic factor of Staphylococcus 18. b. Of the organisms listed, only Streptococcus pneu- aureus is the production of coagulase. This protein moniae exhibits these characteristics. The two is responsible for the formation of fibrin clots in the laboratory tests are bile solubility, which is the host. Coagulase is found in two basic forms: bound characteristic of S. pneumoniae isolates lysing to the surface of the cell, also known as clumping when a solution of bile is dropped on the colony, factor, and free in the medium. The bound form and resistance to optochin. The optochin test is can be detected with several tests that demonstrate performed by streaking a lawn of organism onto clumping of the bacterial cell or sensitized red a blood agar plate and then placing a standardized blood cells, and the free form is most often detected disk containing the chemical optochin onto the by clot formation with rabbit plasma. plate. After incubation, S. pneumoniae will not grow up to the disk—that is, growth is inhibited, 13. d. The initial testing of an isolate of a gram-positive demonstrating that the organism is susceptible to cocci is catalase to differentiate the staphylococci the chemical agent. Viridans group streptococci, (catalase-positive) from enterococci and strepto- S. pyogenes, and enterococci, do not autolyze in cocci (catalase-negative). If an isolate is the presence of bile and are resistant to optochin. catalase-positive and fits the general characteristics of a Staphylococcus spp., the coagulase test 19. b. Of the organisms listed, only Streptococcus pyo- is used to differentiate S. aureus from the other genes exhibits these characteristics. Clinically, species of the genus. S. pyogenes is the most important bacterial cause of pharyngitis. All isolates are susceptible to pen- 14. a. Staphylococcus saprophyticus is normal flora of icillin at this time. In the laboratory, b-hemolytic the mucous membranes of the urogenital tract. streptococci that are susceptible to bacitracin, 314 APPENDIX A Answers and Rationales to Certification Preparation Questions

also known as the A disk, are presumptively iden- 24. d. Of the organisms listed, only Streptococcus sp., tified as S. pyogenes, or group A b-hemolytic not groups A, B or D meets these criteria. Strepto- Streptococcus. The PYR test also can be used, coccus pyogenes is PYR positive and bacitracin because this pathogen is PYR-positive. susceptible. S. agalactiae is CAMP and hippurate positive. Enterococcus faecalis is bile esculin and 20. c. Of the organisms listed, only Streptococcus aga- salt positive. lactiae exhibits these characteristics. Clinically, this organism, also known as group B Streptococ- 25. b. Some bacteria that are environmental have a cus, is an important cause of neonatal meningitis broad range of permissive growth temperatures. and sepsis. Of the b-hemolytic streptococci, it is Among potentially pathogenic, gram-positive unique in being hippurate hydrolysis–positive bacilli, the ability of Listeria monocytogenes to and CAMP test–positive. Latex or other grouping grow well at refrigerator temperatures can be tests are usually used in clinical laboratories rather used to select for this organism. than hippurate or CAMP testing. 26. a. Among the gram-positive bacilli, several basic 21. a. Rapid antigen detection tests are often used to aid characteristics can be used to help differentiate in the diagnosis of group A pharyngitis. The pre- the groups, including catalase, acid-fast, and sent generation of tests are rapid, approximately modified acid-fast stains; bacterial cells that 15 minutes, and reasonably sensitive—65% to branch; and spore formation. Of the organisms 85% in general. Therefore (1) they can be useful listed, only Corynebacterium is a catalase- in quickly identifying most cases of streptococcal positive, gram-positive bacillus that is not acid- pharyngitis. (2) Because of limits to the sensitivity, fast, does not branch, and does not form spores; a negative test does not mean that the patient does the genus Bacillus forms spores, Mycobacterium not have the bacteria present and additional testis acid-fast, and Nocardia cells branch. ing is often needed, so this statement is not true. 27. c. This clinical description is of diphtheria. Coryne- (3) As stated, the major limitation in this test bacterium diphtheriae grows on tellurite medium, method is the limits to detecting small numbers grows as black colonies with brown halos in Tins- of organisms—sensitivity. (4) The rapid antigen dale medium, and shows gram-positive bacilli in tests for group A streptococci is only for those bac- irregular clublike shapes on gram-stained smears. teria and is not useful for the detection of viruses. Rapid antigens for viruses may detect some, but 28. d. The genus Corynebacterium is a catalase-positive, not all, causes of pharyngitis and are in general gram-positive bacillus that is not acid-fast, does designed for bronchitis, bronchiolitis, and not branch, and does not form spores. It shows pneumonia. gram-positive bacilli in irregular clublike shapes on gram-stained smears. Group B Streptococcus 22. c. The ability of streptococcal species to produce would be seen as cocci in chains on gram-stained compounds that can alter the appearance or integ- smear and would be catalase-negative, Erysipelo- rity of the red blood cells in 5% sheep blood agar thrix is H2S-positive, and Listeria is motile. can be used to help presumptively identify potential pathogens. a-Hemolysis is the appearance of a 29. c. The clinical description is classic for Erysipelothrix “green” band of red blood cells around a colony. infection. This organism is positive for the This greening is due to the conversion of hemoglo- production of H2S on triple sugar iron (TSI) slants. bin to methemoglobin. b-Hemolysis is the actual The genus Corynebacterium is catalase-positive, lysis of the red blood cells resulting in a clear area gram-positive bacillus. Group B Streptococcus around the colony, g-hemolysis is a term that his- would be seen as cocci in chains on gram-stained torically was applied to colonies that did not alter smear and would be catalase-negative and b- the red blood cells, hence no zone around the col- hemolytic. Listeria would be motile and show ony. k-Hemolysis is a nonsensical term. This hemolysis on blood agar. image shows greening and thus a-hemolysis. 30. c. Although the clinical disease has little overlap, in 23. a. Two easily performed laboratory tests allow the the laboratory Erysipelothrix and Listeria monocy- differentiation of clinically important isolates of togenes can appear similar. The most common tests the genera Enterococcus from Streptococcus: for differentiating these organisms include: CAMP growth in presence of 6.5% salt and growth on test (Listeria, positive; Erysipelothrix,negative), bile/esculin medium. Streptococci and enterococci hydrogen sulfide production (Listeria, negative; are catalase-negative, do not produce coagulate, Erysipelothrix, positive), and esculin hydrolysis and will grow on PEA medium. (Listeria, negative; Erysipelothrix, positive). APPENDIX A Answers and Rationales to Certification Preparation Questions 315

31. b. Although Streptococcus agalactiae and Listeria 38. c. Nontuberculous mycobacteria (atypicals, myco- monocytogenes may cause somewhat similar dis- bacteria other than tuberculosis [MOTT]), can eases and appear similar on 5% sheep blood be classified by a system first described by Ernest agar, they can be differentiated with gram- Runyon in 1959. This system classified organisms stained smear (S. agalactiae, gram-positive cocci into four Runyon groups, based on: in chains from L. monocytogenes, gram-positive Presence or absence of pigmentation? bacilli), catalase (S. agalactiae, negative from L. Pigment production light dependent or not? monocytogenes, positive), and motility (S. aga- Growth rate slow or fast? lactiae, negative from L. monocytogenes, Runyon I: Photochromogens—slow growing, and positive). produce a yellow-orange pigment when exposed to light. 32. b. Endospores are unique to Bacillus sp. among aer- Runyon II: Scotochromogens—slow growing, obic gram-positive bacilli. All Bacillus spp. are and produce a yellow-orange pigment in light catalase-positive. Once the genus is established, or in the dark. confirmation of B. anthracis requires additional Runyon III: Nonchromogenic—slow growing, testing such as demonstration of a capsule and and do not produce pigment. Note that some lysis of the strain by specific bacteriophages. may produce very pale yellow, buff, or tan pigment but do not intensify upon light exposure 33. d. Bacillus anthracis and Bacillus cereus can be dif- Runyon IV: Rapid growers. ferentiated in the laboratory by a variety of different test results. Of the combinations listed, 39. b. In identification of mycobacterial isolates, the motility (B. anthracis nonmotile and B. cereus Tween 80 test involves testing for lipase using motile) and b-hemolysis on 5% sheep blood agar polysorbate 80 (Tween 80). Certain mycobacte- (B. anthracis nonhemolytic and B. cereus hemo- rial species possess a lipase that splits polysorbate lytic) best differentiate these two potential 80 into oleic acid and polyoxyethylated sorbitol. pathogens. The test contains phenol red, which is stabilized, when the polysorbate 80 is hydrolyzed, the pH 34. d. In a food-borne outbreak, Bacillus cereus can be shifts and the phenol red indicator changes from found in the food. It does not cause a systemic yellow to pink. infection but rather a toxin-mediated disease. 40. b. For the organisms listed, the Enterobacteriaceae 35. c. The single biggest challenge for the laboratory iso- are, in general, oxidase-negative (this entire listed lation of Mycobacterium tuberculosis from respi- group). Traditionally, various biochemical charac- ratory samples is the presence of normal teristics are used to differentiate members of the organisms that grow more rapidly. Therefore the large group of organisms. Salmonella enteritidis decontamination of these sputum samples with is oxidase-negative, nitrate-positive, indole- processes such as decontaminating the specimen negative, citrate-positive, methyl red–positive, with N-acetyl-L-cysteine (NALC)–sodium urease-negative, H2S-positive; Klebsiella pneumo- hydroxide mixture is critical for the successful iso- niae is indole-negative, citrate-positive, usually lation of M. tuberculosis. methyl red–negative (variable), urease-positive, H2S-negative; Escherichia coli is indole-positive, 36. c. The clinical description, the presence of acid-fast citrate-negative, methyl red–positive, urease- bacilli (AFB) in the sample, and the lack of growth negative, H2S-negative; Shigella sonnei is indole- in the laboratory, would raise suspicion that this negative, citrate-negative, methyl red–positive, sample contained an unusual and potentially non- urease-negative, H2S-negative. cultivatable AFB, such as Mycoplasma leprae. The other organisms listed should grow within 41. c. For the organisms listed, the Enterobacteriaceae 8 weeks on standard medium. are, in general, oxidase negative (this entire listed group). Traditionally, various biochemical char- 37. d. The Mycoplasma tuberculosis complex is made acteristics are used to differentiate members of up of several closely related organisms that pri- the large group of organisms. Proteus aerogenes marily cause disease in humans. This group is not a valid name. Escherichia coli is indole- includes: M. tuberculosis, M. bovis (historically positive, H2S-negative, and, although usually found in cattle), M. bovis BCG (vaccine strain motile, not described as swarming. Proteus mir- for the BCG vaccine), and M. africanum (found abilis and Proteus vulgaris can be differentiated in Western Africa and clinically similar to M. using indole; P. mirabilis is negative and P. vul- tuberculosis). garis is positive. 316 APPENDIX A Answers and Rationales to Certification Preparation Questions

42. a. A significant cause of worldwide clinical disease is 50. c. Bordetella pertussis infects the upper airway. diarrhea. Cholera is an important cause of diar- The best specimen for identification of this path- rhea and results in massive fluid loss, “rice water ogen has been shown to be collected from the stools” (white blood cells and blood absent), and nasopharynx. often death within 24 hours. Shiga toxin is a cytotoxin that causes inflammation and ulcerative 51. c. When stained using the Gram stain, members of lesions, destroys epithelial cells, and results in the genus Brucella usually appear as gram- bloody, mucous-laden stools. negative coccobacilli.

43. a. Thiosulfate-citrate-bile salts-sucrose agar (TCBS 52. a. Legionella pneumophila requires specialized agar) is selective medium designed to aid in the medium, 35 C, carbon dioxide, and extended isolation of Vibrio spp. TCBS agar contains incubation; hold for 2 weeks. sodium thiosulfate and sodium citrate to inhibit members of the Enterobacteriaceae. Gram- 53. c. Of the media listed, only chocolate agar provides positive bacteria are inhibited by bile salts. Ferric the X and V factors required for the growth of citrate is used to detect hydrogen sulfide produc- Haemophilus spp. tion. Sucrose is included as a fermentable carbohydrate to help differentiate V. cholera from 54. a. Most members of the genus Haemophilus require other species. Bromothymol blue is included as X and V factors, and these factors are a key char- an indicator of pH changes. acteristic in the identification of this genus. The factor requirement test involves inoculation of 44. b. C. jejuni is the most common human pathogen of unsupplemented media with a light suspension this genus. It is associated with food-borne gastro- of the organism and placement of X and V factor enteritis because of its presence in poultry, raw disks on the agar surface. Growth around only the milk, and water. It is one of the most common X/V combination supports the identification of causes of human bacterial gastroenteritis in Haemophilus spp. numerous parts of the United States. 55. a. Eikenella corrodens is an asaccharolytic, 45. a. The urea slant can be used to aid in identification oxidase-positive bacillus that does not grow on of enteric gram-negative bacilli. The test detects MacConkey agar and that produces a bleachlike the ability of bacteria to hydrolyze urea to ammo- odor. Eikenella has been referred to as part of the nia and CO2; ammonia release causes pH change. HACEK group of organisms along with Hemo- A positive test yields a bright pink slant. philus, Actinobacillus, Cardiobacterium, and Kingella. It is most often associated with 46. b. Salmonella enteritidis grows well on XLD agar wounds of the mouth or teeth or other wounds and is a nonfermenting organism that produces originating with contact with the mouth or teeth, H2S, resulting in the black pigment. Shigella son- but has been known to cause subacute bacterial nei would appear as clear colonies, Serratia and endocarditis. Klebsiella are both lactose fermenting and would appear as yellow colonies. 56. c. Reporting of a sexually transmitted infection (STI) from a child younger than 12 years of age 47. c. Growth on CIN agar with this characteristic usually has significant social and legal ramifica- colonial morphology is indicative of Yersinia tions. Because other Neisseria spp. can be incor- enterocolitica. rectly identified as Neisseria gonorrhoeae, particular care and additional confirmatory test- 48. d. Decarboxylation of the amino acids lysine, ing are often the most appropriate course of ornithine, and arginine results in the formation action. of the following amine end-products: Ornithine decarboxylase (ODC), ornithine ! putrescine; 57. a. Of the species of Neisseria found in humans, lysine decarboxylase (LDC), lysine ! cadaverine; N. meningitidis is a glucose-positive, maltose- arginine dihydrolase (ADH), arginine ! positive, sucrose-negative, lactose-negative major citrulline. cause of bacterial meningitis.

49. b. Triple sugar iron (TSI) slants have traditionally 58. b. Of the species of Neisseria found in humans, been used to help identify and differentiate stool N. gonorrhoeae is an oxidase-positive, glucose- pathogens. These reactions are characteristic of positive, maltose-negative, sucrose-negative, Shigella spp. lactose-negative major cause of venereal disease. APPENDIX A Answers and Rationales to Certification Preparation Questions 317

59. b. When seen in a gram-stained smear, Nesseria spp. culture, antigen detection, and serology. These are gram negative diplococci. methods are all much less sensitive than nucleic acid amplification methods, which have become 60. a. The Hugh-Leifson oxidative-fermentative (OF) the standard detection methods for this infection test shows a pH shift in the open glucose tube in most situations. and no pH in the closed tube. This indicates that this organism is an oxidizer. 73. c. Because of the clinical signs and symptoms, including the rash, the most likely of those listed is Rocky 61. b. The oxidase test detects the presence of cyto- Mountain spotted fever. Additional testing may be chrome oxidase. This enzyme is part of the energy required. Rocky Mountain spotted fever is the transfer cascade of intermediate metabolism. most common rickettsial disease in the United States. The disease is characterized by abrupt onset 62. d. In the laboratory, Stenotrophomonas maltophilia of fever, chills, headache, and myalgia. A rash is has unique characteristics, including growth on common and usually begins on the hands and feet blood agar and chocolate agar with a green- and spreads toward the trunk. Diagnosis is often lavender or yellow pigment. Colonies are non– based on the clinical disease but fluorescent anti- lactose fermenting on MacConkey agar, body (FA) or polymerase chain reaction (PCR) oxidase-negative, catalase-positive, and esculin- for antigen in skin biopsies can be performed. positive. This gram-negative bacillus strongly oxidizes maltose but only weakly oxidizes glucose. 74. a. Primary syphilis is characterized by a chancre that typically heals within 6 weeks. 63. b. Melioidosis is caused by Burkholderia pseudomallei. 75. c. Of the tests listed, only the VDRL is a valid nontreponemal test. 64. a. Stenotrophomonas maltophilia is oxidase-negative, and Burkholderia cepacia is oxidase-positive. 76. b. This patient’s signs and symptoms are classic for Lyme disease. Laboratory testing is often per- 65. b. Acinetobacter and Stenotrophomonas are oxidase- formed to support the diagnosis. negative. The soluble green pigment differentiates Pseudomonas aeruginosa from Burkholderia. 77. c. The characteristic lesion, lack of demonstrable organism in the lesion by gram-stained smear, 66. a. Key tests can be used to differentiate Acinetobac- and the reactive rapid plasma reagin (RPR) test ter from Pseudomonas. Two easy tests are oxidase all support the diagnosis of syphilis (Treponema (Pseudomonas is positive and Acinetobacter is pallidum). negative) and motility (Pseudomonas is positive and Acinetobacter is negative). 78. c. Based on the gram-stained smear and basic biochemical reactions, the best identification of this 67. a. Cystic tryptic agar (CTA) sugar reactions are used organism is Bacteroides fragilis. to differentiate Neisseria species. If Moraxella catarrhalis is tested in this system, the isolate will 79. a. Pseudomembranous colitis caused by Clostridium be glucose-negative, maltose-negative, lactose- difficile is best confirmed by the presence of toxin negative, and sucrose-negative. in the stool. In some unusual circumstances, the organism may need to be isolated from the stool 68. b. Of the organisms listed, only Pseudomonas aeru- and then tested for its ability to produce toxin. ginosa produces the pigment pyocyanin. 80. 69. b. Of the tests listed, the oxidase test is the only test a. Lecithinase production, double-zone hemolysis that exhibits a dark purple color. on sheep blood agar, and gram-stained morphology are key criteria in the identification of Clos- 70. c. An important characteristic of Mycoplasma and tridium perfringens. Ureaplasma is that both genera have only a cell 81. membrane with no cell wall. c. The most common way to confirm that an anaerobic system has the correct gas mixture is to use the 71. c. Nongonococcal urethritis (NGU) is an infectious change in color of the methylene blue indicator. clinical condition that is most often associated with Ureaplasma urealyticum. 82. b. The presence of sulfur granules in a clinical specimen from the neck, mouth, or respiratory tract of 72. c. Traditional methods for the detection of Chla- the patient strongly indicates the presence of mydia trachomatis infections included cytology, Actinomyces spp. 318 APPENDIX A Answers and Rationales to Certification Preparation Questions

83. a. Both the E test and agar disk diffusion or Kirby- 95. b. The term asceptic meningitis describes meningi- Bauer test are used for antimicrobial susceptibility tis that is characterized by negative bacterial and testing and are based on the diffusion of an anti- fungal culture. biotic into an agar-based medium. In the medium, both systems establish an antibiotic gradient. 96. c. Most patient samples for bacteriology cultures only can be semi-quantitated, because of sampling 84. b. The most important clinical need for minimum and plating limitations. Urine cultures, however, inhibitory concentration (MIC) testing is when are routinely quantitated to aid the differentiation the organism isolated does not have a predictable of true infection from contamination. pattern or the organism is isolated in an isolated 97. body site such as the central nervous system. b. Quality control practices ensure that the test results are accurate. Therefore Gram staining 85. b. The best definition of minimum inhibitory con- and reading a glass slide with a mixed smear centration (MIC) is the lowest concentration of of Staphylococcus and Escherichia coli along an antibiotic in a dilution series that inhibits with each gram-staining run of specimens exam- growth of the bacteria. ined within the microbiology laboratory that day is an example of quality control. Quality 86. c. When comparing minimum inhibitory concentra- assurance is the overall process of ensuring the tion (MIC) testing to Kirby-Bauer or disk diffu- quality of the results reported by the laboratory sion testing, the lower the MIC, the larger is the and includes quality control, employee training zone of inhibition. and certification, etc. 98. d. Tracking the rate of skin organism contamina- 87. a. The antibiotic concentration in a disk used for tion among a laboratory’s blood culture results Kirby-Bauer testing is designed to give a specific on a monthly basis and introducing specific zone of inhibition for control stains of bacteria. training to phlebotomists when rates exceed If the organism is able to grow closer to the disk the norm would be an example of quality assur- than expected, the concentration of the antibiotic ance. Quality assurance is the overall process of in the disk is too low. ensuring the quality of the results reported by the laboratory and includes quality control, 88. a. Of the definitions listed, only urethritis refers to employee training and certification, etc. an infection of the lower urinary tract. 99. b. Infectious substances are substances known or 89. c. Streptococcus pneumoniae commonly associated reasonably expected to contain pathogens. with Otitis Media; its identification includes the Optochin test. 100. d. Because of the need to quantitate the sample and the fact that bacteria grow in urine, making 90. b. Streptococcus pyogenes causes impetigo, of the storage and transport of sample critical, a 24- organisms listed. hour urine sample for bacteriology culture is inappropriate. 91. a. Staphylococci produce hyaluronidase which can break down connective tissue, allowing for the CHAPTER 2 spread of infection. 1. c. In contrast to bacteria, yeasts reproduce by a pro- 92. d. Because of the need to isolate potentially fastidi- cess known as budding. The process in the mother ous pathogens and anaerobic bacteria, routine yeast cell begins with a weakening and outpouch- cultures of cerebrospinal fluid should include ing of the yeast cell wall and then formation of a 5% sheep blood agar, chocolate agar, or thiogly- cell wall septum between the mother and daughter colate broth. Other clinical conditions may war- yeast cells. When the cells separate, there is a “bud rant additional specialized media. scar” left on the surface of the mother cell.

93. c. The clinical symptoms exhibited by the patient 2. d. The loose intertwined network of basic structural are consistent with a potential diagnosis of units of the molds that grows in a matlike struc- meningitis. ture is known as the mycelium.

94. a. In cases of vaginitis, clue cells are a common finding. 3. c. Molds that form hyphae and reproductive struc- Clue cells are epithelial cells covered with small tures with little or no pigment are often referred gram positive bacilli found in vaginal discharge. to as hyaline molds. APPENDIX A Answers and Rationales to Certification Preparation Questions 319

4. b. Certain molds form two types of conidia: macro- 12. d. Cryptococcus neoformans can be differentiated conidia and microconidia. Macroconidia are from other Cryptococcus species through the large, multiseptate and club-shaped or spindle- use of the Niger seed test; C. neoformans is Niger shaped spores, whereas microconidia are small, seed–positive (dark colonies). nonseptate teardrop-shaped spores. 13. c. The most common phenotypic test for the identi- 5. d. The structure indicated in the image is a conidio- fication of Candida spp. is the germ tube test. C. phore of an isolate of Aspergillus. albicans is defined as germ tube–positive. The standard procedure needs to be followed because 6. c. Historically, the differentiation of dermatophytes it is well described that changes in incubation con- was accomplished using a variety of morphologic ditions can significantly affect the test. Candida characteristics and growth characteristics. The albicans and Candida tropicalis can be used a pos- characteristics listed support the identification of itive and negative control, respectively. Microsporum audouinii. The most suggestive of these characteristics is the rare distorted macroco- 14. a. The classification of Pneumocystis jiroveci has nidia and rare microconidia. been contested. It has been considered many different things over the years, including a fungus 7. b. Blastomyces dermatitidis is endemic to North and a protozoan. It is known to cause opportu- America, specifically the Mississippi river valley. nistic infections in the immunocompromised When seen in tissue samples it appears as large, patient. Diagnosis requires demonstration of spherical, thick-walled yeast cells 8 to 15 mmin the organism in tissue, lavage, or sputum. It can- diameter, usually with a single bud that is con- not be cultured except in animal models. In the nected to the parent cell by a broad base. laboratory, Grocott’s methenamine silver (GMS) stain is commonly used to identify the 8. c. Coccidioides immitis is endemic in hot, semi-arid organism, which appears as “cups and saucers” climates such as the southwestern United States or “deflated balls.” and northern Mexico. It is a saprobe in mold form (desert soil). It is the most virulent of all 15. b. The appearance and biochemical characteristics of agents of human mycoses. Coccidioides causes this organism support the identification of Can- mild infection in everyone who inhales it, but is dida albicans. The germ tube test is a key indicator. usually asymptomatic and self-limiting. Dissemina- tion in the immunocompromised population is 16. b. Malassezia furfur is normal skin flora in 90% of much higher than found for other fungal agents. humans. Clinically it is responsible for tinea versi- The part of its life cycle in the mammalian host is color and catheter-related infections in patients highlighted by the endosporulating spherules in on long-term intravenous lipids. Macroscopically tissue. the organism appears as cream/brown wrinkled colonies that grow better in the presence of oil. 9. a. These are the characteristic large, spherical, thick- Microscopically it grows as yeastlike cells, often walled yeast cells 8 to 15 mm in diameter showing with distinct collarettes. broad-based budding. 17. c. Conidiation of dematiaceous fungi includes 10. b. Sporothrix schenckii is found worldwide (soil three basic types of structures: (1) Cladosporium saprobe). It is a thermal dimorph. Infections with type—resembles a tree, in which conidiophore this fungus is an occupational risk and is often is the trunk and branched chains of conidia referred to as rose gardeners disease. The clinical form the branches; (2) Phialophora type—short disease of sporotrichosis usually involves chronic conidiophores plus phialide, vase-shaped cutaneous and subcutaneous mycosis character- conidia extruded from phialide and then clus- ized by ulcers and abscesses along lymphatic tered; and (3) Rhinocladiella type—stalked channels. conidiophores that become knobby as conidia are produced. Conidia are produced sequentially 11. c. The most common phenotypic test for the identi- until a Cladosporium-type conidiation is fication of Candida spp. is the germ tube test. reached. C. albicans is defined as germ tube–positive. Torulopsis glabrata (Candida glabrata) is urease 18. b. Acremonium sp. is a mold that displays a white negative and unable to assimilate dextrose, malt- cottony macroscopic morphology and, on micro- ose, or sucrose. CMT agar morphology shows scopic evaluation, exhibits hyaline, septate blastoconidia only. hyphae, and “toothbrush”-like conidiophores. 320 APPENDIX A Answers and Rationales to Certification Preparation Questions

19. a. Penicillium sp. is a mold that displays a velvety, require a moist or liquid environment; when such gray-green colony morphology. On microscopic an environment is not present, the trophozoite has evaluation, flask-shaped conidiophores arranged the ability to convert to the cyst form. in a brushlike formation are seen. 32. b. Polyvinyl alcohol (PVA) is most often used for 20. a. The macroscopic morphology, color, temperature fixed smear preparation. It is less effective when tolerance, and growth rate, combined with the used in concentration methods than is formalin- microscopic characteristics, supports Aspergillus ethyl acetate. Formalin fixatives do not preserve as the genus and A. fumigatus as the species. the morphology of organisms well enough for use in fixed smear preparation. Merthiolate- 21. b. The protein coat that surrounds the nucleic acid is iodine-formaldehyde (MIF) is not used for the called the capsid. same reason.

22. b. Viral envelopes are acquired as the virion buds 33. a. Taenia solium, the pork tapeworm, is known to through the membrane of the mammalian host cause cysticercosis. The oncosphere form can cell and picks up components of the membrane. migrate through the bloodstream and invade tissue, forming a cystlike structure. 23. b. Prions are unique infectious proteins with no associated nucleic acids. 34. d. Strongyloides stercoralis is not often recovered in the ovum stage in feces. The rhabditiform larval 24. a. Viruses have different structures than bacterial or stage is the primary diagnostic stage for mammalian cells. The viral nucleocapsid is the strongyloidiasis. protein coat that contains or covers the nucleic acid (genome) of the virus particle. 35. c. Identifying multiple parasites in fecal specimens is common, so a full examination of each slide is 25. c. Norovirus is an important cause of gastroenteritis necessary. in several settings, including closed environments such as cruise ships and long-term care facilities. 36. a. Necator americanus, Ancylostoma duodenale, None of the other viruses listed are associated and Strongyloides stercoralis all pass through with gastroenteritis as a major symptom. the human lung during their life cycles.

26. b. The severe acute respiratory syndrome (SARS) 37. a. Morphologically, Acanthamoeba spp. appear as a virus arose as a previously unrecognized organism wrinkled, double-walled cyst and are known to causing serious respiratory tract infections. This cause corneal keratitis. virus has been shown to be part of the coronavirus group. 38. d. Trichuris trichiura is known as whipworm because of the appearance of the adult larva. 27. c. This is the description of the coronavirus group. Adult larvae are typically 2.5 to 5.0 cm. in length. The posterior end of the larva is large, and the 28. d. Rotaviruses are responsible for significant out- anterior end is slender and long, resembling a breaks of gastroenteritis and are an enteric virus. whip handle and whip. Additionally, the adult The specimen of choice for detecting this virus male larva possesses a curved tail. is stool. 39. b. Schistosoma haematobium ova are typically 110 29. b. Nucleic amplification assays such as polymerase to 170 microns by 40 to 70 microns and possess chain reaction are the most sensitive method for a large terminal spine. S. haematobium ova are the detection of many/most viruses and patient recovered in urine. samples, including cerebrospinal fluid for the diagnosis of meningitis. 40. c. Echinococcus granulosus is found in areas where sheep are raised in close proximity to dogs; the 30. a. This typical fibroblastic cell line is showing the dog is the true definitive host. Sheep and humans rapid development of foci of rounding and degen- are accidental intermediate hosts. E. granulosus erating cells. This is typical of herpes simplex larvae are typically 4.0 to 5.0 mm in length and virus in culture. comprise a scolex and three proglottids.

31. d. The trophozoite form is the motile, feeding, and 41. b. Entamoeba coli trophozoites are typically 15 to 50 multiplying form of amoeba. Trophozoites microns in diameter. Morphologic criteria include APPENDIX A Answers and Rationales to Certification Preparation Questions 321

one nuclei with an eccentric karyosome and irreg- and serum transferrin provides a measure of how ular peripheral chromatin, vacuolated cytoplasm, many binding sites are available to bind iron and is and broad pseudopods. used with transferrin saturation, the percentage of sites available to carry iron. 42. c. Giardia lamblia cysts measure approximately 8 to 19 microns in length. Morphologic features 2. a. Iron-deficiency anemia resulting from the include an ovoid shape, two to four nuclei, two decreased mean corpuscular volume (MCV) and to four median bodies, and a smooth wall. mean corpuscular hemoglobin (MCH), indicating hypochromic microcytic cells. Vitamin B defi- 43. 12 c. Entamoeba histolytica cysts measure approxi- ciency typically exhibits macrocytosis. Anemia mately 12 to 15 microns in diameter. Morphologic of chronic inflammation tends to exhibit a mild features include a round shape, two to four nuclei, anemia, with a hemoglobin value of approxi- and a rounded chromatoid bar. Clinically, blood mately 9 to 11 g/dL. Hemochromatosis is not an may be seen in stool samples of individuals with anemia and, untreated, exhibits a normal to ele- an E. histolytica infection. vated red blood cell count. 44. a. c.; b. d.; c. a.; d. b. 3. b. The inclusions suggest the presence of excess 45. d. The diagnostic criteria of Wuchereria bancrofti storage iron. Sideroblastic anemia is a disorder include the presence of a sheath and nuclei that characterized by elevated iron stores resulting do not extend to the tail tip. The organism also from an inability to incorporate iron into heme. demonstrates nocturnal periodicity. The inclusions suggest the presence of excess storage iron. Ringed sideroblasts may also be 46. d. Necator americanus rhabditiform larvae possess a found in bone marrow examinations. Iron- long buccal cavity and a small genital primor- deficiency anemia shows a microcytic hypochro- dium. Neither the rhabditiform or filariform lar- mic anemia; however, it is characterized by a lack val stage has a notched tail. Strongyloides of iron. Pernicious anemia is a megaloblastic ane- stercoralis rhabditiform larvae possess a short mia. Thalassemia minor shows a microcytic hypo- buccal cavity and a prominent genital primor- chromic anemia; however, it often has an elevated dium. Filariform larvae possess a notched tail. red blood cell count with hypochromic, microcytic cells but would not normally show iron 47. a. The morphologic appearance of B. hominis is dis- inclusions (although transfusion-dependent thal- tinct. The organism typically measures 5 to 30 assemias may exhibit transfusion-associated microns in diameter. The center of each comprises iron excess. a large vacuole that generally stains a teal color with the trichrome stain. The central vacuole is 4. a. Iron-deficiency anemia is most likely, because surrounded by a thin rim of cytoplasm that con- sideroblastic anemia and hemochromatosis show tains two to six small nuclei. increased iron and ferritin with a decreased total iron-binding capacity (TIBC) whereas anemia of 48. a. c.; b. d.; c. a.; d. b. chronic inflammation has decreased serum iron and percent saturation but normal-to-increased 49. a. Medically important known human tapeworms serum ferritin. include Dipylidium caninum, Diphyllobothrium latum, Hymenolepis diminuta, Hymenolepis 5. c. Gastrointestinal disease may lead to malabsorp- nana, Taenia saginata, and Taenia solium. D. tion, which could possibly affect iron absorption. latum is the only one with operculated ova. Alcoholism and lead poisoning can lead to a secondary sideroblastic anemia, and primary sidero- 50. a. Trypanosoma spp. are hemoflagellates that can be blastic anemia may be seen in myelodysplastic identified in peripheral blood smears as a trypo- syndromes, such as refractory anemia with ringed mastigote form. Trypomastigotes assume a C, S, sideroblasts (RARS). or U shape in blood. Each one is characterized by a central nucleus, a posterior kinetoplast, 6. b. Intrinsic factor antibodies would be present in and a full-body undulating membrane. patients with a true megaloblastic anemia, because it is characterized by the destruction of parietal CHAPTER 3 cells, which produce the intrinsic factor needed for B12 absorption. Homocysteine is elevated in 1. c. Serum ferritin concentrations reflect the body’s both vitamin B12 and folic acid deficiencies. storage of ferritin. Serum iron measures free iron, Diphyllobothrium latum can cause megaloblastic 322 APPENDIX A Answers and Rationales to Certification Preparation Questions

anemia because it competes for vitamin B12 in the 13. c. Haptoglobin is a protein that picks up free hemo- intestines; however, testing for ova and parasites globin, and it frequently decreases as it is used up alone will not define the diagnosis. A bone marrow when free hemoglobin (Hgb) is present in excess examination could determine that megaloblastic of haptoglobin’s carrying capacity. Bilirubin, both features were present; however, it would not be total and unconjugated, is increased with the specific for pernicious anemia. increased red blood cell destruction. Plasma Hgb and urine hemosiderin are also increased 7. b. Megaloblastic anemias result from deficiencies in because of the excesses of free Hgb. vitamin B12 and folic acid. Both are needed for normal cell maturation. Iron and hepcidin play 14. b. Macrocytic, normochromic cells with increased a role in anemias with iron problems, whereas polychromasia are present in most cases of hemo- decreased free erythrocyte protoporphyrin (FEP) lytic anemia, because reticulocytes are being is seen in some porphyrias. Decreased liver func- released prematurely from the bone marrow to tion, alcoholism, and severe hypothyroidism can replace cells being destroyed. Microcytic and cause macrocytic anemia, but the anemia is not hypochromic cells are usually seen in disorders megaloblastic. of iron/heme metabolism.

8. c. Pernicious anemia is a megaloblastic anemia that 15. d. Anemia of chronic disease is characterized by a results from defective DNA synthesis from lack of block in iron incorporation and is a mild anemia, vitamin B12, often showing dysplastic changes in not characterized by increased cell destruction. the cells and sometimes requiring a bone marrow Sickle cell, active glucose-6-dehydrogenase examination to confirm the deficiency, particu- (G6PD) deficiency, and autoimmune hemolytic larly to differentiate from myelodysplastic syn- anemia are types of hemolytic anemia. dromes. The others do not have a need for bone marrow examination. 16. b. The direct antiglobulin test (DAT) would be the best test to begin determining the cause of anemia, 9. b. Vitamin B12 and folic acid are the best place to because it can help determine if spherocytes are start in further investigating this patient’s ane- the result of immune activity or if they are due mia, because this will determine the specific to abnormal red blood cell skeletal protein follow-up most valuable to the physician. Iron interactions. Osmotic fragility will be decreased studies could be performed, but a deficiency is in the presence of spherocytes. Glucose-6- unlikely given the macrocytic appearance of the dehydrogenase (G6PD) activity would be useful red blood cells. Bone marrow examination is only if G6PD deficiency was present. Vitamin not usually performed unless confirmation of B12 is used to determine the cause of macrocytic other testing or rule out of myelodysplastic syn- anemia and does not usually result in spherocytes. drome (MDS) is needed. Intrinsic factor antibody assays may be used to further work up the case if 17. a. The PIGA gene codes for glycophosphatidylinosi- vitamin B12 levels are decreased, because the rea- tol (GPI)-anchored proteins. Paroxysmal nocturnal son for the decrease would need to be confirmed hemoglobinuria shows a mutation in the PIGA or ruled out. gene, which results in deficiencies in GPI proteins, indicated by a negative CD55 and CD59. 10. a. Approximately 70% of acquired aplastic anemia cases are idiopathic (Rodak, 2012). It can occur as 18. a. Glucose-6-dehydrogenase (G6PD) deficiency shows a result of other stimuli, including various drugs, lack of enzyme activity that is needed for the reduc- radiation exposure, and viral infections. tion of glutathione, which in turn works to deal with protecting hemoglobin from oxidant damage. 11. c. The absolute lymphocyte count is most likely to Defective globin chains can be seen in hemolytic be normal, because lymphocytes may also reside hemoglobinopathies or thalassemia. Antibodies to in lymphoid tissue beyond the bone marrow. red blood cells are present in immune-mediated The others and their precursors are primarily hemolytic anemias, and abnormal protein structure found in the bone marrow and tend to have a is seen in disorders such as hereditary elliptocytosis shorter life span in circulation. or hereditary spherocytosis.

12. a. Fanconi’s anemia is characterized by mutations in 19. c. All are microangiopathic hemolytic anemias, with a group of genes that lead to fragile chromosomes, the exception of traumatic cardiac hemolytic ane- which break easily and may not be able to repair mia, because they feature intravascular hemolysis themselves. resulting from red blood cells (RBCs) shearing APPENDIX A Answers and Rationales to Certification Preparation Questions 323

when they contact microclots in the circulation. definitive listing for the Hgbs present. Peripheral Traumatic cardiac hemolytic anemia is macroan- smear review may show the presence of sickle cells; giopathic, because the hemolysis occurs when however, it will not determine if Hgb S is present in RBCs move through implanted cardiac devices the case of someone heterozygous for Hgb S. or patients with cardiac valve issues. 27. b. The crystals are likely hemoglobin (Hgb) C, 20. d. Thrombotic thrombocytopenic purpura (TTP) is because this abnormal Hgb tends to polymerize the most likely cause of the laboratory results, in short hexagonal crystals. Hgb S will polymerize because it is consistent with the anemia and into sickle cells. Hgb SC polymerizes into forms thrombocytopenia with the presence of schisto- that are a hybrid of Hgb S and Hgb C that look cytes. The patient is exhibiting normal coagula- like fingers or birds. Hgb E does not form specific tion results, which would be increased in crystalloid shapes. disseminated intravascular coagulation (DIC). Chronic myelogenous leukemia could show 28. c. This patient most likely has thalassemia minor, in decreased red blood cell and platelet count; addition to his sore throat. The patient has an ele- however, this is a younger patient with normal vated red blood cell (RBC) count with a dispro- white blood cells. Sickle cell disease is unlikely portionately low hemoglobin and hematocrit, in a previously healthy female, and the decreased which is often seen in thalassemia minor. Warm platelets and schistocytes point more to a micro- autoimmune hemolytic anemia would lead to a angiopathic hemolytic anemia (MAHA). decrease in the RBC count. Results are not consistent with a cold agglutinin. 21. b. The majority of warm autoimmune hemolytic anemia cases involve IgG antibodies, although 29. a. Hemoglobin H is the result of a three-gene dele- other antibodies, such as IgA or IgM, may be tion on the a gene. A two-gene deletion would implicated in rare cases. result in a-thalassemia minor.

22. b. Idiopathic onset is an unknown cause of warm 30. b. This is likely a case of b-thalassemia because autoimmune hemolytic anemia (WAIHA). Sec- hemoglobin (Hgb F) (a2g2) and Hgb A2 (a2d2) ondary WAIHA is usually associated with chronic are increased. Thus a chains are able to be pro- lymphoid disorders, viral infections, and autoim- duced; however, b chains are lacking so no Hgb mune disorders. A(a2b2) is present. The other disorders listed are all covered in the a-thalassemia group. ! 23. a. b6Glu Val is the mutation seen in sickle cell ane- ! mia. b6Glu Lys is the mutation seen in hemoglo- 31. c. This is consistent with Pelger-Huet anomaly, which ! bin (Hgb) C, b26Glu Lys is the mutation seen in is characterized by mature neutrophils, but hypo- ! Hgb E, and b63Glu Arg is seen in Hgb Zurich. segmentation in the majority of neutrophils. Alder-Reilly anomaly features abnormal granula- 24. d. All of these conditions are associated with sickle tion, leukocyte adhesion deficiency shows rela- cell disease; however, vasoocclusion is common tively normal looking but functionally abnormal and leads to painful crises that often result in hos- neutrophils. pital visits. 32. a. Acute bacterial infection is the most likely cause 25. a. Patients with sickle cell trait usually have no clin- of these results, with an elevated white blood cell ical symptoms or abnormalities on their complete count and shift to the left. Although some similar- blood count, although they may exhibit occa- ities may exist in the complete blood count picture sional target cells. Under extreme stress or hyp- for chronic myelogenous leukemia (CML), the oxia, patients may have serious complications patient is relatively young for the diagnosis. Addi- similar to those seen in actual sickle cell disease. tionally, the patient appears to have normal red blood cell (RBC) and platelet counts, which 26. b. Hemoglobin (Hgb) electrophoresis, high- may decrease with the neoplastic clone in CML. performance liquid chromatography, or isoelectric Viral infections usually show elevations in lym- focusing is the best means for determining the spe- phocyte numbers. Refractory anemia is unlikely cific Hgbs present in a patient sample. Solubility because the patient has normal RBC counts. testing is a good screen to look for abnormal Hgbs, but does not determine specific Hgb presence or 33. a. a-Naphthyl acetate esterase can be used to exhibit approximate quantities. Bone marrow analysis is positive esterase activity in monocytes, whereas an invasive technique and does not provide a neutrophils and lymphocytes usually stain 324 APPENDIX A Answers and Rationales to Certification Preparation Questions

negative. Naphthyl AS-D chloroacetate esterase 39. c. All of these disorders are classified as myelodys- shows positive activity in granulocytic cells plastic syndromes with the exception of chronic with negative or weak reactions in monocytes. myelomonocytic leukemia (CML), which was Periodic acid–Schiff stains glycogen and muco- moved into the World Health Organization classi- proteins, and staining patterns may be used fication of myelodysplastic syndrome/myelopro- to help in identification of various cell types. liferative neoplasms, along with juvenile Myeloperoxidase activity is strong in neutrophils myelomonocytic leukemia, and atypical CML. from the promyelocyte stage through maturity; however, activity in monocytes is negative 40. a. Intermediate B-cell acute lymphoblastic anemia or weak. (ALL) is the most likely diagnosis because of the B-cell markers (CD19 and CD22), in addition to 34. c. A positive tartrate-resistant acid phosphatase CD10, which are specifically seen in common (TRAP) stain is indicative of hairy cell leukemia, ALL (cALL), also known as intermediate B-cell because hairy cell lymphocytes produce large ALL. This is not a T-cell ALL, because no positive amounts of acid phosphatase isoenzyme 5, which T-cell markers are indicated in the results given. is inhibited in the presence of tartaric acid. Most other cells of various lines are positive for acid 41. a. Prognosis is currently the best in children, as phosphatase; however, they are not resistant to opposed to in infants and adults. Elevated blast the addition of tartaric acid because of normal counts or hypodiploidy are associated with a or decreased levels of isoenzyme 5. poorer prognosis. The presence of the Philadel- phia chromosome has an unfavorable prognosis 35. b. The JAK2 V617F mutation is present in numerous in acute lymphoblastic anemia. cases of myeloproliferative neoplasms, including polycythemia vera, essential thrombocythemia, 42. b. The clinical presentation of disseminated intravas- and primary myelofibrosis. BCR/ABL mutations cular coagulation (DIC), along with the peripheral are seen in CML, and PDGFR mutations are seen smear findings, are consistent with acute promye- in neoplasms with eosinophilia. RUNX mutations locytic leukemia (AML with t[15:17]; PML- may be seen in some cases of acute leukemias. RARa). Acute lymphoblastic leukemia (ALL) is unlikely because of the suggestion of a disorder 36. b. Although leukocyte alkaline phosphatase (LAP) of the myeloid line because of the presence of Auer scores tend to be decreased in chronic myelogenous rods. AML with t(9:11); MLLT3-MLL is a disor- leukemia (CML) and myeloid cells are present, der involving the monocytic line, although DIC karyotyping for the presence of the Philadelphia may be associated with this disorder. chromosome (9;22 translocation) is required for the confirmation of a diagnosis of CML. 43. c. Acute myelomonocytic leukemia is the most likely because of the presence of both myeloid (CD13, 37. a. A bone marrow biopsy would help in confirming CD33) and monocytic cell lines (CD4, CD14, a diagnosis of primary myelofibrosis, which is a CD11b, CD11c, CD64, CD36). possible diagnosis suggested by the dacryocytes, left shift, and abnormal platelets. If the patient 44. b. This smear and bone marrow picture is typical of had primary myelofibrosis, the bone marrow chronic lymphocytic leukemia (CLL) with numer- would likely show areas of fibrosis, in addition ous mature small lymphocytes. Acute lymphocytic to increases in megakaryocytes and abnormal leukemia (CALL) would show the presence of blasts; platelets. Splenomegaly would be explained by hairy cell leukemia shows a hypocellular, fibrotic extramedullary hematopoiesis, which is suggested bone marrow with hairy lymphocytes. Acute mye- by the presence of dacryocytes. Leukocyte alka- logenous leukemia (AML) is not indicated, because line phosphatase (LAP) staining and karyotyping there is no evidence of myeloid cells and blasts. for the Philadelphia chromosome would be more useful in determining a diagnosis of chronic mye- 45. c. Se´zary syndrome is a disorder of T lymphocytes, logenous leukemia. whereas the others are plasma cell disorders.

38. d. Myelodysplastic syndromes are characterized by 46. a. CD2, CD3, CD4, CD5, CD7, and CD8 are asso- anemias refractory to normal treatment and ciated with T lymphocytes. CD13, CD14, CD15 abnormal cellular appearance resulting from dys- are associated with granulocytic/monocytic cells. poiesis in the cell lines. Vitamin B12, folic acid, CD19, CD20, CD22 are associated with B cells. and iron levels usually are normal; however, cells CD34 and CD117 are immature cell markers, do not mature normally. and CD71 is an erythroid marker. APPENDIX A Answers and Rationales to Certification Preparation Questions 325

47. c. Bone marrow core biopsies are the best indicator Eosinophils, neutrophils, and monocytes all of bone marrow architecture and cellularity, contain granules; however, they are not associated because they provide a visual representation of with IgE. the hematologic cells, fat, and vascular structure. Although a general idea may be obtained by 56. b. The patient most likely has a bacterial infection, examining the aspirate, it is better used for look- because the white blood cell (WBC) count is ing at the specific cell morphology. The buffy slightly elevated with increased neutrophils, coat or concentrated smears concentrate any cells including the presence of 10% bands. Do¨ hle bod- present, particularly in cases of hypocellular ies and toxic granulation, although not exclusive samples. to bacterial infections, are toxic neutrophil changes that may present in these cases. Strenuous 48. c. Fibrotic or hypercellular marrow is seen in all of exercise may lead to a transient elevation in the following except multiple myeloma, in which WBCs and the mobilization of neutrophils from sheets of plasma cells may be present. the marginating to circulating pools; however, toxic granulation and Do¨ hle bodies are not usu- 49. b. Megakaryocytes, with diameters up to 50 mm, are ally seen. If the patient had a parasitic infection, the largest cells present in a normal bone marrow elevated numbers of neutrophils would be sample. Lymphoblasts and pronormoblasts are less expected. than 20 mm in diameter. Osteoblasts, although large cells, are not hematopoietic cells, and are 57. c. Chediak-Higashi syndrome is characterized by used in the formation and modeling of bone. giant fused granules in the white blood cell cytoplasm, with neutropenia and thrombocytopenia 50. a. Hemoglobin (Hgb) A is characterized by pairs of a as the disease progresses, and patients often die and b chains, a2b2. a2d2 is Hgb A2, a2g2 is Hgb F, in infancy or early childhood because the granules and a2e2 is Hgb Gower 2, an embryonic Hgb. normally released to aid in the killing of bacteria cannot be released to aid in the kill process. A 51. a. The bone marrow is the site of intramedullary complete blood count with giant platelets and hematopoiesis. The liver and spleen are sites of Do¨ hle-like inclusions in the granulocytes is more hematopoietic activity in the embryo, and hema- characteristic of the May-Hegglin anomaly, and topoietic activity may be renewed in cases of large, dark granules are more associated with bone marrow compromise, such as primary Alder-Reilly anomaly. myelofibrosis. The thymus is a site for lymphoid development. 58. a. Patients with infectious mononucleosis often exhibit an increase in lymphocytes, along with 52. b. Erythropoiesis is stimulated by erythropoietin, the presence of reactive lymphocytes. Neutrophi- which is produced in the kidney, and renal failure lia with a left shift is typically seen in bacterial can decrease the production of erythropoietin. infections or other acute infections.

53. c. The Rapoport-Luebering shunt is involved in the 59. b. CD4 and CD8 markers are monitored in patients production of 2,3 diphosphoglycerate, which with acquired immunodeficiency virus infection. helps regulate oxygen delivery to the tissues. CD33 and CD34 would more likely be used for The overall Embden-Meyerhof pathway is used investigating a suspected acute myeloid leukemia in the production of adenosine triphosphate (AML) case. (ATP); the hexose-monophosphate shunt functions in reducing glutathione; and the methemo- 60. c. Atypical chronic myelogenous leukemia is classi- globin reductase pathway is involved in the fied as an MDS/MPD, because it has characteris- reduction of methemoglobin. tics of both disorders. Acute promyelocytic leukemia is an acute leukemia, and chronic lym- 54. a. This is most likely a normal child, because chil- phocytic leukemia is a chronic lymphoid disorder dren usually have higher relative lymphocyte that affects a different cell line than seen in MDS/ counts than adults. The patient has normal total MPD. Essential thrombocythemia is classified as a white blood cell, red blood cell, and platelet myeloproliferative disorder. counts and normal differential results. 61. b. Lymphocytes are derived from the common lym- 55. a. Basophils have IgE receptors on their surface phocyte progenitor cell, whereas the other cells membranes. Once IgE is bound to the receptor, are derived from the common myeloid progenitor it allows the release of the cell’s granule contents. (or CFU-GEMM). 326 APPENDIX A Answers and Rationales to Certification Preparation Questions

62. c. Elevated eosinophil counts are often seen in para- counts while the erythropoietin levels are sitic infections, particularly those caused by hel- decreased. Secondary polycythemia shows ele- minths. Aplastic anemia shows decreases in all vated erythropoietin levels, often as a response cell counts. Bacterial infections tend to have to tissue hypoxia resulting from the patient’s ini- increased neutrophil numbers, and viral infections tial condition. tend to have increased lymphocytes. 69. c. The mutation seen in chronic myelogenous leuke- 63. c. Antibodies are produced by lymphocytes, specifi- mia (CML) is t(9;22)(q34;11.2), resulting in the cally B cells in the form of plasma cells. None of Philadelphia chromosome, with the translocation the other cells can produce antibodies. creating a fusion gene, BCR-ABL. t (15;17)(q22; q12) is seen in acute promyelocytic leukemia, t 64. b. The nitroblue tetrazolium test will reduce nitro- (8:21)(q22;q22) is seen in acute myeloid leukemia blue tetrazolium in normal neutrophils. In cases (AML) with maturation, and t(11;14)(p15;q11) is of chronic granulomatous disease (CGD), the seen in precursor T-cell acute lymphoblastic phagocytic cells cannot make nicotinamide ade- leukemia. nine dinucleotide phosphate (NADPH) oxidase, which is used in the kill mechanism of neutrophils. 70. d. All are myeloproliferative neoplasms with the Leukocyte adhesion deficiency (LAD) involves exception of Waldenstro¨ m’s macroglobulinemia, problems with adhesion to endothelial cells, and which is a plasma cell disorder. both May-Hegglin and Pelger-Huet anomalies have normal neutrophil phagocytic function. 71. b. More than 15% ringed sideroblasts must be present in the bone marrow for a diagnosis of refrac- 65. c. Myeloperoxidase (MPO) and terminal deoxnu- tory anemia with ringed sideroblasts (RARS). cleotidyl transferase (TdT) would be good initial Ringed sideroblasts must have at least five iron markers to use. MPO is positive in myeloblasts granules, surrounding at least one third of the and promyelocytes, and TdT is positive in early nucleus of a nucleated red blood cell. lymphoid cells. Leukocyte alkaline peroxidase (LAP) is used for mature neutrophil activity, non- 72. c. Do¨ hle bodies are found in neutrophils. specific esterase (NSE) is positive in monocytic Multinucleate red blood cells (RBCs), nuclear cells, Sudan black B is used only for myeloid cells, bridging, basophilic stippling, siderotic granules, and brilliant cresyl blue is not used to determine and macroovalocytes may be seen in dyserythro- blast origin. poietic RBCs.

66. a. Imatinib mesylate and related drugs are used to tar- 73. d. Although iron granules may be seen in some mye- get p210 formed as a result of the BCR/ABL fusion lodysplastic syndromes, siderotic granules are gene in chronic myelogenous leukemia (CML). All- found in red blood cells, not in neutrophils or trans retinoic acid therapy is often used in cases of platelets. Pelgeroid nuclei in neutrophils and acute myeloid leukemia (AML), and 2-CDA/ abnormal granulation patterns in neutrophils and cladribine is a drug used for hairy cell leukemia. platelets are features of dyspoiesis of myeloid cells and platelets. 67. b. This is most likely a case of polycythemia vera, because the patient exhibits more than two of 74. c. Megaloblastic anemia, resulting from a deficiency the diagnostic criteria required by the World of vitamin B12 or folic acid can look similar to Health Organization (hemoglobin >16.5 g/dL, myelodysplastic syndrome, because decreased 9 platelet count >400 10 /L, and, additionally, vitamin B12 or folic acid can lead to a similar white blood cell count >12 109/L, although appearance, such as nuclear-to-cytoplasmic asyn- it’s not apparent from the information given chrony and a hypercellular dysplastic bone mar- whether an infection is present). Chronic myelog- row. Iron-deficiency anemia and a-thalassemia enous leukemia is less likely because of the lower minor exhibit microcytic/hypochromic cells with numbers of immature neutrophils and the higher relatively normal white blood cells (WBCs), and red blood cell count. Although bacterial infection warm autoimmune hemolytic anemia is a hemo- is not ruled out from this information, the combi- lytic anemia with normal WBCs, although red nation of the other elevated parameters is usually blood cells usually show macrocytosis, polychro- seen in cases of polycythemia vera. masia, and spherocytes.

68. b. Polycythemia vera differs from secondary polycy- 75. a. The most common abnormalities of chromo- themia because it exhibits elevated red blood cell somes in myelodysplastic syndrome (MDS) occur APPENDIX A Answers and Rationales to Certification Preparation Questions 327

in chromosomes 5, 7, 8, 11, 13, and 20. Although meningitis, infectious mononucleosis, or multiple multiple chromosome abnormalities have been sclerosis. If the physician suspected infectious implicated in MDS, currently 5q syndrome is mononucleosis, serologic examination specific the only abnormality specific to one disorder sub- to infectious mononucleosis would be ordered, type in the World Health Organization 2008 not a lumbar puncture. classification. 82. b. The patient most likely has Prolymphocytic leuke- 76. d. All are recurrent genetic abnormalities in acute mia (PLL), because prolymphocytes typically myeloid leukemia (AML) except AML with t look like lymphocytes with a prominent nucleo- (1;19)(q23;q13), (E2A/PBX1), which is an abnor- lus. Se´zary syndrome has lymphoid cells with a mality seen in some cases of precursor B-cell ALL. convoluted nucleus, and plasma cells are the cells seen in multiple myeloma. 77. c. Monocytic precursors are associated with 11q23 (MLL) abnormalities. Eosinophils are associated 83. d. Multiple myeloma shows all of the following with inv(16)(p13;q22) and t(16;16)(p13;q22), laboratory features except for decreased immuno- whereas neutrophils are associated with a variety globulin. Immunoglobulin is increased, because of other genetic abnormalities. Erythrocytes are the disorder is a monoclonal gammopathy, with currently not associated with a specific generality. increased IgG.

78. b. T-cell acute lymphoblastic leukemia (ALL) most 84. b. Reed Sternberg cells, sometimes described as commonly affects teenaged males who present “owl eye” cells, are present in cases of classic with a mediastinal mass, although it also may Hodgkin lymphoma. Plasma cells are seen in occur in adult patients in some cases. B-cell multiple myeloma and other plasma cell ALL is typically seen in children. neoplasms.

79. c. Acute promyelocytic leukemia falls under the clas- 85. c. Se´zary cells are lymphoma cells characterized by sification of acute myeloid leukemia (AML) with cerebriform nuclei and irregular nuclear outlines. recurrent cytogenetic abnormalities, because it manifests with t(15;17)(q22;q12), (PML/RARa). 86. b. The hexose monophosphate pathway is used to The others currently do not have a specific recur- reduce glutathione, which is used to help the cell rent cytogenetic abnormality and all are included combat oxidative damage. Adenosine triphos- in the “not otherwise classified” category. phate (ATP) is produced via the Embden- Meyerhof pathway. 2,3 diphosphoglycerate (2,3 80. a. This is a case of acute myeloid leukemia (AML) DPG) results from the Rapoport-Luebering with minimal differentiation. Early myeloid cell pathway. precursors and stem cell markers are present, while the patient is negative for terminal deoxy- 87. a. A normal reticulocyte count is 0.5% to 1.5%. nucleotidyl transferase (TdT), which rules out Values above 1.5% show the patient is responding lymphoid involvement. The patient also is nega- to an increased need for red blood cells (RBCs) by tive for myeloperoxidase and nonspecific ester- pushing out increased numbers of immature ase, in the presence of the early myeloid CD RBCs to the bone marrow. markers, which is consistent with AML with minimal differentiation. AML without matura- 88. d. Bone marrow examination is not indicated in the tion would have a similar flow cytometry profile, diagnosis of a standard case of iron deficiency, in but would have a positive myeloperoxidase which diagnosis can be made by using less inva- result. sive measures such as peripheral blood indices and serum iron chemistries. 81. b. The lumbar puncture would be ordered to determine if there was leukemic involvement in the 89. d. The myeloid-to-erythroid (M/E) ratio looks at central nervous system (CNS), because the the number of myeloid cells (M) to nucleated patient’s other results are consistent with possible erythroid cells (E) in the bone marrow. A ratio acute myeloid leukemia (ALL). If blasts are seen of 1:1 would mean the patient was producing in the CNS, intrathecal chemotherapy would be approximately the same number of red blood indicated in addition to a standard therapy regi- cells (RBCs) and myeloid cells. A ratio of 2:1 men. Orders for a bone marrow examination is in the normal range. A ratio of 1:2 indicates and flow cytometry are also indicated. Blasts the patient is showing erythroid hyperplasia, should not be seen in the peripheral blood in producing larger numbers of nucleated RBCs 328 APPENDIX A Answers and Rationales to Certification Preparation Questions

than white blood cells, which is often seen in proteins and protein 4.1R, which will lead to ineffective erythropoiesis. Patients with chronic instability in the red blood cell membrane. Spec- myelogenous leukemia (CML) will have ele- trin mutations may also appear in hereditary vated M/E ratios, because they are producing spherocytosis, in addition to mutations of sev- large numbers of myeloid cells with the malig- eral other proteins. nant cell clone. 98. d. Acid b-glycerophosphatase, cathespins, defen- 90. b. Macrocytosis without megaloblastosis can pre- sins, elastase, myeloperoxidase, and proteinase- sent in patients with liver disease, chronic alco- 3 are found in the primary granules of neutrophils. holism, and hypothyroidism and in other cases b2-Microglobulin, collagenase, gelatinase lacto- in which reticulocyte production is elevated. ferrin, and neutrophil gelatinase-associated Hemochromatosis is a disorder that leads to ele- lipocalin are found in the secondary (specific vated red blood cell production, not anemia. granules). Acetyltransferase, collagenase, gelatinase, lysozyme, b2-microglobulin are found in ter- 91. a. Diagnostic criteria for aplastic anemia include a tiary granules. Alkaline phosphatase, cytochrome hypocellular bone marrow, absolute neutrophil b558, complement receptor 1, complement 1q 9 count less than 1.5 10 /L, platelet count less receptor, vesicle-associated membrane–2 are 9 than 50 10 /L, and hemoglobin value less than found in the secretory granules. 10 g/dL, with decreased reticulocyte response (Rodak, 2012). 99. c. This sample, based on the intracellular red blood cell inclusions and boomerang-shaped or banana- 92. b. Mutations in a-thalassemia occur as a result of shaped structures is suspicious for a malarial deletions of one or more a-globin genes, and infection, particularly based on the patient travel mutations in b-thalassemia occur because of history. The sample should be referred to the reduced or absent expression of one or more b- pathologist or technical supervisor for confirma- globin genes. The a-globin gene is found on chro- tion and speciation. mosome 16, and the b-globin gene is found on chromosome 11. 100. b. The Donath-Landsteiner test can be used to demonstrate proximal cold hemoglobinuria by 93. d. This is a patient with a-thalassemia minor. An identifying glucose-6-phosphate dehydrogenase increased red blood cell (RBC) count would be deficiency. The osmotic fragility test and direct expected with hemoglobin, mean corpuscular antiglobulin test (DAT) would be less useful volume, and mean corpuscular hemoglobin for differential diagnosis. G6PD activity is used values lower than expected for the number of to identify cases of glucose-6-dehydrogenase red blood cells present. (G6PD) deficiency. 94. c. Hemoglobin (Hgb) F is present in larger quantities in fetal development and after a baby is born. As CHAPTER 4 the baby makes more b-globin chains than g-globin 1. chains, usually between 6 and 9 months of age, the b. When damage occurs to the endothelium, primary Hgb F decreases and the expression of the missing hemostasis occurs first, resulting in the formation or mutated gene becomes clinically apparent. of the primary platelet plug. Secondary hemostasis occurs next, which results in the formation of a 95. b. The thymus is associated with lymphocyte devel- stable fibrin clot. The last action is fibrinolysis, opment for precursor T cells, whereas the others which results in the breakdown of the clot. all develop in the bone marrow. 2. c. Under normal circumstances, vessels are non- 96. c. The equation used is number of cells counted/ thrombotic. Factors that contribute to this include (number of squares counted area of square a negatively charged surface; the inhibition of depth of square) reciprocal dilution to get platelet activation through prostacyclin, nitric cells/mL. Cells/mL 106/mL /L will convert the oxide, and ADPase; and the inactivation of throm- answer to cells per liter. For this patient, 105/(8 bin through heparin sulfate and thrombomodulin. 1mm2 0.1 mm) 20/1¼2625 cells/mL, Once damaged, tissue factor is one of the sub- which converts to 2.6 109/L. stances released that favors the formation of clots. Other prothrombotic substances include the 97. c. Hereditary elliptocytosis results from an auto- secretion of platelet-activating factor and von somal dominant mutation in the spectrin Willebrand factor. APPENDIX A Answers and Rationales to Certification Preparation Questions 329

3. a. Aspirin inhibits cyclooxygenase, which blocks 13. b. One of thrombin’s many actions is to cleave thromboxane A2 (TXA2) synthesis, thereby mak- fibrinopeptides A and B from fibrinogen. This ing platelets nonfunctional for the life span of the step results in the formation of a fibrin monomer, platelets. which can then continue to aggregate with other fibrin monomers. Factor VIII participates 4. c. Both fibrinogen and von Willebrand factor (vWF) in the common pathway. Tissue factor is released bind to platelets. Fibrinogen binds to platelets via by the endothelium during the initial stages the GbIIb/IIIa receptor. vWF binds through the leading to primary aggregation. Factor XIII is GpIb/IX receptor. involved in the covalent cross-linking of D domains of fibrin to form a stable fibrin clot. 5. a. Platelet agonists include collagen, adenosine diphosphate (ADP), thrombin, epinephrine, 14. b. Factor VII is involved in the extrinsic pathway of thromboxane A2 (TXA2), and arachidonic acid. coagulation, along with tissue factor. 6. b. Aspirin inhibits cyclooxygenase, which blocks 15. c. Protein S is a cofactor for protein C. thromboxane A2 (TXA2) synthesis, thereby making platelets nonfunctional for their life span. 16. d. Factor XIII covalently cross-links the D domains of fibrin to form a urea-insoluble clot. Factors II, 7. a. For secondary aggregation to occur, sufficient stim- V, and XI are involved in other parts of the hemo- ulusmustbepresent.Thestimulusoccursafterinter- stasis pathway. naladenosine diphosphate and calcium arereleased, alongwiththesynthesisandreleaseofthromboxane 17. c. Von Willebrand factor circulates as the vWF/VIII A2 (TXA2). Primary aggregation is reversible. complex. If circulating alone, factor VIII will be 8. b. Factor II is also called prothrombin. quickly degraded.

9. a. A deficiency of factor XII, as well as prekallikrein 18. b. A deficiency of factor IX is called hemophilia B. and high-molecular-weight kininogen, does not Hemophilia A is associated with a deficiency of usually manifest with clinical bleeding. Deficien- factor VIII. Hemophilia C is associated with a cies of factors IX, VIII, and VII all generally pre- deficiency of factor XI. sent with clinical bleeding depending on the degree of deficiency. 19. c. Fibrinolysis is the process of breaking down a fibrin clot. The activation of plasmin by plasmin- 10. d. Vitamin K is required for the g-carboxylation step ogen activators begins this process. of the formation of factors II, VII, IX, and X and proteins C and S. In this step, an additional car- 20. c. Plasmin is a serine protease with broad specificity boxyl group is added to the g-carbon of the gluta- against proteins that are susceptible to trypsin mic acid residues on the factors. Without this step, degradation. In terms of the hemostatic pathway, the factor is formed, but is not functional because plasmin has an effect against fibrin, fibrinogen, binding to a negatively charged phospholipid sur- and factors V and VIII. face cannot occur. 21. c. There are physiologic and exogenous plasmino- 11. b. The intrinsic pathway of hemostasis can be mon- gen activators. The exogenous plasminogen acti- itored through the partial thromboplastin time vators include streptokinase and staphylokinase. (PTT) assay. The prothrombin time (PT) assay The physiologic plasminogen activators include can be used to monitor the extrinsic pathway. tissue-type plasminogen activator and The thrombin time test assesses the formation urokinase-type plasminogen activator. of fibrin. The fibrinogen assay is used to measure fibrinogen levels. 22. d. When plasmin cleaves fibrin, the breakdown products that are formed include fragment X, 12. a. The prothrombin time (PT) assay can be used to fragment Y, fragment D, and fragment E. Frag- monitor the extrinsic pathway. The intrinsic path- ment X has a limited binding ability for thrombin. way of hemostasis can be monitored through the Fragments Y, D, and E all inhibit fibrin polymer- partial thromboplastin time (PTT) assay. The ization and inhibit platelet aggregation. thrombin time test assesses the formation of fibrin. The fibrinogen assay is used to measure 23. a. Plasminogen activator inhibitor-1 has a significant fibrinogen levels. role in limiting the activation of plasminogen. 330 APPENDIX A Answers and Rationales to Certification Preparation Questions

24. c. a2-Macroglobulin has wide specificity against deficiency of a factor in the intrinsic pathway. many proteases. It generally is not used until a2- Given the sex, age, and type of bleeding, evalua- antiplasmin is consumed. tions of factors VII and IX should be performed to check for hemophilia A or B. A normal PT rules 25. c. There are both positive and negative feedback out the possibility of a common pathway defi- mechanisms in the control of hemostasis. Positive ciency, which rules out a deficiency of X or V. feedback mechanisms include the thrombin acti- The symptoms and laboratory results are not sug- vation of platelets, release of platelet factor Va, gestive of a platelet abnormality. Factor V Leiden exposure of the negatively charged surface, and is a condition associated with thrombosis. activation of factors Va and VIIIa. Negative feedback mechanisms include thrombin’s involvement 32. a. In dysfibrinogenemia, the structure of fibrinogen in the protein C pathway, tissue factor pathway is abnormal. Fibrin still forms, resulting in nor- inhibitor (TFPI) inactivation of factor Xa, fibrin mal PT and PTT evaluations. As a result of the binding to thrombin, and the interference of fibrin structural abnormality, the thrombin time that degradation products (FDPs) in fibrin formation assesses fibrinogen to fibrin formation is and polymerization. affected.

26. c. In the protein C pathway, protein S serves as a 33. a. Because of compensation of other factors in the cofactor C. Protein S circulates bound to C4BP. hemostatic pathway and the sensitivity of the In the presence of protein S and calcium, activated reagents, factor levels must be reduced to 30% protein C inactivates factors Va and VIIIa. or less before prolongation is observed.

27. a. Acute idiopathic thrombocytopenic purpura 34. c. Disseminated intravascular coagulation is a disor- (ITP) is typically observed in children (ages 2-4) der of consumption. Coagulation proteins, and follows viral infections. Affected patients including fibrinogen, and platelets are all con- may be asymptomatic or have severe mucosal sumed in thrombi, resulting in a prolongation of bleeding. Patients will have a decrease in platelets the prothrombin time (PT) and partial thrombo- and increase in lymphocytes and eosinophils. plastin time (PTT) and decreased fibrinogen and Patients with chronic ITP are usually adults. platelet count. The D-dimer is significant and can help rule out other conditions, because it indi- 28. d. Bernard-Soulier syndrome and Glanzmann’s cates that thrombi are being formed. The D-dimer thombasthenia are both qualitative platelet disor- would be negative in fibrinogenolysis, fibrinogen ders. The bleeding time will be prolonged in both deficiencies, and vitamin K deficiency. In vitamin conditions. The platelet count may be affected in K deficiencies, the fibrinogen assay, platelet count, either disorder. Platelets are not assessed using pro- and D-dimer assay would be normal. thrombin time (PT). The conditions can be differentiated using platelet aggregation tests. In patients 35. d. Factor V deficiency results in a bleeding condi- affected with Bernard-Soulier, responses to adeno- tion because it is part of the common pathway. sine diphosphate (ADP), collagen, and epinephrine Both the prothrombin time (PT) and partial will be normal, whereas the response to ristocetin thromboplastin time (PTT) would be abnormal. is abnormal. The Glanzmann’s abnormal Protein C deficiency, antithrombin deficiency, responses are observed with ADP, collagen, and and the factor V Leiden mutation are all associ- epinephrine, but normal with ristocetin. ated with thrombosis.

29. c. The pathophysiology of Glanzmann’s throm- 36. c. Heparin is an anticoagulant that functions by basthenia is the deficiency of GpIIb/IIIa. significantly accelerating the activity of antithrombin. 30. d. In D-storage pool deficiency, platelets have a decrease in or absence of dense granules. This 37. d. Fibrinogenolysis is a condition in which plasmin results in the lack of a secondary wave of aggrega- is generated without the generation of thrombin tion when the agonist adenosine diphosphate or thrombi. As clots are not formed, D-dimers (ADP) is used. Aggregation with collagen and epi- are not produced. In disseminated intravascular nephrine is also deficient. A normal response to coagulation, thrombi are formed, and after fibri- ristocetin is observed. nolysis, D-dimers are measurable in the blood.

31. a. The abnormal partial thromboplastin time (PTT) 38. a. An abnormal prothrombin time (PT) will be and normal prothrombin time (PT) suggest a observed in deficiencies or in the presence of an APPENDIX A Answers and Rationales to Certification Preparation Questions 331

inhibitor. When patient plasma is mixed with (vWF) is not assessed in the partial thromboplastin normal pooled plasma, the deficient factor is time (PTT) assay, vWF also carries around factor added back, resulting in a correction of the PT. VIII in the circulation to protect it from degrada- If an inhibitor was present, the PT mix with tion. Therefore a decrease in vWF sometimes normal pooled plasma would not result in a results in a prolongation of the PTT assay because correction. of the lower factor VIII levels, which are corrected by the addition of normal pooled plasma. 39. c. The principle of the platelet aggregation assays is that the formation of platelet aggregates will 47. c. International normalized ration (INR) results of decrease the optical density of platelet-rich 1.75 and 1.74 are essentially the same. The INR plasma when an agonist is added. If added to is used to standardize the prothrombin time platelet-poor plasma, aggregation would not (PT) assays among laboratories and is compared occur, because of the lack of platelets. to assess degree of anticoagulation when the testing is performed at different laboratories. 40. b. The goal of the platelet aggregation assay is to assess platelet function. If platelets are not functional, as is 48. c. It is likely that this patient has a deficiency of one the case when aspirin is ingested, the results will not of the contact factors (factor XII, prekallikrein, or be reflective of the patient’s platelets. Fasting is not high-molecular-weight kininogen). Although the required. Freezing the plasma will cause the plate- deficiency of these factors results in a very pro- lets to aggregate before evaluation. longed partial thromboplastin time (PTT), bleeding complications are not observed. 41. b. Fibrinogen is an acute phase protein that will cause levels to increase in conditions in which 49. d. In a patient with a history of miscarriages, one an acute phase reaction is observed. In dissemi- concern is the presence of lupus anticoagulants. nated intravascular coagulation, fibrinogen is a To assess this condition, a panel would include protein that is consumed. Fibrinogen levels are the following tests to rule in or rule out the pres- absent in afibrinogenemia. In liver disease, fibrin- ence of such an anticoagulant: dilute Russell viper ogen production is decreased. venom time (DRVTT, to indicate an anticoagulant against phospholipids), prothrombin time 42. c. The thrombin time test is performed by adding an (PT) and partial thromboplastin time (PTT), excess of thrombin to the patient specimen to and PT and PTT with normal pooled plasma (to assess fibrinogen-to-fibrin formation. Thrombo- rule out a factor deficiency and demonstrate the plastin is used in the prothrombin time (PT) assay. presence of an inhibitor). The 5 M urea solubility test is used to assess factor XIII levels. 50. d. The activated protein C resistance (APCR) test is a screening test that will be abnormal in the pres- 43. c. Factor IX–deficient plasma contains all coagula- ence of a factor V Leiden defect. It is a clot-based tion factors with the exception of factor IX. If test based on the inability of activated factor V to added to asses a patient’s factor VIII level, the fac- be inactivated that is quick to perform and tor VIII present in the deficient plasma will result cheaper than the molecular-based factor V Leiden in an analytical error. test. The other tests listed (protein S, antithrombin, and prothrombin 20210) are all associated 44. c. Calcium chloride and partial thromboplastin are with deep vein thromboses if abnormal, but will the needed reagents for the partial thromboplastin be normal in the presence of the factor V Leiden time (PTT) assay. defect.

45. a. The international normalized ratio (INR) is CHAPTER 5 reported out in conjunction with the prothrombin time (PT) result. It helps to standardize the PT 1. b. The ultrafiltrate that enters Bowman’s capsule results for variations in instrumentation, reagents, from the glomerulus has the same specific gravity and personnel. as protein-free plasma, roughly 1.010.

46. c. von Willebrand’s disease is a qualitative platelet 2. a. Bacteria will continue to proliferate unless refrig- disorder. In this condition, the platelet aggregation erated, reducing urine nitrates to nitrites. response to ristocetin is abnormal. The bleeding time is increased as a result of the qualitative defect 3. d. Orthostatic or postural proteinuria is character- in the platelets. Although von Willebrand factor ized by an elevated protein while a person is in 332 APPENDIX A Answers and Rationales to Certification Preparation Questions

the upright (standing) position and normal pro- 14. d. Urobilinogen is a product of bilirubin metabo- tein excretion in a sitting or reclined position. lism. During hemolysis, red cells are broken down and the bilirubin is converted to urobilinogen. 4. c. A timed urine collection, such as a 24-hour collection, involves voiding and discarding the specimen 15. b. For specific gravity detection, in the presence of at the beginning of the collection and voiding and cations, protons are released by a complexing collecting the specimen at the end of the timed col- agent to produce a color change. lection. If all urine collected during that time is not calculated into the total volume, constituents pre- 16. c. Yeast is often associated with patients having sent may be falsely decreased. diabetes mellitus, where results are consistent with acidic urine and positive glucose on the 5. a. The method of detection includes two indicators, dipstick. methyl red and bromothymol blue, which produce a color change from blue or blue-green to yellow. 17. d. The presence of dysmorphic red blood cells (RBCs) in urine suggests glomerulonephritis. Dys- 6. c. Testing for protein is based on the research findings morphic RBCs are misshapen because they have bySorensen and calledtheprotein errorofindicators, been distorted when passing through the abnor- which is the ability of protein to alter the color of mal glomerular structure. some acid-base indicators without altering the pH. 18. c. Renal tubular cells are seldom seen in urine and 7. a. Glucose detection is based on the enzymatic oxi- are found only in the renal tubules. The urinary dase/peroxidase method, in which glucose oxi- tract from the pelvis down the ureters to the blad- dase catalyzes the formation of gluconic acid der and the proximal urethra is lined by transi- and hydrogen peroxide from the oxidation of glu- tional epithelial cells. Squamous epithelial cells cose. The second enzyme, peroxidase, then cata- originate from the distal urethra and vagina. lyzes the reaction of hydrogen peroxide with tetramethylbenzidine to form a colored complex. 19. a. The proteins involved in cast formation are secreted by the lining cells of the distal tubules 8. d. Glucosuria is glucose present in the urine. When and the collecting ducts. no hyperglycemia is present in the patient, it can be due to an acquired or inherited defect in the 20. c. Ethylene glycol poisoning can result in the presence glucose transport or another renal tubular of calcium oxalate monohydrate crystals in urine. disorder. 21. d. Triple phosphate crystals are present in alkaline 9. d. Ketonuria is the presence of ketones in the urine. urine, so the discrepancy lies in the acid pH or When carbohydrates are unavailable, fatty acids the crystal type. are used for energy, resulting in the production and excretion of ketones. 22. c. Goodpasture’s syndrome is an autoimmune disease that attacks the kidneys, leading to kidney 10. a. Hemoglobin (Hgb) and myoglobin catalyze the failure. Anti–glomerular basement antibodies are oxidation of the chromogenic indicator by the antibodies to the kidney membranes involved in peroxide in the test pad. This is due to the strong the disorder. pseudoperoxidase action of Hgb and myoglobin. 23. a. Calcium oxalate is the most common renal crystal 11. c. Myoglobin is a protein found in heart and skeletal observed. muscle. When muscle is damaged, myoglobin is released. It may be detected after muscle trauma 24. c. The additional finding of white blood cell casts or strenuous exercise. signifying infections within the tubules is a primary diagnostic indicator for both acute and 12. c. Renal calculi are small crystals or stones present in chronic pyelonephritis. the kidneys and causing obstruction. Microscopi- cally, red blood cells can be seen. 25. c. Henoch-Scho¨ nlein purpura is a disease occurring primarily in children after upper respiratory tract 13. a. Bilirubin accumulates as a result of a block in the infections and includes symptoms of raised red bile duct. When bilirubin builds up, it will be pre- patches on the skin. Renal involvement may sent in urine. This is direct, or conjugated, biliru- include mild proteinuria with hematuria and red bin and is a result of the obstruction. blood cell casts. APPENDIX A Answers and Rationales to Certification Preparation Questions 333

26. b. Focal segmental glomerular nephritis affects 38. d. Also called the Ropes test, the extent of polymer- certain areas of the glomerulus and comprises ization of hyaluronic acid is determined. The IgM and C3 deposits. It is associated with heroin stronger the clot, the greater the viscosity of the abuse, analgesics, and acquired immunodefi- synovial fluid. ciency syndrome. 39. b. In the mucin clot test, the presence of a tight 27. c. Waxy and broad casts are associated with chronic rope–like clot indicates the polymerization of glomerulonephritis. All the other choices are often hyaluronic acid in the presence of acetic acid. associated with both acute and chronical glomerulonephritis. 40. a. Monosodium urate crystals appear needle shaped under polarized light and are yellow when aligned 28. d. Renal tubular epithelial cells are present in renal with the slow vibration of compensated polarized tubular necrosis, not an inflammation of the blad- light der (cystitis). 41. d. An effusion is an accumulation of fluid in a body 29. c. Cerebrospinal fluid is produced in the choroid cavity. plexus of the third and fourth ventricle and the lumbar ventricles. 42. d. A transudate is typically clear to pale yellow, with a ratio of fluid to serum lactate dehydrogenase of 30. b. A traumatic tap can be identified by having the less than 0.6, fluid-to-serum protein ratio of less largest concentration of red blood cells in tube than 0.5, and a total cell count of less than 1 and decreasing red cell concentrations present 1000 mL in tube 2 and tube 3. 43. b. Increased neutrophils in a pericardial fluid exu- 31. a. IgG index values greater than 0.8 are indicative date indicate a bacterial infection. of IgG production within the central nervous system. 44. c. Thoracentesis is the process of removing fluid from the pleural space, the space between the 32. a. Low protein values in cerebrospinal fluid indicate lungs and the chest wall. the central nervous system is leaking fluid. 45. a. Peritonitis is an infection of the peritoneum, the 33. b. In cerebrospinal fluid, increase in lactate abdominal lining, caused by a bacterial infection. can be associated with bacterial, fungal, and Cirrhosis is a chronic degeneration of the hepato- tubercular meningitis at levels greater than cytes along with inflammation in the liver and can 25 mg/dL. be associated with alcoholism or viral hepatitis. The presence of neutrophils would support 34. c. In protein electrophoresis of cerebrospinal fluid, a peritonitis. transferrin band for t transferrin is present, which is a protein produced mainly by the central ner- 46. a. Amniotic fluid for fetal lung maturity testing vous system. should be placed on ice on collection and refrigerated up to 72 hours before testing. If testing will not 35. c. Protein electrophoresis is performed on cerebro- occur within 72 hours, the specimen can be frozen. spinal fluid to detect oligoclonal bands migrating in the g region, unlike the bands detected during 47. d. a-Fetoprotein is determined using maternal serum protein electrophoresis. Detection of the serum; this value is then converted to multiples oligoclonal bands aid in diagnosing multiple of the median (MOM), in which the maternal sclerosis. value is related to the median fetal gestational age at the time of testing. Values greater than 36. d. The concentration identifies the amount of sperm 2.5 MOMs are considered positive screen for neu- per microliter. To identify the total amount of ral tube defects. sperm present, multiply the concentration (12,000/mL) with the total volume (3 mL) to yield 48. c. Steatorrhea, or fatty stool, is characterized by pale a total sperm count of 36,000 mL. color,bulkiness or frothiness, or a greasy appearance.

37. c. Fructose comprises 99% of normal reducing 49. d. The acid steatocrit is a rapid test to estimate fat sugar in semen; with normal levels of semen, fruc- secretion via stool and is used to monitor therapy tose is 13 mmols or greater per ejaculate. or screen for steatorrhea. 334 APPENDIX A Answers and Rationales to Certification Preparation Questions

50. c. Elastase 1 is an isoenzyme of elastase, a pancreatic complement activation can have chemotactic enzyme, that can be detected in stool at concentra- properties. Complement proteins also act as opso- tions 5 times higher than pancreatic secretion. It is nins to enhance phagocytosis to help clear debris measured by enzyme-linked immunosorbent from inactive and dead cells. (ELISA) testing. 11. d. C3 convertase cleaves C3 to C3a and C3b. C3a is CHAPTER 6 released into the plasma and can act as an anaphy- latoxin. C3b is released to combine with C42a in 1. c. Low acidic environment and enzymes in secre- the classical pathway or C3bBb in the alternate tions, coughing, and gastrointestinal tract and pathway of complement activation to form C5 skin bacteria are natural barriers to invading path- convertase. Extra C3b proteins can act as an ogens and antigens. opsonin by coating pathogens and attachment to the Fc portion of immunoglobulins. 2. d. The primary organs are where lymphocytes reside to mature. Once mature, they leave the primary 12. d. Certain cytokines produced can enhance the differ- organs and migrate to the secondary organs, entiation of lymphocytes to maturity. Cytokines where they await activation. can have many different effects (pleomorphic) on cells outside of the immune system. Also, different 3. a. Toll-like receptors (TLRs) are molecules on cytokines can have the same effect on the same cell, phagocytic cells that recognize certain substances thus making them redundant. or molecules that reside on surfaces of some bacteria. The TLRs recognize and bind to these 13. b. Variations in the variable regions of the heavy and substances, enhancing the phagocytic process. light chains of an immunoglobulin molecule define the idiotype. 4. a. Once bound to a specific antigen, antibodies can act as opsonins. The Fc portion of the immuno- 14. globulin molecule attaches to the receptor mole- c. Mannose-binding lectin (MBL) of the lectin cules on monocytes and neutrophils to enhance pathway of complement activation is found in phagocytosis. circulation complexed with proteinases. It is considered to be similar in structure to C1q of 5. c. CD10 appears early in the B-cell development and the classical pathway. The MBL-proteinase com- is lost after the immature stage, making this an plex does not require antibody for complement early B-cell marker. CD20 begins to appear during activation. the immature stage of B-cell development; it is found on the later stages and is a marker for the 15. c. Viruses use cellular mechanisms and processes for later stages of B-cell development. replication. These antigens synthesized inside a cell are expressed on the cell surface in the form 6. a. At the double-positive stage of development, this of major histocompatibility complex (MCH) class T cell expresses both CD4 and CD8. I molecules, which are then presented to CD8 cytotoxic T lymphocytes for cell lysis. 7. a. Natural killer (NK) cells are lymphocytes (cells of the adaptive immune system) that have a cyto- 16. b. Precipitation reactions occur between soluble toxic effect against cellular pathogens without antigen and soluble antibody that produce a visi- prior known exposure (innate system characteris- ble end result typically in the form of a visible line tic). Thus these cells have been considered a bridge of precipitate. Agglutination reactions occur between the systems. when the antigen is particulate or coated on a particulate such as latex beads. 8. d. T cells are involved in cell-mediated immunity, whereas B cells that make antibodies are involved 17. c. In agglutination tests where postzone is occurring, in humoral immunity. there are so many antigens in the test system, that each antibody reacts singly with the antigen, so no 9. b. Proteins that are made up of amino acids can be lattice formation occurs and false negative reac- much more structurally and conformationally tions are seen. complex than the sugars of carbohydrates. 18. d. Antibodies that react against different antigenic 10. d. The complement pathways result in cell lysis. determinants (epitopes) of the same specific anti- Other complement proteins released after gen are considered to be polyclonal. APPENDIX A Answers and Rationales to Certification Preparation Questions 335

19. b. In radial immunodiffusion, known antibody is 29. c. The presence of anti-HBs in patient serum is indic- added to the gel. Patient serum contains the anti- ative of recovery from a hepatitis B infection. It also gen to be tested. may indicate immunity resulting from vaccination.

20. a. The patient serum contains the antigen to be 30. b. Human leukocyte antigen (HLA) genes are inher- tested. The gel contains known specific antibody. ited as haplotypes; one haplotype from each The area of the precipitation ring around the cen- parent. ter well is directly proportional to the concentration of the antigen in the sample tested. 31. c. Visible agglutination and precipitation reactions depend on the antigen and antibody concentrations 21. d. The indirect antiglobulin uses commercial red that are in equivalence. It is at this point that pre- cells with known antigens to test for unknown cipatation and lattice formation occurs. Too much antibody in a patient serum sample (e.g., antibody antigen results in postzone phenomenon, and too screening test). The direct antiglobulin tests much antibody results in prozone. patient red cells coated with antibody that occurred in vivo (e.g., hemolytic disease of the 32. b. Type I hypersensitivity occurs as a result of the newborn). release of the granular contents of mast cells when bound to IgE antibodies cross-linked with 22. a. Indirect tests are performed in two steps. The antigen. first step is incubating (cytomegalovirus [CMV]) antigen with specific antibody (patient serum). 33. c. Anti-dsDNA autoantibodies are present in The second step is adding anti-human globulin patients with systemic lupus erythromatosus (antibody to CMV antibody) coupled with the and, when identified, can be considered diagnostic fluorescent dye for visualizing the reaction. of the disease.

23. d. Nepholometry measures light at angles. The light 34. d. The rheumatoid factor is antibody directed source used for detection is placed at an angle against the Fc portion of the IgG molecule. from the detection device. Turbidometry detection devices are placed directly across from the 35. b. Rh disease of the fetus and newborn is not an light source and measures the intensity of the light autoimmune disease but is classified as a type II as it passes through solution. hypersensitivity reaction.

24. a. A crossed line in the Ouchterlony diffusion indi- 36. c. Autoantibody to the thyroid-stimulating hormone cates the antigens do not share epitopes in com- receptor ultimately causes release of thyroid hor- mon and is therefore considered nonidentity. mones and a hyperthyroid condition.

25. a. In a twofold serial dilution, a rise in titer results 37. d. Antithyroglobulin is a common autoantibody in between the initial titer and subsequent titer of Hashimoto’s thyroiditis resulting in elevated more than two tubes or fourfold, the results are levels of thyroid-stimulating hormone and considered diagnostic of infection. hypothyroidism.

26. c. On activation, certain subsets of T lymphocytes 38. d. Type IV hypersensitivity is the delayed-type produce growth-enhancing cytokines that act on hypersensitivity. Skin testing for tuberculosis B cells to mature to antibody-secreting plasma causes a delayed-type hypersensitivity to intrader- cells. B cells would not be able to mature if a defi- mally injected antigens in individuals previouly ciency of T cells existed. exposed to the organism.

27. b. In the serum of a 2-week-old baby, a titer of IgG 39. c. The fluorescent treponemal antibody absorption anticytomegalovirus would be representative of (FTA-ABS) test is a confirmatory test that detects the maternal antibody that crossed the placenta specific antibodies to Treponemal pallidum in and not the baby’s antibody. patient specimen.

28. b. Both rapid plasma reagin (RPR) and Venereal 40. c. The most likely answer is acute hepatitis B Disease Research Laboratory (VDRL) tests are because of the presence of IgM anti-HBc in com- nontreponemal tests that detect antibody to cardi- bination with the hepatitis surface antigen. Typi- olipin. The RPR test is a macroscopic agglutina- cally the presence of IgM indicates the presence tion, and the VDRL test is read microscopically. of an acute phase of a disease. 336 APPENDIX A Answers and Rationales to Certification Preparation Questions

41. a. Lymphomas are generally classified as malignan- 50. c. The hyperacute tissue graft reject occurs within cies of lymphoid tissue. Leukemias are generally minutes to hours of a transplant and is typically classified as malignancies of hematopoietic cells associated with transplantation across ABO of the bone marrow or peripheral blood. Both blood groups and anti-ABO antibodies. can be classified as acute or chronic.

42. b. Bruton’s aggammaglobulinemia is typically seen CHAPTER 7 in infancy. These patients present with frequent recurring infections, especially after protective 1. c. N-Acetylgalactosamine (GalNAc) confers type maternal antibody is gone and normal levels of A specificity. D-Galactose (Gal) confers type circulating T cells. The syndrome is a genetic B- B specificity. L-Fucose (Fuc) confers type O specific- cell enzyme deficiency in which the B cells fail ity. The type A and type B sugars are built on Fuc. to differentiate and mature to antibody- producing plasma cells. 2. d. The A2 subgroup is described as having both qualitative and quantitative differences when com- 43. d. Hereditary angioedema is characterized by recur- pared to the A1 subgroup. This means that there rent swelling. The condition is genetic or can be is less A antigen found on the red cells of people acquired and is the result of a deficiency of the with A2, and their A antigen looks “different” complement protein C1 Inhibitor. when compared to that of people with type A1. Therefore the red cells from those with type A2 44. a. DiGeorge syndrome is the most likely cause. In will not react with anti-A1 lectin. Approximately this syndrome the thymus fails to develop before 20% of those with type A have the A2 phenotype, birth. These patients also show a marked decrease and between 1% and 8% of those individuals in T cells. make anti-A1. There is no anti-A2 reagent, and people with the A2 phenotype would not react 45. d. Severe combined immunodeficiency is a genetic with A2 cells, because that would imply an auto- condition diagnosed in infancy. Both B-cell and antibody is present. T-cell development/function are arrested, resulting in no cell-mediated or humoral immunity. 3. b. People with the Bombay phenotype do not express the H antigen. An individual with the 46. b. Chronic granulomatous disease is an inherited hh genotype would not express H antigen. disease that impairs the neutrophil’s ability to kill certain bacteria. The neutrophils lack the enzyme 4. d. The RHD gene encodes for the D antigen. nicotinamide adenine dinucleotide phosphate The RHCE gene determines C, c, E, and e speci- oxidase, easily demonstrated by the failure to ficities. Therefore the correct answer must include reduce nitroblue tetrazolium or produce a blue both genes. end result. These patients have normal levels of lymphocytes. 5. a. In indirect antiglobulin test (IAT) phase, a control tube is included to show that agglutination 47. b. Because common variable immunodeficiency is detected in the patient test tube is appropriate. commonly diagnosed in early adulthood, this is In weak D testing, the control tube uses saline the most likely explanation for this case study. rather than anti-human globulin (AHG) reagent. Bruton’s, X-linked agammaglobulinemia, and Agglutination in the control tube suggests that DiGeorge’s syndrome are all typically identified the patient cells are already sensitized with immu- in infancy. noglobulin, so a positive reaction in the patient test tube should be invalidated and investigated. 48. d. Waldenstro¨ m’s macroglobulinemia is a disease resulting from an overproduction of an IgM pro- 6. d. Individuals with the D phenotype may possess ducing B-cell population. The monoclonal IgM more D antigen because they have inherited a non- can be seen as a spike on electrophoresis. functioning RHCE gene. Basically, the RHD gene of D individuals hasno competition whenbuild- 49. c. Inflammation is caused by the increased blood ing D antigen, so they end up with more. Rh-null flow and subsequent influx of neutrophils and individuals do not express any Rh antigens. D- other cells to an infected site. The increased blood positive individuals possess the D antigen, but with- flow and activity of the cells, including cytokine out knowing the genotype, the amount of D antigen production, results in the redness, pain, and swell- cannot be estimated. A person with the dce/dce ing to the area. genotype would be considered Rh-negative. APPENDIX A Answers and Rationales to Certification Preparation Questions 337

7. c. The Duffy blood group system contains two also called the Donath-Landsteiner antibody. It is a codominant alleles, Fya and Fyb, as well as Fy3, biphasic hemolysin that attaches to P-positive red Fy5, and Fy6. The Fya,Fyb, and Fy6 antigens cells at lower temperatures. Complement is are sensitive to ficin, and antibodies made against attached, and when the red cells are warmed to the Duffy antigens can show dosage. Approxi- 37 C, hemolysis occurs. The other antibodies mately 68% of the black population is Fy(ab). listed are typically RT reactive antibodies.

8. b. The I antigen is a precursor to the H antigen, so 14. c. Coombs check cells are used to verify that anti- individuals who express the H antigen are pre- human globulin (AHG) has been added to reagent sumed to have the I antigen. Therefore, if an indi- tubes and that it is active. When the check cells do vidual expresses anti-I, it is typically considered not agglutinate, either AHG was not added or it to be an autoantibody. Anti-I typically presents was somehow inactivated. This usually occurs as a cold-reacting, clinically insignificant IgM because of inadequate washing. If the check cells autoantibody. do not work, the entire test should be repeated.

9. a. Dithiothreitol (DTT) disrupts the tertiary structure 15. a. Because enzyme treatment removed the reactivity of proteins, and denatures the Kell system antigens noted in the original panel, we can infer that the on red cells. Chloroquine diphosphate (CDP) can unexpected antibody is directed to an antigen that be used to dissociate antibodies from red cells. is sensitive to enzymes. Of the antigens given in the Anti-human globulin (AHG) reagent is used in list, only the Fya antigen is sensitive to enzymes. the indirect antiglobulin test (IAT). Low ionic strength solution (LISS) is used as a potentiator. 16. b. Whenever an unexpected antibody is currently reactive or noted in a patient’s history file, an indi- 10. c. The Kidd blood group system contains three anti- rect antiglobulin test (IAT) crossmatch must be gens, Jka,Jkb, and Jk3. Antibodies made against performed. An immediate spin or electronic cross- this blood group system are typically IgG, are best match is performed only on patients with no evi- detected by indirect antiglobulin test (IAT), are dence of clinically significant antibodies, enhanced by treating reagent red cells with currently and historically. enzymes, and can show dosage. Antibody titers have also been found to increase and then quickly 17. d. A positive direct antiglobulin test (DAT) implies decrease in patients. that the patient has an IgG antibody attached to the red cells. An elution procedure would dissoci- 11. d. The Le gene adds fucose to either a type I precur- ate the antibody from the red cells and collect it so sor chain to make the Lea antigen or adds fucose the specificity can be determined. An adsorption to the H structure to make Leb. Type II chains removes red cell antibodies from plasma by never express Lewis antigen activity. Newborns adsorbing antibody onto red cells. Neutralization express the Le(ab) phenotype. Antibodies is performed to inactivate an antibody present in against the Lewis antigens are typically room tem- plasma. A titration is performed to determine perature (RT) reactive, IgM class, and not clini- how much antibody is present in plasma. cally significant. Because they are typically IgM class, they cannot cross the placenta. 18. b. A positive rosette test is a qualitative indicator that a fetal bleed has occurred. This is important to 12. a. The U antigen is located near the red membrane on detect in Rh-negative mothers who have an Rh- glycophorin B (GYB), so is always present when S positive baby. To provide the correct dosage of or s is inherited. The amino acid structure on GYB Rh immunoglobulin, a quantitative test must be is the same as the first 26 amino acid sequence on performed to quantify the amount of bleed that glycophorin A (GYA), so the only individuals who occurred. The Kleihauer-Betke test looks for fetal can make anti-N are those who lack GYB. Anti-M hemoglobin in a sample collected from the mother. is typically an IgM class antibody, usually considered to be clinically insignificant. The effect of 19. a. Thrombotic thrombocytopenic purpura (TTP) is enzymes on the S antigen is variable. characterized by thrombocytopenia, microangiopathic hemolytic anemia, renal dysfunction, and 13. b. Allo-anti-P is a rare antibody made by individuals central nervous system involvement. Basically, k with the P2 phenotype, so most examples of anti-P giving platelets to a patient with TTP provides seen in the blood bank are actually autoanti-P. fuel that would exacerbate the condition. The Autoanti-P is associated with paroxysmal cold other conditions listed are all appropriate indica- hemoglobinuria. This antibody is an IgG antibody, tors for platelet transfusion. 338 APPENDIX A Answers and Rationales to Certification Preparation Questions

20. a. In disseminated intravascular coagulation, plate- 27. c. In vitro, complement is detected through the use lets and fibrinogen are inappropriately consumed, of anti-C3b or anti-C3d reagents. When detected, and so transfusion therapy should be targeted this indicates complement proteins have been toward replacing those elements. Cryoprecipitate attached to the red cell surface as a result of the contains in a concentrated form most of the coag- activation of complement’s classical pathway. ulation factors found in fresh frozen plasma. In vivo, complement activation may proceed to These include von Willebrand factor, fibrinogen intravascular hemolysis if conditions are right, (I), factor VIII, fibronectin, and factor XIII. Cryo- which would result in the lysis of red blood cells. precipitate is primarily used clinically for patients with deficiencies of factor XIII and fibrinogen. 28. c. Hemolysis as a reaction end-point indicates the presence of a complement-activating antibody. 21. b. The best answer to this question is genetic inher- This reaction is especially important to recognize itance, which includes but is not limited to the because it is an indicator of an antigen–antibody mother’s blood type. Inheritance of the ABO anti- reaction. When hemolysis occurs, the red cell but- gens are driven by the ABO, H, and Se genes. ton is often smaller than buttons in other tubes Genetic inheritance, environmental factors, and and the supernatant may appear to be pinkish immune function would influence the presence or reddish. of antibodies against certain ABO antigens. 29. a. Mixed-field (mf) agglutination indicates the pres- 22. e. The patient can receive all of the blood types listed, ence of two red cell populations when noted: one but good blood management would dictate the that is agglutinating, and one that is not. Of the sce- order in which they were transfused. Generally, narios presented, a person undergoing a delayed the best course of action would be to transfuse type hemolytic transfusion reaction is most likely to A first because it is usually more plentiful than type have two cell populations in circulation—his/her B. Patients with O type can receive only type O own red cells and those from the transfusion. In blood, so it is best to conserve type O when possi- a delayed transfusion reaction, an antibody has ble. If type A is not plentiful, type B can be given. been stimulated against the transfused cells. A pos- Once either type A or type B is given, types should itive direct antiglobulin test (DAT) in this scenario not be mixed, to help avoid potential reactions. If indicates the antibody has attached to the trans- type A or B is exhausted, it is then appropriate to fused cells but not to the patient’s own cells; there- move to type O. fore mixed-field reactivity is noted.

23. d. An amorph, or silent, gene does produce a detect- 30. d. Patient’s with hypogammaglobulinemia have an able antigen product. Examples of amorph, or overall reduction of g-globulins and may not be null, phenotypes include Rhnull, O, and Lu able to reverse grouping antibodies at levels detect- (ab). able by ABO typing tests. IgM antibodies, other than those normally detected in reverse grouping 24. d. Most blood group systems genes exhibit codomi- tests, or cold autoantibodies may interfere with nant expression, or equal expression of both reverse grouping tests, making the results invalid. traits. In an autosomal recessive inheritance pat- One example of an IgM antibody that may inter- tern, a trait is observable only when not paired fere with reverse grouping is anti-M. with a dominant allele. X-linked and Y-linked inheritance patterns are complex and not typi- 31. b. Cells that are direct antiglobulin test (DAT)- cally seen in most blood group systems. positive or already have IgG attached would give a false-positive reaction when tested by the indi- 25. d. This answer is best explained through the use of a rect antiglobulin test (IAT). The IAT is used to Punnett square: detect antibody bound to red cells in vitro. IAT is a two-stage procedure. In the first stage, anti- AO BABBObodies are encouraged to combine with their cor- OAOOOresponding antigen during an incubation step. If DAT-positive red cells (cells that have antibody 26. a. IgM is classified as having a large pentamer struc- already attached) are used in an IAT test, the first ture. Thus only one IgM is required to initiate the step, in effect, has already occurred. The attached classical pathway in the complement system. In IgG molecules will be detected in the second IAT comparison, it takes two IgG molecules to acti- step, the agglutination step, and give a false- vate complement. positive reaction. APPENDIX A Answers and Rationales to Certification Preparation Questions 339

32. a. An elution is a process that dissociates antigen– 39. a. People with the AB blood type are considered to antibody complexes on red cells. An adsorption be universal recipients because they possess all is a process that uses red cells to remove red cell possible ABO antigens; therefore they do not antibodies from a solution. Prewarming is a tech- make any antibodies to ABO antigens. People nique in which all reagents and patient samples with AB who are also negative for the D antigen used in a test procedure are incubated to reach may receive any type of blood as long as they have 37 C (or the preferred testing temperature) not made an antibody against the D antigen. before the test is conducted. Neutralization combines a soluble antigen with antibody in vitro and 40. b. Transfusing 1 unit of red cells usually increases is used as an antibody identification technique. the hemoglobin (Hgb) by approximately 1 g/dL. Therefore the best answer is elution. Therefore transfusing 2 units of red cells to a patient not actively bleeding should increase the 33. d. Before a reagent is used, it should be assessed to pretransfusion Hgb by 2 g/dL. determine if it meets preset acceptable performance criteria. For a red cell antiserum, an exam- 41. d. A patient with IgA deficiency and clinically signif- ple of acceptable performance criteria might icant anti-IgA requires washed red blood cells if a include reactivity with antigen-positive cells and transfusion is necessary, because washing no reactivity with antigen-negative cells. When removes plasma proteins. It should be noted that testing antigen-positive cells, cells with heterozy- washing is associated with a loss of about 10% to gous inheritance are generally recommended for 20% of the original unit. use because this would detect the weakest expression of antigen (single-dose). 42. b. Of the types listed, the enzyme that converts H antigen to A1 antigen is the most active. There- 34. a. M and N are antithetical alleles in the MNS sys- fore group A1 has very little unconverted H anti- tem. A cell with heterozygous expression of the gen. The order of blood types possessing the most M antigen would therefore need to also express H antigens to the fewest H antigens is O>A2 > the N antigen. In other words, it would need to B>A2B>A1 >A1B. be M+N+. Cells that express only M or N antigen (M+N or MN+) would be presumed to be 43. b. Anti-Fya, anti-K, and anti-S all preferentially react homozygous for either the M or N antigen, at the anti-human globulin (AHG) phase. Although respectively. anti-M also can be found to react at the AHG phase, many examples react only at room temper- 35. d. Neutralization combines a soluble antigen with ature (RT) phase. antibody in vitro and is used as an antibody identification technique. Commercially prepared Lewis 44. c. A patient with R1r possesses D, C, c, and e anti- substance is available for purchase, so of the list gens. R2R2 blood possesses D, c, and E antigens. provided, only anti-Lea could be neutralized. To determine what antibodies could be made on exposure, determine what is different between 36. d. O red blood cells possess only the H antigen, the two, or what foreign antigen could be intro- and O red cells used for reagent purposes typically duced to the patient. In this case, the E antigen also lack D antigen. O-negative reagent red would be foreign and could potentially stimulate cells do not react with anti-D or any ABO antibody production. antibodies. 45. c. To determine the combined phenotype for blood 37. c. Ethylenediaminetetraacetic acid (EDTA) chelates negative for the Jka and Fya antigens, multiply the calcium ions to form a soluble complex; therefore percentages of person negative for each antigen. it prevents the assembly of C1. 0:23 0:34 ¼ 0:0782 or 7:8% 38. a. IgG-sensitized red cells (check cells or Coombs control cells) are commercially prepared with 46. c. Persons of the Oh phenotype (Bombay) type as IgG antibodies attached. Proper control of anti- group O, but also possess potent anti-H. This human globulin (AHG) tests systems (indirect anti-H is usually detected in tests using group O antiglobulin test [IAT] or direct antiglobulin test cells (antibody screen or crossmatch), but may [DAT]) require check cells to be added to negative be noted in ABO grouping, because the reverse tubes to ensure that AHG reagent was added and cells could be hemolyzed instead of just is active. agglutinated. 340 APPENDIX A Answers and Rationales to Certification Preparation Questions

47. c. 54. c. • H gene: Produces H antigen on type II chains in • RoRo ¼Dce/Dce secretions and H antigen on red cells • R1R1 ¼DCe/DCe • A gene: Encodes for a glycosyltransferase that • R2R2 ¼DcE/DcE produces A antigen • Rr¼dce/dce • O gene: Silent gene Therefore anti-E will react only with R2R2 cells • Le gene: Encodes for a fucosyltransferase that because they are the only ones in this list that pos- produces Lea antigen sess the corresponding E antigen. • se gene: Amorph allele; inheriting two se genes means that the person is a nonsecretor 55. b. Antibodies to Duffy system antigens are clinically Le and secretor gene interaction: If Le is inherited significant (associated with hemolytic transfusion without Se, only Lea will be found on red reactions) and have typically been shown to cells and in saliva. Because this person is a non- cause mild hemolytic disease of the fetus and secretor, only Lea antigen will be present in the newborn (HDFN). Because the Fya and Fyb anti- secretions. gens are sensitive to enzymes, anti-Fya and anti-Fyb would not react with enzyme-treated 48. d. N-Acetylgalactosamine (GalNAc) confers type A panel cells. specificity. D-Galactose (Gal) confers type B specificity. L-Fucose (Fuc) confers type O speci- 56. b. Anti-Ch and anti-Rg are usually IgG and react ficity. The type A and type B sugars are built weakly. Neutralization of these two antibodies on Fuc. with pooled plasma is often used as part of antibody identification when either or both anti- 49. b. The DCe/dce (R1r) phenotype is found in approx- bodies are present. imately 35% of whites and 15% of blacks. 0 Although DCe/Dce (R1Ro) and DCe/dCe (R1r ) 57. a. Potential donors who have been transfused in are also possibilities, they are statistically less the last 12 months are deferred because of the pos- probable than DCe/dce. It is not possible for this sibility of exposure to diseases. Although viral person to be DCe/DcE, because she tested nega- marker testing has increased the safety of the tive for the E antigen. blood supply, some diseases have a window in which markers are below the threshold of 50. a. Individuals who inherit the D antigen with weak- detection. ened or missing epitopes are described as having partial D antigen and often present with weak- 58. d. For autologous donors who weigh less than ened expression of the D antigen. Because the par- 100 lb, the volume of blood collected and the tial D antigen is essentially incomplete, if an amount of anticoagulant used should be propor- individual with partial D antigen is exposed to tionately less when compared to a donation complete D antigen, the person would theoreti- from a person weighing more than 110 lb. There cally be able to make an alloantibody against is no age restriction for autologous donors. the parts of the antigen that were foreign. Hgb concentration in a potential autologous donor should be no less than 11 g/dL. Therefore 51. a. The k antigen is present in 98.8% of white individ- the only condition that would preclude donation uals and 100% of black individuals, so it would be is current bacteremia. Blood collected while a very rare to encounter someone who lacked the k patient is septic could cause harm if antigen. transfused later.

52. a. Expression of the Xga antigen is controlled by an 59. c. Cytomegalovirus (CMV) can be transmitted X-linked gene, and prevalence of the antigen is through transfusion via intact white cells con- higher in females than in males. Anti-Xga is usu- tained in cellular blood products. Leukoreduc- ally IgG and the Xga antigen is sensitive to ficin. tion of blood products reduces the risk for CMV transmission because the CMV virus 53. d. Kidd system antibodies are usually IgG, are resides within intact white cells. enhanced by enzymes, and do not store well. The titer of Kidd system antibodies in individual 60. b. Plasma protein fraction (PPF) is prepared from patients can rise and fall quickly, meaning that large pools of human plasma. Although PPF they might not be detected in an antibody screen can transmit infectious agents, the risk for doing if the titer is below the detection point of the test so is reduced because certain viruses are inacti- system. vated or removed during preparation. Of the APPENDIX A Answers and Rationales to Certification Preparation Questions 341

components listed, PPF is the only component permanent. If it is not recorded, it did not happen, treated in this manner. so documentation after the fact is not allowed.

61. c. To prepare platelets, whole blood is first centri- 69. d. Kpa is an antigen in the Kell system that is present fuged at a light spin, and platelet-rich plasma is in 2% of whites and found only rarely in blacks. expressed off the red cells into a satellite bag. The platelet-rich plasma is then centrifuged at a hard 70. b. Vel is an antigen of high prevalence. Vel exhibits spin, and plasma is expressed off of the platelets. variable antigen expression on red cells and is resistant to treatment with enzymes. 62. b. Of the antibodies in this list, anti-K is the one most likely to be IgG and able to cross the pla- 71. a. A 1+ reaction has numerous medium and centa. Anti-I, anti-Lea, and anti-N typically pre- small agglutinates with a turbid background. A sent as IgM class antibodies when encountered 2+ reaction has many medium-sized agglutinates in patient specimens. with a clear background. A 3+ reaction has several large agglutinates and a clear background. 63. c. Group O red cells are most generally used for A 4+ reaction has a solid agglutinate with a clear intrauterine and neonatal transfusions. Rh- background. negative blood is used for fetuses and neonates when the blood type is unknown or Rh-negative. 72. e. A and B genes in the ABO system have codomi- In this case, because the mother has anti-D, Rh- nant expression, and the O gene is a silent allele, negative blood must be used for the intrauterine producing no detectable gene product. For an transfusion. individual to have a B phenotype, a B gene needs to be inherited either in a homozygous fashion or 64. c. Rh immunoglobulin (RhIG) is given to D-negative along with an O gene. mothers to prevent the formation of anti-D. If a mother has already formed anti-D, then RhIG will 73. b. The forward type shows no agglutination, mean- offer no protection. ing the individual does not possess A or B antigens. The reverse type shows reactivity with 65. b. To quantify the amount of fetomaternal hemor- both A1 and B cells, meaning the individual pos- rhage (FMH), the percentage of fetal red cells sess antibodies to A and B antigens. This is char- counted is multiplied by the mother’s blood acteristic of the O blood group. volume. The mother’s blood volume can be cal- Forward Type Reverse Type culated based on her height and weight, but (Patient RBCs) (Patient Serum) Interpretation

often the average of 5000 mL is used for Anti-A Anti-B A1 Cells B Cells calculation. 00+ +O +00 + A ðÞ0:6=100 5000 mL ¼ 30 mL 0++ 0 B ++0 0 AB 66. c. An O mother (genotype OO) would contribute an O gene to her child, so regardless of the 74. b. This patient forward types as group A and reverse father’s type, an O mother could not have an AB types as group O, so an ABO discrepancy is pre- child. A clerical error has likely occurred and sent. In the event that the patient needs blood should be eliminated as a cause of potential before the discrepancy can be resolved, group O error before further serologic studies are conducted. must be transfused.

67. b. Any antibody attached to a baby’s red cells 75. d. Group A and B individuals predominantly make would have to come from the mother. Sample IgM ABO antibodies, but small quantities of from the mother is easily acquired and should IgG antibody can be seen. Group O individuals be readily available. Although an eluate could tend to make predominantly IgG ABO antibodies. be performed on the baby’s cells, that is a time- This is important to understand why ABO hemo- consuming procedure requiring a large sample. lytic disease of the newborn is more commonly The best procedure is to perform an antibody seen in group O mothers. panel on the mother’s serum. 76. d. This patient forward types as group A and reverse 68. d. Use of whiteout obliterates the original results, so is types as group O, so an ABO discrepancy is pre- not allowed. Use of pencil results in a record that sent. You could not report out results until the dis- could be changed—that is, the record is not crepancy was explained. The A1 cells are reacting 342 APPENDIX A Answers and Rationales to Certification Preparation Questions

only at 1+ strength, so this points to possible extra 81. b. Receipt of the rubella vaccine results in a reactivity in the reverse type. Extra reactivity in temporary deferral of 4 weeks. Because this vac- the reverse type can be caused by rouleaux, cine was administered 2 months ago in this indi- cold-reactive antibodies (autoantibody or alloan- vidual, this would not cause a deferral. tibody), or passively acquired antibodies. In the Transfusion of blood, components, human tissue, case of a group A individual showing weak reac- and/or plasma-derived clotting factor concen- tivity with A1 cells, this is often seen in group A2 trates results in a temporary deferral of individuals who have made anti-A1. Testing the 12 months, so this individual would be temporar- cells with anti-A1 lectin would tell us if the patient ily deferred for 10 months based on the time of possesses A1 antigen, and testing the serum with transfusion. A2 cells would tell us if the extra reactivity noted in the reverse type is likely caused by anti-A1.If 82. a. Use of a needle to administer nonprescription the discrepancy could not be resolved, it would drugs is a condition for indefinite or permanent be advisable to request a new specimen. deferral. Casual contact with a person with an infectious disease generally is not a reason for 77. d. A directed unit is donated for a specific person deferral. Potential donors who have ingested aspi- identified by the donor. This is not limited to rin are deferred (48 hours) only if they are donat- blood relatives. A therapeutic phlebotomy is per- ing apheresis platelets. formed on individuals with polycythemia or other blood disorders, as ordered by a physician. When 83. d. Required tests for infectious disease screening an individual donates blood for his or her own include: use, this is called an autologous donation. • HBsAg • Anti-HCV 78. b. The only difference between a directed donation • Anti-HBc and a volunteer donation is that a directed dona- • HCV NAT tion is reserved for a specified individual. Testing • Anti-HIV-1/2 for transfusion of a directed unit is no different • HIV NAT from that conducted on a volunteer unit intended • Anti-HTLV-I/II for transfusion. • Syphilis – RPR or hemagglutination • West Nile Virus (WNV) NAT 79. a. Allogeneic donors must meet the following phys- • IgG antibody to Trypanosoma cruzi (Chagas ical examination criteria before donation: disease)

Physical Examination Criteria 84. a. The HBsAg test detects the surface antigen of the Acceptable Limit hepatitis B virus in the blood. In an infected per- Criteria Allogeneic Autologous son, this antigen can be detected before the anti- Age Applicable state law Determined by body to the core antigen (anti-HBc) is produced. or 16 medical director Blood Systolic 180 mm Hg Determined by 85. a. Cryoprecipitate (CRYO) is defined as the cold- pressure Diastolic 100 mm Hg medical director insoluable portion of FFP thawed at 1-6 C and Pulse 50-100 beats per minute Determined by (bpm) without medical director is suspended in 10-15 mL of plasma. pathologic irregularity, <50 bpm acceptable 86. a. Platelets must be gently agitated during storage by if an otherwise healthy the use of a rotator to prevent the pH from athlete decreasing below 6.2. Temperature 37.5 C (99.5 F) if Deferral for measured orally, or conditions 87. a. Thawed cryoprecipitate components are stored at equivalent if measured presenting risk for room temperature after thawing. by another method bacteremia Hemoglobin/ 12.5 g/dL or an Hct value 11 g/dL or an Hct 88. c. FFP is thawed at temperatures of 30 Cto37 Cor hematocrit of 38% value of 33% in an FDA-approved device. (Hct) 89. b. Frozen red blood cells are prepared for transfu- 80. c. Receipt of human pituitary growth hormone sion by first thawing the unit at 37 C. Next, (PGH) requires indefinite or permanent deferral the glycerol cyropreservative must be removed because of the theoretical risk for transfusion- through a stepwise decreasing osmolar solution transmitted Creutzfeldt-Jakob disease (CJD). of saline. APPENDIX A Answers and Rationales to Certification Preparation Questions 343

90. a. As frozen units need to be thawed and deglycero- 99. b. Per AABB Standards, if an automated temper- lized prior to transfusion, use of frozen units in an ature recording device is not used, then tem- emergency transfusion situation is not practical. peratures of the blood component storage Group AB Rh-negative patients can receive O, A, environment must be measured manually every B, or AB Rh-negative units, so transfusion needs 4 hours. could likely be handled from available refrigerated units of blood. There are special considerations that 100. b. As suggested by AABB, red cell reagents, anti- must be made for pregnant women who requiring sera, and antiglobulin serum should have quality an intrauterine transfusion, but a frozen unit of control performed each day of use. red cells would likely only be used in the event of an antibody to a high frequency antigen. Therefore CHAPTER 8 the best answer for this question is A. Red cell units are usually frozen for long-term preservation to 1. c. Chylomicrons, low-density lipoprotein (LDL), and maintain an inventory of rare units. high-density lipoprotein (HDL) are considered to be lipoproteins that transport lipids throughout 91. c. Indications for transfusion of cryoprecipitate the body; cholesterol is classified as a lipid. include von Willebrand’s disease, Hemophilia A, to control bleeding associated with fibrinogen 2. d. Dextran sulfate precipitates all Apo B–containing deficiency, and Factor XIII deficiency. lipoproteins (chylomicrons, very-low-density lipoprotein [VLDL], intermediate density lipo- 92. b. Indicationsfor transfusion of red blood cellsinclude protein [IDL], and low-density lipoprotein treatment of anemia in normovolemic patients and [LDL]) leaving high-density lipoprotein (HDL) physician decision based on the clinical status of the (Apo A–containing lipoprotein) in the superna- patient. Patients that are massively bleeding, have tant. HDL is then mixed with the reagent choles- bone marrow failure, or have decreased red blood terol esterase and cholesterol oxidase to cell survival have clinical situations that would war- quantitate HDL concentrations. rant transfusion becauseof the need for hemoglobin replacement. In patients with compensated anemia, 3. a. Lipoproteins are characterized by size and density. the body has adapted to allow for adequate tissue High-density lipoprotein (HDL) is the smallest, oxygenation. Transfusion should be withheld in most dense lipoprotein, carrying 50% of its this case until the patient shows clinical signs of weight as protein. inadequate tissue oxygenation. 4. d. Triglycerides are most adversely affected by recent 93. b. FFP has no quality control or minimum require- food intake, and therefore a fast is always recom- ments. Cryo must have80 IU of Factor VIII mended for triglyceride analysis. and150 mg of fibrinogen. Leukocyte reduced red cells must have <5106 residual leukocytes 5. c. Apo E is a ligand for the low-density lipoprotein and 85% of original red cells retained. There (LDL) receptor, reverses cholesterol transport, are no quality control or minimum requirements and is a regulator of cell growth and immune for the number of red cells in platelets. responses.

94. a. FFP is stored at-18 C for 12 months, 6. c. Two enzymes are responsible for esterifying cho- or-65 C for 7 years with FDA approval. lesterol, lecithin-cholesterol acyl transferase (extracellular), and acetyl coenzyme A (acyl- 95. c. Per AABB guidelines, packed red cells are stored CoA) cholesterol acyltransferase (intracellular). at 1 Cto6 C. 7. a. Although lipoproteins can be assayed using a vari- 96. c. Platelets made from a single whole blood donation ety of anticoagulants, the preferred anticoagulant (random platelets) should contain5.51010 is ethylenediaminetetraacetic acid (EDTA) platelets in 75% of the units tested. because it preserves lipoproteins over time.

97. d. RBCs Frozen with 40% glycerol are stored 8. b. Triglycerides are transported throughout the body at-65 C for 10 years. by means of two lipoproteins: very-low-density lipoprotein (VLDL) and chylomicrons. VLDL 98. c. In the IAT, incubation takes place at 37 C for carries endogenously derived triglycerides, and a specified amount of time to allow antigen- chylomicrons carry exogenously derived antibody reactions to occur. triglycerides. 344 APPENDIX A Answers and Rationales to Certification Preparation Questions

9. b. Low-density lipoprotein (LDL) cholesterol is cal- Symptoms and a random plasma glucose culated as follows: 200 mg/dL or LDL ¼ TC ðÞHDL + TG=5 Fasting plasma glucose 126 mg/dL or LDL ¼ 300 ðÞ50 + 200=5 2-hr OGTT 200 mg/dL or LDL ¼ 300 90 Hbg A1c 6.5% LDL ¼ 210 mg=dL 19. d. Insulin lowers glucose levels by increasing the 10. b. Low-density lipoprotein (LDL) is currently uptake of glucose into the cell and through the only lipoprotein or lipid that is recommended increased glucose metabolism. for use by physicians for therapeutic lifestyle changes. 20. b. The data presented indicate that the glucose and low-density lipoprotein (LDL) 11. b. Both forms of hyperbetalipoproteinemia (types are mildly elevated, and the blood urea nitro- IIA and IIB) are due to either a defect in the gen (BUN), creatinine, and microalbuminuria low-density lipoprotein (LDL) receptor (type results are moderately elevated. This informa- IIA) or a defect in Apo B-100 (type IIB). tion together indicates that the patient is at most risk for the development of diabetic 12. d. All enzymatic methods to measure triglycerides, nephropathy. regardless of the enzyme used, begin with the conversion of triglycerides to glycerol and fatty acids 21. d. The renal threshold for glucose is 160 to in the presence of the enzyme lipase, followed by 180 mg/dL. Once plasma levels of glucose hit that the conversion of glycerol to glycerol-3- threshold, it will spill over into the urine. phosphate in the presence of the enzyme glycerol kinase. 22. c. Blood urea nitrogen (BUN) and creatinine are markers of kidney function; however, they 13. c. Marked increases in triglyceride levels, between are not sensitive enough markers to detect 1000 and 2000 mg/dL have been associated early diabetic nephropathy. The best test to with increased risk for the development of use to detect diabetic nephropathy is the pancreatitis. microalbuminuria test. 14. a. Apo A-I is the predominant apoprotein associated with the high-density lipoprotein (HDL) mole- 23. b. An obese, elderly patient with report of increased cule, activates (lecithin cholesterol acyltransferase urination at night, increased thirst, and increased [LCAT]), and is associated with reverse choles- appetite is indicative of a diagnosis of diabetes, terol transport. As a result, it is protective against most likely type 2 diabetes in this case. With the coronary artery disease. patient being 68 years of age and obese with only mildly elevated levels of glucose, the diagnosis of 15. c. The recommended fasting state for the study of type 1 is unlikely. Patients with diabetes would lipids involves nothing but water for 12 hours have increased glycated hemoglobin. With a fast- before the blood sample collection. ing glucose of 210 mg/dL, the assessment of hypoglycemia is unwarranted. 16. c. Using Beer’s law, the concentration of cholesterol in the patient (Smithers) serum is determined as 24. c. According to the American Diabetes Association, follows: 0.679 A1c results between 5.7% and 6.4% indicate an impaired state (prediabetes). Concentrationcholesterol ¼ Absorbanceunknown=Absorbancestandard Concentrationstandard Concentrationcholesterol ¼ 0:729=0:679 200 mg=dL 25. b. Whole blood glucose levels are 12% to 15% Concentrationcholesterol ¼ 209 mg=dL lower than plasma glucose levels. Therefore a plasma glucose level of 100 mg/dL would 17. d. Sucrose upon hydrolysis yields fructose and roughly correspond to a whole blood level of glucose. 85 mg/dL.

18. d. According to the American Diabetes Association 26. a. Of the three methods to measure glucose, glucose criteria for the diagnosis of diabetes (below), this oxidase, hexokinase, and glucose dehydrogenase, patient would most likely be classified as having the hexokinase method is considered virtually type 2 diabetes. specific for glucose. APPENDIX A Answers and Rationales to Certification Preparation Questions 345

27. b. The cutoff points for normal, impaired, and diag- measures solute concentration as the solute’s den- nostic states are described below. According to the sity, which is subject to interference from large values below, the data suggest an impaired state. molecules such as glucose and proteins. Osmolal- Normal Impaired Diagnostic ity, on the other hand, measures solute concentra- Resting <200 mg/dL 140-199 mg/dL 200 mg/dL tion as the number of molecules present by plasma glucose measuring the number of molecules per kilogram Fasting <100 mg/dL 100-125 mg/dL 126 mg/dL of water. plasma glucose 2-Hr oral glucose <200 mg/dL 140-199 mg/dL 200 mg/dL 35. c. The osmolal gap (difference between calculated tolerance test and measured osmolality) when increased is indic- A1c <5.7 % 5.7%-6.4% 6.5% ative of the presence of osmotically active substances present other than sodium, blood urea 28. d. Acute glomerulonephritis is often associated with a nitrogen (BUN), and glucose. Other osmotically recent group A b-hemolytic Streptococcus infec- active substances may include ethanol, methanol, tion. It is hypothesized that immune complex devel- ethylene glycol, lactate, or b-hydroxybutyrate. opment associated with a group A b-hemolytic Streptococcus infection directly injures the glomer- 36. c. According to the National Kidney Foundation, a ular basement membrane of the glomerulus. glomerular filtration rate of 60 to 89 indicates mild kidney damage. 29. a. The classic Jaffe reaction involves complexing of creatinine with an alkaline picrate solution to pro- 37. d. Dubin-Johnson syndrome and extrahepatic duce a red complex (Janovski complex). obstruction are conditions that cause elevations in total bilirubin, with the major fraction increased 30. c. The kidney is responsible for acid-base balance being the conjugated fraction (direct). Gilbert’s dis- through the removal of H ions via (1) the reac- ease is a condition resulting from genetic mutation tion of the hydrogen ions with bicarbonate, (2) in the gene that produces the enzyme uridyl diphos- the reaction of hydrogen ions with filtered phate glucuronyl transferase, the enzyme responsi- buffers such as disodium salt, (3) reaction with ble for the conjugation of bilirubin. Therefore ammonia, and (4) excretion of the free Gilbert’s disease will manifest as unconjugated hydrogen ions. hyperbilirubinemia.

31. b. 38. b. Severe liver disease is the most common cause of altered ammonia metabolism. Therefore the mon- Urine creatinineðÞ mg=dL Urine volumeðÞ mL= min itoring of ammonia levels may be used to deter- Serum creatinineðÞ mg=dL mine prognosis.

1:73 m2 39. a. Hemolysis and neonatal jaundice would manifest Surface area of patient with elevations in total bilirubin primarily as a result of the unconjugated fraction. Urine volume : 1:75 L=day 1000 mL=1L 1hr=60 min Biliary obstruction manifests as conjugated ¼ 1:22 mL= min hyperbilirubinemia. 120 mg=dL 1:22 mL= min 1:73 m2 ¼ 117 mL= min 1:2mg=dL 1:80 m2 40. c. Crigler-Najjar syndrome is an inherited disorder of bilirubin metabolism. Neonates with Crigler- Najjar syndrome have no uridyl diphosphate 32. c. 2 Sodium (mmol/L)+Glucose mg/dL/18+Blood glucuronyl transferase and therefore cannot con- urea nitrogen (BUN) mg/dL/2.8 jugate bilirubin for excretion. In neonates this increased unconjugated bilirubin will cause ker- 33. c. Blood urea nitrogen (BUN) and creatinine are con- nicterus without aggressive treatment. sidered markers of kidney function. Renal failure can cause elevations in BUN and other prerenal 41. a. When bilirubin becomes conjugated it can enter conditions such as dehydration or a high-protein the intestines. In the gastrointestinal tract, bacte- diet that raise levels of BUN without affecting cre- ria convert conjugated bilirubin to urobilinogen. atinine levels. Three things will happen to the urobilinogen: (1) it is excreted in the feces; (2) it enters extrahe- 34. a. Osmolality and specific gravity both measure the patic circulation, where it is absorbed and recir- solute concentration of a solution. Specific gravity culated to the liver and excreted in the feces; 346 APPENDIX A Answers and Rationales to Certification Preparation Questions

and (3) it enters systemic circulation and is low levels of bicarbonate (HCO3 <22 mol/L). excreted in the urine. Because it is an uncompensated condition, the respiratory parameter would be in the normal 42. c. The enzyme responsible for bilirubin conjugation range (PCO2 35-45 mm Hg). is uridyl diphosphate glucuronyl transferase. 51. b. A decrease in ventilation would cause the accu- 43. a. Aspartate aminotransferase (AST) and alanine mulation of the acidic gas PCO2, causing respira- aminotransferase (ALT) are the most sensitive tory acidosis. markers of hepatocellular damage. g- Glutamyltransferase (GGT) and alkaline phos- 52. d. The bicarbonate–carbonic acid buffer system is phatase (ALP) are markers of hepatobiliary dam- the most important physiologic buffering system age. Ammonia, although it may be seen in severe in the body, because it is the buffer system that cases of liver damage, is not a sensitive marker of immediately counteracts carbon dioxide nor- hepatocellular damage. mally produced by cell metabolism. 44. c. Dubin-Johnson is an autosomal recessive disorder 53. c. This process of exchange between bicarbonate resulting in a defect in the ability of the liver cell to and chloride as bicarbonate leaves the red blood secrete conjugated bilirubin into the bile. cell is termed the chloride shift. 45. c. UDP-glucuronyl transferase is the enzyme that is 54. responsible for the conjugation of bilirubin. a. PCO2 is an acidic gas controlled by the lungs (respiratory system) and when in increased concentra- 46. c. Aspartate aminotransferase (AST) and alanine tions will cause respiratory-driven acidosis. aminotransferase (ALT) are markers of hepatocel- 55. lular damage, whereas g-glutamyl transferase a. Room air contains a PO2 of 150 mm Hg and a (GGT) and alkaline phosphatase (ALP) are PCO2 of approximately zero. When a blood gas markers of hepatobiliary damage. sample is received in the laboratory uncapped or if it contains air bubbles, gas equilibration 47. b. Unconjugated bilirubin is insoluble in water and between the air and the blood will occur, causing must be transported by albumin to the liver for a decreased PCO2, increased PO2, and increased further metabolism. Albumin binding prevents pH. Blood gas samples from a patient without unconjugated bilirubin from crossing cell mem- oxygen supplementation with a PO2 greater than branes and accumulating in cells. Extremely high 110 mm Hg should be investigated for air levels of unconjugated bilirubin can exceed contamination. albumin-binding capacity and allow unconjugated bilirubin to cross the immature blood-brain 56. d. Ideal blood gas collection should include collection barrier in neonates, causing kernicterus (toxic in a plastic syringe containing dry heparin; it should damage to the cells in the basal ganglia). be collected, delivered to the laboratory, and assayed within 15 minutes at room temperature. 48. d. With chronic alcohol abuse, the amount of ala- If the process will exceed 15 minutes, samples are nine aminotransferase (ALT) declines, which stable for 45 minutes stored on crushed ice. results in an increased ratio of aspartate aminotransferase (AST) to ALT. g-Glutamyltransferase 57. d. The pH may be calculated using the Henderson- (GGT) is a membrane-bound enzyme that is Hasselbalch equation for the bicarbonate– markedly increased with ethanol use, and alkaline carbonic acid buffer system as follows (NOTE: In phosphatase (ALP) is slightly elevated with plasma and at body temperature [37 C] the alcohol use. pKa of the bicarbonate buffering system is 6.1): pH ¼ pK + logð HCO =ðÞ0:03 ðÞPco 49. b. Primary biliary cirrhosis (PBC) is an autoimmune a 3 2 disorder that targets the bile ducts. PBC is associ- pH ¼ 6:1 + log28=ðÞ0:03 ðÞ45 ated with antimitochondrial antibodies and the pH ¼ 6:1 + log20:7 presence of lipoprotein X (an abnormal lipopro- pH ¼ 7:42 tein produced in individuals with obstructive biliary tract disease). 58. c. In healthy individuals when the kidneys and lungs 50. a. Acidosis will manifest with a pH less than 7.35; are functioning at full capacity the ratio of bicar- because it is metabolic, it would be driven by bonate to carbonic acid is 20:1. APPENDIX A Answers and Rationales to Certification Preparation Questions 347

59. d. Reference intervals for arterial blood gases are as TSH levels are normal is it unlikely that a thyroid follows: abnormality exists. The only abnormal result pH 7.35-7.45 provided is the total T4 levels, which inherently PCO2 35-45 mm Hg are subject to false elevations in individuals with PO2 80-110 mmol/L abnormal binding proteins. This is why total HCO3 22-26 mmol/L levels of thyroid hormones (TT3 and TT4) are not routinely obtained in clinical practice. 60. c. In uncompensated metabolic acidosis the pH would be decreased (<7.35), and this decrease 68. c. Two essential components needed for thyroid hor- would be driven by the metabolic component mone production include tyrosine and iodine. (HCO3 ). HCO3 would be decreased < ( 22 mmol/L). Because it is uncompensated, the 69. c. The term subclinical refers to a condition that respiratory component (PCO2) would be normal can be detected using sensitive laboratory tests, (34-45 mmol/L). but has not progressed to the point of producing symptoms or abnormal thyroid hormone levels. 61. a. The anion gap, especially when elevated, is useful In subclinical hypothyroidism, the thyroid- in diagnosing the type of metabolic acidosis and in stimulating hormone will be decreased but the indicating if a mixed disorder exists. thyroid hormone levels will still be normal. 62. c. The predominant feedback system associated 70. d. The three main noniatrogenic causes of Cush- with the endocrine system is negative feedback, ing’s syndrome (hypercortisolism) include (1) a in which increased levels of hormones feed back pituitary tumor (Cushing’s disease), (2) an adre- negatively to the hypothalamus and pituitary. nal adenoma (Cushing’s syndrome), and (3) ectopic adrenocorticotropic hormone (ACTH) 63. d. All results are normal except for the result for production. Cushing’s syndrome driven by an thyroid-stimulating hormone (TSH), which is adrenal adenoma is ACTH independent, result- increased. Because TSH is produced at the pitui- ing in an adrenal adenoma producing cortisol. tary, increased TSH results indicate a hypothyroid Because the pituitary gland is functional, it is still condition. responsive to negative feedback to the pituitary because of the increased cortisol levels and the 64. a. The three main noniatrogenic causes of Cushing’s ACTH result will be decreased. During syndrome (hypercortisolism) include (1) a pitui- a bilateral inferior petrosal sinus sampling the tary tumor (Cushing’s disease), (2) an adrenal ade- gradient between the inferior petrosal sinus and noma (Cushing’s syndrome), and (3) ectopic peripheral venous site after corticotropin- adrenocorticotropic hormone (ACTH) produc- releasing hormone administration in Cushing’s tion. Pituitary-driven Cushing’s disease is ACTH syndrome is less than 2. dependent and results from a benign pituitary adenoma that produces ACTH. Therefore ACTH 71. values are increased. During bilateral inferior b. Releasing and inhibiting factors are produced petrosal sinus sampling the gradient between the from the hypothalamus, and trophic hormones inferior petrosal sinus and peripheral venous site are produced from the pituitary. after corticotropin-releasing hormone adminis- 72. tration in Cushing’s disease is greater than 3 (often b. The free thyroxine (FT4) index is calculated as close to 50). follows:

FT4I ¼ TT4 Thyroid hormone binding ratio=100 65. b. Hypothyroidism is a condition in which thyroid hormone levels are low; therefore because of pos- 73. itive feedback the thyroid-stimulating hormone a. Assuming that a screening test is abnormal, a will be increased. definitive stimulation test is often performed. The definitive test involves looking at the growth 66. c. Thyroid-releasing hormone (TRH) is a tripeptide hormone response after intravenous insulin produced from the hypothalamus. TRH acts directly infusion. on the pituitary gland to produce TSH, which 74. acts on the thyroid gland to produce T3 and T4. b. Pheochromocytoma is a rare and usually benign tumor arising from the adrenal gland that 67. a. Thyroid-stimulating hormone (TSH) is the most results in the increased production of catechol- sensitive indicator of thyroid dysfunction. When amines (epinephrine and norepinephrine). Total 348 APPENDIX A Answers and Rationales to Certification Preparation Questions

catecholamine levels are not recommended for 83. d. The recommended sample of choice for analysis diagnosis because catecholamines are highly labile of creatinine kinase (CK) is serum or heparin and increase as a result of stress and pain. There- plasma. Anticoagulants other than heparin may fore the recommendation is to assay plasma free inhibit CK activity. metanephrines (metanephrines and normetane- phrines), which are the metabolites of epinephrine 84. c. Creatine kinase (CK) is an enzyme found in the and norepinephrine heart, brain, skeletal muscle, and other tissues. Increased amounts of CK are released in the blood 75. a. Thyrotropin-releasing hormone (TRH) is a tri- when muscle damage occurs, such as the muscle peptide produced from the hypothalamus that damage seen in acute myocardial infarction and acts on the pituitary to produce thyroid- Duchenne’s muscular dystrophy. stimulating hormone (TSH). When exogenous TRH is administered to a person and there is no 85. c. Elevated alkaline phosphatase (ALP) levels may response, it is indicative of a nonfunctioning pitu- be seen with bone and liver disorders. In addition, itary gland. Thyroid gland disorders arising from ALP will also be increased physiologically during a problem with the pituitary gland are referred to periods of bone growth, when bone fractures are as secondary conditions. healing, and in pregnancy. 86. 76. a. Thyroid hormone production occurs in five steps: a. Enzyme assays are recommended to be performed (1) iodide trapping, (2) organification, (3) cou- during the linear phase, so that a consistent pling, (4) storage, and (5) secretion. change over time can be used to calculate the enzyme concentration. 77. d. Pheochromocytoma results in the excess produc- 87. a. The best tube to use for testing these enzymes is a tion of catecholamines, which increase cardiac red-top tube. The other anticoagulants may cause output and blood pressure to divert blood to mus- issues with some of the enzyme assays. cle or brain, mobilize fuel from storage, and cause hypertension. If pheochromocytoma is untreated, 88. b. Amylase is an enzyme that can degrade complex death may occur from cardiovascular carbohydrate molecules such as starch. complications. 89. c. Parathyroid hormone (PTH) is responsible for 78. b. Estrogens are a group of steroids responsible for maintaining calcium levels by acting on bone the development of female sex organs. The estro- and the kidneys and by activating vitamin D. gens include estrone, estradiol, and estriol. Estra- PTH acts on the bone to release calcium and phos- diol is the predominant form of estrogen. phorus through osteoclastic activity. Once released from bone, PTH acts on the kidneys to 79. b. The general relationship among an enzyme, its allow absorption of calcium and excretion of substrate, and its product is represented using phosphorous. High levels of PTH would most the Michaelis-Menten theory, which is repre- likely manifest with hypercalcemia and sented as E+S $ ES!E+P, where E is the hypophosphatemia. enzyme, S is the substrate, ES is the enzyme substrate complex, and P is the product. 90. b. Calcium exists in three forms, 45% unbound (ionized), 45% bound to albumin, and 10% bound to 80. d. Lipase and amylase are both markers of pancreatic other anions. Only the ionized (unbound form) is function; however, lipase is considered more spe- the physiologically active form. cific because it remains elevated longer in acute pancreatitis. 91. d. Parathyroid hormone (PTH) is responsible for maintaining calcium levels by acting on bone 81. c. Alkaline phosphatase (ALP) belongs to a group of and the kidneys and by activating vitamin D. enzymes that catalyze the hydrolysis of phosphate PTH acts on the bone to release calcium and phos- esters in an alkaline medium. ALP catalyzes the phorus through osteoclastic activity. Once conversion of p-nitrophenyl phosphate to a col- released from bone, PTH acts on the kidneys to ored p-nitrophenol product. cause the kidneys to absorb calcium and excrete the phosphorous. 82. b. One international unit of enzyme activity is the amount of enzyme that uses substrate at the rate 92. d. Hemolysis is the most common cause of of 1 mmole/min. hypercalcemia. APPENDIX A Answers and Rationales to Certification Preparation Questions 349

93. b. Calcitonin is a hormone produced by the C cells all DNA sequences) are expressed as proteins, and of the thyroid. Calcitonin is involved in calcium specific sequences of nucleic acid make up a gene. regulation and inhibits bone breakdown; thus it decreases calcium and phosphorus. 4. a. The coding sequence in a gene is divided into regions calledexons.Thesecodingregionsspecifytheamino 94. c. Sodium is the predominant plasma cation and acid sequence in the resulting protein. Introns are maintains osmotic pressure. the noncoding regions of a gene. The promoter region regulates initiation and rate of transcription. 95. a. Diabetes insipidus is a condition resulting from a deficiency in antidiuretic hormone that mani- 5. c. DNA polymerase III synthesizes a daughter strand fests with excessive thirst and excretion of large in the 50 to 30 direction only, because nucleotides amounts of severely diluted urine. Because the are only able to be added to the 30 carbon end body cannot conserve water, sodium levels rise of the sugar. and dehydration may occur. 6. d. mRNA with its three-nucleotide codon will bind 96. c. In a state of acidosis there is an increased amount to the tRNA molecule’s anticodone, which then of hydrogen ions that combine with albumin. carries the attached amino acid to a ribosome to Because H ions are combining with albumin, less be added to a growing peptide chain. albumin is available to bind to calcium; therefore ionized calcium levels increase. 7. d. Mitochondrial DNA does not follow the Mende- lian inheritance pattern. 97. b. The anion gap is often reported with electrolyte measurements as a means of identifying shifts 8. b. The lagging strands are discontinuously assem- that may occur in these assays. Because the anion bled and require ligase to join the fragments. gap is a calculation parameter, it is an inexpensive and easy quality control check. 9. a. RNA differs from DNA in that it has uracil that replaces thymine. RNA is also single stranded. 98. b. Hemolysis can often result from a tourniquet being on the arm too long. 10. a. Response a is the only correct answer; b is incorrect because the gene-rich regions are euchroma- 99. d. A potassium value of 15 mEq/L is too high to be tin; answer c is incorrect because telomeres and considered physiologic. The sample should be centromeres are heterochromatin; d is incorrect checked for a potential preanalytical interfer- because chromosomes are composed of DNA. ence, including hemolysis and incorrect collection tube (contamination via an anticoagulant). 11. d. Restriction enzymes are sequence-specific endonucleases that cut dsDNA into fragments, result- 100. a. The major intracellular cation is potassium. ing in either blunt-ended or sticky-ended pieces. Sodium is the major extracellular cation. These endonucleases are isolated from specific bacteria that use the enzymes as protection from CHAPTER 9 invasion by foreign DNA.

1. b. The sequence of a single strand of DNA gives rise 12. c. Polymerases catalyze the synthesis of complemen- to the sequence of its complementary strand. Dur- tary nucleic acid polymers using a parent strand as ing replication the complementary strands are the a template. In vitro, these enzymes can extend an “daughter” strand. Replication is semiconserva- oligonucleotide primer that is annealed to a tive, meaning the new dsDNA is made up of a par- template strand. ent and daughter strand. 13. b. Alleles linked when inherited are called haplo- 2. b. Nucleic acids include a sugar moiety (2- types. The location of a gene on a chromosome is deoxyribose in the case of DNA), a phosphoric the locus, and the possible alternative forms of acid, and purine or pyrimidine base; deoxyribo- the gene at the locus are referred to as alleles. The nucleic acid is DNA and ribonucleic acid is variations in alleles are known as polymorphisms. RNA. Nucleosides comprise a base and a sugar. 14. c. Messenger RNA (mRNA) is produced in the 3. c. The “central dogma” proposes that DNA replicates nucleus of the cell. After processing, the mature from DNA, DNA is transcribed to RNA, and RNA mRNA leaves the nucleus and enters the cyto- is translated to produce proteins. Only genes (not plasm, where it is translated. 350 APPENDIX A Answers and Rationales to Certification Preparation Questions

15. c. In the organic extraction method, cellular debris electrophoresis, and transferred to a solid sup- and protein are separated by organic solvent port, followed by selective visualization of frag- extraction from the hydrophilic soluble DNA ments by hybridization of labeled probes. fraction. The DNA will be in the aqueous phase of the biphasic solution. 25. a. Hybridization probes are those that reversibly change fluorescence on duplex formation. Fluo- 16. a. Purity of extracted RNA and DNA can be evalu- rescence resonance energy transfer techniques ated by assessing the ratio of the absorbances at depend on the proximity between two distinct 260 nm and 280 nm (A260/A280). A ratio of fluorescent labels. When the two labels are 2.0 indicates a pure DNA extract. brought closer together through hybridization, fluorescence is released. 17. c. Solid-phase extraction methods are amiable for high-throughput and adaptable for automated 26. d. Real-time polymerase chain reaction (PCR) mon- processing. itors the amount of product formed each cycle by systematically quantifying the fluorescence sig- 18. d. The sizes/molecular weight of the genomic DNA nal. The fluorescent signals depend on the amount fragment can be estimated by gel electrophoresis of target DNA present in the sample. using size standards. 27. d. Ultraviolet-induced cross-linking of DNA can min- 19. a. Signal amplification techniques use nucleic acids to imize the effects of amplicon contamination during increase the signal for detection. The branched- sample setup; laboratory surfaces should be regu- chain DNA (bDNA) method is one of these larly cleaned using amplicon-decontaminating techniques. agents, such as solutions of bleach, and closed analytical systems (i.e., reaction tubes that are analyzed 20. b. Denaturing the target duplexes into single strands without being opened) should be used to reduce is the first step in polymerase chain reaction contamination with amplicon. (PCR). Next, the sample is allowed to cool and the primers anneal specifically to the complemen- 28. c. The migration of a nucleic acid fragment through tary sequences on the target. The primers are agarose gel depends on the size, shape, and charge extended by the polymerase. of the fragment.

21. c. RNA targets can be amplified into DNA sequences 29. b. Polymerase chain reaction (PCR) requires sev- when they are initially transcribed into cDNA eral components: DNA polymerase, a buffer using reverse transcription. Some thermostable for the polymerase, primers, the four deoxynu- enzymes have both DNA polymerase and reverse cleotide triphosphates, and a source of template transcription activities, so both steps can be per- DNA (the target). DNA endonuclease is not formed in the same tube with the same enzyme. required for this method.

22. d. Primers are provided in great excess and specifi- 30. c. The purpose of primer extension is to produce cally anneal to a complementary sequence on amplicons, which are the resulting fragments from the target DNA. Once the primers have annealed, a polymerase chain reaction (PCR) cycle. The the action of the polymerase synthesizes two addi- DNA polymerase takes the individual nucleotides tional DNA strands containing the primers as the and adds them to the 30 end on each primer that 50 ends. has annealed to the target DNA strands. The target DNA strands act as the reference for the 23. a. The negative control or blank (all reactants minus polymerase. target DNA) is one of the most important controls when providing quality control for a polymerase 31. c. Ethidium bromide binds to nucleic acids by inter- chain reaction (PCR) run. If the blank demonstrates calating between bases. When ethidium bromide DNA bands, the master mix has likely been con- is irradiated with ultraviolet (UV) light it fluo- taminated with PCR reaction product from a resces. This fluorescence can be detected by hold- previous run. ing a UV lamp next to the gel or using visualization software. 24. c. Southern blotting is the classic method for detecting large segments of DNA that are not easily 32. a. The term DNA microarray refers to a microscopic amplified. The original sample DNA is digested grouping of DNA molecules attached to a solid by a restriction enzyme, separated by support mechanism. A DNA microarray is also APPENDIX A Answers and Rationales to Certification Preparation Questions 351

referred to as a DNA chip or gene chip. A DNA will be identical regardless of cell origin. However, microarray allows for the detection of gene DNA from peripheral blood white cells is the eas- expression or possible deletions using compara- iest tissue to obtain. Mature red blood cells cannot tive genome hybridization (CGH). be used because they do not have a nucleus and therefore do not have any DNA. 33. a. Genomic sequencing has led to an increased understanding of protein interrelationships and 41. d. A short tandem repeat (STR) consists of expression in cells. Proteomics is the study of pro- DNA sequences that have a core sequence of teins on a cellular level. Like genomics, proteo- two to seven base pairs, which are repeated in mics is a large-scale process, but it is probably tandem several times. An example of a trinucle- more complicated than analysis at the gene and otide repeat occurring four times is 50 GTG- transcriptional levels. GTG-GTG-GTG 30.

34. c. The size of the target’s genome does not affect 42. b. A locus is a location of a gene where polymorphic hybridization, but the target nucleic acid sequence alleles are found. The alleles at a locus are the does, along with pH and temperature. genotype for the locus.

35. a. Primers should not be larger than 30 base pairs 43. a. Genes encoding the class I molecules, which long. Primer design should take into account melt- includes HLA A, B, and C are considered class I ing temperature (Tm) for both the forward and genes. Class II consists of DR, DQ, and DP. reverse primers. 44. b. Hybrid capture is a signal-amplification assay that 36. a. After the generation of terminated fragments in detects cytomegalovirus (CMV) and human papil- the cycle sequencing steps, fragments are tagged lomavirus (HPV) DNA in specimens. Hybrid cap- with a fluorescent dye labeled terminator ddNTPs ture methods use a bound antibody that is specific then separated by denaturing polyacrylamide gel for RNA-DNA hybrid molecules that are formed or capillary electrophoresis. during solution-phase hybridization of a DNA sample and an unlabeled RNA probe. The signal 37. d. In pyrosequencing, each incorporation event is from the complex is amplified and detected. accompanied by release of a pyrophosphate (PPi), so the quantity of PPi produced is equimolar 45. b. Molecular techniques are designed to achieve high to the amount of incorporated nucleotide. The specificity for the organism that the test is designed conversion of PPi and adenosine 50 phosphosulfate to detect; however, the techniques are subject to into adenosine triphosphate (ATP) by the ATP false-negative results because of the presence of sulfurylase allows ATP to drive the conversion of inhibitors. Additionally, detection of nucleic acid luciferin into oxyluciferin, which generates visible of a pathogen does not ensure that the organism light. The light produced is proportional to the is the cause of the disease. The organism might number of nucleotides incorporated. be forming part of the normal flora, colonizing a specific area, or causing infection but not disease. 38. c. In autosomal recessive disease, two abnormal alleles at a given locus (by receiving one mutant 46. d. The typical retroviral genome contains at least allele from each carrier parent) are necessary to three genes: gag, pol, and env. produce the disease phenotype. Affected individuals may be homozygous with two copies of the 47. d. The translocation involving chromosomes 9 and same mutation. 22 known as the Philadelphia chromosome is a hallmark in the diagnosis of chronic myelogenous 39. b. Imprinting refers to the differential marking of leukemia (CML). The resulting protein has specific paternally and maternally inherited enhanced tyrosine kinase activity. t(9:22) may alleles during gametogenesis, resulting in differen- be seen in acute lymphoblastic leukemia (ALL) tial expression of those genes. with a poor prognosis.

40. c. DNA extracted from peripheral blood (or bone 48. a. In a fluorescence in situ hybridization (FISH) marrow) white cells is the preferred specimen assay, a fluorescent DNA probe is hybridized to for molecular techniques when they are used to a specific locus to produce a colored signal. A diagnose inherited disorders. In inherited disor- translocation would appear as the color produced ders, the DNA from any cell could theoretically from the combination of two different fluores- be used because the DNA at the molecular level cently labeled probes. 352 APPENDIX A Answers and Rationales to Certification Preparation Questions

49. d. The proteins encoded by many tumor-suppressor 7. c. genes are involved in the regulation of progression through the cell division cycle or in the regulation 8. b. of DNA repair. 9. a. The Centers for Medicare & Medicaid Services 50. b. Oncogenes are derived from protooncogenes, (CMS) is the government agency responsible for which are normal cellular genes. The protoonco- the administration of CLIA certification. The genes become oncogenic and are often associated U.S. Food and Drug Administration is the govern- with cancers when they have been altered by dom- ing body for approval of new clinical laboratory inant mutations. testing procedures and methods. The Department of Transportation regulates shipments of human CHAPTER 10 specimens, among other guidelines. 1. d. Effective communication of change requires direct 10. a. A job description lists the key activities defined and active speaking and listening with all individ- through a job analysis and is the basis for evalua- uals involved in the change. Response a limits the tion of the individual performing that job. employee number and limits the effective listening of those employees. Responses b and c are one- 11. directional communication. Response d has the b. Conventional wisdom states that a starting salary key component of “discussion” with all employees should not be listed in a job advertisement, and is therefore the best answer. because the organization should be able to “sell” the position through the professional benefits of 2. b. Communication is the most appropriate answer the position. However, in this competitive envi- for a manager, because this individual must coor- ronment, some advertisements now include start- dinate multiple activities and people to accom- ing salary. Yet b is still the correct answer, because plish the responsibilities of the profession. the other listings are key components that are Informal discussions and decision making follow required for any job advertisement to be effective the foundation of communication. and informational to the reader.

3. a. Phase 1 deficiency requires a response to the CAP 12. d. Effective evaluations of individuals performing within 30 days of the citation, which indicates job tasks require an overview and knowledge of that responses b, c, and d are not appropriate. the job and work performed, the ability to interact with and observe the person on occasion (proxim- 4. d. The mission of the National Accrediting Agency ity), and a timeliness in scheduled review periods. for Clinical Laboratory Sciences (NAACLS) is to offer approval and accreditation of educational 13. b. To evaluate a job, the evaluation instrument programs in the clinical laboratory sciences. should contain components to assess the key activities of the job description. 5. a. The COLA agency services include education, consultation, and accreditation. CAP also offers 14. a. Maslow’s hierarchy of needs demonstrates that education, consultation, and accreditation. The effective recognition can be perks such as reserved Joint Commission offers accreditation, certifica- parking spots or conference attendance for con- tion, and standards. AABB offers professional tinuing education. development, accreditation, and standards. ASCLS offers education, professional networking, 15. d. Many accepted leadership tenets from historical and the body of knowledge for CLS. observation show that individual and team recognition generate a sense of accomplishment, increased 6. b. COLA offers accreditation services for the communication, and relief and well-being of whole laboratory. The Substance Abuse and employees. Mental Health Services Administration offers guidelines and standards for monitoring of drug 16. d. Budget reviews require a return on investment for abuse and testing. AABB specializes in blood justification of capital expenditures. Replacement transfusion services. Clinical and Laboratory of old or nonfunctioning equipment, an operating Standards Institute offers educational and train- cost-reduction if a new instrument is purchased, ing tools for laboratories. Because this question or new services and technology offered through relates specifically to drug testing, SAMHSA is new instrumentation are all appropriate justifica- the best answer. tion categories for capital expenditures. APPENDIX A Answers and Rationales to Certification Preparation Questions 353

17. b. The Family and Medical Leave Act supplies 27. c. Managed health care plans reimburse by diagnosis guidelines for medical leave of absence. CAP, codes assigned to each patient encounter or case. CLMA, and CLSI are agencies or organizations for professional laboratory networking, stan- 28. b. Standard business practices and effective leaders dards, or guidelines for accreditation. and economists recommend a monthly review of budget variances. 18. a. The Code of Ethics as defined by ASCLS: “They contribute to the advancement of the profession 29. b. To accurately assess a cost per billable for a test, by improving the body of knowledge, adopting a total test volume is required to perform this scientific advances that benefit the patient, main- calculation. taining high standards of practice and education, and seeking fair socioeconomic working condi- 30. c. The Health and Human Services Office of Inspec- tions for members of the profession.” tor General (HHS OIG) developed the Medical Compliance Plan (MLP). 19. b. The three Rs of customer service are relationships, response, and results. 31. a. The Centers for Medicare & Medicaid Services (CMS) regulates all laboratory testing (except 20. b. Legal personnel issues is the key in this statement. research) performed on humans in the United Reimbursement does not fit into a personnel situ- States through the Clinical Laboratory Improve- ation, whereas medical leaves, termination proce- ment Amendments (CLIA). In total, CLIA covers dures, and interview questions are all applicable approximately 239,000 laboratory entities. to personnel issues. 32. a. The Office of Inspector General (OIG) set a tem- 21. b. The Fair Labor Standards Act (FLSA) regulates plate for laboratories to follow to ensure appro- overtime pay. priate billing practices are followed and to curb fraud and abuse. “What is ordered, is tested, 22. c. Diagnosis Related Groups (DRGs) is a system to reported and billed.” classify hospital cases into one of approximately 500 groups for reimbursement based on the 33. b. The Clinical Laboratory Standards Institute pub- nature of the illness. lishes guidelines for technical procedures and is often referenced by the accreditation agencies 23. a. An effective manager will schedule the appropri- (COLA, CAP) to laboratories. ate personnel to the workload requirements. Scheduling is an immediate and ongoing process 34. b. A 24-hour,7-days-per-week service contract can be versus workload management, which is a long- expensive, but if an instrument goes down with no range planning process (b), and a free-for-all backup, the vendor must supply a repair technician in a democratic schedule can create gaps in a immediately to remedy the problem and keep the schedule. A balance of experienced personnel instrument down time to a minimum. Response on all shifts is an appropriate scheduling time will be slower for the other scenarios listed. process. 35. a. 10,000 billable tests in a 2-week pay peri- 24. b. An interview purpose is to assess the qualifica- od¼260,000 annually (26 10,000). The time tions of an applicant and not to establish a friend it takes to produce the unit annually is 130,000 network (a) or to determine the religious back- (260,000 0.5 unit). Each full-time equivalent ground (c). Generally an applicant with a prior (FTE) is equal to 2080 hr/yr. 130,000 divided by arrest is not considered for an interview, but if 2080¼62.5 FTEs. interviewed, the goal is to determine the qualifications for the job. 36. a. The definition of productivity is the measure of the efficiency of the person in converting inputs 25. d. Annual budget assumptions are based on direct to useful outputs. This is measured by the actual and indirect costs to estimate expenses and pro- hours worked by the paid or earned hours of jected reimbursement for tests and procedures the individual employee (which would include to estimate revenue for the budget year. paid time off, holiday, sick, vacation, etc.).

26. c. Ambulatory Payment Categories are the U.S. gov- 37. b. Clinical laboratory has confidentiality guidelines ernment’s method of paying for facility outpatient (HIPAA), procedures (CLIA accreditation docu- services through the Medicare program. mentation), and chain of custody (drug screening 354 APPENDIX A Answers and Rationales to Certification Preparation Questions

documentation SAMSHA or similar). Posting of 45. a. The key to this answer is to apply the “most help- available jobs or open positions in the clinical ful during the interview” aspect to the answers. laboratory does not fall under institutional legal All answers listed could be perceived as a correct issues. response, but the best answer is to observe how the applicant addresses problem solving by giving 38. a. PRN is a Latin term, pro re nata, that translates to each applicant the same scenario and recording “as needed” or “as the situation arises.” their verbal and nonverbal response rather than In situations in which workload fluctuates, as- a yes/no answer. needed employees are very useful in scheduling for unexpected situations, to avoid overtime and 46. d. Best educational practice includes the inclusion of allow for schedule flexibility. learning objectives for each instructional unit. Learning objectives should explicitly describe 39. a. The Fair Labor and Standards Act of 1938 set what the student should be able to do after suc- forth guidelines for hourly employees and regula- cessful completion of the unit. tions on payroll classifications. If a payroll is following the 8 and 80 guideline, the employer is 47. a. Bloom’s taxonomy describes the various learning required to pay an employee overtime for any levels within the cognitive domain. Those levels work more than 8 hours in 1 day. are knowledge, comprehension, application, analysis, synthesis, and evaluation. 40. c. Management of employees in an effective and productive fashion requires much flexibility 48. c. The three learning domains are cognitive, psycho- and creative scheduling. If all highly trained motor, and affective. Affective traits are those and experienced personnel are concentrated on associated with behavior and attitude. one shift for a laboratory that is operating 24 hours per day, 7 days per week, it will place 49. undue stress, confusion, and inexperience on b. Well-written learning objectives should be explicit the other two shifts, improperly allocating and measurable. A student’s ability to list the cor- human resources and resulting in overtime costs, rect steps in a procedure in the correct order is an turnover of staff, and higher labor costs for those explicit, measurable learning objective. two shifts. 50. c. The taxonomic levels that define examination 41. a. Through guidelines of the U.S. Bureau of Labor questions are types I, II, and III. Type I questions and Occupational Safety and Health Administra- are simple, recall questions. Type II questions tion, all employers must offer the hepatitis B involve the ability to apply learned knowledge vaccine at employer cost to any employee who to answer a question. Type III questions require may have exposure to blood-borne pathogens. applying and assimilating knowledge to solve a complex problem such as making an accurate 42. b. A written technical procedure requires the meth- diagnosis given a set of laboratory test results. odology, quality control, and media and reagents needed to perform the test. Although a cost per billable could be added to any procedure, it is CHAPTER 11 not required or necessary. 1. c. Each liter contains 10 dL, so multiply 4 dL 43. 1 L/10 dL (or 0.1, which is equivalent) to cancel d. The College of American Pathologists, The Joint out the deciliter units. Commission, and the state health departments 1L all have legal authority for laboratory inspection 4dL ¼ 0:4L and accreditation. 10dL

44. b. Human resource guidelines on appropriate 2. b. Each liter contains 100,000 mL, so multiply 3 mL interview questions are based on U.S. laws, and 1 L/100,000 mL (or 0.000001, which is equiva- it is illegal to ask a person’s age, family status, lent) to cancel out the microliter units. or health care issues that could discriminate 1L unfairly against any applicant technically and 3mL ¼ 0:000003 L professionally proficient to perform the job listed. 100, 000 L APPENDIX A Answers and Rationales to Certification Preparation Questions 355

3. c. Each gram contains 1000 mg, so multiply 10 mg This means that 75 mL of the stock solution was 1 g/1000 mg to get the number of grams. Then used. This 75 mL was diluted to a final volume of multiply the number of grams by the number of 300 mL, so to determine the amount of diluent micrograms in each gram (106) to get the number needed, subtract the amount of stock solution from of micrograms present. the final volume:

6 300 75 ¼ 225 1g 10 mg 3 mL mL mL diluent needed 10 mg ¼ 10 10 g or 10, 000 mg 103 mg 1g 10. c. Molarity is the number of moles per liter of solu- 4. c. Each kilogram contains 1000 g, so multiply tion. In this problem, milliliters are given as units 0.85 kg by 1 103 g/1 kg to get the number of so convert this to L (1000 mL in 1 L): grams. 2 mol 1000 ¼ 5M 400 mL 1 103 g mL 0:85 kg ¼ 0:85 103 g 850 g 1kg or 11. a. Multiply molarity (which is converted from M to mol/L) by volume to solve for moles. 5. c. Using the equation to convert degrees Celsius to degrees Fahrenheit: 2 mol 1L 300 ml 2M ¼ 300 mL ¼ 0:6 moles F¼9/5 C+32 1L 1000 mL F¼9/5 25+32 F¼9/5 C+32 12. c. Multiply the volume of the solution by the molar- ¼ F 45+32 ity (written as mol/L) by molecular weight (g/mol) ¼ F 77 to solve for grams:

6. 1:5 mol 58:5g b. First convert degrees Fahrenheit to degrees 0:50 L ¼ 44 g Celsius: L mol C¼5/9 (F 32) C¼5/9 (80 32) 13. a. Knowing that 4 M equals 4 mol/L, solve for vol- C¼5/9 (48) ume by multiplying the weight of the sample C¼26.7 by the inverse of the molarity by the inverse Then convert the degrees Celsius to Kelvin: of molecular weight (to isolate liters in the K ¼ C+273 numerator): K¼26.7+273 ¼ 1 mol 1L K 299.7 125:6g ¼ 0:421 L 74:5g 4mol

1= 7. b. A 5 dilution is equivalent to 1 part of urine in a total of 5 parts. The remaining 4 parts are the dil- 14. b. 1= uent, so to prepare a 5 dilution of a urine sample, MW 1 mol ¼ ¼ 49:0g use 1 part urine and 4 parts diluent. Gram equivalent weight valence 54:2 8. b. ¼ ¼ 1:11 Eq 49:0 50g 1 1 1g 1:11 Eq ¼ N ¼ ¼ 0:56 N L 5 10 L 2L

9. b. When making a dilution that changes concentrations, use the following formula (where 1 repre- 15. b. First determine the amount of osmoles in the 3-kg sents the stock solution and 2 represents the solution by setting up a proportion: working solution): 0:67 osmol ¼ X osmol 1 3:0 V1 C1 ¼ V2 C2 kg kg V1 0:8 ¼ 300 0:2 3:0 0:67 ¼ X osmole V1 ¼ 75 mL X ¼ 2:01 osmole 356 APPENDIX A Answers and Rationales to Certification Preparation Questions

vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u The original solution contains 2.01 osmoles. NaCl uSðÞ 2 t x x dissociates into two osmotically active particles per n 1 molecule in solution. To determine the number of sffiffiffiffiffiffiffiffiffiffi moles in the 2.01 osmoles, use the following formula: 4051 ¼ 21:2 9 osmoles ¼ moles activeparticle 2:01 ¼ moles of NaCl 2 Moles of NaCl ¼ 1:005 22. c. The midrange is the average of the lowest and Then convert the number of moles to grams: highest values in the data set: 1:005 moles of NaCl ¼ Xgrams of NaCl 1 mol=58:5g M ¼ ðÞ4+14=2 X ¼ 59:1g M ¼ 9

16. b. The formula used for the calculation of the mean 23. a. When rounding, if the last digit is 5 and the pre- is: ceding number is even, the final retained digit Sx stays the same. x ¼ n 24. c. For addition and subtraction, the answer must Where x equals the mean, Sx is the sum of all values and n have the same number of decimal places as the equals the number of values in the set. starting value with the fewest number of decimal places. In this data set, 3.45 has the fewest number 17. b. The mode of a data set is the number or value that of decimal places, with two decimal places. The is seen most frequently. sum of this data set would be 36.556, so the answer is rounded to 36.56 to reflect two decimal places. 18. b. The standard deviation is the square root of the variance. 25. c. The product of the significant figures is 24 (4 6). The sum of the exponents is 5 (3+2). The correct 19. b. The formula used for the calculation of the mean answer is 24 105. is: Sx 26. a. Accuracy is the closeness of an individual result x ¼ n (or test value) to the true value. Where x equals the mean, Sx is the sum of all values, and n equals the number of values in the set. 27. c. The data from plots 5, 6, 7, and 8 all exceed the 1683 1 standard deviation mark, thus violating the ¼ ¼ 168 x 10 41s rule.

28. a. 1 is indicative of random error. The others are 20. 3s a. The mode is the most frequent result. In this data indicative of systematic error. set, there are three results of 150. qffiffiffiffiffiffiffiffiffiffiffiffiffi 29. a. The sensitivity of a test is the ability of a test to 2 21. ¼ SðÞxx minimize false-negative results. The specificity c. Standard deviation n1 Where SðÞxx 2 is the sum of the difference between of the test describes the ability of the test to min- each value and the mean squared and n 1 is the imize false-positive results. number of values (n) minus 1. 30. In this problem, the mean (x) is 168 ([180+200+150 a. The clinical sensitivity of a test is calculated +170+150+165+205+150+168+145]/10). by dividing the true positives (TPs) by the total of the patients with the disease (the sum of the x (x x)(x x)2 TPs and the false negatives [FN]). Response b 180 12 144 represents the specificity of the test, c the 200 32 1024 positive predictive values, and d the negative 150 18 324 predictive value. 170 2 4 150 18 324 165 39 205 37 1369 150 18 324 168 0 0 145 23 529 2 S (x - x) ¼4051 APPENDIX B Mock Examination

1. What is the preferred specimen for molecular tech- proteolytic enzymes and hemolysins that destroy niques for diagnosis of inherited mutations? cells and tissue are factors that contribute to patho- a. DNA extracted from peripheral blood mature genicity of which of the following organisms? red cells a. Pseudomonas aeruginosa b. DNA extracted from peripheral blood white cells b. Burkholderia cepacia c. RNA extracted from peripheral mature red cells c. Ralstonia pickettii d. RNA extracted from fresh serum d. Burkholderia mallei

2. The biochemical tests performed on a gram-positive 7. Which of the following factors binds to platelets via bacillus were consistent with those of Corynebacte- the glycoprotein Ib/IX receptor? rium diphtheriae. The MLS should now: a. von Willebrand factor a. Perform a spore stain of the colonies b. Factor II b. Determine if the isolate is toxigenic by performing c. Fibrinogen an Elek test d. Thrombin c. Perform an agglutination test to confirm the organism’s identity 8. The respiratory culture of a patient with cystic fibro- d. Subculture the organism to Hektoen enteric sis yielded gram-negative bacilli with the following medium and examine for black colonies reactions: Oxidase: Positive MacConkey agar: Positive 3. A decrease in serum haptoglobin accompanies which Glucose OF open: Positive Gelatin hydrolysis: Positive of the following? Pigment: Metallic green Arginine dihydrolase: Positive a. Extravascular hemolysis Growth at 42 C: Positive b. Intravascular hemolysis Which of the following is the most likely identifica- c. Extramedullary hematopoiesis tion of this organism? d. Suppressed erythropoiesis a. Burkholderia cepacia b. Pseudomonas aeruginosa 4. The presence of waxy casts in a microscopic exami- c. Acinetobacter baumannii nation of urine is consistent with a diagnosis of: d. Stenotrophomonas xylosoxidans a. Strenuous exercise b. Pyelonephritis 9. Which of the following cells can be described as neo- c. Glomerulonephritis plastic lymphocytes with noncleaved clumped nuclei d. Chronic renal failure and very basophilic cytoplasm with prominent vacuoles? 5. The decreased release of thyroid-stimulating hormone a. Reed Sternberg cells (TSH) would result in which of the following actions b. Mantle cell lymphoma cells from the hypothalamic-pituitary-thyroid axis? c. Small cell lymphocytic lymphoma cells a. Decreased release of TSH from the pituitary gland d. Burkitt’s lymphoma cells b. Increased release of TSH from the thyroid gland c. Decreased release of thyroid hormones from the 10. When preparing fresh frozen plasma for transfusion, thyroid glands what compatibility testing is performed? d. Increased release of thyroid hormones from the a. Perform a reverse grouping on donor plasma thyroid glands b. Perform a reverse grouping on recipient plasma c. Plasma must be HLA-compatible 6. Production of exotoxin A, which kills host cells by d. No testing is required inhibiting protein synthesis and production of several

357 358 APPENDIX B Mock Examination

11. The following OGTT results are indicative of what 17. A child presents with an infected fingernail. The state? sample of the wound grows a pure culture. The Fasting serum glucose: 124 mg/dL gram-stained smear showed small gram-negative 2-hour post load serum glucose: 227 mg/dL bacilli. a. Normal Additional characteristics of this organism include: b. Diabetes mellitus Requires increased CO2 for Oxidase-positive c. Addison’s disease growth d. Hyperinsulinism Catalase-, urease-, Pits the agar during indole-negative growth 12. A Prussian blue stain of a bone marrow shows blue The most likely identification of this organism is granules present inside of macrophages. Which of which of the following? the following disorders can be ruled out by this result? a. Weeksella virosa a. Chronic disease b. Legionella spp. b. Lead poisoning c. Iron deficiency c. Brucella melitensis d. Eikenella corrodens d. Iron overload

18. All of the following hormones increase serum glucose 13. A gram-negative diplococcus isolated from a levels with the exception of: patient’s CSF specimen gives the following results: a. Glucagon Chocolate agar: Growth Oxidase: Positive b. Cortisol SBA: Growth CTA test: Glucose, yellow; maltose, yellow; lactose, c. Epinephrine red; sucrose, red d. Insulin

This organism is most likely which of the following? 19. a. N. meningitides Based on the following results, what is the most likely b. N. gonorrhoeae diagnosis? c. N. lactamica ALP and GGT moderately increase d. M. catarrhalis ALT, LD, and total bilirubin minimally increase a. Cirrhosis 14. What characteristic red cell shape is associated with b. Biliary obstruction extravascular hemolysis? c. Infectious hepatitis a. Burr cell d. Mumps b. Schistocyte c. Spherocyte 20. Which of the following conditions is usually associ- d. Target cell ated with thrombosis? a. Factor XIII deficiency 15. Two units of blood are ordered for a patient. Previ- b. Factor V Leiden mutation ous blood bank records indicate the patient had an c. Vitamin K deficiency anti-K 3 years ago. What is the next course of action d. PAI-1 deficiency for completing this order? a. Select three random units of ABO-compatible 21. An antibody panel shows a pattern of anti-S, but blood and crossmatch anti-E cannot be ruled out on this initial panel. b. Perform an immediate spin crossmatch on two Which of the phenotyped cells below would be best K-negative units to help in confirming only an anti-S is present? c. Antigen type units for the K antigen and only a. EÀe+SÀs+ crossmatch units positive for K b. E+e+S+s+ d. Antigen type units for the K antigen and only c. E+e+S+sÀ crossmatch units negative for K d. E+eÀSÀs+

16. The antibody associated with type IV hypersensitiv- 22. The most critical step in obtaining accurate Gram ity is: stain results is the application of which of the a. IgA following? b. IgE a. Safranin c. IgG b. Crystal violet d. IgM c. Gram’s iodine e. None of the above d. Acetone/ethanol APPENDIX B Mock Examination 359

23. Which cell is capable of both phagocytosis and anti- c. Chocolate agar is not appropriate for use in the gen processing? X and V factor requirement test a. Basophil d. The chocolate plate should not have been b. Lymphocyte incubated in CO2 c. Macrophage d. Segmented neutrophil 29. Which of the following conditions is associated with abnormal von Willebrand factor (VWF) cleaving 24. People who have anti-A and anti-B blood group anti- protease? bodies detected in their serum are said to be of which a. Disseminated intravascular coagulation blood group? b. Hemolytic uremic syndrome a. AB c. March hemoglobinuria b. O d. Thrombotic thrombocytopenic purpura c. A d. B 30. In salicylate overdose, what is the first acid-base disturbance present? 25. Which of the following is true regarding indirect a. Metabolic acidosis bilirubin? b. Metabolic alkalosis a. It requires an accelerator to react with the diazo c. Respiratory acidosis reagent d. Respiratory alkalosis b. It is also called delta bilirubin c. It is calculated by adding the total bilirubin and 31. Which of the following red cell indices support direct bilirubin together microcytic hypochromic red cell morphology? d. It is elevated in liver obstruction MCV (fL) MCH (pg) MCHC (g/dL) a. 118 35 34 26. The examination question “What color is a posi- b. 75 30 33 tive spot indole test?” falls under which taxonomy c. 92 30 34 level? d. 67 22 27 a. Level I b. Level II 32. According to AABB Standards, what is the minimum c. Level III information required to be on a sample label for d. Level IV transfusion testing purposes? a. First and last name of patient and date of 27. A positive result was obtained when a Venereal Dis- collection ease Research Laboratory test was performed on a b. First and last name of patient, date of collection, patient’s serum. The MLS should now: and identification of phlebotomist a. Report syphilis antibodies present c. First and last name of patient, date of collection, b. Report positive for syphilis identification of phlebotomist, and unique c. Perform a treponemal test identifying number d. Perform a nontreponemal test d. First and last name of patient, date of collection, identification of phlebotomist, unique identifying 28. An X and V factor requirement test was performed number, and date of patient’s last transfusion on a Haemophilus isolate using the following protocol: 33. The L-pyrolidonyl-b-naphthylamide (PYR) hydroly- 1. A TSB suspension of the organism was prepared sis test is a presumptive test for which of the follow- 2. The organism suspension was swabbed to a choc- ing streptococci? olate agar plate a. Groups A and B 3. X and V factor disks were placed on the agar b. Streptococcus pneumoniae and group C plate c. Group A and Enterococcus 4. The plate was incubated for 24 hours at 35 Cin d. Group A and Streptococcus bovis CO2 On examination, the plate showed growth over the 34. Which of the following ketones is(are) not detected entire plate. In troubleshooting the protocol and using the reagent strip? results, the correct determination is: a. Acetoacetic acid a. Organism should be identified as H. b. Acetone aegyptius c. b-Hydroxybutyric acid b. TSB is not an appropriate medium for prepar- d. All of the above are detected using the ing the organism suspension reagent strip 360 APPENDIX B Mock Examination

35. What is the transport temperature of packed red 41. How do sickle cells or spherocytes interfere with the blood cells? erythrocyte sedimentation rate (ESR)? a. Maintained at room temperature, not to exceed a. Prevent rouleaux formation, so falsely decrease 25 C b. Encourage rouleaux formation, so falsely b. Transported at temperatures below 6 C increase c. Transported on ice not to exceed 10 C c. Agglutinate and increase red cell mass, so falsely d. Transported on dry ice at temperature below increase 25 C d. Decrease plasma viscosity, so falsely decrease

36. An anaerobic, box-shaped gram-positive bacillus is 42. What is the most likely cause of the ABO discrepancy positive for the reverse CAMP test. This organism below? would also be: a. Lipase positive Anti-A Anti-B A1 cells B cells Autocontrol b. Indole positive Patient 3+ 0 1+ 4+ Negative c. Lecithinase positive d. Spore negative a. Subgroup of A b. Cold-reacting antibody 37. If the cytoplasm of a cell is very basophilic or blue c. Neonatal patient when stained with Wright stain, which of the follow- d. Rouleaux ing does it contain? a. Increased concentration of lysosomes 43. What is a point mutation? b. Increased number of Golgi bodies a. Mutation of the stop codon c. Increased number of ribosomes b. Replacement of one nucleotide in the normal gene d. Increased number of mitochondria with a different nucleotide 38. c. Addition of one nucleotide in the normal gene Which of the following is the best way to handle CAP d. Deletion of one nucleotide in the normal gene Proficiency Testing samples? a. Be extra careful and run the test samples several 44. times before reporting to make sure your results Hemoglobin electrophoresis is performed on a are correct patient with known homozygous Hb S who has b. Ensure that quality control and maintenance are received red cell transfusions in the past week. Which acceptable before performing both patient testing hemoglobins would be expected on the gel? and PT testing a. Hb S and Hb F c. Double-check your answers before mailing by b. Hb A and Hb F checking your results with those from another c. Hb S, Hb A, and Hb F laboratory d. Hb S, Hb A, Hb F, and Hb A2 d. The most experienced MLS should do all proficiency testing to make sure it is right 45. Which of the following is the correct location of enteric “H” antigens? 39. A fastidious organism gave the following results: a. Flagella SBA growth: Growth: After Niacin: Yellow b. Capsule Negative 7 days c. Cell wall Acid-fast stain: Colonies: Buff 68 C catalase: No d. Cytoplasm Positive color bubbles The organism is most likely: 46. You receive a blood gas sample on a patient who is in a. M. scrofulaceum the emergency room for assessment of unexplained b. M. xenopi vomiting for the past 4 days and abnormal respira- c. M. bovis tions. Which of the following interpretations best d. M. tuberculosis describes the patient’s blood gas results?

40. The most probable explanation for a patient who pH: 7.50 PO2:85mmHg P : 55 mm Hg HCO À: 35 mmol/L presents with an elevated osmolal gap, metabolic aci- CO2 3 dosis, and calcium oxalate crystals in the urine is: a. Methanol intoxication a. Partially compensated metabolic acidosis b. Ethanol overdose b. Partially compensated metabolic alkalosis c. Ethylene glycol intoxication c. Compensated metabolic alkalosis d. Cyanide poisoning d. Compensated metabolic acidosis APPENDIX B Mock Examination 361

47. The agency that is responsible for protecting a. Accelerated phase workers’ health and well-being is: b. Dormant phase a. CAP c. Mobile phase b. TJC d. Terminal phase (blast crisis) c. NAACLS d. OSHA 53. This type of resistance mechanism modifies the antibiotic targets and results in reduced affinity of anti- 48. A patient’s sample shows a large spike on serum pro- biotics for their microbial target sites. tein electrophoresis in the g region. On further testing, a. Cell-wall inhibition the spike is shown to be mostly IgM. Which of the fol- b. Protein synthesis modification lowing is true regarding this patient’s diagnosis? c. Enzyme modification a. Further testing is necessary to determine if the d. Nucleic acid modification patient has multiple myeloma b. The patient had a high-protein meal just before 54. Which of the following tests is used to monitor the test patients for early signs of renal disease? c. Most likely has Waldenstro¨ m’s macroglobuli- a. Glucose measurement using the reagent strip nemia b. Protein measurement using the reagent strip d. May be in the early stages of combined variable c. Specific gravity measurement using a immunodeficiency disease (CVID) refractometer d. Microalbumin measurement using a sensitive 49. Which one of the following tests on donor units is not reagent strip required? a. Anti-EBV 55. A serum protein electrophoresis was performed on a b. Antibody screen patient. The MLS noted a sharp peak in the gamma- c. Anti-HCV globulin fraction. This peak was similar to the albu- d. West Nile virus min peak. This pattern is consistent with that of which of the following? 50. The white count on an adult patient is 2.5Â109/L. a. Cirrhosis When the blood film is examined, the following b. Acute inflammation results are obtained: 15% neutrophils and 75% lym- c. a1-Antitrypsin deficiency, severe emphysema phocytes. Reference ranges for these cell types, both d. Monoclonal gammopathy relative and absolute, are as follows: 56. A person of the genotype R1R1 could potentially pro- 9 Cells Relative % Absolute no. (Â10 /L) duce antibodies to which of the following Rh Neutrophils 48-70 2.4-8.2 antigens? Lymphocytes 18-42 1.4-4.0 a. D What conclusion can be made about the number of b. e lymphocytes for this patient? c. C a. They are relatively increased but normal in abso- d. E lute numbers b. They are both relatively and absolutely increased 57. Which of the following groups of organisms is most c. They are relatively decreased but absolutely correct for use in performing quality control on a TSI increased agar slant? d. They are both relatively and absolutely decreased a. Escherichia coli and Salmonella, Shigella, and Pseudomonas spp. 51. Which of the following conditions is caused by an b. Escherichia coli and Klebsiella and impairment of an enzyme needed to conjugate Pseudomonas spp. bilirubin? c. Proteus mirabilis, Enterobacter spp., and Escher- a. Gilbert’s disease ichia coli b. Physiologic jaundice of the newborn d. Shigella and Salmonella spp. c. Dubin-Johnson syndrome d. Hemolytic jaundice 58. Which of the following red blood cell precursors has a nucleus that can be described as pyknotic? 52. The most recent CBC results for a patient who has a. Basophilic normoblast been treated for chronic myelogenous leukemia b. Orthochromic normoblast (CML) for several years show a blast count of 38% c. Polychromatophilic normoblast in the differential. The CML is now in which phase? d. Pronormoblast 362 APPENDIX B Mock Examination

59. When testing the patient sample, it was reactive with 65. All of the following characteristics are consistent all panel cells tested, including the autocontrol. Pre- with the appearance of normal cerebrospinal fluid vious records indicate the patient underwent transfu- except: sion 1 month ago. What is the next course of action a. Crystal clear for pretransfusion testing? b. CSF protein of 20 mg/dL a. Perform an enzyme panel c. IgG index of 0.70 or less b. An autoadsorption test is necessary because of the d. WBC count greater than 100/mL patient’s autoantibody c. An alloadsorption test should be performed to 66. Which of the following could result in a false- determine if an alloantibody is present negative result for a DAT? d. It is necessary to perform both an adsorption and a. Improper washing of cells enzyme panel b. AHG reagent not added c. Low pH of saline 60. Alkaline phosphatase values are expected to be d. All of the above increased in all of the following situations except: a. Child undergoing a growth spurt 67. b. During a time of increased bone remodeling An organism isolated from a stool culture gives the c. After a myocardial infarction following reactions: d. Paget’s disease Lactose: Colorless VP: Colorless Lysine: Purple TSI: Red/black Citrate: Blue Indole: Colorless 61. A patient comes to the emergency room with report of Which of the following is the next step in identifying an upset stomach. He has a rash covering his body,and the organism? he is becoming increasingly hypotensive. The blood a. Report Salmonella present samples that you drew for culture were positive within b. Report no enteric pathogens isolated 24 hours for gram-positive cocci in clusters. The most c. Perform serogrouping with Salmonella antisera likely causative organism is which of the following? d. Perform serogrouping with Shigella antisera a. Group B streptococci b. Staphylococcus aureus 68. Which of the following is true about enzymes c. Enterococcus sp. and their effect on red cell antigens and antibodies? d. Staphylococcus sp. not S. aureus a. The Rh, Duffy, MN, and Ss system antigen and antibody reactions are enhanced 62. A 13-year-old boy reports bone pain and headaches. b. The Duffy, MN, and Ss system antigens are Â 9 His white count is 68.0 10 /L, platelet count is destroyed, so antigen and antibody reactions Â 9 57 10 /L, and hematocrit is 34%. His differential are not detectable shows mostly nucleated cells that are identified as c. Only the Duffy system antigens are affected by blasts. They are CD2, CD4, and CD8 positive. enzymes Which disorder does he most likely have? d. The Rh, Duffy, MN, and Ss system antigens are a. Acute myeloid leukemia without maturation destroyed, so antigen and antibody reactions b. Acute myeloid leukemia with maturation are not detectable c. Immature B-cell acute lymphoblastic leukemia d. T-cell acute lymphoblastic leukemia 69. The underlying cause for anemia of chronic inflam- 63. A patient sample tested positive for anti-dsDNA anti- mation is that: bodies. The patient most likely has which of the a. Acute phase reactants impair iron mobilization following? b. Growth factors prevent iron incorporation into a. Rheumatoid arthritis protoporphyrin b. Syphilis c. Inflammation slows down cell division c. Infectious mononucleosis d. Iron stores in the bone marrow are depleted by d. Systemic lupus erythematosus acute phase reactants

64. Which of the following analytes is helpful in distin- 70. In patients with developing subclinical hypothyroid- guishing a condition affecting the liver from bone dis- ism, TSH levels will likely be ______, and fT4 will ease in the presence of an elevation of ALP? likely be ______. a. AST a. Decreased, increased b. ALT b. Increased, decreased c. ALP c. Decreased, normal d. GGT d. Increased, normal APPENDIX B Mock Examination 363

71. Which of the following would result in a permanent 77. Which of the following lipid measurements is esti- deferral for a whole blood donation? mated using a formula, and not measured? a. Jaundice as a small child a. Total cholesterol b. Temperature above 37 C b. HDL c. Recipient of human growth hormone c. Triglycerides d. Accidental needle stick 1 year previously; nega- d. LDL tive for infectious diseases 78. Which of the following would be an acceptable alter- 72. A 30-year-old pregnant female has an OGTT chal- native for a red cell transfusion if ABO group-specific lenge test (screening test) performed at 26 weeks ges- blood was not available? tation. Her serum glucose result is 100 mg/dL at a. Group A recipient with group B donor 1 hour. What should occur next? b. Group O recipient with group AB donor a. Nothing, this confirms the diagnosis of GDM c. Group O recipient with group A donor b. This is suspicious for GDM, a diagnostic OGTT d. Group AB recipient with group B donor test should be performed 79. The laboratory manager’s most appropriate c. Nothing, this is the expected glucose level in a approach to performing employee evaluations could pregnant female best be described as? d. This is suspicious for hypoglycemia, a 5-hour a. Positive reinforcement OGTT should be performed b. Destructive criticism c. Retribution 73. A PT and PTT on a patient fails to correct when d. Threatening mixed with normal pooled plasma. Which of the following is a possible explanation? 80. A latex agglutination test for cardiolipin is performed a. The patient has a factor deficiency of factor X on a patient suspected of having which disease? b. The patient has thrombocytopenia a. Rheumatoid arthritis c. A circulating inhibitor is present in the patient b. Syphilis d. This is a normal finding c. Infectious mononucleosis d. The patient may be pregnant 74. Which of the following sets of results most closely indicates an exudate? 81. Before release of a unit of red blood cells for transfu- a. Clear, ratio of fluid to serum LD of 0.8, ratio of sion, which records must be checked? fluid to serum protein of 0.7, WBC count of a. Donor’s first and last name on the donor label 1000/mL b. The intended recipient’s first and last name, b. Cloudy, ratio of fluid to serum LD of 0.4, ratio unique identifier number, and ABO/Rh of fluid to serum protein of 0.5, WBC count of c. Name of person who performed the crossmatch 800/mL d. Date and time the blood was issued c. Cloudy, ratio of fluid to serum LD of 0.8, ratio 82. of fluid to serum protein of 0.7, WBC count of Laboratory diagnosis of hookworm is usually made by: 2500/mL a. Filariform and larvae in stool d. Clear, ratio of fluid to serum LD of 0.45, ratio b. Serology of fluid to serum protein of 0.40, WBC count of c. Symptoms of nausea, malnutrition, diarrhea, and 800/mL anemia d. Ova in feces 75. Immunoglobulins function by all of the following 83. Which red cell inclusion is often found in lead poi- except: soning cases? a. Coat invading cells to act as opsonins a. Basophilic stippling b. Reneutralize toxins produced by viruses b. Cabot rings c. Activate complement cascade of cell lysis c. Heinz bodies d. Lyse viruses by activating T cells d. Howell Jolly bodies

76. Irradiated red blood cell transfusion would not be indi- 84. The immunoglobulin typically found in secretions is: cated for which of the following patient diagnoses? a. IgA a. Exchange transfusion b. IgD b. Bone marrow transplant c. IgE c. Severe combined immunodeficiency syndrome d. IgG d. Warm autoimmune hemolytic anemia e. IgM 364 APPENDIX B Mock Examination

85. Both blood and urine cultures are positive for an component is most appropriate to prevent this reac- oxidase-negative, gram-negative bacilli that gave tion in the future? the following growth characteristics and biochemical a. Red blood cells reactions: b. Irradiated red blood cells MacConkey agar: Colorless Citrate: Positive c. Leukocyte-reduced red blood cells colonies d. CMV-seronegative red blood cells TSI reactions: Alk/Acid, no Lysine decarboxylase: gas, no H S Positive 2 92. Indole: Negative Ornithine An employee’s demeanor falls under which of the fol- decarboxylase: Positive lowing domains? Urea: Negative Motility: Positive a. Psychomotor b. Cognitive These reactions are consistent with which of the fol- c. Affective lowing enteric pathogens? d. Didactic a. Serratia marcescens b. Klebsiella pneumoniae c. Yersinia enterocolitica 93. Features used in identifying Cryptococcus neofor- d. Proteus mirabilis mans should include which of the following? a. Capsule formation, mucoid, creamy-white to 86. The HIV virus belongs to which of the following yellow colonies, urea positive groups of viruses? b. No capsule formation, produces mycelial forms, a. Rhinoviruses urea positive b. Herpesviruses c. Capsule formation, mucoid, creamy-white to c. Retroviruses yellow colonies, urea negative d. Arboviruses d. No capsule formation, budding yeast cells, mucoid, creamy-white colonies, partially acid-fast 87. Which of the following is a CAP service that accounts for workload recording per billable test plus nonbill- 94. The growth factor erythropoietin is produced by able expenses? which organ? a. CLIA a. Bone marrow b. COLA b. Kidney c. HFAP c. Spleen d. LMIP d. Thymus

88. Which of the following can occur days to weeks after 95. A neonate is to be transfused for the first time with transfusion? group O red blood cells. Which of the following is a. Transfusion-associated graft-versus-host disease performed for compatibility testing? b. Hemolytic transfusion reaction a. Perform an antibody screen and crossmatch with c. Posttransfusion purpura mother’s serum d. All of the above b. Perform an antibody screen and crossmatch with baby’s serum 89. Which of the following is associated with a decrease c. Crossmatch is not necessary if initial anti- in cerebrospinal fluid glucose? body screen of mother’s or baby’s serum was a. Meningitis negative b. Increased serum glucose level d. An antibody screen or crossmatch is not neces- c. Decreased use of glucose by brain cells sary, issue group and Rh compatible blood d. Damaged blood-brain barrier glucose transport 96. ______protect from viral infection by recognizing 90. Which of the following vitamins is less likely to accu- and destroying infected cells and ______protect us mulate at toxic levels? indirectly by producing cytokines to stimulate B a. Vitamin A cells. b. Vitamin C a. CD4+ cytotoxic T lymphocytes; CD8+ T helper c. Vitamin D lymphocytes d. Vitamin E b. CD8+ cytotoxic T lymphocytes; CD4+ T helper lymphocytes 91. A patient transfused with several units of red blood c. NK cells; CD8+ T helper lymphocytes cells developed a febrile transfusion reaction. What d. NK cells; CD4+ cytotoxic T lymphocytes APPENDIX B Mock Examination 365

97. Pernicious anemia can be distinguished from folate calcium in the blood by enhancing absorption deficiency by which of the following findings? through the intestines, stimulation of osteoclasts, a. Presence of hypersegmented neutrophils and suppressing loss in the urine? b. Mean cell volume a. PTH c. Bone marrow findings b. Calcitonin d. Presence of autoantibodies to intrinsic factor c. Vitamin D d. Cortisol 98. Which of the following pairings of crystals and causes is not correct? 100. Which of the following patients would be a candi- a. Calcium oxalate crystals and antifreeze ingestion date for RhIg? b. Uric acid crystals and patients receiving a. O-Positive mother who had a B-negative baby, chemotherapy first pregnancy, anti-K in mother c. Cystine crystals and not clinically significant b. B-Negative mother who had an O-positive baby, d. Amorphous phosphate crystals and not clini- second pregnancy, no anti-D in mother cally significant c. A-Negative mother who had an O-negative baby, fourth pregnancy, anti-K in mother 99. Which of the following hormones involved in cal- d. AB-Negative mother who had a B-positive baby, cium regulation acts by increasing the amount of second pregnancy, anti-D in mother 366 APPENDIX B Mock Examination

ANSWERS TO MOCK EXAMINATION

1. b 35. c 69. a 2. b 36. c 70. d 3. b 37. c 71. c 4. d 38. b 72. c 5. c 39. d 73. c 6. a 40. c 74. c 7. a 41. a 75. d 8. b 42. a 76. d 9. d 43. c 77. d 10. d 44. d 78. d 11. b 45. a 79. a 12. c 46. b 80. b 13. a 47. d 81. b 14. c 48. c 82. d 15. d 49. a 83. a 16. e 50. a 84. a 17. d 51. a 85. a 18. d 52. d 86. c 19. b 53. c 87. d 20. b 54. d 88. d 21. d 55. d 89. d 22. d 56. d 90. c 23. c 57. a 91. c 24. b 58. b 92. c 25. a 59. c 93. a 26. a 60. c 94. b 27. c 61. b 95. c 28. c 62. d 96. b 29. d 63. d 97. d 30. d 64. d 98. c 31. d 65. d 99. a 32. c 66. d 100. b 33. c 67. c 34. c 68. b APPENDIX C Examination Preparation Worksheet

This worksheet is designed to help you formulate a study plan to prepare for the certification examination. To begin this process, it’s important that you complete a mock examination to identify areas of strengths and weaknesses.

My score on the practice examination: ______

Content Areas: Hematology, Microbiology, Immunohematology, Clinical Chemistry, Hemostasis, Clinical Fluids, Immunology, Molecular, Laboratory Math Rank the areas that are your areas of strength based on your performance on questions: Rank the areas in terms of your comfort level:

1. 1.

2. 2.

3. 3.

4. 4.

5. 5.

6. 6.

7. 7.

8. 8.

9. 9.

Part of the challenge in preparing for the certification examination in determining where to start! Most individuals tend to spend more time reviewing the subjects and topics that are most familiar and enjoyable; however, it’s the other topics that need to be the focus in the early stages of preparation.

List the content areas that are the most challenging:

367 368 APPENDIX C Examination Preparation Worksheet

Making a Plan

Anticipated examination date: ______

Time (in weeks) to examination date: ______

The amount of time that you should spend reviewing in each area will vary depending on your schedule and how much review is needed. One to two weeks per content area is ideal if you have time each day to set aside as review time. An example of a 12-week plan is included below. It can be modified based on your schedule. Using a calendar to schedule specific study times will help you to stick with the plan. Some individuals may find it helpful to spend the review time at a library, bookstore, or coffee shop to help them focus on the review and minimize distractions. For each content area, complete the following tasks: • Complete practice questions during first 1-3 days of each period • Prepare list of specific content items that were missed • Spend the rest of this period reviewing those specific content areas • Prepare mini-study guides of each topic. • Critically review the questions that were missed. How can each answer choice be ruled out? Write a few sentences to explain the topic. • Prepare charts, diagrams, and flow charts. • Teach it to a friend. • Keep all of these notes in a notebook or in a binder to review again during the last week!

Examination Date: Beginning to prepare • Complete practice examination. • Identify areas of strengths and weaknesses. • Using a calendar, schedule study times leaving up to the examination date.

11-12 weeks out Clinical Chemistry concentration

9-10 weeks out Hematology concentration

7-8 weeks out Immunohematology concentration

5-6 weeks out Microbiology concentration

4 weeks out Immunology and Lab Math concentration

3 weeks out Hemostasis and Molecular concentration

2 weeks out Clinical Fluids concentration Begin reading through the notes compiled during the review process.

Last week! At the beginning of the week, complete a practice examination. Identify the questions that were missed and review those topics. Keep reading over the notes compiled during the review process.

Day before Be sure to have all of your materials needed to take to the exam. Double-check the location, date, and time. Get some rest! APPENDIX C Examination Preparation Worksheet 369

My Plan!

Examination Date: Beginning to prepare • Complete practice examination. • Identify areas of strengths and weaknesses. • Using a calendar, schedule study times leading up to the examination date.

11-12 weeks out

9-10 weeks out

7-8 weeks out

5-6 weeks out

4 weeks out

3 weeks out

2 weeks out

Last week!

Day before Be sure to have all of your materials needed to take to the exam. Double-check the location, date, and time. Get some rest! APPENDIX D Color Insert Figure Credit Lines

Plates 1, 2, 3, 4, 5, 8, 9, 10, 12, 13, 14, 15, 16 Plate 18 Courtesy Joel Mortensen, PhD. Photograph by Dr. Mae Melvin, courtesy the Centers for Disease Control and Prevention, Public Health Image Plate 6 Library, http://phil.cdc.gov/. Photograph by Dr. W H Ewing, courtesy the Centers for Disease Control and Prevention, Public Health Image Plates 17, 19, 20, 21, 22, 23, 24, 25, 26 Library, http://phil.cdc.gov/. Courtesy the Centers for Disease Control and Prevention.

Plate 7 Plates 27, 28, 29, 30, 31, 32 Photograph by Dr. V R Dowell, Jr, courtesy the Centers From Rodak BF, et al: Hematology: clinical principles for Disease Control and Prevention, Public Health Image and applications, ed 4, St Louis, 2012, Saunders. Library, http://phil.cdc.gov/.

Plate 11 From Public Health Photo Library (PHL 960), http://phil. cdc.gov/phil/details.asp.

370 INDEX

A Aerobic gram-positive bacillus, 9–12 Aerobic respiration, 4 A antigen Aeromonas hydrophilia,22 antibodies to, 201 Aerotolerant anaerobe, 4 biochemical structures of, 201f Affective learning, 290 ABO blood group, 200–203 Affective learning objective, 290 ABO blood typing, 202t Affinity, 186 ABO hemolytic disease of fetus and newborn, 218, 218t Afibrinogenemia, 149t Absidia species, 57 Agar, 52 Acanthamoeba species, 74 Agglutination, 186 Acanthocyte (spur cell) tests and assays, 186–187 cell descriptiom and diseases associated with, 100t Agglutination reaction Acanthocytosis, 113 grading, 207, 208f Accreditation, laboratory, 286 Aggregatibacter aphrophilus,25 Achromobacter species, 29 Aggregatibacter segnis,25 Acid-base disorder, 235t Alanine aminotransferase (ALT) Acid-base status, 233 distribution and clinical significance of, 252t Acidic urine, 165 reaction catalyzed, 250t Acinetobacter species, 28 recommended ranges for, 252t Acquired aplastic anemia, 109 Albumin, 241 Acquired hemostastic disorders with bleeding, 149 A/G ratio and, 241 Acquired immunodeficient syndrome (AIDS), 55 measurement of, 242t Acquired platelet function disorder, 146 specimen considerations for, 242 Acremonium species, 54–55, 60 Albumin/globulin ratio (A/G ratio), 241 Actinomadura species, 65 Alcaligenes faecalis,29 Actinomyces israelii,35 Alcaligenes species, 29 Actinomyces species, 64 Alcoholic liver disease, 244 Activated protein C resistance (APCR) test, 153 Alder-Reilly anomaly, 119 Acute anemias of ambiguous lineage, 126 Aldosis, 229 Acute erythroid leukemia, 126 Aldosterone, 248 Acute glomerulonephritis, 167t Alkaline phosphatase (ALP) Acute hemolytic transfusion reaction, 117 distribution and clinical significance of, 252t Acute intersititial nephritis, 167t reaction catalyzed, 250t Acute lymphoblastic leukemia, 124 recommended ranges for, 252t Acute megakaryoblastic leukemia, 126 Alkaline urine, 165 Acute monoblastic/monocytic leukemia, 126 Alloimmune hemolytic anemia, 117 Acute myeloid leukemia (AML), 124–126 Ambulatory payment category (APC), 288 with maturation, 126 American Association of Blood Banks (AABB), 286 with minimal differentiation, 126 American dog tick, 80 with myelodysplasia-related changes, 125–126 American Osteopathic Association (AOA), 286 not otherwise specified, 126 American Society for Histocompatibility and immunogenetics with recurrent genentic abnormalities, 125 (ASHI), 286 without maturation, 126 Amino acid, 264 Acute myelomonocytic leukemia, 126 mRNA translation during protein synthesis, 264t Acute myocardial infarction, 251t Aminoglycoside, 38 Acute pyelonephritis, 167t Aminostat-FLM, 173 Acute tubular necrosis, 167t Ammonium biurate crystals, 165 Adaptive immunity, 181–185 Amniotic fluid, 172–173 cells of, 181–182 Amoeba, 74–75 humoral components of, 182–184 genus and species of, 72t Addison’s disease, 250 Amorphous phosphate crystals, 165 Adenine, 264f Ampicillin crystals, 165 Adenovirus, 68 Amylase Adrenal cortex, 248–250 distribution and clinical significance of, 252t Adrenal cortex disorder, 248–250 recommended ranges for, 252t Adrenal cortex hormone, 248 Anaerobic bacteria Adrenal gland, 248 identification of, 19b Adrenal medulla, 248 isolated from infection, 33–35 Adrenal medulla hormone, 248 Anaerobic culture, 3 Adrena medulla disorder, 250 Anaerobic gram-negative bacillus, 36–37 Aerobacter species, 21 identification of, 19b Aerobic culture, 2 Ancyclostoma duodenale,73

Note: Page numbers followed by “f” refer to illustrations; page numbers followed by “t” refer to tables; page numbers followed by “b” refer to boxes.

371 372 Index

Androgen, 248 Autoimmune disease, in immunology, 190–191 Androstenedione, 248 Autoimmune hemolytic anemia, 116–118 Anemia, 103–104 characteristics of, 116t types of, 104 Autoimmunity, 190 Anemia of chronic inflammation, 104, 106 Autosomal dominant disease, 271 Anemia of chronic kidney disease, 109 Autosomal recessive disease, 271 Aneuploidy, 276 Autosomal recessive gene, 268f Anion gap, 235 Avidity, 186 causes of increased/decreased, 238t pathologic processes for, 238 recommended ranges for, 236t B Anisocytosis Babesia, 72t,78 cell descriptiom and diseases associated with, 100t Babesia, 115 Annelloconidia, 50 Bacillus anthracis, 10 Annual review, 287 Bacillus cereus, 10 Anode, 241f Bacillus species, 9–10 Anthrax, 10 Back-up broth, 3 Antibody, 183–184, 184f Bacteremia, 19 neutralization substances for, 210t Bacteria phase of reactivity and, 209t fungus-like, 64–65 Antibody M, 205 groups of, 4 Antibody N, 205 growing in laboratory Antibody S and s, 205 media for, 1–3 Antibody screen requirements for, 3 identification and, 206–211 hemolysis and, 115 potentiators and methods, 206t serology and, 187 Antibody screen antigen sheet, 206t, 209t structure and metabolism of, 3–5 Anticoagulant therapy, 150 taxonomy for, 5 Antidiuretic hormone, 166f, 167f in urine, 164 osmolality and volume regulation, 236 virulent factors of, 5 Anti-DNase B titer, 187 Bacterial genome, 274 Antigen, 184–185 Bacteroides fragilis, 36–37 in ABO blood group, 200 Balantidium coli,75 immune response to, 185 Band neutrophil, 95t Antigen-antibody reaction, 186 normal range values for, 98t grading, 208f B antigen Antigen D, 204 antibodies to, 201 Antiglobulin crossmatching, 211 biochemical structures of, 201f Antimicrobial agent, 37–38 Bartonella henselae,31 Antimicrobial susceptibility testing method, 37 Bartonella species, 31 Antinuclear antibody test, 191 Base excess, 235t Antiplatelet therapy, 150 Basidiospore, 50 Anti-streptolysin O (ASO) titer, 187 Basophil, 93 Antithrombin, 146, 150 normal range values for, 98t a1-Antitrypsin, 241 Basophil granule, 98b Aplastic anemia, 108–109 Basophilia, 118 Apoprotein, 228–229 Basophilic normoblast, 92t classes of, 230t B-cell lymphoblastic leukemia/lymphoma, 124 Arbovirus, 71 B-cell lymphoma Arenavirus, 69 morphologic and immunophenotypic features of, 127t Arthritis, 171t Bedbug, 80 Arthroconidia, 50 Beef tapeworm, 76 Arthropod, 72t Bench (clinical) teaching, 291 Artifact, 165t Bernard-Soulier syndrome, 148t Ascaris lumbricoides,73 Bicarbonate, 234, 235 Ascospore, 50 pathologic processes for, 238 Asparte aminotransferase Bilirubin, 162 distribution and clinical significance of, 252t measurement of, 244t recommended ranges for, 252t processing in hemoglobin degradation, 162f Aspergillus species, 56 recommended ranges for, 245t A subgroup, 200 specimen collection and handling, 244 Atypical chronic myeloid leukemia, 124 Bilirubin crystals, 165 Atypical mycobacteria, 13–15 Bilirubin metabolism, 242, 243f Auditory learner, 291 disorders of, 243–244 Authoritative leader, 283 Billable test/FTE, 288 Autocontrol, 208 Biochemical mediators, hemostasis and, 146 positive, flow chart for, 210t Bioterrorism agent, 275 Autocratic leader, 283 Biovar trachoma, 30 Index 373

Black piedra, 58 Burkholderia species, 28–29 Bladder fluke, 78 Burr cell (echinocyte) Blake, Robert, 284 cell descriptiom and diseases associated with, 100t Blanchard, Ken, 284 Blast crisis, 120 Blast forms, 169t C Blastocystis hominis,76 Calcitonin, 239t Blastomyces dermatitidis,63 Calcium Bloastoconidia, 50 measurement of, 240t Blood pathologic processes for, 240t staining of, 98–99 recommended ranges for, 239t urinalysis and, 162 specimen considerations for, 239t Blood agar, 2 Calcium oxalate crystals, 165 Blood-borne pathogen, 289 Calcium pyrophosphate dihydrate crystals, 171 Blood cell count, 168–169 Calicivirus, 70 Blood donation, 212–213 Campylobacter jejuni, 21 Blood flow Camylobacter species, 21 hemostasis and, 146 Cancer Blood fluke, 78 molecular genetics in, 275–276 Blood gas, 233–234 tumor markers and, 194t recommended ranges for, 235t Candida albicans,61 Blood parasite, 72t, 78–80 Candida glabrata,61 Bloom’s taxonomy, 290 Candida kreusi,61 B-lymphocyte maturation, 181 Candida parapsilosis,61 Body fluid, 168–169 Candida species, 61–62 amniotic, 172–173 Candida tropicalis,61 cerebrospinal, 168 Capital expense, 287 pericardial, 172 Capsid, 66 peritoneal, 172 Capsomere, 66 pleural, 172 Carbapenem, 37 seminal, 170–171 Carbohydrate, 229–233 serous, 171 Carboxyhemoglobin, 93 synovial, 171 Case study basede teaching method, 291 Bombay phenotype, 200 Catecholamine, 248 Bone, 238 Catecholamine excess, 250 Bone homeostasis, 238t Cathode, 241f Bone marrow Cat tapeworm, 77 acute myeloid leukemia and, 125 Cell. See also Red blood cell; White blood cell chronic myelogenous leukemia and, 119 morphology and arrangement of, 4 collection of, 102–103 size of, 90 essential thrombocytopenia and, 121 Cell count, 168–169 hematopoiesis and, 91f Cell counter, 100 myelodysplastic syndrome and, 121, 122t Cell membrane, 4 plasma cell myeloma and, 128 of fungal organism, 50 staining of, 98–99 Cell membrane inhibitor, 38 types of, 90 Cell wall, 4 Bone remodeling, 238 of fungal organism, 50 Bordetella pertussis,24 Cell wall synthesis inhibitor, 37 Borrelia, 32–33 Centers for Medicare & Medicaid Services (CMS), 285 Boss-centered leadership, 283 Cephalosporin, 37 Botulism, 36 Cerebrospinal fluid (CSF) Botulism neurotoxin (BaTN), 36 appearance and significance of, 168t Branched-chain DNA (bDNA), 270 chemistry tests on, 169 BRCA 1 gene, 272 hematology tests on, 168–169 BRCA 2 gene, 272 microbiology tests on, 169 b-g Bridge, 241 physiology of, 168 Broadfish tapeworm, 76 specimen collection and distribution, 168 Brucella, 26–27 Cerebrospinal fluid (CSF) cell, 169t Brugia malayi,79 Cerebrospinal fluid (CSF) electrophoresis, 169 Bruton’s agammaglobulinemia (X-linked agammaglobulinemia), Cerebrospinal fluid (CSF) glucose, 169 192 Cerebrospinal fluid (CSF) glutamine, 169 B subgroup, 201 Cerebrospinal fluid (CSF) IgG index, 169 Budget preparation, 287 Cerebrospinal fluid (CSF) lactate, 169 Bulbourethral gland fluid, 170 Cerebrospinal fluid (CSF) protein, 169 Bunyavirus, 69 Ceruloplasmin, 241 Burkholderia cepacia,28 Cestodes, 72t, 76–78 Burkholderia mallei,29 Chagas disease, 80 Burkholderia pseudomallei,29 Chain termination, 264 374 Index

Chediak-Higashi syndrome, 119 Codocyte (Target cell) Chemical hygiene plan, 289 cell descriptiom and diseases associated with, 100t Chemiluminescence assay, 187 Codon, 264 Chemistry test Cognitive learning, 290 on cerebrospinal fluid, 169t Cognitive learning objective, 290 on feces, 173 COLA, 286 of pleural fluid, 172 Cold agglutinin disease, 116–117 Chickenpox, 189 Colistin, 38 Chilomastix mesnili,75 College of American Pathologists (CAP), 286 Chinese liver fluke, 77 Colony-stimulating factor, 182t Chlamydia species, 29–31 Combined DNA index system (CODIS), 273 Chlamydia trachomatis, 30, 274 Common pathway, for hemostasis, 142–144, 144f Chlamydoconidia, 50 Common variable immunodeficiency, 192 Chlamydophila pneumoniae,30 Communication, 284–285 Chlamydophila psittaci,31 Competitive assay, 187 Chlamydophila species, 29–31 Complement, 182–183 Chloride, 235 components, pathways and activities of, 184t pathologic processes for, 238 Complement activation pathway, 182, 183f recommended ranges for, 236t Complement deficiency, 192–193 Chocolate agar, 2 Conflict management, 287 Cholesterol Congenital adrenal hyperplasia, 249 measurement of, 229t Congenital dyserythropoietic anemia (CDA), 109 recommended ranges for, 231b Conidiophore, 50 Cholesterol crystals, 165 Conjugated bilirubin, 245t Choroidal cell, 169t Conn’s syndrome, 248, 249 Christmas disease, 149t Contact factors, for hemostasis, 143t Chromoblastomycosis, 59–60 activation of, 142 Chromosome, 4, 262–265 Continuing education, 289 abnormalities in, 276 Continuous quality improvement (CQI), 283 Chromosome 6, 194 Contract staff, 288 Chronic eosinophilic leukemia, 121 Cooperative learning, 291 Chronic glomerulonephritis, 167t Coronavirus, 70 Chronic granulomatous disease, 119, 192 Corrective action/discipline, 287 Chronic kidney disease, anemia of, 109 Cortisol, 248 Chronic lymphocytic leukemia (CLL), 126 Corynebacterium diphtheriae, 11–12 Chronic lymphoid neoplasm, 126–128 Corynebacterium species, 11–12 Chronic myelogenous leukemia, 119–120 Cost allocation, 287 Chronic myeloid leukemia, 276 Coumadin, 150 Chronic myelomonocytic leukemia (CMML), 123 Creatine kinase (CK) Chronic neutrophilic leukemia, 121 distribution and clinical significance of, 252t Chronic pyelonephritis, 167t reaction catalyzed, 250t Chylomicron remnant, 228 recommended ranges for, 252t Ciliate, 72t,75 Creatine kinase-myocardial bound, 251t Circulating lymphoma, 128 Crigler-Najjar syndrome, 244 Clacium, 239 Crossmatching, 211–212 Cladophialophora carrionii,59 Cryogenetic analysis, 276 Cleavage-based amplification, 270 Cryoprecipitated AHF CLIA (Clinical Laboratory Improvement Amendments), 285 storage and expiration of, 215t CLIA 88 law, 285, 289 transfusion therapy and, 214 CLIA program, 285 Cryptococcus neoformans, 169 Clinical (bench) teaching, 291 Cryptococcus species, 61–62 Clinical Laboratory Improvement Amendments (CLIA), 285 Cryptosporidium parvum,75 Clonal hematopoietic stem cell disorder, 119 Crystals. See also specific types of Clonorchis sinensis,77 in urine, 164–165 Clostridium botulinum,36 Crytococcus neoformans,61 Clostridium difficile,36 Ctenocephalides canis, 80 Clostridium perfringens, 35, 36, 115 Ctenocephalides felis, 80 Clostridium species, 35 Cunninghamella species, 57 Clostridium tetani,36 Current procedural terminology (CPT), 288 CNA, 3 Cushing’s syndrome, 139, 248 Coagulase-negative staphylococcus, 6–7 Customer service, 287 Coagulation cascade pathway, 142 Cyclospora cayetanensis,75 Coagulation factor Cyst, 74 grouping of, 142, 142t Cystic fibrosis, 271 Coagulation factor VIII, 271 Cystine crystals, 165 Coccidia, 72t,75 Cystitis, 167t Coccioides immitis,63 Cytokine, 182 Code of ethics, 284 activities of, 182t Index 375

Cytomegalovirus, 69, 189, 275 Duffy blood group system, 204 Cytoplasm, 50 Dwarf tapeworm, 76 Cytosine, 264f Dysbetalipoproteinemia, 231t Dysfibrogenemia, 149t D Dyskeratosis congenita, 109 Dacryocyte (teardrop cell) cell descriptiom and diseases associated with, 100t Daily quality control, 289 E D antigen, 203 East African sleeping sickness, 80 Day-to-FTE ratio, 288 Eastern/Western equine encephalitits, 71 Deaminase, 265 Echinococcus granulosus,77 Deer tick, 80 Echinocyte (burr cell) Defective TXA2 synthesis, 148t cell descriptiom and diseases associated with, 100t Deficit Reduction Act, 288 Ectoparasite, 80 Dehydroepiandrosterone (DHEA), 248 Education, 289–290 Dehydroepiandrosterone sulfate (DHEA-S), 248 Ehlers-Danlos syndrome, 139 Delayed hemolytic transfusion reaction, 117 Eikenella corrodens, 25–26 Delegative leader, 283 Electrolyte, 236–237 Dematiaceous fungus, 58 pathologic processes for, 236–237 Democratic leader, 283 recommended ranges for, 236t Deoxyribonucleic acid (DNA) total body water compartments and, 236t molecular composition of, 262 types of, 235 specimen collection and storage, 266t Electrophoresis, 266–268 structural organization of, 263f from anode to cathode, 241f Deoxyribonucleic acid (DNA) isolation, 265 Elliptocyte Department of Health and Human Services (DHHS), 285 cell descriptiom and diseases associated with, 100t Department of Transportation (DOT), 285 Embden-Meyerhof pathway, 94f Dermacentor variabilis,80 energy and, 93 Dermatophyte, 60–61 Employee-centered leadership, 283 identification of, 53 Employee corrective action and discipline, 287 Dermatophytic mycosis, 51 Employee evaluation, 287 genus of, 51t Employee orientation/training, 287 Dermatophytosis, 60 Encephalitis, 71 Det Norske Veritas (DNV), 286 Endocrinology, 244–246 Diabetes mellitus Endolimax nana,74 diagnostic criteria for, 232, 232t Endonuclease, 265 gestational, 232t Energy type 1, 191 Embden-Meyerhof pathway and, 93 Diagnostic related group (DRG), 288 source of, 231 Diamond-Blackfan anemia, 109 Entamoeba coli,74 Diarrhea, 173 Entamoeba hartmanni,74 Dientamoeba fragilis,75 Entamoeba histolytica,74 DiGeorge’s syndrome, 192 Enteric fever, 19 Diiodotyrosine (DIT), 246f Enteric gram-negative bacillus, 17b Dimorphic fungus, 62–64 Enteric pathogen, 21 Diphyllobothrium latum,76 Enterobius vermicularis,73 Dipylidium caninum,77 Enterobius vermicularis, 164 Direct antiglobulin test (DAT), 211 Enterococcus species, 9 positive, 202 Enterotoxin, 6 Direct bilirubin, 244t Enterovirus, 70, 275 Direct expense, 287 Envelope, 66 Direct fluorescent assay, 187 Environmental Protection Agency (EPA), 285 Disaccharide, 229 Enzyme, 250 Discussion, 291 distribution and clinical significance of, 252t Disseminated intravascular coagulation, 114, 149 measurement of, 251 Distance learning, 291 reaction catalyzed, 250t DNA chip, 270 recommended ranges for, 252t DNA hybridization array, 270 Enzyme abnormality, 113–114 DNA/RNA inhibitor, 38 Enzymology, 250–251 DNA. See Deoxyribonucleic acid (DNA) Eosin, 99 DNA synthesis, 107f Eosinophil, 93 DNA virus, 68–69, 68t normal range values for, 98t Dog tapeworm, 77 Eosinophil granule, 98b Do¨ hle body, 118 Eosinophilia, 118 Dracunculus medinensis,74 Ependymal cell, 169t Drug-induced hemolytic anemia, 117 Epidermal growth factor receptor (EGFR), 276 Dubin-Johnson syndrome, 244 Epidermophyton floccosum,60 Duchenne’s muscular dystrophy, 272 Epigenetics, 265 376 Index

Epithelial cell, 164 Fee for service, 288 Epstein-Barr virus, 69, 189 Fermentation, 4 Equine encephalitis, 71 Fetal bilirubin, 173f Erysipelothrix, 11 Fetal distress test, 173 Erythrocyte Fetal maturity test, 173 glucose metabolism in, 94f Fetus, hemolytic disease of newborn and, 117–118, 218 Erythrocyte inclusion ABO versus Rh, 218t description, composition and disease states of, 101t Fibrin, 145f Erythrocyte sedimentation rate (ESR), 99–100 Fibrin clot, 145f Escherichia coli, 20 Fibrinogen, 145f Essential thrombocytopenia, 120–121 Fibrinogen group, 143t Ethics, code of, 284 Fibrinogenolysis, 149 Eukaryote, 3 Fibrinolysis, 144–146 Euthyroid sick syndrome, 247 inhibitors of, 145 Excess glucocorticoid, 139 Fiedler, Fred, 284 Exon, 264 Fiedler theory, 284 Exonuclease, 265 Filariae, 72t,79 Exophiala jeanselmei,59 Filovirus, 70 Expense, 287 Fisher-Race genetic theory, 203f Extracellular fluid compartment, 236t Fixed cost, 287 cations and anions in, 236t Flagella, 5 Extracellular water (ECW), 235 Flagellate, 72t,75 Extravascular hemolysis, 117 Flavivirus, 70, 190 Extrinsic pathway, for hemostasis, 142, 144f Flea, 80 Exudate, 171, 171t, 172 Flow cytometry, 103 Eyeworm, 79 lineage-associated markers analyzed in, 103t Fluid volume regulation, 235, 236 F Fluke Factor IX deficiency, 149t genus and species of, 72t Factor VIII deficiency, 149t types of, 77 Factor V Leiden, 271 Fluoroquinolone, 38 Factor V Leiden mutation, 153 Focal segmental glomerulosclerosis, 167t Facultative anaerobe, 4 Folate Fair Labor Standards Act (FLSA), 285 DNA synthesis and, 107f Familial breast cancer, 272 Folate deficiency, 107 Family Aeromonadaceae, 22 Folded cell Family Alcaligenaceae, 23–24, 29 cell descriptiom and diseases associated with, 100t Family Bartonellaceae, 31 Follicular cell, 246 Family Brucellaceae, 26–27 Follicular lymphoma, 276 Family Burkholderiaceae, 28–29 Fonsecaea compacta,59 Family Campylobacteraceae, 21 Fonsecaea pedrosoi,59 Family clamydiaceae, 29–31 Food and Drug Administration (FDA), 285 Family Enterobacteraceae, 16–21 Food botulism, 36 Family Enterococcaceae, 9 Forensic DNA typing, 273 Family Francisellaceae, 27 Fragile X syndrome, 272 Family Helicobacteraceae, 22 Francisella tularensis,27 Family Herpesvidae, 68–69 Free thyroxine (f T4), 247 Family Legionellaceae, 26 Fresh frozen plasma Family Leptospiraceae, 32–33 storage and expiration of, 215t Family Medical Leave Act (FMLA), 285 transfusion therapy and, 214 Family Moraxellaceae, 28 Full time equivalent (FTE), 286 Family Mycobacteriaceae, 12–16 Fungal infection, 51t Family Mycoplasmataceae, 31 categories of, 51 Family Neisseriaceae, 22–23 Fungal organism, 50–51 Family Pasteurellaceae, 24–26 Fungus. See also specific types of Family Pseudomonadaceae, 27 dimorphic, 62–64 Family Rickettsiaceae, 32 media for, 52 Family Staphylococcaceae, 5–7 specimen collection, transport and processing, 51 Family Streptococcaceae, 7–8 tissue examination, methods of, 53 Family Vibrionaceae, 21–22 Fungus-like bacteria, 64–65 Fanconi’s anemia, 109 Fusarium species, 55 Fanconi’s syndrome, 167t Fusobacterium species, 37 Fasciolopsis buski,77 Fasciolopsis hepatica,77 G Febrile nonhemolytic transfusion reaction, 217 Gas gangrene, 36 Fecal analysis, 173–174 Gastroenteritis, 19 Fecal carbohydrate, 174 Gene, 264 Fecal enzyme, 174 Genetic code, 264t Index 377

Genome, 265 Health Information Technology for Economic and Clinical Health Geotrichum candidum,62 Act (HITECH), 285 Geotrichum species, 55 Health Insurance Portability and Accountability Act (HIPAA), 285 Gestational diabetes mellitus, 232t Helicobacter, 22 Giardia lamblia,75 HELLP syndrome, 115 Gilbert’s disease, 243–244 Helmet cell (keratocyte) Glanzmann’s thrombasthenia, 148t cell descriptiom and diseases associated with, 100t Globin chan Hemacytometer, 99, 102f hemoglobin and, 97t Hemagglutination, 187 Globulin Hematocrit A/G ratio and, 241 manual, 99 types of, 241 Hematology Glomerular disorder, 167t laboratory findings in, 98–103 Glomerular filtration, 166 normal range values in, 98, 98t Glomerular filtration rate (GFR), 166 manual and semiautomated testing techniques in, 99 Glucocorticoid, 248 Hematology instrumentation, 100–102 Gluconeogenesis, 231 Hematology test Glucose on cerebrospinal fluid, 168–169 analysis of, 231 of pleural fluid, 172 as energy source, 231 Hematopoiesis, 90, 91f measurement of, 232t diagram depicting, 92f pathologic processes for, 232–233 sites of, 91t recommended ranges for, 232b Hematopoietic malignancy, 276 urinalysis and, 162 Hematopoietic neoplasm, 119–121, 275–276 Glucose-6-phosphate dehydrogenase deficiency, 113–114 Hematuria, 162 Glucose control marker, 233 Heme, 91 Glucose dehydrogenase, 232t Heme disorder, 104–106 Glucose metabolism, 94f Hemochromatosis, 104, 106 Glucose oxidase, 232t Hemoflagellate, 72t g-Glutamyltransferase Hemoglobin (Hgb) distribution and clinical significance of, 252t globin chains and, 97t recommended ranges for, 252t measurement of, 102f Glycocalyx, 5 using pseudoperoxidase and guiac, 174f Glycogenesis, 231 normal range values for, 98t Glycogenolysis, 231 production of, 91 Glycolysis, 231 role of, 91 Goodpasture’s syndrome, 167t variants of, 93 Government Accountability Office, 286 Hemoglobin (Hgb) A1c, 231 Graft-versus-host disease, 194 Hemoglobin buffer system, 234 Gram-positive bacillus: Clostridium, 35–36 Hemoglobin C, 110 Gram-positive coccus, 9 Hemoglobin C crystal Gram stain, 1 cell descriptiom and diseases associated with, 100t on cerebrospinal fluid, 169 Hemoglobin degradation, 162f Granulocyte, 215t bilirubin and urobilinogen, 162 Grave’s disease, 191 Hemoglobin E, 110 Gray platelet syndrome, 148t Hemoglobinopathy, 110–111 Group B streptococcal disease, 274 Hemoglobin S, 99, 110 Group D streptococcus, 8 Hemoglobin SC, 110–111 Guanine, 264f Hemoglobin SC crystal Guinea worm, 74 cell descriptiom and diseases associated with, 100t Hemoglobinuria, 162 Hemolytic anemia, 112–118 H from extrinsic defects, 114–118 Haemophilus aegyptius,25 from intrinsic defects, 112–114 Haemophilus ducreyi,25 Hemolytic disease of fetus and newborn, 117–118, 218 Haemophilus influenzae,25 ABO versus Rh, 218t Haemophilus species, 24–25 a-Hemolytic streptococcus, 8 Hairy cell leukemia (HCL), 127 Hemolytic uremic syndrome (HUS), 115 Halophilic Vibrio organism, 22 Hemophilia A, 149t, 271–272 H antigen, 201f Hemophilia B, 149t Haptoglobin, 241 Hemostasis Hard tick, 80 control of, 146–149 Hashimoto’s thyroiditis, 191 important factors in, 143t Hazard, 288 laboratory tests for, 151t HE, 3 primary, 140–141 Health and Human Services, Department of (DHHS), 285 secondary, 142–146 Healthcare Common Procedure Coding System (HCPCS), 288 systems of, 139 Healthcare Facilities Accreditation Program (HFAP), 286 vasculature disorders affecting, 139 378 Index

Hemostatic response, steps of, 139 Hyperlipoproteinemia, 231t Henoch-Scho¨ nlein purpura, 139, 167t Hypermagnesemia, 240t Hepadnavirus, 68, 189 Hypernatremia, 237, 237t Heparin, 150 Hyperphosphatemia, 240t Heparin induced thrombocytopenia (HIT), 148t Hypersegmentation, 118 Hepatitis A virus, 71, 189 Hypersensitivity, 193 Hepatitis B vaccine, 289 Hypertensive crisis, 115 Hepatitis B virus, 189 Hyperthyroidism, 246 serology for, 190t Hypervolemia, 237t Hepatitis C virus, 70, 190, 275 Hypha, 50 Hepatitis D virus, 190 Hypocalcemia, 240t Hepatitis E virus, 189 Hypochloremia, 238 Hepatocyte, 242 Hypofibrinogenemia, 149t Hepeviridae family, 189 Hypoglycemia, 232–233 Hereditary elliptocytosis, 113 Hypokalemia, 237 Hereditary hemochromatosis, 271 Hypomagnesemia, 240t Hereditary hemorrhagic telangiectasia, 139 Hyponatremia, 236, 237t Hereditary persistance of fetal hemoglobin (HPFH), 112 Hypophosphatemia, 240t Hereditary spherocytosis, 112–113 Hypoproliferative disorder, 108–109 Herpes simplex 1 and 2, 189 Hypothalamus, 245, 245t Herpes simplex virus, 275 Hypothyroidism, 247 Herpesviridiae family of viruses, 189 Hypovolemia, 237t Herpesvirus, 68–69 Hershey, Paul, 284 Herzberg, Frederick, 283 I Heterogeneous assay, 187 Identity assessment, 273–274 Heterophid fluke, 77, 80 IgA nephopathy, 167t Heterophyes heterophyes,77 Ii blood group system, 204 Heterplasmy, 272 Immature B cell, 181 Hexokinase, 232t Immune complex autoimmune disease, 190 Hexose monophosphate pathway, 94f Immune-mediated hemolytic anemia, 116 H gene, 200 Immune system. See also Innate system High-density lipoprotein (HDL), 228 viruses and, 188 measurement of, 230t Immune thrombocytopenic pupura (ITP), 148t recommended ranges for, 231b Immunoassay. See specific types of HIPAA (Health Insurance Portability and Immunodeficient disease, 192–193 Accountability Act), 285 Immunoelectrophoresis, 186 Histoplasma capsulatum, 63–64 Immunofixation electrophoresis, 186 Homogenous assay, 187 Immunogen, 184–185 Hormone Immunoglobulin, 185t definition and classification of, 244 Immunoglobulin A, 184, 185t transport proteins and, 245t Immunoglobulin D, 184, 185t Hormone feedback control system, 245f Immunoglobulin E, 184, 185t Hortaea werneckii,57 Immunoglobulin G, 183, 184, 185t H substance, 201b structure of, 184f Human epidermal growth factor 2 (HER2), 276 Immunoglobulin M, 184, 185t Human genome, 265 Immunologic test Human Herpesvirus 6, 7 and 8, 69 on cerebrospinal fluid, 169 Human immunodeficiency virus (HIV), 71, 190 Immunology Human immunodeficiency virus (HIV) type 1, 275 autoimmune diseases in, 190–191 Human leukocyte antigen typing, 273–274 transplantation, 194 Human papillomavirus (HPV), 275 tumor, 193 Human resource, 286–287 Immunoproliferative disease, 191–192 Huntington’s disease, 271 Impedance, 101 Hybrid capture, 270 Imprinting, 272 Hydatid tapeworm, 77 Indirect bilirubin, 244t Hydrogen bond, 264f Indirect expense, 287 Hydrostatic pressure, 235 Indirect fluorescent assay, 187 Hymenolepis diminuta,76 Infancy, transient hypogammaglobulinemia of, 192 Hymenolepis nana,76 Infant botulism, 36 Hyperaldosteronism (Conn’s syndrome), 248, 249 Infection, 33–35 Hyperbetalipoproteinemia, 231t Infectious disease Hypercalcemia, 240t molecular methods in, 274–275 Hyperchloremia, 238 serology and, 187–190 Hyperchylomicronemia, 231t Infectious mononucleosis (IM), 189 Hypercortisolism (Cushing’s syndrome), 248, 248b Inflammation Hyperglycemia, 233 innate system and, 180 Hyperkalemia, 237–238 Inherited aplastic anemia, 109 Index 379

Inherited breast cancer, 272 Laboratory (Continued) Inherited disease, 271–273 regulattory guidelines for, 285–286 Innate immunity, 180–181 workload and productivity in, 288 Innate system Laboratory demonstration, 291 humoral components of, 180 Laboratory education, 289–290 processes of, 180–181 Laboratory Management Index Program (LMIP), 288 Inspector General, Office of (OIG), 286 Laboratory safety, 288–289 Interferon, 182t standard precautions for, 38 Interleukin, 182t Lactate dehydrogenase, 250 Intermediate density lipoprotein (IDL), 228 distribution and clinical significance of, 252t Intermediate metabolism inhibitor, 38 reaction catalyzed, 250t International Classification of Disease 10 (ICD-10), 288 recommended ranges for, 252t Interview, 287 Lactose fermentation, 2 Intestinal nematode, 73–74 Lamellar body, 173 genus and species of, 72t Large intestinal fluke, 77 Intracellular fluid compartment, 236t Latex agglutination, 186 cations and anions in, 236t Lattice formation, 186 Intracellular water (ICW), 235 Leadership Intrinsic pathway, for hemostasis, 142, 144f management styles, 283 Intron, 264 motivational theories/styles, 283–284 Iodamoeba butschlii,74 styles of, 283 Ionized calcium, 239t Lean, 289 Iron, 104–106 Learner, 291 Iron-deficiency anemia (IDA), 104, 105 Learning assessment, 290–291 development of, 105f Learning domain, 290 Iron overload, 218 Learning objective, 290 Isospora belli,75 Lecture, 291 Ixodes scapularis,80 Legionella pneumophilia,26 Ixodes species, 32 Leishmania braziliensis,79 Leishmania donovani complex, 79 J Leishmaniae, 79–80 Janus kinase 2 mutation, 276 Leishmania mexicana complex, 79 Jaundice, 242–243 Leishmania tropica complex, 79–80 Job advertisement, 286 Leptospira biflexa,32 Job description, 286 Leptospira interrogans,32 Job position, 286 Leucine crystals, 165 The Joint Commission (TJC), 286 Leukemia Juvenile myelomonocytic leukemia (JMML), 123 lymphoma versus, 275 sites and classification for, 191 K Leukocyte adhesion disorder (LAD), 119, 192 Leukocyte alkaline phosphatase (LAP), 104t Kaizen, 289 Leukocyte esterase, 163 Kell blood group system, 204 Lewis antibody, 205 Keratocyte (helmet cell) Lewis antigen, 205f cell descriptiom and diseases associated with, 100t Lewis blood group, 205 Ketone, 162 Lice, 80 Ketosis, 229 Ligase, 265 Kidd blood group system, 204 Ligase chain reaction, 270 Kidney, 166–167 Lipase Kinase, 265 distribution and clinical significance of, 252t Kinesthetic learner, 291 recommended ranges for, 252t Kingella species, 26 Lipid, 228 Kirsten rat sarcoma viral oncogene (K-ras), 276 Lipoprotein, 228 Kleihauer-Betke stain, 212 characteristics of, 230t calculation of, 212b Lipoprotein particle, 229f Kupffer cell, 242 Listeria monocytogenes, 11 Listeria species, 10–11 L Liver, 242–244 Laboratory Liver clearance, 146 accreditation of, 286 Liver disease, 149 communication in, 284–285 Loa loa,79 financial aspects of, 287–288 Low-density lipoprotein (LDL), 228 human resources in, 286–287 measurement of, 230t leadership in, 283 recommended ranges for, 231b organizational management structure for, 283 Lp(a), 228 procedures in, 289 Lumpy jaw, 35 professionalism in, 284 Lung surfacant, 173 quality control and assurance in, 289 Lyme borreliosis, 32 380 Index

Lyme disease, 188 Metabolism Lymphatic system, 181 of bacteria, 3–5 Lymphocyte, 98 Metagonimus yokogawa, 77–78 appearance and significance of, 169t Metamyelocyte, 95t changes in, 119 Metapneumovirus, 70 normal range values for, 98t Methemoglobin, 93 Lymphocytopenia, 118 Methemoglobin reductase pathway, 94f Lymphocytosis, 118 Methicillin-resistant Staphylococcus aureus (MRSA), 274 Lymphoid neoplasm manifesting as solid tumor, 128 Methylase, 265 Lymphoma, 128 Methylene blue, 99 clonality of, 275 Metronidazole, 38 leukemia versus, 275 Microaerophilic, 4 sites and classification for, 191 Microalbuminuria, 162 Microangiopathic hemolytic anemia, 114 Microarray, 270 M Microbiology test MacConkey agar, 2 on cerebrospinal fluid, 169 Macrocyte of pleural fluid, 172 cell descriptiom and diseases associated with, 100t Microhematocrit, 99 Macrocytic anemia, 106–108 Microsatellite, 265 Macrocytosis, 108 Microsporidia species, 75 Macrolide, 38 Microsporum audouinii,60 Macrophage, 169t Microsporum canis,60 Magnesium Microsporum gypseum,60 measurement of, 240t Microsporum nanum,60 pathologic processes for, 240t Mineral, 238–239 recommended ranges for, 239t recommended ranges for, 239t, 242t specimen considerations for, 239t Mineral homeostasis, 239t Major histocompatibility complex, 185, 194 Mineralocorticoid, 248 Malaria, 115 Minimal change disease, 167t Malariae, 72t, 78–79 Minisatellite, 265 Malassezia furfur,62 Mite, 80 Malignant cell, 169t Mitochondria, 272 Malignant hypertension, 115 Mitochondrial DNA, 265 Management, styles of, 283 Mitochondrial DNA disease, 272 Management by objective (MBO), 283 Mixed hyperlipoproteinemia, 231t Mansonella ozzaardi,79 MNS blood group system, 205 March hemoglobinuria, 115 Mold Maslow, Abraham, 283 examination of, 53 Maslow’s hierarchy of needs, 283 opportunistic, 54–56 Mass spectrometry, 270 Molecular bacteriology, 274 Mastocytosis, 121 Molecular genetics, in cancer, 275–276 Matrix-assisted laser-desorption ionization time-of-flight (MALDI- Molecular testing, 274 TOF), 270 Molecular virology, 274–275 Mature B cell, 181 Mollicute, 31 Maxam-Gilbert chemical sequencing, 270 Moniodotyrosine (MIT), 246f May-Hegglin anomaly, 119 Monoclonal antibody, 185 McGregor, Douglas, 283 Monoclonal gammopathy of unknown significance, 192 Mean corpuscular hemoglobin (MCH) Monocyte, 97 definition and calculation of, 101t appearance and significance of, 169t Mean corpuscular hemoglobin concentration (MCHC) normal range values for, 98t definition and calculation of, 101t Monocytosis, 118 Mean corpuscular volume (MCV) Monosaccharide, 229 definition and calculation of, 101t Monosodium urate crystals, 171 Measles virus, 70 Moraxella catarrhalis,28 Medical laboratory. See laboratory Moraxella species, 28 Medicare & Medicaid Services, Centers for (CMS), 285 Mosquito, 80 Megaloblastic anemia, 107–108 Motivational theory/style, 283–284 diagnosis and treatment of, 108 Motivator-hygiene theory, 283 Membranous glomerulonephritis, 167t Mould54. See Mold Mendelian inheritance, diseases with, 271–272 Mouton, Jane, 284 Meningitis, 61 M protein, 187 differential diagnosis for, 170t M-spike, 242 Messenger RNA (mRNA), 264 Mucor species, 56–57 translation to amino acid during protein Mucus, 164 synthesis, 264t Multiple myeloma, 191 Metabolic acidosis, 235t, 236 Multiple-rule quality control, 289 Metabolic alkalosis, 235t Multiplex PCR, 269 Index 381

Mumps virus, 70 Neutrophil, 93 Mutation, 276 abnormalities in, 118 Mycelium, 50 appearance and significance of, 169t Mycetoma, 59 development of, 95t Mycobacteria other than tuberculosis (MOTT), 13–15, 16 inherited abnormalities of, 119 Mycobacterium abscessus,15 normal range values for, 98t Mycobacterium avium,15 Neutrophil granule, 97b Mycobacterium avium complex (MAC), 15, 16 Neutrophilia, 118 Mycobacterium fortuitum-chelonae complex, 15, 16 Newborn, hemolytic disease of fetus and, 117–118, 218 Mycobacterium gordonae,15 ABO versus Rh, 218t Mycobacterium kansasii,15 New World hookworm, 73 Mycobacterium leprae, 15, 16 Next-generation sequencing, 270 Mycobacterium marinum,15 Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Mycobacterium scrofulaceum,15 192 Mycobacterium species Nitrite, 163 collection guidelines and diagnosis, 15–16 Nitroblue tetrazolium test, 192 isolation and identification of, 14b Nocardia species, 64–65 types of, 13–15 Nocardiopsis species, 65 Mycobacterium tuberculosis, 13, 274 Noncompetitive assay, 187 Mycoplasma pneumondiae, 116 Non-Hodgkin lymphoma, 128 Mycoplasmataceae homonis,31 Nonintestinal nematode, 74 Mycoplasmataceae pneumoniae,31 Nonmalignant macrocytic anemia, 108 Mycosis, 50 Nonmalignant white blood cell disorder, 118–119 Myelin basic protein, 169 Non-Mendelian inheritance, diseases with, 272 Myeloblast, 95t Nonpathogenic Neisseria species, 23 Myelocyte, 95t Nonspecific esterase (a-naphthyl acetate or butyrate esteraste), 104t Myelodysplastic neoplasm, 123–124 Nontuberculous mycobacteria, 13–15 unclassifiable, 124 Normoblast, 92t Myelodysplastic syndrome, 121–123 Normovolemia, 237t bone marrow and peripheral blooding findings in, 122t Northern blotting, 268 Myelodysplastic syndrome, unclassified, 123 Nuclease, 265 bone marrow and peripheral blooding findings in, 122t Nucleic acid Myelodysplastic syndrome with isolated 5q-deletion (5q- physiology and function of, 263–265 syndrome), 123 specimen collection and storage, 266t bone marrow and peripheral blooding findings in, 122t structure of, 262 Myeloperoxidase (MPO), 104t Nucleic acid amplification, 67 Myeloproliferative disorder (non-BCR-ABL), 276 Nucleic acid enzyme, 265 Myeloproliferative neoplasm, 119–121, 123–124 Nucleic acid isolation, 265–266 unclassifiable, 124 Nucleic acid sequencing, 270 Myers-Briggs Type Indicator (MBTI), 284 Nucleocapsid, 66 Myoglobinuria, 162 Nucleotide, 262

N O Naeglaria fowleri, 74–75 Obligate aerobe, 4 National Accrediting Agency for Clinical Laboratory Sciences Obligate anaerobe, 4 (NAACLS), 286, 289 Occupational Safety and Health Administration (OSHA), 286, 288 National Institute for Occupational Safety and Health (NIOSH), Office of Inspector General (OIG), 286 285 Old World hookworm, 73 National Labor Relations Board (NLRB), 285 Onchocerca volvulus,79 Necator americanus,73 Oncogene, 275 Negative feedback amplification, 146 Oncotic pressure, 235 Neisseria gonorrhoeae, 23, 274 Operational expense, 287 Neisseria meningitidis,23 Opportunistic fungus, 51 Neisseria species genus of, 51t nonpathogenic, 23 Opportunistic mold, 54–56 pathogenic, 23 Oral glucose tolerance test, 232t Nematode, 73–74 Oriental lung fluke, 78 genus and species of, 72t Orientation, 287 intestinal, 73–74 Orientia,32 nonintestinal, 74 Ornithodoros species, 80 Neonatal alloimmune thrombocytopenia (NAIT), 148t Orthochromic normoblast, 92t Nephelometry, 186 Orthomyxovirus, 70 Nephron, 166 Osler-Weber-Rendu disease, 139 Nephrotic syndrome, 167t, 242 Osmolality, 235 Neubauer counting chamber, 168 recommended ranges for, 236t Neuroacanthocytosis, 113 regulation of, 236 Neutropenia, 118 Osmolarity, 160 382 Index

Osmotic pressure, 235 Pheochromocytoma (Continued) Ouchterlony diffusion, 186 types of, 250 Oval fat body, 164 Phialoconidia, 50 Oval macrocyte Phialophora verrucosa,59 cell descriptiom and diseases associated with, 100t Phili, 5 Ovalocyte Phosphatase, 265 cell descriptiom and diseases associated with, 100t Phosphate bond, 262 Oxacillin-resistant S. aureus,6 Phosphorus, 239 Oxygen, 4 measurement of, 240t Oxygen dissociation curve, 97t pathologic processes for, 240t Oxygen saturation, 235t recommended ranges for, 239t Oxyhemoglobin, 91 specimen considerations for, 239t Picornavirus, 70–71, 189 P Piedraia hortae,58 Paecilomyces species, 55 Pinworm, 73 Paecilomyces variotii,55 Pituitary gland, 245 Pancytopenia, 109 Pituitary hormone, 245t Papillomavirus, 69 Plasma, 213t Para-Bombay phenotype, 200 Plasma cell, 169t Paracoccidioides brasiliensis,64 Plasma cell dyscrasis, 191–192 Parafolicular cell, 246 Plasma cell myeloma, 128 Paragonimus westermanni,78 Plasma cell neoplasm, 127–128 Parainfluenza virus, 70 Plasmid, 4, 274 Paramyxovirus, 70 Plasmin, 144 Paraprotein disorder, 139 Plasminogen, 144 Parasite, 188 Plasminogen activator inhibitor (PAI), 145 classification, genus and species of, 72t Plasmodium falciparum, 78–79 in urine, 164 Plasmodium knowlesi,79 Parasitology, 71–80 Plasmodium malariae,79 Parathyroid hormone Plasmodium ovale,79 effect on bones and kidneys, 239t Plasmodium species, 115 recommended ranges for, 239t Plasmodium vivax,79 Paroxysmal cold hemoglobinuria, 117 Platelet Paroxysmal nocturnal hemoglobinuria, 113 ABO and Rh compatibility, 214t Partial pressure of carbon dioxide (pCO2), 233 activation of, 140–141, 141t measurement of, 234 components, functions, and structure of, 140t recommended ranges for, 235t function of, 140 Partial pressure of oxygen (pO2), 234 normal range values for, 98t measurement of, 234 role of, 139–140 recommended ranges for, 235t shape change, 141, 141f Participative leader, 283 storage and expiration of, 215t Parvovirus, 69 transfusion therapy and, 213 Pasteurella multocida,24 washed, 214 Pasteurella species, 24 Platelet adhesion, 140, 140f Pathogenic Neisseria species, 23 Platelet aggregation, 141 Patient day-to-FTE ratio, 288 Platelet granule, 141t P blood group, 205 Platelet secretion, 141 PEA, 3 Plesiomonas shigelloides,21 Pediculus humanus capitis, 80 Pleural fluid, 172 Pediculus humanus humanus, 80 Pneumocystis jiroveci,62 Pelger-Huet anomaly, 119 Pneumocystis pneumonia, 62 Penicillin, 37 Poikilocytosis Penicillium species, 55–56 cell descriptiom and diseases associated with, 100t Peptostreptococcus,35 Point mutation, 276 Pericardial fluid, 172 Polychromatic erythrocyte, 92t Periodic acid-Schiff (PAS), 104t Polychromatic normoblast, 92t Peritoneal fluid, 172 Polycythemia vera, 120 Personal protective equipment (PPE), 288 Polymerase, 265 pH, 233 Polymerase chain reaction (PCR), 67, 268–269 measurement of, 234 components of, 269t physiologic buffers for, 234 controls and contamination issues, 269–270 recommended ranges for, 235t cycles of, 269t of semen, 170 diagram of, 267f Phaeohyphomycosis, 59 variations on, 269 Phagocytosis, 97b Polymerase chain reaction (PCR) kinetics, 268–269 innate system and, 180 Polymorphism, 265, 276 Pharmacogenetics, 276–277 Polymyxin B, 38 Pheochromocytoma Polysaccharide, 229 clinical features of, 250b Pool staff, 288 Index 383

Pork tapeworm, 76 Reagent strip, 160f, 161 Poroconidia, 50 sources of error and correlations, 161t Porphyromonas species, 37 Real-time PCR, 269 Positive feedback amplification, 146 Red blood cell, 163 Postzone phenomenon, 186 abnormalities and disease states for, 100t Potassium, 235 ABO compatibility for transfusion of, 213t pathologic processes for, 237–238 crossmatching and, 211 recommended ranges for, 236t maturation of, 92t Poxvirus, 69 metabolism and physiology of, 93 Prealbumin, 241 normal range values for, 98t Pre-B cell, 181 production and destruction of, 90–91 Precipitation, 186 storage and expiration of, 215t tests and assays, 186 substance H and, 201b Prevotella melaninogenica,37 transfusion therapy and, 213 Primary adrenal insufficiency, 250b washed, 214 Primary hemostasis, 140–141, 146 Red blood cell count, 168–169 Primary myelofibrosis, 121 Red blood cell disorder Prion, 66 anemia and, 103–104 PRN (pro re nata) staff, 288 Red blood cell index, 101t Pro-B cell, 181 calculation of, 99 Problem-based learning, 291 Red blood cell membrane disorder, 112–113 Productivity, 288 Red cell distribution width (RDW) Professionalism, 284 definition and calculation of, 101t Progenitor cell, 90 Red marrow, 90 Prokaryote, 3 Red marrow hematopoiesis, 90f Prolymphocytic leukemia, 126–127 Refractometer, 160 Promoter, 264 Refractory anemia with excess blasts, 123 Promyelocyte, 95t bone marrow and peripheral blooding findings in, 122t Pronormoblast, 90, 92t Refractory anemia with ringed sideroblast, 123 Propionibacterium,35 bone marrow and peripheral blooding findings in, 122t Propionibacterium acnes,35 Refractory cytopenia with multilineage dysplasia, 123 Prostate fluid, 170 bone marrow and peripheral blooding findings in, 122t Protein, 239 Refractory cytopenia with unilineage dysplasia, 123 function of, 239–242 bone marrow and peripheral blooding findings in, 122t classification by, 240t Reimbursement, 288 serum protein electrophoresis regions, 241t Renal blood flow, 166 specimen considerations for, 242 Renal disease, 167t urinalysis and reagent strip, 162 Renal function test, 166–167 Protein C, 150 Renal glycosuria, 167t Protein S, 153 Renal lithiasis (calculus), 167t Protein synthesis inhibitor, 38 Renal tubular concentration, 166, 167f Prothrombin 20210 mutation, 153 Renal tubular disorder, 167t Protozoa, 72t Renal tubular epithelial cell, 164 Prozone phenomenon, 186 Renal tubular reabsorption, 166, 167 Pseudallescheria boydii,59 Renal tubular secretion, 166 Pseudomonas aeruginosa,27 Renal tubular secretion test, 167 Pseudomonas species, 27 Renin-angiotensin-aldosterone system, 166, 166f Psychomotor learning, 290 osmolality and volume regulation, 236 Psychomotor learning objective, 290 Reovirus, 71 Pulmonary disease, 61 Reproduction Pulse-field gel electrophoresis (PFGE), 268 of fungal organism, 50 Punnett square, 268f, 271 Resource-based relative value scale, 288 Pure red cell aplasia, 109 Respiration, 4 Pyrosequencing, 270 Respiratory acidosis, 235t Pyruvate kinase deficiency, 114 Respiratory alkalosis, 235t Respiratory distress syndrome, 173 Q Respiratory syncytial virus (RSV), 70 Reticulocyte count, 99 Quality assurance, 289 Retrovirus, 71, 190 Quality control, 289 Reverse transcriptase, 265 Quantitative disorder, 148t Reverse-transcriptase PCR, 269 Quantitative fecal fat, 174 Reye’s syndrome, 244 Quantitative white blood cell disorder, 118–119 Rh (D) typing, 204 Rhabdovirus, 71 R Rh antibody, 204 Rabies virus, 71 Rh blood system, 203–204 Radial immunodiffusion, 186 Rh deletion, 204 Rapaport-Luebering pathway, 94f Rhesus (Rh) hemolytic disease of newborn (HDN), 117 Rat tapeworm, 76 Rheumatoid arthritis, 191 384 Index

Rh genetic theory, 203f Serologic assay, 187 Rh hemolytic disease of fetus and newborn, 218, 218t Serology RhIG, 214 infectious disease and, 187–190 Rhinovirus, 70–71 Serous fluid, 171 Rhizomucor species, 57 Serum marker Rhizopus species, 57 measurement of, 167 Rh null, 204 Severe combined immunodeficiency, 192 Rhodococcus equi,65 Sheep liver fluke, 77 Rhodotorula species, 62 Shift (stress) reticulocyte, 92t Rh polypeptide model, 203f Shigella species, 20 Ribonucleic acid (RNA) Short tandem repeat (STR), 265 molecular composition of, 262 Shwachman-Diamond syndrome, 109 specimen collection and storage, 266t Sickle cell disease, 110 Ribonucleic acid (RNA) isolation, 265 cell descriptiom and diseases associated with, 100t Ribosomal RNA (rRNA), 264 Sickle cell testing, 99 Ribosome, 4 Sicle cell trait, 110 Rickettsia, 187 Sideroblastic anema, 104, 106 Rickettsia rickettsii,32 Signal amplification, 270 Rickettsia species, 32 Six Sigma, 289 Rifampin, 38 Skeleton, 90f Ringworm, 60 Small lymphocytic lymphoma, 126 RNA retrovirus, 275 Sodium, 235 RNA. See Ribonucleic acid (RNA) pathologic processes for, 236–237 RNA virus, 68t, 69–71 recommended ranges for, 236t Rocket electrophoresis, 186 Soft tick, 80 Rocky Mountain Spotted Fever (RMSF), 32 Solid tumor Role playing, 291 molecular genetics of, 276 Rosette test, 212 Southern blotting, 266 principle of, 213t Specific esterase (naphthol AS-D choloacetate esterase), 104t Roto’s syndrome, 244 Specific gravity, 160–161, 160f Roundworm, 73 Spermatozoa, 170 RSV, 70 Spherocyte Rubella virus, 71, 189 cell descriptiom and diseases associated with, 100t Rubeola, 189 Spindle shaped cell, 169t Spirochetes, 188 S Spliceosome, 264 Saccharomyces cerevisiae,61 Sporangium, 50 Safety, 288–289 Spore, 5 Safety officer, 289 reproduction of, 50 Sanger sequencing, 270 Sporobolomyces species, 62 Sarcoptes scabei, 80 Sporothrix schenckii,64 Scalded skin syndrome, 6 Spur cell (acanthocyte) Scedosporium apiospermum,59 cell descriptiom and diseases associated with, 100t Scheduling, 288 Squamous cell, 164 Schilling test, 108 Staffing, 288 Schistocyte (schizocyte) Stained smear, 1 cell descriptiom and diseases associated with, 100t of blood and bone marrow, 98–99 Schistosoma haematobium, 78, 164 Staining technique, 104t Schistosoma japonicum,78 Staphylococcus aureus, 5–6, 7 Schistosoma mansoni,78 Staphylococcus epidermis, 7 Schizocyte (schistocyte) Staphylococcus saprophyticus, 7 cell descriptiom and diseases associated with, 100t Stenotrophomonas maltophilia,29 Schmidt, Warren, 283 Stomatocyte Scopulariopsis species, 56 cell descriptiom and diseases associated with, 100t Secondary hemostasis, 142–146 D-Storage pool disease, 148t disorders of, 149t Strand displacement amplification, 270 Se gene, 200 Streptococcus, 187 Segmented neutrophil, 95t Streptococcus agalactiae, 8 normal range values for, 98t Streptococcus pneumoniae, 8 Semen Streptococcus pyogenes, 7–8 analysis of, 170 Streptomyces species, 65 postvasectomy, 171 Strict anaerobic bacteria, 33 components of, 170 Strongyloides stercoralis,73 Seminal fluid Subcutaneous fungus, 58 physiology of, 170 Subcutaneous mycosis, 51, 58–61 specimen collection and analysis, 170 genus of, 51t Senile purpura, 139 Substance Abuse and Mental Health Services (SAMHSA), 286 Septate, 50 Sudan black B (SBB), 104t Septicemia, 19 Sugar, 262 Index 385

Sulfhemoglobin, 93 Thyroxine (T4), 246 Sulfonamide crystals, 165 function of, 246b Superficial fungus, 51 synthesis of, 246f genus of, 51t Tick, 80 Superficial mycosis, 57–58 Tinea nigra, 57 Superoxide (SO) anion, 33 Tinea versicolor, 57 Superoxide dismutase (SOD), 33 T-lymphocyte maturation, 181–182 Synovial fluid, 171 Togavirus, 71, 189 Syphilis, 33 TORCH testing, 188 stages and tests for, 188 Torulopsis glabrata,61 Systemic autoimmune disease, 191 Total bilirubin Systemic lupus erythrematosus, 190–191 measurement of, 244t Systemic mycosis, 51 recommended ranges for, 245t genus of, 51t Total body water compartment, 236t Total carbon dioxide, 236t Total cholesterol T measurement of, 229t TACO (Transfusion-associated circulatory overload), 217 recommended ranges for, 231b Taenia saginata,76 Total protein, 245 Taenia solium, 76–77 measurement of, 242t Taenia species, 76–77 Total Quality Management, 289 TA-GVHD (transfusion-associated graft versus host disease), 217 Total quality management (TQM), 283 Tangier disease, 229 Toxic granulation, 118 Tannenbaum, Robert, 283 Toxic shock syndrome, 6 Tapeworm, 72t, 76–78 Toxin, 6 Target cell (codocyte) Toxoplasma gondii,76 cell descriptiom and diseases associated with, 100t T protein, 187 Tartrate-resistant acid phosphatase (TRAP), 104t Training, 287 Taxonomy, 290 Transcription-based amplification, 270 T-cell lymphoblastic leukemia/lymphoma, 124 Transferrin, 241 Teaching method, 291 Transfer RNA (tRNA), 264 Team building, 287 Transforming growth factor, 182t Teardrop cell (dacryocyte) Transfusion-associated circulatory overload (TACO), 217 cell descriptiom and diseases associated with, 100t Transfusion-associated graft versus host disease (TA-GVHD)), 217 Temporary staffing, 288 Transfusion-associated sepsis, 217 Terminal deoxyribonucleotidyl transferase (TdT), 104t Transfusion-related acute lung injury (TRALI), 217 Test/FTE, 288 Transfusion therapy, 213–216 Tetanus, 36 reactions to, 117–118 Tetracycline, 38 reaction to, 216–218 Thalassemia, 111–112 Transient hypogammaglobulinemia of infancy, 192 Theory X, 283 Transitional (urothelial) cell, 164 Theory Y, 283 Transplantation immunology, 194 Therapy-related myeloid neoplasm, 126 Transplant rejection, 194 Thermal dimorphism, 62 Transportation, Department of (DOT), 285 Thirst, 236 Transport protein, 245 Threadworm, 73 hormones and, 245t Thrombin-activatable fibrinolysis inhibitor (TFPI), 146 Transudate, 171, 171t, 172 Thrombocytopenia, 146 Trematode, 72t,77 Thrombocytosis, 146–149 Treponema pallidum,33 Thrombolytic disorder, 150 Trichina worm, 74 Thrombolytic therapy, 150 Trichinella spiralis,74 Thrombosis, 150–153 Trichomonas hominis,75 Thrombotic thrombocytopenic purpura (TTP), Trichomonas vaginialis,75 114–115, 148t in urine, 164 Thrush, 61 Trichophyton mentagrophytes,60 Thymine, 264f Trichophyton rubrum,61 Thyroid, 246–248, 247t Trichophyton schoenleinii,61 recommended ranges for, 246t Trichophyton tonsurans,61 sample collection and handling, 248 Trichosporon beigelii,62 Thyroid disorder Trichuris trichiura, 73–74 groups of, 246 Triglyceride laboratory findings in, 247t measurement of, 229t Thyroid hormone, 246 recommended ranges for, 231b function of, 246b Triiodothyronine (T3), 246 Thyroid hormone binding ratio (THBR), 248 function of, 246b Thyroid hormone synthesis, 246f synthesis of, 246f Thyroid-stimulating hormone (TSH), 246 Triple phosphate crystals, 165 Thyrotropin, 246 Trophozoite, 74 Thyrotropin-releasing hormone (TRH), 246 Troponin, 251t 386 Index

Trypanosoma brucei gambiense,80 Virus particle replication, 66 Trypanosoma brucei rhodesiense,80 Visual learner, 291 Trypanosoma cruzi,80 Vitamin B12 Trypanosome, 72t,80 DNA synthesis and, 107f Tubular disorder, 167t Vitamin B12 deficiency, 107 Tubulointerstitial disorder, 167t Vitamin C deficiency, 139 Tumor immunology, 193 Vitamin D Tumor marker, 194t effect on intestines and kidneys, 239t Tumor necrosis factor, 182t recommended ranges for, 239t Tumor-secreting hormone (TSH), 247 Vitamin K deficiency, 149 Tumor suppressor gene, 275 Vitamin K-dependent factor, 142, 143t Turbidimetry, 186 Von Willebrand disease (VWD), 148t Tyrosine crystals, 165 W U Waldenstro¨ m’s macrobulinemia, 192 Ureaplasma urealyticum,31 Wangiella dermatitidis,59 Uric acid crystals, 165 Warm autoimmune hemolytic anemia, 116 Urinalysis, 159 Washed platelet, 214 renal disease and, 167t Washed red blood cell, 214 Urinary cast, 163, 163t storage and expiration of, 215t Urine Wegner’s granulomatosis, 167t changes to unpreserved, 159 Weil-Felix test, 187 crystals in, 164–165 West African sleeping sickness, 80 examination of Western blotting, 268 chemical, 161 Western/Eastern equine encephalitis, 71 microscopic, 163–165 Westgard, James, 289 physical, 159–161 Westgard Rules, 289 nephron function in production of, 166 West Nile virus, 189 pH of, 161–162 Whipworm, 73 Urine clarity, 159–160 White blood cell, 164 Urine color, 159 changes to morphology of, 118–119 clinical correlation of, 159 normal range values for, 98t Urine specimen, 159 types of, 93–98 Urine volume, 159 White blood cell count, 168–169 Urobilinogen, 163 White blood cell disorder. See Nonmalignant white blood cell processing in hemoglobin degradation, 162f disorder White piedra, 58, 62 V Whole blood ABO compatibility for transfusion of, 213t V617F, 276 storage and expiration of, 215t Vacuolization, 118 Wiener genetic theory, 203f Vancomycin, 38 Workload, 288 Vancomycin-resistant enterococcus (VRE), 274 Wound botulism, 36 Vanillylmandelic acid (VMA), 250 Wright-Giemsa stain, 99 Variable cost, 287 Wuchereria bancrofti,79 Varicella-Zoster virus, 69, 189 Vasculature X disorders affecting hemostasis, 139 XLD, 2 role of, 139 X-linked agammaglobulinemia (Bruton’s agammaglobulinemia), Vasculitis, 139 192 Veillonella,35 X-linked disease, 271–272 Very-low-density lipoprotein (VLDL), 228 X-linked disorder, 113 Vibrio, 21–22 Vibrio alginolyticus,22 Y Vibrio cholerae,22 Vibrio parahaemolyticus,22 Yeast Vibrio vulnificus,22 identification of, 54 Viral genome, 274 types of, 61–62 Viral-load testing, 274 in urine, 164 Virion Yellow marrow, 90 characteristics of, 65 Yersinia species, 20–21 definition of, 66 Virus, 188–190 Z characteristics of, 66 Zero order kinetic, 251f culture methods for, 66–67 Zone of equivalence, 186 groups of, 68t Zygomycetes, 56–57 herpesviridiae family of, 189 Zygomymycosis, 56 nonculture methods for, 67–68 Zygospore, 50 properties of, 65 Zymogen, 142 This page intentionally left blank PLATE 1 PLATE 2

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