DOCUMENT RESUME BD 200431 0 TITLE Medical. Service Climi Laboratory ProcedureHematologi INSTITUTION Department of the Air Force, Washingon,t, Department of the'Army,- Washington, D.C. EEPORT 110 AFM-150-51; -TH-B-227-4 PUN:. DATE 5 Dec 73 NOTE- 151p.; Contains colored pictures which may reproduce Well

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DESCRIPTORS , *Allied Health OCcupations: *Armed Forces;_Biology;-- Government Publications; Health Occupations; Higher Education; Instructional Materials; naberatory Manuals: *LaboratorY Procedures; 'Medical Education; *Medical. Technologists; riaterials-zr, Science IDENTIFIERS- *

.ABSTRACT Presented are laboratory studies focusing onblood- cells and theomplete scheme of . Forked is'the basin for the following types :of laboratory operations: (1) distinguishi4.the morphology of normal and abnormal blood cells; (2) measuring theapcentrations or number of blood cells; (3) measuring

concentration a** abnormalities'detecting of heloglobin; (4) . measuring defecesin coagulation; and (5) performing a few specific disease-related-tests which involve blood cells. Types, of equipment needed, actual stkavise perormance of tests, and reagents needed,= as well as established, minimum levels of accuracy are included. lAuthor/CS)

********************************* ****** -*4***** roductions dupplied by EDRS are the.best can be from th6 original docdment. ****************************************** --

D&;ARTM-ENTS. OF THEAIR FO10E_ AF MANUAL -160-51 _ . THE 'ARM TM- 8-227-4 Washington DC 20330_ 5 December 1973- Medical Service CLINICAL LABORATORY PROCEDURES-1

The purpose of this-manual is to present the laboratoi-y steadiesw concern the blood cells and the complete: scheme of blood coagulation. It forms thebasis -for the following types- of laboratorli operations(1) clighiguishing the Morphology of normal' and abnormalblood. cells, ,(2)imeasuring:.--ti the concentrations.or number of bloodcells; (3) measuring concentra- tion driOr,.t4 tecting abnormalities of ; -(4)measuring defects in.boagulation; and (5) pei-forining a Lei- specific disease-related testa which involve-blood cells. It enumerates types of e uipment needed, actual stepwise performanceof teSts,..and= reagents- needed, as well as e'tablishes rniiijmum levels ofaccuracy:

1

-ChaPtdr..-1Blood Section ATbe Composition of, Blood. Introduction- -- , . t 1-1 eneral Cellular Structure 6 1-2 'Cellular Constitu'ents. 1-3 Liquid Constituents _ L 1-4 1-2it ection BFormation of Blood Cells _mbryonic Heinatopoiesis, 1-5 1-2n r ostnatal Hematopoiesis_ 1-6,-, 1-3 on CNormal Cell Mtu eneral Features_ Cytoplasm ;Nucleus' SeCtiorl: DAbliormal General Features Cytoplasm Nucleus. Section' E--Functions Introduction 1-13 1-6 Erytfirocytes 1-14 1-6 Leukocytes' _ 1 1-15 _ 1-7 .= _ 1-16 1-7 Chapter 2---Material employed in He atolo`gy 'Section ALaboratory Reagents Preparation _ 2-1 2-1 Labeling Reagent Containers '2-2. 2-1 Safety Precautions- . _ _ _ _ _ .- Supersedes AFM 169751,1 May 1962 and TM 8 -227 -4, 1 Maych 1963. (For summary of -evised, deleted, or added.rhaferial; se.--siga:6,De page.) OPR: SGPSE (14repar0 by SHCS/MST)' DISTRIBUTION (Page 7-10) -AFM 60751/TM -2 7 5 Dec mbei 1973.

page Section BLaboratory Glassware Diluting .Pipets 2-2 Unopettes - , 2-3 Calibration of Sahli Pipets _ 2-6. 2-3 Blood Cell Counting Chambers'.. __ _ 2-7 . 2-4 , Cleaning -_ , 2-8 2-5 7Preparation of Siliconized cilassware _ _. 2-9 2-6 Section CCompound Microscope Introduction = 2-10 , Magnification 2-11 . Illumination = 2-12- Focusing 2-13 Care-of the Microscope = 2-14 es 2-15 Section D-Centrifuges Types ; 2716 2-10 Precautions 2-17 t 2 -14 Section EAutomated Equipment Introduction 2-1a 2 -12 Dilutors-'_ - - = 2119 2-12 I 2-12 Slide Stainer =t _ Counting Devices - -,...2,21 2-12 IL Hemoglobinometer 2-22 2-17.. Fibrometer 2-23 2-17 , Hematological Systems 2-24 .217 Maintenance of Automated Hematological Equipment 9_95 2-18 Chapter 3Basic Hematological Procedures Section ACollection of-Blood Specimens' Introduction VOnipuncture Capillary- Puncture Anticoagulants Section BPreparation and Staining of-Blood Smears

Introduction . Slide Meth-etl Coverglass Method Section CBone Mprrow Specimens, Introduction _ Processing of Bone Marrow Specimens for Exa inaion Chapter 4Morphology of Blood Cells Section A---General Information -Basic .Concepts Of Cell M.orphology 4-1 General Rules of Cell Identification _ 41 Section BErythroeytes IntrOduction 4-1 Erythrocytic Series 4-2 4-2 Variations in Erytocyte§ = AFt1L-1600,17I'M December --- 973 - iii pier Seetion C-Leukocytes Granulocytic Series 6 -15 Agrant31putes 4-7 .--LymphoeyticBeries _ 4-8 Monoelytic -SerieS:- 4-9 Plaamoc3-iic/Series = 10 VarrationS-of Leukocytes ion1:1-=Thrembocytes Introduction_ 4-14 Megakaryocytic Series 4-13 4:45 Chapter' 5Method§ in Hematology -Seetion -AManual Cell Counts i-1 5-1 Count (Hemacytomete 5-2 51 White-Blood-Cell Count (HemacytOmeter) 5-3 5-6 Total Count 5-4 /5-8 Absolute Count (Cooper a- 5 i 9:

Reticulocyte _Count 5-6 0 -

Cerebrospinal Fluid Cell Counts _ 5-7 -- 5--11

Semen Analysis r =8 5712 Section B---Hernatecrit

Introduction- . 5-9 5-14

Madrohernatocrit (WintrObe) , 5-10- 5-14 kg Microhematocrit -- ( - . 5-11 5-16 ,- Section CErythroyte Sedimentation --Rate--.

IntrOduction _ _, _: 5-12. 5i16 Detern ination of Sedimentation Bate (Wintrobe-Landsbe 5-13 5-47 Seetion D rnoglobin General nformation 5-14 -18 Coinpoun s of Hemoglobin 5-15 - 51_8 Variatio of Hemoglobin 5-16 L18 Hemoglo nometry 5-17- 5718 Cyanmeth moglobin Method 5-18_ 5-18 Detectieof Hemoglobin S andon S Sickling ' 5-19 5-29

Demonration of the Sickle Cell Phenomenon _ 5-20- 5-21 obin Electrophoresis (Cellulose Acetate) 5-2k 5-22

Fetal Hemoglobin .() = 5-22 5-24 Section EExamination of Blood Smears Introduction I 5-23- 5-25 Examination of Peripheral Blood Smears 5-24 b-26 Examination of Bone Marrow Smears 5-25 5-28 Section FErythrocyte Indices and Fragilitp_Tests Erythrocyte Indices . 5-26 5-28

Erythrocyte Osmotieirragility Test (Dacie ) _ 5-27 5-29 Ham' Test for 5-28 5-.31 Section GDemonstration of L.E. Cells General information 5-29 5-32

Rotary Bead Method .= 5-30 5-32 v AVIV 160-51/TM S-227 1973

Section H--Special Stains .. Kaplow) ,- 5-31 - :5-L33 Peioxidase'Stain 1 LeukocyteAlkatine*Phosphatase K(Kapp')l 2 5734 Heinz Body Stain 5-33/ 545 Siderocyte Stain, __ I, 5 z34 - 5-36 Section IMisepllaneous Teas' . - ScreeningTest for Glucose-6-Phosphate Dehydrogenase Deficienty 5-35 5-36 Shipment of Cytogenetic Specimens. 5-36 5-37 Chaptor 6--Blood tCoagulation Section AIn tfOduction HemOstilsis -6=1 Coagulation 2 SectiO,BCoagulation Factors Blood. Factors Plafelet -Factors Fibrinolyfic Factors Section--CThe _Coagulation Mechanism Introduction TheModern Theory CoagulatiOrt Ihhibitors . Section pCoagulatidli Studies Introduction 6-9 6-5 Blood Collection 6710 6-5 Bleeding Tirne = 6-11 6-6 Whole- Blood 'Clotting Ti 6,-12 6-7 Clot Retraction Test 6.13 .6-8 Tourniquet Test 6-14 6 -8 Partial Thromboplastin Time (Activated 6-15 6-9 Qpe-Stage 6-16 6-10 Prothrombin Consumption Time 6-17 6-11 Thromboplastin -Generation Time 6=18 -6-12 6-19 6-14 Assay (Semiquantitative) 6-20* 6=15 Plasma Recalcification Time - 6-21 6-16 Detection of a-Circulating Antidoagulant 6-22 -6-16 Detection of Factor XIII Deficiency = 6-23- 6-17. Count. (Rees-Ecker) 6-24 .6-17 Platelet Count (Phase 6-25 6-18 Macroglobulia Distilled Water Screening Test 6426 6-19 Cryoglobulin Scteening Test 6-27 .6-29 e Chapter 7Quality Control Introduction 7-1 7-1 Definition of Terms 7-2 7-1 -Statistical Evaluation of Hematology* 7-3 7-2 Control in Blood = Collection 7-4 72.6 Control in Hemoglobin Deterrnrtions 7-5 Control in cell Counting (Hemacytometer) 7-6 7-7 Control in Electronic Counting 7-7 7-8 AFM 160-51/TM. , 227 ember 1973

_ram? ., ?age Control-in -Hernatocrit Determination .7 7-8 Control in Differential Counts . _ 7L9 7-9- COntrol, in Coagulation Studies 7-10 -7-9 Control, in Other Teats : 7-11 7-9 Stun/nary, 7-12 : 7-9 Attachments 1.Bibliography A-1-1 2.Glossary A2-1 3.-Prefixes, Suffixes, and Stern only Used in. Hematology: = A3-1 4.Index A44- Figures 1-1.Typical Cell Structure 1-2 1-2.Hernatopoietic Syeterti in anAdult 18,---20 Yea of Age 1 1-3.Development of Blood Cells

Hematological Pipets .

2-2. _ Unopette System 2-3.Rulings on a Hemacytorneter, 2-4.Compound Microscope.. 2-5. u. Microhernatocrit Centrifuge and Microcrritocrit Reader =- 2-1.1- 2-6.Automatic Dilutor . -2-13 2-7.Automatic Slide Stainer 2-8.Coulter Csunter Model FN 2-15 2-9,Schematic tIsiagiarn of the Coulter Counter 2-16- 2-1N. Schematic Diagram of a Photoelectric Particle Counte 2-16- 241. Fibrometer 2-18 2-12. Coulter Model S _ 2-19

-1.Site of Venipuncture _ 3-1._ 3-2.Venipuncture Procedure 3-3 Vacutainer System _ _ - 3-5 3-4.Capillary Puncture Procedure 3 -6 3-5.Slide' Method for Preparation of Blood Films 3-10 3-6.Coverglase Method for Prep_ aration of Blood Films -13 4- 1.Erythrocytic Series 4-2.Variations in Erythrocytes

4-3.Granulocytic Series _ 4-4.Lymphocytic Series and Monocytic Series 4-1 Plasmocytic Series 4=13 4-6.Variation's in Leukodytes 4-15

- 4-7.Megakaryocytic Series, _ 4-10'' 5-1.Processing Blood for Manual Cell Counts 5-2 '5-2.charging the Hernacytbrneter 5-3.Hemacytometer Counting Chamber. Ar ed1 5, are used to count red blood cells _ 5-4 5-4.Hemacytometer Counting Chamber. Areas Marked A, B, C,and D are used to count white blood cells 5-7 Morphology of Spermatozoa-

ty -Figures 5-:-6. Wintrobe Iternatocrit- Tube 5-15 5,7.Dithionite Tube Test 1fiterpreta 521: 5-8.Hemoglobi n Blectrophoretie Scp.nk., 5-23 5-9.Examination of Peripheral Blood Smears 5-26 5-10. Erythrocyte Osmotic Fragility Curve : 5-31 6-1.Modern Theory of Coagulation . ',- 6-4 7-1.Classical Gaussian (Bell Shaped) .Curve __ 7--2 72.Exafriple of-a Monthly ±Standard Deviation Calculation 7,4 7-4.Example of a Quality Cootinl Char(' . _ 7,5

Tables _ Nomenclature of Coagulation Factors Diagnostic' Coagulation Tests AFM 160=5 /TM 8-227-4 member 1973

ECTIONTHE .CQMPOSITION. OF the nucleus is ,a network cirTmclear fibrils BLOOD Made Lip of DNA (deoxyribonucleic acid) protein called chromatin.. It is thought that -1.-Introduction. -Blood is a complex and the decreasing growth activity of a cell dur- unique fluid_ of variable composition circulat- maturation is regulated by chromatin. ing-through the Vascular-system of the body. Atypical cell tineleus ni Itis a tissue in which' cellular constitu- ents are suspended in a= liquid .rafdiumper- forming specialfzed fUrictions.- The prime c. Stirrolinding the nucleus is a mass of function of blood is to -maintain oxygen and protoplasm called cytoblasnt contained-With in the cytoplasm are numerous granules, food supply for 'tthe bodyy- cells and prevent accumulatton- of waste products.- Blood also filaments, and globules '.These structures are protects against -causative Lgents of disease diVided into two group§ known as organoids and supplies sUbstances which-fpromote the (otmanelles) and inclusions. The organoids clotting proces.hen injury causes bleeding are thought to perform most of, the metabolic or hernorrhage.---TO "perforrn_ these_ complex functiOns of the cell. Mitochondria, Golgi funetions, the -ceinposition of blood must be apparatus, fibrils, centriales, and the chroma- complex. Formed - elements account for ap- tin SubstarIce- are.classified as organelleS. proximately 4515er.ettit,Otthe blood volume. Cytoplasm inclusionS: are usually seen as The remaining 55 -Peitentlis _a liquid of a granulation. This granulation is an aCcu- very clrnplex nature called plasma. .Plasma mulation /of proteins, lipids, carbokydrates, is composed mostly of water but it also con-p pigments, 'and secretory granules. = tains other important` substances. 1,3. Cellular _Constituents: 1-2. G6eral Cellular Structure; a. -Erythrocytes. =Anerythrocyte_(red a. A typical cell is composed of a single'blood cell)is an elastic, nonnucleated, bi- nucleus embedded in cytoplasm_ living concave disc having a-diaineter;of apOroxi- substance of the cell is a grayish viscous -mately 7.2 microns The mature_ red cell con- liquid called 'protoplasm. Protoplasm is en- .tains about 34 pereent hemoglobin (a. com- closd in the cell ibterfaces.by a cell mem- plex iron-bearing pigment which transports, brane which selectively/regulates the inter- oxygen). Hemoglobin is contained 'in the in- change of materials between- the -cell and terror of the cell, and the outer surface of the its environment. A typical cell is diagramed cell is surrounded by a cell membrane. When: in figure 1-1. unstained, the cell has a pale, greenish- b.The nucleus is a spherical or oval body yellow appearance; and light orange with an sti7T-ounded by a' thin nfmbrane (nuclear accented central zone of pallor when stained embrarie). Contained in the nucleus is a h Wright's stain. The production of eryth, 4there called.the nucleolus. The nucleolus is rocytes, or , occurs primar- thought to be an organizing 'center for the ily in the l-ed marrow of the spongy bones cell and can have, the capacity for cell pro- (see figure 1-2). An adult female has ap- iluction. The absence of nucleoli, signif es the pi-oximataly 4.8 million/comin-red 'cells, and end of cell developnient. Alsb found within an..adiilt male has approximately 5.4 million/ 5 December 1973

CYTOPLASM

CENTRIDLE

NUCLEAR MEMBRANE Figure 1-1. Typical Cel cu mm red cells. The erythrocYtes have an of the blood before- doting occurs average life span of 80 to 120 daks. m plus fibrinogen). Thechemical. na-. ilre of plasma is very complexl Plasma is b. Leukocytes. Leukocytes_ are commonly .composed mostly of water, but it also.contains knocri as white. blood cells because of their dissolved Wises (oxygen, carbon dioxide,'and lack of color in unstained preparations. They nitrogen), -proteins (albinnin, globulin, and are nucleated cells which have an average di- . fibrinogen), carbohydrates -.(glucose), lipidt ameter of 8 to 12 microns. Leukocytes are di- (fats, lecithin, and cholesterol), nonprotein vided into three groups: (1) ,-.nitrogenous substances, and inorganic salts. (2) lymphocytes, and (3) . They are differentiated by the specific nuclear and b. Serum. Serum is the fluid portion of cytoplasmic staining properties., blood after the clotting process is complete. Fibrinogen the _precursor of fibrin,is re- c Thrombocytes.. Thrombocytes are most moved from plasma to form the framework- commonly known as platelets. Ptatelets are detached fragments of the cytoplasm of their. of the blood clot. precursor cells--makaryoeytes which are found in the b(3ne marrow. Unstained plate- SECTION BFORM- TION O LOP lets appear as small hyaline structures with CELLS adipeter of appioxirnately .2microns. When stained .with Wrightstain, they have '1 -5.. Embryonic Hema.topoiesis: a pale blue cytoplasm with a dark granular center. 'Platelets are very fragile and live for --- a. The primary source of blood cells is the a perioof about 3 to 5 days mesenchyme connective tissue hi' the embryo. Three phases of embryonit herhatopoiesis -1-4. Li1quid -Constituents: _rherg&,4 resulting in the formation of blood. Plasma. Blood platina is the fluid por-, b. During the first 2 months- of embryonic 8-227-4 5 -December 1973

development the mesoblastic phase occurs. is red bone marrow and It is actively erigaged The blood.,cells are formed in -the_ blood is- iii hematopoiesis.- At about 5 years of- age:- lands of the yolk sac. Immature- blood cells,- a nonhernatopoietic type of marrow (fatty develop, having -large nuclei containing .-a- yellow bone rnarrow)rwhish isreserve po- Vesicular Chromatin meshwork The nuclei tential tends -:to replacernqat of the red bone are.surroUnded by a thin rim of cytoplasin.- marrow. This partial replacement Of red mar: Thcells gradually develop hemoglobin- to row is. complete when the indiViidual reaches be&me a' niteleated red blood cell. Mitosisoc- maturity (about 18 years of age) at which curs and daughter cells contain more -hemo- itne, active hematopoietic cenIers in bone tis- globin. Finally the nucleus is lost and the sueare limited almost exclusively to the ster- mature erythrocyte-,..` is produced. num, pelvic area, vertebrae, skull, ribs, clavi-. c. Atg.hhhth!thh hepatic phase begins. cies, scapuli, and the ep/physes of the long Blood cell development shifts to the body bones (figure 1-2). of the fetus as.the organs of thereticuleen- Exti edullary heniatopoiesis is blood. dothelial system (liver, spleen,- thymus, etc.) productionhich occurs in sites other than are fated. -Duringthis-phase,ced cells begin the bone w. Active sites are the spleen, their normal development. GrAnufacytes and thymus, lymph -nodes, and other, lymphoid thrombocytes are-lormed in the- liver while tissues-figure1-2).Cellproductionis, -lymphocytes -and mono-cytes -,are formed- in largely limited to lymPhopoiesis. The lymph the spleen and thylnus. nodes are the primary source of lymphocytes d. The'rageloid Pha.ge begins during the and Plasmocytes. fifth month of gestation. Blood 'cellsare formed air they bone marrow and lymphatic':SECTION C NORMAL CELL' system which- at the time of birth.cOnstitute MATU4ATION the total sour cos of hematopoiesis: The bone marrow is the princiPle source of production 1-7.General Features.Inc' the course o of erYthrocytes, _granulocytes, and thrornbo- blood cell, -maturtition, certain specifie lea cytes. The lymph nodes are the primary sites, tin-es are developed. Each of the, component, of production of ilymphocytes, monocytea, parts of the cell 'undergoes a transformation and plasmocytes. during. maturation. The. immature cell or blast cell contains a- large nucleus, a small Post -natal Hematopoiesis: . _aniount of cytoplasm,and no granules. As a. Blood for at birth Confined:the cell ages, the cytoplasm becomes less bas.o-, -primarilytothebonne. marrow',(central(central philic and nuclear chromatin betomes hedv- medullary. structure of the bone). Blood cells ier (darker stain). Reduction in size and loss multiply by .rnitosii and then mature to a of nucleoli occurs as the cell becorhes-older. specific cell type. '-17ie mature cells lose the The three types of granulation.(neuifo- ability to repraduceand develop a definite life philic, basophilic, 'atid eosinophilic) beeorne- span.:Regeneration-of blood cells after birth more specific and arrialler as the cell ages. involve multiplicationof precursor: cells, Maturation, in, general, involves: (1) cyto-, evolution d the definitive- ehliracteristics of _plasmic -differentiation, (2) nuclear mature- each type, and .release of mature cells. tion," and (3) reduction in cell size (see figure

= 1 -3 b. .is the -production of broad cells byv the bone marrow (medullary site of 1-8. Cytoplasm. The basaphilia of a blast production). The- red bone marrow is the cell is. proportional to the ribonucleic_ acid principle source 9f production =of red cells (RNA) content. As the cell matures the and, white cellsofvthe grantilocytic series. At RNA content decreases and the cell becomes_ birth the central medullarY structure of bones a paler blue. In the rnyeloid cells a specific $ AF11160-51/TM = 8227-1 December 1973

Skull RED.BONE ARROW LYMPH NODES

PRODUCES: PRODUCE:

- Sternum- Humerus Lymphq es Moncrcytes Neu rOphils - Ribs LIVER

PRODUCES: Prethrombin, fibtinogen

. EXCRETES:BilirObin

STORES: Erythrocyte picturing factor.-- *ACTS ds Otramedullary Stomach.- Sple n hematopoietic center on Erythrocytes d.em nil,.

Lymph-nodes

Pelvis

Femur STOMACH SECRETES: Intrinsic erythrocytic Metiocytes maturing factor

Lymphocyte ELLOVBONE MARROW DESTROYS: Worn out blood cells.- ACTS as exlramedullary hornatopoietic ACTS as- center on cleniand. hern;topoietic cehter orr demand

dult 18-20 Yeaisof Age. AFM TM 8-227 Deeebilier 1973 MESENC1-1YrsiE; BLOOD ISLANDS YOLK'SAC

LIVER --SPLEEN

RETICULOENDOTHELIAL CELL. : (PARENTCELL OF'ALLBLOOD CELLS).

RED BONE MARROW

My.eloblast Rubriblast

-,RtornYelocy e

Rub icyte Basophilic

Metarub cyte

BaSophilic Eosinophilic

. MeternYelocyte Megakyocyte Neuophilic Band,

usely .Segmanted Eosinophi4BasophilicBasophilic NeufroPhil Band Band-- Erythrocyte

MOnocyte Lymphocyte 'Eosinophil Basophil Erythrocyte Hypersegmented Thrombocyte Figure 1=3._Development of Blood Cells. AFM 160-51/TM 8-L-7-4 5 December 1973 type of 'granulation occurs. When grannies. chronous development. Since the normal se- appear they are pinkish red and few in quence of cell maturation is upset: atypical number: The granules increase in number-and cells will be present. Abnormal cells can be differentiate into three types upon matura- recognized -by:(1)abnormal cytoplasmic tion:- As the cell matures it -develops an of -. maturation, (2) Abnormal nuclear matura- finity for the acid or basic portion of the tion, and (3) abnovnal size. . stain (.1,VriOht's, stain). -Basophill0. granules ...stain blue, eosinophilic granules:stain bright 1-11. Cytoplasm. Asynchronism of the cyto- orange,and neutrophilicgranulesstain plasm is most commonly seen in the granu- rpinkishpurple. Lymphocytes are usually de- locytes. Granulation can he primitive or ab- void of cytoplasmiegranulation but they can sent. In some instances the granules fail to posseSsnonspecific a-zurophilic (dark purple) differentiate. Erythrocytes show basophilia granules, usually characteristic of monocytes late in the series and retarded. hemoglobina- and plasinocytes.- Upon maturation, theeryth- tion. Inclusions, in the cytoplasm, such as rocyte develops -a light orange :respiratory Dohle bodies(infectiousdiseases), Auer pigmeat Called-hemoglobin. hods (leukemia), and toxigranulation (in- fection affecting the m w ) ° are seen in 1.9."'Nucleus. The nucletis of the young'. cell the abnormal white cells. is large, round, and occupies most of the cell. As the Cell. Matures the size of the nu:. 1-12. Nucleus. Abnormal'` often show dens decreases.' Nuclei` of early or -Primitive two nuclei in severe disturbances, such as cells. usually have one or more nucleoli, The leukemia. Nucleoli have a retarded reduction: latter are''stnall, round, homogeneous- areas The nucleus'can have an irregular outline or which usually stain light blue with a darker indentation (Rieder Cells). Hypersegmented boundary. In appearance nucleoli are some- nuclei occur in the insepsis and in what like craters in the nucleus. They are Pernicious anemia. Abnormal, maturation of surrounded by strands of chromatin. These the nucleus often results in variation in cell nucleoli, plus- a'delicate reticular network of size: .chromatin, are the principal indicators of bood cell -immaturity_ As the cells mature SECTION EFUNCTIONS OF BLOOD the nucleus gradually becomes smaller, stains CELLS darker, and the chromatin meshwork become ','coarse" with the strands of chromatin les,- -13. Introduction. Each component -of blood fine and lacelike. In the,course of cell dev'el- is uniquely. capable of performing one or opme-pt the nucleus changes its shape, par- More functions. Together, these compon- tictilaly in the granulocytic series, where it ents provide the maintenance of a relatively becomes indented, lobulatecl, Segments, or stable environment .of, the body by a variety fragnienteci. As maturation or development of mechanisms. This Maintenance of n rela- progresses, the ,nucleus, if. still. intact, be- tivebiologicalconstancy 'orintegrityis "comes small, compact, usually , dark, and known as homeostasis. Once the blood cells structureless and can:completely 'disappear. reach full maturity they enter the blood- The loss or shrinking of the nucle#S is ac- stream and begin fulfilling their funetionS. 'compinied by a decrease in cell size. 1-14. Erythrocytes. Hemoglobin is the main fttnetioning component of the cell. It carries SECTION 13...ABNORMAL CELL out the :transportation of oxygen to the tis- MATURATION sues and the :removal of carbondioxide. Hoinoglohin 6.1So aids in the maintenance of 1-10. General Features.Abnormal cell the delicate acid-base buffer system of the maturation is asynchronous development as body. The erythrOcyte must also -supply., opposed to normal cell maturation or syn- energy to accomplish. the active transport AFM 16041/TM 1-7

of gluposeartdions agains dient- a.oss are also thought to transport, or at least con- the red eell membrane. tain, lysins which act- on fibrin. It is sug- gested that limit the action of 1-15, \Leukocytes. Leukocytes remove substances such as histamine. How- this -is vading antigens(forexample,bacterial.. acomplislred is not yet ,clear. The mobilila- and to some extent transport and 'distrib- tion of epsinophils from their reserve in the. ute antibodies:- Monocytes andallof the bone marrow is at least in part under hor- granulocytes have been shown_ to demonT monal control. If the adrenal cortex is -func- strate,directional movement. Their move- tioning properly,- an injection of adrenocor- Menlis :subject to chemotaxis, which . is de- ticotropic hormone (ACTH) results iin fined as the response of .living protoplasm Marked decrease in the number of circulat- to a chemical stimulus. This is a m'eaus of ing eosinophils and in the number of dr; attracting cells- .to substances which they eulating, lymphocytes. On the other hand,: must either transport' or 'engulf. The process there is an increase in the 'number of cir of -eng4ng and destroying bacteria,or culating neutrophils. ,- is a prime fUnction of leuko- cytes. asophils-. The function of basophilS in ,rnan leis not been ascertaine-They quite pos7 -a.' Monocytes. These cells will engulf 'bac- sibly represent a vestige of evolution. Their teria and larger materials, including even granules have been found*eontain hepari, protozoa and red cells, and are called macro? anrj =these cells frequently appear diving the phases. In this- regard, 61monocytes are per- clot dissolution: phase of an injury.. Henee, haps the most efficient of all the it has been suggested they may be involved cells. -s contain many of the in clot absorption. enzymes that- are foundinrnicrophages (granulocytes); In addition, monocytes con- e. LymphocyteS. The lYmphocyte is now tain lipase_s whieh enable them to dissolve believed to be directly: connected wtth anti the lipoid capsule§ of .certain bacteria. body production. Undoubtedly, the lympho- cyte, performs i'mpoiant immunologic,-func- Neutrpphils.Neutrophilicleukocytes tions.According veryrecentstudies, are ex' ellerit microphages. That is,. they en- many of the activities previously thought gulf bacteria and other microscopic particles. to take, place in the reticulpendOthelial (RE) The particles are first surrounded by cellu- systerfi-actuallY take plaCe in lymphocytic lar pseudopodia and then incorporated into a cell vacuole. There the foreign bodies -mix with silbstanceS released fromthe cytoplasm bf leukocytes. In this way theeukocyte is 1-16. Platelets. Platelets -possesS metabolic not injured by whatever "combat activity" systems, expend energy, and respond to stim- is taking, Place in the vacuole. Neutrophils, uli. They contain many enzymesand undergo are ..-full3Ndeveloped (mature) cells that are respiratory activity and glycolysis. They-,pos- Sess coagulation factors usually designated incapable of 'mitotic- division, They carry on as PF-1, PF-2, and on, through .PF-7. The active metabolism. Eventually the granulo- cells contain -fibrinogen and vasoconstrictor cytes disintegrate and in inflammatory proc- substances, calcium, and many other com- esses aresucceeded by monocytes. ponents which are either known or pre-. c. Eosinophils. Eosinophils are found in sunned to participate in the clotting mecha- tissue .fluid as well as in peripheral blood, nism.- Clot-promoting lipoprotein's are also especially in areas where there is an aller- 'found in platelets. In addition, well-defined gic reaction. Current thinking -holds ,that. eo- antigens have been found :in platelets. The sinophils are involved in antigen-antibody role of plateletsinthe blood coagulation- reactions, and have been shown to phagocy- echanism will be described in more det '1 tize antigen-antibody reactants. Eosinophils in chapter 6. 'A 160751/TM 8 -227-4 5,, 'D ember 1973

Chapter 2 MATER AL EMPLOYED IN HEMATOLOGY

SECTION ALABOR-ATORY REAGENTS never be poured baCK. because it can On- taminate the remaining reagent. 2-1. Preparation. Vat ous stains and solu- tions are utilizedih' routine hematological b, Reagents are preferablystaed in alphat ex;Aminations. These stains and solutions betical order on shelving protected from must be prepared with the -utmost, care and dust, moisture, and.:direet sunlight. ,precisely according to formulations. Detailed= c. Never use a reagent which cannot be directions for the preparation of all reagents_ claarly identified from the label on the con, which are required for performing proce--' tamer. Discard all reagentS which cannot be clures outlined throwhok this manualare accurately identified. contained in the respectiVe procedure. Care- ful attention -should be given to preeisemeas- d. Always read the label before dispensing urements, order in which reagentS4e added, a reagent. control of temperature where indicated, fil- e When working with`rnewly -prepared r -- tration, and aging. .Particular attention must agents, 'especially stains,' ascertain whether be giKen to storage of reagents particularly desired results are being obtainrd. enSatis- withreference to requirements for refrigera- factory solutions should be discara.ed and re- tion incubation, and protection- from' intense placed, ght, f, All mixing containers, stirring rods, and 2-2. Labeling Reagent Containers-Proper containers used for storageof -reagents labeling of reagents is an extremely impcir- shO"Uld be chemically cleaned prior touse. _ taut. detail. Labels should be domplete, g. During mixing ,aiid peparition,as well curely attached, and neatly and legibly wat-: as in stoi7age, itis :'good practice' to avoid.: ten or preferably, typewritten. Items recorded contact. of reagents with metals, Many re- on the label should include' all constituents agents, contain substances which- will react and quantities utilized, date of preparation, chemically with metals and produce: changes initials of the individual who 'prepared the which, will render them unusable for labOra- reag,ent;Thrid- expiration date if the solution tory .work, deteriorates with age. Labels should be pro- tected against damage from 'water or, other h. Do not allow inexperienced personnel to fluids by covering with a protectikFe coating prepare reagents without close §4ervision. of cellophane tape over the surface of the i. Certain reagents are poisonous (that is, label. Drabkinis solution and bichloride of mer- cury); and adequate precautions should be 2-3. Safety 'Precaut on's. There arevarious taken to prevent aceideny poisoning, precautions' which must- be taken in handling highly toxic: reagent§ Should 1,:apeconspicu- reagentsintheheniatologylaboratory. ously labeled "POISON" and should Iv stored, Among the most important are the follow- in a Separate cabinq in the laboratory. ing: j. Commercial reagents should be checked a. Once a portion of a reagent has been re- with standards for purity, Record alllot moved from the original -container, it should numbers in case a reagent is not pure. 99 AFM 160-5I/TM S-227-4 .- pece>Irl

lowed btseVeralo,vashin with distilled wa- -Retrieveprotectiveplasticshield, ter. If pllets ,.become plugged through neglect, -coveringcapillary.Obtainblood. sample

the ha !1.,,be opened. with a:fine steel from free -flowing . finger puncture (or, use

wire, ± the above procedure's: may then be thoroughly mixed venous blood specinien). carrieout When necessary, pipets may be 3) Holding capillary, almost' horizon- dried pidly bydrawing a small amount-of tal, touch, tip to blood. Capillary action fills-.: alcoh- .,_through, them and then ether Or ace- the pipet. Carefully Wipe excess blood off tone kellewed by a stream of air- until the the tip without removing any: blood froM glaSslliendiv moves frly and no Moisture- re- theduside of fhe_tube_ (4) Squeeze reservoir slightly. .Cover e s should be inspected each time .opening of overflow chamber with index o as to prevent the use .of equip- finger and :seat holder in reservoir neck. e .iliac is dirty or that haochipped 'ends. .Release pressure on reservoir and remove 2-51 Unopettes: finger from overflow chamber opening; Site- tiOn draw WOO into diluent. The Unopette system (Becton- ) queeze reservoir gentlp two or nson and Co,) consists of a disposable three tithes to rinse capillary tube, forcing m- bore glass capillary pipet with an dilitent.aup into, but not out of, overflow ched plastic tab for handling. The pipet chamber, releasing pressure each time to Me figure 2-2)is attachedto- plastic return diluent to reservoir. Mix blood sam- regervoir in which predetermined amounts ple with diluent by 'inversion. ut.ing fluids, depending on their pur- pe, can be placed. The blood aspirated (6) To dispense, reverse capillary tube, the diluting fluid can be mixed and seal overflow chamber .end onto reservoir, flippensecl with great ease and speed. inVert--. andsqueeze.ProCeduresforthe Unopette system. are available in the manu- qh. The Unopette system can be used for facturerls instriiction booklet. of variety 'of hematological proceduirs. In .(7) Always follow manufacturer's in- oral, the Unopette systemisused as ws: structions. 1) Using the pointed end of protec- 2-6.- Calibration of Sahli Pipets: plastic. shield of the capillary. holder, p- ly firm and= even pressure and puncture a. Introduction. Sahli hemoglobin -pipets. )sealin top of plastic reservoir. are volumetric pipets (see figure 2--4). The volume is "supposed" to be 20 lambda as gisterea on thf side of the,. pipet. Nor- mally, because this volfune i8 not an accurate measurement, standardization of the pipet is necessary. This is usually done with mer- cury and the procedure is necessary to as- certain the correction factor for each pipet. Procedure: (1) :Heavily lubricate the barrel of a 2 ml syringe with.Stopcock grease._ (2) Tightly fit the tip of the syringe into the broad end of a1 -holed rubber stopper, size 0. (3) Clamp the stopper to a heavy re- Figure -24. UnopetteSystem. tort stand: insert the base of the pipet into 5 December t :theother end of the rubber stopper Until Mercury Temperature (weight of 1 pl)* it is in contact. with the tip-of the'syringe.- (degrees Chtjgrade) Adjust the assembly .; so, that- the pipet-.oc- 22.0 cupies a vertical position. 23.0 4: 2.1,0 `13.5-1 ( -1) Allow the mercury and the pipet 25.0 .13.51 toreach : room temperature before starting. 26.0 j;3.53 : the calibration. 27.0 13.53 (5) ,Determine e temperattire of the 28.0 13.53 - mercury. *These values do not indicate the true densities and weights of water andmercury. but ipelude Aor- (6), Slightly wi w the plunger of rections for various factors, such as the coefficient the syringe. of expansion of glass and the bdoyant effect of air (7) Hold. the beaker containing mer-: on the fluid weighed. Bury so that the tip of tte pipet is immersed. (4) ,fk-FThe 'Volume of the pipetisob, (8) Withdraw the plunger of the sy- tained by calctilating the volume of riser- ringe until mercury reaches the 20 'ell mm gory. mark. Example: T microlit lig. , (9) Hold thebarrelof thesyringe Wt. of, mercury in )4:No. mg at T'-- vol. :1=ig' . steady and quickly pull the beaker of mer- in microliters. cury .away from the tip of the pipet. (Slight *.All Hg fluctuation in the column of mercury at (Wt of delivered Hg268 mg lig, x -13.5-5 Hg.. this point has no appreciable significance ,,1,9.8 pl vol s provided there is no losS of mercury from The expected weight for 20 cubic micro- the tip of the pipet.) liters of mercury is 270.8 mg. . (10) Expel the mercury from the pipet NOTE: For -routine purposes, 'it is ACept- into -a weighing bottle by ,manipulation of able tO..use pipets 'within ± 1 ..percent .-of - the syringe. (The weight of the weighing 20 microliters without making a correction. ; bottle: is precisely determined before star- Therefore, pipets which ..deliver 268,1to ing the calibration.) 27:3..5 mg of mercury would be acceptable. ,. ( I.1) Weigh the mercury: and the weigh- Discard unacceptable pipets... . e t ottle on an analytical balance. 2-7. Blood Cell Counting Chambers: c. Calculations: (1) Determine the weight of the pipet- a. The most common type of hemacytorn- eterconsistsof two .counting chambers te) mercury by subtraction:(weight of-separated by grooves or . canals. On the weighing bottle --K mercue?) (weight smooth- -glass surface of -thecounting,cham- of weighing bottle) -. AS an example -consider ber are straight lines etched into, glass in the weight of Dthe mercury .to be 0.268 a gridwork pattern. The Neubauer ruling, grams. preferred for hematological work, consists Convert grams toilligra of a gridwork with dimensions of 34= 1000nIg 0.268 .g Hg. x 2687mg by 3 mini% It is further divided into 9 smaller 1 g squares -with dithensions- of 1 mm by!l,rrtin-L (3) 'Select the temperature correction 4 of these squares are used for the white ' factor (that is, the weigh_ t of1 midioliter count. The 8 outer squares are further sUb- rcury in milligrams) from be10A` i-'-ur divided into 16 squares 0.25' mm on a side. Mercury Thecentral'. $quareisd-vided- into25 Temperature weight of 1 * squares, 0.20 mm.on. a sid-, which are used (degrees Centigrade) mg 20.0 13.55 forthered -cellcount. Thus the .large 21.0 13.55 squares are 1square inm, the 16 small AFM 160-51/TM 8-927-1 5 December

squares in the.outer -Urge sgiiares are: 1/16 can used '. to remove bloo in- the square mm and the 25 central squares are )re of pipets. Dilution pipets ire dry when 1/25 square mm (see figure ? ) a the bead at reely rn the bulb. GlassWare b: The coo ei glass must :be free of to used r coagulation studies must be defects and must be optically° plane on both scrul ulouslyclean, This glassware should sides .within 0.002 m# according to the bec nonorganic detergents and rinsed Well US- Bureau of Standards. Wheri'thecover itlr distilled water.: The etched surface- -of . ass is placed on the platform the sOce thehernacyto,meter. should- he , betwe9n it and the ruled platform :should rinsed in water and blotted dry with lens be 0.1 mm: paper to avoid marring or further etching of the lines on the surfaces.- A method of 2-8. Cleaning. Glassware can be cleaned in Cleaning- small here. tubes and pipets (such hot, -soapy ,Water and. thoroughly rinsed in as WititrObe sedimentation rate tubes) is to distilled water., Blood dilution -pipetScan attach a' capillary pipet, by a rubber hose, be% washed' by flushing water and acetone to a water' type suction. pump. Attach the through them. Ordinary..,household- bleach tube to the flat dal of the pipet and hold

mmilrimmomminsounmiimmEsimumminil eimmouraiimmiu=it 111111MENINIMMEMI simommEmimuiiMINKMMIONNIMINIAMM I MIMMEALMOMMENIMIll micNolommnmFmn.....1! Vensuimmimmimionairli OWELMIIIIMMIMMEIMIling =1111111111!!IIMMINEMELIONEIlmm

111111111 111111

111111111 Figure Rulings on a He _acytomete OF 160-31/TM -8- 7 74 5 December 1 973 the tribe under .water. Blood is drawn from ), All- siliconized glassware- should be the tulle aswater is drawn in. When the prep9.re6, washed, and stored in n.n area

tube isclean,- -invert the tube;remove the :- separate frOmregular glassware: residual water'by suaion, and alloW to -dry. t. '(1) Asthe.silicpne ages or a new hatch ,Occasionally,the tube should be cleaned isprepared, perform a clotting time on %ith dilute sodium hypochlorite (household no' mal whole blood to eh on each). to remove deposits of residual blood. talnlity" of thecoated re. Inaddl tion, water should have a flat or convex 2-9. Preparation of Silieonized Glassware:`meniscus in a.properly coated. tube. a. Coating,Nraterialg:. (I) Silicone(SC-87. 'Dri-Fil avail- SECTION CCOMPOUND MICROSCOPE. able from Ge eral Electric Corpora Sil- icone Prod sDivision, 'Waterford,- Nd.'w 10f Inti.oduction..A modern microscope for York)_ use.in the hematologylaboratory is equipped with .an illMminator system, a substage con- (2)Xylene. denser; system; an objective system, a pro- (3)10c',Silicone Add106- jector (eyepiece or ocular system), an iris.. . ml of lconcentratecl silicone .Solut 900 diaphragm, -nicol prisms, a tubular barrel, mlofxvleneinagean groan (monocular orbinocularbodies):,and a 6toppered bottle. ThiS solution can be used Mechanical stage (see figure A- corn- :lefinitely. pound microscope uses a combination of lenses,theobjective. lens (lens cloSer to the h. Procedure: object) and the ocular lens -(lens clOser to (1) Handle the 10:silicone' solution the eye) to project the image ,to -the retina with rubber glovesand work ina. hood of tho eye. The Objective lens acts Much equippediVitll an exhaust fan.. .like a sniall projection lens which projects (2)--Syringes,barrelS,andplungers an enlarged primary. image near the top are coated wAh10% silicone solution in a of the tubular bara This image, formed large beaker. in air, is known, as an "aerial image." This object is viewed.. through' the, ..Projector or fillonetest (3) To coat testtitbeS, eyepiece which acts -like a magnifier except tube with the .10% silicone solution. Rotate that itfria0ifies an- aerial object instead -the . tuba and transfer to : other test tubes. of an actual object. The final image -pro- ( =1) Pipets are coated by placing tip jected on -the tretina of the eye is called a of the pi-pet about 2 inches intothesolution. "virtual image" because the light rays ap- Draw the solution up- with asuction bulb pear to 'come fzorn the image. The rays an inch below themouthend. are actually crea.ted by an increase in mag- (5) Allow -the' glassware to drain. for nification by the lens system. 20- minutes after coating. Rinse well in tap water and then in three or four rinses of Z-11.Magnificatio

distilled water. Dry. in an oven at 120° C. OfMagnification in a microscope-- is liin- that). can c.Discussion: .itedto the useful manification he achieved, thatis, the ability to obtain (1).. Glassware can be desiliconized by finedetail of the object' being examined. soaking in saturated sodium hydrOxide for This ability to render visible the fine 'detail, 10 minutes. Rinse in distilled water and is the resolving' power.of the microscue, dry in an oven. The'resolving power of a microscope de- (2) Silicone glassware can be used sev- pendent on the numerical: aVerture (N.A.) eral times before recoating. of the objective lens ancondenser lens. 5.1/TM 8- -474 Decenther 1973

OCULAI ([YEP ESCES)_

ULAR FOCUSING TROL

CONTROL

IECTIVES

CONDENSER LOCK-

IRIS - DIAPHRAGM

STAGE#CONTROL SUB STAGE CONTROL- Figure Compound 'Microscope.

Therefore; " properadjustmentofthese of the ocular by'the power of the objective lens- is essential in order to obtain useful g.ves the degree of magnification of the rrm ation. eject under observation. The degree of magnificationisexpressedindiameterS b. Microscopes- in genetal uSe in Medical (refers to an increase in diameter-Y. ,The laboratories are provided ,with'ithile'e objec- ocular Magnification; the millimeter length' tives with focal lengths of 1:9 mm, 4 mm, of the objective,its magnification poWer, and 16 rdm, respectively. Microscopes are and the total apparent increase obtained usually providea. with 5X and 10X (most using oculars and objectives of the poWers common.)ocUlars.Multiplying the power shown are given (below:.. N Deceniker- 1973

Ocular Objective Magnification specimen. The.. size of the 'opening in the 10X rn (10X 100 diameters condenser- together with its position up-.or 10{ m (43X) 430 diameters down controls the light entering the- system. 10X 1.9 mm (97X) 970. diameters When the. is close to the. stage," Magnification increased -inpractice by concentration- oflightisgreater; as .:the using a jiigher powev objective_ Most .micro -; condenser. is moved ,downward-, lesslight scopes afeggnipped with a ,reyolving nose-nose -.up.u, ugh the. -object under pieee,, and selection Of adiobjectivelens is done observation. With ea-se e., Improper ,illuminationisindic ted when: (1) dark points or shadows appear 2-12. -Illumination:' in the field;(2) the outline of an ob. :ct Cori of .theobject is bright on one side and dark on the: tincler study is an extremely 'important- de- or (3). the object .appears tyr"be in a glare sail. -.Incorrect- lighting of the object can of jight; This can usually be tovrected by` -rate results and conclusions. changing the position of the mirror, by Correct illumination can be obtained frii1n1 reducing the amount 'of light by adjusting a concave mirror or substage light. the size of the opeling intheirisdia- phragm, or by raising or lowering the con- b. Illumination entering the microscope denser., can be central er. oblique. l'o.obtain central illuminati'oii the light from the source must _2-13. Focusing,. Focusing cap be defined as be ,reflected from the mirror directly into- the adjustment of the relationship between ,the microscope tube. Obligne . lighting mint the optical system and the object so that' be .avoided because an object in-the,eente-r ear image is obtained.-- Sevefal jrniportant ,of the fieldwill sway from to. Sid& les, to be observedhen loCusing-the'mi- rvherrthettple. adjustment is rotated, e_ croscope on the p'ifeParation light is rusnally; ne-2Problem with .;,:Sitbstage alluminatorSi . la. After the objectis mounted on the stage, the objective to he used is turned c. Regulattoll of the amount of light into line 'with the eyepiece. -Mitted by-- theirisdia... . pliragm in'the substage condenser. The size h. Movement of the objective is acco of-the opening in the diaphragm is -controlled- plished :by revolving thenosepiece. The '. ? i by anever on the ,side of the condenser. nose-pi,,._.eceisprovIded ill order to enable The lever of the iris diaphragm should never rapido, :convenient substitution. Of one ob- he forced to the full limit either direc- jective7Qr another. This change is effected_ tion.Doing may damage the delicate by gi;asping:two oftilipele objectives betw,een7!:. .leaves .Qf the .:diaphragm. Generally, when t hethii filly, a.nkforefin:--r , of the trig-III liancl." ob:S`ervingii liquid, ,.preparationsunder-lOw and 'i.otael-rig theintintil vie, -41esir0 'objec7. 'power, the diaphragm, opening, should 'be We is brought irifo' .line --with thethe axis of --_ ------,--.'-.-- --,': ,-. partially 'dosed. Under the -high- dayeh- , the. body tube.It- is, vet'-y -ttri-portant that jectiNe,,the diaphragm is generally ,oPened..exact --alinemetit be obtained.. The correct to a greater degree to alioW more '.light setting is indicated ----bya-- -slight --,"click" as toPass:, through- the material. When 01)- the objective comes intb14-0-Ton. serving stained preparations under the oil immersion 'Objective, the iris diaphragm is c. Whenever the nosepie isravolved4' its movements should he o_braved to make usually opened-wide. certain that the objectives-- o not come into d.:- The.. substage condenser-. functions to contact with the -object.. Some microscopes direct a light beam of the desired numeri- are not parOcal; that is, objects -in focus cal aperture (N.A.) anctfield size onto the under low 'power will- not, be in focus when , 160751/T_ e- ber 1973 1 the nosepiece is. rotated to. a higher power' a. -,No un tztrized ,person nia-,_ of magnification. It may, tl-ie-reforer, be neces =-'Apulatethe nncroscope. sary to refocus whey changing. to higher magnification. In microscopes which are par- h. Keep the microscope as free from diet focal, it is t ossible to swing other objectives. and- -dust as possible) Dusty lenses produce into place without touching the coarse .a- foggy images, while dust in the focusing justrnent and with only a slight turn of mechanisms causes excessive wear' of those the fine adjustment knob required to .re- parts. store perfect focusing. c. -The Microscopeshowfi alWaysbe cov- ered when` not in use. d. -To bring an object into focus,- watch from the side and use the coarse a.diust- d Care -should.--be taken to prevent all ment to lower the objective until it is below parts of the' microscope from corning into the point at which. the object would nor- contact with acid, alkali, chloroform: alco- mallyheexpected?ocome -intoview. hol, or other substances 'hick corrode Metal NPTE: To avoid damage to slide or micro- ordissolvethecementing, substance by scope, view from. side for focus- means of which the lenseS are secured' into ing. Then, Using,7 the coarse adjustment and, the objectives and ocular's. . at the same time looking through the ocular, e. Always *carry the microscope wish two . - raige the Objective very slowly until the hands by the arm and base. field comes into view. Further, adjust to the best image, rising only the fine adjust f. Avoid sudden jars, such as plading the ment. microscope on the table with undue force. e. In fociising upward With the fine ad- g. No dust should be permitted to settle justment, the object willfirst appear on the lenses nor, 'should the finger come in _contact with any of the surfaces. faultoutline,thengradually moredis- 1inetly.._and finally, sharply defined. If the h. Tile lens system should never be sep- adjustnt goes beyond the point of sharp arated, as the lenses are liable to become definition, return to the point of greatest clecentered and dust can enter. clarity by using the fine adjustment. i. Avoid all violent contact of

f. Never move an objective downward - :bye lens aud the cover glass. while looking through the eyepiece:. Wheo j. Keep -yepieces in the :c ogeope' at the objective is moved downward; -alWaYs, all tiiries to keep free of dust. observe the downward motion with the eye: held leVel. with thei.microscopetag-e. Failure k. -To remove dusti.brtish the len's g with ,to observe these precautions can result in camel's hair brush:` Avoid hard wiping, damage to the lens of the objective or the as dust is often hard and abrasive. object under study. 1. Xylol is the only agent which should he used -in cleaning lenes or removing 'OH 2-14. Cote' of the Microscope. The micro- from objectives.Only small amounts of . scope isan instrument of .precision with xylol are ,necessary and should he used with many delicate parts, and it must he-handled care. with, the utmost care. Care should not be confined to the optiCal elerunts gene. The When sewinmachine oilis used to microscopOs a combination of optip1 and lubeicate moving parts of the microscope, mechanical'`' excellence, one complementing- all excess should he wiped o'ff.VO prevent the collection of grit and dust the-other. The following precautions should ,-, .always he observed in the care of the micro -n. The microscoPe 'should be protected pscope: against direct sunlight and moisture. FIVI 16O 51/TM 8-227-4 5 . D embjr 1973

o. In very warm, humid .climates, miard- Phase. 131- microscopy becornpiZ scopeS sh4ld be stored in dry- cabinets increasingly prevalent in platelet counting. when' not in user Such 6abinets should- be,.In bright -yieldllurnnirnation, a complethly reasonably airtight, equipped with' ''a light transparent :specimen is difficult td'" see in bulb to supply heat, and several' cloth bags any detail. By using phase eontrag, trans- containing .a hygroscopic salt such as cal- .paent living objects can be*studied. Phase cium chloride to absorb moisture. In warm, Microscopy operates on the principle that htimid -climates, the lenses iff unprotected a portion .43f light is treated differently microscopes canheattackedo. by certain from the4rest, and caused to interfere with",

'fungi 'Which et-eh glass and ruin the .lenses:, . the rest, it produces a visible image of an After use, al#ays turn the nosepiece otherwiSe invisible transparent specimen; o a position which brings the low power Phase contrast accessoriesare-rrvrtilable objective into direct line with the- opening from the standard optical Companies in the substage condenser. If- .this-precau- , don is not take-n,c-the. longer. higher :powered SECTION ,DCENTRI,Fij.GE.S ebjectiVes can accidentally come -into -eon- tact with the condenser lens. a..72-16. Types: a. Table4:op' Models. These unitsare q. ,,The entiremicroscopeshould be mounted on rubber feet which absorb vi- cleaned frequently to remove, dust, finger bration. The speed 1S controlled by means of marks, off, ,grease, and remnants of speci- a rheostat on the -frent panel, Top speeds wrens. All parts .the microscope should . of cents ifuggs will- vary and the top -speed 4pe kept scrupulously clean;at all times. 6f. a particular instrument should be- known r. Never tper with any of ,the partS in -order to- use the speed control device. ofthe micr_cope. the instrument does .Those -centri-ifuges have adapters to hold not seem to12e -functioning properly, im- tubes-and-adapters for 12 tubes. mediately call the matter to the . attention -,..._. b. FloOr-MountedModels.The heavier of the laboratory officer. -. - floor-mounted models accommodate a large s. Maintenance of the micrbscope should -number of.tubes one time. The top be done in accordance with the manufac- speed of these instruments is higher'' turer's booklet of instruction. that of table models. Because of tyir in creased inertia, they, are equipped 'Nvith t. Immediately .'after use, the oil jminer- brake to facilitate stopping. In. these units, sion objective must be wipd clean of 'oil the tubes are placed in balanced receptacles with a soft, absorbentlens pa are- Mounted on spokes' ernanating, from a central hub.. Types: c. Microhematocrit Centrillrge. This renz__ a. Binocular. This type of microscope is ifugeis. a specialtype. of ,,highspeed preferred since it gives a more natural and centrifuge. employed to spin capillary tubes restful condition of ob/rvatipn. A beam- . 'see figure 2-5). Thecireular tube holder dividing prism and three inirrorg-livid on this centrifugeisflatc,..' .S'urrounded by the light equally, sending half to the left a rubber ring. It has a.idapacity of 24 cap- eye and half ft) the right eye. The coating illary tubes. After a catiillary tube is filled on the mirrors '4S- enhanced by aluminum with blood, itis closedwitha- commercial to increase the reflectiyity. The binocular. plastic sealing materiafy Duririg:-centi.ifuga, body is protected by coverglass seals to keep tionthe Sealed endf is,' always placedin dust from entering. The binoctilar, corn- position -facing tows rd the outsig# of the pound microscope is the preferredYinicro- holder plate. Mosf:,4ntrifuges of this type scope for routine hematology. spin the tubes at 1(060 rpm. Figure 2-5. Alierohematoerit Centrifuge and ohernatocrit Reader.

2-17. Preeatctions. Inallinstances where, isite each other, they must be tical ceptrifugation is required, careful attention. -ight,'. and the must cm the same miff be given to balancing -the-Jfnits. This amounrof fluid. If at all possi centrifuges means that tubes must be pittded exaetlk should Q- equipped with tachoe ei.s so that Ant 160-51/TM 8-227-4 5' December 1973 'Speed may-be checked and controlled. Certain draw_ s the sample, the i3ther syringe meas- procedures,. such as Tiematoerits, require- a ures the diluent. Both syringes can _be set to critical relative centrifugal force (RCF or G). draw-a metered amount repeatedly within an To determinethe RCF (or, G) for thesepro- alloivable range of error. A. daily calibra- -tedures, consult the = serology .manual or a- tionch.cck of this - instrument isrecom- .nomograph. The inside of 'the, centrifuges mended-to insure accuracy. should be -occasionally, cleaned to prevent dust particles from beingblown into speci- 2-20. Slide Stainer. The automatic slide mens. The lid on the: centrifuge should be stainer is a coordinated three-phase opera- closed and locked liefore and durinir operation.- tion timt fixes, stains, buffers, rinses, and °Thy 'open, the lid ,when' .centrifuge.. has -dries differential slides at a rate of approxi- stopped rotating. mately. one perminute. Stain packs are available with this instrument, but satis- -.--SECTION MAT_ E factory Wright stain and buffer can-be-pre-- - EQUIPMENT pared in the laboratory. The slide stainer is illustrated in figure 2-7. 2-18- Introduction. In recent yearsstru- . mentation. has been developed to automate 2-21 Counting Devices: Many hematological analyses The use of automated equipment in the laboratory has a. Introduction. Two automated methods increased the number. of .analyses performed are available for counting- -blood cells. The by decreasing the man-hours spent on the 'optical system is based upon the production analyses., These instruments perform various Of light impulses. In this system, the cells function's such as: dilutiops, staining, cell are dilnted and drawn through the counting counts,,hemoglobi,, coagula- zone lay tpiisitive displacement metering- tion studies, and indices. The type of instru, pump. As cells pass through the counting rent used by`a particular laboratory de- area, they prodnce photoelectronie. impulses

pends on the size Of the -daily .workload, Which can be counted. Another method of the complexity of the method used, and the automating bloodcellcounts utilizes the technical ability of the technician- Perform- principle of resistance in an -electrical field. ing the analysis: If the number of hema- Since blood cells are poor electrical con- tology requests received is 30-40 .a day, ductors, they act as resistors to current flow. would be advantageous to have automatic As more cells pass into the electrical field diluters, pipetters, and electronic gel] count- they offer corresPondingly more resistance. ers for blood cell counts and hemoglobin The change in the current flow caused by the determinations. When the number of routine change in resistance is sensed and counted requests approaches 100-150 per day, an by a digital.: counting apparatus. The digital automatic hematology system should be con- counter is designed to produce numbers in a sidered. range- which approximates the number of blood cells.that cause the resistance. 2-19. Dilutor& Automatic dilutors assist in the processing of large numbers of sped-- b. Coulter Counter (Model FN): mens by reducing technician time per test. (1) The Coulter Counter (see figure 2- Repetitive dilutions can be accomplished 8) operates on the principle _of resistance. rapidly with an instrument similar to the A suspension of blood cells in an electrolyte one illustrated in figure 2-6. The concept of (that is, saline) is drawn through an aper- an automatic dilutor involves the sampling ture with electrodes on each, side to form frOm a larger volume and the dilution of the an electrical circuit. As theblood cells pass sample. The automatic dilutor is essentially through the aperture, the mass of the cell two syringes that can be operated in a sys- changes the resistance between the elec- tematic manner: One syringe measures and trodes. The change of resistance alters the rtst-ien Di11.11.-1 kal/111

Figure 2-6. Automatic Dilutor. rent flow and causes electronic pillses in the sample being counted. By arrariging as a 'resultof the variation in the field. The the pulses, the sampling volume, and the changes are amplified, inspected, and counted mpling time, the Coulter. Counter reports electronically. The end product is .a number directly the number of cells per_ cubic -milli- which 'represents the number of blood cells meter. A schematic of the 6peration of the Figute 2-7. Automatic Slide Stainer. '- Coulter Counter is illustrated in figure quickly into, the apex When the valve isopened, thb mercury eter needs cleaning fall-s, thus creating a, vacItuni in the sampler. (b) ROcord a baCkground count with This change in pressure causes cells in the diluent and diluent -plus saponin at the \VBC. sample to be sucked through the aperture threshold setting. . and into the sampler, The removal of cells (c) Retord thecontrol suspension from the sample causes a change''in electrical counted at the RBC and W.Bc- setting. conductivity between the two electrodes. (d) Flush orifice with dilute scidium This change is amplified and displayed on hypdchibritp(bleach),Flush the system the scope.. It is further ,amplified and re- thoroughly s.with distilled water and then gistered on the digital counter. The higher saline. the count,. the greater the probability :that In addition, to the daily mainte- more than one cell will enter the aperture nance, the following maintenance should be at one tine` (coincidence passage), For. this performed once a week: reasonWB.Cs over 10,000 and 'all 'RBCs are (A) Oil the vacuum pump. corrected for coincidence passage. (b) Clean the Orifice; be very (2) Adequate maintenance of the Coul- with the orifice insert. ter Counter will reduce incidence of instru- (c) Check thregholdr.gero.g ment failure. The following Should be done on .a daily basi (4) ChOck the threshold plat (a) Observe the mercury traveltime month., in the manometer, If the mercury column (5) .Once every .6 months perform the does not .move, moves erratically, or flows following maintenance: ite..S1193..... j sessityrry

COULTER COUNTER,.

i &Ic, I- THRESHOLD

CIRCUIT

SAM?LER PULSE BRIGHTENING SIGNAL --- ABOVE THRESHOLD

MERCURY

SAMPLE APERTURE Figure 2-9. Schematic Diagiam the Cou a) Change the latex tubing. (2) False impulses resulting from elec- (b) Clean the manometer. tronic problems such as -interference from (c) Cheek the calibration factpr other machines can cause erroneous results. the instruinent. p) Contaminating particles in appar- c. FisherAutocytometer.Thisinstru- ently clear diluent are another source of ment is a photoelectronic counting device mpulse. Background counts must be made which uses an optical sensing system and an daily on all diluents. electronic counting system. A schematic pre- (4) White blood cellcounts are not sentation of this arrangement appears in accurate over 10,000 per cu min and must figure 2-10, The bloodcells, are diluted;`'fie diluted with saline. syringe fed into the counting area, and in- spected by a reversed dark field microscope '(5) High platelet counts or nucleated arrangement. When no particles are present erythrocytes can cause erroneously elevated in the .sample, the narrow light beam from counts. A correction must the lamp passes straight through to a dark- be made if these cells are observed on the fielddisc, which blocks further passage. . However, any suspended particles, suchas SQUARE APERTURE DEFINES PHOTomuLTIPLIER -"blood cells, will interrupt the lightbeam. SIZE OF LIGHT REAMS TUBE MICROSCOPE Scattering of the light beam due to the IA- LENSES mass of each blood cell causes a light flash to pass around the dark-field disc. The light LAMP 111 flash is focused into a detection system and CONDENSING COLLECTING FLOW LENSES converted into an electrical pulse thatcan CELL LENSES be coun ?ed. DARK FIELD DISC TO ELECTRON C d. Problems of Electronic Counter COUNTING CIRCUIT (1) Dilution inaccuracies often resul Figure 2-10. Schematic Diagram of a Photo- rroneous results.' electric Particle Counter. 051/TM- 8-227-1 5 DeceMber 1973 = 2=1.7. / (6) Standardizationisrequired with perform routine coagulation tests. This in- hernacytoineter results. strument consists of a precision coagulation (7) Bubbles must-be avoided since they- timer, a 37.2' .1:0.7°C incubati3r block, and will be counted as cells_ an automatic pipet. The automatic pipet, (8) The ',orifice of the counter can be- attached to the Fibrometer by an electric- come plugged.' All equipment. used With the cord,dispensesreagents or plasma and Counter should be cleaned. automatically activates the Fibrometer. A plastic cup beneath the probe on the Fil3ro_ (9) The equipment has a high -initial meter is charged withreagents. When the cost ;meticulous ,,.careis required for the plasma is delivered with the switch on the proper maintenance of the counter. pipet in the "ON" position, the timer is started and the electrodes drop into the 2-22. IL liemoglobinometer:, mixture. The first fibrin formation on the a.-This-instrument-utilizes-a direct digital electrodes cloSes the circuit and stops-the vresentation for a rapid readout of hemo-..timer. The elapsed time in.seconds, is read iglobirt concentration. It contains a high- directly frpm the digital readout register precisio( interference filter witha. wave - on the timing device. This instrument lengthof548:5nm.Inprinciple,this manufactured by the Baltimore Biological instrument measures the three naturally Laboratory. occurring hemoglobin pigments (oxyhemo- globin, reduced hemoglobin, and carboxy- 2-24. Re tologicgl Systems. Complete he- hemoglobin). This instrument is not suitable rnatological systems have been developed for rapid Measurement of cyanmethemo- thatperformsevenhematology param- obin and hence cannot be effectively used eters- red blood cells count, white blood cell for certain artificial standar& or survey count, hemoglobin concentration, hemato- ampler which contain variable quantities crit, , mean cor- of methemoglobin. puscular hemoglobin, and mean= corpuscular hemoglobinconcentration.Theseinstru- b. Operation of the, machine is simple. ments automatically dilute and analyze the Prior to actually testing a specimen, the specimen, and tabulate the results o_ n data machine must be-Set using a baseline Saline forms. solution. ThiSis done by running saline .through the machine and adjusting to zero The Technicon SMA 7A. This system with the zero control, Next, the machine is a sequential multiple autoanalyzer' con- calibrated using an amaranth dye solution: sisting of: a sampler containing 40 cups, The machine is adjusted to 15 grams per a proportionating pump which precisely pro- dl with this calibration solution. The ma- pels reagents and samples, a manifold con- chineis now ready to accept the actual sisting of constant diameter plastic tubing specimen. AD internal pump draws the speci- and mixing coils, an optical-electronic cell men up through the long slender sampler on counter, an electrical conductance ,flow cell the front of the machine. An aliquot of a to determine hematocrit, a colorimeter for hernolyzing solution is then pumped from hemoglobin determination,= and recorder to the reservoir. The specimen is mixed, hemo- measure the data. The sample- is split into lyzed, and passed through a filtered cuvet four streams to perform (1). BBC count, forreading.The readinggoestoan (2) WBC count, (3) hernatocrit, and (4) analog computer which converts it into a hemoglobin. The instrument performs four numerical reading that is displayed on the separate determinations at a rate of 60 digital readout register. samples per hour or 240 tests per hour. Fibrometer. The Fibrometer (see figure b. MOdel S CoulterCounter. This instru- 2-11) is an automatic instrument used to ment (see figure 2-12) Utilizes automatic 160-51/TM S-227-4.

"..1.11111

Figure 2-11. Fibrometer. dilution analysis to perform-the seven l enia- identification card is submitted to': An auto- tologyparameters. The RB,C and WBC matic printer. The instrument is activated, counts are performed in triplicate. If there it aspirates a sample, it perforrns the neces- is any -disagreement due to debris or other sary dilutions, and the results are printed error, the .technician will be alerted by an automatically on thepatient'sidentifica- alarm system. The patient sample is intro- tion card. The Coulter Model S analyzes duced to the sample aspirator and the patient and. records the data quickly and accurately.

2-25.Maintenance of Automated lima-to- logical Equipment Operation of all laboratory equipment according to the manufacturers' instructions is imperative for valid _results. In addition, periodic maintenance and clean- ing following manufacturers' and internal op- erating procedures must be performed to keep the equipment in operating condition. All op- eration and maintenance must be performed by competent personnel. ek.

r 1 'I BASICHEMATOLOICAL;PROcEDURES

SECTION ACOLLECTION OF BLOOD 3-2.. Venipuncture: SPECIMENS a. Site To obtain blood by venipuncture,- 3-1.. In trod uction : draw the specimen directly fyom a patient's vein with -a sterile hypodermic. needle and a. Hematological laboratoryprocedures 437i11-0 or a _vac44411?144d i41P0e:AeYice ire based, upon the examination of blood In adults use the veins located in the -prox specimens. To obtain validtestresults, irnal forearm or antecubital space as illus- specimens must be properly collected, proc- trated in figure In infants employ the essed, andt recorded. Blood specimens are jugular of femoral vein for the venipunc- usually obtained by either venous or capjl- ture. The vein selected should be large, lary puncture. The source of the specimen readily accessible, and sufficiently close' to is determined chiefly by the quantity of the surface to b'e seen and palpated. If Beni blood required to perform the laboratory puncture Poses a problem due to the age of procedures and the age and condition of the patient sclerotizaticin due to repeated the patient. venipuncture, or any other unusual circum b.' There, ig' generally little difference in stance,the technicianshouldconsulta blood counts performed on venous or capil- physician concerning the procedure. UNDER lary blood if a free-flowing capillary blood NO CIRCUMSTANCES SHOULD A TgcH- specimen is obtained. Valid blood counts NICIAN WITHDRAW BLOOD FROM. A cannot be made when capillary specimens SAGITTAL SINUS, JUGULAR VEIN, OR are not taken from a free-Bowing sample FEMORAL VEIN. This should be left to or when they, are obtained from cyanotic or calloused areas or areas of local stasis. White blood cell counmade on blood obtained CEPHALI C from such sourc- can vary as much as VEIN. 1000-150b cells pe mm from their real value. For. general purposes, however, ve- nous samples are preferable since they allow for multiple and repeated hematological ex- aminations and provide a sufficient quantity of blood for performing any other required laboratory procedure. Further, with venous blood the chances of error are reduced be- mErilAtiANTE-. cause operations are made under ideal condi- BRACHIAL VEIN tions and repeat operations are possible. In situations where there are limitationson the quantity of blood which can be obtained, that is, in small infants, or extensive burn cases, mieroquantitative methods are satis: factory for performing an analysis ona specimen obtained by capillary puncture. Figure 3-1. Site of Venipunctu e.. the discretion of the physician in charges leave the vial covering over the needle when !Occasionally, the best vein is. -found not in use. hand,- leg,; car foot. These areas are more (4) Inspect the needle bevel and point iensitive, 'and 'the veins are not as firmly against a-light background. The bevel should, nchored as those 'of,- the 'arm. -Veins can be smooth and -shiny and the' point sharply me distended and.easier: to enter by al- tapered. Discard damaged needles. owing the arm to hang down for 2 or (5) Check` to be sure that the skringe minutes, by Massaging the.blood vessel toward the body, or by gently slapPing the wiIrks smoothly and that the needle -is -}riot plugged or burred. The syringe, and needle site of puncture:Young and vigdrous persons must he dry to avoid hetnotsis of the red usually have elastic veins well filled with , blood. Elderly or debilitated pergons' ,can cells. The ,plungerrnust-q-na ch the syri have -,selerdled or fragile_ veins, .which. are and must be. pushed firmly to the bottom of hard to enter-or which collanse the ckiiiiciei to pre-Vent' inieCtion ofair=into the vein. b. Equipment. All Syringes, needles; cets,.and other instruments used for the c 1 - Syri-nge -Procedure: lection of bloOd specimens iritist- be sterile. (1.) Place a tourniquet -around the pa- Disposable syringes or blood collection sets,tient's arrnaboVe the elbow tie tly 4nough with vacuum tules.-_ are available through to check venous circulatiqn,but noso normal supply channels. These should be tightly as to stop arterial now. CAUTION': used whene4r possibleASentic technique Do not allow thetourniquet to remain in place is necessary to prevent the.possible trans- for more thim g minutes. Check the pulse missi'dir of homologous serum hepatitis: The at the. wrist to make sure that arterial cir- following equipment is necessary to perform culation is not cut off. a vehipuncture: (2) Instruct the -patient to open, and (1) Isopropyl alcohol, 70%. Dilute 726 clo§e the fist several times to increase -cir- ml of 95% isopropyl' alcohol to 1 liter with culation. distilled water. (3) By inspection and palpation locate (2) -Tourniquet. the desired vein, deterinine the direction of its course, and estimate its size and depth 3) Alcohol sponges. (see figure 3-2a). (4) Sterile syringes or vacuum blood (4) Cleanse the skin over the selected sample devices. vein With gauze soaked in 70 percent iso- Gauze pads, 2.x 2 inches. propyl alcohol: Wipe off excess alcOhol with a sterile dry' gauze. Do not contaminate the Needle, 1 to 1 1/2 inches long, area after cleaning (see figure 3-2b). 23 gauge. (5) Have the patient "make a fist" and* (7) Suitable blood collection tribes and straighten the arm. Frequently it is helpful labels. to have the patient place the ,clenched fist of c. Preparation: the other hand under-the elbow to straighten the arm. (1)' Cleansehandsthorovghlywith soap and water. (6) Grasp the syringe intheright hand and place forefinger on the hub of the (2) Place' an identifying label on the needle to guide it. Grasp the forearm with blood collect- ing tube. the left hand about 2 inches below the area (3) Assemble thesterileneedleand to be punctured and hold the skin taut with syringe. If a vacuum system -is used, screw the htimb (see figure 3-2c). the needle into the plastic: holder. Always- 7) With, the needle bevel Up, parallel 1227-4 5 &ember, 1973

he vein

d. Insert _needle into the, veih.

Figure 3-2.Venipuricttir6 Pocedure. to, and alongside the vein, insert the' needle 8) Aspiration of the blood is accom- quickly' under the skin and then. the plished by gently pulling upon, the syr Fe vein. The insertion into the skin and vern- plunger (see figure 3-20. The Syringear- can he performed M one complete Motion I should be held steady duringrthis-proc- '(see figure .32d). After entry int.o the veipt ess.Withdraw the desirgd quantity.. blood. will appear in` the needle hUb. Do not probe or move the needle, horizontaily, as (9) Reinke the tairniqUet (see figure discomfort and possible nerve damage may 3-2f). This must be done prior to with-

esult. drawing the needle from the vein. , e. Aspirate -e-mov.e Urn iotiet

h Have the patient extend the the arm and maintain light pressure on the sit-- Figure 3-2.Continued.

(10) Place a sterile gauze -pad over the .and maintain light pressure on the gauze .poine where the needle:enters the skin and .pad over venipuncture site (see figure 3-2h). deftly withdraw the needle- simultaneously e. _Vacutainer Prodedure: putting pressure on the siteAsee figure 3-2g). (1) Place the Vacutainer tube in the (11) Have the patient extend' the arm holder Until the rubber stoker reaches the:.. kFNI 160-5 6) When repeated venipunctures have to be performed -.on one patient itis arl as- -abletoselectdifferentsitesfor blood witli-drawah Figure 1-3. Vicutainer System. (7) .Reinove the tourniquet as early as possible once a food flow.pf -blood- has been

guideline. The short _needle should tre' ern- established.. Prrilonged application of the` bedded in the stopPei: but,the neegle _Must tourniquet results in Partial stasis of blood not break-the,variuum (see figure and changes many quantitative values of (2)4ollowsteps. 1-6inparagraph ,blood compOnents. ).:Blood drawn by veniPuncture inter the vein with the needle par,_often.,stored fora period of time before it is allelto and alringside the veinProbing- analysed Foi thiseasun c- general horliental movement of the needle while-;,precautions. must he.folloyed .in order to in- under the skin must be avoided. Release _the . . sure a valid 'analysi-s-. -Before, withdrawing : = tourniquet. blood from its contain, make sure _pie (4).- :after entry into the vein prfih the bloodsampleisthoroughlybutgently tube all :the-way into the, hold0,;, vacuum is mixed. Blood containers should be tightly': broken,' and blood ilOws freelY'1ritri th'e' tube. stoppered at all times to prevent drying or 'f5) Other tubes can be inserted in the contamination. Store the blood specimen_ in ,saina manner if needed. the refrigerator. Blood counts should be (6) Place a sterile, gauze pad .river the done within 3 hours after colleption. Under point where the' needle enters the `Skin and no circumstances should bloodtalc for deftly withdraw the needle, placing pres- hematological examinations be s od over- sure on the site. night. (7) Have the -patient exteria the arm and maintain light pressure on the gauze 3-3. Capillry. Puncture: pad over-The venipuncture a. Site. Several different siteare suitable for' capillary puncture. Becaus4 it is the most f.. DisstSsion'. accessible, the palrriar or lateral surfaqe of (1) deanliness is ess the tip of the finger .(preferably ring finger) forming a venOuncture: the most common site in adults. However, (2)Itt is -most. important that crec C'eitailii =problems can be encountered such technique be practiced inirder. to avoid un-. heavy calloused areas or exeris .sive tissue necessary pain to the patient, preyent tissue fluids (edema) that,tend to result in non- darriage, secure a good representative blood. representative samples.. The'lobe of the- ear specimen, and prevent contamination rif, the can be used for capill-ArY plincture. However, spedMen or infection of the patient. differences incell concentration ',do...occur Syringes.aneneedles must be thor- when blood.isobtainedfrointhiS'` site, oughly inspeaed for damage or malfunction. primarily becauseof higher lymphocyte (4).If difficulty is experiAnc,ed in en- 'concentrations in the ear lobe. Because of terIng` the vein or ,a hernatdrica begin's to the sill -amount of tissue on the fingers rrn,: releas'e th0 tourriiquet and prornptly of infants, the preferred site is the heel or withdraW the .needle and apply pressure to big toe. A modification of "the normal tech- the wound. nique _that. has -proven quit4 satisfactory (5) Vigorous pulling on the plunger of when working with.the heel of infants is -to the syringe' can collapse the vein, produce make two incisions in a criss-cross fashion of the blood specimen, or. -cause. Or "T." -air to enter the syringe. NOTE: To be a valid report, 'AFiVIE 160-51/T31 8-227-4 5 December 1973

capillary" blood. Must_, be :frOm a ,FREE- Procedfire: FLOWING puncture wound; (1) The puncture site should be warm -t- to assure good cirmilation of blood. If it is ii-3M-ent- `old,- apply warm water (38°-40° C)fer a .GAuze pads_ 2 inches. _ few minutes. If blow is to he drawn Blood Ian the eak,"-'-the edge of the lobe, not the fiat _Glass slides,,heparinizecl- capillary sides, Should be ptinctured. tubes, and other devices to receive the (2) The Site': fo be punctured.isfirst specimen. rubbed with al&avol soaked' gauze to remve (4) Isopropyl alcohol, 70' percent: Di- dirt and epi,thliaV debris, increase circtila- lute 726 ml of 95 percent isopropyl_ alCohol to tiori, and tinder the areat_reasonably diSin- 1 litdpwith distilled water. ed (figure 3=1a).

p pre-ssure =.- Figure 3-4. Capillary Puncture Procedure. AFM 60-51/TM -227-4 5 December 1973 3-7 (3) Allow sufficient time for the c the lateral rather than the ventral aspect of culation to equalize. the finger. (4) While making a finger puncture, apply .gentle pressure to the finger to hold 3-4. Anticoaglants: . the skin taut Hold the finger in one hand and the lancet in the other. The puncture is a. Anticoagulants are used to prevent the made perpendicular tothelinesof the coagulation of the blood speCimens and the fingerprints, which results in a more free- reagent empleyed should not bring about alteration of blood components. Unfortu, flowing wound (see figure 3-4b). nately, many anticoagulants can alter cell. (5) The first drop of blood that ap- structures as well as coagulation. The anti- pears is wiped away before specimens are coagulants most often used are ethylene taken (see figure 3-4c). diamine-tetra-acetate (EDTA), ammonium- (6) The blood must not be squeezed out :potassium oxalate (Heller and Paul double since this dilutes .it with fluid from the tis- .oxalate), -and heparin. sues, thus altering the ratio of cellular ele- ments tofluid,as well asthe ratio of b. The 'choice of anticoagulant will de- cellular elements to each other. pend on the analysis to be made. Ethylene- (7) After the desired specimens have diamine-tetra-acetate (EDTA) is the anti- beencollected,have the patient holda coagulant of choice for most hematological sterile dry gauze pad over the wound until analyses.Thisanticoagulantcausesa bleeding stops (see figure 3-4d). minimum of distortiontothecellsand platelets.It does not dissolve quicklyin d. Discussion blood, however, so the tube must be inverted (1) A disposable lancet is a very satis- four or five times when blood is added. The factory instrument for puncture of the skin. dipotassium salt is prepared as a 1 percent However, if this is not available a sharp solution in distilled water, and a final con- pointed surgical blade is quite suitable. centration of 0.5 ml of anticoagulant for each 5 ml blood is used. Another Common (2) Do not use the finger on a hand anticoagulant is ammonium-potassium oxa- which has been hanging over the side of the late( Heller and Paul double oxalate). This bed as it is likely- to be congested. Edema- combination of oxalates does not shrink or tous or cyanotic areas should not be used. enlarge the red blood cells appreciably. It is (3) The finger should be thoroughly essential, however, to add an optimal volume dry prior to puncture ;blood will not well of blood to the oxalate, no less than 3.5 nor up on a finger that is moist. Furthermore, more than 6.0 MI. the alcohol or other antiseptic used can coag- ulate the blood proteins causing cell clump- c. Heparin does not alter the size of cellu- ing and erroneous values as well as dilute lar components. It is, in fact, the standard for cell volumes, This will result in incorrect comparisonofanticoagulantdistortion. counts and differentials. Heparin is more expensive and dissolves less (4) Finger punctures should be made readily than double oxalate salts. Approxi- along the lateral aspect of the fingertip mately 0.5 to 1.0 mg is required to anti- More nerve 'endings are locatedonthe coagulate 5 nil of blood for 72 hours. The fingerprint area of the fingers, therefore, quantity of anticoagulant noted above in more pain results from punctures in this each case is sufficient to prevent clotting of area. Scars can also form in these sensitive the blood specimen. On the other hand, an areas, and difficulty may be encountered in excess of anticoagulant should he avoided puncturing a callous. All of these difficulties because too much will result in distortion of are eliminated by drawing the blood from cellsandhemolysis.Ideally,differential AFM 160 -31 5 Deco-fiber.: 1973 blood smears should not be prepared from ntiatescells:Wright'sstain. powder blood that contains an anticoagulant. composed of eosin and methylene blue- that d. If oxalate is added to vials and dried stain selectiVely. The methylene blue is in an oven, take great care to avoid tempera- posed of several staining. components. The tures above 80° C. Oxalates are converted eosin stains cell ytoplasm, and the methyl- to carbonates by prolonged exposure to ele- ene blue stains. nuclear material, grannies, vated temperatures. Under normal circum- and inclusions. Both stains oxidize rapidly _ becausethey inalkalinesolution. stances,itshouldnotbenecessaryto are prepare your own oxalate solutions since Giemsa,apurifiedpolychrome stain,is prepared anticoagulant vacuum tubes are added to compensate forthisdefect by availablefromFederalmedicalsupply maintaining the azurophilic staining prop:- sources. erty of the mixture. e. A correctly anticoagulated blood sam- 3-6. Slide Method:' ple is essential to the proper performance of a blood cell count. The cellular constituents a..Priticiple.- A small drop of bloodis must remain free in the plasma and should placed near one end of a clean, glass Slide. be as similar as is possible to those remain- Using a second slide as the spreader, the- ing in the patient's circulation. blood is streaked into a thin film and -allowed to dry. Itis then fixed and stained with -- modified Wright's,stain. SECTION BPREPARATION AND STAINING OF BLOOD SMEARS b. Equipment:

. (1) Venipunctttre or finger puncture 3-5. Introduction: aterial. a. Invaluable information concerning a (2) Clean glass slides. patient's condition can be gained from the c. Reagents: examination of a blood smear. Slides and coverglasses must be perfectly ,clean and (1) Wright's: Stain Buffer: free of grease, oil_ or dust to prepare (a) Solution A: Dissolve 9.47 grams good blood smears. It is best to use new disoclimn phosphate (Na-,HP0,)(dibasic), slides, but sometimes it is even necessary to anhydrous, in a 1-liter volumetric flask con- clean new slides in 95 percent alcohol. There taining about 750 ml of distilled water. Di- are two basic methods for the preparation lute to the mark with distilled water. of blood smears: the coverslip and slide (b) Solution B: Dissolve 9.08 grams methods. The coverslip method has certain potassium acid phosphate (KH,P0i) (mono advantages over the slide method; distribu- basic), anhydrous, in a 1-liter volumetric tion of cells is more like that of the in vivo flask containing about 750 ml of distilled circulation. The principal disadvantage of water. Dilute to the mark with distilled the latter method is that coverslips are very water. f.g-ile and easily broken during processing. (c) Mix 27 ml of the Na.,11130 _Solu- Many stains and techniques are used tion (Solution A) with 73 ml of the to stain blood films. Two types of stains are solution (Solution B); Qs. to 1liter 'Fjnal pH should be 6.4. ingeneral usethose which stainfixed cells, and those which will stain living cells (2). Wright's Stain Solution: (supra-vital stain). The panoptic (differen- (a) Add9.0gramsofWright's tiating,) stains generally used in hematology powder stain, 1.0 gram of Giemsa powder are Giemsa and Wright'sstains. When stain, and 90 ml of glycerin to a mortar of optimal staining conditions exist, Wright's suitable size. Triturate this mixture thor- stain is very satisfactory and easily differ- oughly for 15-30 minutes. AFM 60-51/TM 8-227-1 5 December 1973

(b) Transfer the mixture to a large prncilas if. dissolves during the staining open-mouth jar (a kitchen spoon is ideal for process. . _ this purpose). Cover the jar and - incubate (9) Coverh slidecompletely with theglycerin/stain -mixtureovernight at Wright's stain and allow it to-remain -on the 37° C. smear for about 2 minutes to fix. the bleed (c). Transfer the 'glyaerin/stain mix- cells. The stain should. cover =the slide but re to a large brown bottle and add 2,910 should-not beiallowed to overflow the edges ; ml of acetone-free methanol. the -stain must be re lenish d should it be- (d) Age the Wright's stain' solution- gin to evaporate. approximately 2 weeks in the dark. Mix daily to assure that the Wright's stain powder. is (10) Add an equal volume of Wl -ig completely diSsolved. stain buffer directly to the stain and blow the mixture gently to aseure,maxinitun mix .d. Procedure: ing. Allow it to remain for: about 4 minutes. (1) Make a finger puncture oveni- NOTE: The times recommended for stain- puncture in the usual manner. ing and buffering are approximate .and 42) Touch a. drop of blood to a clean should be adjusted with each fresh batch of glass slide at a point midway between the stain to give the most satisfactory result's. sides of the slide and a short distance from (IA ) Using tap water, float off the-mix- one end. If a venipuncture is made, dispose ture of stain and diluent from the' slide to the drop ,of blood directly from the needle. -avislid the deposition of metallic scum on the If a finger puncture is made, dispense the smear. The scum appears after the addition drop of blood from the puncture site after of 'theThuffer to the,..stairt., Wash the slide ,discarding the first drop. thoroughly undercold, slowly-running NOTE: The drop of blood should be no water. larger than 1/8 to 3/16 inch in diameter see figure 3-5a). (12). Air dry the smear and wipe the excess stain from the tinder surface of the (3) Lay the specimen slide, on a flat slide. surface and hold it securely. Place 'a sniooth, clean edge of the spreader slide on the speci- . Discussion men slide at an angle of about 30° from the (1) A properly prepared. blood_. smear horizontal (see figure 3-5a). ismargin-free; - has- no lines,-ridges,or (4) Pull the spreader slide toward the holes;Y. is placed-centrally on the slide ;- has drop of blood until contact is made within an adequate thin area; and has .a uniform the acute angle formed by the two slides as distribtition. of leukqcytes. shown in figtire 3-5b. (2) It is preferable that blood smears. (5) Allow the blood to spread toward_not he made from blood containing an the sides of the slide.. coagulants- since the leukoCytes change their (6) Push the spreader slide smoothly staining Characteristics, 'develop, vacuoles, and lightly toward the 'opposite end of the engulf oxalate crystals, and show nuclear specimen slide, drawing the blood behind it deformities.However_, .satisfactoryslides into a thin film (see figure 3-5c). are made with blood anticoagulated with (7) Allow the blood filmto air-dry EDTA. completely. Do- not blow on the slide in an 3)' Avoid the,,following errors: effort to enhance drying. (a)' Thick films made from an excess (8) Using:a lead pencil, write the name amount of blood placed on.the slide.. \ (or identification)of the patient, inthe (b) Delay in transferring the blood thick area of the smear. Do not use a wax to the slide. a. Place spreader slide at an b. Contact the blood with the angle of about 300 from the spreader slide horizontal

c:Push spreader slide to the opposite end of the slide drawing the blood behin'd it into a thin film Figure 3-5. Method for Preparation of Blood Films. 0-51/an 8-227-4 5 December 1973 (c) A spreader slide that -has. dam- (I'). A gOod quality smear should mac- aged orunpolished ends.. -roscopiCally he buff in hue. It :should not be (d) 'The use of dirty; dusty, greasy, bluergreen, or red. Microscopically, the red Or scratched. slides: blood cells Should he a light orange. and the If slides cannot he stained immedi- white blood cells bluish if they display their ately, they should- be dried and then fixed in true staining color. methyl alcohol-for 30 minutes. (14If the RI3Cs are ..bluish or green, ,() .Iacasesof marked leukoPenla, this indicates that the stain is too alkaline. -smears can he .prepared from the whiteicell With an alkaline 'stain,--the- WBCs stain layer ("huffy coat") obtained by centrifug- heavily andf generally ..display fairdistin-, ,.: ing the, blood 'slowly in a Wintrobo--hemato-, guishingcharacteristics.: However, -any ab- crit tube At 5.00 -800 rpm for 5-'minutes. normalities of the RBCs, are masked by the heavy stain .:11eavy staining can be caused (6) !Lis it t-ant that the blood film by: he- completely dried before staining; other- which too -Wise the wet areas will wash off the slide. (a) Blood are thick. (7) Protect' blood slides from insects Overstaining(prolongedlitiffei such asflies,cockroaches,. etc, :They can action "clean". raw blood slides.very rapidly. -c) Evaporation of the Methanol in* (8-)Protect. slides from areas of high.:thastain, humidity. Excessiye moisture te.nds.to hemo.- (d) Stain or diluent which is al lyze red blood cells. (9)4Slides should be' stained as.soon as (e) Alkaline fumes. iiikssible -after _preparation, White cells.tend (15) If the red blood cells are bright red, to -become distorted and to disintegrate very the stain is too acid. In this condition they rapidly, 'thuS causing considerable difficulty in identification. stain well but the white blood cells (except, eosinophilic granules) stain very poorly if (10) Very: littleactual staining takes at all. Thus, the stain is of no value for dif- . place during the -fixation stage: Most of the ferentialstudies.Tendency towardacid 'staining actually occurs during- the buffer- . .. staining is caused by : A ing stage: . Incomplete drying before stain-, (-11) During the buffering stage,itis in g. important that only amounts of buffer equal (b) Insitflicientstaining (instiffi- to the stain he added, otherwise there is a -cientoffer action). tendency to overdilute, causing. the smear to verdilution of the ain stain weakly. .(c) buffer. (12) After the staining is complete, do (d) Prolongedwashing of the slide not blot the smear but air-dry it. To speed after staining. up the drying process, the .smear can, he placed in the heat of the substage light. It (e -)Stitin-or bufferhich is acid. is important that the slide not he heated too ( f) Acid fumes. intensely or too long since overheating tends (16) The staining reactionof blood are to darkenthe-staining reaction. as follows: 3-12 AFM 160 -51 /TM 8-227-4 5 December 1973

Type of blood cell or component Good stain Acid stain Alkaline stain Erythrocytes Buff pink Bright red Blue or green ;All nuclei Pu4sle-blue Pale blue Dark blue .-Eosinophilic granules Granules red Brilliant red, distinct Deep gray. oeblue NeutroPhilic 'granules Violet-pink Pale- Dark, prominent Lymphocyte cytoplasm Blue Pale blue Gray or lavender (17) The technician should strive 'for a slips by placing a drop of blood on one staining reaction which is neither tcto alka- coverglass, placing a second coverglass upon line nor too acid. Such a stain gives good it, and deftly pulling the coverglasses apart. distinguishing features for all the cells of >,. the blood system. b. Equipment: (18) If the staining reaction is exces- (1) Capillary or venipuncture material. sively alkaline, this can be corrected by de (2) Coverglasses, No. 2, 22. x 22 mm. creasing time of staining or neutralizing the stock stain solution with 1 percent acetic c. Reagents: acid or 1 percent hydrochloric acid._ Add the (1) Wright's Stain Buffer. See p acid a drop of a time. Check the results graph 3-6c. after the addition of each drop of acid with (2) Wright's Stain Solution. See para- trial slides. graph 3-6c.. (19) If the staining reaction is exces- sively acid, this may be corrected by in- d. Procedure: creasing the time of staining or neutralizing (1) ,Prepare patient for- a illary punc- the stock 'stain solution with 1 percent po- ture or venipuncture, and per the punc- tassium bicarbonate or a weak solution of tdre. ammonia water. Add the ammonia water or (2) Without touching the skin, bring .a potassium bicarbonate one drop at a time. coverslip into contact with the blood speci- Check the results on trial slides after the men (figure 3 -6a). If the venipuncture tech- addition of each drop of the neutralizer. nique is, employed, dispense a drop of blood (20) Stainingreactionscanalsobe directly from the needle onto the coverslip. varied by adjusting the proportions of diso- (3) Quicklyplaceanothercoverslip dium phosphate and potassium acid phos- over the drop of blood (see figure 3-6b). phate used in preparing the buffer. For Let the blood spread untilit has almost example, Increasing the proportion of diso- stopped spreading dium phosphate will make the 'buffer more alkaline; reducing it will make the buffer (4) Pull the two coverslips apart in sliding fashion. As the coverglasseS are More acid. , pulled apart they must be, held in an abso- (21) A pciorly stained upear can some- lutely horizontal axis (see figure 3-6c). 'Any times be saved by washing rapidly with tendency to a, vertical pull ruins the prep- 95 percent alcohql, washing quickly in water, aration, since holes in the smear may re- then restaining. sult. (22) Automatic stainingdevices(see (5) Place the preparation (smear side chapter 2) are currently being used in many USAF hospitals. up) on a clean paper and allow to dry in the air. 3-7. Covergiass Method: (6) Stain and examine according to a. Principle. A blood' specimen is spread the method described for the slide method in a uniform even layer between two cover-. above. 5December_.1973

a. Bring. a coveslip into contact b. Mac? anothecover slip over

A with the blood cimen the drop of blood

c. Pull the.two cover ...glasses apart in a sliding fashion

Figure Cover lass Method for Preparation of ood 3-14 Anil 160-51/TM 8 5 December 1173 e. Discussion: (3) Formalin Fixative, 10%. Dilute 10 (1.) The coverglass methodgivesa ml of 40% formaldehyde (R.G.) to 100 rid much better distribution of white cells and with distilled water. is the method preferred by many in making c.Procedure: differential white counts. (1) From the first aspirate of marrow (2)he slide method renders satisfac- (apprOximately 0.5 ml) prepare several Minas tory differential counting difficult because on coverglasses or slides. the larger white cells are pushed to the edges (a) Quickly place a drop of marrow andthelymphocytesremainscattered on the end of a slide or coverglass. through the spread. (b) Remove excessbloodwith When red cells are to be examined capillary pipet or a syringe leaving the gray for parasites, the slide method is saperior, marrow particles and a small amount of inasmuch as more fields are available for blood behind. study and red cell distribution is as good (c) Prepare thin films by the slide oi- as with the coverglass method. coverglass method. . (4) Some disadvantages of the cover- (d) Optional slides are made at this glass method are that coverglasses are awk- time for various staining procedures. ward to handle, easily broken, and must be (2) Imprint, slides- are prepared by mounted ori a glaSs slide before they can be placing a drop of aspirated marrow on a examined. slide or coverglass. The particles are picked out and transferred-to anpther slide or cover- glass. The particles are crushed gly and SECTION CBONE MARROW Smeared. SPECINIENS (3) The clot remaining inhe syringe 3-8. Introduction. Examination of the bone , isplaced in a wide mouth jar containing marrow is an important diagnostic tool when the formalin fixative solution for histologi-. a disease affecting the hernatopoietic sys- cal examination. tern is present or suspected.' Bone marrow (4) The physician may obtain a second a.spiration' or biopsy is; always perfotmed by aspirate of marrow (approximately 1 nil). a physician. In most cases the laboratory This is .added to a small tube containing technician will assist the physician by Pre-' EDTA and.centrifuged,in a Wintrobe:Iierna- paring the bone marrow material for ex- tocrit tubs at 2500 rpm for 8 Minutes. As-

. amination by a pathologist. pirate off the plasma. Remix the plasma and all of the buffjr cellular layer 'and prepare 3-9. Processing of Bone Marrow Specimens smears of the mixture. for Examination:. (5) Staintherequiredslideswith a. Principle. Several bone marrow samples Wright's stain.Slides should be fixedin are smeared on a series of slides, stained, methyl-alcohol if`delay is experienced be- And examined, microscopically for. cytoloki- fore staining:- cal detail. The procedure 'outlined is to be (6) Process other slides for additional modified as required. ins as required. h. Reagents: (7) In certain cases the marrow as- piration is performed for bacteriologic diag-- (1) Wright's Stain Buffer. See pal nOsis... Express the aspirate into a sterile graph 3-6c.... tube:coritainingl ml of 1 percent. sodium (2) Wright's Stain SOlution. See para- citrate.Bacterioldgic examination can be graph 3-6c. made as required: AFM 160-51/TM 8-227-4 5 December 1973 3,15

d. Discussion: (3). Allslides beaccurately labeled. A properly prepared hone marrow smear should meet the following criteria: (4) They should be air-dried immedi- ritely. a) Thin smearto 5 cm in length (5) As soon as they are thoroughly and 2 cm wide. dry, bone marrow- slides should be fixed in (b) Nucleated cells almost touching. methanol to prevent disintegration of the more fragile cells. Visiblefattyareasandhigh concentration of marrow foci. (6) -Since interpretation of findings in bone marrow examinations is yery:difficult, (2) The procedure described above is even when performed by troika persons, it an outline, and deviations from it shouldf. is of utmost importance that the smears and be in accordance with instructions from the stains be carefully Prepared using scrupu- pathologist. lously clean equipment. AFM 60 51JTM :8- 227 -4 December

Chapter 4 MORPHOLOGY OF BLOOD CELLS 'SECTION AGENERAL INFORMATION f. If there is doubt in the identity_ cell, classify to the more mature form. 4-1. Basic Concepts of Cell Morphology: In this chapter all normal and most ECTION BERYTHROCYTE S commonly seen abnormal blood ',cellsa.re morphologically deScribed. Although general Introduetion: rules foridentification are given along with representative photographs and drawings, it' In the normal development and matt a- is important to realize that no biological Mon, (erythropoiesis)ofthe, .eryth entity fits the guidelines precisely. series, the red blood cell undergoes a ridtia-_ tion of morphological changes. Th._ cell dez b. The present classification of bloCid cells velopment is a gradual transit. n and six is man's attempt at identifying stages of differentstagescanbeidentified.The aturation by assigning artificial steps to a nomenclature used to describe red blood . ntinuing grocess.' The process is a smooth, cells is recommended by the American Society continuing one and therefore no one cell of Clinical Pathologists and the American ever precisely fits the criteria for a specific Medical Association. The terms with some of c.stage. These stages are. artificial classifica- their synonyms are as follows : tions which exist to simplify identification... ASCP re ology Synonyms 4-2. General Rules of Cell Identification. Rubriblast Pronormoblat Certain general rules are applied to all cell Prorubricyte. Basophilic normoblast maturation(hemopoiesis),eitherinthe Rubricyte Polychrornatophilic erythrocyte, leukocyte,thrombocyte,or normoblast plasmocyte series.. Although these rules are Metarubricyte Orthochromatic broken by individual cells, they are an aid normoblast to classifying cells. Diffusely basophilic Polychromatic . erythrocyte erythrocyte a. Immature cells are larger than mature Erythroc Normocyte cells and become smaller as they mature. b. Erythropoiesis is regulated by the in- b The relative and absolute size of the take of substances to build the cells, the nucleus decreases as the cell Matures. In some storage ,ofthesesubstances,andtheir cell series the nucleus disappears. properutilization. When normal erythro- polesis occurs, both the cytoplasm and the The cytdplasm in an immature cellis nuclei of the cells, grow at a synchronizP quite blue in.,color and _lightens as the cell 'rate.Individual differences in physiology matures: and physical. structure of the 'erythrocyte A. The young nucleus is reddish and be- account for minor morphological changes comes bluer as the cell ages. so often encountered. In certain diseases, - these morphological changes rnay.ary to p. e Nuclear chromatin is fine and lacy_ in greater extent. These variations occur the inimatiire cell.It becomes coarse and size, shape, staining, and inclusions in the clumped in the more mature -cells. erythrocyte. 5. December' 1073

4-4. Erythrocytic Series. The erythrocy oPlasm: The cytoplasm appears series is illustrated in figure 4 -1. _ band around the nucleus. It a. ,-Rubriblast: --orange-red due to the 'hemoglobin content.. Size: 14 to 19 Cytoplasmic granules are absent. eter. e. Diffusely. Basophilic Erythrocyte: .(2) Nucleus:Thiscellhas large ) 'Size: 7 to'-9 microns in diameter. round to oval purple nucleus :which 2ccupie's Nucleus: the nucleus is absent. most of the cell. The nuclear chromatin is Cytoplasm: The cytoplasmstains, arranged in a close mesh network -forming a bluish buff with Wright's stain and there re:ticular appearance. There are 0-2 light is;!i1.0 centrala light pallor ai'in,..the erythro- blue Mieleicilf-present within the nucleus. cyte.' With supravital staining this cell 'will (3) Cytoplasm: The cytoplasm is dark show light blue reticulum strands in the blue, granule-free, and limited to, 4---thinyilin*Cytoplasm and is called a retialocyte. around l'hd nicleus. There evidencevidence- of hemoglobin formation. f. Erythrocyte:, (1) Size: 6 to 8 microns in diameter. b. Prorubricyte: (2) Nucleus: The nucleus is absent. Size,: 10 to .15 m crons'4in diam- (3) Cytoplasm:. The cytoplasm of the periphery is light orange with a central zone ucleus: The nucleuS is generally of pallor. The appearanceof the central zOne round, dark purple, and smaller than the of pallor is due to the biconcave morphol- nucleus of the rubriblast. The chromatin is ogy I of the cell, which allows more- li_ coarse and clumped giving the nucleus a through the center than through the mar darker stain. Nucleoli are usually not pres- areas. but ,when they, are, they appear- more proininent than in the rubriblast. 4_ Variations in Erythrocytes. These var- 3) Cytoplasm: `The cYtoplaSin iat ons are illustrated in figure 4-2. blue and more abundant than in the rubri- a. Size: blast. Cytoplasmic granules are not present. (1) Anisocytosis.Anisocytosisisa c. Rubricyte: variation in the size of erythrocytes beyond )Size: 8 to 10 'microns in diameter. the normal limits. Cells of varying size are Itis a frequent (2) Nucleus:Thegiucleusisdark, seen in the same fields. round or oval; and s`maller than the pro- finding in leukemia, pernicious anemia; 'and rubricyte nucleus. The, chromatin material other anemias. )' Macrocytes. Macrocytes- are eryth is found. in denSe, irregular clumps 'Nucleoli __ rocytes larger than 9 microns in diameter. are not present.. , (3) Cytoplasm: The cytoplasm is mo These cells can be found in healthy newborn abundant than in -the precursor cells. It is tants,pern ciousanemia,andother blue-pink (polychromatic), the pink result- ane .w.ias. ig from thefirStvisible appearance of .(3) Microcytes. Microcytes are erthro hemoglobin'. Cytopf Ygranules are ab- cytes smaller than 6 microns in diameter. sent. These cells are frequently seen in iron de- andMediterranean d. Metarubricyte:' ficiency,spherocytic, Size: 7 to 10 microns in diameter. hemolytic anemia. Nucleus:Thiscellhas .a b. Shape: round, and blue-black degenerated naleus. (1) Poikilocytosis. This term describes a marked variation in the shape uf,erythro- The nuclear chromatin is clumped and uni- , formly dense. Bytes;Polkilocytescan `'4 bepear-shaped, Rubriblost Metorubricyte Prorubricyte e. Rubricyte Erythro6ite

Figure- Erythrocytic Seri AFM 160-51 fT 5 December -1973

comma-shaped oval-shaped, rvarious pathological significance except when cre- other bizarre, -forms. 'These Cells are .en- nated cells are found in spinal fluid as dis7 .-cpuntered in pernicious anemia and many cussed in the chapter concerned with .per- - other types of anemia. forming cell counts on cerebrospinal fluid. (2) Sickle Cell (Drepanocytes). Sickle.. (9) Schistorytes., These cells,are frag-

cells are abnormal .erythrocytes which as- ments of erythrocytes. Frequently these cells me a crescent or sickle-shaped ippearance- have a ,hemispherical shape (helmet cells). finnder4-onditiokiS:-Of:iedtieed oXygen tension Schistoclytes-Can'be.lound in severely burned The presence of sickle cell-s an inherited patients and many anemias, . abnormality due to the pr ence of hemo- (10) Rouleaux Formation: This phenom- globin S. Sickle cell anemia is encountered enon is adherence of erytlic-ocytes to one primarily -in Negroes. another presenting a stack-of-coins ate (3) Spheracytes, These are abnormal an'ce. It oceurs in conditions characterized erythrocytes which are 'spheriCal in shape, by increased amounts of fibrinogen and .--haying a diameter smaller than norrnal,,and- globulin. a- darke'r stain (Without central pallor) than - normal erythrocytes. These cells are. c. Staining: the erhernorytic dixeases and are particularly (1) Hypochromia. Hypochromiaisa characterfstic of congenital hemolytic jaun- condition in which the normal Central pallor dice, a hereditary disorder. , iS increased .,.due to 'decreased hemoglobin 1; ( 4 )OValOCYteS ( Elliptocytes) .These content. This condition is chargderistic of cells are abnormal erythrOc3rtes which have many anemias. an ovalillor "sausage" shape. Ovalocytosts is (2) Polychromatophilia. This term de- inherited and can be seen in thalassernia and scribesnonnucleatederythrocyteswhich :sickle cell anemia. show- bluishcolorationinstead! oflight (5) Target', Cells. (Leptoeytes).ATarget or Polychromatophiliq is due to the -cells-- are erythrocytes which have deeply fact that the cytoplasm of these cells does stained (pink) centers and borders, sepa- not mature, resulting in the abnormal per- rated by a pale ring, kiVing them a target- sistence of the basophilic cytoplasm of the like appearance. These cells have a greater earlier nucleated stages. than normal diameter. The cells can be seen in various types of hemolytic anemias, in d. Inclusions: liver disease, and after a splenectomy. (1) Howell-Jolly Bodies., These are nu- (6) Burr Cells. Burr cells are train u clear remnants .found in the erythrocytes of or crescent-shaped erythrocytes with o e or the blood in various anemias, They are round, more spiny projections on the periyhery. dark violet granule,s about 1 micron in diam- These are 'seen in small numbers in uremia, eter. Generally, only one. Howell-Jolly body carcinoma of the stomach, and peptic ulcer. will he found in ariY one red cell. How.ever, (7) Acanthrocytes. Acanthrocytes are two or 'more may sometimes be present. irregularly-shaped erythrocytes with long Howell-Jolly bodies generally indicate ab- spiny projections. This is a congenital ab- sent or nonfunctioning spleen. normality associated with low serum concen- (2) Cabot'sRings(RingBodies). tration of low density (beta) lipoproteins. These are bluish threadlike rings found in (8) Crenated Erythrocytes. These cells the red cells in the blood of patients with have serrated or -prickly outlines resulting severe anemias. They are interpreted as rem- from shrinkage of the cells. This condition nants of the nuclear membrane anda4pear occurs when blood films dry too slowly and as ring or "figure-eight" structures. Ugually the surrounding plasma becomes hypertonic. one such structure will he found in This deformityis'artificialand has no one red cell. 60-4-1-/TM 8-227-4 5 December 1973 Basophilic Stippling. Rbund, yaungi cells of this series are riarned,,jiy blue-purple granules of varying size in the adding the suffik-"perniciotis anemia,eityp- cytoplasm of the red cell represent a con- that is, rubricYte, perniCious'lanemfitype, ensation of the immature basophilic stib-. etc. stance (seepOl.rhromatophilia) which nor- rn ally .disappears-with .matin;i%y.-.Thisis known as basophilfc Stippling. It can. be ECTION C,LELTItOCYTES demonstrated by standard staining tech- . 4-6. ni; ques in contrast to reticulocytefilaments Granulocytic Series. The stages in the require a. special stain. Stippling oc- netemal maturation of the granulocytes are: curs in anemias and h avy metal Poison- (neu- ing (lead, zinc, silver, niercuiy, .bismuth,) trophilicr-eosinophilic, and basophilic), met- anddenotes immaturity of the cell. amyelo-cyte' (neutrophilic, eosinophilic, and basophilic)), (neutrophilic, eosirio-,:, (4) Heiii -Ehrlic-Bodies.- These, are philic, and basophilic), and segmented cell 11 inclusions ogy d 'prirn'arily in those (neutrophilic, easinophllic; and basophilic). hemolytic anemiasi-nduced by toxins. They As° the granujocyteS mature, the granules are.. round, refractilebodies.inside the eryth- increase in number. These granules later be- rocyte %a.ndarevisibleonlyinUnfixed come specific and differ in the aflnity :for smears. It is thought that they are proteins arious_dyes. Neutrophilic granul6 do not whichave been. denattired and that they-gain intensely:, with either Basophilic re aindication of erythrocyte. injuiw. ._ granules have an affinity for the basic or (5) Siderocytes..TifeSe are erythrocyteS blue dye; Eosinophilic stain red with an

. containing-, irondeposits. .These, deposits affinity for .the acid dye. The criteria for indicate tin incomplete reduction of the iron identification of the various stages of tl from ferric to the ferrous state which is nulocvtic series are: size of. eell, nucleuS'- normally found in hemoglobin. Prussian blue °plasm ratio, nuclear shape, number of stain must be used- to readily demonstrate cleoli, and the types and size of these cells. plasmic granulation.`khe granulocytic :sari . is illustrated in figure 4-3. e. MekaloblasticErythrocytes.The - v lopment of.-megaloblastic cells is caused by : deficiency of vitamin B, or,folicacid. (1) Size:' 12-, o microns in diameter. Pernicious anemia is a disease considered to be due to a deficiency in vitamin "B,., and/or (2) Nucleus: The nucleus is /Tama or certain related growth factors. With This ovoid and stains .,predpminantlyreddish- deficiency the erythrocytes do not mature purple, The interlaced 'chromatin strands. normally and are generally larger thannor- are delicate, Nvell-defined:.and evenlYstained. mal. The most notable characteristic of this Two:or,- more pale blue nucleoli are demon- abnormal maturation is a difference in the arable. The nucleus occupies--most of the rates of maturation of the cytoplasm and cell with a nticleuscytoplasm ratio of 6t1. the nucleus. The development of the nucleus It is separated from the cytoplasm by a defy is slower than that of the cytoplasm, so that nite nuclear membrane. in the more mature of the nucleated foims (3) Cytbplasm:The- cytoplasm a Spongy nucleus as well as an exceptionally narrow, deep blue,- riongranular rim a large size may he observed. Nuclear chroma- the nucleus. tin inAhe .megaloblast is much finer and is . b. Promyelocytei without the clumps observed, ih the rubri- last. Such development is termed asynchro- (1,)Size: 12 to 20 microns in diamJter. nism. The .mature cellislarge (about 10 (2) .Nucleus:' The nucleusisround or microns) and is termed a megalocyte. The ovoid with coarse4clumping, plc chrorna- Metorubricytes Anisocytosis Poikilocytosis:. tickle Cells 0 ychrornasio SOherocytosis

Hipochromic Mocrocytic Erythrocytes Marked Poikilecytosis tknisocytosis and Target Cells.

Metorubricyte1 Metorubricy HowellJolly Bodies bTarget Cell '..,'Crenated .RBC Figure 4 2. Variations in Erythrocytes. Crenated RBCBurr Cells

Acant..ocytes 2 .Leu-k ocy tes- .

96 :2..,=:,- a.Rubric /es.( Anemia) Metarubricyte Figure 4-2. Continued. (Pernicious Anemia) AFM 16941/TM 8-227-4 5 December973

fin material. One to three oval: light-blue gutrophilio -Band nucledli are, usually 'present.. The nucleoli -Size: -10. to 16 microns iri diameter. are less --,distinct than in the myeloblast. (2). Nucleus:. The nucleusisshaped This cell -has a.. nucleus - cytoplasm.ratio, of like a horseshoe with -a dark pYknotic mass at each pole of the nucleus where the lobes (3) Cytoplasm: .The 'cytoplasm is light develop. The nucleus-cytoplasm ratio -is ap- purple and contains varying numbers arid proximately .1:2. sizes/ of dark nonspecific. granules which stain red to purplish-blue. The granules us- Cytoplasm: The cytoplasm contains_ uaily overlie the nucleus. many small, evenly distribute& light pink granules. c. Myelocyte. In the my_ eloeytic stage the granules, are definite and so numerous that g. Neutrophilic Segniented Cell: frequently.they_ obscprenucleardetail. (1) Size: 10 to 15 microns in diameter. --- While prornyeIorytes.-are sometimes= distin (2)- Nucleus: The nucleus -IAA- definite guished asneutrophilic,eosinophilic;or lobes separated by a very narrow filament basophilic, the differentiationis .generally or strand. The nucleus-cytoplasm ratio is considered as firit occurring in the myelocytic approximately 1:3. stage. (3) Cytoplasm: The cytoplasm is light d.' Neutrophilic Myeloc pink and the small, numerous, and evenly (1) Size: 12 to 18 microns in diameter. distributed neutrophilic granules have a light pink color. (2) Nucleus: The nucleusisround, oval, or flattened on one side. The chromatin h. Development of the. Eosinophilic Group.- strands are' light purple, unevenly - stained, Cells of the eosinophil group are character- thickened .i -Nucleoli are usually absent. ized by relatively large, spherical, cytoplas- The nucleus is smaller than the earlier cells micgranuleswhichhavea --particular of this series with a nucleus - cytoplasm 'ratio affinity for the eosin stain. The earliest eosin-: of 2:1. ophil (myelocyte) has a few dark spherical (3) Cytoplasm:.The oplasm granules with reddish tints which develop bluish-pink and contains a small relatively among the dark, nonpeeific -granules. As the light area of ill-defined, pink granuleawhich eosinophilic cells pass through their various develop among the dark, nonspecific, azuro- developmental stages, these granules become philic granules of the . As the less- purplish -red. and more: reddish- orange. myelocyte ages, the dark granules become The dark blue, nonspecific grantiles, charac- leAs prominent -and the light-link-colored teristic of the prornyelocyte and the early myelocyte stages, disappear. Because the per- neutrophilic.'granules predominate. centage of eosinophils is usually low in bone e: Neutrephilic Metamyelocyte: marrow or peripheral blood smears_ , no use- (1) Size: .10 to 18 microns in diameter. ful clinical purpose is served by routinely separating the eosinophils into their various (2) 'Nucleus: The nucleus is indented inyelocyte, metamyelocyte, band, and seg- or kidney shaped. The nuclear chroinatin mented categories. On the other hand, in situ- stains dark purple and is condensed into ations such as eosinophilic leukemia in which irregular strands. Nucleoli are absent. The the eosinophils are greatly increased, an nucleus-cytoplasmratioisapproximately analysis of the incidence of the various stages. 1.5:1. would be useful in diagnosis. (3) Cytoplasm: The cytoplasm pinkish-blue' and- covered with many small, Mature Eosinophil: light pink granules. (1) Size: 10 to 15 micronsin diameter. 160, 22'74 5 Deeernber 19

yeloblast Metarnyelocyte Baud. Neutrbp.bli'

-Promyelocyte Prornyelocyte with Auer Body

Myelocyte Figure Gran locytic Series. (2) Nucleus : The nucleus. has-definite- Ly-rnphoblas lobes separated'. by a very narrow filament Size: 10 to 18 microns in diameter. and. Seldom'. does an eosir;ophil have more than two lobes. (2) Nucleus: The nucleus has an oval or round shape and stains. reddish-purple. 3) . Cytoplasm:The cytoplasmcon. The nuclear chromatin_ is fine, well distrib- bright reddish-orange, distinct gran- uted, and coarser than in the myelobla.st. ules. the granules are spherical, .'nnifotnr. Chromatin is cOndensed at the edges of the in size, and evenly distributed throughout the nucleus to.form a definite nuclear manbrane. cytoplasm, but rarely overlie the nucleus. One or more nucleoliare present. The j. Development of the Basophilic Group. -nucleus is prdrninent with a nucleus-cyto- plasm ratio of 6:1. These.. have, round, indented, band, or lobulated nuclei and are classified according (3) Cytoplasm: The cytoplasm is deep to the shape of the nuclei, as basophilic mye- blue With a frequent perinuelear clear zone. locytes;--- metarnyelocyteki bands; and seg- mented forms: These cells are -so few in b. Prolymphocyte: peripheral blobd ancibone marrow that there (1) Size: 10 to 18 microns in diameter. is little value in differentiation of (2) Nucleus: The nucleus is oval and the various maturation stages. slightly indented. The nuclear chroniatin is k. Mature Basophils: coarse, slightly clumped, and dark purple. One light blue nucleolus is usually- present. Size: 10 to 15 microns in diameter. The nucleus-cytoplaim ratio is 5:1. Nucleus: The nucleus has definite (3) Cytoplasm :The cytoplasm varies lobes separated by a very narrow filament from light blue -to dark blue and it can show or `strand: The nuclear details are obscured a few red-purple (azurophilic) granules. by the cytoplasmic granulation. c. Lymphocyte: (3) Cytoplasm: The cytoplasm is cov- ered by many blue to black granules. These. (-1)Size: The mature cell of this series granules are unevenly distributed and vary varies greatly in size. Sinall lymphocytes are in number, size, shape, and color. 7 to 9 microns indiameter. The large lymphocytes are 1.0 to 18 microns in diain- 7. Agranalocytes. Fare. leu- der. kocytes devoid of specific granulation. These (2) Nucleus: The nucleus is round or cells generally originate in the lymphatic-sys- oval and can be slightly indented. The nu- tem, but can be fdund in normal bone mar- clear'-'7natin is markedly condensed, dark row. Agranulocytes include the lyrnphocytic purple -blue, and clumped. Nucleoli are ab- series, monocytic series, and plasrnocytic se- sent and a definite nuclear membrane is pres- ries. ent. The nucleus-cytoplasm ratio is approxi- mate]1.5:1.0. 44. Lymphocyt,eries. The stages in the Cytoplasm: .The cytoplasm is light developMent of the lymphocytic series are blue to blue with a perinuclear clear zone lymphoblast, prolyrnphocyte, and -lympho- around the nucleus. A few azurophilic gran- cyte. These cells are fragile and can show ales can be seen in the cytoplasm of larger shape variants. Lymphocytes usually have round contours, blue cytoplasm, and eccen- lymphocytes. trically located round nuclei. Cells of this se7. ries are differentiated on the basis of the nu- 4-9. Monocytic Series. The Stages in the de- clear chromatin. The lymphocytic series is velopment of the monocytic series are mono-. illustrated in figure 4--4. blast,. prornonocyte, and monocyte. Cells of yniph.o:brosi-.

5-rii,146-c9:11-,

ymphociie romonotyle-

Lymphocyte: Neutrophil (L e nd) Azurophilic Granulation b.Monocyte Figure 4-4. LyruphoeytieSeries and MeneenieSerie AFM160-51/TM 8-227-4 5 December 1973 the series are slightly larger than gran ulo- the monocyte is, dull gray-blue Iv' bilethe cyto- They. round with smooth margins plasm: of the neutroPhils in the adjaNnt and seldo how shape variants. The. mature fields is definitely lighter and is pink ratter- monocyte is differentiated from the lyrripho---Ethan gray-blue. The Uonstiecific granules': of and metamYelOcSite by the !very fine, the monocyte are usually firie'and evenlidis- light staining nucleus_ The monocytic series tribUted, giving to the cell a dull, opaque or is illustrated in figure 4-4. ground-glass appearance; In addition to the badlcgroundpf evenly distributed nonspecific Mondilast: granules, there may be a few ,unevenly dis- (1) Size: 1to 20 microns in diameter. tributed larger azurophilic . granules. --Vacti (2) NucleuThe nucleus is round or -oles are_ten --demonstrable in cyto- oval and red-pu'rple in color. The nuclear plasm. chromatin, is fine and well distribute-O. One 4.10. Plastnocytic Series. Plasmdcytes. con - .to two nucleoli are present. -The nucIe4s- -approximately .1- percent ofthe cytoplasrn ratio is white cellsin the normal bone marrow. (3) CytoPlasm: The Cytoplasm is a clear, These cells- can be present in the peripheral deep blue and forms a thin rim around the blood in chronic infections, -granulomatous nucleus. and allergic diseases, an -multiple myelorng.. 12/. Promonocyte: The stages of development are: plasma- blast, proplasmoCyte, and plas-mocyte.- These (1)SiZe:11.2 to 2.0 -microns in diameter, cells are illustrated in figure 4-5. 1 (2) Nucleus: The nucleus is irregularly- a. Plasmoblast: shaped and light purple. The nuclear_ chroma- tin is fine 'and spongy. A nucleolus can be (1) Size: 14 to 24 present. The nucleiii-cytoplasm ratio is 5:1. (3) Cytoplasm: The_ cytoplasm is gray- (2) Nucleus: The nucleus is large, oval bluewithfinered-purple(azurophilic) or round, and contains within the fine, pur- globules. plish, reticulated chromatin one to three blue- nucleoli. The nucleus-cytoplasm ratio varies ,Monocyte: from 2:1 to 11. )- Size: 12 to 16 microns in diameter. (3) Cytoplasm: The cytoplasm is rela- (?) Nucleus: The nucleus is round` or tively abundant and, stains dark blue. No kidney-shaped, but can be deeply indented granules or vacuoleS are found in the cyto- or have two or more lobes. One ofthe most plasm. distinctive features of the motiocyte- is the presence of superiMposed lobes, giving the b. Proplasmocyte: _nucleus the appearance of brainlike. convolu- (1) Size: 14 to 22 microns in diameter. tions. Heavy lines' marking the edges of the (2) Nucleus: The nucleus is ovoid and folds and grooves are features which are not locatedeccentrically.Thechromatinis seen in other cells. AnOther featureof the purple, coarser, and, more clumped. One to nucleus, which is of value in diagnosis, is the two nucleoli are present. The nucleus-cyto- tendency for the nuclear chromatin to be plasm ratio is 2:1. loosewithlight spacesinbetweenthe chromatin strands, giving a coarse linear (3) Cytoplasm: The cytoplasm is pale blue, with pale reddish-blue vacuolation. -A pattern in contrast to the lymphocyte which pale perinuclear zone or "halo" is usually . has clumped chromatin. Nucleoli are absent. The nucleus-cytoplasm ratio is approximately present. 3:1. -Plasrnocyte: -(3) Cytoplagm: The cytoplast (1) Size: 8 to 18 roue in-diameter. Afl$1 160-51/TM a-227-4 5.. December'.1973

(2). NiAeus:-The nueleus is small, ovoid; brilliant translucency. In oes onalells and eccentrically ideated: The -chromatin is the globules. a-be quite promipent coarse, lumpy, and ptirple: Nucleoli are not a rect.or bluish-red stain. Such globules Are Usually present. The micletii-O oplasm called- Russell. orfuchsin bodies, or epsina- tio is 1;i2. phi& globules, '-Vacuoles of .gal ioug.'isizes arei=requently demonstrable. _ (3) CytoPlasin The cytopla's_fn. adjacent

o the nucleus is lightly stained in contrast, to _ 'Variations of Leukocytes. Variations the periphery of the cell which hasa high of leukocytesoccur as a 'result Of abnormal saturation .of red and blue dyes. In some cells maturation: of the nucleis andfOr cytoplasm. the dark cytoplasm has -. agreeniSh or These variations are -induced by leukemic larksptir7blue 'color. The cytoplasm contains states;infectiousdiseases,andtoxicity: multiple small and relatively Unstained glob- Many variations in leukocytes have been ob= 'Ule's embedded bluish-reds lilainentous served; Irowever; tlikrnanbal describes-- only matrix, it-is the -:presence of these tapiocalike---=tilerniost fi'equently--occurring globilles in the dark Surrounding medium A more complete study of leukocyte varia- .which giVesAthe Plasrnocyte its characteris- obtained from the referendes tic mottled. and foamy aPpearalice and .its listed in the tiblipeaphy of 'this manual.

Plosmobiost

Figure Plasrnocytic Series. Ant 160-5iVT31 8-227-4 ,5- December 1973 Variations in leuko in appearance.s.-Downey and -Mcignley. de- figure 4-6. scribed three =types of atypical- lymphocytes, but this classification has no real clinical a; Dohle Dohle bodies are light purpose. blue or blue-gray,: small, round= inelusions found in the cytoplasm -of, neutrophilic leu: (1) UP to 20 =1 icocytes. This variation' may occur in toxic diameter.. conditions such as severe infections; burns, (2). Nucleus: The nucleus is oval or and scarlet bitten:. 'kidney shaped ,with very coarse chromatin ,strands not as lumpy anormal lympho- b. Auer Rods: Auer rods are rod or e..- spindle-shaped, cytoplasmic incluaiona. They stain red-purple and are 1 to 6 microns long (3). Cytoplasm: The cytoplasrn s blue to and leis -than 'L5 microns thick. They are dark bine. Often it is vacuolated which gives requently found in letikerni4s. rise to a foamy appearance. c.- Toxic Granulation. Toxic granulation ,LE: 'Cells:. occurin the neutrophilie metamyelocyte, -(1) Pergons having lupps e hernato- band, and. segmented cells. These 'granules;sps,- one of the"collagen" diseases, have an '- are distinguished. frorn the normal rranula- abnormal plasma profein which causes swell- 'tion because they are coarser and stain a inK and breakdown of certain blood cell nu- dark purple': The variations, occur .toxic clei in vitro. This degenerated nuclear mate- states and severe infection.'` ial attracts phagocytic cells, particularly d. Xasket.Cell. A basket cell is a ruptured egmented neutrophils, which engulf this leukocyte which has a network appearance. clear mass- The resulting and in- These cells result from a partial breakdown elusion material is termed an "L.E. "- cell. of the immature and fragile leukocytes. Bas- 2) The nucleus of an L.E. cell is ad-, ket cells- are found predOminantly in. dis7; jacent to the peripheraloutline of the inclu- eases with an acute shift toward hnniature sion material. The inclusion is smooth and forms, for example, leukemias. smoky. or light purple and has no visible e. Vacuolat6c1 'Cell. A vacuolated- cellis chromatin network. a degenerated cell with holes or vacuoles in i. Rosettes. Rosette formation is the in- the cytoplasm,- Vacuola.ted cells -canibe seen termediate stage in the formation of an in severe infections, poisoning, and leuke- L.E. celLA rosette formation consists ,of . mias, and in cells that have been in Hell& & neutrophhicleukocytessurroundingfree Paul oxalate too long. masses of lysed nuclear. material. f. Ily.persegrrientation. A normal neutro- Tart Cells. A tart cell contains lysed philic segmented cell ha's a nucleus with an nuclear material within its cytoplasm. It average of three lobes or segments. In a hy- differs from an L.E. cell because the inclu- persegmentecl cell the nucleus is broken up sion 'retains characteristic nuclear structure. into. five to ten lobes or segments. This cell This inclusion is not smooth and haS a darker usually has a larger diaineter than a normal staining periphery. neutrophilic segmented cell. Hypersegmeri- tation is often. seen in pernicious anemia. 1ECTION D=THROMBOCYTES 4-12. Introduction: g. Atypical Lymphocytes (Virocytes, Turk Cells);These lymphocytes are characteristic a. The general pattern of thrombocyte of infectious mononucleosis but they may also maturation is slightly different from that be seen in apparently-healthy individuals and of leukocyte maturation. The cells of the 4hose, with certain other diseases. Atypical megakaryocytic series tend to grow larger as lymphocytes are larger than normal and vary they mature until there iscytoplasmic frag- Band Neutrophil-Toxic Granu 1a i on

s.. :Hypersegmenf Atypical LYnphacytes: -Infectious Mononucleosis Figure 4-6. Variations in Leukocytes. rientatron for breaking off) to form the cy- . Megakaryocytic Se _topldsmic thrombocytes seen in the periph- eral blood. g a. : b. Azurophili tanulation begins to ap- (1) Size: 15 to 30 microns in diameter. pear in the second stage of developthent and (2) Nucleus: One to two large oval or continues until it almost obscures -the nukidney-shaped nuclei are present. The red- clear- lobes. The nucleus develops from a dish nuclear chromatin has a granular ap- fine single lobe to Multiple ill-defined lobes. pearance.Severalnucleoliare The stages in the normal maturation of the present megakaryocytic series are megakaryoblast, within the nucleus but these are difficult to prornegakaryocyte, me2akaryocyte, xnetameg- see. The nucleus-cytoplasm ratio is approxi- akaryocyte, and thrombocyte. These cells mately 10:1: areillustrated-in figure 4-7. (3) Cytoplasm: The cytoplasm is blue, 60-51111 f 8.227.1 -5' 04ceriiher 1978 none anular, and y ehow blunt Prou--- (2) Nuclens:Two...tosixteennuclei sion. are iiresent land- these. are -supentriposed. on and attached to each other_ narrow fila- b.- Prorneokao e:' ments,. Nucleoli are indistinct. Nuclear chre: 1) Size:i 40 to 100 3 microns mn -Alarm-. ;a:Min is lumpy' and coarsely linear. er. (3) Cytoplasm The -cytoplasm is cov- (2:). Nucleus:' One two indented round-ered with numerous, small blue-purple gran- or oval nuclei are present The nuclear chro- ules, No evidence of Platelet fragmentation matin is purple, coarse,- and granular. Nu- is present. . dleoli-are present bid indidtinct. ,(3) Cytoplasin: The cytoplasm is,..blue d. yetarnegakaryo and has . blunt, bubbly extensions. Alum- (1) Size: 40 to 100 microns in diame-, philic granules are usually present. ter.

.: (2) Nucleiii.: Same as megakaryacyte. size:` 40' to Cytoplasm: The cytoplasm is much ter. like the megakaryacyte but shows evidence.

egakaryocyte

Thronibacy-tes Figure1. Megakaryocytic Series. ofagmentation or sloughing of thrombo. light. blue with centrally located azurophilic cytes from the-cytoplasmic margins. (red-pUrpIe)granules, NOTE :Normally, Thrombacytes: megplcaryacyte-s- and tnettirileg-akaryncytes do -- not appear in the ,periphe;a1; Wood ; low- Size1. to 3 ;microns in d mete ever, they can be sierf 'in sonic leukemias. (2)' Nucleus: None. In bone- smears, there should be little difm ficulty recognizing cells:Jmbeingf- this' cytoplasm: The cytoplasm appears series because of their enormous size. 16051',Pr141 5 December 1

,Chapter 5- MET IN HEMATOLOGY

SECTION OUNTS . 2. ,Red Blood Cell- Colint (tlethacytometer):

_ 5-1. Introduction: a. Principle. =A sample of blood is diluted a. Blood cells are subject to quantitative with a sPecial isotonic solution in a micro= variations as well as the qualitative varia- pipet. After adequate mixing, a portion of the sample is introduced into the counting tions described. in chapter 4 Some diseases stimulate the production of blood cells while chainber (heniacyto-ifieter).- The red others prevent or diminish the production cells (erythrocytes) in a known volume are of blood cells, For this `reason a cell count then counted; gives valuable -information to the physician b. Reagents. The most common diluting -° concerning his patient's condition: Further- fluids used for the RBC count are Gower's more, in the case of the leukocyte Count,' solution. and. Haye-rn's. solution. Either solu- the total count is necessary, to calculate ab- tion' can-be used and the preparation of 'both. solute counts _for each type of leukocyte. is given below. Normalsaline may also be used. This is done by multiplying the total count by the percentage of the' particular 'cell type. (1) Gower's Diluting Fluid. Add 12.5 grams of sodium Sulfate (Na2S01); b. Cell counts can be performed by a anhydrous, and 33.3 ml glacial acetic acid Hay methods. Erythrocytes and leuko, to a 200-ml volumetric flask. Dilute to the cytes are counted by manual methods or ma_rk- with distilled water. NOTE : Add the automated methods. Other cell counts are wter slowly-and mix thoroughly. performed only by manual methods. It important when performing a cell _count to (2) Hayern's Diluting Fluid. Add 0.5 maintain 'good quality control. Great care gram mercuric chloride (I-IgCl,), 5.0 grams should be taken when performing any cell sodium-sulfate (Na2SO4), and 1.0 gram sodi- um chloride (Nadi) to a 200-m1 volumetric count. flask. Dissolve the contents with distilled c. The following paragraphs outline pro- water and 'dilute to the mark with distilled cedures for red blood cell(RBC): count, water.Filter thesolutionseveral times white yood cell (WBC) count, total eosino- through the same filter paper- NOTE De- phil count, absolute basophil count, reticu- terioration of this solution can occur after locyte count, cerobrospinal fluid (CSF) cell three weekts. This solution must be discarded count, and, sperm count The RBC and WBC if a precipitate forms. counts are routinely done; they are per- formed either by the hemacytometer meth- c. Procedure: ods (manually) or by automated methods. (1) Draw well -mixed capillary or ve- Total eosinophil and ablute basophil counts nous blood to the 0.5 mark on the red blood are performed by a hernacytometer method cell diluting pipet. The blood column must using special diluting fluids to accentuate be tree of bubbles. (See figure 5-1a.) these cells. are dernonskated (2) Wipe off the excess blood from the by using a supravital stain. Semen Aaxis outside of the pipet to avoid transfer of cells and CSF counts are included in this section to the diluting fluid. When wiping, take care because a hemacytorneter is used to perform not to touch the end of the pipet with gauze. the appropriate counts. ( See figure 5-1 b. )

6 -1- g2-27-4 ember1973

b .'Wipe offextnssblniad9dh.iiing :tbe,.tip, thepipet::

c..Inert pipetip oppropei .-type of-dilutiog::fluid dra 1.3.-Ef. fluid to proper 'ma tking, on pipet -tube Figure 5t Processing Blood for M Manual llCo n Immediately draw diluting fluid to (short end) of the pipet, hold the pipet at a the 101 mark, at the same time rotating the,. 45° angle, and touch the pipet tip to the pipet between the thumb and forefinger to junction of the coverglass and the counting mix the specimen and diluent. Hold the chamber (see figure 5-2). pipet 'upright to prevent air bubbles from (10) Allow the mixture toflow under flowing into the bulb (see figure 5--1c). the coverglass until the chamber is com- (4) Place a finger:over the tip of the pletely charged. Similarly fill the opposite pipet and remove the rubber suction tubing. chamber of the hemacytometer. If overflow- (5) Mix the contents of the pipet for ing into the moat or air bubbles occur, clean about 10 secondg by holding the pipet on a and dry the chambers, remix the contents of horizontal plane between the thumb and the pipet, and refill both chafnbers. forefinger of one hand and rotating in a (11) Allow the cells to settle for about ,"figure-eight."(See figure 5-1d.) A me- 3 minutes. Under low-power magnification, chanical shaker is 'rased to further mix the focus on the ruled section of the counting contents. chamber and.locate the center one-sq-mm (6) Place a clean hemacytometer cov- .area for counting the red cells. erglass on the counting chamber. Moisten- (12) Observe for even distribution ing the shoulders on which the coverglass cells. rests aids in keeping: the thin coverglass in (13) Switch to high-power Magnifica- place. tion and locate the square in the upper left- (7) Mix the specimen in the pipet [as hand corner (see figure 5-3)... in step (5)] for about 3 minutes to assure (14) Count all the red cells lying within even distribution of cells. this square and those touching the center (S) Expel the unmixed and relatively line of the upper- and right-hand triple cell-free fluid from the capillary portion lines. The red cells which touch theleft- of the pipet (usually two to three drops). handandbottom-centerlinesarenot (9) Place the forefinger over the top counted.

Coverslip Depth equal- 0.1rare

Figure 5-2. Charging the Hemacytometer. AFM 160-5T 8-2 )7 eCeMber 1973

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1 -1

Figure 5-3. Hemacy oeter CountinChamber. Areas .marked L 2, 3, 4, and 5 are usedto count red blood cells. (15) In the same mariner, count the red RBC diluting fluid. All the blood is washed cells in the remaining four small squares, as into the bulb of the pipet (which has a vol- illustrated. A variation of more than twenty- ume of 100). Therefore,0.5 volumes of fivecells between any of thefive ayeas bloodarecontainedin100 volumes of counted indicates uneven distribution. dilutingfluid.The resultingdilutionis Shatuld thisoccur,repeat theprocedure 1:200. These figures are arbitrary and refer starting with step (7). _strictly to dilution and not to specific volu- (16) Count the second chamber in th6 metric measurements. same manner. (2) The depth of the counting chamber is 0.1 mm and the area counted is 0.2 sq ,Calculations: mm (5 squares are counted each with an (1)- . Routinely, blood is drawrr to the area of 0.04 sq mm; therefore, 5 x 0.04 0.5 mark and diluted to the 101 mark, with sq mm d total of 0.2 sq mm). The volume counted is area x depth e volume or 0.2 (5) Failure to expel two to three drops _sq mm X _0.1 mm a 0.02 cu in the pipet tips before charging the hema- cytometer. -(3) The formula is as follows: (6) Overfillingthe chambe ofthe Average (of two chambers) number of hemacytoineter, which causes erroneously- dilution (200) high counts. RBC's counted x volume (0.02) (7) Wet or dirty lases and herna- (4) Example: cytometers. First Chamber Second Chamber (8) Uneven distribution of cells in the Cells counted Cells counted counting chamber causes erroneous results-. in each square in each square (9) Inaccuracy or carelessness in malc- 110 99 i ng counts. 100, 102 95- 87 (10) Diluent which is cloudy or contains. 115 95 debris. 90 107 tomixanticoag lated 510 RBCs counted 490 RBCs counted (11) Failure blood thoroughly before use. Average of the two chamber counts f. Discussion: 510 1000 500 RBCs counted 490 2 (1) The error resulting from the dis- 1000 tribution of cells in the hemacytometer in- 500 x 200 cluding field and performance errors is ±7 to 0.02 5,000,000 RBCs per cu mm 11 Field errors are minimized by count- e. Sources of Error: ing large numbers of cells. Performance er- ors are minimized by good technique and (1) Improper collection of blood speci- equipment. mens causes variable results. (2) The minimum blood sample_ recom- ) Wet or dirty pipets. mended for performing routine red blood ) Poor condition or inaccurate cali- cell counts is that obtained using one pipet bration of pipets. Pipets must be in good and 'counting two chambers asoutlined condition and calibrated to have a maximum abqye. However, when the red, blood cell error of ± 1%. count. Is to be used in the calculation of (4) Poorpipettingtechniquecauses blood- indices, greater accuracy is required. high or low counts. Poor pipetting technique In tins case, the procedae is to use two includes separate. pipets and fill two chambers from (a) Undershooting desired line with each pipet. The average count is then ob- blood or di luting fluid. tained and is used in the calculation of the blood indices. (b) Overshooting desired line with (3) When venous blood is use, it must blood or diluting fluid. be mixed properly with an anticoagulant at (c) Air bubblesin the column on the time the blood is drawn and again just bulb. prior to pipetting to assure uniform distri- (d) Failure to wipe tip free of blood. bution of 'cells. Vigorous mixing should be (e) Too slow manipulationfollow- avoided since hemolysis can result. ing the withdrawal of the specimen thus al- (4) If capillary blood is used, the tech- lowing some of the blood specimen to coagu- nician must work rapidly following the late. puncture, otherwise coagulation of the,speci- (f) Failure to mix the blood and men and settling of cells in the specimen diluent properly. are likely to.begin before the blood is mixed 160-51/TM 8-227-1 5 Decembe with the diluent. This results in' a low cell (2) Adult Female: 3.6-5.0 trillion count and/or a clogged pipet. To avoid in- RBCs per, cu mm. troducing excess tissue fluids that result (3) Childhood : 5.4 million BBCs from "milking" the finger, do NOT massage per en mm. the finger after puncture has been made. (4) Birth:. 4.7-7.1trillion RBCs per (5) It is of the utmost importance that cu mm. clean, dry diluting pipets be used so that, hertiolysis is precluded. 5-3. White Blood Cell Count (6) Hayern'ssolutionshouldnot be (Hernacytometer): more than 3 weeks old and should be filtered a. Principle. A sample of blood is diluted' frequently. if the solution stands for any with solution which lyses nonnucleated red length of time,it becomes cloudy which, blood cells. Following adequate mixing, the makes it useless as a red blood cell diluting specimenisintroducedintoacolinting fluid. . chamber where the white blood cells (leuko- (7) Occasionally/blood "leaks" into the cytes) in a diluted volume are counted. diluting solution from inserted pipets and b. Reagent. 2% Acetic Acid : Add 2 nil gla- causes serious errors. Thus, routine filtra- cial acetic acid, to a 1,00-m1 volumetric flask. tion is necessary. Dilute to the mark with distilled water. Add (8) Pseudoagglutinatipnofthered 1 drop of,,1% gentian violet and mix. cells occurs in cases of abnormal plasma proteins (myeloma, . Hodgkin's disease) or c. Procedure: with an increase in cold agglutinins (viral (1) Draw well-mixed capillary or ve- pneumonia). This clumping is preVented by nous blood exactly to the 0.5 mark in a white adding 0.01 g of gelatin to 100 ml of Hay- blood cell diluting pipet. This blood column em's solution. Heating of the Hayem's solu- must be free of air bubbles. tion to 64° C counteracts the effect of cold ) Wipe the excess blood from the agglutininswithouthemolyzingthered outside of the pipet to avoid transfer of blood cells. It is claimed that Gower's solu- cells to the diluting fluid. Take care not to tion prevents pseudoagglutination of erythro- touch the tip of the pipet with the gauze. cytes. (3) Immediately draw 2 percent acetic (9) In polycytheia the number of red diluting fluid to the "11." mark' while cells may be too great to permit acccurate rotating the pipet between the _thumb and -enuineration if a dilution of 1:200 is used. forefinger to mix the specimen and diluent. Blood is therefore drawn to the 0.3 instead Hold the pipet upright to prevent air bubbles of the 0.5 mark and the sample diluted to in the bulb. (See figure 5-1.) the 101 mark in the usual manner. The result- (4) Mix the contents of the pipet for ing dilution of 1:333 is used in the calcula- 3-5 minutes and fill the counting chamber tion. as described in the technique for red blood (10) In anemia the number of red cells cell counts in paragraph 5-1c. may be too small to permit accurate enumer- (5) Allow the cells to settle for about ation if a dilution of 1:200 is used. Blood 3 minutes. Under low-power magnification is therefore drawn to the 1.0 instead of the and reduced light, focus on the ruled area 0.5 mark and the sample diluted to the 101 and observe for even distribution of cells. mark in the usual manner. The resulting di- (6) Count the white cells in the four lution of 1:11)0is used in the calculation. 1-sq-nimcornerareascorrespondingto g. Normal Values:, those marked A, B, C, and D of figure 5-4 (1) Adult Male: 4.2 -5.4 million RBCs in each of two chambers. per cu rntnr (7) Countallthe whitecellslying AFM 160, 1 5-7 se mumps illiiimpripi IF!hopitop

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IIVIPROVED NEUBAUER RULING

Figure 5-4. Hcniacytometer Counting CharnbeAreas marked A, B, C, and D ar' used to count white blood cells. within the square and those touching the 0.5 mark and diluted to the 11 mark with upper and right -hand center lines.- The white WBC diluting fluid. All the blood is washed ceilk which touch the left-hand and bottom into the bulb of the pipet (which has a lines are not to be counted. In each of the volume of 10). Therefore, 0.5 volumes of fou r 'areas conduct the count as indicated blood are contained in 10 volumes of dilut- by the "snake-like" line in the figure 5 ingfluid. The resulting dilutionis A variation- of 'more than 10 cells between These figures are arbitrary and refer strictly any of the four areas counted indicates un- to dilution and not to speCific volumetric even .distributio-nandrequiresthatthe measurements. procedure be repeated. The depth of the counting chamber d. Calculation: is 0.1 'mai and the area counted is,4 sq mm -Routinely, blood is drawn to the (4 squares are counted, each with an area 160 51/61 8=227-4 5 December 1973 of 1.0 sq mm; therefore, 4 X 1.0 sq inm mark and the diluting fluid (2 percent acetic a total of 4 sq mm). The volume counted acid) is drawn to the 101 mark. The result- is: area -x depth = volume. Four sq mm x ing dilution is 1:100. 0.1 mm s 0.4 cu mm. (6) In cases of leukopenia, the white (3) The formula is as follows: pipet should hfilled to 'the 1.0 mark and 'mark with 2 percent acetic Average (of two chambers) number of diluted to the acid. The resulting dilution is 1:10. dilution (20) WBCs counted xvolume (OA) WBCs per cu (7) If nucleated erythrocytes are pres- ent, the count is corrected by the folllowing (4) Example: formula:

First Chamber . Second Chamber 100 Cells counted- Cells counted observed count x in each square in each square 100 + 5 nucleated erythrocytes 35 45. The percent nucleated erythrocytes is ob- 40 37 tained from the differential count (see para- 36 graph 5-2b). 44 (8) Unopette procedures are available 158 WBCs counted 162 WBCscounts for RBQ and WPC counts. Average of the two chamber counts: g. Nounal Values: 158 329 160 WXCs, 162 2 (1) Adults (both sexes ) :4,50041,500 320 WBCs per en mm. 160 x 20 0.4 8,000 WBCs per cu (2) Childhood: 6,000-14,000 WBCs per en e. Sources of Error. See paragraph 54c. Birth: 9,000-30,000 WBCuper f. Discussion: MM. (1) The allowable. error when four large squares are counted is ±20%. 5-4. Total Eosinophil Count: ing eight large squares decreases the error to. ±4570. a. Principle,' A. sample of blood is diluted with a solution which. selectively stains the (2) The importance of clean, dry dilut- eosinophils and eliminates all other leuko- ing pipets cannot be stressed too much as cytes and erythrocytes from view. Following the greatest source of error in the counting mixing, the specimen is introduced into the of both white and red blood cells is the use counting chamber and the number of eosino- of wet and/or dirty pipets. phils in .a known volume of blood is counted. The counting chamber must be scrupulously clean and free of debris which b. Reagent. Pilot's Solution: Add 50 ml might he mistaken for cells. propylene glycol, 40 ml distilled water, and 1.0 ml sodium carbonate 410% aqueous) to (4) The minimum blood sample recom- 4 150-mi beaker. Mix well and filter. Add mended for performing routine white blood 10 ml of phloxine % aqueous) just prior cell counts is that obtained using one pipet to use. The- addition of 10 units of heparin and counting two chambers as previously to this solution prevents clumping of cells. outlined. (5) In cases where the white cell count c.Procedure: isexceptionally high, as in leukemia, the (1) Draw capillary (or venous blood) dilution should he made in the red blood cell to the 1.0 Mark in each of two white cell diluting pipet. The blood' is drawn to the 1.0 diluting pipets. FM 160 -51 /TM 8-227-4 5 December -1973 5 -9 -(2) Draw -Pilot'ssolution tothe Lion renders the erythrocytes invisible;, and- mark- of. each the sodium carbonate causes lysis of all the (3) Mix by gently shaking the pipets- leukocytes except, the eosinophils. The phlox- for 30 seconds. ProlOnged and harsh Shaking ine stains the eosinophils.%:, will tend to cause rupturing of the eosino- (6) In the -Thorn. test an eosinophil phils. count .1/ma made 'prior to the initiation (4) Expel the cell -free liquid, from the of the test proper. This establishes the pa- -capillary pOrtion of the pipets. tient's total eoSinophil count, to which the (5) Using one pipet, charge both.cham- response of the adrenal cortex to ACTH can hers of a hernaCytorneter and with the other be judged. ACTH is then injected and.at pipet charge both chambers of the second an ,interval of 4 hours, another' eosinriphil. hemacytorrieter. count is made. The interpretation. of this test is as followS: (6) Allowbothhemacytometersto stand fOr 15 minutes to permit staining of NormalapProximately a 50:percent the eosinophils. To prevent evaporation, the drop in eosinophils. hemacytometers are placed on a damp towel Cushing'S disease (hyperadrenal- and covered with Petri dish. covers. ism)-0:30 eosinophils per cu .mm. Addison's disease (hyPOadrenalism) (7 Under low-powermagnification, no change in eosinophil count. count the red stained eosinophils in the en-- tire ruled area (9 sq mm) on each of the (7) Nasal smears are also submitted;for -four chambers (a total area of 36 sq 'mm). eosinophil. evaluation:- TheSe-smearsare . The chamber has a depth. of 0.1 mm so the stained with Wright's stain and examined total voluMe is 3.6 cu mm. for the presence of eosinophils. Calculations: g. Normal. Value: 150 -300 eosinophils cu Number of eosinophils counted x 7dilution (10) volume (3.6) eosinophils per cm, Trim 5-5. Absolute Basophil Count (Cooper): e. Sources of Error. See paragraph 5-2. Principle. Erythrocytes' are lyged with f. Discussion: cetylpyridiniutn chloride.. The-.,basophils are rendered .insoluble in ammonium sulfate and (1) Fuchs-Rosenthal or -Levy hemacy- stained -with toluidineblue. The stained torneter is preferable to a standard counting'basophils are then countedin .aFuchs- chamber sinceits greater volume(4.0 x Rosenthal counting. chamber.. 4.0 x 0.2 mm) allows for counting of more cells, thereby reducing the statistical error. h. Reagents: Counting two of these chambers is equiva- (1) Solution 1., Add 100 mg of EDTA lent in accuracy to seven standard chamber (disoclium salt) to a 100-ml volumetric flask. counts: Dilute to the mark.with 0.85% NaCI, s (2) In eosinopenia, it is necessary to set (2) Solution2.Mix 25 ml of 0.5% up more chambers to provide an optimum eetylpyridininm chloride solution (aqueous), number of cells,,to be counted. 20 ml of 0.8%, toluidine blue in 5% alumi- 3) The eosin-acetonedilutingfluids num sulfate, and- 20 ml of distilled water are unsatisfactory and should not be used. in a.'beaker. Filter and transfer-to a reagent, bottle. Estimationofeosinophilsona stained blood smear are too inaccurate for c. Procedure: e because of poor cellular distribution. (1) Add 0.08 ml solution 1 to a 10 x 75 (5) The propylene glycol in Pilot's solo- trim test tube. 160-51/TM 8-227-4 5 December, 1973

Add- -0.02 ml blood from a slain punc- blue, 1.4. grams potassium :oxalate, and 0.8 ture and mix. g -NaCI in,,distilled.water. Dilute, to 400:ml. Add .0.1:ml-solution 9, Filter before use. Fuchs- Rosenthal chambers c. Procedure:. ng a capillary pipet. (1) Add -.3or' drops of ,new methyl (5) Place --the- chambers: in a moist:at- ,ene blue and- 3 or 4 drops Of blood (venous mosphereand. allow the cells to settle for 5.- or capillary) to 'a small test tube or mix in a microhematocrit.J.ubeMix, well and let stand for 15 minutes. (6) Count the basophil_ in all sixteen arge squares. The.basOphils are purple-red,. (2) Place a small drop on a clean glass rrietachrornatically-stained cells. slide and prepare:a thin smear- (see' chapter: 3, paragraph 3,6). d. CalcUlations..The charnber, has an area (3) After the slide: has dried, :foc . of 16 sq,.mm and-a depth.of,0.2- mm; there- on the smear under low:-power magnifica- -forei the. volume mint. equals 16 r 0.2 or tion and-locate the thin portion of 'the smear:: 3.2 cu Mm;The formula le.r.as follows: (4) Switch to oil immersion magnifica- Total basophils counted Basophils per -cu ntrn. tion and count 200 erythrocytes, including .reticuloayteS in each of five different areas e. Sot-frees of Error. See paragraph 5-2e. on the smear (a total -of 1,000 cells). Re- f. Discussion: cord the number of retieulocytes. observed. (1). in absolute basophil count istsed d. Calculation: to study-allergic reactions. Number of reticulocytes counted 'To reticulocytes (2) The EDTA solution prevents plate- 10 let agglutination. e. Sources of Error: (3) The aluminum sulfate acts as (1) Equal volumes of blooC and. stain mordant to improVe the staining qualities give optimum staining conditions.,-An excess of toluidine blue. of blood causes the reticulum to understain. An excess of stain usually obscures the re- to I) -Anotherprocedureemployed tieulurn. perforM a hasophil cOunt is the Neutral Red Technique. It has the following_disadvan- (2) Crenatederythrocytesandrou, tage: EOsinophils can be Stained; basdphils !Num formation make an accurate wont ci . he water soluble, and platelet aggre- difficult to perform, gates can form. Stain precipitated on erythrOcytes causes them to appear as reticulocytes. (5) Basopenia referstoa decreased number of basophils. Basophilia refers to an (4) :Dirty- slides causes uneven spread- increased number of basophils. ,) The dye solution should have ade-. 5-6. Reticulocyte Count: quate time (step 1)to penetrate the cell a. Principle. Nonmicleated immature and stain the reticulum. erythrocytes Jdiffusely basophilic` erythro-, f. -Discussion: cytes)retain some basophilicsubstance. (1) 'Reticulocytes, are nonnucleated With supravital staining this substance' ap- erythrocytes :which exhibit blue reticulum pears as a reticulum within the cell.- This strands within their cytOplasm when stained cell is then known as a reticulocyte. supravitally. Whenstainedonlywith h. Reagent, NeW Methylene 'Blue Solu- Wright's stain, they are buff-pink in color ion.' Dissoli./0- 0.5 grams of new methylene and larger and darker than erythrocytes. 60-51/111 ,-227-1 suer 1973

-.RetiCuloeytes: serve as an index. of (a) With capillary pipet _-i oduec the activity of the bone marrow in blood drop of well-mixed spinal fluid' into one regeneration; As 'such, these counts are of countingchamberofaliemacytometer. value in following anti-anemia- therapy. Sat- (CAUTION: Avoid contamination by care- isfactory response to therapy is evidenced ful, handling of spinal fluid.) by an increase. of :reticulocytes, in the pe- -(b) -Examine the entire ruled area. ripheral blood. "Increased reticulocyte counts for the presence of cellular elements. -If both -also. occur whenever .thereis: rapid- bone marrow activity as in leukemia or blood leukocytes and e'rythrocytes are observed, note the condition of the red cells(fresh regeneration associated with hemorrhage-or..or crenated), (c) Count all cells in the entire ruled 3) Several methods for staining and- area(0.9 en out°. Counting .reticulocAps are in common -use. Compared to the vs& of alcoholic solutions (2) Turbid spinalfluidisset up as Of dye, 'methods employing -saline solutions follows: of new -methylene blue can give slightly (a) Draw cerebrospinal fluid diluti higher values for reticulocytes. Foncompara- fluid to 1.0 mark of the white blood c tive studies, the same method should be diluting pipet. . used throughout the work. . (b) Carefullydraw well-mix d (4) PreCipitated stain is-often confused specimen of spinal fluid to the 11 rruirk with reticulum but can be recogniz-ed by its (c) Shake the pipet for 9 minutes. presence 'throughout the smear. and apart to _the specimen. from the red cells. Precipitation can be elirn- inated as a source of error by frequently (d) Discard the fluidin the capil- filtering the stain. lary portion of the pipet. (e) Charge thecounting chamber g. Normal Values: and allow the cells to settle for 5 minutes. (1) Birth.:. (f) Under low -power magnification (2) AdultS (both, sexes.) : 0.5.4.57 count' all cells in the entire ruled' area (0.9 cti mm). Cerebrospinal Counts: (g) ,Switch to high -power and per- ri rough differential count. a. Principle.Cerebrospinalfluidisde- livered to a counting.chamber and examined (3) For very cloudy spinalfluid- a microscopically for bloodcells.Normally, white blood cell dilution -is made as folio-Ws: spi-nal fluid is clear. If the spinal fluid. is (a) Draw spinalfluidtothe0.5 cloudy, a dilution is made before charging mark in the white blood cell diluting pipet. the counting chamber. (b) Draw cerebrospinalfluiddilut-' h. Reagent. Cerebrospinal Fluid Diluting ing fluid to the 11 mark. Fluid: Add 10. ml of glacial acetic acid and (c) Perform steps (c) through (g) 0.2 grams of crystal violet to a 100mI Volu in paragraph 5- 6c(2). metric flask. Dilute .to the :mail{ with dis- 'tilled water. cL Calculations: (1) Clear spinal fluid: c. Procedure: Number_ of cells counted NOTE: Set up cellcounts on spinal volume (0.9) cells, perCU mm fluids within 30 minutes after. withdrawal \- of the specimen. )Turbid spinal fluid: (1) Clear spinal fluid is set up as .fol- Numiwr- of cells counted X dilution (10/9) lows: volume (0.9 cu mm) cells per cu mm AFM 1607517TM 8=-227-4 5 December 1973

Very clouded spinal fluid: 5-8. Semen Analysis: Number of cells counted x di a. Principle.Serridn-analysisinvolves volume (0.9 en mm) gross examination (volume, color,turbidity, cells per en Tom viscosity, and pH) Wand microscopic. exami, e. Sources of Error. See paragraph 5-2e. nation(motility and spermatozoa count). f. Discussion: b. Reagent. Spermatozoa Fixative Solu- tion: -Add5grams sodiumbicarbonate 1) If more than 100 leukocytes per eu (NaHCO,) and 1 ml, of formalin to a 109; trim. are present, centrifuge the undiluted ml.-volumetric flask. Dilute, to the markwith specimen,- make a smear, and stain with distilled water. modified Wright's stain. Perform a routine differential count and also estimate the ratio c. Collection instruction. Aphysician will of erythrocytes toleukocytes.. NOTE: It usually give the instruction; however, the may be necessary to use egg albuminor- patient should be reminded of several criti- cell-free serum to r the sediment adhere cal points. to the slide. (1) The patient may be required to ab- 12) Normally the spinal fluid is water- stain from intercourse for a period directed It can be turbid if cell count is 500 by the physician. e cells per cu mm. If there is frank (2) The specimeniscollectedina blood, with spontaneous clotting, the indica- clean container that 'has been prewarmed tion-; are those of a bloody tap. Xantho-.to body temperature. ehromia develops after subarachnoid hemor- (3) The specimen should be delivered rhage haS been present for a few hours and to the laboratory within 30 minutes. is due to disintegration of, blood pigments. Xanthochrornia may also develop from tu- (4) The specimen must be kept at body mors, abscesses, and inflammation. temperature (370- C) and not subjected to extremes of heat or cold. (3) Cell counts above 10 are considered to be evidence of intracranial disease. The d. Gross Examination: predominant cell in most viral infections, (1) Record the time ofcollection and syphilis, and tuberculous meningitis is the receipt of the specimen. lymphocyte.Bacterialinfectionsdueto (2) Measure and record the volume. meningococcus, pneumococcus, etc., usually result in a predominance of the neutrophil. (3) Observeandrecordthecolor Cerebral and extradural abscesses as well as (white, gray, yellow, etc.), turbidity (clear, subdural hemorrhages produce a neutro- opalescent,opaque,etc.),andviscosity phiIic response although' bacteria are not (viscid, gelatin, liquid). demonstrated. (4) Determine the pH with a pH rea- gent strip and record this. (4) Biochemical, bacteriological, viro- logical,serological, and hematological ex- e. Motility Exarnimation: aminations are all necessary to reflect the (1:) When the specimen becomes -fluid true condition of, the cerebrospinal fluid. The (within 15 to 30 minutes after collection the current laboratory standing operating pro- semen liquefies by the action of fibrinoly- cedures should give guida.hce to the efficient sin), place 1 drop on a slide(prewarmed to method to accomplish all the necessary ex- 37° C) and place a coverslip on it. aminations. (2) -Underhighdrypower,count g Normal. Value: 0-5 cells per motile and nonmotile spermatozoa in two or (chiefly lymphocytes), more areas. Only those which moveforward AFM160-11/TM8 -2274 5 December 197 5-13 actively are considered motile. Record the sperm are quite uniform in appearance. Any percent of motile spermatozoa seen. sperm with rounded, enlarged, small, or (3) Repeat this procedure in .3 hours bibbed heads are abnormal. Abnormal tails and 6 hours, using a new drop frOm the are enlarged, small, irregular in length, .ab- original specimen each time. sent, or multiple. See figure 5-5 for mor- phology of spermatozoa. . Spermatozoa Count: g. Calculations: (1) Draw the liquefied semen to the 0.5 mark on white blood cell diluting pipet. (1) Number of sperm counted x dilution (20) (2) Draw fixative solution to the 11 volume 0.4) mark on the.WBC pipet. ---,--- sperm per cu trim (3) Let the mixture stand until the (2) Sperm per cu mm x 1000 sperm mucus dissolves. per ml (4) Shake the pipet thoroughly and 'charge a hemacytometer. h. Sources of Error: (1) Delay in analysis results a lower (5) Countthespermatozoainthe percentage of motile forms and a lower same manner as you would count white count. blood cells. (2) Temperature extremes cause sper- (6) After counting the sperm, examine matozoa to die. the morphology and report the percent of (3) See paragraph 5-2e for sources of abnormalforms.Morphologically-normal error when counting. =.

==11110

NORMAL PIN-HEAD IMMATURE

GIANTJAEAD ACUTE TAPERINGFORM AMORPHOUS FORM.

DOUBLE TAIL CONSTRICTED HEAD DOUBLE HEAD Figure 5.5. Morphology of Spermatozoa. 5-14 AFM 160 51/ym$--2274 5 Deceinher :.1073

n: (1)-One hematocrit point ¢ 0.34 (1) Semen analyses are Usually per- hemoglobin per 100'ml" of .blood. formed for sterility studies following a vasec- (2) One hematocrit point 107,000 tomy or 'a barren marriage. erythrocytes per cubic millimeter 'of blood.... (2) Semen analysis can be performed for medico-legal cases involving rape or to 540. Maerohernatocrit (Wintrobe): support or disprove a denial of paternity on ,a. Principle. Whole blood is collected by the grounds of sterility. venipuncture, mixed with an anticoagulant, (3) Semen is derived from the follow- and pipetted into a Wintrobe tube. The tube ing: testes, seminal vesicles, prostate, epi- is then centrifuged to pack the cellular ele- didymides, vasa deferentia, bulbo-urethral ments. The height of the packed red, cell glands, and urethral glands. column is read and reported as a. percentage of the total blood column. This percent is j.. Normal Vallies: the hematocrit or packed-cell volume (PCV). 1) Volunie: 1.5-5.0'ml. b. Procedure: (2) pH: 7.2-7.6. (1) Draw 5 ml of blood by venipunc- (3) Motility: 60-90%. ture, place, the blood in a test tube contain- .(4) Spermatozoa Count. 60-150 mil- , ing EDTA, and mix thoroughly by gently lion per ml. inverting the tube several times. (2) Draw the blood into a capillary pipet.Fill the Wintrobe tube to the "0" SECTION 9-11EMATOCRIT mark by inserting the pipet to the bottom' 9.Introduction of the tube, while holding the tube at .a 45© angle. As the -tube is filled,. slowly withdraw a. The hematocrit or the packed-cellvol- the pipet so that the tip is always just below ume is the percentage Of the total volume the level a the blood. The blood column Must of red blood cells in relation to the total be free of bubbles. volume of whole blood. The procedure is per- formed by filling a tube (capillary or Win- (3) Place the Wintrobe tube in a cen- trobe) with blood and centrifuging at con- trifuge and spin at 3,000 rpm for 30 minutes. stant speed for a constant period of time. The RCF must be 2250 G. The packed-cell volume is then measured. (4) Read the height of the collumri of The hematocrit,hcan also be determined by packed red cells at its junction with' the automated sequential analyzers but isus- huffy coat. This level, as shown on the as- ually a calculated value. Two manual methods cending scale etched on the tube,isthe are presented in this chapter: the macro- hematocrit and is reported in percentage . method of Wintrobe and a rnicromethoci. (see figure 5-6).

h. The hematocrit is the most useful single c.Sources of Error: index of the degree of anemia or polycythe- (1) Failure to mix the blood specimen rnia. It can be the most accurate (2-4 per- thoroughly yields an unrepresentative sam- cent error) of all hematological determina- ple. Always remix the sample immediately tions. In contrast, the direct red blood cell prior to filling the tube. chamber count has a percent error of 8- (2) Prolonged application of the tour- ti.107,.., The hematocrit is, therefore, prefer- niquet causes a concentration of blood cells. able to the red blood cell count as a screening This results in, an elevated .value. testfor anemia. Erythrocyte counts and hemoglobincanheestimatedfromthe (3) The. sample must be spun 30 hematocrit by the following formulas:- minutes at 3;000 rpm. Neither the time nor AFM 160-51/TM S -227 -4 5 Decembe1973. 5-15 2 the speed can be -varied without altering the results. The -TiCF must be 2250 a (Fl) Misreading .the red cell level by in _ . eliding the buffy. coat yieldsan increased result.: d. Discussion: (1) To allow for better sepa ation of the cellular elements in the hematocrit, the tube should stand for at least 15 minutes prior to centrifugation. This separation is adequately accomplished. if a1 -hour sedi- mentation rate precedes the hematocrit. Above the column of packed red cells is a layer of white cells and throm- bocytes called the "huffy coat." This layer, is normally about 0.5 to 1.0 mm in thickness and serves as a rough guide of white cell. and plktelet numbers. It should not be sub- stituted for the white cell countas variations in size of cells influence the number esti- mated. (3) The appearanceoftheplasma might suggest certain abnormalities. Nor- mally, this fluid is clear and straw colored. A golden-yellow colored. plasma might in- dicate the presence of jaundice While a y Ceat creamy-opaque appearance would suggest an excess of fat in the blood (lipernia). (4) Innormal bloodcentrifugedat 3,000 rpm for 30 minutes, trapped plasma comprises an average of 4.3 percent of the red cell _column. This is reduced to 2.5 per- cent by centrifuging for 55 minutes. Anise-- cytosis and spherocytosis do not alter plasma trapping. Variation in the amount of plasma trapping is so small after centrifuging for minutes, even with pathological bloods, that it is recommended 4ver the 30-minute tentrifugation when greater accuracy is de sired. e. Normal Values: (1) Birth:. 44-470 (average 54 %).

(2) Childhood: . 34-41% (average 38%). AdultMales:40-54%(average 47% Figure 5-6. Wintrobe He natn r t Tube. AFM .160?51/TM December. 1973

. . Adult Female 47% (average diVrOper centrifugation leads to 42% varied, results.For. good quality control, main prescribed centrifuge speed and 5.-11. Mierohematocriti time. a. Principle. A capillary tube is filled with nisi-ending the redcelllevel by whole blood by gravity and capillarity to includkfi the buf! coat causes elevated within 1 or 2 cm of the end. The unfilled end is sealed and the tube is centrifuged. -d.. Discusslo After centrifugation, the capillary tube is (11) The microhernatocrit technique is placed in a reading device and the hemato advantageous because of speed, and because it value determined. only a- small quantity of blood is necessary b. Procedure: for the determination. An additional advan- ,(1) If anticaaguld venous blood tage is .the ease with which this procedure the specimen, fill a pla apillary tuber with. rsuarlapted to infants and small children. h ood. If blood` without anticoagulant is used, The .rificrohdinatocrittechniquerequires fi a.hepar.inized capillary tube] ,with the onlyaa simple capillary puncture whereas in blood ..siecimen. A hepIvinizedkicapillAry the .ntrobe method venous blood must he tribe is Identified by a red line on ifs e nothen advantage is the use of dis- (2)- Seal the unfilled end of t posalcapillary tubes. with a cdrnmercial-sealifigiVax or clay- the rniorohematot cannot be ., read promptly, the capillary tubes must be (3) Place the cipillary tithe in one of' properly identified and placed in a vertical the numbered slots in the centriftige head in such a manner that thkl,ealed)end is in position. Slanting of the cell layer, will occur contact with the peripheral 'Ailajt the cen: if tubes are left in a horizontal position for trifuge head. CAUTION: Failure to have the'more than 30 minutes. capillary tube in contact with the rim might e. NormalNalues: result in a shattered tube as the centrifuge- (1) Birth: 44.64% (aN1 rage 54%). speeds up. (2) Childhoqd: 34 -41 %© (average (4) Screw theflatcentrifuge head '38% ). cover in place. (3) AdultMales: .40-54%(average` (5) -Centrifuge the capillary tube at 47 ). 10,000 rpm for 5 minutes. (4) Adult Females: 38 =47%© (average (6) neterminethehernatocrit, value 42%). with' the aid of a microhematocrit reader. NOTE: Since there are a variety of readers SECTION C.ERYTHROCYTE available, it is necessary that the technician SEDIMENTATION RATE carefully follow the directions of the ripriu- facturer for the particular device utilized. 5-12. Introduction. The erythrocyte sedi- mentation rate (ESR) measures the rate of c. Sources of Error: settling of erythrocytes out of the cellular- (1) Impro.per sealing of the- capillary plasma suspension. The ESR is complex bio- tube. causes the blood to blow'out of the logical phenomenon influenced by a variety capillary tube during centrifugation, of flictors. Size and shape of erythrocytes (2) Capillary tubes must, Abe properly cause the ESR to fluctuate. Microcytes tend identified. Numbered holders for 'capillary tosettle slower than normal cells,while tubes are available. Place a tube in a slot on macrocytes fall more 'rapidly.. An increase in the holder and record the riumber on the spherocytes and/or bizarrely-shaped red cells retards the sedimentation rate. With a de- laboratory request slip. .;40 AFM 160-51/TM 8-227-4 5 December '1973 5-17

creased hematocrit there is .less retnrdation , ontte descending scale etchedon the tube. of sedimentation- by the erythrocytes them- Each mark equals 1.0 mm while each mania selves and they tend to settle,faster.'Vorrec- ,bered mark equals 10 nim (1 cm). The figure tions for anemic blood are available; how- obtained:is reported in mirrper houras the . ever, 'most- experts consider .thiscorrection "uncorrected".erythrocytesedimentation useless and invalid. Always correct' for rate. anemia when requested to do so by a 1311Y- (6) If a "Corrected" sedimentation siciariAn increasedhematocritlabove 'requested, perform a hematocrit Cala-11001's )retards the sedirhentation rale. to correct for sedirrientation rate are avail- certain ase proCesses.therels an increase able in the Federal Supply. Catalog'. in fibrinogen and/or globulin. The creased concentration of these substances causes-the SOUrces of Error: erythrocytes to settle- faster, The determina- (1) The blood specinien must be pro tion of the erythrbcyte sedimental on rate erly mixed kith the -proper anticoagulant to is Performal by many methods.- -The most obtain an undiluted-representative sample. popular, the Wintrobe-Landsberg, is Outlined (2) DelaY in performing, the test be-

below.. yond 2 hours after the blood,is drawn de- - creases the sedimentation rate.-- 5-13.- Determination of Sedimentation Rate (3) Increase in temperature accelerates (Wintrobe-Landsberg): the rate.Desirable temperature range is 22° C to.27° C. a. Principle.Anticoagulated blood placed -in a narrow tube. The blood cells (4) The tube must be vertical. .k 3' settle out -.of the suspension% leaving clear variation from -the vertical accelerates the plasma above them. The distance that 'the- rate by 30%. erythrocytes fall within a, given interval 6f (5) Dirty Wintrobe tubes or &pillary time is measured. pfpets can decrease the rate. b. Procedure: (6) Tubes should be placed free from vibration or disturbance.. (1) Draw 5 ml of blood by venipunc- ture and place in a test tube -containing' d. Discussion: EDTA (lavender top vacuum tube), (1) The erythrqcytesedimentOion -.(2) Thoroughly Mix thebloodandlrate is a nonspecific test whith suggests the anticoagulant by gently inverting the tube possibilitys of 'a disease process and tissu -,several times, being careful not to causebub- damage in the body: It is not cl anostic .but .bles fs extremely -useful in following the course if- Draw thebIood into the capillary of some diseases. pipet and fib the Wintrobe tube to the "0 (2) 'The- rate is usually increased in in- mark. This is done b_ y inserting a capillary fiammatory infections, toxemia, cell or tissue destruction, severe anemia, `active -tuberculo- pipet to the bottom of the Wintrobe tube, , while holding it, at an angle of 45°. As -the' sis,syphilis,acutecoronarythroinbosis, tune. is filled, slowly withdraw thegiipet so rheuinatoid arthritis, and malignant pros- tht the tip is .always just bekiw the level of esses. the blood.. The blood cblumn must be free of (3) ° Sicklecellanemia, polycythemia, -bubbles. hypofibrinagenemia and certain drugs usu- ally decrease the rate. -(4). place the--filled Wintrobe tube- in' ack in an exActly vertical position and Normal :Values: e the ti gie and room temperature. (1) \Nign : 0 mm. (5) At the end of exactly 1 hour, -read ) -Females: 0-20 mm. the level to which, the red -cells have settled- Children-1'3 mm. 6-18 .AFM. 160-51/TM 8-227-4 '5 December 1973

SECTI ON 1-311EMOGIATINT differences are 'genetically controlled the normal hemoglobin components are hemo- 5-14. General Informs:06n. Hemoglobin is a globin A. (HbA), hemoglobin A, (HbA,),: conjugated protein composed of the .basic and fetal' hemoglobin (HbF). HbA consti protein globin linked to 4 hem_ e molecules. totes most of the hemoglobin of a4 normal Ninet y-eight percent of all the .iron found adult while HbA, constitutes a much smaller in the blocidis contained in ;hemoglobin. amount. IMF is present during the first 4 Hemoglobintransports' oxygen andid carbon to 6 months of life and not normally present dioxide. This important substance reacts in adults. Hemoglobin S and hemoglobin 0 with. oxygen, to forth oxyhemoglobin. In the.are the most commonly occurring abnormal. tissues oxygen is released and reduced hemo- heoglobins.m Others (D, E, H, etc .Y are globin formed. Hemoglobin canreact with fOund in "rare occurrence associated with acids,bases, and oxidizing and reducing agerits.It also can exist in a variety of several types. of anemia. The various types forms. These heinoglobin compounds and of hemoglobin are separated. by elearopho- variants are .discussed. briefly. iii the follow- resist ing paragraphs. For more detailed informa- . tion, _refe,_ to the standard. _hematological_ 0.Ilemoglobinometry. The hemoglobin texts. concentration is directly proportional to the oxygen-combining capacity of blood. There- 5-15. Compounds of Hemoglobin: fore, the measurement of the hemoglobin concentration in- the blood is important as a a. Oxyhemoglobin. Oxygen combines loosely with the iron (ferrous state) hem. Greening test for cliAensea associated with oglobin. The loosely attached, xygen diffuses anemia and for following the response of, -into-the-tissues for oxidative processes The these- diseases to .treatment. There -are four basic ways to measure the hemoglobin cpn- hemoglobin then binds carbon dioxide and centration : (1) measurement of the oxygen- exists as reduced hemoglobin. combining capacity of blood (gasometric b. Carboxyhernoglobin. Hemoglobin com- (2) measurement of the iron content .(chern- bioes with carbon monoxide to form car- ical method), (3) colorimetric measurement boxyhemoglobin, Carbon monoxide has an.-of speci gravity( gravimetric method), affinity 200 'times greater for hemoglobin and (4) co metric measurement of a col- than oxygen,' does. Hemoglobin in this corn- fired derivae of, hemoglobin. The fourth bination is incapable of oxygen transport. method is t most widely used; The .cyan- in methodisthe method-- of c. Methemoglobin.Thiscompoundis thethemogl formed when the ferrous state of the heme choice a s recommended by the,,Technical Subco eeon ,Haemoglobinometryofd is oxidized to the ferric. state. This com- Stand- pound is incapable of dlci g en transpbrt. theInernationalCommittee, for a_rdizat n in Haematology.`_ d. Sulfhemoglohin.0.1', hiscompoundre- suits from the combination of inorganic sul- fides and hemoglobin. This compound is in- 5-18. Cyanmethemoglobin Method: capable of oxygen transport. a. Principle. flood is diluted with 'a- di-: -e. Cyanmethemoglobin.Thiscompound bile solution of .potassium ferricyanide and results when methemoglobin combines With potassium cyanide at a slightly alkaline pH. the cyanide radical. This compound is used The ferricyanidtconvertS the-.hemoglobin to in hemoglobiaometry: methernoglobin. The cyanide thenreacts with the thethemoglobin to forp the stable 16.Variations, of. Hemoglobin. The va- cyanmethemoglobin. The- con- riations of hemoglobin' occur due to struc- tent is then determined in a spectrophotom- tural differences in the globin protein. These eter. Reagents: calibrated Sahli pipet." Use of a- properly cal-. (1) Drabkin's Rsagent (Cyanmethern- ibrated auto-dilutor is recommended. oglobid Reagent). Available 'inpellets or (3.) Wipe the excess bide' from the powder in the Federal.SuPply Catalog. outside of the pipet and add the cbntents of (2) Cyanmethemoglobin Standards. the pipet, to the covet containing the Drab-T- Stable, accurate standards- -.cif cyanmethemo- kin's solution. globin. are available cornmercially. These so- (4') Rinse the pipet several times with lutions usually contain about 80 mg per al the contents of the covet to assume' complete. hemoglobin as cyanmethemoglobin. For ex- delivery of the blood sample. Mix well and ample, if a.1:301 (0.02 ml to 6.0 rn1). dilution allow to stand- at room temperature for 10 is used it the procedure, an 80 mg per dl minutes. standard corresponds to: 80-, x 301/1000' (5) At a wavelength, of 540 rim, Set the 24 g hemoglobin per dl in undiluted blood. spectrophotometer to zero absorbance with Standards of other concentrations and dilu- the blank covet. The blank contains 6 ml of tions may be calctlated by this Same for- fresh Drabkin's solution. mula.- . (6) Read and record the absorbance of the unknown. c. Example of Preparationof it Calibre-, ton 'Curve: e. Calculations. Obtain the grains he o;

(1) With an .80 mg per dl standard, globin per dl from the calibration curve. , set up standards as follows: f. Sources of Error: Cyanntethento Standardglobin Diluent Cone accurate calibrationof Sahli pi- 0 mi 6 MI 0 pets re u. in errors in accurcy .and pre-_ 2 1.5 ml 4.5 ml 6 g per di cision. 3 s ml ml 12 a per dl (2) The 4 4.5 'nil. 1.5 ml 18 g per dl spectrophotometer -mustbe 5 6 ml 0 ml.- 24.g per dl standardized accurately to obin valid -re- sults. (2) Place tube 1 in the well of the spec- (3) Cuvetsthatare ' dirty,wet, trophotometer at a wavelength of 540 nrn. scratched, or mismatched" are sources of er- Adjust the spectrophotometer to zero. ab- ror. __sorbance. (3) Read and record absorbance for (4) Lipemic blood produces turbidity tubes 2-5. in solution which can 'elevate results. (5) Heavy smokers usually have - (4) Plot the absorbance versus hemo- sultS 10 percent lo vier because of the pres- globin concentration on linear graph paper. ence of carboxyhe*globin. Carboxyhemo- (5) Check the, calibration daily with globin requires a full hour to be converted one of the prepared standards NOTE: The to eyanmethemoglobin. hemoglobin standaNs. should .be capped and (6) Trakl_dn's solution deteriorates stored in the refrialitor untiused. When rapidly in light----4-tRould be stored in a used to calibrate for hemoglobin determina7 dark brown bottle and refrigerated. tions, they are allowed to warm, up to "rod temperature to minimize condensation of g. Discussion: moisture from the air on the civets: (1). Precautions in the Use of Cyanide:' Procedure: (a) 'Always fillpipets with. suction bulb, and never by application of section by )Pipet (using suction bulb) exactly mouth. Use automatic dilutor& to dispense 6 ml of Drabkin's solution into a covet. Drahkin's solution whenever prissible.. (2) Ekaw well-mixed venous or capil- - (h) Immediately clean up any Spille lary blood exactly to the 0.02 mark, of a Drabkin ution with a wet cloth. Dispose' 5-20 FM 16041/TM 8-2274_ 5 December 1973

of the so on with a wet eloth.--Dispose-=of (2) Childhood: 10-5 g hemoglobin per the solu on by flushing, it down the sink dl. With w e (3) Adult Males :14-17 .g hemoglobin c) Store' Drabkin's reagent pellets per. dl. or po der in a locked cabinet. (4)- Adult Females: 12-16 g hemoglo, d) Exercise extreme care when pre- bin per dl. parg Drabkin's aolution. This.sol'utiorr is 'poinous. Take precautions to avoid getting 5-19. Det,tion of Hemoglobin S and Non tholution into the mouth and inhaling the Sickling' Hemoglobins: mes. (e) Antidotes .for cyanide with out- a. Principle,- Erythrocytes are introduced into a phosphate buffer splution containing -lined procedures for administration should lytic agent. The red be posted in a conSpicuous place in the lab- a reducing agent and oratory. Speed in giving the antidote treat--cells are lySed and the. hemoglobin isre- ment is essential. Below is a suggested pro- duced. Reduced sickling types of hemoglobin cedure which may be adopted: are insoluble in phosphate buffer and tur- _ bislityresults._Qn addition of urea_ herrioglo- Sum mon a doctor; but start treatment-= bin S dissolves. inimecliatel Give amylitrite by inhalation (break b. Reagents: capsule ; hold Under nose). (1) Stack' Phosphate Buffer Solution. Open window forfree circulation of air. Dissolve 160.48- grams anhydrous potassium (2) Cyantnethernoglobinistheniost dihydrogen phOsphate (KH2PQ4), AR, and able of the various hemoglobin pigments 28.88 grarris anhydrous potassium' --monohy- drogen phosphate (.1(21-1P0;), AR in a 1-liter showing- no evidence of deterioration after refrigerator. The volumetric flash containing. 500 ml of dis- 6 years of storage in with distilled availabilityofpreparedstandards-isa tilled water. Dilute to 1-liter distinct advantage of this techniclue.All water.. hemoglobinderivativesare convertedto (2) Dith-oriiteReagent.Add .20.0 cyaiimethemoglobin with the exception of grams_ dithioe (Na2S,O,211,0),:and 0.25 sulfhernoglobin. grams saponi' to a 100-m1 volumetric flask. (3) This method is highly accurate and-.'Add 80 ml of k phosphate buffer solu- is the most direct analysis available for total tion. Miwell. D lu'td the mark with dis- heroin or hemoglobin iron. Its disadvantage till&I w This rea ent remains stable un- is the use of cyanide cornpolinds which, if der ref rige titian at C Ter approximately handled carefully, should present little haz- 1 month; WAR G. Sodium dithionite h- agent may ignit allowed to beCome damp. ard. Use only clean dry u s in handling. (4) For accuracy in hemoglobin deter- minations,itis- absolutely necessary .that (3) Urea (USP). the spectrophotometer and. Sahli pipets be c. Procedure: accurately calibrated. (1)- Pipet 2 ml of dithionite reagent in (5) Venous samples give moe constant 12 x 75 mm test tube. values than capillary samples. (2) Add 0.02 nil of well-mixed anti- (6). If the procedure is performed prop coagulated blood (collected in EDTA). erly, the degree of accuracy is ±2 to 3%. (3) Mix the contents and alio to h. Normal Values: stand at room temperature for 5 minutes. (1) Infants at birth: 18-27 g hemo- (4) After 5 minutes examine the tube globin per dl. for turbidity a lined reader (see fig- AFM 160--,51/TM 8-227-4 5 December 1973 1 of hemoglobin found in Negroes and people from Mediterranean areas. (2)'` The degree of erythrocyte- sickling is dependent on the 'concentration of hemo-

_globin. SS, SC, and .SD cells sickle more rapidly than AS cells. NewbornS with sickle cell .anemia, have erythrocytes more resistant to sickling due te the presence of hemoglobin

(3) The dithionitetestalsodetects other sickling types of hemoglobin: Urea causes heLnoglobin S (end structural van -, ants of hemoglobin 5) to dissolve. Other hemoglobins remain turbid in the Presence of urea. (4) _This test_ is_a rapid _screening test for hemoglobin S. All positive 'tests should be electrophoresed for confirmation. f. Interpretation. Hemoglobin 5 causes turbidity i be. Hemoglobin A is solu- POSITIVE NEGATIVE ble in the phos d buffer. TEST TEST 5-20. Demonstrationf the Sickle Cell Figure 5-7.Dithionite TubeTest Phenomenon: Interpretation. a. Principle.Erythrocytesofpersons with sickle cell anemia ot trait will assume 4 sickle shape when the oxygen tension is tire 5-7). Hemoglobin S, if present, produces . lowered. This may be demonstrated by mix- rbidity in the tube. ing a drop of blood with a reducing agent d.:Sources of. Error: such, as sodium metabisulfite. (1) The use of 10 x 75 rnm test tubes b. Reagent.Sodiu a._./4- _eta.bisUlfite,2%. could cause a false ,negative result. Add 2 g of sodium m ab sulfite to a 100-m1 -) The dithionite reagent has a lim- volumetric flask. Dilut =to the mark with ited stability. The freshness of this reagent distilled water. This so_ution,, if stored at must be checked with positive and negatilie 3° Nor 4° C remains effeCtive for about 1 controls. The test should show the blue-pink week. color of reduced hemoglobin and adequate , c. Procedure: lysis of erythrocytes. (1) Place one drop of capillary(or )Unstoppered tubes containing di- venous without anticoagulants) blood On a thionite reagent decompose when left out at _clean glass slide. tie room temperature. (2) Add one or two drops of 2 percent (4) False negative results could occur aqueous sodium metabisulfite and mix. if the bloodample for testing _;is drawn- within four months of transfusion: (3) Place a coverglass on the prepara- tion and express the excess blood by gently e. Discussion: pressing the coverglass. The gentle pressure -Hemoglobin S an inherited type will produca film thin enough to permit AFM 1.60-51/TM .8-227-4 5 December 1973

examination of individual red cells It not (4) Aeetic Acid, 5%. Dilute 50 ml of. necessary to seal the preparation. glacial acetid acid to- 1-liter with. distilled (4) Obluve immediately and at inter- water. Mix well_ and storeina glass- vals of 15 t730 minutes after preparation. stoppered bottle. for si us of sickle cell information.: (5) Clearing Solution (25%. Cyclohex- anone). Dilute 12.5 ml cyclohexanone to (I: _iscussion: -50 ml with- mix: Prepare fresh (1) The likling phenomenon, is a eon, daily. seqdence of Ainherited abnormal 'type Of hemoglobin(hemoglobinS). The severity of (6) Chloroform, A.R. the anemia- as compared to the trait will c. Preparation of-Hemolysate: vary with the proportion of defective to nor- mal hemegIbbin. This 'abnormal hemoglobin (1) Place 5 ml of blood into. a tube -can-be identified electrophoretically. con ainingan, anticoagulant. (2) When a- fresh solution of sodium (2) Centrifuge and remove the plasma. metabisulfite is used, positive- cases should Wash four times with 0.9% so-. show-10-to -75 . percent-'- sickling within -15 *umchloride.- minutes. There .is ositive correlation be- (4.) Centrifuge,removefinalwash, ween the degree of kling and the severity freeze 1 hour (keep frozen if specimen is of-the disease. to be analyzed at a later time), thaw, and Normal blood treatd in.- th y add an .equal amount of- TEB buffer. MiX -.shows no sickling. well. ;Let stand for 15 minutes .at room HeMoglobin Elec roPhore Cellulose temperatureCentrifuge at medium speed- 'Acetate)': for JO minutes. Transfer supernate to an- other tube, discard precipitate. a.. Principle.Hemoglobin.-fractionsare separated by the rate of their .protein migra- (6) Add an equal volume of chloro- tion in an electrical medium. The fraction4 form, shake vigorously for minute, and are stained with Ponceau S and quantitated centrifuge. on a densitometer. The order of mobility (7) Decant ; the supernate. Determine from the cathode'toward the anodp is > thseh:mogloi7 concentration of the hemo; A, > F b. ,Reagents: (8) Adjust, to a value of 7 g hemo- (1) TEB . Buffer (pH 8.8).Dissolve globin per dl using an appropriate quantity 16.5 g tris (hydroxyrnethyl) aminomethap of distilled Water.- The amount of distilled (Tris),1.56 g disoclium EDTA and 0.92 water to add to -1.0 ml of hemplysate is g boric acid in -900 ml distilled water ; dihite determined by the formula: to 1-liter and. mix. Store refrigerated in nil Distilled water required 'polyethylene bottle. Discard when microbia Cone. of original hemolysate_ growth appears or the pH changes. 7 (2) Sodium Chloride .Solution, d. Procedure :.. Add 9 g of sodium chloride. (NaCI) to a (1) F,ill the cell with TEB buffer. 1-liter flask. Dilute to 1-liter with distilled ,(2) Soak the cellulose acetate mem- Water. brane- in a tray of TEB buffer for 10 Min- )Fixative-DyeSolution. Dissolve utes:,Gently blot the membrane to wipe 2g of Ponceau S, 3.0 g of trichloroacetie off excess buffer. acid, and 3.0 g. of sulfosalicylic acid -in 100 3) Place the membrane on- the cell ml of distilled,water. accordi,ngto manufacturer's instructions. AVIV!, 16051/111_ 8 7 fleeeimber .197 4) Apply the unknown and contro the membr hernolysates to the cathode side of thememo blade. brane. .-(11) Scan and integrate the area on (5) Electrophorese for 50 minutes at densitometer.(Follow the manufacturer's 375 volts. instructions for the operation of a densitom- (6) Immediatelyremovethe .rnem- eter.) Examples of scans are shown in figure brans from die cell and transfer' to a stafn- 5-8. ing tray containing 50 ml of fixatiVe-dye ,(12) Report the hemoglobin gehotype solution: Stain for 10 minutes. (AA, AS, 4C, SS, C5,-. or CC) and the percent.: A to the nearest tenth- percent (7) Rinse the membrane in three suc-, (0.1).' alkali deriatUration test is also cessive washes of .5% acetic acid. Drain-performed on all specimens and fetal hemo-, excess liquid: the inside wall of the ,globin reported. On all specimens migrating tray.. -,on 5, a dithiOnite test is performed to rule (8) Place a ,glass plate- in a tray con- out the possibility of its -being a D. The tainingclea'ringsolution. Transferthe dithionfte_ test`is not fePOrted unless the re7_ nierribLne tetheilearing' solution forf' sults are, inconsistent with 8. minute. Agitate gently during clearing. e. Calculations: (9) Remove the glass plate from the (1). Estimate the nunther of.square clearing solution with, the Membrane po- milliineters for each fraction: -TOtall the' in- ioned on the glass plate. Drain any clear- dividual area. ing solution from the membrane and re- move any air !nibbles by squeezing. Dry (2) __Calculaterelative. the membrane at approximatelY 100? C for each. fraction as 10-15 minutes. Fraction area X 100 (10) -Remove the plate from the oven Total area and allow to coal.Place the plate.in a ourceS of Error: humidity chamber for 30 minutes. Remove 1) A heavy applict on of

F .A1' A3 NORMAL Figure 5Hemoglobin Electrophoretic Scans. AFM- 160-51/TM 87.227,4 causes a thick electrophoretic pattern after sOlution than adult hernaglobin (HbA). Al- staining. Such a pattern must be repeated. kali 'converts HbA to -alkaline hernatin. Al- (2) Handlingthecelluloseacetate kalirte hernEitin is insoluble and precipitates. embrsne with the hands interfeies 'with' HbF is quantitated by :measuring the hemo- the electrophoretic pattern. .AlWays handle globin concentration before and- after de- .the membrane With tweezers.. naturation. - (3) Some acetate _membrane's have de- b. Reagents: fects.- If any membranes are -defective, dis- (1) Drabkin's Reagent card the entire lot. 5-18b(1). g. Discussion: _ (2) Sodium Hydroxide (NaOH), 0.10

(1)- Ae hemoglobinmigrates _ identi- N. Dilute 5.55 ml 'saturated NaOH to 1- cally to he,moglobinC. They- are distin liter, with, distilled water. Mix well and guished by the quantity present. If this band store in a polyethylene bottle at room tem- is 40% or more of the total hemoglobin, perature. -itis- C..A2 hemoglobin should always be (3) AmmoniumSulfate,Saturated. --less tilan'20%1 Add 400 g ammonium- sulfate, AR, to 500 (2) '.TwoIL slow-moving, nonhemoglobin ml of distilled water. Stir mechanically for components e seen using this technique. 10 minutes. Heat until all the salt has dis These fractionare carbonic anhydiaseS,I solved and filter rapidly.through a. What- II (CAI an CAR). man #1 Alter paper. Store in a polyethyl- Hemoglobin A. is elevated in thall ene bottle., a minor, P Preparation of HernoTysate See para- (4) Genotyp'e SS is found graph 5-21c. With sickle cell aneniia. d. Procedure: (5) Genotype. AS is found in palients (1) Dilute 0.2 ml of hemolysate with with sickle cell trait. 1.8 ml of Drabkin's reagent and mix gently. (6)- This method separates . hemoglobin (2) Place 2.5 ml of 0.1 N NaOH in a iA.,., in, the presence of hemoglobin S in 13 x 100 mm test tube. At zero time, add -patients manifesting sickle-thalassemi a dis- 0.5ml ofdilutedhemolysate ,and mix ease. rapidly. (7) Hemoglobin F is quan stated by (3) Exactly 2 minutes after addition the alkali denatnration test becau e it mi- of the hemolysate, add 2.0 ml of saturated grates close to the hemoglobin Al fraction ammonium sulfate. Mix by Inverting 'six on- the electrophoretic pattern. es. DO NOT SHAKE! (8) Include knowi and C-, (4). Filter through a 7 or 9 cm What- trols in each analysis. man #42 filter paper. If the filtrate is not h. Normal Values: crystal clear, filter again. This is the fetal (1) A3 Hemoglobin: 1.5-3.4%. hemoglobin solttion. (2) Genotype: AA, (5) Prepare .total hemoglobin soln- ) F Hemoglobin0-2% (except in tion by adding 0.05 ml of diruted hernolysate infants).. to, 5.0 ml distilled water. Mix well by` -. sion. DO NOT SHAKE ! 5-22. (Alkali. Denatura- (6) Prepare a fetal hemoglobin blank tion Tiit.): by adding 0.5 ml of, Drabkin's reagent to a. Prikiple. Fetal hemoglobin; (HbF) is, 4.5 ml of distilled water. Mix as above. more resistant to denaturatfon inn alkaline (7) Preparea.total hemoglobin con- -. AFM- 160-51/TM 8,227-4 einber97 3 5,25

.trol by. adding 0.05 ml of Drabkin's reagent h, Normal- Values: to 5.0 ml of distilled water. Mix as above.- (1) _Infanta, (leis:than Months) (8) Measure-Ohe absorbances- of the to 80%. ' fetal and total hemoglobi oluti against :-(2) Adulta:.0-2%. the appropriate blanks. e. Calculations: N OF BLOOD- SECTION EEXAMINATI \ Absorbance fetalhemoglobin,solution SMEARS 10 Absorbance total hemoglobin solution_ X 23, Introduction: - fetal hemoglobin Report % to nearest tenth (0.1). a. The critical examination of a blood f. Sources of Error:- smear includes the following: quantitative (1) If the hemoglobin concentration and qualitative study of platelets, differen- of the .herriolysate is less than 5 g per (11,.tial count quantitating the, three types of the sensitivity of the procedure is decreased. leukocytes (granulocytes, lymphocytes, monocytes)'and morphological character- 21) Foor --pipetting-teehnique-causes 'sties of erythrocytes-1_ and leukocyte& Stain-- significant error due to the small volumes ing the blood smears is a critical part of involved. the examination. The procedure for stain- (3) If . the absorbanceofthe. fetal ing is described in chapter 3. To accurately hemoglobin Solution 'is- greater not 0.700, perform the differential count it is neces- dilute 1.0 rill ofSe fetal -hemoglobin sOlu7 sary for a tech_ nician to recognize all the tion with 9.0- ml distilled water: Read- the characteristics of normal blood cells. This includesnorinal biological variation. For absorbance against. the. totalhemoglobin blank. Multiply the calculated resuk'by 10., instance, not every lymphocyAe is exactly the same size, nor do all 'lymphocytes have g. Discu3siOn exactly the same number azurophilic gran- (1) The alkali denaturation test can ules. be performed on bloody .rectal discharges b. Certain morphological and histochem- from infants. This test is used to deter,-_iCal characteristics are utilized to differen- mine whether the discharge is due to in- tiate blood cells. A review of the significant gested maternal, blood or. a gastrointestinal features promotes a better understanding lesion. The procedure is as Tollows: of blood differentials. Cellular characteris- (a) Add a small amount of bloody tics such as r_ elative size, shape, cytoplasmic discharge to 10 ml of distilled water. granulation: nuclear-cytoplasniic ratio, nu- cleal- configuration, chromatin or nucleoli Add 0.2 ml 10-% .sodium hydrox- are very important. These features are dis- ide soldtion. cussed in chapter 4. (c) If the solution turns pink, it is fetal blood.- .A muddy brown -solution- indi- c. Experience is the foremost teacher in cates blood of maternal Origin: hematology. It is. readily acquired in a busy hematology section where the opportunity (2) Fetal hemoglobin constitutes' ap- for differential analysis occurs frequently. proximately 80% of a newbvn- infant's Experience can be diversified. And interest- hemoglobin.Thisdecreasestoapproxi- ing if- proficiency slides and material 'from mat4ly 5% at six months. cases of confirmed diagnoses are maintained_ 3) Fetal hemoglobin is increased in as study sets. This study material should adults with thalassernia major and minor, be available to all technician_ s in the la_ bo- hereditary persistence of HbF, erythroleu- ratory. kemia, aplastic anemia, and spherocytosis. d. Allroutine blood smears shOuld b until the- physicians have ro vievied fferential rep -period is. usually adequate. Oeca.sionally, a review of a:specific 'problem .slide results in findings whiCh were not originally apparent and reinforces confidence in the -laboratory by the- medical staff .This practice also -ads to the experience' Ind proficiency of technician. igure5-9.ExaminationofPeripheral 5-24. ExaminationofPeripheralBlood Blood Smears. Smears;- a. Principle. The stained blood smear values, may be calculated by multiplying, the permits the study of the appearance and the percent value by the total leukocytic count. identification of the different kinds of leu-' kocytea,- and-tire- -appearance -of erythrocytes-- c._ Erythrocyte Morphology. and thrombocytes (blood platelets). (1) Study the erythrocytes and report any evidence of rouleauxformation or signs b. Differential Leukocyte Count: of immaturity. (1) Inspect the smear under low power (2) Report the erythrocyte morphol- magnification. Locate the thin end of the ivy with reference to size, shape, s aing ear whereth.ere no.. overlapping of characteristics, and inclusions. Reportthe erythrocytes. degree of the specific morphological char- (2) -Switch* to oil immersion. Identify actaristic(for example, moderate hypo and count 100 consecutive leukocytes and chromic). record each cell type separately on the dif (3) If = nucleated,erythrocytesare ferential counter. Begin at the thin end of found (usually ,these are metarubricytes), the smear and count the white cells' obreportthenumber per100leukocytes served as the slide is moved in a vertical counted. direction. 'When near theedges of the d. Qualitative Platelet Evaluation: smear, move the. slide horizontally for a distance of about two fields, then proceed (1) Observe the thrornbocytes in sev- vertically back across the smear. See figure eral oil immersion fields to obtain a rough . 5-9. Continue' this "snake-like" movement estimation of their numbers (normal, in- until 100 leukocytes have been counted and creased,or decreased) Normal is an aver- field._ lassi fled. age of 4-6 per oil immersion (2). Note any abnormality in morphol- (3) Ifthe WBC countisbetween ogy (giant platelets, etc).If the throml3o-. 20,000 and 50,000 per cu mm of blood, cytes appear to be significantly,decreased, count and classify 300 leuko-cytes.'When the fhrombocyte count and/or a clot retrac- count is greater than 50,000 per cu Irim of blood, count and classify 500 leukocytes. tion test may be indicated. (4) The number of each type of leu7 e. Discussion: kocyte is expressed as -a percent of the total (1) All abnormal white cells (for ex- number of white cellscounted. Absolute ample,mmature, hypersegmented, toxic, AVM 160- lyTm 8-227-4 5 December 1973 atypical- lymphocytes, etc.), should be clas- philic (blue), eosmophilic (red), 'and neu- .sified or -described and reported-in percent, trophilic (pink) granules--are quite obvious separately : Celiac that are ruPtured, frag in routine 'blood smears. The presence, ab-. mented, or degenerated are-- not included sence,-type, and quantity: of granules are- in- the differential count; but .should, be noted characteristic attributes used to differen- seiaratelY and reportedas the:number seen tiate leukocytes. per 100 leukOcytea. LIft (6) The size ratio of nucleus- to cyto- (2) in view of- the gradual transition _plasm (N:C) .is a differentiating °chara,c- from .the metamyeloeyte to the band neu-' teristic. For instance, a.cell, with a nuclear trophil and then, to the segmented neutro- Mass equal to the cytoplasmicmass would Mill, exact classificlition is sometimes diffi- have an N:C. ratio of 1:1. The total' cell cult. In such_cases, classify the cell according mass is usually greater in the more imma- to the Mare `mature form. ture cells and decreases as the cell matures. (3) Size considerationsin' differen The nuclear mass usually decreases also as-. ating blood' cells requiie a' defined. lin the cell matures. Of- course, lymphocytes ----standare-The micron:001 -rern)--is- usually are the exception to thigenerality.- used in reference to microscopic dimensions. (7) The nuclear configurations of leu- Ocular micrometers are available_ through kocytes help distinguisf these cells. Round, Federal medical supply -Channels and are -ovalindented,- band,orsegmentedare easilycalibrated, using a hernacytorneter terms used to describe variations in shape. which has standardized dimensions. In rou- These normal configurations can be dis- tine screening of blood smears, an experi- torted.- by physical and _chemical factors. enced technicia.n relates the size of a normo- Some of the leukocytes are so 'fragile that cytic erythrocyte (seven to, eight microns) in thick 'blood smears their normal config- to the size :of the white cell to be differen- uration may be distorted by the pressure tiated, since erythrocytes' are usually pres- Of erythrocytes fOrced against them. These ent tfriroughout- the microscopicfield.Fi- artifacts should be recognized as such in nally,qt_should be understood that personal an intelligent 'evaluation of blood differen- visuat discrimination is an inaccurate gauge tials. of linear measure. Some eference measure (8) In addition to nuclear shapC and should be employed.. size, the internal nuclear morpholagy shows (4) The shape of bloo'd cells often de- differential inclusions. The chromatin ap- pends -Upon the smear and staining tech- pears finely reticulated in some cells, or as nique. Variations that have .no clinical sig- a coarse network, oit...even clumped, in oth nificance occur from physical and .chemical ers. The parachromatin, a lighter staining distortion that result from technical error. material beside the chrornatin, is .scant' or These variations are avoided with careful. abundant. The appearance of the chromatin technique. Pach routine smear should be and, the quality. of parachrornatin are uti- scanned initially to evaluate the smear and lized- to differentiate blood cells. The' pres- stash quality beforedifferentialanalysis. ence, absence, and number of. nucleoli- in (5) Cytoplasmic granulationneu- the nucleus are the most distinctive cha , trophilic, basophilic' or eosinophiligis an acteristics of immature nuclei in blood cells.

important morphological observation. Dif- _(9) All abnormal blood smears -should ferences in granule color in Wright-stained be examined by another trained person for preparations are caused by the variable dye confirmation of the results. affinfty of specific granules.. The intensity

- of colors and the relative blueness or redness f. Ntirmal Differential Value's: of the erythrocytes is used to evaluate the -11) Neutrophilicmetamyelocy es:0- quality of 'the- stain. The familiar baso, .AF1Y1 160,--51/TM 8-227-4

-(2) Neutrophilic bands: 3-5 (a) : 0-5%. 3) SegmentedneutrOphils: (b) Prorfiyeloc :p-8 (4) _EosinophilS: 2-4%. ). Neutrophili -:.' 5) Basophils: 0-1 6). Lymphocytes : 20-35%. (d) Neutrophilic (7) Monoces: 2-67 Neutrophilic bands: -1.(1-35% Examination of Blood MarrOwSnmais: NeutroPhilicsegmentedAll ' 7-30 .Principle.Nucleated bloodcellsare g) Eosinophilic Ails'''. 1-4 counted and classified from a bonemarrow h) Basophilic cell's: 0-1%. Smear stained withModified. Wrjght's stain. LYMPhoe s5-1* b. Procedure; Monocytes: (1) Usingoilimmersion magnifica- k) Plasitocytes: 0-1%. 300-500- nucleated _ on,. count and classify Erythrocytes: cells. a) Rubriblasts: 0-1%. (2) glassify all blood cellsaccording tct cell tyPe and various stagesof matura- b) Prorubricytes: 1470 ) Rubricytes: 3-10: 0. (d) Metarubricytes; 5L25% (3) Calculate myeloid-erythroid by -dividing the numtier of nucleated= _Megakaryocytes: 0-3%, rotytes into the number of, granulocytip (4.) Myeloid-ErythroidRatio(M:E): (mYeloid)- cells, -4:1. (4) A peripheral blood .aluation us- ally accompanie's the bong marrow reports. SECTION-l'ERYTHROCYTE IN WES This evaluation usually included anerythro- AND FRAGILITY TESTS cytecount, .leukocytecount,differential -5726: Erythrocyte Indices: count, hernoglobin, hernatocrit and a retie- 'accurately deter- locyte count. a. Principle., By using mined red blood cell counts, hemoglobinand .Discussion: hernatocrit values, the size andhemoklobin (1) The differentiacell count on a content of the average red cell in agiven bone. marrow smear is carried out by a blood sample calculated. The values ob- hematologist, Pathologist, or trained'tech- tained are the erythrocyte indices andaid -nician, in the classification and study ofanemias. (2) Since interpretation of findingsin h. Calculation of Erythrocyte Indices, bone marrow examinations is verydifficult, (1) Mean Corpuscular Volume (MCV) it is of utmost importance that the smears The average volume of the individualred and stains are carefully preparedusing blood cell. pulously clean equipment. Hernatoerit (percent) X 10 cubic micro (3) The laboratory technician Red cell count'(iiiillions)-millions) ually responsible for preparing bone mar- Example: HernatOcrit 45 percent smears, staininx the smears, checking Red count 5,000,000 per cu 45 x 10 the quality of the stained smear,add cover- 5.0 90 cubic microns (normal) slipping the slides. (2) Mean Corpuscular Hemoglobin d. Normal Values: CH)The average weight of hemoglobin feukocartcs. of the individual red cell. AFM 1qX61 / l4lf8-227-4 Dec b 549

Hemoglobin (gm per ill) X 10 d. Normal Values: Red cell cvvnt (in, my romicro flennigfobin15 gin per dl Mean Corpuscul Volume: 8292 Red COunt 4,000,000 per Cu mm bic microns. X 10 30 mieromicrograma (normal) () MeanCorpusctilar'Hemoglobin: -. 27-31 micromicrograms. Man Corpuscular Hemoglobin Cancen ation (MCHC)The, percent raf (3) MeanCorpuscular Hemoglobin hemoglobin in the average red cell. Condentration: 3246%. nernoglobin (grjuier dl) X 100 iNpercent .5-27. Brithrecyte -Osmotic Eragility Test 77-14ematoerit Icerernt) (Dade): Triple: Hemoglobin 15 g per dl flematocrit-45 percent a. Principle.. A specific amount -of blood X -100 33 percent (normal) is introduced into. a-series of tubes con-. 46, taming different concentrations of buffered sion:. salt solutions. The ability of the erythrocytes Accaratet7 individualdetermine- to resist heir olysis -in--such-solutions- is- (10=-7 tiOnS of hernoglobin,:.hernatacrit, and eryeh- termined-spectrophotornetricallyon'the, recyte count insure reliableindices. The basis of free hemoglobin present. following procedures are recommended: b; Reagents: (a) Erythrocyte counttwosepa- pipets and 2-41 :counting chambers, or (1.) BufferedSaline(Equivalentto electronic cell counting. 10=%®_ NaCI). Add ,180 g of dry sodium (l.) Hemoglobinprecise reagent' chloride(NaCl),27.31 g mono-hydrogen ., sodium phosphate (Na21-1P0,), and., 4.-86 -standUrds and accurate instrument calibra- dihydrogen sodium phosphate (NaH2PO4) to a 21iter volunietric flask. Dilute to 'the atochitWintrobe, mark with distilled water. fuge at 3,000 rpm -for 55 minutes.-:' (2) WorkingSolution(1%NaC1). (2.)It isuseful-- to compare the cal- Add 10 ml of the buffered saline to .a 100-m1 cUlatedindice with a stained peripheral volumetric flask. Dilute to the mark, with blood smear. distill&I water. 3)-- ntrobe- classified anemias into' the following groups-- on the basis of the c. Procedure: indices: (1) Set up two series of 12 tubes n a Anemia. M rack; label one series PATIENT and the Maerocric 95-160 other CONTROL. -40 Microcytic 72-79 (2) allowing dilutions: Microcytic llypochro 50-79 Normocytic 80-94 Tube Distilled NaCI. Number Water Concentration. (4) The MCHC cannot exceed theor= 4.25 nil 0.75 ml mal, value, since the erythrocyte cannabe 3.75 ml 1.25 ml 0.75% 1A5 0.65% supersaturated with hemoglobin. The IVICIIC_ 3.25 ml is the most valid of the indices since it does 4 3.00 ml 2.00 ml 0.60% not require the erythrocyte count in its dexi- 5 2.75 ml 2.25 ml 0.55% 6 2.50 ml 2.50 ,ml 0:50% vation. It is a good index of iron deficiency. 2.2 2.75it 0.45% (5) The MCV and MCII are increased 2.00 ml 3.00 ml _0.40% at birth and fall to low values during child- .1.75 nil 3.25 ml 0.35% 10 1.50 ml 3.50 ml. GM% hood. The MCIIC is fairly constant for all 11 1.00 nil 4.00 ml 0.20% ages. 12 0.50 ml 4.50 ml 0.10% AFM 160 1 TM 8-227-4 A, 5 December 1973 = Draw- 5 ml ofvenous blood from 4Rough handling of the blood spe e patieftt and deify hto a tube contain- men es hennolysis which leads to ini ing heparin. results. (4) Pipet' 0.05 ml blood into each tube Discussion: f the- PATIENT series. Mix ilnmediately (1) In .hypotonic salt sdlutions, erytl

_ (5) Similarly collect- 5 ml of venous roeYteA take up water, swell, to a' dpheroid blood from a normal .individual and deliver shape and .burst In -congenital splieroaytiC- into a tube containing heparin. Pipet 0.05 (anemia the red cells,with defective structure: ml of blood into each tubpf the CONTROL more readily ruptuye at. salt *concentrations series. Mix immediately. r closer- to .isotonicity (0.85 p ercent).7 These (6) Allow the ,tubes tostand at- room sells thus show an increased osmolic frag temperature for,30 Minutes.- ity. In contrast, the flat or thin but other- (7) Mix akain and centrifuge at 2,000' wise normal red cells of hy-pochromic anemia rpm for 5 minutes. show a decreased osmotic fragility and do -- not-- hernolyze-- until lower .salt-- concentra- (8) Transfer the iupernates to .covets tions are teached.. 1 as and read. at 545 rim using tube numb (2) When hemolysis begins beyond the a blank and number 12 as 100 %© hernolysis. range of the- prepared solutions or when ('9) Read and record the absorbances termediate dilutions are desired,nit for each tube of the CONTROL series and tiorial dilutiais are readily prepared, aing each tube of the PATIENT series. the 1 percent ,sodium Chloride stock sPlution. (10) Calculate the percent hemo at (3) In easel where, the results kf the each salt concentration for both sees of fragility test are borderline,' the .folrowing ttes. procedure is recommended to enhance any (11) Plot the curves of the patienVs and latent abnotAalityinfragility..Irictibate control's osmotic :fragilitY using- the percent samples of defibrinated blood (control and hemolysis as the vertical ordinate and the patients) 'at 37° C for 24 heoirs under salt concentration as the horizontal abscissa sterile conditions and controlled p14 (7.35 -.' (see figure 5-9 for an example curve). 7.50). The test is then performed as de- scribed aboye. d. Calculation: (4) Decreases in pH/ increase osmotic Absorbance of a particuipr tube fragility. The reagents are buffered to main- Absorbance of tube 12 tain a constant pH of 7.35 -7.50. x 100

= . (5) This test may also be run visually, Sources of Error: with some sacrifice-of accuracy, by allowing (1) The concentration' of the Nael in the blood-saline dilutions to stand at 20° C-" the aolntions is critical. The salt must be for 45 minutes. The tubes are then lightly * chemically pure and: dried before weighing. centrifuged (1,000 rpm for 3 minutes) and It is visable to _dry the salt in a 100° C observed for signs of initial and complete oven ad store it in a desiccator. Store the; heniolysis. A slight pink' coloration. Of the NaCI solutions in a glass-stoppered tightly supernatant fluid indicates initial- hemt;lysis sealed bottle. and a clear red solution, free of .sediment, indicates coMplete, liernolysis. The salt co/1-s, Inaccui-ate preparation of the .dilu- centrations in these- two tubes are noted tiorts'a sea inticcu rate results. and recorded. The control should always be '(3). Maintain the pH of the solution at reported alpng with results of_ patient's tubes. an interval . of 7.35-7.50 A different pH range causes invalid results. g. Normal Values: -December 1973

RAGILITY

CREASE)

CONGENITAL- ..HEAOLY-rie ANEMIA

0.24 0..30 0.40 0.50 0.60 0.70 0.00 Q,E3

Perelifkt

Figure 5-10. Erythrocyte Osm Fragility Curve. (1) 0.30% Saline: 97-100% hemolysis. Erythrocytes in this form of anemia- ,lyre (2) 0.35% Saline: 90-99% hemolysis, easily in slight variations in the pH -(acid). In this test, the. erythrocytesare subjected (3) 0.40A Saline: 50-95% hemolysis, to pH values ranging fronl 6.5 to 7:0 at (4) 0.45% Saline: 4-45% hemolysis. 37° C. (5) 0.50% Saline: 0-6% hemolysis. b. Reagen (6.) 0.55% Saline: 0% hemolysis. (1) Hydrochloric Acid," 0.33 N. Add 29 nil concentrated HCI td' a 100-m1 volumetric . Ham' Test for Erythrocyte .Fragility-: flask. Dilute,to the mark with distilled water. a. Principle. This test is positive in par- Mix. oxysmal nocturnal hemoglobinuria (PNF1). (2) Sodium Chlorid Splution (Saline),- 160 = 1J 8-22V-4- piker9

I = , 0.9%. Add 9 -grams Aodium. chloride to a 1- congeii tar spherocyjf anemia -liter volume-Vic flask."-Diltite to the Mark is suspected, est ,should hie, repeated, 'th1itilled water. !, acirlified,ser nigreviously inactivated at 56 CfiN- 30 miites, : ocedure: A :Since erhrocytes of P,1%-r1-1 require., 1) aregilly- withdr ti arildefibri- complement. fol.- h molysis the -modified test -nate 10 .mf.of venous blOod,from the patient: (item atiove)will :lie -negative PNH (2) Carefully withdraw- anddeflbri-1 and will remain positive in spherocytosis. nate 10 rnl=ofi verious frorn a normal Interpretation. Hernolysis in the acidi- individdh-1. s is uloe is indicative o- paroxysmal noe- (3). Centiifuge both [bloodpecimens al hemoglobiriuria. at 1,000 rpm Dar 10 minup.i. -; ; _ (4) Separate serum and cells. _CTION GDEMON STRAJI-QN OF --(5) Wash the- cells ;three times with CELLS = - normal salfne, then prepare a 5 percent sus- pension of cat in normal aline. 29. General Information: (6) Measure -1.0 ml i Of the 5 percent, a: Persons having:Jupus erythernatosus, test cells jntp each -of :4. tubes. Repeat this one of the "collagen" diseases, have an ab- for the contro -normal specimen. normal plasma/ protein which causes sivell- (7) Acidify 1.9 nil Of ,serum by adding ing - b.ride breakdown of :certain blond. cell ..- . nEclear 0.1 ml of 0.33. N hydrochloric' acid..Do this nuclei4,h vitro. This degenerated for both the test, and control serums. material attracts phagocytic Cells, partic- cularly 1egtnented neutrophilS,'Which' engulf, - (8) Into' each of the tubes add test nuclear mass. Title resulting phagocyte serum, control serum, acidified serum and this '19 nclusidni material is termed an "L.E." acidified test serum as follows: Test Controi Add: b. The ttvo inost po'bular methods of dem 1 1 1:0 ml rest serum 2 1.0 ml control serum onstrating -pie L.E.cell and antinuclear 3 1,0 ml acidified test serum antibodies are the rotary bead method and( 4 1.0 ml acidified &Carol serum fluorescent /antibody. method,- The rotary, bead method is positive in 75-80% of the (9) Mix all eight tubes and incubate withlupuserythernatosus.The for 1 hour at 37° C. Shake every 10 minutes. patients fluorescentJantibodi method is. positive in (10) Centrifuge at to end of-the 1-hour ,95-100% o the patientt with luPus erythem- period and examine for h molysis. atosus. Throtary bead-method- ispre- sented in the next Paragraph.r The fluores- d. .biscussion: cent antibody method requires'. equipment (1) With a positive test the tubes con- that limits its use to larger laboratories. ining-, acidified sera and patient'scells should show.considerablelhernolYsis.. -30. Rotal-y Bead Ilethod: (2),- Normally no tubes 4houldhow a. Principle. Leukocytes are broken.down hemolysis. vitro allowing theall-morn-la plasma pro- -(.3) . Oecasionally., tubes with- ein to react on the altered nuc ear material. fied.sera and patient's cells may show .moder libation enhances the nudkar deteriora- ate helholYsis.- n and phagcytoSis. Slides are pi.epared examined r the peculiar "L.E."cell. (4) A falseositive test issometimes. seen in cOngenitapherocytib anemia. Procedure: AFIVI 60-51/111 8-227--4

-Place 10 gins- beads ent,i and_ diagnosis depends on demons sgcl um heparin(U.SsP.in -a- 16 by, 125 Lion of- the L.E. cell.. Frequently the ea_ les test tubes symptoms appear after intense eXposin 6- to (2):1-0-btain 10-15 m1 of -ArencinS blood sunlight. Leukopenia,thrombocytopenia, from the patient and place,in.the irepared and an elevated sedimentation rate are some of the clinical signs of the disee tubes . . (3) Mir- the blood and antidoagulant Free masses of -lysed nuclear !hate- .gently three-fou times. rial, .{°ithor without polymorphonuclear- leukocytes clustered about- -them (rosette for- (4) Let the- blood specimen iincubate mation),- are suggestive Of the L.E. phenom- at room temperature for ,1 hour. i. enon.- Observing "rasettes" should ericotir (5) Place on any suitable heinatologi, age- technician to repeat examinations cal sample mixer and rotate at a --slew :speed and further search for the-true "L.E." cells. (12-1.5- rpm) for 30 minutes. A. positive report Should not be made with- . . -(6) Place in centrifuge aid -Spin- do wn out .the: identification ',of= thiseell-.he in- 9,000 rpm for 15 minutes. clusion body with the leukocyte homo- geneous and has no chromatin rn. This (7) Removp and discard ernatant 4feature distinguishes the true .E." Cell '- Phi frond the "tart" cell(nucleophagocytosis). With a disposable pipet rmove the This latter cell - contains an engulfed, dam- huffy coat and a -small amount of . _ BCs nrid aged nucleus, usually that of a lymphocyte place. iti a Kahn tube. which still contains a =recognizable chroma- (9) Mix well and fill three nelnlieparin-I tin pattern and -a distinct nuclear membrane. ize.d'capillary tube with themixture, Interpretation:. (10-) Spin capillary tubes: down in a (1) These cells are seen as large polyr mtctohematocrit spinner for 4 minutes. orphonticlear.; (qegmented) leukocytes (1) Etch the capillary tubs with contain large ingested-nuclear fing- diamond point pencil at the junetlnof the en,,sin their cytoplasm. IlBC-buffy-coat layer and break tai 4 tube. (2)- The inclusion body is a purplish- (12) Tap out the huffy coati acid a small staining, smoky, :homogeneous mass of ma- amount of plasma on a slide arid, rnix,until terial which is so large that it usually pushes the cells are homogeneous.;Smeak the cells the nucleus to one side of the cell. out in the same manner as for a differential smear anClet air -dry. Prepare t.-o smears.. SECTION II SPECIAL STAINS (13) Stainthe . smears with modified Wright'sstain and examine the smear s 31. Peroxidase Stain (li{aplow) under oilimmersion for the piiesence of the lupus erythematosus phenomenlon. a. Principles-:-The members' of, the granit4._ locytic spies contain an- enzyMe, peroxidase, (14) Examine knownposiqve :slides along with the patient slides. which:liberates the -oxygen from hydrogen peroxide. This enzyme is' Mere, prominent c. Discussion: in mature forms.-:A benzidine derivative is. (1) Lupus erythematosus is- la chrenic, used as an indicator of .peroxidase activity. sometirrieS fatal, disease of unknbwn The indicator- is oxidized, and .preCipitates ogy. The peculiar skin eruption licross the in the forth of brown to bluer anules. nose and cheeks (butterfly rash) and arthri- tis can be accompanied by va!iot s visceral b.- Reiigents: manifestation-s. Often the rash is not pres- : (1) -FormalinEthanel Solution,

, s . AFM 160- 51 /TM- 8-227-4

i Mix a ml of IformaldehYde! with 90 ml:'of thour of obtaining the specimen and stained atso ute ethanol. ta -- within 3 hefts after they are prepired; (2)Stain'.Mix the following geiits -(3) The inonocytei thOught tobe in the order kited. slightlyperoxidasepolitivethroughthe . 1 phakocytization of peroxidase positive gran- 30'7, Ethanol 100.0' ml Benzidine dihydroehloride _ 0.3 I,- g _tiles of ruptured cells.- 0.132 M ZnS0..71120(3.8% W/V) - 1.0ml ( 4) Myeloblasts;,.can -showeak peroki- Sodium Acetate (/Ti:tCTF130. 3110) 1.0- g' dase activity using this method.' 3t7e- Hydrogen peroxide _ 0.7ml` 1.6 M sodium hydroxide 1.5ml (5) Addition of 4.9 mg of sodium gyp.- .,Sdfranin 0 g nide to the stain inhibits peroxidase activity Mix well after each addition. Final pH is in all granulocytes except eosinophils. 6.0 0.05. Store the stain in' a .screw -capped (6) If greater nuclear detail is required,. jar.The- stainisstablefor aboutsix counterstain with 1% aqUeous cresS/1 violet --Months.' . acetate-for-1 :minute or-in-freshly prePared Giemsa stain for 10 minute-s. c. Procedure: (7) Giemsa stain is prepared, as fol- (1) Prepare blood smears from fresh lowS: Mix 3.8 g Giemsa stain -powder. and blood or bone rridrrow aspirates. DO NOT 200 ml glycerin. Incubate at 60° C for two use anticoagulated blood. hours. Add 312 ml absolute methanol;' dilute' (2) .Fix the smears in -10% formalin-.the staining solution 1:10 with .1% sodium ethanol solution for 60 seconds. carbonate before use. -(3) Wash of fixative gently for 15-20 seconds .in running water. -Shake off excess.5-32. Leukocyte Alkaline Phosphatase water. (Kaplow (4) Place the slides in a Coblin, jar a. Principle. Blood smears are fixed and containing the stain for 30 secoCids at -room stained for alkaline phosphatase activity. temperature. _b. Reagents: (5) Wash the slides for 5-10 seconds in running water and dry.- Examine the (1) )-Vixative Solution. Mix 10 ml form= smears under, oil immersion. aldehyde and 90 ml absolute methyl alcohol. Store in the freezing unit of a refrigerator. d. Interpretation. Peroxidase positive cells (2) Propanediol Stock Sokition, 0.2 M. are identified by yellow-green to. blue and Add '10 g 2-amino-2-methy1-1, 3-propane- brown-green granules. Cells of the granu- did in -a 500-m1 volumetric flask. Dissolve locyte series from the promyelocyte through in, _and dilute to the mark with, distilled the segmentedneutropliilare peroxidase water. Store in the' refrigerator. positive. The degree of pe'oxidase activity increases a the granulocytes mature. Mono- (3) Kropa.nediol Buffer, 0.05 M, (pH cytes may'khow a weak reaction. All other 9.75). Add 25. rill' X0.2 M propanediol stock _cells are negative. solution and 5 ml of 0.1 M hydrochloric acid to a 100-m1 flask. Dilute to the mark Discussion : with distilled water. Store .in the refrigera- (1) ,The oxidizing enzyme in the gran- tor. ules of the granulocytic leukocytes deterio: (4) Substrate Mixture. Dissolve 20' mg rates rapidly in vitro. It is, therefore, nec- naphthol AS-BI phosphate (Sigma Chemi- essary to use fresh blopd in making the caVompany) in 1.0 ml of dimethylforma- preparation. midk. Add 250 ml of 0.05 M propanediol (2) Smears shoilld be prepared within buffei;: to the solution. Pipet 10 ml aliquots of A.FM 160a1/TS-227-4r his solution into' test tuheS, stone (1) Count two slides(100 celli p freeze.' slide)on each patient, . rating the ;!_seg- (5) Stain. Solution. Thaw isubstrates mentecKneutrophils according to how much tubes to room temperature (as needed).Add black staining of, i the granules. is. -cibseivel, 4pigs of fast red-violet-- saltr,T:B g If -no-staining- is noted; the rating is -0; if Chemical Cotripany) t(K'eachs 10 nit of; "gu Slight black Staining is noted, the rating is strate solution. Mixvigorously=for 3se_copd_ 1--F; if a -medium amount of black staining'is fiTtr and USE IMMEDIATEL ndted, the rating is 1.? +, if alleavy amount 'of , dark black staining is observed,_thkrating'is (6) Counterstain' (Harris, HernatoXy-'-3'+, and if there is heavy likick staining 'coz- ering all the cpoPlairnythe rating is 4+. (a ) Dissolve 100 g of potassium or lin- (2) After( 10,0i cells -per slide are. rated, , ammoniu_m .aLtimin_ 1000 Tni of distilled. figurethescore-I.viii cellscounted as water, Heat until the Solid dissolves.- -' O.. no scorecellsated.-as .11+ gek4i,. Scscore (b) .ThssOlve_5 g of hernatoxylirf crys-'_ - ,tals in 50 ml of 95 c ethyl-alcohol. of Peach; cellsraed as'2+4efii.--idore 2 each,. ate. ' 1 -(c) Mix the alum andhematoxyl'n , (3) Repprt the total number of cells, solutions: Heat to boiling and add.25 g mercuric oxide. giving their rat_ gs-, and 'searfReport. (d) -Reheat the solution for abo total score,for ch individnaslide. Aver- minute until dark purple. ;age the two totalscoiesancfcreport the aver- (e) Rapidly cool in- cold wa age Also, alays- repo_r'.the normal- score (f) This stain is stable range. perature far .3 to 4 months. _ ,-,d. Discussion:- --_ - poly- ProcedUte: (1)r IPatientswithinfections, A cylhemia and miTloproliferative disordert (1) Prepare fresh blood smears and air-dry. cletrionstrate increased -- alkaline phoSphatase activity. -, (2) Prepareneg-ativecontrolsfrom (2). In patients with acute- or chronic _ normal human blood. ,granulocytie leukemia, -alkaline phosphatase _ (3). Positive smears are- by activity is dedrea,ed: 'collecting bloOd smears from 'obstetric pa- tients during-the first 24 hours after delivery. f. Northal iralues. ScOreS of 13-130 have been obtained in heay adults. However, (4) Irrimerse the slides in the fixative the attending physi should -interpret : 0 to -10° C for 30 seconds. Whether vaclues-ar_e- atorabnormal. (5) GentlY wash the smears in run- ning water gnd 5-33. Heinz BodyiStain:.,

- (6) -Place the slides in the stain -Solu, a: Prl eil B mixed -with methyl tion for 10 minutes at'roornmperature. violetgni-in d a smear is prepared. (7) Repeat step (5). Heinz bodiestxstained with, methyl violet are (8) COunterstain h hematoxylin puiple, round or oval granules, one-two stain for 3 to 8 minutes) microns in diameter within the erythrocytes, (9) Repeat step ( b. Reagent. Methyl VioletuSolution. Dis- solve 0.5 g methyl violet in 100 in] of saline (10) Mountwithglycerin-gelat'ln Perrrionnt (Fistr Scientific Company) and (0,.9 J, NaCl} examine under oil immersion: c. Procedure: 1) Mix- equal - AVM 1.6951/TM 5 December 1973 methyl violet solution in- leukocyte diluting SECt'ION IMISCELLANEOUS TESTS pipet.. (2) Let stand at room temperature for 5-35. Screening Test- forGlucpse-6-Phos- 15 minutes. U phate Dehydrogenase Deficiency: (3) Prepay a- smear and examine a. Princifde:._Blood is- oxygenated -to con- under oil immersion. vent hemoglobin to oxyhemoglobin. Sodium ii. DiscuSsion: ascorbate in- the presence of OxyhernogItobin 4 G-6-PDL -cde- (1) Ileinz. .bodfasareinviaLibilein yields hydrogen_ rteraxide, ficient individuals, hydrogen- peroxide re- Wright:stained Preparation. .4 4 acts with hemoglobin to form a brown to (2) They- can to - observed in reticn- , locYte Preparation8 and by the use of phase green heme chrome. microscopy. b. Reagents:

' __.(3) -Heinz__ bodies_. _._are thought_thought be (1) Glucose Sodium ,Ascorhate. _Mit__ ,_ natured heinoglobin.-They are usualldeity- -1.0 g glucose and 2.0 g sodiftnyascorbate in onstrated_ in hemolytic. anemias caused by . .. . a mortar. Transfer .15 mg of-the mixture toxic agents. . to a seriesof 16- x 100 nim test tribes arid -stopper: These tubesare stable indefinitely 5-34. Siderocyte Stain: when stored at 720'13 C. ' a. Principle._ Siderooytes are e,hrocytes (2) Phosphate Buffer, _1/15 M (pH containinif iron granules. The granules .are 7.4), Disgohre 0.9 g--Patassibm dihyd-rOgen blue i.vhki Stained with Prusian blue. phosphate(10-1,P0,)and 3.81 g slitirn b. Reageuts:- .rnonohydrogen phosphate (Na,I1P0,-721i,b) (1) Pfussian Blue.Dissolve 4 g of in distilled water to make 50.0 ml. potassium ferrocyanide in 29 ml distilled -,(3) Sodium Cyanide SolutionDissolve water. Add concentrated 1-1C1 until a white 500 mg sodium cyanide in apptaximately precipitate forms. Filter to remove the pre-450 ml tilled water. Add 20 nal 1/15 _M cipitate; phospfia ebuffer, Add 1 M HCl until the-PH (2) Safranin Solution, 1%. Add .0.1 - is 7;4, a out 19 ml). Dihite to 100 ml.with (100 mg) gsafranin to 10 ?al distilled water. distilled water. C'Procedure c: Procedure: (I) Cover blood or bone smears with 1) Collect a b ood sample in EDTA 'Prussianblue. for30 minutes(NOTE: or heparin from the patient ,and anormal Wright-stained preparation can be used). individual.. (2)' the Prussinn blue off the slide (2) Aerate the blood samples to a bright with distilled water. red color by gentle swirling under air. (3) Counterstain)vith % sgfranin 'solution for 2-3 seconds] Add 2 ml aerated blood to each to staining the glucose sodium ascor- d. Interpretation. Iron granules present bate in erythrocytes stain blue. (4). Add- 0.1 ml of the sodium cyanide e. DiscussionE solutio4Ce tubes. (1) Sideracytes -occur in several (5), well and, incubate in 37° C anemias, lead poisoning, and after splenec-. water bath for 4 hours. Agitate the blood torny. suspensions every hour. (2) On Wright-stained preparation the granules are .bluish- purple and are called d.Interprefition. Bloid deficient in G- Pappenhemer bodies. 6-PD turns brown after 1-3 hours of in- ARM.- 160- TM 8-2n 5 December 1973 -37 cubation. blood remaips bright 10 ccsteriledisposablesvririrr,es during thistin e _or exarnple, Glaspak or -Plastipak- by Becl ton-Dickinson J. e. Discussion: (2) Sterile needles. Dosiurn; (1) ONO. late --anr _agulants- cannotbe (3) Heparin (Lionoemin j.ised, as rapid Ofscoloration ,occurs inoth Organon). (If substitution is necessary, do abnormal and n-ormal. subjects. not use heparin in which phenol used as a preservative.

(2) Blood-.stored' ACD at 4° C. n --c. -Blood Collection.. NOTE:- It is essential .be tested or Q-6 PD deficiency: ip to four- teen days afteficollection.-' that sterile -conditions be maintained while. drawing. :and :transferring 'blood to mailing 5-36. -Wproen1 of ,Cytogene . (1) ,Scrub the venipuneture area ' at . , least.twice_with gauze padg-soaked With (1)' Contact the laboratory to be per 70_%'isopropylalcohol(other-antiseptic arming the cytogenetic studies and reeniest itgent-S--- may- be toxic to cultures). media andinstructions,for the test. Follow Allow: alcoholto '-dry 'corriPlete ekplicitly the instructiong of this laboratory. .and,-, without .additional .'palpatiOn ,(which / may introduce contamination) draw $-10 cc! (2) Send blood specimeninspecial venous blood into a sterile syringecontain= media which is supplied by the Cytogenetith' ifig.1000 units of heparin. Laboratory.(Due toinh.erent instability of culture media, fresh kits are supplied only Withdraw needle. and immediately on,request. remove needle from syringe. (4). Immediately empty syringe into Specimens on; each patient must bloodseparation vial and seal tightly, be-,-accompanied by KaryotypeiReckuest Forni. (obtained from the receiving 8 laboratory) (5) Mark vialwithpatient's-name, with brief clinkal history, -phisical findings, time, andadate specimen was di and most recent white_ blood, Eell count and d Mailing Instructions.. Ship irnmediately differential. yia. AIR MAIL . SPECIAL DELIVERY. (41' Under normal circumstances, re- Specimens are usually viable fitirup., to 72 sults of analysis are awilablef four to, six hours _after drawing. 'Wheneverpossible,' weeks after receipt of specimen.. therefore, specimens. should not be shipped Thursday or -Fritlay .to avoid 'weekend ar b. Supp Needed: -rivalS; SECTION AINTRODUCTION physiology, and interpretation of tests re- main controversial. When bleeding ,occurs, 6L : elotting'is initiated by aggregation orplale- lets. The platelets congeal to plug the site a. Coagulation of the blood- -iS unly one of theinjury.The congealing_ (viscous of the components in _the larger function Metamorphosis) processisstimulated by of_stopping= blood flow _known: as hemosta- contact with Collagen or by the formation sis. Hemostasis is a complex process in Nhich of thrombin. Iiemostasis_ 'isnot achieved levet-fa factors work together_ or in sequence without .- theSimultaneousfol'inationof to stop the flow of blood from an injured fibrin. Platelet and plasma factors are acti- blood vessel. The three main mechanisms vated, and by a complex process a fibrin involved in this process are grouped as fol- clot is formed. The arrest of bleeding is lows:extravascular,vascular, and iritr- attained, when a firm fibrin network seals vavular. the blood vessel wound with enough strength . - b. The. extravascular mechafiism involvys to withstand, the impact of intrayascular the physical effects of the tissues(skin, . pressure. muscle, etc.) which surround blood vessels. b. . Bleeding. disorders: occur in the:foroW. The effectiveness of this mecharlism in slow- ing areas: injury to the vascular :system, ing the .flow of blood depends on the tissue's inadequate Platelets, inadequate fibrin clot thickness, weight, tightnesA, and ability to ting mechanisms, and inadequate fibroblas., contract.This mechanismalsoprovides tic'.repair. The laboratory performs :a :variety- chemicalfactors-(tissuethromboplastin) of tests which assist the ''PhYsician in his which aid in the lotting process. investigation of blood coagulation. Several:' c. The vascular mechanism involves :the of these - tests measure the.'overall, coagula-- veins, arteries, and capillaries themselves:. tion process. The bleeding -disorders :are due Their effectiveness depends on thickness of :to one or several of the many factors :in-. the vessel wall and its structure, coritracti- volved in :this' process. In most instances bility, and retractibility. prolonged, bleeding is dile to a deficiency o one factor or another. 'However, in so d, The intravascular mechanism invols inStanees, it is dile to therapeutic anticoag coagulation of. the blood. This is a 'highly lants that are intentionally injected to complicated mechanism that requires four terfere with the coagulation mechanise, stages for completion:- Thsse stages are the fev rare instances prolonged -bleeding' is genepation, of thromboplastin,the genera- e to a natural or antigenically-stimulated tion of thrombin, the production of fibrin, increase in circidating anticoagulants pro and the dissolution of the fibrin clot. (-leed in the body. J 6-2. Coagulation: SECTION BCOAGULAON FAC'T'ORS a. Blood coagulation is the formation Of a clot from liquid blood. Itis a complex Blood FactorsSee -Table for -a- - subject and many aspects of nomenclature, complete list of factors' Arict their .l ecember 1973 TABLE 6-1.1CTOMENCLAT serum calcium sulent- to interfere with TION FACTORS. flood anagulation is incompatible. with.life. , Factor e. "Factor-. V (Labile Factor Proaccelerin, Numeral 'Description. Name Acceleratoif Globulin): Factor V: is derived- Fibrimigen from pla snia globulinand iti acts as an ac--. Prothrombin Thromboplastin celerator in the =conversion of -prothrombin - Calcium to -thrombin in th -f? presence of :tissue throm- V Labile Factor, PrE)ccelerin, boplastin. Factor:---V is not-present in serum AC-globulin because' it is consumed during the clotting VII = Proconvertin, Stable Factor of blood. VIII AntihemophiliFactor (SHF') Antihemophilic Globulin (ARC) f. Factor VI (Acceierin)Factor VI has IX Plasma Thromboplastin Component been eliminated as an entity by tare Inter.: (FTC), Christmas TFactor -- -X.---- stuaitPrower Factor national -Cornrnitteem Blood Clotting-- Pax- XI Plasma Thromboplastin tors..

Antecedent (.PTA) . XII Hageman Factor, Glass Factor Factor' 'II (Stable Factor, Proconver- XIII . Fibrin Stabilizing Factor tin). Factor VII is stable to both heat and (FSF), Fibrinase st(irage. It is thought to act as. an accelerator in the conversion of 4prothrombin ta throm- Factor I(Fibrinogen). -Fibrinogen,- a _ , plasma protein, converted into fibrin i bin. Factor VII is not consumed in the clot-- Ling_ prakcess; -therefore, it has a high con- -the presence of thrombin. The majerz.sonce centration in gerurit Severe deficiency .of of fibrinogen is the liver, Bleeding (hie_ to- fact6r VII causes-adecreage in.prottirombin ,a fibrinogen deficiency does not usually be.--activity. come manifest until the plasma concentra-. tion,is below 75 mg per dl.. Factoi! V Z (yAntihimophilic Factor, - b. Factor ie()Thine Globulin). Factor VIII is es- II(Prothrombin).. This sub- the formation of intrinsic blood stance is a stable glyaoprotein, . synthesized thq)mboplastin in the first stage of clotting. in the liver if ari adequate amount ofvflamin is available. Prothrombin Is the inactive DeficieftcyFf facto VIII results in the re- duction of-thromboplastin as.well as .de- precursor of thrombfii. . creasecl conversion of prothrombin. Fac,tor, -..e. Factor. III (Thromboplastin).:Throm- VIII deficiency is a hereditary sex-linked dis- boplastinis a variety of liPoproteins de- order trapsmitted by females and mani- rived from platelets,- the brainthe lungs, fested almost exclusively in males (Hemo- and other tissueS..periVed.from the tissues, philia A). thrembophistin requires:Calcium and factors- V, 'VII, and X to.'convert prothrombin to i. FactorIX(PlasmaThromboplastin thrombin. plasma'. thromboplastin (formed Oomp'onent, Christmas Factor). Factor IX -.- during the..'actnal clotting process froM fac- influences the amount of thromboplastin tors formed. This faCtor is not consumed in the IX, XI, .XII, and platelets) re- --clotting, process; therefore .":quires careittm and facterS V and X to -con-- it is present serum: Deficiency of faacir IX iseither. vent prothrombin to throb-thin. hereditary or acquired and is known as d: -Factor.: IV (Calcium).. Calcium rs an Hemophilia B or Christalas disease. inorganic _40ri wtich is. necessary for clot- finto oecur :-C,alciurrr functions as an,.ticti- j. FactorX (Stuart Prower:Factor). Far vator.;of thrornhoplastie products (,Stage I), for X aids in the prorript conversion ofpro- a cocatalySt in the conversion of- prothrom- thrombin to thrombin-Deficiency of factor bin to thrombin andin- the for X is either acquired or hereditary./ mation of fibrin (Stage -A dec'rease in k.Factor XI (Plasma Thromboplastin AFIV1,160751/TM 8-22774. 3,5 December 1973

. Antecedent). Factor -XI aids in the forma- .Platelet Factor -7.Th tion of plasma thrombOplastin, This. factor sorbed blood factor VII. is stable and -is found in plasma or serum. Deficiency of- factor XI is -probably, heredi- FibrinolyticFactors- is the tary and -results in a miltilemophilia. dissolution of a fibrin clot. The process- is 1, Facto (Hageman Factor, Glass a. necessary activity following clot forma- tion. The mechanism of clot dissolution' is = Factor)- factor isnot required for normal hernostasiS,- -but it is important in complex and 'involves a variety_ of factors: In active circulating. plasma profibrinolysin the various in vitro assays of the elating , 'rnifhanisms. It is a plasma contact- factor (plasminogen) is .conVerted toits active i--=-with-glassand onto glass. FactoFactor'formi-fibririolysin (plasmin), 'by tissue a.cti., vators,streptokinas'e,urokinase, and un 'XII is related 6, factor XI in the activation . thromboplastin, and behaves like an en- known activators. Fibrinolysin acts locally zyme for which one substrate is factol. XI. to dissolve the clot.. nn.. Factor XIII (Fibrin Sabilizing- Fac- tor,,Fibrinase). Factor XIII converts a SECTIONrCHE COAGULTION loosely. linked, fibrin clotin the presece MECHANISM the calcium ions) into a tough fga Its tivityisgreatly reduced Serum (as 6-6. Introduction. The classical theory of comparedlvith plasma) because of its strong.'Morowitz proposed that four components -adsorption of fibrin. interact to form a clot as fo,,Ilows:. Thromboplastin -6-4. Platelet Factors. Platelets are active bin , Thrombin hi blood coagulation, They perform the fol- Calcium' lowing functions:aidinvasoconstriction Thrombin Fibrinogen Fibrin clot and ,the formation of A hemostatic plug,. thromboplastic. activity, and clot retraction. From this concept , tire modern theory was Whein platelets contact -a wettable surface, devised. The mtodern 'theory is based on-four at first they adhere to one another and then stages :(1)the formationofthrombo-' rupture, releasing chemical factors. plastin, (2) the conversion of prothrombin to thrombin, (3) forrnation -of an insoluble a. Platelet Factor 1. This factor acceler- fibrin clot through the interaction offibrino- ates prothrombin conversion and is actually gen and thrombin,throbin, and (4)- the lysis of the blood factor y adsorbed on platelets. fibrsin ,clot by fibrinolysin Theie stages are b. .Platelet ,Fietor 2. Factor 2 accelerate_s illustiatecl in figuke 6-1. fibrinogen clotting of thrombin. 6-7. The Modern Theory: c.-!Platelet Factor 3. A phospholipid sub-. stance involved in, prothrombin activation. a. Stage I. Stage I involves, the foymation This is the most important factor and prob- of intrinsic (plasma), thrornboplastin, This . ablyis an actual intrinsic component of. stage is ,initiated by the platelets adhering platelets. and rupturing, releasing platelet factor 3. Platelet *tor 3 reacts with AliF (factor I. Platelet Factor4. ThiS factor reacts VIII) and.-PTC (factor IX) in the presence o neutralize heparin. . of calcium (faaor. IV_ ), PTA (factor XI),. factor XII, factor V, and factor X;to forth., . e. Platelet Factor. 5. This 'factor is ad- sorbed intrinsic fibrinogeri.' intrinsic 'thromboplastin. Tissue thrombo- ....,. . I plastin on extrinsic thromboplastin isre-s_ f. Platelet Factor 6. This factor reduces' leased by the affected tissues. The average- fibrinolytic.activity, time of this reaction is 9 Minutes. INTRINSIC THROMBOPLASTIN SYSTEM EXTRINSIC THROMBOPLASTIN SYSTEM.

FACTORS.

'STAGE I VIII,IX, XII PLATELET FACTOR 3

TISSUE THROMBOPLASTIN

RELEASED BY AFFECTED.TISSUE

PIT

PROTHROMBIN

. Plasma ThrambdplasIm Ti .s m ..ThromboOlastin Calcium ++,..FaelorS: ,Calciumt+,FaOtots

X: :VII, & X

THROMBIN THROMON

THROMBIN

'FIBRIN

STAGE. IV AF14 1.60-51/TSI',5-,227-4 -.5 December 1973 6-5 b. Stige II. In the intrinsic systempro- study. Specific tests to measure the various thrombin is converted*to thrombin in the coagulation functions are listed in Table presence of-plasma thromboplastin, calcium, 6-2. and factors V and X. The extrinsic system - requires the presence .of an additional fac- TABLE 72. DIAGNO4STIC: COAGULATION Cot, factor VII, for 'the conversionof pro TESTS thrombin, to thrombin. This stage takes: place Coagulation Function in 12-15 sOonds. Vascular Function , Tourniquet c. Stage' III. Alter the thrombin is gert- Test erated, it quickly reacts with Platelet Function Bleeding Time, Clot Retrac- fibrinogen to tion, Tourniquet Test; Pro- form fine fibrin clot. The fibrin clot plus . thrombin Consumption, calcium ions- and factor XIII, the fibrin- Thromhop.lastin Generation stabiliz-inx factor, form a stabilizedclot. Test, Platelet Count, The clot, is characterized by its insorubility Clotting; Ability Clotting Time, Paitial in 5 M urea. The stage takes place-inone Thromhoplastin second. Thrombin Time Stage.I De e Prothrombin Consumption d. Stage IV. This stage its rinolyti Test, Thromboplastin Gen- dissolutionof the clot. Circulating plasminqr eration Test gen is converted to it. active form plasniin. Stage II Defects One Stage Prothrombin Time - The clot in the- presence, of plasmin is dis--- Stage lit Defects One Stage Prothromhin solved. Time, Fibrinogen Level St ge. IV Defects Clot Retraction, Thrombin Time, Fibrinolysin Assay 6-8. Coagulation Inhibitors. In addition to .the factors necessary for clot formation, in- or hibitors. are resent which control but do 6-10. Blood Collechon: not prevent cgulation. Natural inhibitors a. Two-Syringe Technique. To avoid con- haVe been desibedforvirtuallyevery tamination'of blood by tissue juices the two clotting ft-I.c' tor. Clo g activity 'is also in- syringe venipuncture isusually employed hibited by the admini ion' of ant oaga- (ekcept"fortheone-stageprothrombin lants such as heparin an. me) for coagulation tests using venous blood. The technique is as follows: (1) Cleanse the site with 70% i opro- SECTION DCOA IGULAT STUDIES- 'by! alcohol and allow to dry. (2) Place a tourniquet on the arm. 6-9.Introduction.Thepartialthrombo- plastin testis Immediately insert the needle and they most useful screeningremove the tourniquet. (Quick removal of method for the detection cif blood coagua- don disorders. This test encompasses all the tourniquet prevents stasis.) three stages of coagulation and indieates ('1) Withdraw about 2 ml of blood. Re: an abnormality in all of the clotting factors, move'the syringe and discard it. with the.excoption of factor VII and plate- Attach a second syringe and with- lets. The, prothrombin time and thrombo- v therequiredquantityofblood: plastin generation time detect. deficiencies (6) Blood samples for anticoagulation in other stages. The prothrombin timede- tects abnormalities in stages II and III while should be immediately mixed with the anti- the thromboplastin generation test detects coagulant. abnormalities in state I. A systematicap- b. Anticoagulants. Anticoagulants for cq- proach should be followed ina. coagulation agulation 'studies can be, obtained in corn- 160 -51 /TM 8-22774 5 December 1973 mercially,prePared vacuum tubes orpre-- stopwatch immediately after the puncture pared in the laboratory. The preparation is made. If a good puncture is made, you and use of anticoagulants is as follows: will hear a click of the bladt% touching the (1) Sodium Citrate, 3.S76,,' Dissolve'3,8 glass slide. g of sodiuni citratein 100 ml distilled (5) At intervals' of 30 seconds, gently water.Refrigerate.c As:. an anticoagulant, blot the blood from the wound with a piece, combine one part 38% sodium citrate with of filter paper,. being careful not to touch

9 parts blood., the skin. , I. (2) Sodium.' Oxalate, 0.1 M. Dissolve (6) At the time ivhen blood fails to ap- .34 g sodium oxalate in 100 ml distilled pear on the filter paper, stop thestOpl,Vatchl water.Refrigerate. As an anticoagulant, cotnbine one part 0.1 M sodium oxalate, c. Ivy Method: 'th 9 parts brood. (1) Place a blood pressure cuff on the 3) EDTA. Dissolve 10 g of 'EDTA41 patient's upper arm and inflate it to 40 rrirri salt in 100 ml distilled water. Pipet 1 ml of this solution, into a -suitable test tube. Cleanse the forearm with 70% iso- Allow to dry in the oven at low tempera- propyl alcohol 'and allow to dry. ture. For use as an anticoagulant, add 5 ml (3) After the area has dried, make of venous blood and mix well. three incisions mm deep and 3 mm long c. Glassware. All glassware for coagula- with 'a sterile surgical blade, Avoid super- tidn studies must be scrupulously clean. ficial veins. Used glassware should be free 6f chemicals (4): Start a stop 'watch and blot the and any traces of human blood components. three :punctures gently at 15 second -inter- All glassware should be cleaned in deter- vals.' gents free of organie solvents and rinsed (5) Continue 'until the bleeding stops several times with distilled water..Never use from all wounds. Record the longest time. . test, tubes or. Pipets that are damaged. The use of disposable syringes and needles elimi- = d. Sourcesof Error: nat'es the need for siliconizing glassware. (1) An Inadequate punctureresults in a poor flow of blood.. Squeezingthe site to 6-11. Bleeding Time: Obtain a free flow of blood must not bedone. (2) Low skin temperature produces a isthe a. Principle. The bleeding time constriction - of the capillary vessels result- time it takes for a small standardized wound to stop bleeding. It is dependent upon the ing in decreased blood flow.' elasticity of the skin and capillary vessels, (3) A standardized puncture- is neces- the efficiency of tissue 'fluids, and the Me- sary for valid results. Too deep awound pro: chanical and chemical action_ of the' plate- longs the bleeding tirrie, while a shallow lets. wound shortens the bleeding time. b. Duke Method: e. Discussion: (1)' The puncture site is about. 15-20 (1) The Ivy Bleeding Time is the meth- mm above the rounded fatty portion of the od of choice ,because the blood pressure on the vessels is constant, the incision is uni- ear lobe. forM, and the arm offers an area for multi- (2) Clean the area on the ear with ple determinations. 70% isopropyl alcohol and allow to dry. (2) The bleeding time dependspri- ;3) Place a glass slide behind the ear. marily on extravascular and vascular fac- (4) Puncture the thinnest part of the' tors and, to a lesser degree, on the factorsof with a surgical knife blade. Start the coagulation. The chief factorcontrolling AFM 160-51/TM 8-227-4 5 December 1973 6-7

bleeding from a small cut is the constriction clots. Then- tilt_ tube 2over2,, 30 econdsu. of the minute vessels following injury. Ac- it clots.- When tubeclots, tilt tubein the curacy in this test is enhanced by blotting same manner. the drops of blood at shorter intervals of ( 5) .'Report, thetime - takefor the time as the drops of blood becoine progres' blood in the 'last- toe to cl6t, the blood. sively smaller. does not clotwithin60 minutes .repOrt the', (3) Throthbocytes play an -important Lee-White tune 'as geater: than 60 minutes. part in the forMation of the hemostatic plug Hold all tub _Hours for observa, which seals off a wound. In thrombocyto- ri:of clot retractio lot lysis, -ancreryth- penie purpura them is a decrease in platelets rocyte fallout. resulting in a prolongecy)leeding time due to a defective platelet plug.'-An additional fac- c.Sources of Error.: tor prolonging the bleedink time in this con- (1):Thefollowing yaiables',tend to de- dition is a defect in capillary contraction. crease -the clotting time: rough handling of (4) In hemophiliatihe?leeding tiMe-is the blood specimen, presence:of tissue normal., This is explained by the fact that (traumatic Venipuncture), Irequent tilting _there,are no vascular. r extravascular ab- of the tube, and untlean tubes. normalities. However, the test should not he (2) The following variables tend to in performed on a known hemophiliac, for de- crease the clotting time: extreme increases in layed oozing of blood is a real hazard. temperature, variation in plir'and perform- f. Normal Values: ance of the test at roomteMperature. (1) Duke Method: d. Disc-tisSion: (a) One to 3 minutes. The sensitivity of the test is in- (b) Three to 10 minutes border ne. creased by- the use . of polystyrene or (2) Ivy Method: Up to 7 minutes. conized test, tubes. The.-test is .perfprmed in the .same manner and 'Checked every_ minute 6-12. Whole Blood- Clotting Time after 25. minutes has.-elapsed. White): (2) An. activated :clotting time is per- formed using:celite as an activator; a. Principle. The whole blood clotting time (3) This test is of value primarily as it is a rough measure of all -intrinsic clotting' is used to follow heparin therapy. Its use factors in the absence of tissue factors. Var- iations are wide and the teSt'sensitivity. as a .Screening procedure is ,limited due to limited. -poor sensitivity. (-4)-: A prolonged clotting time immedi- b. PrOcedure: ately indicates impaired coagulation, but (1) Draw 5 ml of venous blood With a normal clotting time does not exclude many disposable plastic syringe, using the two- serious clotting defects. syringe method. (5) In hemophilia there is a prolonged (2) Label 3 Rahn tubes 1, 2, and 3 and clotting time. This is due to a deficiency of place 1 ml of blood in each tube: Set a clock factor VIII. for 60 minutes and start the clock as soon as (6) The coagulation time is normal in the blbod starts into the second syringe. A thrombocytopenic purpura. ThiSis.explained stopwatch can be used. by the fact that only a small number: bf (3) Place the tubes ina. 37' C water thrombocytes need be present for normal co- bath as soon as possible. agulation to take place. (4) After 5 minutes tilt tube 3 (the (7) When heparin is administered as a last tube filled) every 30 seconds until it therapeutic anticoagulant,' its effect may be 16041/Tti9227 5 D -eniber' 73 determined by the degree of prolongation 'of -Small clot which dan be interpreted as ade',, the coagulation time. quite :retraction even: though itisinade- (8) The method of measurement of the 'quate. coagulation time by the useof- capillary (2) Active fibrinolysins cause the clot blood drawn into a capillary tube. is com- to dissolve The presence of. erythrocytes in.- pletely, -unreliable and should never be done, the expressed serum suggests the presenco of since the sample contains a large. amount of fibrinolysins. sire -fluid.: Furthermore, -, the volume of (3) Certain anemic patients, with a blood in a canillary, tube. is difficult to con- low hematocrit value, show increased. clot re- trol, A-capillary tube technique using- venotts traction due to the formation Of a small clot. blood has recently been described. as having a high degree of accuracy. d. Discussion: e. Normal Values: (1) Poorclot ,retractionoccursin thrombocytopenia, qualitative platelet defi- (1) Glass Tubes: ciency, and in cases of increased red cell (a) Five to 15 minutes at 37° C. mass. (b) Up. to 20 minutes at room tem- pera ure. (2) The cl6t retraction' is normal in hemophilia since there are a normal number (2) Polystyrene Or Siliconizeci Tubes: of platelets. However, the onset of contrac- Twenty-five to 45 minutes. tion is often ,delayed in blood samples from (3) : Up 2 hemophiliac patients. minutes.15 seconds. (3) -Tubed frorfl the completed venous coagulation time can be used to perform 61.3., Clot Retraction. Tes this test. a. Principle. Whole bloods allowed to clot (4) Attempts to quantitate the clot re and as it clots it expressesserum. The clot traction test -are generally unsuccessful. retracts due to the action-of platelets on - fibrin. e. Normal Values. A 'normal- clot retracts " fro_ m the sides and bottom of the test tube b. Procedure: within 1 to 2 hours and,expresseS 45-60% (1) -Withdraw 2 ml of venous blood of serum after. 24 hours: sing the two-syringe technique., (2) Place, I ml of blood into, each of 2 6-14. *Tourniquet Test: glass test tubes and immediately incubate in a. Principle. The fragility of capillaries is a 37° C water bath. Inspect the tubes. in 1 determined under increased pressure. The and 24 hours. pressure partially obstructs the venous re- (3) Examine the tubes for retraction turn from the arm and increasep intracapil- aftjr incubation. Separation of the clot from larypressure; The °number ofpetechial the test tube is coniplete retraction ( +4). hemorrhages reflects the degree of capillary (4) At the end of 24 hours you may fragility. wish to measure the amount of expressed b. Procedure: serum. The. per cent Serum .expressed is cal- (I) Place a blood pressure cuff on the culated as follows: patient's arm and inflate it to a point mid . ml serum way between the systolic and diastolic. pres- x 100 . ml blood sure. c. Sources of Error: Remove the cuff and wait 2' min- A fibrinogendefiency causes a utes. A FM 160:17Tal3=227 1 / (3) Examine a representative area (a Calcium Chloride, 0.025 M. Add circle about 2.5 centimeters in diameter)on 2.77 g calcium chloride to a 1-liter volu- the hand or arm for the presence of pete- metric flask. Dilute to the mark with dis- chiae. tilled water. (4) Grade the number of pet chiae as (3) Control Reagent. Obtain a suitable follows: 0-10 .= 1+, 10-20 2+, 20-50 normal control from the Federal Supply Cat- 3 41, and over 50 equal 4+. 'alog or use plasma from a normal subject. c, Sources of Error: Abnormal controls are also -available from 11) Mistaking skin blemishes or p ete- the Federal Supply Catalog. i a increases the number. Note s in blem- c. Procedure: ishes before the test. (1) Combine 9 parts of freshly col- (2) Capillary fragility variest differ- lected blood with one past 3.8% sodium cit- ent sites. rate. (3) Maintaining the pssure too long Mix well and centrifuge foy 5 min- uses false positives. utes at 2000 rpm, immediately after draw- = ing the blood. Remove supernataht plasMa d. 13iscussion and refrigerate until ready to use. Plasma '(1) The tourniquet test is -a crude test, must be tested within 3 hours. to determine the ability of blodk vessels to withstand trauma and should rivt be used (3') Place tubes of activated partial thromboplastin and 0.025 M calciurh chloride as a screening test for surgery. , (2) Increased in 37° C water bath. Incubate for about 5 vascularfra ilityis minutes. sometimes found in qualitative anduanti- tative 'platelet defects, vitamin C fieiency, (4). Pipet 0.1 ml of activated partial dietary ascorbic acid deficiency,nd rn the thr'ornboplaAin ,into the desirenumber of various purpuras. The term ` "pia pure is not tubes (prepare 'three. tubes each tin- specific, but applies to a nurnber of affections known and one for the control characterized b' bleedin-g into tissue. (5) Add 0.1 ml of patient's plasma or (3) The tourniquet test is most often control toithe tubes. Shake well and incubate performed by the phyaician. for 2-4.minutes at 37°C. e. Normal" Values: 0-10 petechiae per 2.5 (6) ,After the 24 minute incubation areas period, shake well ; then fin.bly blow 0.1 of 0.025 M csalcium chloride in 0 the test mix- 6-15. Partial Thr nthoplas in Time ture and simultaneously start a stopwathh. (Activated): ('7) Return the tubes to the incubatmt and after 30 seconds tilt the tube. and ob- a. Principle. Norrnal.citrated plasma con- serve for clot formation. tains all clotting factors .except calcium ions and platelets. Calcium, ions and a partial (8) At -least two answers should agree thromboplastin (platelet-like substance)is within one Vecand. Take an average for the added to the plasma and the elotting time final answer:Always report the control. yecorded., An activator is added to. make a. Sources of Error: C------A activation of the plasma independent of the surface of the tube. ( 1 ) Plasma must be.prepared with care without disturbing the bully coat. b. °Reagents: (2) If plasma is left at room tempera- (1) Activated Partial Thromboplastin. e, the test. must be performed within 45 This `reagent is available from commercial minutes. Otherwise plasma can be stored at sources. 4° C for 3 hours. AFiVI160-51/TM S-22+4 5' December 1973 -Clean glassware and accurate -ma. are mixed under. carefully contolled pettinzare'essential for valid results. conditions, fibrin strands will normally form (4) --Do not use sodium. oxalate:as -an within a-matter of seconds. The interval anticoagulant. Sodium citrate is a better during which time reaction occurs. is -the- preservative and activates.plastna faste,. prothrombin ;time. This:- test measures th6 overall prothrombip -activity of plasina.t (5) Tubes must be shaken. tho-- -".before, and after incubation to inrir°eitide- b. Reagents: -""" .quate (1) Thromboplastin. :from e. Discussion: the Federal Supply 'Catttlbg.."-Calcium added. (1) A partial thromboplastin is unable (2y Plasma, Human, :Abnormal, Ding' to Compensate for a deficient. plasma 'factor: nostic. Available from the .Federal .Supply A tissue thromboplastingives. a normal Catalog, value in this test: with a deficient factor. (3) Plasma,HumaiL Noernal,Diag- (2) The .activated partial, thromboplas- nostic.Available from the Federal Sup- 'tin reagent pr oVideS maximum surface ply Catalog. pesure forthe test to be (4) Bloocr Collecting Tube' With Sodi- um-Citrate Available from the Federal Sup, (3) This test is an over test . for:, all three .stages of coagulation with the ply..Catalog. Contains. 0,5 nil-of 3.8% sodium a,ception of factor VII, calcium and platelet citrate. factor 3. Procedure,: (4) l'he' partial thromboplastin, time is Add exactly 4.5 nil of blood to the prolonged in all deficiencies. of prothrombin e cainiig.0 .5 of 3.8% sodium citrate. and factor-. V,_ as well as deficiencies 'of all the plasma i'att-ors in- the intrinsic.. system. (2). Centrifuge 5 minttes at 2,000 rpm 'To exclude an abnormality in the extrinsic _as' soonas' possible.Immediately remove system, a Prothrembin time test should be plasma:to12 x 75 nftri test -tube and rdriger- .performed on all abnorinal Plasma. ate until ready to use. (5) In conjunction with th6:0-16throm-. (3) Preparethromboplastinasde- bin time- the :following deficiencies can he scribec'Lby package insert, determined: (4) .Incubate th% thvombophist n,pa- . tient plasma, and control plasmas 'for 5 min - F7T PT peficievey utes in a 37° C water bath-Plasma may not Abnormal Normal Stage I: factors VIII, be incubated for more than 10 minutes, as IX, XI, XII Ileat destroys factor VIII. Abnormal Abnormal Fibrinogen, Prothrom- bin,,factor V, and X (5) Pipet0.2mlofthromboplastin, Normal Abnormal Factor VII into the desired numbei; of tubes. A differential partial- thromboplas- (6) Blow in 0.1 ml of patient or con tin time is'Jperforrned to he specific trol plasma, simultaneously starting a stop- deficiency. watch. Normal Values: Less that 35:0 seconds, 7) Incubate at 370 C for 5-6 seconds. en using-citrate anticoagulant. 4 '(8) To read': 'wipe the tube dry and hold toward an 'adequate light source. Tilt 6-16.- One-Stage Prothrornhin Time: tube very gent& once or -twice and. observe Principle. When optiimal amounts of for appearance of the fibrin web which is thromboplastin, calcium,./and. citrated plas- -the endpoint, NE% 16(1.-.51VTIVI 87227-4 5, :December 641

(9) Run the patient plasma and con- e. Discussion:: trol i-n triplicate. The, three. determinations 1)- The -proth ximbin .activity -rnAy b should agree as- follows: epo'rtecl iii percentage as well as in seconds Froth ramb in o by applying- the -patient's prothrombin- time 1.2-20 seconds .0.5 second (inseconds).to,a potbombin. activity 20-30 seponds 1.0 second Curves-.for different normal controls 01.)'er 30 seconds- 2.9 second are, supplied. f)y the manufacturer..if the d, Sources of Error: plasma :dilution ...curveisto be prepared, whole normal, slasma must be diluted with (1) TiMing is 'critical. The stopwatch prothrombin must be started the instant the plasma conL fr=ee plasma.Salineshould tacts the .thromboplastin, and -never be used In making the dilutions, The:- it must be percentage :activity should never be calcu- stopped immediatel- vhen the-clot forms, lated by 'dividing ., the fibrilla] time by the (2) Thromboplastin must be prepared patient's prothrombin time. as stated in the manufacturer's directions. (2) The prothrombin activity of -a pa-- ) Avoid traumatic venipuficture. tient's.plasina has important diagnostic ,and. (4) Tourniquet application must not .Prognostic significance in diseases of the be prolonged.. liver ,'in vitamin N deficiency, and in .the of dicintaiol as a therapeutic lanticoagitlant/ (5) The' test must be.accomplished Within 3 IfOlurg after collection of the blood , (.3)Prolongation of the one -stage ,pr . specimen. If the test is not run immediately, thrombin time does not measure prothronw refrigNite the plasma' and thromboplastin. bin deficiency alone but rather inolicatesso Refrigratecl plasma is stable for a maxi- failureof conversionof prothrornhinr to mum of 4 hours. thrOmbin. Specifically, the 'test detects':defi7 cencies of -factors I, V, VII or X. Varying (6). The blood must be added to the reductions44- in, any one,.- or cornhination, 'coagulant immediately to avoid ,clot for- ion. these factors prolong the one-stagenrothrom- bin time. (7) The test must be ,performed at the temperature of 37°T; incubation must not (4) This -well -Adapted for be prolonged. brbrneter use. (8)-clean glassware is essential. After Normal Values: 12 15 seconds. eachuse,- the glassware.. must be scrupu- lously cleaned with soft soap and la test tube Prothronibiti Consumption Time: . , _brush and thoroughlyirinsed. Synthetic de- a. Principle.Trothrombinispartially terNnts should not bused. Unscr;atched' used up in the -- normal clotting process. The ..glasswa,re reserved ely for prothrombin mount of PrOthrombin remaining in serum determinationsX cl be utilized. indicates the ..adequacy of the thromboplas- (9) The use of-sodium oxalate as anti- tin'comPle, The time required for a clot to coagylant decreaseTAhe activity of/factor V. form wheifoptimal amounts of thi.omhoplas- Use sodium citrate as stated in the proce- tin, fibrinogen, factor V, calcium, and ser m 4ure. are rni,e(y, is the' prothrombin consumpt_on, (10) The trromboplastin reagent must. time. be tested for constant. potency. This is de- Reagents: termined by analyzing the normal cOnt..

plasma, . _Thomboplastin, See paragraph 6 (11) Hemolyzed plasma must not be 16h(1). used. .(2) Normal Adsorbed Plasma Reagent. 160-51/11I 5 December 1973

9. parts of freshly collected,: Time Vgrintion h -1 part 3.8% .sodiiirn cit- Over 30 seconds 2.0 seconds 2p-30 seconds 1.0 second_ itrifuge tat 2,000 rprn for 5 12-20 seconds 0.5 second minutes and remove supernatant-Vasnm d. Soiirces Error.Seepara raph (c) For each ml of plasma, add .100 of chemically 'Pure bariumasulfate (see Fec(ral Supply Catalog). .e. Discussion: (d) .Mix- well for 3 minutes and al- (1) Patients havin decreases or de- low'to stand at room- temperature for an ad- fects in the thromboplastin complex (throin- ditional 2 minutes to complete bacytopenia, hemophilia, deficiency- in (e) Centrifuge the material and the plasma thromboplastin antecedent or corn- upper three-quarters of the plasma.. ponent),_ will not uge- up normal amounts of (f) Perform a prothrombin time on prothrombin in the clotting process. The ex' the adsorbed plasma. The prothrombin time cess of residual prothrombin in the serum should be greater than 60 "'secdnds; if .tult, will result in a Seriim prothrombin time of repeat the absorption; NOTE: : Barium sul- less than 20 seconds. fate-adsorbed plasma is available commer- (2) Administration' of'heparinwill cially; AlsO available is a reagent containing serve to shorten the prothrombin consump- fibrinogen,factor V, thromboplastin, and tion time aswellas prolong the "Lee calciuth.. White" clotting time.Dicumarol, onthe' c. ProCedure: other hand, will have no effect upon the serum brothroMbin time, even while pro- (1) Draw 5 ml oflood by a Lion 13- longing the plasma prothrombin time. This matic-venipuncture. indicates that dicumarol has no influence (2) -Place the .blakd in a test tube and upon the thromboplastin coMplex. zIllow it to clot at room temperature. (3) NorMal results are considered valid (3) Place the clotted blood in a 37° Conly if the onestage prothrombin time is t wa er bath for 1 hour. normal. (4) Centrifuge the specimen at .3,000 (4). The .test abn'ormal in platelet rpm for 3 minutes, and' transfer the serum iencies and Idefi den ci es ,of factors_VIII, to another test tube. X, XI, and (.5) 'Placethe Serum, thromboplastini Itis 'important that the barium reagent, and barium sulfate - adsorbed plas sulfate -4 clsorbed plasma be deprothrombin- ma: in a 37° C water bath for 5'minutes to ated. -su_ iciently to give a prothrombin time equilibrate to that temperature. of oil` 1 _minute. This reagent is a source .(6) Pipet 0.-1 ml of serum, 0.1 ml of of:fibrinogen and factor V, since they are barium sulfate-adsorbed plasma, and 0.2 mi. ..ifsed up in the formations of a clot. of thromboplastin reagent into a Kahn tube in that order, starting a stopwatch simulta- f.Normal Values: 1) Longer than 30 seconds: Normal. (7) Determine the time for.the clot to Twenty-30 seconds: Doubtful. form as in the one-stage prothrombin time. 3) Less than 20 seconds: Abnormal. (8) Repeat the procedure, commencing with step 6, for two other determinations. 6A8.Thro boplastin Generation Tiine: (9) The three determinations :should Princirile. A potent thromllastin is agree as follows: geherated when platelets, protlof ibin -free AFM 160-51/TM 8-221 5 December 1973 6-13 plasma; serum, 'and calciumare .Pipe '0,1' 'tillof (1,025: M calcium , AftergenerationofthethrOmboplastin, Chlo into three 12 x 74 nun- test tubes.- all' factors .necessary to proiluce a ,clot are ( -2) Place Jest, tubes: the folloWing present except. for factor ,I and factor reagents in a 37°:.p water bath,: When these factors are added to a -normal (a) Partial thromboplastin- renge thromboplastin generation mixture, a- clot-is (b) Normal plasma substrate. deteected within 71.6,Seconds, .With -ab- normal \time, deficient factors are -identi (c) Normal adsorbedtplasma. (d) Normal aged serum. fied.by (dsorbedpatient plasma oraged patient serum. (e).0 ml of 0,025 M ,calciuM chlo- ride. b Reagents: ur tubescontaini 01 ml'-of 0.025 M calci chloride. ,(1) -Normal : Plasma Substrate.. Avail able from-the Federal Supply Catalog. This (3) Prepare'. a generatioc mixture by control is used as the source of faders I, adding the ,following reagents to 'a clear 12 II, V, VII, and X. x 75-mm test tube: (Platelet- (a) 0.2mlpartialthromboblastin (2) Partial Thromboplastin reagent. life Substance). Available from commercial (b) 0.2- normal adsorbed plasma sources. . reagent. (3) Normal Adsorbed Plasma -Reagent: 0.2 ml normal aged serum rea- Seeparagraph6-17b(2).The ,adsorbed plasma, is the source of .factors V, VIII, XI, (d) 0.2 ml of 0.025 M calcium chlo- -and XII. Dilute normal adsorbed plasma 1:5 ride, simultaneously starting a stopwatch in saline (0.85% NaCI) aid allow,to stand. or automatic timer. -.for 1 hour at .4° C, or in an ice bath before' used At , 2 minutes pipet 0.1 ml -of- the generation mixture. into the first tube con- (4) Normal Aged Serum. Add 2 ml of taining 0.1 ml of 0.025 M calcium chloride. freshly-drawn blood to a clean 13 x keU Immediately add 0.1 ml of norrnal plasipa tube and allow to clot at 37° C for 4 hours. substrate to the tube, simultaneously start- After the incubation, centrifuge and rernave ing a stoplkatch. , the clot. Dilute the serum 1:10. with 0.85% NaCI (saline) and allow to stand for 1 hour (5). -Check for the first formation of before using. a clot using the tilt-tube method. (If avail- able, substitute a )a ibrometer for detection (5) Calcium Chloride,0.025 M. Add of clot formation.) 0.277 g anhydrous .calcium chloride to a (6) Repeat steps 4-5 at 4,6, apd 8 100-MI volumetric flask. Dilute, to the mark minutes. with distilled water. (7) The normal range for the control (6) Patient'sAdsorbed Plasma Rea- reagents is 7-16 seconds foP clot formation. gent. Prepare in the same manner as normal This range is obtained within the _2to 8_- adsorbedplasma,,substitutingpatient's minute generation time. plasma for normal plasma.-- d. Test for PatieiDeficiencies (7) Patient's -Aged Serum- Reagent. Prepare in the sale manner as normal aged (1) Repeat the procedure for standard- , serum substituting patient's Serum for nor- ization of control reagents, substituting pa- mal serum. tiert's aged serum for normal aged serum and patient's adsorbed plasma for normal c. Standardization Control Reagents: adsorbed plasma. 6-14 AF 1 160 -51 /TM .S-227-4 .5 December 1973

()If there is a marked difference be- adsorbed- plasma; one ata'. time, for the tween the normal generation time and the patient's reagent in 'the generation mixture: patient's generation time, repeat the, test, substituting normal aged serum and normal Interpretation:

Adsorbed Aged Delieleil Plasma. Serum PT PTT F'actor Abnormal Normal_ AbnOrmal Abnormal V Normal Normal Abnormal Normal VII

Abnormal Normal. Normal. Abnormal VIII Normal Abnormal Normal Abnormal IX Normal Abnormal Abnormal Abnormal. X Abnormal Abnormal Normal Abnormal XI AbnorMal Abnormal Normal Abnormal XII Abnormal Abnormal Abnormal Abnornfal Inhibitor f. Sources of Error: (4) Adsorbed --plasma,.contains factbrs XI, and XII. If the TGT is pro- (1) -All reagents must be freshly pre- . pared each clay. longed when patient's adsorbed plasma is substituted for normal ads-orbet. plasma, Tubes of plasma si bsti--ite . left at -deficiency. in one or more of these factors . room temperature are unreliable.' is indicated, All U.S.P. barium sulfate prepara-.. Aged serum contains factors IX, tions are not standard in reaction. Factor IX rid XII. If the TGT is prolonged times may be incompletely removed. when patient's aged serum issubstituted

(4)Antithromboplastinastin- . activity . in far norrnal aged serum: -a deficiency in one bipod prolongs TGT, and accounts for some or more of these factorsis'indicated. , . - double deficiencies. (97.Factors XT and are y.present This isnOt a routine laboratory in adsorbed, plasma: and aded serum; A de- proi, e; considerable technologist skillis, ficiency ill eith&i of::-.theae factors prolongs: required.., ', the TGT'. Determination of the specific fac7.y, (6) A lot canformwhen the gener`a7 tor deAciency is done by the physician. oft :. bon mix- tt-e is prepared. This- is removed the baSis of cliniCal.grounds with a wooderi'applicator stick. h.' Normal -Valites.Clot formation time (7-) Otherrsourcesoferrorcanbe of 7-16 seconds --within S minutes genera ound in paragraphs 6-15d and 6-16d. tion time. g. Discussion: 6-19. Thrombin Time:. (1) A platelet deficiency can be de- tected by the TGT. This is done by prepar-,_ a. Principle: A known amount of- throrn- ing platelet-rich plasma" and subtituting it bin .is , added to :pjasma and the time re- fori.the partial thromboplastin reagent. An quired for clot formation is recorded. abnormal generation time is noted in a pa- iieent with platelet deficiency. `b- Reagents; k, (2) The TGT reveals abnormalities es- (1) Thrombin,.5000:Unitspermi.. sen'tial to the development of thromboplastin Availathe from. the- Federal Supply -Cata- activity in the intrinsic blood system. log% (3) The TGT should be correlated with (2) StockThrombinSolution, 500 the PT and PTT to _determine the specific Units per Reconstitute -late thromthn abnormality. ,with 10 ml of saline (0.85 percent NaC1).. -AFM.- 160-51/TM 6-1

Working Thrombin Solution,10 ammonium sulfateancl. measured with Units per. ml. Dilute the stock solut-on 1;50 spectrophotemeter.. with saline (0.85-percent NaCI). . b .,Reagents-: (4) Control Plasma. Available'ederal Parfentjev Reagent. Add 133.33 g Supply Cata ammonium sulfate, 10.0 g NaCI, and 0025. Pr.ocedure g merthiolate to a Witer _volumetric flask. (1) Obtain 4.5 ml of venous blood and Dilute-to-the mark-with distilled- water. d to. 0.5 ml sodium citrate. Mix well, (2) Saline (0.85 Percent NaCI). Add . (2) Centrifuge and separate the 8'.5 .g sodium chloride to. a. 14iter Volumetric plasma. flask. Diltite to the mark with distilled water. (3) ..`Incubatepatient -plasma,control (3), -Fibrinogen, Available plasma, and thrornbin.solution irr37° C water the Federal. Supply Catalog. bath for 3 minutes. (4) Fibrinogen, Stock Standard,500 (4) Pipet 0.1 mlof control plasma mg per dl. Ada 500 rak of fibrinogen to a- and 0.1 ml of thrombin solutionginto a clean 100-rri1 volumetric flask..,Dilute, to the mark test tube, simultaneously starting- a stop- with saline. watch. Observe for' clot formation. c. 'Calibration'ettr-ve: (5) Repeat step (4) for the patient's'. .( lb Prepare- the follolving dilutions ..of the stock standard: d. irces of Error. See paragraph, 6 FikrogenStok 16d. Stanard Saline Concentration 10.0 ml -500 mg' per 41 eDiscussioni, 350- (1) The throMbin time is03 longed in 5.0 ml 9 mg .per-01:: fibrinogen- levels below 100 mg per dl; PreS- 2.5 nil' 7.5 ml 125 rag per vuce, of fibrinolysins,, and preselCe-_ef cir- 4(2) Set up the following cuvets for each culating anticoagulants standard: (2) Low concentrations of ;heparin in . Blank .Cuile Cuvet the patient's 'plasma may not be observed 0.6 ml 'standard 0.5,m1 standard by this test. To -detect ' small amounts of -4.5 ml saline 4.5 Farfentjev reagept heparin, thrombinisdiluted, and normal and patient's plasmas thrombin times are (3) Three minutes: after addition of determined. A prolongation of the patient's the Parfentjev reagent, shake the cuvets thrombin time over that of the normal at vigorously, and read the -absorbarices of the some dilution indicateS an antith'rOmbic sub- test cuvets at 510 nm with' fhp blank set,t stance. zero absorbance. , (3) To perform the antithrombin test Plot absorbancd versus colleen dilute, the workfifg thrombin 1' :2, 1:, 1:8, ti6in on linear. graph paper 1:16, and 1:32 with saline. Then follow the d. Procedurp:' procedure for the thrombin time test using (1) Draw 4.5 ml of fresh venous blood .1' the dilutions. and add to a test tube containing 0.5 ml f. NOrmal yalues: 11-15 Seconds, 3.8% sodium citrate.' (2) Centrifuge and separate the 6-20. Fibrinogen Assay (Semiquantitative): plasma. a. Princinlel, Fibrinogen, a Plasma glob- (3) 'Set up the following cuvets for each din formed- -n the liver,is salted out by. unknown plasma: 6-16 5. December 1973

Blank inkno-zun. (1) -Draw 4. « rnl of lyes1 venous bloOd 0.5 ml plasma . 0.5 ml plasma add. to 0.5. rnl of 385, sodim citrate. 4.5 Parfentjev reagent . . (.2) Ceritrifuge at :2,590 rpm for 20

Three minutes 'after /Addition of minutes and separate the plasma. . the Parfentjev reagent; shak6: the cuvets Add- 0.2..m1 of Ypatien read the.absorbances of the and 1(.-).2' ml of normal control Plasm test- cuvets at510 nm 'With' the blank set at test tubes- itul,31?__Clc_aterbatTL_ zero absorbance l ,;; (5) Obtain; the 'fibrinogen (4) Add 0.2 .ml. of 0.025 M. calciUm - concentra- chloride to each tube,. Simultaneously start- tion-flora the calibrationcurve. ing dstopwatch. e. Sources of Error: .Tilt the 'tube every 30- seconds for (1) The Parfentiev 'reagentdeterio- the first' 90 seconds. Remove the tube and rates after two weeks of storage. Prepare observe forclbt formation. fresh-every-2 1,vdeks.-- d. SourceS of Errol (2) The' fibiinogen has a potency. for 60 months. Do mot use outdated fibrinogen (1) Platelet-pour plasma must be used to prepare standards. In-the test. Platelet rich plasma shortens the recalcification time. The procedure is limited dependent on time, anticoagu- (2) The test is dependent on the plate- lant, Ind concentration of other proteins. let count, concentration of plasma clotting factors, time of storage in glass, and the pres- Di-scussion: ence of circulating anticoagulants.' (1) Although the procedure is_li- (3) Seepar agrabh 6-15dforother a rapid determination can be obtained when sources Of error. .adecinate fibrinogen,leveare prey e. Discussion: 2, (2) Screening procedures, -in kit form, (1) This _testisthe basis for other are available commercially.,\ coagulation procedures. ,Fibrinogenis esSe41a1 to clot for- (2) An abnormal result. occurs in de- mation in tage 3 of the clotting m1echanism. ficiencies of factoVIM IX, X, fibrinogen,. Bleeding encountered- 1,vh6n\ the plasma and the presence tulatini antitd- filzriegen leVel falls below 75 m per dl. agulant. 'colorimeteric fibrinogen pro e= f. Normal.,V. 90420's:eau-ids. F tlu9e is

. , , Normal Valties20.0H-400 Ing,Per-dl.' 6-21,Ditection of a Circulating Anticoagulant: 6-21. Plasma Recalcificat un Time: a. Princ1ple. An abnorinal recalcificatibn a. Principle. Calcium. _chloridersadder(- time isnot. corrected -'l Y the addition p plasma and the clottingrtirne recorded! normal plasma if a circulating anticoagn- is present. b. Reagents: b. Reagents. See paragraph 6-21b. ((1) CalciumChloride, .0.025 M. See paragraph 6-181)(5). c. Procedure: (2) Sodium Citrate, 3.& Percent. Avrt l- (1) Obtain citroted plasma from a nor- ahle from the Federal-Supply Catalog. 1 donor and from the patient. Set up the following mixture in 12 c. Procedure: x 75 mm test tubes: 21iF11 160- :x % 5 December 1 73 6-17

Nor, Patit (5)After the onloesen the Tube No, Pltisr, Plasma Plaerrur, = clo a add 3 ml urea on - 0 nil 100-q 0.15 ml 0.05 n 7 Observe ;tile: clot 3 0.10ml SOz hour intervals. 0.05 ml 0.15 ml , 0.02 ml 0.18 till 10/7, dIrateretatiop. A clot, that lyse 0 0.20 rill CLIntrol hours isificient in factor XIII. Add 0.2 -rnl of..0.025 M CaC1, to 6-24. P-latelet Count (Rees-Ecker): e anal'' determine the plasrna.-`recal.7. time :(see 6.-21) on each,tube.. a.',Princip10:..,A.-sample of bood is diluted ' in an isotonic Anticoagulalit. The anticoagu- Interpr lant employed i':contains a. stain and serves (1). The 'abiulftnal- recalci le the dual purPose: of being bOth 'fi'xing is 'corrected.by rayrnal plasma if circulat- diluent' and a staining medium. 'After ,MixT, ing anticoagulant is present. ing, the specimenisidtreduced into the 7, counting charrilier:and the number of throm- (2) A. circulating _anticoagulant pres- ent in patient's .plasma prolongk the recal-'*ocvtes in a: knOwn voltrrrie.is c9iinted. 'ficaticirr time of norrrial -plasma. ,b, Reagent. Rees -Ecker Diluting _Fluid. Add 3.8x sodium citrate, 0.2 ml 40 % form- Detection of Factor XIII Deficiency: aldehyde, 0-1 g bi lk rot cresyl,..blue..- to 100 ml volhmetrie.aask,'Dilute'fQ the mark a. Principle.-FactorXIIIconverts a 1th distilletwater::Fitter thetselutiOn,Sust loosely linked, fibrin clot On the presence Prior to use ,,I eep,iglassHstoppered bottle of c4leittrii ions) into A tough gel. The welk in the reftigeratOr::Pretia're,the solution fre-' lormed clot is insoluble in 5 NI urea. In the quently since in'.641-..-SoltitioiWtherlermal-de-- absence of factor XIII: the clot lyses within ,hyde oxidizes s-to formic acid, hernoly2ing the., 3 hours.. red cells.. lieagentS: c...ProCedure: (1) Sodium Citrate, 33%. Available (1), Fill a red- c011... diluting, pipet with., the Feder al.Supply Catalog.. freS111,y4iltered Rees-Ecke solutioch -And ex- leaving a film of :the .solution` Calcium Chloride, Lie pipet. paragraph 6-181)(5). (2.)DrIrce-.4flood tO the 0.5 'n the Tirea:-Selution, 5. M. .Add 30 o .red cell pipet. urea to-Li,1 00-Ml volumetric flask. Dilute to the mark with distilled, water. (3) Wipe the excessillood from the out- side of the .pipet and dilute to the 101mark

. c. Procedure: with Rees-Ecker diluting-fluid. (1) Draw 1.5 ml of fresh venous 'blood (4) Shakethe p-ipet for-,several dd to 0.5 ml of 3.3 sodium citrate. minutes. Discharge ...a few drops from the pipet: thei-ehitrg0 both chamberS.ofa hem- (2) Centrifuge , at2,5.00rpm for '5 acytottieter., rtes and sepaiate plasma. (5) Allow' the thromboey e tie (3) Vipet ml of plasma and .0'1!: of --for minutes. To preventdrying,' the AI calcium chloride to a clean 13 x 100, counting ChaMber-'inay be placed. on a damp test tube. towel' and cover eel a Petri dish -covey. . (4) 'Incubate' the_lot for. 30 minutes (6). Under, highpower -magnification, in a 37° C,.i,vater batl count all .:of.,7th-e=throraboeytes :seen in t center-1 -sq-tmni, area of each' of the two -esentatimeandprefei abletothose eharged chambers of the .1-emacytometer, obtained by eqpirtiry puncture 'Direct KO.: -With p-roper adjustment of the light* and edures,ho ever,. require .greater and continualfinefocusing,- tte thronbocytes" eXveience for 0.0 per -Performance ...and 'in- are "-S'Seenacsblue, highly:refractile bRdies, trpretation.- The error in a single count `has- may fe rciiirid;-oval, comnia-_ estimated to:-be-about 10 percent, :Ind' shaped: They size from o'nefive---ithis of:'/error can reduced by miero.p_ muitiP1 counts.. A major disadvantage... ofd the di- d. 'et lculatisms_ T Volume in botli:c _, m- reet cu mmarea x depth = 2 sq ombocyte :count' LS that the platelets .mm ='Ocu mrn). The formula .roust unted.: under high -power -.rather . t)i r1oilirinnersiOnnlagnification. as is used for ealatilation-of direct platelet 'count is', as fallOwS: _a indirect 'method. Becatise of :the ,ex, ely --small size of blood-platelets ';= it Total n:atelets counted x dilution \ - pia elets, per con-mon error ,tb`l ton fUse them -Avith.".'yeasts;.- volume del fid P-reebated stain. ";1' cu MM Due fo the Thheren iris in this procedure; it is recoinrriendjcl thf.Ort-v- ounted x 200 platets peru hop,,te count 'be performed bn nOrinal. person' as a control. --e. -Sources of Errar::,,, Values: 150,-000-p,0,000 Platelet aggIutinatesvalidate the/platelets per cu rim NOTE: ?.ThiS range. count. ThiS_ is prevented,'by using. ticrupq- is generally acceptable; -hut it must he rei-i14'-- lousily-clean glassware. Therefore, the fbjlow-, ized that there-is a great variance- in iioi- ingr precautions al;e. recommended: / values which differ xvith.eae4_ technique; Boilcountingchambersaicl laboratory, And technician _verichns fol!-..2 hours in distilled water to which a small amount of sodium bicarbonate 6-25. Platelet eoull hase):' ,has been added. a. Principle. :Blood is diluted with 'amt. -PipetS ninst he very caefu monium .oxalate, and. the diluted specimen cleaned. is introdUced!.into a .counting chamber.:.. The -diluting fluid must' be -itelets.japPear round a oval, pink, PurPle,-- quent1filtered or centrifuh-ed aud,aud, stored ....meven: back mat& phase ..condenser. to 4° C. 1x: 1%. (P) Other sources of -error as listed IJeagerit /Ammonium,'oxalate, -4 7.10d ammonium oxalate to a 1.00-m1' under -red :ail(t blood-Cells counts hTso volurreti lc' ilask. Dilute to the mark with apply!_ziCluding -)ipettingeri'orA__ Theseiyro- distilled. water. Storeinthe refrigerator cedutzs should be cousiffted. ..,. and filter before use. ore accuracytray be obtained thrtit4 use of the phase-Contr_ast micro- c. Procedure: scope. (1) Draw 5 ml of venous blood and . iminediately place in a test tube containing thscussion: EDTA. ( If eaPil lark,. blood is used, immedi- No one method for the enumera- ately filla red blood cell diluting pipet to n of )th rombocytes is satisfactory in every the 1 mark.) t. In expel tenced hands the dill ect`pre ,(2)Fill a red.bIdod cell aiR.i.ting 'pipet is more accurate than14.,the indirect to the 1mcl.rkiantl. ;then di -aW:.AMmonium rne and venous blood samples are more late.to-:7the' 101 mark. . 160-51/TIVI -S-227-4 5 December 73 3) Shake. the- pipet 3 5 minutes. (3) This Method is more accurate' than 4 (4). Mscard from 1/3-to 1/2the_ Volume 'the Rees-Ecker. The main drawback to this from an RISC pipet, then charge a special method is- that special equipment' is.re- flat bottom -phi counting chamber; Place' qnired. the-counting ehamber-,iii a- -Petri dishcon- ) See .paragratilis 6 -24f for further taini'rig moist gauze. and 'let stand 15 to discuSsidn. 20. minutes .to insure complete settling of platelets. . Normal Values::150,060 to 350,000 per' (5) Count the platelets in all 20 squares-I of the area normally used for the red cell 6-26. Milweroglobulin istilled -Water count. Multiply.the_results:iby LOGO,. S_creen in g -Tes t Both sides. of each chamber are filledith: the same sample--; the count. from a Pri'nciple. lVfacroglobulins are precip- eith r side; should not ,°.deviate more` than itated when brought into= contact with dis- 10% frorn- the other count. If a' greater tilled water. than 10% deviation occurs, repeat the count b.Procedure: using a -fresh dilution. -and a second cham- (1) Collect 5 ml of blood the ber. patient by venipuneture. (7) Report the average of the two (2) Set the blood aside for1 hour sides counted. o clot,, and then separate the serum by d. Sources of Error; centrifuging at 2,000 rpm for 5 minutes.. NOTE:' Oxalated, citrated, or heparinized (1.) The diluting fluid must befreSh plasnia is also satisfactory for Vie, perfor- acrd free of bacterial contamination. mance of this test. (2) Platelet, clumping occurs if there (3) Place 5 ml- of distilled water in is_ delay in adding blood to the anticoagu- a Wassermann tube. lant-or if the mixing is inadequate. Platelet clumps cause inValicrresUltS. (4) With a 1. ml pipet draw up 0.5 ml of patient's serum or plasma. Holding (3) Occasionallyextraneousmaterial the pipet tip close to the meniscus of the is mistaken for platelets. water, add 0.2 :rrtl of the patient's serum (4) Glassware that is not scruplously or plasma to the distilled water. clean causes platelets to attach to the debris (5) Interpret results. on the glassVare. Interpretation: (5) See paragraph 6-24e. other sources of error. -(1) Observe the behavior of the drop of serum or plasma sinking in the water e. Discussion:. and the modifications of color of the solu- (1) Plateletssometimesshowden- tion. dritic processes:\ StrUctures suchasdirt, (2) If macroglobins are present: crystals, and WBCs are refractile.. Plate- (a) The drop will sink leaving lets are not. The white cellsare normally __y. trace:. lysed;. however, with patients that demon- (b) The entiredistilled --water me- strate excessive high white counts some cells' dium i will 'promptly assume a whitish colo,- will .be observed in the platelet counting due tothe. condensation of transparent, prep. slimy ::masses. forming -a preeipitate which (2) Ammonium oxalate insuresclear- quickly sinks to the bottom. ing of the background by hemolysis. 3) The separated precipitate will dis AFM 16051 227 aDecember 1973 solve well whenransferred to ate_: ube (6) Incubate one serum .specimen and containing normI sal-ne. one plasma specimen at '4° C (refrigerator temperature) for 4 hours. 6-27. Cryoglobulin Screening Test: (7) --Incubate one serum specimen and a. Principle. Cryoglobulins in serum or one plasma specimen at 37° C for 4 hours.. plasma are precipitated at 4° C and dis- (8) Read results. solved by temperature elevation to 37° C. Interpretation: b. Procedure: (1) The test is positive if the serum (1) Collect 10 ml of blood by veni- or plasma incubated at 37° C shows no punctureand.place a 5 ml portion in a tube chlingesandthe serum or plasma-incubated containing anticoagulant(citrate,oxalate, at 4° C shows the following changes: or heparin )',and the other 5 ml portion (a) The serum or plasma appear into a clean sterile test tube. clotted in tOto. (2) Incubate both tubes immediately (b) The plasma- or'serum appear divided into. two layers ; the upper one con,- for 1 hour at 37° C. taining normal plasma ok. serum, faintly (3) One hour later, separate thesuper- dined by hilirubin ;. the lower :one- repre- natant plasma from --the tube containing sented by the Cryoglobulin, whitish in color, the anticoagulant and the serum from the which has precipitated to the bottom of the clot in the untreated tube.. test tube-. (4) Transfer aliquots of 1 ml of serum (2) The test is confirmed if the serum two cleansot eri le Wassermann tubes. or plasma, incubated at 40 -Cis brought (5) Transfer aliquots of 1 ml of plasmahack to a temperature of 37° C and its into two clean sterile Wassermann tubes. appearance returns to normal. 7-1. Introduction: simply'. put, the closeness of the observed a. Quality control is a means to insure-value to the actual value. Accuracy of,a the reliability of the tests performed. result is dependent on(1) preparation of reliable results :mislead the PhYsiiian and patient,- (2)- technique-used- for collection- could.quite possibly harm:the patient. Qual- -.of the blood speCitnen, (3) interval of time between collectiow,and testing of the spec- ity control can tell_the laboratory the degree . . of uncertainty which always goes with a imen _(4) standardized and 'Controlled rea- result. The object of a good quality control gents and .equIpment, and .(5.) elim nation progria'm is to, keep this degree ofuncer- of random errors. tainty within -narrow and usable limits The b. Precision: The.: with vehich clinical laboratory must adapt certain sta- a series of replicate measurenients. agree tistical procedures which aid in detecting with each -other: Precision is achieved when inaccurate results.All data sUbmitted. by to or more replicate measurements fall the laboratory should be substantiated by within the range ,of allowable variation. sufficient statistics to indicate that -itis within reliable limits. There is no substitute c. Arithmetic Mean: A simple "average." for statistical validation of a result. The arithmetic mean of a series- test results. is obtained_ by averaging the test b Qualitycontrolinhematologyhas results. :three objectives: accuracy of results, pre- d.. Standard Deviation (S.D.): The range cision of results, and detection of random of -dispersion (distribution) of values about, errors. TheSe , objectives are accomplished their mean. S.D.- is ft descriptive tool which in two ways: (1) By careful, diligent, and condenses the frequency distribution of a intelligent performance with due recogni- series of values into a single. measurement tion of the litnitations and sources of error unit. If a sufficiently la.rge number of similar of each procedure; (2) By statistical eval- determinationsis -perform-ed on a single uation of results and the measures.necessary s'imp'le and a distribution curve is plotted to keep specimens "in control' The meas- of the frequency of test results, the curve ures ,necessary involve~ instrument checks, ideally forms the classical 'Gaussian °Sell, technician performance, choiceof proce- sbapecl) curve found in figure, 7-1. The peak dure, etc. of .the curve isat or near the average (mean ofallthetest .7-2. Definition of Terms. The science of sta- resultsplotted). Algebraically.,standarddeviatiOnisthe tistics deals with 'the study of data and square root of the sum of the squared differ- provides the basis .for.'applicatiOn and in- terpretation of a -quality control program. ence' frOrri the mean value divided by the Statistics, like all other sciences, has evolved total number of values less -1. It is usually certain terms and expressions which -expressed as qUire definition and understanding to get maximum use of this valuable tool. where: fl. Accuracy: The degree of approxima- diffefenee betwe 0i:each .-value And the mean tion of a result to the actual value,or, value 7 -2 160-51/TM .8-227:4

1.7d' sum of the squared differen e the av- j. Reproducibility: A measure of how erage much a test result varies from established n =-thenumber' of.` determinationp 6 ed. mean values if the test is carried out -,in (N-1 is used only when N30: diiierent laboratories by different person- - nel using similar equipment and. procedure. It also measures variation produced ivhen a test is performed N;ithin a laboratory by different technicians,' The survey programs you participate in provide a method 6f de- termining the reproducibility: of a testpro- cre.edu .k. Proven consistency in ob- 2 3s taining accurate and preciSe results.Re-. Figure 7-1. Classical Gaussian (Bell-shaped) liability is established. by checking the test Curve:=L-=--- procedure at frequent -intervals- with con- trol -St-keit/lens.In ,doing- so,--not only is confidence Limits. or Range of Allow- the_relia.bility of the test procedure estab able Variation. The limits of a . procedure fished but also that of the reagents and the are: equipment used. (1) ± 1 mean standard deviation in- 1. Ranglomization :The avoidance cal eludes 68% of all test results. fixed pattern _ki the placement of control tests within a system, ) 2 mean standird deviation eludes5(;-"G of all test resulf: M. Random Errors : Errors which oecur (3) ± 3 mean :standard deviation ins without direction or rule. These errors are -cludes 99.7% of all test results. usually due to instrument malfunction or personal factors. f. Median: That value Which divides a distribution so that an equal number of n. Validity: Testresults are valid if they values are on either side of, it. For example, are a measure of what was inten-ded and with values such as 6r300,6,400,7,000, are accurate, precise, and reproducible. 7A00, and 7,200, 7,000 is the median. ,Statistital t valuation of ilematology: g. Mode: The most frequently occurring value found directly beneath the peak of a. General Gonsiderations. It must be ac- .a distribution (curve). It is not necessarily knowledged that exact reproduction of tests synonymous with median, is not attainable. Variation of resultsis a fact. The ultimate attainment of quality h. Coeffi'cient of Variation (C.V. ) ;This control in the laboratory is to understand is an expression of the 'variation in percent this variation so that confidence limits. can rather than an absolute value. Algebrai- be established, erroneous results detected, cally, it is expressed as: and appropriate ,corrective action initiated S.D. to. correct any signifieani disctepancy., An meanx. 10 adequate quality eontrol system indicates i. Allowable Percent Variation: This is the confidence of the test,and- provides an expression of the limits within which -ample warning when a procedure is "out .a. value must fall, in percent. Algebraically, of control." The' approach to controlor, it is expressed as: statistical evaluation of hematology istai employ the standard deviation as: a tool. Allowable percent variation C.V. xonfid nee li b. Calculation of Standard Deviation. 0 (1) Reconstitute the control`accordin g is.de rmined by: subtaating two-- to instructions. NOTE. Commercial- controls arc-1 doilatione to the :average value. for2red blond cell counts, white blood cell pa,Lion of Quality Control t harts: = counts, coagulation studies, hemoglobin des terminations,andhernocritdetermina- When control limits for various thematology_ tions are available. Aernoglobin control' procedures have 'been established, pre are scaution can be pre-red using outdated quality' control charts :reflecting the 2 blood. The- method is 'included standard 'deviation range for each test, as Shown in steps (I) through (5). in this chapter. . . (2) Mix the sample thoroughly by re- (I). On"- the left border -of a ,sheet of Oeated inversion graph prtper, .._,prepare_vertical -scaleo_ , (3) Perform the desired analysis 'on appropriatetestunit 'Valdes,The scale the control: should extend symmetrieallv above and be- low the average value,- -:ad'dquately encom- (4) Conflinueanalyzingthecontrol passing-2 standard deyiafion values. (See for an overall total of at least 30 example,- figure 7-3-.) consecutive daYs. 2) Draw a solid black- line ex ending (5) Coniptite : the ,ifverage value and from the average value completely across standard deviatiOn for each control meas- the chart. Label the line "average" at the ured, as shown. in figure 7-2. right border. ( a) In c c\lumn A, record values ob- (3) Similarly extend lines from values tained from- daily analyses of the control. representing ,±7- 2standarddeviations (b) Total the values and record in (S.D.) in parallel to the averaget line. Ap- block B. propriately label these lines ± 2 S.D. (c) Determine the average of col- . urnnA by dividing the total(block B) (4) Across the bottom of the graph, by 30 or the number of times thee control recotd the days of the montt, using two was analyzed. Record the average (block squares for each day. C). (5) Label the chart with the name of d) Compute and record in column the analysis, control lot number, and the D the_ individual differences of each test Month for which the control is to be used result from the average (block C). in monitoring the test. (e) Square each individual difference d. Quality Control Application and record in column E. (f) Total column E arid. record in (I) On the morning of each duty day block F. allow -a control to warm up to room tern' perature. (g) Calaulatethe standarddevia- tion; as, shown in block G. Consult a table (2) After thorough mixing,analyze of square' roots in determining this value. a control sample, along with one or more (h) Calculate the acceptable range clinical specimens, with each applicable pro- for the control based -± 2 'standard devia- cedure. Both the control sample and patient tions--( block 11). Some laboratories maypre- specimen must be processed by the same fer to convert this to the coefficient of varia- technician under the same conditions for tion, any given procedure. (i) Record the control limits for the (3) If the control value falls within test procedure under blockIThe upper 2 standard deviations (S.D.), report 'the limit is determined by- adding two_ standard results of patient specimen analysis. deviations to the average value. The lower, (4) Record the control sample result 7 AFM

PROCEDURE:, -CONTROL POOL NO.: TECHNICIAN(S):

rn of Squared Differences From Average:. . Test. lue rence Results From Ave rage lculation of rulard Deviation 50):

, SUrn.of _Snuared_Difference From Average 'No. Tests

Calculation of Acceptable SD Range: Acceptable limits X SD or: Z X .

Test.Control Lirmite

suit:

Consul

Figure 7-2. Example of a Monthly ± 2 Standard Deviation Calculation. on the appropriate quality control ,chart for this probably indicates improper handling the day concerned, as shown in figure 7-3., of the controlsample during the first run. (5) If the control value iS outside the (6) if the second control analysis is -±2limits,recheck testcalculations.If out of limit, a.basic flaw in the.teSt should this is not the cause, repeat' the procedure he suspected. Carefully check for possible on all specimens, including a new control causes such as deteriorated- reagents, im- sample. If the control sample falls within proper pH, inaccurate temperature, defec 2 S.D. on the second run and clinical tive instrumentation, etc. specimen values remain largely unchanged,- (7) Reportclinicalspecimenresults '-I

II

b. 7-6_ AFM 160-51/TM 8-227-4 . 5 December 1973- Obtained with an accurate control reading introduce considerable variation in results., - after correction of. the -test discrepancy. Oc, Studies have shown.that massaging the casionally this may necessitate re-collection finger or forcing blood frdm an inadequate of specimens from patients. puncture significantly affects both red and (8): keep a 'permanent record of all white count. The blood must be immediately control sample values falling outside ± 2 in the desired- anticoagulant and S.D. in the day-to-day performance of pro- placdmix Differentialslides should be cedures.- Enter the cause, if deterinined, and _ prepared froni- fresh- blood or 'from -EDT4 corrective action taken for each iirocedure anticoagulated blood very soon after colle& concerned. This data may prove "useful in tion. All specimen containers must be prop: erlyidentified with thepatient'sname. troubleshooting.,future analytic problems, Record all "out-of-limit" results on quality Proper collection of blood spe'cimens pre- -control charts -Ance these vahles -Should be vents one 'source of random errors. used in calculating monthly standard 'devia- ticins as described below. 7-5. Control in Hemoglobin Determinations: (9) When 1 -month has elapsed, inspect a. Preparation of Control Solutions: -''the quality control charts (figure IA) for )Centrifuge at least 20 ml of a-n- the various methods_ Calculate. the _monthly ticoagulated blood having a high hemo- standard deviation for each procedure that globin content -. Remove the plaSma. exhibited more or less equal distribution of (2) Wash the cells 3 tithes in 0.9% _ control values above and below the mean.- This is accomplished using the chart values NaCl(physiologicsaline),removingas accumulated during the month's run. much saline, as possible following- the - last centrifugation. (10) Prepare new quality control charts reflecting the adjusted ± 2 S.D. range com- (3) Freeze the washed cells. overnight puted in step (9). to pbtain hemolysth. If complete hemolysis is not obtained, refreeze for a second-nigitt. (11) If monthly result` trends of any prodedure indicateconsistentprogression (4) To the thawed hemolyzed cells add. toward one control limit or the other, re- 1/2 volume of chloroform and shake vigor- . study the method. Following any correc- ously fcir -1 minute. tions in technique, reagents, or instrumen- (5) Centrifugeimmediately tation, redetermine the ± 2 S.D. limits on minutes at 3,000 rpm. a new series of 30 daily control analyses. (6) Removethesuper atanthernd- (12) Continue analyzing the control in -globin solution to graduatedubes and re- conjunction with clinical specimens, and re- cord the volume. cord .control results on the new quality con- (7) Analyze thesolutionfor heo- rol charts serving the new month. globin concentration in g per dl. (13) In the manner described above, (8) Adjust the hemoglobin concentra- .prepare quality control charts for each suc- tion to approximately 14.0 g per dl with cessivemonth,- always using ± 2S.D. glycerin. The glycerin is also a preserva- ranges compUted from control sample values tive for the sorittion. The volume of glycerin of the previous month. In actual practice, added is calculated as fellows: this should progressively narrow the accept- able range of analytic error for each pro- actual concentration sedure. 14.0 1 X volume of control solution 7,-4. Control in Blood Collection. The method (9) 'Mix thoroughly after addition of by which a blood specimen is collected can glycerin. Store in the refrigerator. This so- X227-4 ember 1973

lution is stable up to 10 months in. the re- :d) Colorimeter and spectrophotOrne- frigerator: ter problems including precalibratcon,_ obso- (10) Use as whole blood to verify rou- letecalibration,incompletecalairation tine hemoglobin determinations. .. (satisfactory at one end of the curvenot. atthe other), inadequate length bf -the scale, b. Calibration: unstable electrical compOnent§, unmatched (1) Sahli Pipets. The procedure for covets,dirtycuvets, and poor standard (evaporation and deterioration). calibratiOn of Sahli pipets, is outlined in .- paragraph 2-6. At least -3 pipets should be (2) Personal,Errors:. _calibrated -to --the .nearest are-: (a) r Incomplete mixing-of-the sample reference pipets All new pipets can bom- .(b) Inacciirate: andcareless :pipet, pared 'colorithetricalry with the ting; ,reference pipets. An accurate pipet must -- producea (c) inaccurate adjustment of the col- hemoglobin solution in Drabkin's identical orimeter. to soltitions prepared. from reference pipets. (d) Inaccurate obserVation The allowable error is 0.5% T. machine- readings idssfiess, fatigue; bias). (2) Dilutors or -Diluting Pipets_ These (e) Inaccurate conversion of- absorb, instruments are checked with 5 ml volu- ance of% T to g per dl of hemoglobin. metric ftasks. The volume must be -5 mi. (f) Recordingerrors. Duplicator hemoglobin solutions prepared. (g) Transcriptioa errors. ,from these 'instruments must agree within 0.57a T. . 77-6.. Control in Cell Counting (3) Spectrophotometers. Calibration (fletnacyt9rnetet.) : curves are prepared with certified cyanmet- a. Sources of Error. There are.many vari- hemoglobin standards in the concentration ables contained in these methods. of 5, 10, 15, and 20 g per dl. Each standard is run in triplicate and the %© T mustagree (1) Instrument Errors: within 0.5%. The curve is plotted And (a) Inaccurate diluting pipets. checked daily 'with one of the hemoglobin )Inaccurate counting chamber. standards. Any change in reagent, pipets c) Contaminated diluent fluid. _ or spectrophqtometers requires preparation (d) Irregular cell. distribution (field of new standard cifrves.' errorl (2) Personal Errors: c. Sources of Meror. Regardless of statis- tical methods, the maximum allowable -(a) Insufficient xin of the error sample. for a hemoglobin must not exceed 0.5g. Prevention of the following errors main- (b) Inaccurate pipetting of the sam-. tains good quality control. pie and diluent. (c) Insufficient or excessive shaking (1) Instrument, Errors: of the sample pipet. (a) Inaccurate. sample Pipcalibra- (d) Inaccurate filling of the. count- ion. rig chamber. (b) Improper calibration of diluent (e) Inaccuratecounting(fatigue, dispenser including volumetric pipets, self- bias, etc.). filling pipets, and-automitic-dilutors. (3) Limits of Precision. (c) Failure of automatic dilutors to (a) RBC (at 5,000,000) _curate maintain calibration caused by insecure vol- 16%. ume. 4djustrrient,dirt in the valves, and (b) WBC (7,000 ±1 %, (Berk- . 'bubbles in the chamber. sun, Magath, and Hurn). -, _- AFM 160-5 8-227-4 5 December 1973 b. Minimizing Errors: (k) Incomplete hemolYsis (1) Sampling ih duplicate pipets. WBC) (1) Containinated diluting fluid: ( ) Filling duplicate chambefi. gin)Ethessiv.e number of lailge plat (3) Doubling the number of cells to .be counted. (2) Persdnhl Errors:- (4) Calealato, anddeviation (a) Inaccurate Pipettirig.. the dupliCate analysis method. (b) -Incornplet9:-..rnixing of origina saninle=and of diluted satnpie. (a) Run = duplicates on 20 routine , (c)- F4lure_tOtadju§tAbe instrument (b) Sum tin differences between the controls properly= duplicates of the `20 specimens. (d) ErOrs in recording counts. .- e),,Brrors in reading correction fac- (c) -CalCulate S.D. busing the followt- ing formula: (f) Failure to correct WBC counts, Sum (differenees)3 above S.D. number of pairs (g) Failureto.correctcellRBC (drCell coUn maintained counts. within the standard. deviatiOn. 7-8. Control in Hematocrit Deter-minations: 7-7. Contred iii Electronic Counting: a. Standardization of the Method. Cpntrols a. Controls. Controls for RBCs and liVBCs, Ito,. Validate, hematocrit determinations are are available commercially.Statistical analy- available from 'commercial sources.Itis sis Must be performed to establish confi- important to use capillary tubes of the dence limits of the instruments. best quality to-minimize errors due to varia- tion in the diameter of the tubing. The tubes b. Calibration. These instruments must be -nest be .adequately sealed to prevent loss of . .1. calibrated and maintained according to the blood during centrifugation. -Centrifugation anufacturer's instructions. -speed and time -must be able to Rroduce c. Sources of Error. Electronic cell count- constant-volume of .,cell ,packfleinatocrit' ing can be done with much greater preci- readers must provide-accurate positioning Or sion, but possible sources of error are 'even- the' blood column and_ha.ye an easily read more numerous. and accurate scale.

1) .Instrument Errors; b. .Sources of Error: (a) Inaccurate pipet calibration. ( 1 ) Instrument Errors: fb) Inaccitrate calibration of diluent (a) Variation in jaliber ofcapillary "dispenser, -tubes. (c) Inaccurate' calibration 'of current (b) Inaccuratelycalibratedmacro- and threshold' settings, hematocrit tubes. (d) Electronic malfunction. (c) Insufficient_ centrifuge speed (e )' Defective or dirty manometer. failure to check-brushes routinely. f) Leaking seals in the glass com- (d) Insnfficifntrunning time. -1.-- ponen s. (e) Leaking tube seals. (g) Partially plugged aperture. (f) Poorly designed (h) Dirty glassware. reader.

- (i) Foaming (detergent re (2) PerSonal Errors: aponin).- I (a) Ingomplete mixing of the sample. (j) Loss of cells on standing. (b) Incomplete sealing Of tubes. AFB' 180-51/TM 87227-4 5 December 1973

(c) Reading errors. Clean Venipunctu re. yenipqnctures ; (d) Tra.nscriptionerrors. e_:nontraumatie. Contamination With thromboplastin leads to erroneops re- sults. ! 779,-.Control in Differential Co &--'711-eading .fltiterential-slides requires deal of ex- Anticoagulants. od- mustbe tep:nical ,skill:' A technicians placed in proper amounts of-the desired anti- should haviAti i*training to be competent coagulant- immediately and mixed well. in readinc irential sinears. However, in (6) Delay, in TesUng. Testing must be certain instances -these -slides_ must ke re- -performed as-Soon-asossible- after-= receiv-- -viewed by another`technician, laboratory su- in the specimen. pvisor, or-pathologist. The instances are: (7) Accurate. Timing.gtarting_ the a. Counts below 4,000 cells- per. cu mm stopwatch must be coordinated with the-ad- and above 15,000 ,cells per cu iritri. clition of the final reagent or ingredient, and b. A lymphocyte-. count above 40 % for stopping the watch, with appearance of the adult and 60% for-a fibrin clot. (8) Reagents. Reagents must be fresh--.- c. The observation of abnormal or a ., cal cells. andstable::.Expitation esmust 'be checked. _ i. d. The observation cif any nucleated will- rocytes. -(9) TeghnicalSkill.-,. A great deal of skill is-required in the perfermance of coagu- e_ Platelets. which- aPpear increased , 4- : lation tests. .Directions must be followed ex- creased, orfahnorrnal. plicitry. V10. Control in Coagulation Studies: c. Fibrometer. The Fibrometer has elim- Controls: Citrated plasma controls are inated many variables in coagulation test- available from the Federal Supply Catalog. ing.Itis recommended that Fibrometer Controls are run with each tapplicable ,test methods,be employed if possible._ grid. Statistical analysisis_ performed. _To adequately_ control these procedures all- sped- 7-11. Control in,Other_ Many proce- mom, /not be performed induplicate,. or dures areThttlined iii chapter whIgh are' eventriplicate:The ,differences :,lietWeen -not performed as frequently as CRCs. How- -these replicate-analyses must be within the evr, quality _control can 'still be eStablished confidence limits of the laboratory.,... rthe-Se kocedures, -For example, abnormal and normal specimens can be analyzed in b. Sources of Error:. the same manner as the patient's specimen. (1) Gllssware. Glassware must be clean This will indicate if a particular stain or re-' and, ofRtandard size. Coagulation .glassware agent is detecting; what it is designed to de should'never 4-ber' used for {other' testsAll tect. In some ca.,W, controls can be difficult pipets must be accurate and clean. Precau-- to obtain. For "'example controls for ,herno- tions ninst be taken when._pipetting to avoid &bin electrophoresis_ should include AS, cross -clentamina ionof specimens 5 rre- SS, SC, and AA hemoglobins ; however, all agents. . but AA hefficiglobin can be difficult to oly (2) Lighting.. Insufficient._ lighting can,.4ain. Larger laboratories may be the source ea sea failnre.ttrobserve'an-tsendPoiritclot' of§upply for theie'COntrols. formation). 3) Incubation. The water- bath tem- 7.42. Summary. An ex echnician, perature must be controlled to 37° C. Varia-,well-informed in quality cdrit principles tion of this teMperature procluces invalid re- and proceahres, shofild direand monitor sults:- the quality control programof any given 7-10 AFM 8-227-4 clinical laborafbry. An effective quality con- attention, interest, and consistency of _trol program for clinical hematology requires plication on the part of allassigned persq

BY ORDER OF THE SECRETARIES -OF THE AIR FOKEANpe, OFFICIAL GEORGE S -tRtiw Chief of St'd

JACK R. BENSON, Cotomi rUSA

, Director of Administration !-

Official- CREIGHTON W. ABBAMS ' General,-United States A, oily

VERNE. L.BOWERS Major General, United ,§ta The Adjutalt General SUMIVIARY OF REVISED, D LETED, OR ADDED MATERIAL Revision encompasses a'rearrangenien't. and additions to,the subject terial. The introductory chapter consists of the basic concepts of hematology. The chapter on blood cell counting chambers and dilution pipets is enlarged to include equipment employed, in hematology with emphasis on a.utornatioThe clrapter covering the collection and processing of blood specimens .ation-rind_ processing: The morphology and maturation of erythro- include tone marrow a .

cytes, leukocytes, and thr mbocytes are included in one chapter. The-methodology of hematology,:.g. is cornlyjnedr.into anoth chapter. The chapter, on blobdoagulation studies is enlarged to include all pertinent tests aiding the sludy.efblood.coagulation. A. chapteris inclutled to present qu lrty Control aspen ti matqltikk.- Distribution

Air Force F; X: USAFSchool of Health Care Sciences, Sheppard AF TX 76311 . 1 1,000 Army: To be distribu in, acct rdance with DA Form-43/1, Require e t for' Medical Tech- ni1 Manual AFM 160-51/tM achmen t -1. 5 Deieraber 1973 .A1-1.

BIBLIOdRAPHY . 1. Ackerrpann.- Bauer; J. a and Tb of Quality Control Specimens-Sp Erythrp-

G. Tray'.'Clinical' Labbratory Methods.'.%. cyte Countipg, Hematacrit and Hempito- 7th eclition-i-Saint Louis: C. y. Mosby Co., bin Determinations," 'America'n 1968.. of Clinic* Pathology, vol 53 (1 pril 2: Albert-S. N. and others. The Hernatoerit 1970), 474:A80.-- .'in Clinical Practice; Springfield, Illinois: Brittin, G. M. and others.-"Stability of Charles C. Thoinas Co:, 1965. . Blabd- ,in ;Used., Ariticoagu- . .... - , _1pts)"471104)4.::jourial. ofClinical -. .Alperin,J.B.;Lehmann, H.,and Schneider, R. b. "Sickling Tests: Pitfalls vkl52-(1969),.690-694. in ;Performs. ce - andInrpretation," Britton, C_,T, C. Disorders of the Blood. ortlia.. American Me leal Associa-, New York:Grune and Stratton, 1969, n, vol 202, 5 -(30- October 1967), 12. Caruana, J.,. Cohen, N.- A., and Kennedy? ,419-421. ;. 'S J. B. "A Simple Method- for Preparing a arnett, R. N. and Pinto, C. .L. "Repro Hemoglobin Control Solution'," American. ducibility of _Prothrorabin Time .Dete Journal of Medical Technology, vol 36 mina ions Between Tedhnologists: Co (1970), 562. ,parison 'of the Fibrinometer and Tilt-T Methods," American Journal of 13. Coagulation Procedures. Dide:..kivisipn: dihology, vol 46 (1966) 148-151 merican Hospital Supply- CarporAion?-, _. 1970.- .Satsaksis,41. G., Briere, R.40, and. Golias, T. "Rapid Qualitative and Quantitative 14, Coagulation Procedures. Raritan, New Hemoglobin ',Fractionation,"American Jersey: Ortho Diagnostics, 1968. Journal of Clinical Pathology vol 44, NP. 15 CrahW.rMicalIdentificaUor H. ,Li, C . ancl;Yai, L T. (December 1965), 695-701.. Mocytes ach, D. J. and Pow eel, Modi and Granulocytes," Arnerican :Journal of _ fication of the !Sickledex' Test .foie Hemo- Clinical Pathology, vol 55 (Mareh 1971), globin S," Clinical. Chemistry, vol 17, No.' 283 -290 : 10 (1971), 105,5-1056. 16. ordicicshank, Dodds, T. L and Mc-, eutler, E. -"A Series oT New. Screening Donald, G. A. Atlas of Haematology. Balti- roc lures for 'Pyruvate Kinase Defi- more: Th4 Williams and Wilkins Com- pant, 1965. cien Glucose-6-PhospkateDebydroge- nase Deficieney,'and Glutathione Reductase 17. Dacie, J. V. and Lewis, S. M., Practial Deficiency," Blood, vol 28, No 4 (October Haematology.. 4thedition,NOW- York 1966) 553-562. . Grune and Strattop, Inc., 1968. S.Bripyhous, AC; Graham, J. B.and 18..Darmady, E. M., and Davenport, S. G. iti ye, S. W. "The Partial Thiomboplastin T. Haeinatalo'gical Technique. 3rd edition, "Tine as a Screening Test-for the .Detec- New York: Grime and Strattoni 1963. tion of Latent Bleeders," The American Journal of the Medical Sciencei- vol 243, -.19, blav clsiln,J, .and -Henry, B. Todd- No. 3 (March 1962), 279-287; Sanford Clinical Diagnosis-1)y Laboratory Methods. 14th edition, Philadelphia: W. B. Brosious, E and Zucker S. "Preparati n Saunders company, 1969. A1-2 ; .AFM 160-51/TM 8 -227-4 Attr chment 1 5 December 1973 20. Delvaux, .T. C. and Lewis, J. F. "Revi- 31. Haserick, J.R. "L. E. Phenomenon," sion of a Method for Performance of Total Journal of the American Medical Associa- and Differential Cells Counts in Cerebro- lion, vol 146, No. 16 (1951), 16-20. spinalandSerousFluid,"American Journal of Medical Technology, vol 33, No. 32. Kaplow, L. S. "Cytochemistry of Leuko- 4 (July-August 1967), 336. cyteAlkalinePhosphatase,"Anierican Journal of Clinical Pathology,vol39 Diggs, L. W. and others. The Morphol- (1963), 439. ogy of H Milan Blood Cells. Chicago : Ab- bott Laboratories, 1970. 33. Kato, K. Atlas of Clinical Heniatology. New York: Grune and. Stratton, 1969. 22. Dorland's filustrated Medical. Dictio ary. 24th edition,Philadelphia: W. 34. Kazal, L. A. and Tocantins, L. M. Blood Saunders Company, 1985. Coagulation, Hemorrhage, and Thrombo- _ sis. New York: Grune and Stratton, 1964. 23. :Dorsey, D. B. "What Can Quality Con- trol Do ForHematology," American 35. King, J. W. and Willis, C. E. "Cyan- . Journal of Medical Technology, vol 31 methemoglobinCertification.Program of (1965), 150-153. the College of American Pathologists," American. Journal of Clinical Pathdlogy, 24. Dorsey, D. B. "Quality Control in Hema- vol 54 (1970), 496-501. tology," American Journal ofClinical Pathology, vol 40 (1963), 457-464. LaFond, D. and Miale, J. B.Prothrom- binTimeStandardization,"American 25. Eichelberger,James W.Laboratory Journal of Clinical Pathology, vol52 Methods in Blood Coagulation. New York: (1969), 154-160. Hoeber MedicalDivision,Harper and Row, 1965. 7. Leavell, B. B. and Thorup,- D. A. Funda-- entals of Clinical Hematology. 3rd edi- 26. Engstrom, A.. W. "Effect of Hemolysis tion, Philadelphia: W. B. Saunders Com- onthe One-Stage Prothrombin Time," pany, 1971. American Journal of Clinical Pathology, .vol 49 (1968), 742-743. Lenahan,J.G.andPhillips,G.E. Seminar on the Coagulation of Blood. 27. Frankel, S., Reitman, S. and Sonnen- MorrisPlains,New Jersey:Warner- .wirth, A. C. Grady ohl's Clinical Labora Lambert Research Institute, 1969. tory .Methods and Diagnosis, vol. 1, 7th edition, Saint Louis:. The. C. V. Mosby 39. Levinson, S. A. and McFate, R. P. Clin- Co., 1970. ical Laboratory Diagnosis. 7thedition, Philadelphia: Lea and Febig-er, 1969. 23. Freudlich, M. H. and Gerande, H. W. "A New Automatic Disposable System, 40. Linrnan, J. W. Principles of Hematology for Blood Counts and Hemoglobin," Blood, New York: The Macmillan Company, vol 21, No. 5 (1963), 648-655. 29. Gambino, S.R. and Waraksa, 41. Loh, Wei-Ping. "Evaluation of a Rapid "An Automatic Instrument for the Meas- Test Tube Turbidity Test for the Detec- urement of Hemoglobin Concentration," tion of Sickle Cell Hemoglobin,' Ameri- American Journal of Clinical Pathology, can Journal of Clinical 'Pathology, vol `vol 52 (1969), 557-564. (1971), 55-57. 30. Gregory, D. M., Matusik, J. E., and Pow- 42. Maher, D. J. Medical Technelogy. Vol 1, ell, J. B. "Rapid Solubility Test for De- 6th edition, Berkeley, California: Berkeley tection of Hemoglobin S," Clinical Chem- Scientific Publication, 1969. istry, vol 17, No. 11 (1971), 1081-1082. 43. A Manual of Methods for the Coa ila- AFM 160-51/TM 8-227-4 A ent 1 5 December 1973 A1-3

LionLaboratory.Baltimore:Baltimore ogen, American Journal of Clinical Path- Biological Laboratory Division, B-D Lab- ology, vol 52, No.. 6 (1969), 114-118. oratories, Inc., 1965. 51. Platt, W. R. COlor Atlas and Textbook. of 44. Martinek, R. G. "Review of Methods for Hematology. Philadelphia: J. B. Lippin- DeterminingHemoglobin in Human cott Company, 1969. Blood,"JournalofAmerican. Medical Technology, vol 32 ( 1970.), 37-63. 52. Powell, W. J."SickleCell.Disease," Journal of American Medical Technology, 45. Miale, J.B. "The Fibrometer System vol 8'3 (1971), 613-619. for Routine Coagulation Tests," Ameri- can Journal of Clinical Pathology, vol 43 53. SAM-TR-67-58, Laboratory Control of (1965), 475. AnticoagulationTherapy.BrooksAir Force Base, Texas, June 1967. 46. Miale, J. B. Laboratory Medicine: Hema- 54 Sandoz Atlas of Haematology. Basle, tology. 3rd edition, Saint Louis: The C. V. Mosby Co., 1967. SwitZerland: Sandoz LTD., 1952. 55. Seiverd, C. E. Hematology for Medical 47. Miale,J.B."Routine Evaluation of Bleeding Tendency in Surgical Patients," Technologists. 3rd edition, Philadelphia: Lea and Febiger, 1969. . The Journal of the American Medical As- sociation,vol 185 (7 September 1963), 56. "Technical Subcommittee on Haemogio- 752-756. binometry of the International Commit- 48. Nalbandian,andothers."Dithionite tee for Standardization in Haematology," Tube TestA Rapid, Inexpensive Tech- Blood, vol 26 (1965),.104. nique for the Detection of Hemoglobin S. 57. Thompson, R. B. A Short Textbook of and Non-S Sickling Hemoglobin," Clinical Haematology. 3rd edition,Philadelphia: Chemistry, 17, No. .10 (1971), 1028- -J. B. Lippincott Company, 1969. 1032. rA. Wilbourne R. L. Procedui.e Control 49.. Owens J. B. and others. A Rapid. Micro- the Coagulation Laboratory. Dade Divi- TechnicforHemoglobinEleotrOphore- sion, 'American Hospital Supply Corpo- Sise" American Journal' of Clinical Pa- ration, 1970. tholday, vol 46 (1966), 144 L147. Wintrobe, M. M. Clinical Hematol6gy-. 50. Phillips,. L. L. and others, "Comparison 6th edition, Philadelphia:- Lea and .Feb- of 2 Methods for Determination of Fibrin- igere 1967: AFM 160-51 TM 8-227-4 Attachthen

GLOSSARY

alocyte: A leukoc,yte Without, definite nucleus appears as a. pale staining smear eYtOPlasinic granules. without prescribed form or'shape. AgranulotYtosis: complete or nearly com- Basopenia': An abnormal decrease- in the plete -absence of the granular leukocytes number of basophils.. froni the blood and bone marrow- Basophil: A granular leukocyte, the gran- Aleukemic Leukemia: A fatal condition of tiles of which have an affinity for the basic the blood-forming tissues, characterized, by dye of Wi'ightiS stain (methylene blue). marked proliferation of -immature cells in The 'granules are large,irregular and the bone marrow, without their presence, blue-black in color: iii any great numbers, in.the blood stream. Basophilia: An abnormal increase in the Anemia: A condition- in which the blood is number of basophils. deficient in quantity or quality of erythro, Basophilic:Stainingreadilywithbasic cytes: dyes, for example, blue with Romanovsky Anisocirtosis:,Variationrnsizeofthe type stains.. erythrocytes:'- Binary Fission: Simple cell division. Anomaly: Abnormality. Bleeding Time The time required for a small Arioxernia: Lack of normal proportion of standardied wound, made in the capillary oxygen in the blood. bed of the finger or ear lobe to stop bleed- Antecubital Space:. The area on the fore- ing. arm frontal to the elboW. Blood Dyscrasia: A disease of the blood or Anticoagulant: A substance that prevents blood-forming organs. the coagulation of blood. Commonly used Buffy Coat: The layer of leukocytes that .ones are potassium oxalate, sodium oxa7 collects immediately above the erythro- late, sodium citrate, EDTA, and heparin. cytes in sedimented- or centrifuged whole Aplasia: Incomplete or defective blood de- blood. velopment ; cessation of blood cell -forma- Cabot's Rings: Lines in the fent' of loops. or tion. figures-of-eight seenin erythrocytesin Aplastie Anemia: Anemia characterized by severe anemias; incomplete ordefectiveblooddevelop- Centriole: A minute cell organoid within ment. the centrosome. Asynchronous: Uncoordinated develop- Centrosome: An area of condensed cyto-' ment. plasm active in mitosis. AzurophilicGranule:Rounded,discrete, Chemotaxis: The phenomenon of mevemerct reddish-purple granule, smaller, than the of leukocytes caused by a chemical in- granules of neutrophils; 0-10 are com- fluence. mon in lymphocytes, and they are .very Chromatin: The more stainable portion of numerous, and smaller, in the cytoplasm the cellnucleus, contains genetic mate- of monocytes. rials. .Band Form: In the Schilling, classification, Clot Retraction: The rate and degree of con- aneutroPhilwith thenucleusunseg- traction of the blood clot. mented and ribbonlike; also stab, staff, Coagulation Time The time required for. nonfilamented. - venous blood, in the absence of all tissue Basket Cell: A degenerated primitive cell f-actors, to clot in glass tubes under con- Which has ruptured and in which the cell trolledconditions.

133 AF 160 -51 /TM :8-227-4 : Attachment 5 December- 1973

Cocatalyst: substance that works in tan- Eosinophilic: Readily stained` with dem with ati'dther group of chemicals to red-orange stain. accelerate a reaction velocity without be- Epigastric: Pertaining' to the upper middle ing used up in the reaction. portion of the abdomen. Color Index: The ratio between the amount. Erythreniia: A disease marked by persistent of hemoglobin and the number of -red polYcythemia and increased blood volume; blood cells. also polycythemia vera.

. : A hematology study Erythrocyte: Red blood cell. which consists of a red cell count, white Erythrocytosis: An increase in the total cellcount, hematocrit, hemoglobinand number -of erythrocytes. blood smear study including difierntial Erythrogenic: Producing erythrocytes. white cell count. Erythroleukerriia: Anbn s mal condition Congenital: Born with a personexisting. charadterizedy piferation of erythro- at or before birth. blastic and eloblastic Cooleyls Anemia (Mediterranean Diseaie or Erythropenia A decrease in the number of Thalassemia) : A chronic preigressive ane- red cells ithe bloOd. mia commencing early in life anecrharac- Erythropoie is: The production of erythro- terizedby Many normoblastsinthe cytes. blood, unusual facies, milenomegaly and familial and racial incidence. Target type' Etiology: e theory of the causation of a red blood cells are often present fr in the disease. peripheral blood. Extravascular: Occurring outside, of the Crenation: The scalloped or notched appear- blood vessels. ance of the periphery of erythrocytes Extrinsic: Originating outside of the partic- found when the cells are sUspended in a ular area. hypertortic solution. Also found in smears, Fibril: A microscopic filament often com- .caused by dirty glassware, slow drying posed of fibrin. and poor smearing technique. Fibrin: The end product of the clotting Cytoplasm: Protoplasm of a cell excluding mechanism which forms a network of fib- the nucleus. ers that enmesh the formed elements of DNA: Deoxyribonucleic acid. blood. Differential Count: An enumeration of the Fibrinogen: The precursor of fibrin which tyPeeef white blood .c,ells seen on a stained is-present normally in the plasma and blood smear. /, produced by the liver. Discrete: Separate. Fragility Test (Osmotic) : A test devisgd to Dyscrasia: Abnormality. measure the resistance of the erythrocytes Ecchymosis: Subcutaneous extravasation of to break down (hemolyze) when subjected blood covering a large area. to varying' concentrations of hypetonic Endrithelianeukocyte:Monocyte. salt solutions. Eosinopenia : An abnormal decrease in eo- Fulminating : Sudden and severe. sinophils. Edsinophil: A granular leukocyte, the,.gran- Golgi Apparatus: A meshwOrk of lipid con ides of which have an affinity for the acid taming fibrils Within the 'cytoplasmic por- dye of `Wright's stain (eosin). The gran- tion of a cell. ules are large, round, uniform in size, red- (: A white blood cell that con- nge.incolor and are shiny and re- tains' specific cytoplasmic granules (neu .ctiie. -' trophils,eosinophils,andbasophils) ; EosinophiliaA relative or 'absolute. leuko- these granules are peroxidase positive. cytosis inwhich the main increase is in Granulocytosis: The presence' of increased eosinophil. numbers of granulocytes in the blood. AFM 160-5 22'7-4 dA4p.chinerkt 2 5 December 197:3 A2-3

'Grantdocytopenia(Granulopenia ) :A de- Heterozygous: Derived- from gerfli _11s. crease in the number of granulocytes in like in respect to one oymore faetbrs. the blood. HomeostaSis:Stability.. in normal.body.

-Ctranulopoiesis : The production of granulo- states. . cytes. Homozygous: Derived from germ cells.which Hemacytometer: A calibrated chamber in e. alike. which blood cells are counted: Howell-Jolly Bodies: 'Small basophilic par- - Hematin: A brown or blue-black amorphous tides sometimes found In erythrocytes, iron substance which unites with glohin remnants of raidlear material. and forms hemoglobin. HygrOse epic :Readily- -taking up. and- retains ernatocrit: The ,packed cell volume (PCV) ing water,. . of red blood calls obtained by centrifuga: Hyperplasia: An increase in cell! formatiqn... tion of a blood specimdn in a hematocrit Hypertonia:- Greater than isotonic con erl9 tube. - tration. Hematology: The branch of medicine that Hypertrophyi -Enlargement of an organ or deals with the study of blood' cells, blood- part due to increase in the size-of the. con- producing organs and the manner in stituent dells, which these cells and organs are affected Hypochrornia: A -decrease in color of the in disease. erythrocytes, ,herico a decrease in their Hematorna: Subcutaneous effusion of blood hemoglobin content. with resulting swelling, pain, and discol- HYpoplasia: A deereaSe in cell formation. oration, forming a tumorlike mass. Hypotonici Less thanisotonicconcentra- Hematopoietic (}16mopoietic): Blood form-

ing. . Idio_pathic: disease of unknown cause.- Hemoglobin: The colorilt matter of the red Inclusion: Usually lifeless, an. accumulation blood cells. A complex iron-bearing pig- of .fati, proteins, crystals, pigments.Or se-. ment which carries oxygen and carbon cretory granules within a cell cytoplasm. dioxide. Inhibitor : A substance, directed against a co- Hemoglobinuria: The presence of free hemo- .agulation factor or 'factors, which inter ;g-lobin in the urine. fetes with the coagulation process. Hemogram: The blood picture. Intravas.cular: Occurring within the blood Hemolysis :'The dissolution or dissolving of vessels,' ,the erythrocytes. "I Intl ihsiclSituated within the particular: Hemolytib Anemia: That type of anemia part.- characterized by excessive intravascular In Vitro: Within' a test tube (glass, etc.). destruction of red cells. In Vivo: Within the living organism,, in Hemophilia: A hereditary disease character7, life. ized by a prolonged coagulation time and isotonic: 'Solutions with the sarrie osThotic repeated hemorrhages, occurring only in pressure. males and transmitted only by females Jaundice: Yellowness of- the skin and eyes and affected males. The cause is a clefi due to the presence of blood pigments in ciency in a plasma factor (antihernophilic the blciod ; follows'excessive destruction of globulin or thromboplastinogen)result- the blood, obstruction of the bile passage, ing in a .defect in' thromboplastie activity. diffuseliver disease, certain infections, toxidehernical agents and drugs. Hemoptysis,: The spitting of blood; -coughing Juvenile Cell: In the Schilling classification, , up blood, the cell between the myelocyte and band Hernostasis: The checking of the. flow of forms ; also metamyelocYte.. blood_ from -a vessel. KAryolysis: Apparent destruction of the nu- Hepatic: Originating from the liver. cleus otba cell. A2-4 FM 16051/TM Attachment 2 5 D e ber 197:1 yorrhexis:Fragmentation of thenu- Mean Corpuscular Volume V): The vol- cleus; a degenerative process usually fol- ume of the average red blood cell. lowed by karyolysis. gakaryoblast:The. parentCellof the L.F. ,Cell: A large segmented neutrophil or megakaryocytic series. inophilthat containsingested auto- egakaryocyte: An extremelylargecell 'Iyzed nuclear fragments in its cytoplaSm. with an irregularly lobed, ring- orough, Leukemia: An ultimately fatal disease of the nut-shapednucleus whichstainsblue- blood-forming organs characterized by in- purple. The cytoplasm is abundant, light creased numbers of leukocytes and associ- blue and is packed with fine azurhilic anem4i. grannies. This cell gives risetotrombo- Leukemoid Crisis or Reaction: A temporary cytes. appearance of immatutleukocytes in the Meoloblasti The type of red cell. precursor blood stream, with a marked increase in found in pernicious anemia. This differs the total white count. In the laboratory from the normal erythrocyte precursor sometimes temporarily indistinguishab,le (normoblast) in that the megaloblaSt is from leukemia. larger and the nuclear chromatin has a Leukocyte: White blood cell.... fine Meshwork or scroll design. Leukocytosis: An increase in leukocytes in M:E Ratio: The ratio of myeloid to eryth- the blood. roid cells in the bone, marrow, Leukopenia: A, reduction in the number of Mesentery: The fold of ptritoneum which leukocytes in the blood. attaches the intestine to the posterior ab- : LeukoCyte formation. dominal wall. Lymphoblast :. The parent cell of the. lym- Metarnyelocyte: Juvenile cell of Schilling. phocytiC series. Metarubrieyte: An erythrocyte with a:pyk: LymphoCyte: A white blood cell haying a notic, contract d nucleus. Also called or- round or oval nucleus and sky blue cyto- thochromatophi ienorrnoblast. plasm. The nuclear chromatin is densely Methemoglobin: .A spectroscopically de-- clumped but separated by many clear tected comp) and of hemoglobin found in areas giving ,a "hill and valley" effect. A. nitrobenzot, and other poisonings. The fewred-purple( az u rophilic )granules blood is a chocolate brown color to the eye. may be present in the cytoplasm. Microeyte: An erythrocyte smaller than nor- Lymphocytosis: A relative or absolute in- mal. crease in the number of circulating lym- Microcytosis: An increase in the number of phocytes. microcytes. Lymphopenia: An abnormal decrease in the Micron: One-thousandth of a millimeter,, the number of lymphocytes. common unit of microscdPic measurei Lysis: Destruction by a specific agent. Mitochondria :Granular components of a Macrocyte: An erythrocyte larger than nor- cell cytoplasm active in oxidative proc-

mal. . esses- Macrocytosis: An increase in the number 'of Mitosis: A. series of changes through. which' macrocytes. the nucleus passes tin indirect celldivi- : A hasophil or a true tissue cell. sion. A tissue sliowing many cellsin Maturation Factor: A substance which will mitosis indicates-. rapid growth of that cause cells to ripen and come to. maturity. tissue. MeanCorpuscularHemoglobin(MC11): : The parent cell of the monocytic The average amownt of hemoglobin in the series. red bloOd cell, ,. Mohocyte: A large white blood cell with! a Mean Corpuscular Hemoglobin Concentra- pale blue-gray cytoplasm containing fine tion (MCHC) : The average percent hemo- azu rophil isgranules.Thenucleusis globin saturation in the red blood cell. spongy and lobulated. AFM 160751/TM 8-.227-4 Attachment 5 De ember 1_973 '

Monocosis: A relative or absolute increase Organoid: ° Structur present in ,cells

_ in the number of, circulating monocytes. sembling organs.. Mucosa: Mucous membrane. Ovalocyte: An elliptical erythrocyte. Myeloblast: The parent cell of the granulo- Oxyhemoglobin: The bright red hemoglobin cytic or myelocytic series. that is looPely combined with oZygen and Myelocyte: The stage, in ,development of the found in arterial blood. granulocytic series which is characterized Pancytopenia: A : reductioninallthree by the first appearance of specific gran- formed elements of the blood, namely, the ules(eosingphilic, neutrophilic or baso- erythrocytes,leukocytesandthrombo- philic) and a round nucleus. cytes. Myeloid Cells: The granular 'leukocytes and Pathologic Increase (Or Decrease): Due to their stem cells. abnormal function or disease,as con Myelopoiesis: Formation of bone marrow trasted to physiological (due to normal and the blood tells that originate in the body functio'n). bone marrow. , Pernicious Anemia : A chronic, macrocytic Myeloproliferative anemia caused by a defect in production Rapidproductionof of "intrinsic stomach. bone marrow constituents. factor" by the Thereis accompanyingruegaloblastic Necrosis: The death of a circumscribed por- erythropoiesis, poikilocytosis, granulocytic tion of tissue. Simple necrosis is degener- hyperseginehtation,achlorhydria, and ation of the cytoplasm and nucleus' with- 'neurological disturbances. out change in the gross appearance of the Petechiae Small spots -on the: skin formed tissue. by' subcutaneous effusion 'of blood (also : A decrease in the number of purpura and ecchymoses). neutrophils in the blood. Phagocytosis: The destruction of of ganisrn Neutrophil (Poiymorphonuclear Neutrophil and extraneous matter by a process of en- or Segmented Neutrophil) : A granulocyte velopment and absorption. having-fineneutrophilic% - (pink-violet) Plasma: The fluid portion of the blood com- granules in the cytoplasm. The nucleus posed of serum, and fibrinogen, obtained divided into two or more lobes:: each lobe when an anticoagulant is used. is usually connected by a filament. Plasma Cell: A lymphocyte -like cell, with an Neutropha: An increase in neutrophils. eccentrically placed deep-staining nucleus. Normoblast: The nucleated precursor of the The nuclear chromatin is distributed in a normal red blood cell. Also called a rubri, "wheel-spoke" fashion. The cytoplasm blast deep blue with a lighter halo about Normocyte .(Erythrocyte): A red bloodell rincleus. of normal size. Platelet: Thrombocyte. NRBC: Nucleated red cell, usually a metar- Poikilocyte: A red blood cell havizfg 'an ab- ubricyte when seenintheperipheral normal shape (pear-shape, sickle-shape, blood smear. etc.). , . Nucleolus: An intranuclear pale blue body, Poikilocytosis: Increased ,number of .;abnor- surrounded by a dense condensation of mally shaped erythrocytes. chromatin. Polychromasia:Diffuse basophilia of the Occult Blood: The presence of blood which erythrocytes. cannot,be detected except by special chern-, Polychromatophilia: The presenceinthe ical.tests. stained blood smear of immature, non- Oliechreinernia: A decrease in hemoglobin. nucleated, bluish-staining,,red blood cells. Oligocythemia: A decrease in the number of Polycythemia: An increase in the total num- erythrocytes. ber of erythrocytes. (See erythremia. 44' Atilt -160 51/TiV1.' Attachment .2 5 December 1973 Precursor: A substance another Sickle Cell Anemia: This is a hereditary and substance is formed. familial form of chronic, hemolytic ane- PrornyelocYte: The PreeuA'sor of yelo- mia essentially peculiar to Negroes. It is cyte having nonspecific aZuroP (red-- characterized clinically by symptoms of purple) cytoplasmic granille%. anemia, joint pains, leg ulcers, acute at- Prorubricyte: The second stake °develop- tacks of abdominal pain and isdistin- ment.of the red cell. guished hematologically by the presence Prothrombin: The inactivepreeursorof of distinct hemoglobin, peculiarsickle- thrombin which is formed in the liver and shaped and oat-shaped red cdrpuscles, and present normally in the plastno. Its forma- signs of, .excessive blood destruction and tion dePerids upon turequate 'vitaNn K. active blood formation. Punctate loasoviilic ag- Smudge Cell: A ruptured white also gregates in the ei'Ythroeytes that stain basket cell, or degenerated cell. blue with the basic dye of Wright's stain; SPherocyte: A red blood cell which is .snore also basophilic spherical, smaller, darker and more fra- Purpura: Small spots oh the skin finned by gile than normal. subcutaneous effusiuri of blood. Stasis: A stoppage of blood flow. Pyknosis: A condensation and rednction in Supravital Stain:. A stain of low toxicity size of the-cell and its nucleus. which will not cause death to living_ cells ReducedHemoglobin:. A corrihYlirtionof or tissues. hemoglobin and ci rbbn- djoxidQ which is Synchronous: Occurring at the same time fowl(' in venous biooll. and in a regular pattern. ReticulocYte: A red blood cell allowing a Target Cell (Leptocyte): An abnormal, thin reticulurn or network when 5toed with erYthlveyte characteriitie of Cooley' s or vital dyes (for exarriple, brilliant .'Cresyl Mediterranean anemia. The stage i/otween the nucleated Triturate: To grind together. .red cell and the mature erythrOcYte. Thrombin: This is_ an enzyme formed from Reticuiocytesis: Arr increase ahov-e normal prothrombin which convertsfibrinogen valuesofretieolecytesinr)eripheral to fibrin. This is not, present in circulating blood. blood. RNA: Ribonucleic acid. ThroMbocyte: A blOod platelet. Rouleaux Porrriation: The arrangement of ThroMbocytopenia: A decreaseinblood red cells with their flat surf cirrg, in platelets; also thrombopenia. which they appear as figures r Wembling Thrombocytosis: An increase in blood plate- stacks of coins:., lets. .. Rubricyte: Polychrornatophilie pblast. Thromboplastin: The substance that initi- soinientatienRate,t]rYthrocY (ESR): ates the process of blood clotting. It is re- The rate at which red cells willttle out leased from injured tissue and/or formed in their own plasma in a given toe under by 'the disintegotion of platelets in com- controlled conditions. bination with several plasma factors. Serum: The fluid portion of .the blq0d, after Thrombopoiesis: The production of th'on clot formation. bocytes. Shift to the Left: A used to designate Throtribesis: Formation. of a th o bus, or 'that condition which the .immature blood clot.' forms of: the neutrophils are increased Vacuole: space or cavity formed in the above their normal nbrnbef. protoplasm of a cell. Shift to the Right: Increase in mature, PYk- Venipuncture: The act of puncturing a vein notiC, and hypersegni%pte0neutrophils..: in order to remove a sample of blood. Sickle Cell :A sielcie,:or crege.ht7shaped Viscous Metamorphosis: FrictiOn between erythrocYte. molecules resulting in a structural change. AFM 1604 /TM '227 -4 Attachment 5 December 1973

Vitamin K; A vitamin constituent of the liver in the production of prothrombin, normal diet requiring bile salts for ab- Xanthochromia: A yellowish discoloration, sorptibn. This vitamin utilized by the usually associated with spinal fluid. TN achmen 5 December 1973

PREFIXES, SUFFIXES, AND STEMS COMMONLY USED IN HEMATOLOGY

liema °logic Hematologic Usdgo aning Ukaga Meaning a- or an without micro- small aniso- unequal mono- single baso- blue myelo- marrow -blast primitive form ortho- correct or normal chrome color -osis . . -state of .condition ..a crena ,, a toteh crease of '.: -crit to separAte -penis decrease of Yte cell : phago- to eat eosino- ' red-orange -philos attraction for erythro- red plasia a forming or molding.) hem- haern- he a blood -plastic to form hyper-, , excessive poiesis to mak.r-e hypo- deficient poikilo- varied iso- equal poly- much or many leuk- leuko- white pro- :before macro- large reticulo- a net mega- great or huge thrombo- sctlortain meta- after or -next -tonos artension A Blood cell maturation, normal (1-77,.,8-9) =,1-3 cytoplasm (1-8) Acanthrocytes (4-5b(7)) _ _4-4 1-3 Accuracy, quality. control (7 -2a) _ _ = = _ -7-1 general features (1-7}( _ 1-3 Agranulocytes (4-7) 4-10 nucleus (1-9) _ _1-6 Blood collection (3-1) -= lymphoeytic series.;(4-8) _ 4 -10 _ =3-1 monocytic series 4-10 anticoagulants (3-4) 3-7 plamocytic series (4-10) 4-12 eapilitfry puncture (3-3) 3-5 Alkali denaturation: test, fetal hemoglobin quality; control ..(7-1). _ _ 7-6 veriipiineture (5-22).=_ _ _ 5-24 3L1 Alkalnr phosphatase, leukocyte (Kaplow preparation 'of site (3-2c) (5-32) 5-34 syringe procedure (3-2d) 3 -2 vacuts.iner procedure (3-2e) A niscicytosi s (4-5a (1 ))- =:_ . = _ _ _ 4-2 = Anticoagulants .(34) _ : 3-7 Blood, function of (1-13) _1-6 EDTA (ethylene-diarnin7tetra-deetat eryttroYtes (1-14) 1=6 (34b) 3-7 leukocytes (1-15) _ =1-7 Heller and Paul double oxalate (3-4b) 3-7 platelets (1-16)(6-4) 1-7 heparin (3-4c) 3-7 Blood smears: sodiurnf'citrate (6-10b(1)) coverglass method (3-7) 3-12 sodium oxidate_ (6I0b(2)) - 6-6 examination (5-23) _5-25 Auer rods (4-11b) 4-14 slide method (3-6) _ _ _3-8 Autocytometer, Fisher (2 -21c) _ _ 2-16 staining (3-6d) _ _ J3-9 utomuted hcrnatologeal _:equipment (2 -18 Bone marrow specimens: 2-12 xariining slides (5-25) =_ 5-28 -17! slide%preparafrop (3-9c) 3-14 counting -devices (2- 2-212 stain (3-9b) , =3-14 dilirtors (2-19).______2-12 Burr cells (4-5b(6)) _ _ _ _ _4-4 fibronleter (2-23) 2-17 hernoglobinometer, IL (2-22) 2-17 C slide stainer (2-20) 2-12 Cabot's rings (4-5d (2)) 4-4 B Capillary_ puncture (3-3) _3-5 Carboxyhemoglobin (5-15b) 5-18 Basket cell, leuktocyte-varlant (4-d ). ='= 4-14 Cell counts: =9a Basophilic stipplin'g, erythrocyte basophils, absolute (5-5) 5 (4-5d(3)) _ 4-5 cerebrospinal fluid cells :(5--7) _ . 5-11 Basophils (1-15d) (4-6j) eosinophils (5-4) 5-8 4-1Q,. erythroeytes ,,(RFC) (5-2) 5-1 absolute count (5-5).______=_ _ _ _5-9 leukocytes (WBC) (6:3 ) 5-6 Bleeding time (6-11): 6-6 plateletti ((324, 25) 6-17, Duke method (6-11h) 6-6 6-18 Ivy method (6-11c) __ = _ =_ _ _= _ _6-6_. reticulocytes (5-6) _5-10 Blood' cells, formation of embryonito hema- ,- semen (5-8) _5-12 topoiesis (1-5) _1-2 Centrifuges:. hepatic phase (1-5c) . -1-3 floor model (2-16b) 2-10 rnesoblastie phase (175b) __1 -2 rnicrohernatocrit (2-46c) =2-10 myeloid phase (1-5d) .1-3 table-top model (2-16a) 2-10 postnatal hernatopoiesis (1-6) _1-3 .Cerebrospinal, fluid cell count (5-7) _ _5-11 extramedullary (1-6e) Chernotaxisi rlekned (1-15) rnyelopoiesis (1-6h) 1-3 Citrate, sodium (8-10b(1)) 6-6 Blood cell maturation, abnormal (1 -10, 1 _1-6 Clot retraction test (6-13) 6-8 cytoplasm (1-11) 17-6 Clotting time, 'Lee-White (612) nucleus= .(1;12) .6-7 Coagulation (6-2) _6-1. AFM 160-5 27-4 .A December 1973

Page 'age blood lactors (0-i Dilutors,-autdmatic _.2 -12 fibriholytic factors (6-5) Dohle bodies in lCukocyte (4-11a) 4-14 mechanism (6-6, 7) 6- Drabkin's solution (5-18b). = 5-19 platelet factors (6-4) 6-3 Drepanocytes. (sickle cells)(4-5b(2)) 4-4 theory (6-7)__ Duke's bleeding time 16-11b) 6-6 Coagulation studies: 4 bleeding time (6-11) -6-6 blood collection (6-10) _ 6-5 EDTA, anticoagulant (324b) circUlating anticoagulant detection (6-22)_6-16 Electrophoresis, hemoglobin (5-21) clOt retraction (6-13) 6-8 Elliptocytes (see ovalocytes) clotting time, Lee-White (6-12); = - 6-7; Eosindphils-.41-15c, 4-6h, factor XIII deficiency detection (6-23) -==_=6.17. fibrinogen assay (6-20) 6=16 cull count (5A) partial_ thromboplastin time, activated Thorn test (54f(6)) 6-9 Erythrocytes (ABC)... _6 =16 P41 Calcification tiitie-.-(6721) count (5-2 ) platelet _count (6-24, 25) 6-17, defined (1-3a) =1-1 6-18 development (4-4) '4-1 prothrombin consumption time (6-17) _6-11 functions :(1 -14) 1-6 prothrombin .tine, one-stage (6-16) ,==,-,6-10 morphology (4-3) 4-1 quality control (7-10) 7-9 Ei-ythroeYte 'sedimentation rate;(ESP)

thrombin time (6-19) 6-14 (5-12, 13) _ _ . 5-16,, thromboplastin generation time 6-12 5 -17.. tourniquet test (6-14) 6-8 Erythrocytes, variations in (4-5) _ 4-2 'Coefficient of variation, quality. control inclusions. (4-5d) . 4-4 (7-2h) megaloblastie (4-5e) __4-5 -. Confidence limits (allowable variation) (7-2 shape (4-5b) _ 4-2. Control, quality. (see quality control) size (4-5a) ..4.4' Coulter cell counters: staining (4-5c) _ 4-4 Model FN .(221b) 2-12-- Erythrocytic._. series (4-4) - 4-1 Model S (2-24b) _ 2-17 erythrocyte -(.44f) : .1,______....42 Counting chamber, hemocytometer (2-7 2-4. erythrocyte, diffusely basophilic (4-4e) _ 4L2 -. Counting devices, automatic (2-21) _2-12 metarubricyte (4-4d) 4-2 Coulter Counter, Model FN (2-21b) __ _2-1.2 prorubridyte. (4-4b) 4-2

Coulte'r Counter, Model S (2-24b) 2-17 rubriblast (4-4a) . 4-1 Fisher Autocytorneter (2-21c) _2-16 rubricyte ; (4Ac) _ i..- Technicon .SMA 7A (2 -24a) 21.7 Ethylene=iliamine-tptra.ace ate (EDTA), -4 Coverglass method, blood smears (3'7) = _MT (3-4b) _ ' 3-7 Crenatea erythrocyte (4-5b(8)) 4 'CrYoglobulin screening test (6-27) ,6-20 Cyanmethernoglobin method .(6-18) -18 Fetal hemoglobln: Cytogenetic specimens (5-36) 5-37 alkali denaturation test (5-22) 5-24 blood collection . (5-36c) 5-37 Fibrinogen assay (semiquantitative) (6-20)6-15 mailing instructions (5-36d); 5-37 Fibrinolytic factors in coagulation (6-5) 6-3 Cytoplasm (1-2) _ _1-1 Fisher autocytorneter (2-21c) _. 2-16 .abnormal (1-11) 1-6 Formalin:fixative, bone marrow (3-9b(3)) 3-14 normal (14) _1-3 Fragility testi, erythrocyte: am test ,(5-28) 5-31 D osmotic test (5-27) 5-29, DNA (deoxyribonucleic acid) (1-26 1-1 G Dehydrogenase, glucose-6-phosphate, leuko- cyte (5-35) 5-36 Giernsa stain (5-31e(7)) 5-34 Differential counts,' leukocyte: Glassware: 2-3 counting (5-24b) _ - -5 -26 calibration of Sahli pipets (2-6 quality control (7-9) _ _ 7 -9 cleaning (2-8) 2-5 2-4 smear preparation (3-6, 7) = 3-8, counting chambers (2-7) 3-J2 pi pets'( 2-4 ) 2-2 ining (3-6d) =3-9 siliconizing (2-9) 2-6 . TM 8-227,71

Glue --e-6-phosphate dehydrogenase, eon- st-,(5-35) 'dranuloeytes (4 -6) ination,-rnicroseopic (2-12) _ 2-8 basopliils (4-6j, k central or oblique (2-12b) 2-8 development (4-6) improper (2-12e) - _2-8 "eosinophils (4-6h; i) regulation, by iris criaphragm (2-12c) 2-8 beutrophils (4-6dg) substage condenser, function (2-12d) 2-S 'Gfantilocytle 0es (4-6 Inclusions, erythrocytic: Cabot'a,- rings;_(4-5d (2) ) _4-4 Heinz-Ehrlich bcidieS(4-5d(4)) 4-5-

ell-Jolly bodies (4-5d(1)) = _ Ham test for erythioeyte fragilify (5-28 5-31 clerocytes (4- 5d.(5)).- _4-5 ,Hayerre,s diluting fluid (5-2b) 5-1 Stippling, basophilic (4-5d(3 4-5 Heini-thrlich bodies, erythrocytic -stain Indices, erythrocyte (5-26) -28 5d(4))(5-33)- 4-5, Ivy's ,bleeding time (6-11c) 5 -35 . Heller and Paul double 'oxalate_, (3 -4b) 3-7 Hema4crit: r -L.E cells (4-11h, 0 4 macrokernatocrit (Wintrobe) (5-10) 5-14 fluorescent antibody method (5-29b) 2 microheniatOcrit (5-11) = 5 -16 rotary bead-method (5-30) 5-32 quality control (7-8) _ 7-8k Lee Whiteclotting,time (6-12) .6-7 Hematological systems: Leukocytes (see also White blood cells) Coulter Counter, Model S (2-24b) _ _ =2-17 count' (5-3) - 5-6 fiechnicon SMA -7A (224a) 217 development ; ,- 4-5 Hematopoiesisz functions (1-15) 1 -7 embryonic (1-5)_- morphology (4-6-4 11) =.- _ -4,5 rarnedallary-:(1 =6c 4-13 . myelopoiesis (1-6b) Leukocyt ,variations: postnatal _ atypical lYmphocytes 4711g) 4-14 Hernctcytorneter:''` Auer rods (4-11b) 4-14 Neubauer ruling (figure 2-3) ''basket cells- (4-11d .:4 -=14 use in counting:, Dohle bodies (4711a) 4-14 basophils (5-5)== ._ . . _ _=.__,_5-9 hypersegmentation (4-11f) 4-14 eosinophils (5-4) __ 5.-8 L.E. cells (4-11h) _ 4-14 erythrocytes (5-2) 57.1 "rosettes"_ (4-!-11i) 4-14 leukocytts (5-3)_ _ 5-6 "tart" cells (4-11j) 4-14 platelets (6-24, 25) 6-17, toxic geanulation (4-11c) 6-18 vacuolated cells (4-11e) 4-14 Hemoglobin : Lupus erythernatosus (see L.E.

compounds (5-15)' _5-18 Lymphocyte's: . cyanrnethemoglobin method 5-1 _5-18 atypical (4-11g)-- 4-14 :electrophor sis (5-21) _ _5-22 development (4-8) 4-10 -kenetieari'V 'elan (5-16) 5-18 Lymphoeytij series (4-8) '4-10 quality contI, (7-5) _ 7-6 lymphoblast (4-8a) Hemoglobin, fetal: lymphocyte. (4-8c) 4-10

alkali denaturation. test (_=22) _=,;5=24 prolymphocyte (4-86) 4-10 electrophoresis (5-21) _ _ 5-22

genetic variation (5-16) _ 5-18 M Hernoglobinometer, IL (2-22) , = _ _2-17' Macrocytes (4-5a(2)) 4-2 Hemoglobin S: Macioglobulin distilled water screening_ dithionte method (5-19b(2))__ 5-20 (626) 6-10 'electrophoresis (5-21)- 5-22 (1-15a) ______1-7 genetic- variation. (5-16) 5-18 Maturation', bloorl.cell: Hernostasis (6-1) 6-1 abnorthal (1-10, 11, 12) _ 1-6 Heparin, anticoagulant (3-4c) = _ _ ,_- 5-7 normal (1-7, 8, 9) 1-3 Howell-Jolly boclieherythrocytic (4T5d(1)).4-4 Mean, arithmetic; quality control -2c) 7 -1 Hypersegrnentation of, neutrophil. (4-11f) 4-1i Mean' Corpuscular Hemoglobin CH) Hypochromia, erythrocytic (4-5c(1))__== _ 4-4 - (-26b(2)) 5-28 6651- 8-2274..,,A tachment 4

Fag Mean Corpuscular Hemoglobin Concentra- Peroxidase stain (5 -31) -5-33 lion (dcHq):(5-26b PhagocytoSis (1-15) 1f17 Mean Coypuscular..Volu Phosphatase, leukocyte alkaline (5-32) 5-34 (5-26b(1)): Pipets, diluting (2-4 figure 2-1). 2-2 Median, qUality control (7 -2f _ cleaning (2-4d, .e;. 2-8) 2, akaryoeytic'serieS (4 -13) 2-5 gakaryohlasi (4A.3a), Pipets, diluting, 'Use in coon

gakaryocyte (4-13c) = 4-16- hasophils (5-5) 5=9 rriegakiiyacyte (4-13d) CSF cells (5-7) _ 511

egaltarYocyte (4 -13b) = 4-16 eosinophils (5-4) 5-8 athoc'ites(platelets)(4-13e A'-47 platelets (6-24,25) _ -6r17, oblastic phase,. hematopoiesis (1-5b)= ,1-2- 6-18 Metamegakaryocyte (4 13d) 4.-16 red blci"od cells (5-2) , tarubricyte (4 -4d) _4-2 spermatotoa (5-8f) 5-13

crocyte (4-5a(3)) white blood cells (5-3) _ _5-6 ohernatocrit: Pipets, Sahli.,hemoglobinl centrifuge(2-1 _ _2-10 calibration (2-6) 2-3 procedure (5-11 5 -16 use (5-18)- 5-18 reader (figure -11 Pipets,,Unopette (2 5) = 2-3

Microscope, comp Plasma (1-4a) _-_ . 1-2 care (2H14) _ 2-9 Plasmocytic series (4 -10) 4-12 focusing (2-1 2-8 4,1g plasrnoblast. (4-10a) illumination (2-12) 2-8 . plasmo-CYte. (4710e) '4-12 magnification (2-11) 2-5 proplasmocite (4-f0b) _ 4-12 types .12-15) - 2-10 Platelet ,aunt; Microscope,. phase (2-15b) 2-10 phase microscopy (6 -25) = 6-18

in Platelet counts (645 a 16,718 Rees-Ecker (6-24) 6-17

Mode, quality control (7-2g) ._. _ _ 7-2 platelet factors (6-4) 6-3 Monocyte (1-15a, 4 -9c) 1 Platelets (thrombocytes): 4-12 4-15 - formatidn (4-13) Mongcytseries __role in coagulation .(1716; 6-4 rnonoblast (4-9a) 41 2 monocyte (4-9c) Poikilocytosis (4-b(1 ) ) 4-2 promanocyte (4-9b ) =4-1 , Polychromatop,hilia (4-5c(2)) - 4-4 Myeloid:;Erythroid (M: E) o (6-25b) 5-28 Precision, quality control (7-2b) 7-1 Myeloid phase,hematopVe (1 -5d) Prothrornbin consumption time (6-17) 6-11 Myelopoicsis (16b) 1-3 Prothrombin time, one stage (6 -16) 6-10

Neubauer ing, hernacytorneter-gure Quality control: 2-3) , __2 -5 chart preparation 7-3 Neutrophils "(1-15b, 4-6) 1-7 objectivea (7-1b) 7-1 Nucleolus of nucleus (1 -2b; 1-9) 1-1, Quality control in: blood 'collection (7-4) _ 7-6 Nucleus of cell (1-2b) cell counting: abnormal maturation (1-12) electronic (7-7) _7-8 normal maturation (1-9) hemacytometqr (7-6) 7-7 nucleolus (1-2b; 1-9) coagulation studies (7-10) 7-9 differential counts (7-9) 7-9 0 hematocrit determinations (7-8) hemoglobin determinations 47-5) 7-6 Osmotic 'fragility test, erythrocyte (5 -21) 5-29 Quality control, statistical tests used: Ovalocytes (elliptocytes)(4-5b (4) ) _ =4 4-4 accuracy (7-2a) 7-1 Oxalate, amMoniUrn-potassium (3:4h) * 3-7 Coefficient of variation (7 -2h). _ 7-2 P . confidence limits (7-2e) 0 mean, arithmetic (7-2c) 7-1. Partial lastii ctivated median (7-2f) 7-2 (PTT) (6-15) 9 mode (7-2g) 7-2 AFM 160.5 TM .8427 -4 Attachment4 ember 197. A4-5

Page, Faze random errors (7-2M)= 7-2 calculation (7-3h) andomization (7-21) 7-2 Stippling, basophilic (4-5d(3))- 4-6 reliability .(7-2k) Sperrnatoioa (see semen analysis) reproducibility (7-2j) _7-2 Spherocytes (4-5b (3) ) _ _ _ _ 4-4 standard deviatidn (7-2d) 7-1 validity (7-2n) F 7-2 variations, allowable percent (7-21) 77-2 -Targe el leptocytea) -(.4511(5)) 4-4 Tart cells (4-11j) _4-14

Technicon SMA 7A (2-24a) = -Random errors, qualitycontrol (7-2m) 7-2 2-17 Thorn, test (5-4f) _ _Z-9 Randomization, quality control (721) Thrombin- time (6;19) Red blood cell count, hemocitortrieter (5-2) 6-14 Red blood`: cells (see erythrocyteA) Thrornbocytes (see platelets) Thromboplastin generation time(6-1 6-12 control 7-2k) = Tourniquet test (6-14) Turk cells (4-11g) Reptoducibility, quality control (7:-j) Reticulocyte count (5-6) Roirleaux formation (4-5b(10) S Safety piecautions ( Schistecytea (4-5b(9),) Vacutainers (3-2e) -3-4 Sedimentation rate, erythrocyte Validity, quality control (7-2n 7-2 Semen analysis (6-8) =,_, , = _ 5 -12 VenipUncture (3-2)' 3-1 Sickle cells (drepanocytes) (4- =4-4 Viziocytes (4-11g) 4-14 demonstration of (5-20)_ 5-21 Siderocytes (4-5d(5)) stain .(534) 5-36 White blood cell counts Silicanized glasaware, preparation (2-9) electronic (241) 2-12 Stainer, slide, autpiiiatic (2-20) _2-12 hemacytometer (5-3) Stains:. 5 -6 Wintrobe.Landsberg sedimentation- rate Giemsa's (5-31e (7) ) 5 -34 (5-13) z -1 5-17 Heinz body - (5 -33) 5-35 Wintrobe tube (figure 5-6) _ _5-15 leukocyte alkaline phophatase (5-32)_ _5-34 macroliematocrit (5-10) 5=14 peroxidase (5-31) 5-33 sedimentation_ rate (5-13) -5-17 siderocyte (5-34) 5-36 Wright's stain: 'Wright's (3-6c(2)) 3-8 buffer preparation (3-6c(1)) 3-8 Standard beviation (3-0.), quality control stain preparation (3-6c(2)) 3-8 procedure (3 -6d). .3-9

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