MEDICATION-INDUCED BLOOD DYSCRASIAS:

Diagnosis, Treatment And Prevention

Jassin M. Jouria, MD

Dr. Jassin M. Jouria is a medical doctor, professor of academic medicine, and medical author. He graduated from Ross University School of Medicine and has completed his clinical clerkship training in various teaching hospitals throughout New York, including King’s County Hospital Center and Brookdale Medical Center, among others. Dr. Jouria has passed all USMLE medical board exams, and has served as a test prep tutor and instructor for Kaplan. He has developed several medical courses and curricula for a variety of educational institutions. Dr. Jouria has also served on multiple levels in the academic field including faculty member and Department Chair. Dr. Jouria continues to serves as a Subject Matter Expert for several continuing education organizations covering multiple basic medical sciences. He has also developed several continuing medical education courses covering various topics in clinical medicine. Recently, Dr. Jouria has been contracted by the University of Miami/Jackson Memorial Hospital’s Department of Surgery to develop an e-module training series for trauma patient management. Dr. Jouria is currently authoring an academic textbook on Human Anatomy & Physiology.

ABSTRACT

Although drug-induced hematologic disorders are less common than other types of adverse reactions, they are associated with significant morbidity and mortality. Some agents, such as hemolytics, cause predictable hematologic disease, but others induce idiosyncratic reactions not directly related to the drug’s pharmacology. The most important part of managing hematologic disorders is the prompt recognition that a problem exists. The main mechanisms to manage hematologic disorders include vigilance to observe signs and symptoms indicating a blood disorder and patient education of the warning symptoms to alert them of the need to report a condition to their primary care provider or an emergency health team. nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 1 Continuing Nursing Education Course Director & Planners

William A. Cook, PhD, Director, Douglas Lawrence, MA, Webmaster,

Susan DePasquale, MSN, FPMHNP-BC, Lead Nurse Planner

Policy Statement

This activity has been planned and implemented in accordance with the policies of NurseCe4Less.com and the continuing nursing education requirements of the American Nurses Credentialing Center's Commission on Accreditation for registered nurses. It is the policy of NurseCe4Less.com to ensure objectivity, transparency, and best practice in clinical education for all continuing nursing education (CNE) activities.

Continuing Education Credit Designation

This educational activity is credited for 4 hours. Nurses may only claim credit commensurate with the credit awarded for completion of this course activity.

Pharmacology Content is 0.5 hour (30 minutes).

Statement of Learning Need

Clinicians need to know how to manage the risk of hematologic disorders induced by medication. Understanding the risk, recognizing the signs and symptoms that may indicate a blood disorder, and being skilled in how to educate the patient are essential knowledge needs of clinicians to ensure patients, caregivers and health teams are able to recognize the warning symptoms of hematologic disorders.

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 2 Course Purpose

To provide nurses and health team associates with knowledge about medication-induced dyscrasias to better recognize, treat, and educate patients, caregivers and other health team members on acute and long-term management.

Target Audience Advanced Practice Registered Nurses and Registered Nurses

(Interdisciplinary Health Team Members, including Vocational Nurses and Medical Assistants may obtain a Certificate of Completion)

Course Author & Director Disclosures

Jassin M. Jouria, MD, William S. Cook, PhD, Douglas Lawrence, MA

Susan DePasquale, MSN, FPMHNP-BC – all have no disclosures

Acknowledgement of Commercial Support

There is no commercial support for this course.

Activity Review Information

Reviewed by Susan DePasquale, MSN, FPMHNP-BC

Release Date: 5/21/2016 Termination Date: 5/21/2019

Please take time to complete a self-assessment of knowledge, on page 4, sample questions before reading the article.

Opportunity to complete a self-assessment of knowledge learned will be provided at the end of the course. nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 3 1. The complete blood count (CBC) is sometimes referred to as

a. the cellular components test (CCT). b. the peripheral blood count (PBC). c. the EDTA test. d. the three-phase test.

2. Microscopic evaluation of a blood smear is best when the slide is prepared

a. indefinitely if properly stored. b. within 8 hours of collection. c. within 3 hours of collection. d. up to 24 hours after collection.

3. True or False: Freezing of blood samples is essential to preserving the samples for a valid, complete blood count (CBC) test.

a. True b. False

4. The complete blood count (CBC) analyzes

a. concentration of leukocytes (white blood cells). b. volume of RBCs (red blood cells). c. weight of RBCs (red blood cells). d. All of the above

5. ______provides the best morphologic preservation of blood cells and prevents coagulation of the blood specimen.

a. Cold agglutination b. Dipotassium (K2) EDTA c. IgM antibodies d. Romanowsky stains

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 4 Introduction

Certain medication, such as hemolytics, cause predictable hematologic disease, but others can induce idiosyncratic reactions not directly related to the drug’s pharmacology. In the first course of this two part series, it was emphasized that the most important part of management of a hematologic disorder is the prompt recognition when a problem exists. This is done by two mechanisms: firstly, vigilance for signs and symptoms that may indicate a blood disorder; and, secondly, patient education about the warning symptoms that should alert them to the need to urgently contact their medical provider or emergency services if a prompt medical appointment is not possible. This second course focuses more specifically on the identification and management of drug-induced blood dyscrasias, including prevention through vigilant monitoring, hygiene and vitamin intake.

Diagnosis Of Blood Dyscrasia: The Complete Blood Count (CBC)

One of the most common tests to identify and monitor a condition of blood dyscrasia is the complete blood count (CBC) with differential. It is one of the most common laboratory tests performed, which informs clinicians about blood cell production and the ability of the red blood cells (RBC) to carry oxygen and of the white blood cells (WBC) to fight infection. Hematology medicine (concerned with the diagnosis and treatment of blood health and disease) laboratory testing helps to identify disease states that may be associated side effects of certain drugs that cause blood dyscrasias.

The performance of a complete blood count (CBC) has three phases: pre- examination (before testing), examination (testing), and post-examination

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 5 (reporting). The pre-examination phase includes the proper identification of the patient and proper collection and handling of the specimen. The specimen is analyzed in the examination phase.

The CBC (sometimes referred to as the peripheral blood count (PBC)) is a primary screening test that provides information regarding the cellular components of the blood as they circulate in the peripheral blood. The concentration of leukocytes (white blood cell (WBC)), erythrocytes (red blood cell (RBC)), and platelets as well as a categorization of the different WBC subsets is included. Additional information regarding RBCs is also integrated into the CBC and includes, at minimum, the concentration of hemoglobin and the packed cell volume of RBCs, called the hematocrit.

Finally, a CBC can also provide what are known as the RBC indices that are used to depict the volume and the total weight of each RBC and concentration of hemoglobin in it. The CBC can be determined by automated and/or manual methods. The post-examination phase includes reporting and interpreting the data.

Based on the information collected from the CBC, the laboratory professional can provide diagnostic criteria or meaningful recommendations for any follow-up testing (reflex testing) to the medical provider to support quality patient care. The CBC report can often be submitted for medical review in an emergency situation within minutes; however, manual differentials by a trained laboratory professional will take longer. This section highlights the varied phases of the CBC test and final report for medical review, interpretation and diagnosis of a blood disorder.3,49,62-68

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 6 Phases of the CBC

Pre-examination Patient identification

Specimen collection and handling

Examination Automated results

Evaluation and analysis of peripheral blood smear

Post-examination Interpretation of data

Reporting results

Pre-Examination Phase of the CBC

The pre-examination phase of the CBC encompasses patient identification, blood collection, and specimen handling. Briefly, patient identification includes the patient’s name and a second identifier that can be a hospital number but more commonly is the patient’s date of birth. This documentation must be available from the patient at the time of the blood collection. Once the patient has been properly identified, the laboratory professional performing the phlebotomy must be well acquainted with the various collection devices, their safety features and requirements, and the anticoagulants or additives within the sample collection tubes. This individual should have a thorough knowledge of the phlebotomy procedure safety issues including methods to prevent exposure to blood-borne pathogens.

Although other blood collection tube additives can be used for hematologic analysis, almost all specimens for a routine CBC are collected in a purple- lavender-top sample collection tube that contains ethylenediaminetetraacetic acid (EDTA). Dipotassium (K2) EDTA provides the best morphologic

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 7 preservation of blood cells and prevents coagulation of the blood specimen. The anticoagulated specimen allows a laboratory professional to generate multiple blood smears from one tube of blood, a technique that must be performed within 3 hours of blood collection.

Proper transport is of great concern within a clinical facility and is even more important when samples are brought in from locations outside the facility. Samples from outside facilities must be delivered in a manner that complies with limited temperature variations and time restraints. Freezing or excessive heat will damage the blood cells and render analysis invalid. Although microscopic evaluation of a blood smear is best when the slide is prepared within 3 hours of collection, the instrument analysis can be delayed for 6–8 hours without deterioration of the data. Some parameters are valid for up to 24 hours after sample collection if properly stored. Often, however, the hematology instrument’s manufacturer provides the recommended time points for performing the automated CBC to ensure that the analysis produces data for a patient of the highest quality. The information generated from manual and instrument analysis is only as good as the specimen that is tested. Therefore, the pre-examination stage of testing is of primary importance.

Examination Phase of the CBC

Laboratory professionals use automated instruments to determine the CBC information for the majority of patient samples. Proper instrument preparation includes quality control and assessment to confirm the normal function of the instrument. Commercial controls, patient controls, or moving averages are used to determine the analytical reliability of the automated instrument. Once the analytical reliability of the instrument has been confirmed, examination of patient samples can begin.

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 8 Automated Results

Throughout the examination phase, laboratory professionals must follow safety protocols designed to minimize risk of exposure to biohazards, chemical hazards, and physical hazards and dispose of biological waste appropriately. Each hematology instrument utilizes different but overlapping technologies (i.e., impedance, optical light scattering) to determine the reporting parameters of the CBC. A typical panel of CBC parameters includes information regarding the cells produced by the hematopoietic system, the RBCs, WBCs, and platelets. Because other organs and organ systems also affect hematopoiesis, a myriad of disease states can be evaluated from CBC data to determine diagnosis, treatment, and prognosis for a patient.

In addition to the typical CBC parameters, hematology instruments generate scatterplots and histograms that laboratory professionals interpret. Each scatterplot and histogram contains information about the cell populations, interfering substances, and instrument function and therefore serves as forms of quality control for instrument and specimen integrity. For example, the laboratory professional consults the scatterplots and histograms to assess the volume of granulocytes (increased volume indicates immaturity or nuclear hypersegmentation whereas decreased volume of lymphocytes can indicate chronic lymphocytic leukemia). The scatterplots and histograms are also helpful in assessing RBC parameters that can be affected by conditions such as cold agglutination (RBC clumping at temperatures below body temperature) and a severely elevated WBC count. The parameters and reference intervals of atypical mature (adult) CBC are listed in the following table.

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 9 Parameters and Reference Intervals of a Typical Adult CBC

White blood cell count 4.5−11.0×103/mcL (4.5−11.0×109/L)

Red blood cell count 4.0−5.5×106/mcL (4.0−5.5×1012/L)

Hemoglobin 12.0–17.4 g/dL (120–174 g/L)

Hematocrit 36–52% (0.36–0.52 L/L)

Mean cell volume 80–100 fL

Mean cell hemoglobin 28–34 pg

Mean cell hemoglobin 32–36 g/dL concentration

Red cell distribution width 11.5–14.5%

Reticulocyte count Relative (%) I. 0.5–2.0% Absolute II. (×109/L)

Platelet count 150−400×109/L

Mean platelet volume 6.8–10.2 fL

Automated WBC Differential Relative Absolute (×109/L) (%)

Neutrophils 40–80 1.8–7.0

Lymphocytes 25–35 1.0–4.8

Monocytes 2–10 0.1–0.8

Eosinophils 0–5 0–0.4

Basophils 0–1 0–0.2

Male and female reference intervals are combined. For age- and sex-specific reference intervals, see the front cover of this textbook. Additional parameters are dependent upon instrumentation. Data are shown as conventional units (SI units).

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 10 Leukocyte, Erythrocyte, Hematocrit and Hemoglobin Report

The leukocyte count (WBC count), erythrocyte count (RBC count), hematocrit (Hct), and hemoglobin (Hb) are determined using automated instrumentation. The WBC and RBC counts are reported as the number of cells per liter. The WBCs are reported as billions of cells per liter (×109/L), and the RBCs are reported as trillions of cells per liter (×1012/L). The hematocrit measures the volume that the RBCs occupy within whole blood and is reported as a percentage (%) or as the volume of RBCs in liters divided by the volume of whole blood in liters [L/L]. In automated analyzers, the hematocrit is usually calculated from the measured MCV and RBC count using the following formula:

The hemoglobin is measured spectrophotometrically after it has been released from lysed erythrocytes. It is reported in grams per deciliter or grams per liter. The laboratory professional interprets the accuracy of the RBC count, hematocrit, and hemoglobin values using a quick mathematical check called the rule of three. Simply, the RBC count x3 = hemoglobin x3 = hematocrit (%). If the calculated values do not agree within ±3% of the measured values, a measurement error or instrument malfunction could have occurred, or the patient could have a pathology that requires investigation.

A diurnal variation in blood cell concentration occurs in which the value for the WBC count is lowest in the morning and highest in the afternoon,

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 11 whereas the RBC count, Hct, and Hgb are just the opposite; the higher values are observed in the morning.

Erythrocyte Indices

The erythrocyte indices help classify the erythrocytes by their size and hemoglobin content. Hemoglobin, hematocrit, and erythrocyte count values are used to calculate the three indices: mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC).

When calculating the indices, it is important to note that the conversion factors used in the formulas vary depending on the use of conventional units or Systeme International (SI) Units for hemoglobin and hematocrit. These indices suggest how the RBCs will appear microscopically and provide significant diagnostic information (most commonly for the diagnosis of anemias). Laboratory professionals correlate the indices with the Hct, Hgb, and RBC count to ensure that technical problems are identified when they occur.

Mean Cell Volume

The MCV denotes the average volume of individual erythrocytes and is expressed in femtoliters (fL or 10-15 L). It is measured by automated instrumentation and can be calculated from the Hct and RBC count.

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 12 Classification of Erythrocytes Based on MCV

Terminology Description

Normocytic 80.0–100.0 fL

Microcytic Red cells with a reduced volume (<80 fL)

Macrocytic Red cells with an increased volume (>100 fL)

Anisocytosis Increased variation in the range of red cell sizes

The MCV is used to classify cells as normocytic, microcytic, or macrocytic and usually correlates with the appearance of cells on stained blood smears (i.e., cells with an increased MCV appear larger (macrocytic), and cells with a decreased MCV appear smaller (microcytic)). However, it must be remembered that the MCV is a measurement of volume, whereas estimation of the size of flattened cells on a blood smear is a measurement of cell diameter. Cell diameter and cell volume are not the same.

Example:

A patient has an Hct of 0.45 L/L and an RBC count of 5.0×1012/L; 90.0 fL = 0.45×10005. The value, 90.0 fL, indicates that the cell has a volume that falls within the reference interval (80–100 fL) and is therefore classified as normocytic.

Spherocytes usually have a normal or only slightly decreased volume (MCV), but on a stained smear, they cannot flatten as much as normal erythrocytes because of a decreased surface area and increased rigidity. Spherocytes, therefore, often appear to have a smaller diameter than normal cells. On the other hand, codocytes can appear larger due to an increased diameter, but nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 13 the MCV is often normal. Generally, abnormalities in the MCV are clues to disease processes of the hematopoietic system.

Mean Cell Hemoglobin

The MCH is a measurement of the average weight (in picograms, 10−12 g) of hemoglobin in individual erythrocytes. The MCH is calculated from the hemoglobin and erythrocyte count.

Example: A patient has an Hgb concentration of 15 g/dL and an RBC count of 5.0×1012/L. 30 pg = 15×105. The value, 30.0 pg, indicates that the RBCs contain an average weight of hemoglobin that is within the reference interval (28.0–34.0 pg).

The MCH does not take into account the size of a cell; it should not be interpreted without taking into consideration the MCV because the MCH varies in a direct linear relationship with the MCV. Cells with less volume typically contain less hemoglobin while cells with larger volume typically contain more hemoglobin.

Mean Cell Hemoglobin Concentration

The MCHC is the ratio of hemoglobin mass to volume in which it is contained (i.e., average concentration of hemoglobin in a deciliter of erythrocytes, expressed in g/dL). The MCHC is calculated from the Hgb and Hct.

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 14 MCHC (g/dL) =

Example:

A patient has an Hgb concentration of 15 g/dL and an Hct of 0.45 L/L. 33.3 g/dL = 150.45. The value, 33.3 g/dL, reveals that the cells contain a normal concentration of hemoglobin (32.0–36.0 g/dL) and are therefore normochromic.

The MCHC indicates the concentration of hemoglobin in the general cell population and is described by the suffix -chromia, meaning color. Cells can be classified morphologically as hypochromic if the area of central pallor is >1/3 of the cell size. The term hyperchromic should be used sparingly (if ever).

The only erythrocyte that is hyperchromic with an MCHC >36.0 g/dL is the spherocyte. Spherocytes have a decreased surface-to-volume ratio due to a loss of membrane but have not lost an appreciable amount of their hemoglobin. In certain conditions, the indices MCV, MCH, and MCHC can be falsely elevated.

Classification of Erythrocytes Based on MCHC

Normochromic 32.0–36.0 g/dL

Hypochromic <32.0 g/dL

Hyperchromic >36.0 g/dL

Red Cell Distribution Width

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 15 Because the MCV represents an average of erythrocyte volume, it is less reliable in describing the erythrocyte population when considerable variation in erythrocyte volume/size (anisocytosis) occurs. The red cell distribution width (RDW) is the coefficient of variation of the MCV and may be referred to as the RDW-coefficient of variation (RDW-CV). The formula for the RDW- CV, a calculated index from hematology instruments to help identify anisocytosis, follows:

Abnormal increased RDW values (714.5%) indicate an increase in the heterogeneity of erythrocyte size. No known abnormalities are represented by a decreased RDW.

Caution must be used in interpreting the RDW-CV because it reflects the ratio of the standard deviation of cell volume and the MCV. An increased standard deviation (heterogeneous cell population) with a high MCV can give a normal RDW-CV. Conversely, a normal standard deviation (homogenous cell population) with a low MCV can give an increased RDW-CV. Examination of the erythrocyte histogram and stained blood smear gives clues as to the accuracy of the RDW-CV in these cases. When the standard deviation is increased, indicating a true variability in cell size, the base of the erythrocyte histogram is broader than usual. Because of this interpretation issue, automated instruments often report the RDW-CV and RDW-standard deviation (RDW-SD). The RDW-SD is directly measured and not affected by the MCV.

Reticulocyte Count nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 16 Immature, anuclear erythrocytes containing organelles and residual ribosomes for hemoglobin synthesis are known as reticulocytes, which usually spend 2–3 days in the bone marrow and an additional day in the peripheral blood before their RNA is degraded and they become mature erythrocytes. The peripheral blood reticulocyte count indicates the degree of effective bone marrow activity and is one of the most useful and cost- effective laboratory tests in monitoring response to therapy and pathophysiology of anemia. Reticulocytes can sometimes be identified as polychromatophilic erythrocytes (erythrocytes with a bluish tinge) on Romanowsky-stained smears. The polychromatophilia is due to the presence of basophilic RNA within ribosomes mixed with acidophilic hemoglobin.

A supravital stain such as new methylene blue or brilliant cresyl blue must be used to definitively identify the presence of reticulocytes. Although automated methods for reticulocyte enumeration are available on some hematology instruments, many laboratories use a manual method. Test results are expressed as a percentage of reticulocytes in relation to the total RBC count (relative count) or as the absolute number (see the following section). In the automated method, >30,000 RBCs are assessed, so the method is more precise than the manual method (which assesses only 1000 RBCs) and is more accurate when the reticulocyte count is very low.

Absolute Reticulocyte Count

The absolute reticulocyte count is a more informative index of erythropoietic activity than the relative reticulocyte count. When reported as a percentage, the reticulocyte count does not indicate the relationship between the peripheral blood erythrocyte mass and the number of reticulocytes being produced. The reticulocyte count reported as a percentage can appear increased because of either an increase in the number of reticulocytes in the

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 17 circulation or a decrease in the number of total RBCs. Therefore, it is recommended that in addition to the percentage of reticulocytes, laboratory professionals report the absolute reticulocyte count to provide a more useful estimate of reticulocyte production.

Automated analyzers can provide the absolute count and can be calculated when using manual methods for reticulocytes:

Absolute reticulocyte (×109/L) = RBC count (×1012/L)×Reticulocyte count (%)

Example:

A patient has an RBC count of 3.5×1012/L and a 10% reticulocyte count; 350 ×109/L = 3.5×10. The value 350×109/L represents an increase in reticulocyte production since the mean normal value is 90×109/L.

Platelet Count and Mean Platelet Volume

Automated hematology instruments generate the platelet count, which is reported as billions of platelets per liter (number of platelets×109/L). The mean platelet volume (MPV) is similar to the MCV for erythrocytes because it represents the average volume of individual platelets. The laboratory professional utilizes both the platelet count and the MPV to assess thrombopoiesis and pathologic conditions related to platelets. A decreased platelet count generally represents decreased thrombopoiesis, increased platelet destruction, or consumption. Reactive or malignant conditions can cause an increase in the platelet count.

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 18

WBC Differential

The WBC differential is an analysis and enumeration of the various subtypes of WBCs. An altered concentration of one specific type of leukocyte most commonly causes an increase or decrease in the total WBC count. For this reason, an abnormal total WBC count should be followed by a WBC differential, also known as a diff.

The WBC differential can be performed by automated instruments or manually. To perform a manual WBC differential, a blood smear stained with a Romanowsky-type stain (usually Wright’s stain) is viewed microscopically. A total of 100 leukocytes are viewed and each leukocyte subtype is classified. The differential results are reported as the percentage of each cell type counted. To accurately interpret whether an increase or decrease in cell types exists, the absolute concentration of each cell type is calculated using the results of the WBC count and the differential.

The Peripheral Blood Smear

Each testing location and institution sets the parameters that trigger the necessity of a manual morphologic examination of the peripheral smear, but generally a peripheral blood smear is prepared for microscopic examination when CBC values obtained from an automated instrument differ from what is considered normal. A laboratory professional reviews the blood smear for overall quality, the morphology of white blood cells, red blood cells, and platelets, and performs a WBC differential. The peripheral smear is correlated with the parameters reported by the instrument as the culminating interpretation of the CBC.

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Briefly, a wedge smear is made by placing a small drop of blood at one end of a microscope slide and spreading that drop to create a thin smear (or film) of blood. After the blood has dried, the slide is stained using a Romanowsky-type stain. A well-made and properly stained blood smear is required for accurate interpretation of the CBC. The slide is examined macroscopically and microscopically to ensure that the blood was spread and stained properly. The optimal blood smear is pinkish purple in color and transitions to a feathered edge.

Information such as RBC agglutination (appears as a grainy blood smear), lipidemia (represented as holes within the smear), and multiple myeloma (bluish-colored smear) can be suspected during this important macroscopic evaluation of the blood smear and should be noted by the laboratory professional before moving on to the microscopic evaluation. This macroscopic assessment determines whether the blood smear is acceptable for microscopic analysis.

Low-Power Magnification

The laboratory professional first assesses the general appearance and distribution of WBCs, RBCs, and platelets using low power magnification 10x objective (100x magnification). A high concentration of WBCs at the furthest edge of the smear (the feathered edge) indicates poor cell distribution and is sufficient evidence that a new smear should be made. In addition, very large or abnormal WBCs are often pushed to the outer edges of the smear. Cells that have ruptured are called smudge cells. These are often B lymphocytes and their presence is characteristic of pathological conditions such as chronic lymphocytic leukemia. The laboratory professional also performs an estimate

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 20 of the number of WBCs (i.e., WBC estimate) under low power (either 100x or 400x) and correlates the WBC estimate to the WBC count.

On a well-made blood smear, the erythrocytes are evenly distributed and well separated on the feathered edge of the smear. Stacking or aggregating of cells is associated with certain pathologic states. The following table summarizes specifics of the peripheral blood smear examination.

Summary of the Microscopic Peripheral Blood Smear Examination

10×Objective 40×Objective 100×Objective (100×magnification) (400× (1000× magnification) magnification)

WBCs Scan for abnormal or Perform WBC Evaluate leukocyte large cells estimate morphology

Smudge cells Perform 100-cell differential

RBCs Scan for rouleaux and Determine the critical Evaluate erythrocyte agglutination area morphology: Assess size, shape, color, presence of inclusions

Platelets Scan for clumps and Perform platelet satellitism estimate

Evaluate platelet morphology: Assess size and granularity

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 21 In the presence of IgM antibodies (cold agglutinins) directed against erythrocyte antigens, erythrocytes can agglutinate forming irregular clusters of varying sizes. This agglutination forms irregular, grapelike clusters that are readily differentiated from rouleaux.

On automated hematology analyzers, a CBC with an elevated MCV and low RBC count but a normal hemoglobin suggests the presence of cold-reacting erythrocyte agglutinins. In addition, the calculated hematocrit will be falsely decreased and the MCH and MCHC will be falsely increased. The effect of cold agglutinins is overcome by keeping the blood at 37 °C. When performing blood counts, the diluting fluid also must be kept at 37 °C. The following table provides helpful information regarding abnormalities that may be seen in erythrocyte arrangement during laboratory testing.

Abnormalities in Erythrocyte Arrangement

Terminology Description Associated Physiologic State

Agglutination Irregular clumps Due to antigen–antibody interaction of red blood cells

Rouleaux Red blood cells Usually associated with abnormal or increased arranged in rolls plasma proteins (red blood cells can be dispersed or stacks by mixing cells with saline)

Rouleaux is an alignment of erythrocytes one on top of another resembling a stack of coins. This phenomenon occurs normally when blood is collected and allowed to stand in tubes. It can also be seen in the thick portion of blood smears. In certain pathologic states that are accompanied by an increase in fibrinogen or globulins, rouleaux becomes marked and is readily seen in the feathered edge of blood smears. When the erythrocyte assumes

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 22 abnormal shapes, such as sickled forms, rouleaux formation is inhibited. Rouleaux is also inhibited when erythrocytes are suspended in saline. The presence of rouleaux or agglutination is a possible indication that a new smear should be made.

The platelets must also be evaluated using low power magnification. The manner in which the platelets have spread on the slide is checked because platelet clumps can be pushed to the outer edge of the smear, and in some cases, platelets can adhere to neutrophils (a phenomenon called satellitism). This can result in falsely decreased estimation of the platelet count. Platelet clumps and satellitism can be eliminated using sodium citrate as an anticoagulant. Finally, fibrin strands can be observed in this scan of the blood smear, indicating that the blood sample was coagulated (likely due to improper mixing following the venipuncture). In either case, a new sample should be obtained from the patient and a new smear should be made.

The final task at low power magnification is to determine the critical area of the smear that will be used to perform the morphologic examination of cells. This critical area is usually identified using the 40x objective (400x magnification) and is characterized by the proximity of RBCs to each other (the area of the smear in which very few RBCs overlap or touch and are generally distributed in a uniform manner). At high power magnification, the critical area is used to evaluate RBC morphology and perform the WBC differential and platelet estimate.

High-Power Magnification

Following the quick, yet important, scan of the blood smear on low magnification, the laboratory professional evaluates the smear on high power, often at 1000× magnification. Ultimately, interpretation of the nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 23 microscopic findings in the peripheral blood smear and correlation, or lack thereof, with the automated instrument report is made for all parameters of the CBC. A WBC differential is performed in which 100 cells are observed and classified to determine the relative number of leukocytes as a percentage and to identify the presence of morphologic abnormalities.

A platelet estimate is performed and compared with the instrument- generated platelet count; the morphology of the platelets is noted. Finally, the RBC morphology is assessed for size, shape, color, and inclusions using either the 40× or 50× objective and compared with the instrument report for the RBC indices. To evaluate abnormalities including inclusions, the laboratory professional should review the slide with the 100× objective (1000× magnification).

Erythrocyte Morphology

The erythrocyte is sometimes called a discocyte because of its biconcave shape. On a Romanowsky-stained blood smear, the erythrocyte appears as a disc with a central area of pallor surrounded by a rim of pink-staining hemoglobin (the center stains lighter in color compared with the rim). The area of pallor is caused by the closeness occurring between the two concave portions of the membrane when the cell becomes flattened on a glass slide. Normally the area of pallor occupies about one-third the diameter of the cell.

Anisocytosis denotes a nonspecific variation in the size of the cells. Some variation in size is normal because of the variation in age of the erythrocytes with younger cells being larger and older cells smaller. Poikilocytosis is the general term used to describe a nonspecific variation in the shape of erythrocytes. It is important to note that some abnormal morphology can be artifactual because of poorly made or improperly stained smears.

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Anisocytosis

Anisocytosis can be detected by examining the blood smear and/or by reviewing the MCV and RDW (discussed earlier in this chapter). Normal erythrocytes have a diameter of about 7−8 mcM (μm) and an MCV of 80– 100 fL. If the majority of cells are larger than normal, they are macrocytic; if smaller than normal, they are microcytic. If there is a significant variation in size with microcytic, normocytic, and macrocytic cells present, the MCV can fall within the reference interval because it is an average of cell volume. In this case, the RDW is helpful.

An RDW of >14.5% suggests that the erythrocytes are heterogeneous in size, which makes the MCV less reliable. Microscopic examination of the cells is especially helpful when the RDW is elevated. To evaluate erythrocyte size microscopically, the cells are compared with the nucleus of a normal small lymphocyte. Normocytic erythrocytes are about the same size as the lymphocyte nucleus.

Poikilocytosis

Most laboratories report only significant poikilocytosis. The stained smear should be reviewed while keeping in mind the overall context of the laboratory results and the significance of the reported findings. To determine the significance of and to decide whether to report poikilocytes, the following should be considered: 1. Will it assist in differential diagnosis of the disease (likely anemia)? 2. Will it make a difference in the management of the patient? 3. Is the dominant poikilocyte significant in this setting?

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 25 4. Do the specific constellation of findings indicate a particular pathologic state?

Poikilocytes are further subclassified according to the specific cell shape. As much as possible, the CBC report should specify the red blood cell shape of diagnostic significance existing within the umbrella term of poikilocytosis or poikilocytes. Only when there are too many red blood cell shapes or when some cell shapes cannot be described would the term poikilocytosis suffice to identify erythrocyte morphology; otherwise the individual red blood cell shapes should be identified.

Acanthocytes also called spur cells, are small spherical cells with irregular thorn like projections. Often the projections have small bulb-like tips. Acanthocytes do not have a central area of pallor. These cells have membranes with free cholesterol accumulating preferentially in the outer bilayer of the membrane leading to decreased fluidity. Remodeling by the spleen results in spheroidal cells with irregular surface projections. These cells are readily trapped in the spleen.

Codocytes, also called Target cells, are thin, bell-shaped cells with an increased surface-to-volume ratio. On stained blood smears, the cells have the appearance of a target with a bull’s-eye in the center. An achromic zone and a thin outer ring of pink-staining hemoglobin surround the bull’s eye. The typical appearance of these cells is discernible in the area of the slide only where the cells are well separated but not in the extreme outer- feathered edge where all cells are flattened. Target cells can appear as artifacts when smears are made in a high-humidity environment or when a wet smear is blown dry rather than fan dried. Target cells have an increased surface-to-volume ratio of the cell. nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 26

Dacryocytes, also called teardrops, are erythrocytes that are elongated at one end to form a teardrop or pear-shaped cell. The teardrop morphology can form after erythrocytes containing cellular inclusions have traversed the spleen. Erythrocytes with inclusions are more rigid in the area of the inclusion, and this portion of the cell has more difficulty passing through the splenic filter than the rest of the cell. As splenic macrophages attempt to remove this rigid inclusion, the cell is stretched into an abnormal shape. The teardrop cannot return to its original shape because the cell either has been stretched beyond the limits of deformability of the membrane or has remained in the abnormal shape for too long.

Sickle cells, also called drepanocytes, are elongated, crescent-shaped erythrocytes with pointed ends. Some forms have more rounded ends with a flat rather than concave side. These modified forms of sickle shape can be capable of reversing to the normal discocyte. Sickle cell formation can be observed in stained blood smears from patients with sickle cell anemia. The hemoglobin within the cell is abnormal and polymerizes into rods at decreased oxygen tension or decreased pH. The cell first transforms into a holly leaf shape and as the hemoglobin polymerization continues, it transforms into a sickle-shaped cell with increased mechanical fragility. Some holly-leaf forms can be observed on stained blood smears in addition to the typical sickle shape.

Echinocytes, also called burr cells, are usually smaller than normal erythrocytes with regular, spine-like projections on their surface. Their presence is most often artifact in stained blood smears because of the glass effect of the slide. The glass releases basic substances that raise the pH of the medium surrounding the cell and induce echinocyte formation. Plasma

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 27 provides a buffering effect on the cells, and for this reason, blood films made from whole blood may show only certain areas of echinocyte transformation. To determine the in vivo or in vitro nature of echinocytes, a wet preparation can be made in which a drop of blood is enclosed between two plastic cover slips and the unstained individual erythrocytes are observed. If no echinocytes are present in the wet preparation but were noted on the stained blood smears, the cell abnormality occurred as an in vitro artifact.

Echinocytes can appear in blood that has been stored at 4°C for several days. Consequently, blood specimens from patients receiving transfusions can have echinocytes if blood is taken from the patient immediately after transfusion; however, after a few minutes, the buffering action of patient’s plasma causes the transfused echinocyte to resume a normal discoid shape. For true in vivo echinocytes, the characteristic appearance is not related to tonicity of the medium in which the cells are suspended. The shape change is instead thought to result from an increase in the area of the outer leaflet of the lipid bilayer as compared with the inner layer. Echinocyte formation is reversible (i.e., the cell can revert to a discocyte). However, an echinocyte can eventually assume the shape of a spherocyte, presumably because the spleen grooms (removes) the membrane spines; in this circumstance, the cell cannot revert to a normal shape.

Elliptocytes, also called pencil cells and cigar cells, vary from elongated oval shapes (ovalocytes) to elongated rodlike cells. Some laboratory professionals may use the terms elliptocytes and ovalocytes interchangeably, whereas others may use distinct guidelines to delineate the two morphologies. True elliptocytes have parallel sides and a central area of biconcavity with hemoglobin concentrated at both ends. Elliptocytes are formed after the erythrocyte matures and leaves the bone marrow because reticulocytes and

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 28 young erythrocytes in patients with elliptocytosis are normal in shape. The mechanism of formation is not known but is assumed to involve alterations of the erythrocyte membrane skeleton. Elliptocytes are the predominant shape of erythrocytes in hereditary elliptocytosis. On the other hand, ovalocytes are fatter on one end than the other and appear to have an egg shape. Ovalocytes are formed in a manner similar to elliptocytes.

Keratocytes, also called helmet cells, have a concavity on one side and two hornlike protrusions on either end. Keratocytes are produced when a fibrin strand impales an erythrocyte. The two halves of the erythrocyte hang over the strand as saddlebags; the membranes of the touching sides fuse, producing a vacuole-like inclusion on one side. This cell with an eccentric vacuole is called a blister cell. The vacuole bursts, leaving a notch with two spicules on the ends.

Knizocytes are cells with more than two concavities. This cell’s appearance on stained blood smears can vary depending upon how the cell comes to rest on the flat surface; however, most knizocytes have a dark-staining band across the center with a pale area on either side surrounded by a rim of pink-staining hemoglobin. The mechanism of formation is unknown.

Leptocytes are thin, flat cells with normal or larger than normal diameter. Although the cell’s diameter is normal or increased, its volume is usually decreased. The cells have an increased surface-to-volume ratio either as a result of decreased hemoglobin content or increased surface area. The leptocyte is usually cup-shaped like stomatocytes, but the cup has little depth. Target cells can be formed from leptocytes on dried blood smears when the depth of the cup increases.

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 29 Schistocytes are erythrocyte fragments caused by mechanical damage to the cell. They appear in a variety of shapes such as triangular, comma, and helmet-shaped. Because schistocytes are fragments of erythrocytes, they are usually microcytic. They maintain normal deformability, but their survival in the peripheral blood is reduced. The fragments can assume a spherical shape and hemolyze or can be removed in the spleen.

Spherocytes are erythrocytes that have lost their biconcavity because of a decreased surface-to-volume ratio. On stained blood smears, the spherocyte appears as a densely stained sphere lacking a central area of pallor. Although the cell often appears microcytic on stained blood smears, the volume (MCV) is usually normal. The spherocyte is the only erythrocyte that can be called hyperchromic because of an increased MCHC.

Stomatocytes, or mouth cells, appear as small cup-shaped uniconcave discs (in wet preparations). Upon staining, these cells exhibit a slitlike (mouthlike) area of pallor. Normal discocytes can be transformed under certain conditions to stomatocytes and, eventually, to spherostomatocytes. The stomatocyte shape is reversible, but the spherostomatocyte is not. Cationic drugs and low pH cause a gradual loss of biconcavity leading to the stomatocyte and eventually the formation of a sphere.

Stomatocytosis is the opposite of echinocytosis; the shape change in stomatocytosis is thought to be the result of an increase in the lipid content or area of the inner leaflet of the membrane lipid bilayer. Stomatocytes also can appear as an artifact on stained blood smears; thus, care should be used in identifying them.

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 30 The following is a helpful table that lists the varied erythrocyte (red cell) morphologies, cell descriptions (shapes) and disease states associated with the cell description.

Erythrocyte Morphologies

Terminology Synonyms Description Associated Disease States

Poikilocytosis — Increased variation in the See disease states shape of red cells. associated with specific poikilocytes on this table.

Acanthocyte Spur cell Red cells with spicules of Abetalipoproteinemia; (spike) varying length irregularly alcoholic liver disease; distributed over the disorders of lipid surface; no area of metabolism; post pallor. splenectomy; fat malabsorption; retinitis pigmentosa.

Codocyte Target cell Thin, bell-shaped, with Hemoglobinopathies; (bell) increased surface-to- thalassemias; obstructive volume ratio; on stained liver disease; iron blood smears, appears as deficiency anemia; a target with a central splenectomy; renal bull’s-eye, surrounded by disease; LCAT deficiency. achromic zone and outer ring of hemoglobin.

Dacryocyte Teardrop Round cell with a single Myelophthisic anemias; (tear) elongated or pointed primary myelofibrosis extremity; may be (PMF); thalassemias. microcytic and/or hypochromic.

Drepanocyte Sickle cell Contain polymerized Sickle cell disorders. (sickle) hemoglobin showing various shapes: sickle, crescent, or boat shaped.

Echinocyte Burr cell; Spiculated red cells with Liver disease; uremia; (sea urchin) crenated short equally spaced pyruvate kinase cell projections over the deficiency; peptic ulcers; entire surface. cancer of stomach;

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heparin therapy.

Elliptocyte Ovalocyte; Oval to elongated Hereditary elliptocytosis; (oval) pencil cell; ellipsoid cell with central iron deficiency anemia; cigar cell area of pallor and thalassemia; anemia hemoglobin at both ends. associated with leukemia.

Keratocyte Helmet Red cells with one or Microangiopathic (horn) cell; horn- several notches with hemolytic anemias; heart- shaped cell projections that look like valve hemolysis; Heinz- horns on either end body hemolytic anemia; glomerulonephritis; cavernous hemangiomas

Knizocyte — RBC with more than two Conditions in which concavities; on stained spherocytes are found. blood smears has a dark band of hemoglobin across the center with a pale area on either side.

Leptocyte Thin cell Thin, flat cell with Thalassemia; iron (thin) hemoglobin at periphery; deficiency anemia; usually cup-shaped, MCV hemoglobinopathies; liver is decreased but cell disease. diameter is normal.

Schistocyte Schizocyte; Fragments of red cells; Microangiopathic (cut) fragmented variety of shapes hemolytic anemias; heart- cell including triangles, valve hemolysis; commas; microcytic. disseminated intravascular coagulation; severe burns; uremia.

Spherocyte — Spherocytic red cells with Hereditary spherocytosis; dense hemoglobin immune hemolytic content anemias; severe burns; (hyperchromatic); lack transfusion with ABO an area of central pallor. incompatibility; Heinz- body hemolytic anemias.

Stomatocyte Mouth cell; Uniconcave red cells with Hereditary (mouth) cup form; the shape of a very thick stomatocytosis; mushroom cup; on stained blood spherocytosis; alcoholic

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cap smears cells have an oval cirrhosis; anemia or slit like area of central associated with Rh null pallor. disease; lead intoxication; neoplasms.

Variation in Hemoglobin (Color)

Normal erythrocytes have an MCH of approximately 30 pg. However, the MCHC is a better indicator of chromia or color of erythrocytes on Romanowsky-stained smears. Normally, on stained smears, the erythrocyte has a central area of pallor approximately one-third the diameter of the cell. In certain conditions, RBCs contain less hemoglobin than normal and appear to have a larger than normal central pallor (hypochromia). On the other hand, the only erythrocyte that contains more hemoglobin than normal in relation to its volume is the spherocyte.

Hypochromic cells are poorly hemoglobinized erythrocytes with an exaggerated area of central pallor (>1/3 the diameter of the cell) on Romanowsky-stained blood smears. Although occasionally normocytic, hypochromic cells are usually microcytic. Hypochromic cells are the result of decreased or impaired hemoglobin synthesis. When visualizing a blood smear, correlating the automated findings from hematology analyzers to the appearance of cells is important. In the case of hypochromia, the MCHC value will be decreased.

Polychromatophilic erythrocytes (reticulocytes) are usually larger than normal cells with a bluish tinge on Romanowsky-stained blood smears. The

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 33 bluish tinge is caused by the presence of residual RNA in the cytoplasm. Large numbers of these cells are associated with decreased erythrocyte survival or hemorrhage and an erythroid hyperplastic marrow.

Erythrocyte Inclusions

Erythrocytes do not normally contain any particulate inclusions. When present, inclusions can help direct further investigation because they are associated with certain disease states. Basophilic Stippling

Erythrocytes with basophilic stippling are cells with bluish-black granular inclusions distributed across their entire cell area. The granules can vary in size and distribution from small diffuse to coarse and punctate. The granules, which are composed of aggregated ribosomes, are sometimes associated with mitochondria and siderosomes. Basophilic stippling is not believed to be present in living cells; instead, stippling probably is produced during preparation of the blood smear or during the staining process. Electron microscopy has not shown an intracellular structure similar to basophilic stippling. Cells dried slowly or stained rapidly can demonstrate fine, diffuse stippling as an artifact. Pathologic basophilic stippling is more coarse and punctate.

Cabot Rings

Cabot rings are reddish-violet erythrocytic inclusions usually occurring in the formation of a figure eight or oval ring. Cabot rings are thought to be remnants of spindle fibers, which form during mitosis. They occur in severe anemias and in dyserythropoiesis.

Howell-Jolly Bodies

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 34 Howell-Jolly bodies are dark purple or violet spherical granules in the erythrocyte. These inclusions are nuclear (DNA) fragments usually occurring singly in cells, rarely more than two per cell. Howell-Jolly bodies are associated with nuclear maturation abnormalities. They are thought to occur as a result of an individual chromosome failing to attach to the spindle apparatus during mitosis, and, thus, it is not included in the reformed nucleus. When the nucleus is extruded, the Howell-Jolly body is left behind (until removed by splenic macrophages). Variations in Erythrocyte Color

Terminology Description Associated Physiological or Disease States

Hypochromia Decreased concentration of May be present in iron hemoglobin in the red cell. deficiency anemia, thalassemia, Red cells have an increased area of and other anemias associated central pallor (>1/3 diameter of with a defect in hemoglobin cell). production.

Polychromasia Young red cells containing residual Found in increased numbers in RNA. hemolytic anemias, newborns, Stain a pinkish-gray to pinkish- recovery from acute blue color on Wright’s stained hemorrhage. blood smears. Usually appear slightly larger than mature red cells.

Heinz Bodies do not stain with Romanowsky stains but can be visualized with supravital stains or with phase microscopy of the living cell. They appear as 2–3 mcM round masses lying just under or attached to the cell membrane. Heinz bodies are composed of aggregated denatured hemoglobin.

Iron Inclusions refer to particulate iron molecules that can be detected in erythrocytic cells in both normal and abnormal conditions. Intracellular

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 35 siderotic granules represent iron that has not been incorporated into hemoglobin.

Sideroblasts are erythroblasts that contain stainable iron granules whereas siderocytes are non-nucleated, mature erythrocytes that contain stainable iron granules. Sideroblasts and siderocytes can be identified with Perl’s Prussian blue iron stain, which stains iron aggregates blue. The granules do not stain with Romanowsky stains. About 20–60% of all erythroblasts in the marrow contain iron that can be visualized with Perl’s Prussian blue stain. This number decreases in some pathologic states and can be markedly increased in others. Reticulocytes and erythrocytes in the peripheral blood do not normally contain stainable iron aggregates unless the patient has been splenectomized.

Abnormal Erythrocyte Inclusions

Terminology Description Associated Disease States

Basophilic Round or irregularly shaped granules of Lead poisoning; stippling variable number and size, distributed anemias associated throughout the RBC. with abnormal Composed of aggregates of ribosomes (RNA). hemoglobin Stain bluish black with Wright’s stain. synthesis; thalassemia.

Cabot rings Appear as a figure-8, ring, or incomplete ring. Severe anemias; Thought to be composed of the microtubules dyserythropoiesis. of the mitotic spindle. Stain reddish violet with Wright’s stain.

Howell-Jolly Small, round bodies composed of DNA usually Post splenectomy; bodies located eccentrically in the red cell. megaloblastic Usually occurs singly, rarely more than two anemias; some per cell. hemolytic anemias; Stains dark purple with Wright’s stain. functional asplenia; severe anemia.

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Pappenheimer Clusters of granules containing iron that are Sideroblastic bodies usually found at the periphery of the cell. anemia; Visible with Prussian blue stain and Wright’s thalassemia; other stain. severe anemias.

Heinz bodies Bodies composed of denatured or precipitated G6PD deficiency; hemoglobin. unstable hemoglobin Not visible on Wright’s stained blood smears. disorders; oxidizing With supravital stain appear as purple, round- drugs or toxins; post shaped bodies of varying size, usually close to splenectomy. the cell membrane. Can also be observed with phase microscopy on wet preparations.

Pappenheimer bodies are damaged secondary lysosomes and mitochondria variable in their composition of iron and protein. This type of inclusion appears as clusters of small granules in erythrocytes and erythroblasts and stains with both Romanowsky and Perl’s Prussian blue stains. Romanowsky stains reveal Pappenheimer bodies by staining the protein matrix of the granules whereas Perl’s Prussian blue is responsible for staining the iron portion of the granules. Pappenheimer bodies occur only in pathologic states.

Normal Erythrocytic Cell Inclusions

Terminology Description

Reticulofilamentous  Artifactual aggregation of ribosomes. substance  Not visible on Wright’s stained smears; supravital stain (i.e., (reticulocyte) new methylene blue) must be used.  Appears as deep blue reticular network.

Sideroblast  Iron granules found in erythroblasts.  Stains blue with Perl’s Prussian blue stain but not with Romanowsky stains.

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 37 Terminology Description

Siderocyte  Iron granules found in erythrocytes.  Stains blue with Perl’s Prussian blue stain not stain with Romanowsky stains.

Professional Review of the CBC

The role of the laboratory professional is to analyze and interpret the CBC data generated by the automated hematology analyzer and the manual, peripheral blood smear review. The interpretation is essentially a correlation of the various components of the CBC in order to identify the likelihood of abnormal results, pathology, and discrepancies in the generated data. The hematology instrument or laboratory information system produce alerts that include delta checks or that indicate the presence of interfering substances, both of which the laboratory professional must resolve.

Delta checks compare a patient’s current clinical values for a test with previous values. This type of quality control can detect sudden changes in a patient’s physiology or can be useful in identifying instrument error. Delta checks are particularly important in diagnosis and in monitoring therapy. Abnormal results and the presence of interfering substances (i.e., lipemia, hemolysis) must also be noted and corrected. In the event that the data can be correlated for diagnosis, the laboratory professional should be able to recommend subsequent testing to the patient’s medical provider.

Bone Marrow Disease And Examination11,43,63,69-86

The hematopoietic system consists of the bone marrow, liver, spleen, lymph nodes, and thymus. The bone marrow is one of the body's largest organs,

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 38 representing 3.4 to 6 percent of total body weight and averaging about 1500 grams in adults. This section discusses bone marrow disease and indications to proceed with examination, including the procedure of bone marrow aspiration and biopsy.11,43,63,69-86

Hematopoiesis (blood cell production and maturation) can be seen at different anatomic locations (yolk sac, liver, spleen, axial and radial bones), depending on the gestational and postnatal period. In normal adults, hematopoiesis is seen mainly in the bone marrow. The bone marrow–derived pluripotential hematopoietic stem cells, under the influence of various cytokines or growth factors, or both, differentiate into myeloid (granulocytes, monocytes, megakaryocytes, erythrocytes) and lymphoid cell lineages. Benign conditions and malignant diseases related to these cells are called hematolymphoid disorders. Nonhematolymphoid diseases may also involve the bone marrow; therefore, examination of the bone marrow has a wide application in clinical practice. Because hematologic diseases involving the bone marrow can result in morphological abnormalities of the peripheral blood cells, the bone marrow examination should be interpreted in conjunction with a peripheral smear examination. Severe thrombocytopenia is generally not a contraindication to the procedure. In experienced hands and with the needles currently available, the bone marrow aspiration and biopsy carry minimal risk.

The hematopoietic marrow is organized around the bone vasculature. An artery entering the bone branches out toward the periphery to specialized vascular spaces called sinuses. Several sinuses combine in a collecting sinus, forming a central vein that returns into the systemic circulation. Hematopoietic cords, in which hematopoiesis takes place, lie just outside of the sinuses. After maturation in the cords, the hematopoietic cells cross the

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 39 walls of the sinuses and enter the blood. Hematopoietic cell colonies are compartmentalized in the cords.

Structurally, bone marrow consists of hematopoietic cells (erythroid, myeloid, lymphoid, and megakaryocyte), adipose tissue, bone and its cells (osteoblasts and osteoclasts), and stroma. The main function of the marrow is to supply mature hematopoietic cells into the peripheral blood in a steady- state condition as well as to respond to increased demands.

A semidormant pool of pluripotential stem cells maintains a self-renewal property. Granulocytic, monocytic, eosinophilic, erythroid, and megakaryocyte progenitor cells are influenced in their differentiation by colony-stimulating factors (CSFs). CSFs are produced by T lymphocytes, as well as stromal cells, fibroblasts, endothelial cells, and macrophages, when stimulated by monocyte interleukin-1 (IL-1) and tumor necrosis factor (TNF). Some CSFs, such as IL-3 and granulocyte-monocyte CSF, have a broad influence and are required throughout proliferation and differentiation of progenitor cells. Others, which include granulocyte, monocyte, and eosinophil CSFs, are lineage specific, and regulate division and differentiation only of corresponding, committed progenitor cells. In addition, erythropoiesis is influenced by erythropoietin produced in the kidney.

In the process of cell egression from the cords to the circulation, a number of releasing factors are identified. The best characterized of these are granulocyte colony–stimulating factor (G-CSF) and granulocyte–macrophage colony–stimulating factor (GM-CSF), but other factors, such as components of a complement system, androgenic steroids, and endotoxins, may play a role. The endothelial lining of the sinusoids forms a continuous, veil-like wall through which the mature cells migrate from extravascular sites into the

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 40 circulation. This is accomplished by close contact between mature hematopoietic cells and endothelial cells. A transient migration pore is formed during such contact through which the mature cells pass into the circulation without loss of plasma to the extravascular pool. It is evident that the bone marrow is subjected to a complex regulation by many cellular and humoral systems of the body, and any disease that affects these systems is likely to affect hematopoiesis.

Bone marrow studies are frequently used in the diagnosis of hematopoietic disorders. Once a formidable task, obtaining bone marrow tissue has become, with current improved techniques, a standard procedure. Several techniques have been devised, each having its own merits and limitations. Bone marrow aspiration and bone marrow biopsy are usually performed concurrently. Although obtaining the bone marrow for examination carries little procedural risk for the patient, the procedure is costly and can be quite painful. For this reason, bone marrow studies should be performed only when clearly indicated or whenever the medical provider expects a beneficial diagnostic result for the patient.

Hematologic diseases affecting primarily the bone marrow and causing a decrease or increase of any cellular blood elements are among the most common indications. It is not unusual for more than one blood element to be increased or decreased, as occurs in leukemias and some refractory anemias. In these situations, bone marrow study affords specific information, and it usually precedes any other diagnostic procedure. Systemic diseases may affect the bone marrow secondarily and require bone marrow studies for diagnosis or monitoring patients' conditions. Patients having any of the solid malignant tumors may undergo bone marrow studies when the initial diagnosis is established for evaluation of the degree of tumor

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 41 spread and staging of the disease. On occasion, a bone marrow study may result in a diagnosis of unsuspected metastatic malignant tumor. During the course of malignant disease, additional marrow studies may be performed periodically to monitor the status of tumor burden and its therapeutic response.

Infections manifesting clinically as fever of unknown origin may exhibit granulomas, focal necrosis, or histiocytic proliferations. Intracytoplasmic organisms may be seen in the marrow. Material for morphologic studies and bacterial cultures may be collected simultaneously during a single procedure. The suspected diagnoses of disseminated tuberculosis, fungal infections (particularly histoplasmosis and cryptococcosis), and some protozoan infections are frequently confirmed through such studies. Hereditary and acquired conditions occasionally involve the bone marrow histiocytes (i.e., Gaucher's disease, sea blue histiocytosis, hemophagocytic syndrome, and others). A simple procedure such as bone marrow aspiration or biopsy may establish the diagnosis.

Indications for Bone Marrow Evaluation

A primary objective of a bone marrow examination is to assess the quantity and development of hematopoietic cells. Bone marrow evaluation is necessary for diagnosing, managing, making prognoses, and following up a variety of hematologic and nonhematologic disorders. Because the cells in the peripheral blood often reflect changes in the bone marrow, the bone marrow should always be evaluated in conjunction with the results of the peripheral blood count and smear review. Thrombocytopenia, coagulation factor deficiency, or anticoagulant therapy is not a contraindication for the bone marrow procedure. However, a bone marrow biopsy should not be

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 42 performed when a bone marrow procedure will not be useful in diagnosing or evaluating the patient’s condition.

Conditions for Which a Bone Marrow Evaluation Is Indicated

Purpose Condition

Primary diagnosis of hematopoietic  Acute leukemias and lymphoid malignancies  Chronic myeloproliferative disorders  Chronic lymphoproliferative disorders  Myelodysplastic syndromes  Hodgkin’s and non-Hodgkin’s lymphomas  Plasma cell neoplasms

Staging of lymphoid malignancies  Lymphomas and solid tumors

Post-treatment follow-up  After chemotherapy and radiation therapy for neoplasms  After stem cell transplant

Detection of infection and/or source of  Mycobacterium and fungal infections fever of unknown origin  Granulomas  Unknown infectious agents using cultures and special stains  Hemophagocytic syndrome

Primary diagnosis of systemic diseases  Metabolic disorders (i.e., Gaucher’s disease)  Systemic mastocytosis

Miscellaneous  Evaluation of storage iron  Evaluation of unexplained cytopenias

Obtaining and Preparing Bone Marrow for Hematologic Studies

The sites for bone marrow studies in adults are most commonly the posterior superior iliac crest, occasionally the sternum, and very rarely the anterior superior iliac crest and spinal processes or vertebral bodies. Sternal

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 43 aspiration should be avoided in children, as well as in patients with multiple myeloma and metastatic carcinoma, because these diseases can cause thinning and erosion of bone that may increase the chance of perforation and can cause potentially fatal cardiac complications.

Occasionally, when a localized bone lesion is visualized on roentgenogram or computed tomographic (CT) scan, a directed or open bone marrow biopsy of the lesion may be performed by a radiologist or surgeon in an operating room while the patient is under anesthesia. In newborns and infants, a bone marrow sample can be obtained from the upper end of the tibial bone. Before performing the procedure, the physician should inform the adult patient or the parent or guardian of a child of the procedure, its risks, and its expected benefits for the diagnostic process. The bone marrow procedure cannot be performed until a consent form is signed and witnessed by a second person, commonly the patient's nurse. The actual procedure is often performed with the assistance of a clinical laboratory scientist. While the physician performs the procedure and the nurse attends to the patient, the clinical laboratory scientist gives full attention to the processing of the specimens.

It is the clinical laboratory scientist's responsibility to ensure that the samples are adequate. If they are not, the physician is informed immediately so that the procedure can be repeated before the patient is discharged. Samples are preserved appropriately for histologic, flow cytometric, cytogenetic, microbiologic, electron microscopic, molecular, and other studies as indicated in a particular case.

In experienced hands, complications of bone marrow biopsy and aspirate are very rare (0.1%). These rare complications include bleeding or infection at

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 44 the biopsy site, transient neuropathy, and osteomyelitis. After the procedure, the patient is advised to lie on the biopsy site, which should be re-evaluated in 15 to 30 minutes for any bleeding or oozing. Bone marrow aspiration and biopsy can be performed on patients with severe thrombocytopenia, coagulation factor deficiencies (more than 50% activity is required), and in those receiving anticoagulant therapy. The only valid contraindication is failure to meet the indication criteria.

Equipment

The instrument tray used to perform a bone marrow procedure should contain enough equipment to complete the procedure and to prepare the tissues obtained for the appropriate studies. Complete bone marrow trays are sold as disposable equipment, which is convenient, and also avoids the risk of transmitting infectious diseases. Because of potential disease transmission, nondisposable bone marrow trays are rarely used today.

The required materials to perform a bone marrow procedure include:

 30-mL, 20-mL, 10-mL and 5-mL syringes  2% Lidocaine  Iodine prep  Alcohol (70%) prep  23-Gauge and 21-Gauge needles  Bone marrow biopsy/aspiration needle 11-gauge × 4 in.  Filter papers  Buffered formalin 10% with a pH of about 6.8 or other fixative for histologic processing of bone biopsy and marrow particles  Tube containing liquid EDTA anticoagulant  One box of slides  One slide folder nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 45  One rubber bulb  Pasteur pipet  Petri dish  Sterile blades  Gloves (several pairs of different sizes)  Sterile gauze and cotton balls  Applicator sticks  Bandage  Culture bottles for bacterial culture. (Note: Some bone marrow specimen should be saved in a syringe for tuberculosis and fungal cultures, when indicated).  Pencil to label slides  No. 11 Bard Parker blades Several different styles of aspiration and trephine bone biopsy needles or instruments are commonly used. Most instruments used today are patterned on the needle introduced by Jamshidi. These instruments are produced in several sizes for both adult and pediatric patients. Modifications of the original aspiration and trephine needles have been developed by different companies and are manufactured as disposable equipment.

Aspiration of Bone Marrow

A bone marrow aspiration may be performed as an independent procedure or in conjunction with a bone marrow biopsy. The procedure can be performed in the outpatient setting in clinics or in the physician's office. As a rule, children and very apprehensive patients receive a mild sedative before the procedure. The site selected is shaved, if needed, and washed with soap. Then an antiseptic is applied, and the area is draped with sterile towels. A local anesthetic such as 1% to 2% lidocaine (Xylocaine) is infiltrated into the skin, in the intervening tissues between the skin and bone, and in the

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 46 periosteum of the bone from which the marrow is to be obtained. A cut of about 3 mm is made through the skin with a Bard-Parker blade to facilitate piercing skin and subcutaneous tissue.

The physician penetrates the bone cavity with an aspiration needle, assembled with guard and stylet locked in place. When the marrow cavity is penetrated, the stylet is removed, a syringe is attached to the free end of the needle, and the plunger is quickly pulled, drawing 1.0 to 1.5 mL of marrow particles and sinusoidal blood into the syringe. Because the vacuum created in the syringe is important for rapid and efficient suctioning of the cells and particles, the syringe should be 10 mL or larger with a well-fitting plunger.

Despite the use of local anesthesia, the patient normally experiences discomfort during the aspiration process (aspiration pain). Accomplishing the aspiration with a quick and continuous pull on the plunger diminishes the patient's discomfort and decreases the chance of clotting the specimen. A clotted specimen is useless for smear preparation because the fibrin threads strip the cytoplasm off of the cells and hamper their spreading. Keeping the volume of the initial aspirate small also prevents dilution of the sample with large amounts of sinusoidal blood, thus improving the quality of the aspirate.

The first-aspirated material is used immediately for preparing smears. Additional aspirate may be obtained in separate syringes if needed for flow cytometry, chromosome studies, bacterial cultures, and other tests. Once an adequate aspirate is obtained, the quality of the smear depends entirely on the clinical laboratory scientist's skill and speed in preparing the smears and preserving the morphology of the marrow cells. Part of the first aspirate is used for the preparation of direct and marrow particle smears. Another

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 47 portion is placed in an ethylene diaminetetraacetic acid (EDTA) anticoagulant-containing tube for use as a particle preparation. If some aspirate still remains, it can be left to clot. The clot may be fixed in 10% buffered formalin or another chosen fixative and processed for histologic examination.

The preferred anticoagulants for ancillary studies performed on the bone marrow aspirate are EDTA for flow cytometry and sodium heparin for cytogenetics. If the aspiration attempt is unsuccessful (a dry tap), an additional core biopsy may be obtained (placed in saline or RPMI) for flow cytometric studies. In these situations, touch preparations of the core biopsy are useful for Wright-Giemsa stained morphologic evaluation, as well as cytochemical and fluorescence in situ hybridization (FISH) studies, if needed. Preparation of Bone Marrow Aspirate

All necessary materials, preservatives, and slides should be meticulously clean and in readiness to avoid any delay. The aspirate in the first syringe contains mostly blood admixed with fat, marrow cells, and particles of marrow tissue, which should be used for smears. Several direct smears can be prepared immediately, using the technique for blood film preparation. A small drop is placed on a glass slide, and the blood and the particles are dragged behind a spreading slide with a technique similar to that for preparing blood films. Although this method of preparation preserves the cell morphology well, it is inadequate for the evaluation of the cells in relationship to each other and for the estimation of marrow cellularity.

Smears of marrow particles are prepared by pouring a small amount of the aspirate on a glass slide. The marrow tissue is seen as gray particles floating in blood and fat droplets. The particles are aspirated selectively with a plastic dropper or Oxford pipette and transferred to a clean glass slide,

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 48 which is covered gently with another slide. The two slides are pulled in opposite and parallel directions to smear the particles without crushing the cells. Some people recommend an alternate technique using two cover glasses. In this process, the marrow particles are squashed between two cover slips, which are then gently pulled apart.

Techniques for preparing particle smears vary from person to person and from laboratory to laboratory. The aspirate may be transferred into a watch glass and the particles collected with a capillary pipette or the broken end of a wooden stick applicator. With experience, one usually adapts a technique that facilitates production of high-quality slides. The clinical laboratory scientist should prepare an adequate number of slides of smeared marrow particles. In cases of newly diagnosed acute leukemia, no fewer than 10 slides should be prepared. These are needed for histochemical stains such as myeloperoxidase, Sudan black B, naphthyl AS-D chloroacetate esterase, alpha-naphthyl butyrate esterase, and others.

Marrow particle smears are used in the evaluation of cellularity (usually marrow biopsy is ideal) and the relationship of the cells to each other. Well- prepared smears have the added advantage of excellent cell morphology, allowing subtle changes in cell maturation and cytoplasmic inclusions to be recognized easily. All direct and particle smears should be labeled at the bedside with the patient's name, identification number, and the date and then air-dried.

Histologic Marrow Particle Preparation

The leftover marrow particles obtained during the aspiration procedure can be processed for histologic examination. The tissue particles, admixed with blood, may be left to clot, then fixed in 10% buffered formalin and

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 49 processed for histologic sectioning. However, better results are obtained if the blood and particles are transferred to an EDTA anticoagulant–containing tube before clotting sets in. The blood and particles are then filtered through histo-wrap filter paper, and the concentrated particles enfolded in the paper are fixed in 10% buffered formalin. In the histology laboratory, these particles are collected through scraping the paper, and then embedding the particles in paraffin for further processing.

Bone Marrow Core Biopsy

A bone marrow core biopsy is especially indicated when the marrow cannot be aspirated or is a dry tap, owing to pathologic alterations encountered in acute leukemias, myelofibrosis, hairy cell leukemia, and other disorders. A trephine bone marrow core biopsy is also performed for the diagnosis of neoplastic and granulomatous diseases. In multiple myeloma and for staging of lymphomas or solid tumors, bilateral posterior superior iliac crest biopsies are recommended, as increased sampling size enhances the likelihood of capturing a focal process. An adequate biopsy sample is at least 15 mm in length.

When a bone marrow biopsy is performed in conjunction with a marrow aspiration, customarily the biopsy sample is obtained after the aspirate. This sequence is usually achieved by changing the direction of the needle to avoid the aspiration artifact. However, this technique may result in an aspiration artifact with hemorrhage into the area of the biopsy site, leading to difficulties in evaluating cellularity and morphology. Therefore, a core biopsy sample should be obtained before the aspiration or the marrow biopsy procedure is to be performed through a new puncture site in the anesthetized area.

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 50 In some cases when flow cytometry is indicated, and the aspirate is difficult to obtain (i.e., in patients with hypercellular marrow, hairy cell leukemia, or marrow fibrosis) an additional core marrow biopsy may be obtained in normal saline or RPMI (Roswell Park Memorial Institute) solution. Adequate cell suspensions can then be made from this biopsy sample and processed for flow cytometric studies.

Preparation of Trephine Biopsy

Touch Preparation:

The bone marrow core biopsy sample is supported lightly without pressure between the blades of forceps and touched several times on two or three clean slide surfaces. The biopsy core sample should not be rubbed on the slide, because rubbing destroys the cells. The slides are air-dried. The touch preparations are fixed in absolute methanol and stained with Wright-Giemsa stain.

In the absence of a good aspirate smear, the touch preparations may be the only source for studying cellular details and the maturation sequence of the bone marrow biopsy sample. For example, in a case of hairy cell leukemia, dry taps are common and cellular morphology by Wright-Giemsa stained touch preparation may be a useful clue to the diagnosis and allow cytochemical staining procedures, such as tartrate-resistant acid phosphatase (TRAP). Sometimes the touch preparations contain enough cells to obtain differential counts and blast evaluation, and to perform histochemical studies.

Histologic Bone Marrow Biopsy Preparation:

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 51 The biopsy specimen is immersed without delay in B-5 or 10% buffered formalin fixative. Histology laboratories may have a choice of other preferred fixatives such as Zenker's solution, Carnoy's solution, and others. After fixation, the biopsy specimen undergoes standard histologic processing of decalcification, dehydration, embedding in paraffin blocks, sectioning of 2- to 3-µm thick sections, and histologic staining. The advantage of the bone marrow biopsy is that it represents a large sample of marrow and bone structures in their natural relationships.

A variety of different stains can be used to demonstrate marrow iron, reticulum, and collagen. However, because of decalcification, the core biopsy may not be a good method for studying marrow iron stores, as the processing leaches iron from the tissue, which may be underrepresented in the iron stain. Acid-fast organisms and fungi in granulomatous diseases may be detected quickly with specific stains, offering great advantages in diagnosing these infections. For instance, mycobacterial cultures may require weeks of incubation to show growth of organisms, whereas on tissue sections, the histologic and etiologic diagnosis may be made within 10 to 12 hours. When metastatic tumors and lymphomas are found in the bone marrow, immunohistochemical stains can be used on histologic sections to demonstrate specific tumor markers. Thus, a very precise diagnosis of the origin of a tumor can be made without elaborate, expensive, and invasive techniques.

A disadvantage of the bone marrow biopsy is that fine cellular details are lost in the processing; therefore, it is of little value in the diagnosis of myelodysplastic syndromes and subtyping of acute leukemias. In these situations, the Wright-Giemsa stained aspirates or core biopsy touch preparations may supply the missing morphologic details. Multiple touch

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 52 preparations also offer an opportunity for histochemical stains (myeloperoxidase, Sudan black B, naphthyl AS-D chloroacetate esterase, α- naphthyl butyrate esterase, etc.), which are essential in the classification of leukemias. Polymerase chain reaction (PCR) and FISH can be performed on paraffin-embedded, formalin-fixed marrow biopsy specimens. PCR technology may be used to evaluate B-cell or T-cell lymphomas, various leukemias, and minimal residual disease.

Trephine bone marrow biopsy specimens may be embedded in methyl methacrylate, a synthetic plastic medium, and sectioned into 1 to 2 µm thin sections without decalcification. The morphological quality of the cells is extremely well preserved, and a differential count can be done on hematoxylin–eosin (H&E) or Giemsa-stained slides. However, this technique requires specially trained personnel, equipment, and separate handling in the histology laboratory, which increases the cost of the procedure. The processing time of the tissue also increases, which may not be acceptable if rapid diagnoses are required. In addition, tissue embedded in plastic media, instead of paraffin, may not be suitable for immunohistochemical studies of bone marrow.

Bone Marrow Examination

The examination of the bone marrow aspirate smears should start at low magnification with a dry objective of 10×. Scanning the slide permits selection of a suitable area for examination and the differential count. Bare nuclei should be avoided; such nuclei result from destruction of the marrow cells by squashing or stripping of their cytoplasm by fibrin threads. An area is selected in which the cells are well spread, intact, and not diluted by sinusoidal blood.

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 53 When marrow particles are examined, such areas are found at the periphery of the particles. At this low magnification, marrow cellularity is also evaluated. The megakaryocytes are usually noted adjacent to a spicule, about five to ten per low-power field. Nonhematopoietic tumor cells infiltrating the bone marrow may also be seen at this magnification. These are usually larger than the granulocytic or erythropoietic precursors and are scattered in small groups and crowded clusters. Some show a glandular configuration.

After the initial scan, immersion oil is applied to the slide and the examination continues on high magnification (oil immersion objective 50× or 100×). The high magnification provides details of the nuclear and cytoplasmic maturation process. The iron in histiocytes is visualized as brown-blue granules. Cytoplasmic inclusions of a diagnostic nature can be seen in histiocytes and granulocytes. Differential counts of bone marrow are performed with the oil immersion objective.

Estimation of Bone Marrow Cellularity

Cellularity is reflected in the ratio of nucleated hematopoietic cells to fat cells. Bone marrow cellularity normally varies with age, and the estimated cellularity must be compared to age-related normal ranges. At birth the normal marrow cellularity is 100%. Thereafter, the cellularity gradually decreases. Overall marrow cellularity in adults is about 50% (± 10%). The general rule to estimate age-related normal ranges is 100 minus age ± 10. For example, the estimated normal marrow cellularity of a 40-year-old person would be 100–40 ± 10 (i.e., a range from 50% to 70%).

Cellularity may vary from area to area, and, therefore, estimated cellularity should represent the average percentage. If hypercellular (90%) and

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 54 hypocellular (10%) areas are seen, this finding should be mentioned descriptively in the report because in such cases an average cellularity may be difficult to estimate. The immediate subcortical region of adult bone marrow is usually hypocellular as compared with the deeper medullary area. Therefore, sections that contain predominantly subcortical bone are frequently suboptimal for assessing true marrow cellularity.

The bone marrow biopsy specimen is most reliable for assessment of cellularity, because it offers a large amount of tissue for evaluation. However, the evaluation of cellularity can also be done on well-prepared aspirate smears or marrow particles. The best area for examination of cellularity in smears is the area between two uncrushed particles. The ratio of cells to fat is evaluated at low magnification (objective 10×), so that larger areas are included in the field of observation. The empty spaces that result from the spreading of the cells but are not occupied by fat cells are disregarded and treated as an artifact.

The terms decreased or increased cellularity is used when fewer or more than the expected normal number of cells are found. Precise evaluation can be achieved with experience, and good reproducibility can be attained among several observers. The marrow cellularity can be expressed in percentages, but this is best done on histologic sections of biopsy specimens. Marrow cellularity has diagnostic value when it is related to the M:E ratio, which is calculated after a differential count is performed.

It is always important to look for any abnormal changes in the bony trabeculae. Various conditions can alter the morphological appearance of these trabeculae. Marked thickening of trabeculae can be seen in myeloproliferative disorders (myelofibrosis with myeloid metaplasia),

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 55 whereas thinning of trabeculae can be seen in older adults, in patients with acquired immunodeficiency syndrome (AIDS) or other cachectic conditions, and after chronic steroid administration. Various metabolic disorders can also alter the morphology of bony trabeculae. Examples include a mosaic pattern, seen in patients with Paget's disease, and resorption and cyst formation, in persons with hyperparathyroidism and chronic renal failure.

When lymphoid aggregates are seen in the bone marrow biopsy specimen, the differential diagnosis includes benign lymphoid aggregates and malignant lymphoma. Usually benign aggregates are small and well demarcated, nonparatrabecular, and composed predominantly of small, round lymphocytes with plasma cells at the periphery and blood vessels present within the aggregate. Conversely, malignant follicles are usually large with ill-defined borders, paratrabecular, composed of atypical lymphocytes, and lack plasma cells at the periphery. However, a neoplastic lymphoid infiltrate can also be interstitial, diffuse and patchy. In some cases, immunohistochemical stains can be performed on the marrow core biopsy to differentiate between benign lymphoid aggregates and malignant lymphoma.

Bone marrow fibrosis may be found in patients with hairy cell leukemia, in myeloproliferative and myelodysplastic syndromes, sometimes in acute leukemia, after radiation, and after toxic injury to the marrow. On routine H&E sections, streaming of marrow stroma and dilated sinusoids suggests marrow fibrosis; this finding can be confirmed and graded by performing reticulin and trichrome stains. Normally, occasional reticulin-positive fibers may be seen around the blood vessels. The fibrosis can be graded as mild, moderate, or severe. In addition, a description of the fibrosis as fine or coarse, and focal or diffuse, may be helpful, especially when following the

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 56 improvement of a patient with myelofibrosis after treatment or bone marrow transplantation.

Trichrome stain is usually performed to detect any collagenous fibrosis, which if present may indicate irreversible fibrosis. Fibrosis and other bone marrow conditions such as marked hypercellularity in acute leukemias may result in a dry tap when attempting the aspiration procedure. However, if flow cytometry is needed, it is a good practice to obtain two bone marrow biopsy samples. One biopsy sample can be put in formalin and processed for morphological examination. The other core biopsy sample can be put in saline or RPMI medium; a cell suspension is then made for flow cytometric studies.

Bone Marrow Differential Count

A bone marrow differential count is an excellent tool for training a novice in bone marrow morphology and is widely used in diagnosing and following up patients with leukemias, refractory anemias, and myelodysplastic and myeloproliferative syndromes. Because of the compartmentalization of the hematopoietic cells and high cellularity of marrow, at least 500 to 1000 nucleated cells need to be classified for a representative differential count.

In infants during the first month after birth, dramatic alterations occur in the distribution of the different marrow compartments. At birth there is a predominance of granulocyte precursors, which switches within a month to a predominance of lymphoid elements. In early infancy many lymphocytes have fine chromatin and a high nuclear-to-cytoplasmic ratio, and lack

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 57 distinct nucleoli. They are called hematogones and represent normal lymphoid progenitor cells. Hematogones may be misinterpreted as blasts if the observer is unfamiliar with these characteristics. In children up to 3 years old, one-third or more of the marrow cellularity is made up of lymphocytes. The lymphocyte number gradually declines to the normal adult level thereafter.

In adult marrow the lymphocytes are distributed randomly among the hematopoietic cells and within lymphoid follicles. This can introduce significant variation in the differential count from sample to sample in the same patient. The majority of adult marrow is composed of granulopoietic and erythropoietic precursors. For the purpose of the differential count, these are enumerated into different categories according to their stage of maturation. When adequate numbers of cells are tabulated, the percentage of each category is calculated. The ratio between all granulocytes and their precursors and all nucleated red cell precursors represents the M:E ratio. Some hematologists prefer to exclude the segmented neutrophils from the differential count as being part of the neutrophil storage pool of the marrow. The normal M:E ratio in this case is between 1.5 and 3. However, pathologists and hematologists who interpret the bone marrow histologic sections of particle clot and biopsies in conjunction with marrow smears include the segmented neutrophils in the differential counts, because these cannot be excluded in the evaluation of histologic specimens and are part of the marrow cellularity. The normal M:E ratio then is slightly higher and ranges between 2 and 4. The granulopoietic tissue occupies two to four times greater marrow space than the erythropoietic precursors, owing to the shorter survival of the granulocytes in the circulation (i.e., neutrophils, 6 to 10 hours, versus erythrocytes, 120 days). Changes in the survival of granulocytes and erythrocytes are reflected in changes in the M:E ratio.

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Megakaryocytes (large bone marrow cells necessary for normal blood clotting) are not included in the differential count. Megakaryocytes are unevenly distributed, and a differential count is a poor means for their evaluation. Usually five to ten megakaryocytes are seen per microscopic field at low magnification (objective 10×). When clusters of megakaryocytes and promegakaryocytes are seen in every field, it is an indication of megakaryocytic hyperplasia. In a normocellular marrow, finding fewer than two megakaryocytes per field on screening may indicate megakaryocytic hypoplasia. A marked increase or decrease in the number of megakaryocytes is easy to evaluate, whereas slight to moderate changes are difficult to judge and are better estimated on histologic sections of biopsy and particle specimens.

Bone Marrow and Peripheral Blood Interpretation Based on Cellularity and M:E Ratio Changes

A bone marrow aspirate or biopsy sample represents a minute part of a very large and dynamic organ. Its activity and responses are reflected in blood changes; therefore, evaluation of the bone marrow should always be done in conjunction with evaluation of the peripheral blood. In adults with 50% marrow cellularity, about 30% to 40% represents granulopoiesis and 10% to 15% erythropoiesis, with an average M:E ratio of 4:1. An increase or a decrease in marrow cellularity with the normal M:E ratio usually indicates a balanced granulocytic and erythrocytic hyperplasia or hypoplasia, respectively. However, if cellularity changes occur simultaneously with the M:E ratio change, the interpretation requires a broader understanding of hematopoietic tissue physiology and its reactions during disease.

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 59 Cell morphology and the M:E ratio are well represented in random bone marrow specimens. The variations are not significant even when samples are compared from sternal and iliac crest aspirates. However, marrow cellularity is poorly represented in random smears; thus, this interpretation should be considered with some degree of reservation. Even large biopsy specimens may have a great degree of variation in cellularity. For these reasons, in diseases in which marrow cellularity is crucial for the diagnosis (aplastic anemia, marrow hypoplasia), more than one bone core biopsy may be required.

Bone Marrow Iron Stores

The storage iron of the bone marrow is in the form of hemosiderin. The iron content of hemosiderin is higher than that of ferritin. Other components of hemosiderin are protein, ferritin aggregates, some lipids, and membranes of cellular organelles. Hemosiderin can be seen on unstained smears as golden- yellow granules. On Wright–Giemsa-stained smears it appears as brownish- blue granules. However, for more precise evaluation, Prussian blue reaction is used to demonstrate the intracytoplasmic iron of histiocytes and red cell precursors.

The evaluation of marrow iron stores is essential in the diagnosis of anemias and especially in refractory and dyserythropoietic anemias. When the morphological characteristic of the iron particles in the storage nutrient histiocyte and erythroblastic precursors is an important diagnostic consideration (i.e., in sideroblastic anemias), an iron stain is performed on a particle smear. If the overall distribution of the amount of iron is of clinical importance (i.e., iron-deficiency anemia, anemia of chronic diseases, hemochromatosis, and others), then histologic sections of bone marrow biopsy sample, and/or marrow aspirate, and marrow clotted particles are

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 60 stained for iron. The biopsy sample and the particles are a more reliable source of information, because they represent a large sample of hematopoietic tissue. The EDTA chelating method, which does not affect the storage iron, should decalcify bone marrow biopsy samples for iron studies. Rapid-acid decalcifying solutions extract iron and must not be used in these cases.

Bone Marrow Report

The bone marrow report must contain all relevant information and optimally is composed of two components: clinical information and morphologic interpretation. The clinician should provide the patient’s biographic data, clinical differential diagnosis, and relevant therapeutic information. The morphology and interpretation by the pathologist and laboratory professional must include site of sampling (i.e., sternum), types of sample obtained, differential counts from both peripheral blood and bone marrow, and morphologic abnormalities in any cell lineages in the patient’s peripheral blood or bone marrow.

The results must be interpreted in conjunction with any additional studies (special stains, flow cytometry, cytogenetics, molecular studies). If the additional studies are significant in establishing the diagnosis but are unavailable when the bone marrow report is written, an addendum report should mention the significance of these studies. The comparison of the current marrow specimen to the previous tissue samples (marrow or other nonmarrow biopsies) is essential in some situations.

Finally, the pathologist should render a diagnostic interpretation within a reasonable period of time. For example, if 50% blasts were present in the blood or the bone marrow, it is reasonable to call the clinician about the

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 61 preliminary diagnosis of acute leukemia. However, the final diagnosis can be made only after the special stains and flow cytometry studies have been completed. Comments, if needed, should be concise and relevant to the case and can include a recommendation for additional tests and a possible differential diagnosis.

The bone marrow report usually encompasses the following:

1. The name of the laboratory or physician's office from which the report originates. 2. The patient's data, including age and relevant clinical summary or clinical diagnosis. 3. A description of material received for studies, such as smears of aspirate, marrow particles, and bone biopsy (or biopsies). 4. Data from the complete blood count (CBC) and WBC differential count, and a description of the blood smear, preferably from the day on which the bone marrow specimen is obtained. A platelet count should be included, as well as a reticulocyte count, if available. 5. The bone marrow differential count. 6. A description of cellularity, M:E ratio, granulopoiesis, erythropoiesis, and megakaryocytopoiesis. Any change in the nonhematopoietic elements of marrow, such as hemophagocytosis, granulomas, microorganisms, metastatic tumor cells, histiocytic hyperplasia, or the appearance of bony trabeculae, is included in this section of the report. The status of iron stores and special staining procedures performed are reported. 7. A description of histologic sections of marrow particles or bone marrow biopsy. 8. The diagnostic conclusion. This should encompass separate diagnoses of blood and bone marrow even where the same diagnosis is

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 62 applicable to both. For example, 1) blood — pancytopenia, and bone marrow, left posterior iliac spine aspirate and biopsy — myelodysplastic syndrome, refractory anemia with ringed sideroblasts; or 2) blood — acute myeloid leukemia minimally differentiated (WHO classification), and bone marrow, left posterior iliac crest aspirate and biopsy — acute myeloid leukemia minimally differentiated (WHO classification).

Bone marrow can provide a representative picture of disease processes and has wide application in clinical medicine. Marrow examination has a significant role in the evaluation of leukemias, lymphomas, plasma cell disorders, myeloproliferative disorders, myelodysplastic disorders, myelofibrosis, metastatic tumors, various anemias, granulomatous diseases, infectious diseases, metabolic diseases, in evaluating the status of engraftment after bone marrow transplantation, and in assessing chemotherapy effects. The clinical laboratory scientist's contribution in this phase consists of preparing the optimum blood and bone marrow slides and performing the differential count. Examination of the blood and bone marrow, correlation with the clinical presentation, and diagnostic conclusions on each specimen are the responsibility of a physician who has adequate training and experience to integrate all of the available clinical and laboratory information in reaching the correct diagnosis.

Treatment and Management

The most important part of management is the prompt recognition that a problem exists. This is done by two mechanisms: firstly, vigilance for signs and symptoms that may indicate a blood disorder and, secondly, patient education about the warning symptoms that should alert them to the need

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 63 to urgently contact their medical provider or emergency services if a prompt provider appointment is not possible.

Treatment of blood dyscrasias requires specialist expertise. Any drugs suspected of being involved in the reaction should be discontinued immediately and short-term supportive treatments given to aid a potential spontaneous recovery. Such support can include blood and platelet products, antibiotic or antifungal agents and recombinant human hemopoietic growth factors. Aplastic anemia may require immunosuppressive therapy and bone marrow or stem cell transplant. Modern treatments for blood dyscrasias, such as granulocyte colony-stimulating factor (G-CSF), have significantly reduced mortality rates. Hemolytic anemia normally recovers within two to three weeks of drug withdrawal, although corticosteroid therapy may be beneficial. The treatment for medication induced blood dyscrasias typically falls into one (or more) of three categories as reviewed below. This section covers the varied methods of treatment required depending on the type and severity of patient response to medication and disease outcomes, as well as the important of clinicians’ vigilance to be aware of and to educate their patients of the potential for drug-induced dyscrasia, methods of prevention and treatment.81,87

Removal of Drug

If a patient presents with one of the conditions discussed above, it is imperative that the drug causing the reaction be discontinued immediately. In many instances, this will require the provider to identify and initiate alternate treatments for the patient. In some rare instances, the drug may cause withdrawal symptoms in the patient. The provider will have to manage these effects.

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Symptomatic Support

In addition to eliminating the drug, it is often necessary for the provider to provide symptomatic support to the patient. All of the conditions above produce a number of symptoms, many of which are significant. Patients will require some level of treatment to reduce and/or eliminate the symptoms caused by the drugs and the associated conditions.

Immunosuppressive Therapy

In some instances, patients may require immunosuppressive therapy to mitigate the effects of the condition. Immunosuppressive therapy is used in instances when there is a risk that the patient’s immune system may interfere with treatment. This is most common when patients are receiving transfusions or donations of blood, marrow, or organs. Since the patient’s immune system may attack these foreign bodies, it is important to suppress the immune system, thereby allowing the treatment to take effect. The following table provides treatment information for each condition.

Aplastic Anemia Because of the high mortality rate associated with severe and very severe aplastic anemia, it is imperative that drug-induced aplastic anemia be diagnosed quickly and therapy initiated immediately. Treatment should be based on the severity of disease, with the goal of therapy being to improve peripheral blood counts, limit the requirement for transfusions, and minimize the risk for infections.

As with all cases of drug-induced hematologic disorders, the first step is to remove the suspected offending agent. Early withdrawal of the drug can allow for reversal of the aplastic anemia. Appropriate supportive care is also essential because the major causes of mortality in patients with aplastic anemia are infections (bacterial and fungal) and bleeding. Patients must receive transfusion support with erythrocytes and platelets, as well as appropriate antimicrobial prophylaxis or treatment during neutropenic periods.

Routine use of growth factors such as recombinant human nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 65 erythropoietin and granulocyte colony-stimulating factor has not been shown to improve outcome and are not recommended for the management of aplastic anemia. Current treatment guidelines for aplastic anemia recommend the use of prophylactic antibiotic and antifungal agents when neutrophil counts are below 500 cells/mm3 (0.5 × 109/L). If patients experience febrile neutropenia, broad-spectrum IV antibiotics should be started immediately.

Current guidelines do not recommend the use of prophylaxis for viruses or Pneumocystis jiroveci. For patients who have been heavily transfused, iron chelation therapy with agents such as deferoxamine or deferasirox may be necessary to avoid the serious consequences of iron overload.

The clinical course of aplastic anemia is variable. The condition can progress to severe or very severe disease in some patients, although it can remain relatively stable or even resolve in others. The treatment of moderate disease ranges from no clinical intervention to immunosuppressive regimens, and treatment should be based on the degree of cytopenias. For patients with disease requiring treatment, the two major treatment options for patients with drug-induced aplastic anemia are allogeneic HSCT and immunosuppressive therapy. Factors that determine which therapy would be preferred include age, disease severity, and availability of a human leukocyte antigen– (HLA-) matched sibling donor. For patients younger than the age of 40 years, the treatment of choice is allogeneic HSCT from an HLA-matched sibling donor. This treatment modality is associated with potential cure and results in a 5-year survival rate of 77% in adults and up to 90% in children. Unfortunately, most patients do not have a matched sibling donor. For young patients who do not have an available HLA-matched sibling, allogeneic HSCT from an unrelated donor may be considered but is usually reserved for those who fail to respond to upfront immunosuppressive therapy. When used in this setting, the 5- year overall survival rate in these patients has improved to over 50%, primarily because of improvements in HLA typing and unrelated donor selection.

For patients older than the age of 40 years and for those who are not candidates for allogeneic HSCT, the preferred first-line therapy is immunosuppressive therapy. Allogeneic HSCT in older patients is associated with significantly higher transplant-related morbidity and mortality. The highest mortality rate was seen in older patients and those with poorer clinical status at the time of transplantation. Complications of allogeneic HSCT, such as graft- versus-host disease and graft rejection, require all patients to be closely monitored for an extended period of time.

The current standard immunosuppressive regimen for the treatment of acquired aplastic anemia is combination therapy with antithymocyte globulin (ATG) and cyclosporine. This combination nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 66 has been reported to achieve 5-year survival rates between 75% and 85%, but the response rates in older patients are lower. ATG is composed of polyclonal immunoglobulin G (IgG) against human T lymphocytes derived from either horses or rabbits, and it has been a standard component of immunosuppressive therapy for aplastic anemia for many years. In a study comparing the horse versus rabbit product, both given in combination with cyclosporine, treatment with the horse-derived

Antithymocyte globulin product resulted in significantly higher response rates (68% vs. 37%) and 3-year overall survival rates (96% vs. 76%). Although the mechanism for this difference is not completely understood, the greater depletion of CD4+ cells associated with the rabbit ATG as compared with horse ATG may be associated with adverse outcomes. Based on these results, treatment with the horse-derived ATG product is preferred for treatment.

Because response to immunosuppressive therapy is often delayed (3–4 months), patients require continued supportive care until recovery. Patients should be monitored for adverse effects, including serum sickness, which can occur about 1 week after ATG begins.

Cyclosporine plays an important role in immunosuppressive therapy for aplastic anemia. Although cyclosporine monotherapy has been used to treat moderate aplastic anemia, it is more often used in combination with ATG. The addition of cyclosporine to ATG therapy has been shown to increase response rate, improve failure-free survival, and reduce the number of immunosuppressive courses needed. Cyclosporine inhibits interleukin-2 production and release and subsequent activation of resting T cells.

Cyclosporine dosing has varied from 4 to 6 mg/kg per day to 10 to 12 mg/kg per day, with the most frequently reported initial dose of 5 mg/kg per day in two divided doses. Cyclosporine doses are titrated to a target blood concentration that can be patient and institution specific but are usually in the range of 150 to 250 mcg/L (125–208 nmol/L) for adult patients.

Increased relapse rates have been observed when tapering cyclosporine rapidly, and it is recommended that cyclosporine be continued for at least 12 months after response and then tapered slowly. Corticosteroids are added to ATG-based immunosuppression because of their ability to reduce adverse reactions associated with ATG administration. In an effort to improve outcomes, several other agents have also been investigated for treatment of aplastic anemia. The additive benefits of other immunosuppressive agents such as mycophenolate, cyclophosphamide, and sirolimus have been evaluated. However, they have not been shown to be superior to the combination of ATG and cyclosporine, and their place in nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 67 therapy is not defined.

Allogeneic HSCT has long been the established treatment of drug- induced aplastic anemia. Although current practices in allogeneic stem cell procurement generally favors the use of peripheral blood stem cell harvesting, recent experiences have suggested that for patients with aplastic anemia, stem cells sourced from bone marrow may be associated with better outcomes, because of the relative lack of T cells in a bone marrow product, which is thought to confer a decreased risk of graft-versus-host disease. Data to support this theory are largely from single-center experiences, and the benefit of one source has not been proven in well-designed trials. Until it is clearer whether one stem cell source is better than another, the choice of stem cell source should be largely based on donor availability and preference.

Agranulocytosis The primary treatment of drug-induced agranulocytosis is the removal of the offending drug. After discontinuation of the drug, most cases of neutropenia resolve over time, and only symptomatic treatment (i.e., antimicrobials for infection treatment and prophylaxis) and appropriate vigilant hygiene practices are necessary.

Sargramostim (granulocyte-macrophage colony-stimulating factor [GM-CSF]) and filgrastim (granulocyte colony-stimulating factor [G-CSF]) have been shown to shorten the duration of neutropenia, length of antibiotic therapy, and hospital length of stay. Although the use of both agents has been reported in the literature, a commonly reported regimen is G-CSF 300 mcg/day via subcutaneous injection.

The only prospective, randomized trial to date did not confirm the benefit of these growth factors. However, some experts have questioned the validity of these results based on the small sample size (n = 24) and the lower than standard dose of filgrastim used (i.e., 100–200 mcg/day). One systematic review found that patients with a neutrophil nadir less than 100 cells/mm3 (0.1 × 109/L) had a higher rate of infections and fatal complications than those with a higher nadir. Therefore, most clinicians recommend the use of growth factors in patients with a neutrophil nadir less than 100 cells/mm3 (0.1 × 109/L), regardless of the presence of infection.

Hemolytic Anemia Drug-Induced Immune Hemolytic Anemia The severity of drug-induced immune hemolytic anemia depends on the rate of hemolysis. Hemolytic anemia caused by drugs through the hapten or adsorption and autoimmune mechanisms tends to be slower in onset and mild to moderate in severity.

Conversely, hemolysis prompted through the immune complex mechanism (innocent bystander) phenomenon can have a sudden

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 68 onset, lead to severe hemolysis, and result in renal failure.

The treatment of drug-induced immune hemolytic anemia includes the immediate removal of the offending agent and supportive care. In severe cases, glucocorticoids can be helpful, but their use outside of autoimmune hemolytic anemia is not supported by strong evidence. Other agents such as the chimeric anti-CD20 monoclonal antibody rituximab and IgG treatments have been used, but their role is yet to be clearly defined.

Drug-Induced Oxidative Hemolytic Anemia Removal of the offending drug is the primary treatment for drug- induced oxidative hemolytic anemia. No other therapy is usually necessary because most cases of drug-induced oxidative hemolytic anemia are mild in severity. Patients with these enzyme deficiencies should be advised to avoid medications capable of inducing the hemolysis.

Megaloblastic When drug-induced megaloblastic anemia is related to Anemia chemotherapy, no real therapeutic option is available, and the anemia becomes an accepted side effect of therapy. If drug- induced megaloblastic anemia results from cotrimoxazole, a trial course of folinic acid, 5 to 10 mg up to four times a day, can correct the anemia. Folic acid supplementation of 1 mg every day often corrects the drug-induced megaloblastic anemia produced by either phenytoin or phenobarbital, but some clinicians suggest that folic acid supplementation can decrease the effectiveness of the antiepileptic medications.

Thrombocytopenia The primary treatment of drug-induced thrombocytopenia is removal of the offending drug and symptomatic treatment of the patient. The use of corticosteroid therapy in the treatment of drug-induced thrombocytopenia is controversial, although some authors recommend it in severe symptomatic cases. Corticosteroids are sometimes helpful when clinicians are initially trying to distinguish between drug-induced thrombocytopenia and idiopathic thrombocytopenic purpura (ITP).

In the case of HIT, the main goal of management is to reduce the risk of thrombosis or thrombosis-associated complications in patients who have already developed a clot. All forms of heparin must be discontinued, including heparin flushes, and alternative anticoagulation must begin immediately. The direct thrombin inhibitors are the alternative anticoagulants most commonly used in current practice. Three direct thrombin inhibitors are currently available: lepirudin, argatroban, and bivalirudin.

Lepirudin, the first drug that was approved for the treatment of HIT, is a recombinant analogue of hirudin, a natural anticoagulant found in leeches. Lepirudin is renally eliminated and requires dosage adjustment in those patients with kidney dysfunction.

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It is also important to note that antibodies to lepirudin develop in about 30% of patients who receive this agent for the first time, and it is therefore recommended that patients receive only one course of lepirudin.

Argatroban is another IV thrombin inhibitor indicated for the management of HIT. But unlike lepirudin, argatroban is metabolized in the liver and can be used in patients with end- stage renal disease. However, dosage adjustment is needed for patients with significant hepatic impairment. The most recently approved direct thrombin inhibitor is bivalirudin. It is similar to lepirudin in that is a parenteral bivalent analogue of hirudin. It requires dosage adjustment only in severe renal failure. Fondaparinux, an anticoagulant pentasaccharide that inhibits factor Xa, has been proposed by some as a potential treatment for HIT because it does not appear to cause in vitro cross- reactivity with HIT antibodies.

Clinical data, however, to support the use of fondaparinux in the treatment of HIT-induced thrombosis are lacking. The most recent guidelines by the American College of Chest Physicians suggest that fondaparinux is most appropriately used in patients at relatively low risk of having HIT but for whom the use of either UFH or LMWH is not desired. These agents should also be considered for the treatment of patients who have acute HIT without thrombosis because of the increased risk of thrombosis occurring in these patients. Because of the increased risk of venous limb gangrene, warfarin should not be used alone to treat acute HIT complicated by deep vein thrombosis.

Drug induced thrombocytopenia, in most cases, resolves quickly after removal of the offending agent. In some cases, however, thrombocytopenia can persist for weeks or months, especially in the case of chemotherapy-induced thrombocytopenia or thrombocytopenia caused by immune mechanisms. In this setting, limited options are available to maintain platelets in a safe range while awaiting count recovery.

Historically, transfusions were used to maintain platelet counts until bone marrow recovery. The emergence of thrombopoietin analogs such as eltrombopag and romiplostim has raised the question of using drug therapy to treat drug-induced thrombocytopenia.

Current indications for these agents are limited to ITP, but preliminary data suggest a potential benefit in patients with prolonged drug-induced thrombocytopenia. Currently, this treatment cannot be recommended routinely, but future studies can help to elucidate if there is a role for these agents in the management if drug-induced thrombocytopenia.

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Reporting A Drug-Induced Blood Dyscrasia

Because of the seriousness of drug-induced hematologic disorders, it is necessary to track the development of these disorders to predict their occurrence and to estimate their incidence. Reporting during post-marketing surveillance of a drug is the most common method of establishing the incidence of adverse drug reactions. This section briefly reviews aspects of reporting an adverse drug reaction, which is an extensive are of health research and patient care protocols guiding medical treatment and intended to improve patient safety, which learners are encouraged to further investigate and pursue with their health agency of employment should an adverse medication event occur.1,7,36,85-87 The MedWatch program supported by the Food and Drug Administration is one such program. Many facilities have similar drug-reporting programs to follow adverse drug reaction trends and to determine whether an association between a drug and an adverse drug reaction is causal or coincidental. In the case of drug-induced hematologic disorders, these programs can enable practitioners to confirm that an adverse event is indeed the result of drug therapy rather than one of many other potential causes; general guidelines are readily available.

Because drug-induced blood disorders are potentially dangerous, rechallenging a patient with a suspected agent in an attempt to confirm a diagnosis is not recommended. In vitro studies with the offending agent and cells or plasma from the patient’s blood can be performed to determine causality. These methods are often expensive, however, and require facilities and expertise that are not generally available. Laboratory

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 71 confirmation of drug causation is not always necessary to warrant interruption or discontinuation of therapy. Therefore, it is extremely important that practitioners be able to clinically evaluate suspect drugs quickly and to interrupt therapy when necessary.

Throughout the past decades, lists of drugs that have been associated with adverse events have been developed to help clinicians identify possible causes. Unfortunately, these lists are extremely extensive, including a large number of very commonly used drugs, making it difficult to determine the cause of any abnormality. Furthermore, the absence of a drug from such a list should not discourage the investigation and reporting of an agent associated with an adverse event. It is imperative that clinicians use a rational approach to determine causality and identify the agents associated with a reaction. The clinician should focus on the issue, perform a rigorous investigation, develop appropriate criteria, use objective criteria to grade the response, and complete a quantitative summary. A complete, thorough, and detailed drug and exposure history must be obtained from the patient in order to best determine any potential for drug causation. A systematic approach to evaluate the information available in the literature also helps the clinician focus and to intervene to treat the cause of the disorder.

Adverse Drug Reaction Probability Scale

A common tool used by clinicians to rate the likelihood of causality in adverse drug reaction (ADR) investigations is an ADR probability scale (algorithm). One such scale was developed and tested by Naranjo and colleagues. This tool provides a series of scored questions that lead an investigator to the likelihood that an ADR was caused by the suspected medication. Depending on the aggregate score, the causality is rated as

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 72 doubtful, possible, probable, or definite. The scale gives the most weight to the temporal relationship of the reaction with relation to administration of the drug, observations after a rechallenge of the suspected medication, and alternate explanations for the ADR. As mentioned earlier, it is often unethical to rechallenge patients who experience severe hematologic toxicities. Thus, without a rechallenge, it is difficult to achieve a definite causality rating with such an algorithm.

In determining the likelihood that an observed reaction is caused by a particular medication, clinicians should review the medical literature for past reports supporting the observation. Using an evidence-based approach, the investigator assigns greater weight to prospective study designs such as clinical trials or cohort studies than to case reports or expert opinion. This provides a framework for the investigator’s confidence in published literature describing adverse drug reactions.

Summary

Drug-induced thrombocytopenia is the most common drug-induced hematologic disorders. Although drug-induced hematologic disorders are less common than other types of adverse reactions, they are associated with significant morbidity and mortality. Aplastic anemia has been identified as a leading cause of death followed by thrombocytopenia, agranulocytosis, and hemolytic anemia. Similar to most other adverse drug reactions, drug- induced hematologic disorders are more common in elderly adults than in the young; the risk of death also appears to be greater with increasing age.

Although many are idiosyncratic effects, some drugs with a well-known risk of blood dyscrasias are widely used due to a lack of alternative agents with similar effects. In the case of these, following specific monitoring advice may nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 73 help avert adverse effects, but specific advice to patients on how to spot symptoms indicative of blood dyscrasias is important. In the case of new drugs, any suspected case of blood dyscrasias should be reported to regulatory bodies. It is imperative that medical professionals know and understand the risks that medications pose in order to recognize the symptoms of a medication-induced blood dyscrasia at early onset and take the necessary steps to rectify it. Rechallenge with a drug suspected of causing toxicity is usually not advisable.

For some drugs, such as heparin, quinidine, and vancomyin, in vitro testing has been performed and mechanisms for cytopenias elucidated. However, such testing is not always possible given that for most there are no standardized, commercially available assays and that reactions may be related to metabolites as opposed to more easily tested parent compounds.

Because of the seriousness of drug-induced hematologic disorders, it is necessary to track the development of these disorders to predict their occurrence and to estimate their incidence. Reporting during post-marketing surveillance of a drug is the most common method of establishing the incidence of adverse drug reactions. Drug-induced blood disorders are a rare adverse effect, whose deleterious effects can be mitigated by the vigilance of health professionals to promote patient education and prevention of an adverse drug reaction through close monitoring of potential risk factors, early recognition and intervention, and proper reporting.

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Please take time to help NurseCe4Less.com course planners evaluate the nursing knowledge needs met by completing the self-assessment of Knowledge Questions after reading the article, and providing feedback in the online course evaluation.

Completing the study questions is optional and is NOT a course requirement.

1. The complete blood count (CBC) is sometimes referred to as

a. the cellular components test (CCT). b. the peripheral blood count (PBC). c. the EDTA test. d. the three-phase test.

2. Microscopic evaluation of a blood smear is best when the slide is prepared

a. indefinitely if properly stored. b. within 8 hours of collection. c. within 3 hours of collection. d. up to 24 hours after collection.

3. True or False: Freezing of blood samples is essential to preserving the samples for a valid, complete blood count (CBC) test.

a. True b. False

4. The complete blood count (CBC) analyzes

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 75

a. concentration of leukocytes (white blood cells). b. volume of RBCs (red blood cells). c. weight of RBCs (red blood cells). d. All of the above

5. ______provides the best morphologic preservation of blood cells and prevents coagulation of the blood specimen.

a. Cold agglutination b. Dipotassium (K2) EDTA c. IgM antibodies d. Romanowsky stains

6. True or False: Cells that have ruptured are called smudge cells.

a. True b. False 7. The complete blood count (CBC) is a primary screening test that provides information regarding the cellular components of the blood as the components

a. circulate in the peripheral blood. b. circulate in the lymphatic system. c. form in the bone marrow. d. circulate through the liver.

8. Ethylenediaminetetraacetic acid (EDTA) allows a laboratory professional to generate multiple blood smears because

a. it is an anticoagulant. b. it protects against blood-borne pathogens. c. reflex testing is part of the initial CBC. d. most tests are manual.

9. A complete blood count (CBC) can also provide what are known as the RBC indices. which are used

a. to depict the volume of each red blood cell (RBC). b. to depict the total weight of each RBC. c. to measure the concentration of hemoglobin in RBCs. nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 76 d. All of the above

10. True or False: The laboratory professional first assesses the general appearance and distribution of WBCs, RBCs, and platelets using high power magnification.

a. True b. False

11. Because the MCV represents an average of erythrocyte volume, it is _____ reliable in describing the erythrocyte population when considerable variation in erythrocyte volume/size (anisocytosis) occurs.

a. not b. equally c. less d. more

12. The reticulocyte count reported as a percentage can appear increased because of either an increase in the number of reticulocytes in the circulation or a decrease in the number of total

a. leukocytes. b. RBCs. c. anticoagulants. d. stem cells.

13. The laboratory professional utilizes both the platelet count and the MPV to assess ______and pathologic conditions related to platelets.

a. hemostasis b. agglutination c. thrombopoiesis d. avitaminosis

14. Information such as red blood count agglutination appears as a ______blood smear.

a. pinkish, purple b. red c. clear nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 77 d. grainy

15. ______, having a bluish-colored smear, may be suspected during the macroscopic evaluation of a blood smear.

a. Hepatic damage b. Thrombopoiesis c. Cytopenia d. Multiple myeloma

16. The effect of cold agglutinins is overcome by keeping the blood

a. frozen. b. at room temperature. c. at 37°C d. at 2°C 17. The presence of IgM antibodies (cold agglutinins) directed against erythrocyte antigens, erythrocytes can agglutinate forming

a. irregular grapelike, clusters of varying sizes. b. rows that look like stacked coins. c. clusters similar to rouleaux. d. even cell distribution.

18. Rouleaux is an alignment of erythrocytes that occurs normally when blood is collected

a. and smeared onto a slide evenly. b. and allowed to stand in tubes. c. and then frozen immediately. d. and then suspended in saline.

19. True or False: On a well-made blood smear, the erythrocytes are evenly distributed and well separated on the feathered edge of the smear.

a. True b. False

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 78 20. An abnormality in erythrocyte arrangement that appears as rows that look like stacked coins is termed

a. irregular clusters. b. agglutination. c. rouleaux. d. satellitism.

21. The final task at low power magnification is to determine the ______the smear that will be used to perform the morphologic examination of cells.

a. platelet estimate in b. critical area of c. RBC (red blood cell) size, shape and color in d. RBC inclusions in

22. Low power magnification is usually identified using ______magnification.

a. 400x b. 100x c. 40x d. 20x

23. The platelets must be evaluated also using low power magnification, and in some cases, platelets can adhere to neutrophils, a phenomenon called

a. an irregular cluster. b. a morphologic abnormality. c. rouleaux. d. satellitism.

24. A WBC (white blood cell) differential is performed in which ______cells are observed to determine the relative number of leukocytes as a percentage.

a. random b. 100 c. 10 to 20 d. 10 nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 79

25. The ______is characterized by the proximity of RBCs to each other (the area of the smear in which very few RBCs overlap or touch and are generally distributed in a uniform manner).

a. peripheral blood smear b. area of pallor c. the “glass effect” d. critical area

26. To evaluate morphologic abnormalities including inclusions, the laboratory professional should review the slide with the

a. 40× objective. b. 50× objective (500x magnification). c. 100× objective (1000× magnification). d. 400x magnification.

27. On a Romanowsky-stained blood smear, the erythrocyte has a central area of pallor caused by the closeness between ______the membrane when the cell is flattened on a glass slide.

a. the rim and the area of pallor in b. the two concave portions of c. the biconcave shape and cell center within d. the rim and discocyte in

28. To evaluate erythrocyte size microscopically, the cells are compared with the ______of a normal small lymphocyte.

a. nucleus b. rim c. area of pallor d. size

29. True or False: Normocytic erythrocytes are about the same size as the lymphocyte nucleus.

a. True b. False

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 80 30. ______also called spur cells, are small spherical cells with irregular thorn like projections.

a. Drepanocytes b. Codocytes c. Acanthocytes d. Echinocyte

31. Which of the following denotes a nonspecific variation in the size of erythrocyte cells?

a. Poikilocytosis b. The “glass effect” c. Elliptocytosis d. Anisocytosis

32. Poikilocytosis is the general term used to describe a nonspecific variation in the ______of erythrocytes. a. biconcavity b. color c. size d. shape

33. Acanthocytes do not have

a. irregular surface projections. b. a central area of pallor. c. irregular thorn-like projections. d. All of the above

34. Drepanocytes are elongated, crescent-shaped erythrocytes with pointed ends that are also known as

a. elliptocytes. b. ovalocytes. c. sickle cells. d. cigar cells.

35. In the case of hypochromia, the mean cell hemoglobin concentration (MCHC) value will be nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 81

a. increased. b. decreased. c. unchanged. d. static.

36. Cabot Rings are reddish-violet erythrocytic inclusions usually occurring in the formation of a figure eight or oval ring that occur in

a. severe anemias. b. dyserythropoiesis. c. a., and b., above d. None of the above

37. True or False: The bone marrow is one of the body's largest organs, representing up to 6 percent of total body weight in adults.

a. True b. False

38. An artery entering the bone branches out toward the periphery to specialized vascular spaces called

a. colonies. b. cords. c. nodes. d. sinuses.

39. Bone marrow studies should be performed only when clearly indicated or whenever a beneficial diagnostic result for the patient is expected because

a. bone marrow examinations are risky. b. bone marrow studies are not reliable. c. bone marrow studies are painful to the patient. d. of the danger of infection.

40. A primary objective of a bone marrow examination is to assess the quantity and development of

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 82 a. nonhematologic disorders. b. hematopoietic cells. c. leukocytes. d. erythrocytes

Correct Answers:

1. b 11. c 21. b 31. d

2. c 12. b 22. a 32. d

3. b 13. c 23. d 33. b

4. d 14. d 24. b 34. c

5. b 15. d 25. d 35. b

6. a 16. c 26. c 36. c

7. a 17. a 27. b 37. a

8. a 18. b 28. a 38. d

9. d 19. a 29. a 39. c

10. b 20. c 30. c 40. b

References Section

nursece4less.com nursece4less.com nursece4less.com nursece4less.com nursece4less.com 83 The reference section of in-text citations include published works intended as helpful material for further reading. Unpublished works and personal communications are not included in this section, although may appear within the study text.

1. Weiss DJ. Drug-associated blood cell dyscrasias. Compend Contin Educ Vet. 2012;34(6):E2. 2. Haeck PC, Swanson J a, Schechter LS, Hall-Findlay EJ, McDevitt NB, Smotrich G a, et al. Evidence-based patient safety advisory: blood dyscrasias. Plast Reconstr Surg. 2009;124(4 Suppl):82S – 95S. 3. Uggla B, Nilsson TK. Whole blood viscosity in plasma cell dyscrasias. Clin Biochem. 2015;48(3):122–4. 4. McKenzie C. Antibiotic dosing in critical illness. J Antimicrob Chemother. 2011;66 Suppl 2:ii25–i31. 5. Goldman, Lee and Schaefer, Andrew I. (2016). Goldman-Cecil Medicine, 25th Edition. Elsevier Saunders, New York. 6. Wali R, Fadoo Z, Adil S, Naqvi MA. Aplastic anemia: clinicohaematological features, treatment and outcome analysis. J Coll Physicians Surg Pak. 2011;21(4):219–22. 7. Dolberg OJ, Levy Y. Idiopathic aplastic anemia: Diagnosis and classification. Autoimmunity Reviews. 2014. p. 569–73. 8. Rovó a, Tichelli a, Dufour C. Diagnosis of acquired aplastic anemia. 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