Wintrobe's Clinical Hematology 14e SC

When you have to be right

Linking past accomplishments to the present understanding of the science and clinical practice of hematology

Wintrobe’ Clinical Hematology 14e

By John P. Greer, MD, Fred Appelbaum, MD, Daniel A. Arber, MD, Angela Dispenzieri, MD, Todd A. Fehniger, MD/ PhD, Bertil Glader, MD, PhD, Alan F. List, MD, Robert T. Means, Jr., MD, George M. Rodgers, MD, PhD This extensive title, which combines scientific principles with up-to-date clinical procedures, has been thoroughly updated for the fourteenth edition. You’ll find in-depth material on the biology and pathophysiology of lymphomas, leukemias, platelet destruction, and other hematological disorders as well as the procedures for diagnosing and treating them. • Combines the biology and pathophysiology of hematology as well as the diagnosis and treatment of commonly encountered hematological disorders in one volume. • Access the latest knowledge in genetics and genomics • eBook features 300 online self-assessment questions for reviewing material and preparing for Board exams. • Packed with more than 1,500 tables and figures.

ISBN

9781496347428

Publication Month

December 2018

Edition Type

Fourteenth

Product Format Height x Width Number of Pages

Hardback

213 x 276 mm

2432 pages

Language

English

Visit www.wisepress.com for more information

The 14th edition continues Max Wintrobe’s commitment to linking past accomplishments to the present understanding of the science and clinical practice of hematology. There are many stories that are among the most interesting and successful in medicine, including megaloblastic anemia, sickle cell disease, hemophilia, transfusion medicine, anti-thrombotic therapy, and treatment for childhood acute lymphoblastic leukemia, chronic myeloid leukemia, acute promyelocytic leukemia, and Hodgkin lymphoma. We appreciate the expert authors who have provided state of the art chapters and point to future directions. Genetics and immunology are central to many of the topics. Hematopoietic cell transplantation, gene therapy, and immunotherapy are specialized therapies and have focused chapters, as well as discussions in other chapters about their use in improving survival for a variety of hematologic diseases.

There is an emphasis on four components of diagnosis: the morphology of the peripheral smear, bone marrow, lymph nodes, and other tissues; flow cytometry, cytogenetics, and molecular markers and mutations. Therapeutic principles are discussed based on pathogenesis and an accurate diagnosis. The book is divided into eight parts: 1) Laboratory Hematology; 2) The Normal Hematopoietic System; 3) Transfusion Medicine; 4) Disorders of Erythrocytes; 5) Hemostasis and Coagulation; 6) Disorders of Leukocytes, the Spleen, and Immunoglobulins; 7) Hematologic Malignancies; and 8) Hematopoietic Cell Transplantation. There is an emphasis on four components of diagnosis: the morphology of the peripheral smear, bone marrow, lymph nodes, and other tissues; flow cytometry, cytogenetics, and molecular markers and mutations. Therapeutic principles are discussed based on pathogenesis and an accurate diagnosis.

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Who will benefit from this book The audience for Wintrobe’s Clinical Hematology encompasses the entire spectrum of health care providers, including medical students, nurses, residents, nurse practitioners, physician assistants, clinicians, and scientists.

Part 1 LABORATORY HEMATOLOGY

Section 1 Chapter 1  ■ 

Examination of the Blood and Bone Marrow

KRISTI J. SMOCK

INTRODUCTION

Thus, data such as patient age, gender, and time of specimen collection

as well as pertinent correlative clinical information should be noted.

Most often, blood is collected by venipuncture into vacuum collection tubes containing anticoagulant. 7 The three most commonly used antico-

Since the advent of microscopy several hundred years ago, there have been

continual advances in our ability to identify and quantify the components

agulants are tripotassium or trisodium salts of ethylenediaminetetraacetic

of blood and bone marrow. One important advance was the invention

acid (EDTA), trisodium citrate, and heparin. EDTA is the preferred anti-

of the Coulter counter in the 1950s, which allowed accurate automated

coagulant for blood counts because it produces complete anticoagulation

counting of large numbers of cells. In the present time, evaluation of blood

with minimal morphologic and physical effects on cells. Heparin causes

and bone marrow counts and morphology, along with important ancillary

a bluish colorati n of the background when a blood smear is stained

studies, are essential for accurate diagnosis of hematologic disorders and

with Wright-Giemsa, but does not affect cell size or shape. Heparin is

for monitoring disease progression and response to therapy. This chapter

often used for red cell testing and functional or immunologic analysis of

introduces the fundamental concepts and limitations that underlie labora-

leukocytes. Trisodium citrate is the preferred anticoagulant for platelet

tory evaluation of the blood and bone marrow and introduces additional

and coagulation studies. Anticoagulated blood may be stored at 4°C for

testing that may aid in evaluating hematologic disorders.

a 24-hour period without significantly altering cell counts or cellular morphology. 4 However, it is preferable to perform hematologic analysis

Blood elements include erythrocytes (red blood cells [RBCs]), leu-

kocytes (white blood cells [WBCs]), and platelets. RBCs are the most

Laboratory Hematology

as soon as possible after the blood is obtained.

numerous cells in the blood and are required for tissue respiration. RBCs

lack nuclei and contain hemoglobin (Hg), an iron-containing protein that

RELIABILITY OF TESTS

transports oxygen and carbon dioxide. WBCs include a variety of cell

types that have specific immune functions and characteristic morphologic

appearances. WBCs are nucleated and include neutrophils, lymphocytes,

In addition to proper acquisition of specimens, data reliability requires

monocytes, eosinophils, and basophils. Platelets are cytoplasmic fragments

accurate and precise testing methods. Both manual and automated testing

derived from bone marrow megakaryocytes that functio in hemostasis.

of hematologic specimens must be interpreted in light of expected test

Blood evaluation requires quantification of the cellular elements

accuracy and precision (reproducibility), particularly when evaluating

by either manual or automated methods. Automated methods are more

the significance of small changes. Accuracy is the difference between

commonly used, are more precise than manual procedures, and provide

the measured value and the true value, which implies that a true value is

additional data regarding cellular characteristics. Automated methods

known. Clearly, this may present difficulties when dealing with biologic

also require less technical time and minimize the possibility of human

specimens. The Clinical and Laboratory Standards Institute (CLSI),

error. However, the automated measurements describe average cellular

formerly the National Committee for Clinical Laboratory Standards, has

characteristics, but do not adequately describe the variability of individual

developed standards to assess the performance characteristics of auto- mated blood cell analyzers. 8 Automated instrumentation requires careful

values. For example, a bimodal population of small (microcytic) and

large (macrocytic) RBCs might be reported as average normal cell size.

calibration and regular quality control and quality assurance procedures

Therefore, a thorough blood examination also requires microscopic eval-

to reach expected performance goals for accuracy and reproducibility.

uation of a stained blood film to complement hematology analyzer data.

CELL COUNTS

SPECIMEN COLLECTION

As previously mentioned, cell counts are obtained manually or by au-

Proper specimen collection is essential for acquisition of accurate labo-

tomated hematology analyzers. Because blood contains large numbers

ratory data for hematologic specimens. Before a specimen is obtained,

of cells, sample dilution is required for accurate analysis. The type

careful thought as to what studies are needed will aid in optimal collec-

of diluent depends on the cell type to be enumerated. RBC counts

tion of samples. Communication with laboratory personnel is helpful

require dilution with an isotonic medium, whereas for WBC or platelet

in ensuring proper handling and test performance.

Copyright © 2019 Wolters Kluwer, Inc. Unauthorized reproduction of the content is prohibited. Anumber of preanalytical factors may affect hematologic measure- ments, and specimens should be collected in a standardized manner to reduce data variability. For example, patient activity, level of hydration, counts, a diluent that lyses the more numerous RBCs is used to simplify counting and avoid errors. The highest degree of precision occurs when a large number of cells are evaluated. Clearly, automated methods are

superior to manual methods for counting large numbers of cells and

medications, gender, age, race, smoking, and anxiety level may signifi- cantly affect hematologic parameters. 1-3 Similarly, the age and storage conditions of the specimen may affect the quality of the data collected. 4-6

minimizing statistical error. A recent comparison of five common he-

matology analyzers showed good between-instrument concordance for

1

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2 Part 1: Laboratory Hematology— SECTION 1

WBC

RBC

basic blood count parameters, but with less agreement for reticulocyte

counts, nucleated RBCs, and WBC differentials, indicating that manual review remains a valuable tool. 9

REL#

V O L U M E

Manual RBC, WBC, and platelet counts are performed using a mi-

croscope after dilution of the sample in a hemocytometer, a specially

50

100

200 300 f

constructed counting chamber that contains a specific blood volume. This

PLT

process is time consuming, requires a great deal of technical expertise,

and has largely been replaced by automated methods. There are a variety

REL#

of automated hematology analyzers available from manufacturers, such

as Abbott, Beckman Coulter, Siemens, Sysmex, Horiba, and others.

2 10

20

30

f

DF 1

Analyzer selection depends on the volume of samples to be tested and

the specific needs of the laboratory and ordering physicians. The ana-

lyzers range in price and workload capacity from those that would be

RBC 4.56 13.5 40.3 88.3 29.5 33.5 13.4 Hg Hct MCV MCH MCHC RDW PLT 202 8.2 MPV

ID# 1

WBC

6.7

appropriate for an individual physician’s office or point-of-care facility

#

ID# 2 Sequence #

%

to those needed in a busy high-volume reference laboratory.

NE LY MO EO BA

59.4 31.6 7.7 0.7 0.6

4.1 2.1 0.5 0.0 0.0

Automated hematology analyzers sample directly from phlebotomy

DATE:

06/21/96 08:55:45

tubes and use volumes as small as 150 µL for a full complete blood count (CBC) analysis. 9 They perform a variety of hematologic measurements in

Normal WBC Pop Normal RBC Pop Normal PLT Pop S TIME: Cass/Pos

addition to basic cell counting, such as Hb concentration, red cell size, and

leukocyte differentials. They may also perform more specialized testing,

such as reticulocyte and nucleated RBC counts, and flagging of blasts, left- shift, and variant lymphocytes. 9-11 Current analyzers utilize combinations

FIGURE 1.2 Histograms and printout generated by the Coulter automated he- matology analyzer utilizing light scatter and electrical impedance. BA, basophil;

of techniques to detect and differentiate specific cells types, including

electrical impedance, radiofrequency conductivity, laser light scattering,

flow cytometry, fluorescence detection, cytochemistry, and monoclonal antibodies ( Figures 1.1 and 1.2 ). 9,11 Using flow cytometric technologies,

EO, eosinophil; Hct, hematocrit; Hg, hemoglobin; LY, lymphocyte; MCH, mean

corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration;

some analyzers detect specific blood cell populations by antigen expression,

MCV, mean corpuscular volume; MO, monocyte; MPV, mean platelet volume;

NE, neutrophil; PLT, platelet; RBC, red blood cell; RDW, red cell distribution

such as detection of CD34-positive peripheral blood stem cells or leuke- mic blasts. 9,12-14 Integration of data from various sources of information

width; WBC, white bl od cell; DF1, differential; Rel, relative.

has improved the accuracy of the five-part differential and decreased the

numbers of unidentifiable cells requiring manual review for identification,

RED BLOOD CELL PARAMETERS

although analyzers do still frequently generate flags for abnormalities that require further investigation. 15-18 The International Consensus Group for Hematology Review has suggested criteria that should lead to manual review of a specimen after automated analysis and differential counting. 15

RBCs are defined by three quantitative values: the volume of packed red

cells or hematocrit (Hct), the amount of Hb, and the red cell number per

unit volume (RBC). Three additional indices describing average quali-

tative characteristics of the red cell population are also collected. These

Various Angles of Scattered Light

are mean corpuscular volume (MCV), mean corpuscular hemoglobin

(MCH), and mean corpuscular hemoglobin concentration (MCHC).

All of these values are routinely determined by hematology analyzers.

Volume of Packed Red Cells (Hematocrit)

The Hct is the proportion of the volume of a blood sample that is occu-

pied by red cells. Hct may be determined manually by centrifugation

of blood at a given speed and time in a standardized glass tube with a uniform bore, as was originally described by Wintrobe. 19 The height of

the column of red cells after centrifugation compared with total blood

Sample Stream

sample volume yields the Hct. Macromethods (using 3-mm test tubes)

Focused Laser Beam

with low-speed centrifugation or micromethods using capillary tubes

and high-speed centrifugation may be used.

Manual methods of measuring Hct are simple and accurate means

of assessing red cell status. They are easily performed with little

specialized equipment, allowing adaptation for situations in which

automated cell analysis is not readily available or for office use.

However, several sources of error are inherent in the technique. The

spun Hct measures the red cell volume, not red cell mass. Therefore,

patients in shock or with volume depletion may have normal or high

Sheath Stream

Hct measurements because of hemoconcentration despite a decreased

Sample Feed Nozzle

red cell mass. Technical sources of error in manual Hct determinations

usually arise from inappropriate concentrations of anticoagulants, poor mixing of samples, or insufficient centrifugation. 19 Another inherent

error in manual Hct determinations arises from trapping of plasma in

Copyright © 2019 Wolters Kluwer, Inc. Unauthorized reproduction of the content is prohibited. FIGURE 1.1 Optical flow cytometric technology used in automated hematology analyzers. A suspension of cells is passed through a flow chamber and focused the red cell column. This may account for 1% to 3% of the volume in microcapillary tube methods, with macrotube methods trapping relatively more plasma. 20,21 It should be noted that abnormal red cells

into a single cell sample stream. The cells pass through a chamber and interact

(eg, sickle cells, microcytic cells, macrocytic cells, or spherocytes)

with a laser light beam. The scatter of the laser light beam at different angles is

often trap higher volumes of plasma because of increased cellular rigidity, possibly accounting for up to 6% of the red cell volume. 21

recorded, generating signals that are converted to electronic information about

cell size, structure, internal structure, and granularity. (Adapted and redrawn from

Very high Hcts, as in polycythemia, may also have excess plasma

Cell-Dyn 3500 Operator’s Manual . Santa Clara, CA: Abbott Diagnostics; 1993.)

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Chapter 1: Examination of the Blood and Bone Marrow 3

Mean Corpuscular Hemoglobin MCH is a measure of the average Hb content per RBC. It may be calcu- lated manually or by automated methods using the following formula 19 :

trapping. Manual Hct methods have a coefficient of variation (CV) of approximately 2%. 20

Automated analyzers do not depend on centrifugation techniques to

determine Hct, but instead calculate Hct using direct measurements of red × mean red cell volume. Alternatively, some analyzers measure Hct directly by cell number and red cell volume as follows: Hct = red cell number

= hemoglobin (g/L)/red cell count (10 12 /L)

MCH

MCH is expressed in picograms (pg, or 10 −12

g). In anemias sec-

comparing the sum of all RBC size measurements to the volume of the

ondary to impaired Hb synthesis, such as iron deficiency anemia, Hb

specimen. Automated Hct values closely parallel manually obtained

mass per red cell decreases, resulting in a lower MCH value. MCH measurements may be falsely elevated by hyperlipidemia 30 because

measurements, and the manual Hct is used as the reference method for

hematology analyzers (with correction for the error induced by plasma

increased plasma turbidity will erroneously elevate Hb measurement.

trapping). Errors of automated Hct calculation are more common in patients with polycythemia 22 or abnormal plasma osmotic pressures. 23

The CV for automated analysis of MCH is 1% to 2% in most modern analyzers, compared with approximately 10% for manual methods. 20,25 Mean Corpuscular Hemoglobin Concentration

Manual methods of Hct determination may be preferable in these cases.

< 1.5%. 24,25

The CV of most automated Hcts is

Hemoglobin Concentration

The average concentration of Hb in a given red cell volume, or MCHC, may be calculated by the following formula 19 :

Hb is an intensely colored protein, allowing its measurement by spectro-

= hemoglobin (g/dL)/Hct (L/L)

photometric techniques. Hb is found in the blood in a variety of forms,

MCHC

including oxyhemoglobin, carboxyhemoglobin, methemoglobin, and other

The MCHC is expressed in grams of Hb per deciliter of packed

minor components. These may be converted to a single stable compound,

RBCs, representing the ratio of Hb mass and the volume of red cells.

cyanmethemoglobin, by mixing blood with Drabkin solution (contains potassium ferricyanide and potassium cyanide). 26,27 Sulfhemoglobin is not

With the exception of hereditary spherocytosis and some cases of

homozygous sickle cell or hemoglobin C disease, MCHC values will

converted, but is rarely present in significant amounts. The absorbance of

not exceed 37 g/dL. This level is close to the solubility value for Hb,

the cyanmethemoglobin is measured in a spectrophotometer at 540 nm

and further increases in Hb may lead to crystallization. The accuracy

to determine Hb. This technique is used both in manual determinations

of the MCHC determination is affected by factors that have an impact

and in most automated hematology analyzers, although cyanide-free

on measurement of either Hct (plasma trapping or the presence of

methods are used by some. Hb is reported in grams per deciliter (g/dL) of

abnormal red cells) or Hb (hyperlipidemia and leukocytosis), which is methodology specific. 28 The CV for MCHC for automated methods ranges between 1.0% and 1.5%. 24

whole blood. The main errors in measurement arise from dilution errors

or increased sample turbidity caused by improperly lysed red cells, leu- kocytosis, or increased levels of lipid or protein in the plasma. 28-30 Older

As noted earlier, the MCV, MCH, and MCHC reflect average values

analyzers reported spurious increases in Hb levels when white cell counts

× 10 9

and may not adequately describe blood samples when mixed popula-

exceeded 30

/L because of increased turbidity, but this is decreased

tions of red cells are present. For example, in sideroblastic anemias,

with newer flow systems so that Hb levels remain extremely accurate in the

× 10 9

/L. 24

a dimorphic red cell population of both microcytic hypochromic and

face of WBC counts as high as 100

With automated methods,

< 1% (CV). 24,25

normocytic normochromic cells may be present, yet the indices may

the precision for Hb determination is

Laboratory Hematology

be normochromic and normocytic. It is important to examine the blood

Red Cell Count

smear as well as instrument red cell histograms to detect such dimorphic populations. 15 The MCV is an extremely useful value in classification of anemias, 24,32,36 but the MCH and MCHC often do not add significant,

Manual methods for counting red cells have proven to be very inaccurate,

and automated counters provide a much more accurate reflection of red cell numbers. 31 Both erythrocytes and leukocytes are counted after whole blood dilution in an isotonic solution. Because the number of red cells (expressed as 10 12 cells/L) greatly exceeds the number of white cells (by

clinically relevant information.

Red Cell Distribution Width

The red cell distribution width (RDW) is a red cell measurement that

a factor of 500 or more), the error introduced by counting both cell types

quantitates cellular volume heterogeneity reflecting the range of red cell sizes within a sample. 37,38 RDW has been proposed to be useful in early

is negligible. However, when marked leukocytosis is present, red cell

counts and volume determinations may be erroneous unless corrected

classification of anemia because it becomes abnormal earlier in nutritional

for white cells. The observed precision for RBC counts using automated hematology analyzers is approximately 1% (CV) 24,25 compared with a minimum estimated value of 11% with manual methods. 31 Mean Corpuscular Volume

deficiency anemias than other red cell parameters, especially in cases of iron deficiency anemia. 39 RDW is particularly useful in characterizing

microcytic anemia, allowing discrimination between uncomplicated iron

deficiency anemia (high RDWand normal-to-lowMCV) and uncomplicated heterozygous thalassemia (normal RDWand lowMCV), 39-41 although other tests are usually required to confirm the diagnosis. 42 RDW is also useful in

The average volume of the RBC is a useful parameter that is used to

classify anemias and may provide insights into the pathophysiology of red cell disorders. 32 The MCV is measured in femtoliters (fL or 10 −15 L)

identifying red cell fragmentation, agglutination, or dimorphic cell popu-

lations (including patients who have had transfusions, have sideroblastic anemias, or have been recently treated for a nutritional deficiency). 39 Reticulocyte Counts

and is usually measured directly in automated analyzers by dividing the

sum of the individual RBC volumes by the RBC count but may also be calculated from the RBC count and the Hct using the following formula 19 :

= Hct (L/L)

× 1000/red cell count (10 12

MCV

/L)

Determination of the numbers of reticulocytes or immature, non-nucleated

The CV in most automated systems is approximately 1%, 24,25 com- pared to 10% for manual methods. 20 Agglutination of cells, as with cold

RBCs that still contain RNA provides useful information about the ca-

pacity of the bone marrow to synthesize and release red cells in response

Copyright © 2019 Wolters Kluwer, Inc. Unauthorized reproduction of the content is prohibited. agglutinin disease or paraproteinemia, may result in a falsely elevated MCV. 33 Most automated analyzers gate out MCV values above 360 fL, thereby excluding most red cell clumps, although this may falsely to anemia and helps to distinguish between decreased RBC production and enhanced peripheral destruction. Corrected reticulocyte counts or the reticulocyte production index (RPI) can be used to compare the

magnitude of reticulocytosis with the magnitude of anemia to determine

lower calculated Hct determinations. In addition, severe hyperglycemia > 600 mg/dL) may cause osmotic swelling of the red cells, leading to a falsely elevated MCV, which could also lead to a falsely high Hct and falsely decreased MCHC. 23,34,35 Leukocytosis may also spuriously elevate MCV values. 28 (glucose

whether the bone marrow response is adequate. In the past, reticulocyte

counts were performed manually using supravital staining with methylene

blue that stains precipitated RNA as a dark blue meshwork or granules (at least two per cell). 43 Normal values for reticulocytes in adults are

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4 Part 1: Laboratory Hematology— SECTION 1

ensure reproducibility of results between laboratories. It is important to

0.5% to 1.5%, although they may be 2.5% to 6.5% in newborns (falling

scan the entire blood smear at low power to ensure that all atypical cells

to adult levels by the second week of life). Because there are relatively

low numbers of reticulocytes, the CV for manual reticulocyte counting is relatively large (10%-20% or higher). 44-46

and cellular distribution patterns are recognized. In wedge-pushed smears,

leukocytes tend to aggregate in the feathered edge and side of the blood

To increase the accuracy of reticulocyte counting, automated detection

smear rather than in the center of the slide. Larger cells in particular (blasts and monocytes) tend to aggregate at the edges of the blood smear. 56 The

methods using fluorescent dyes that bind to RNA allow for many more

cells to be analyzed, thereby increasing the accuracy and precision of counts. 47,48 Most hematology analyzers offer automated reticulocyte counting and can report reticulocyte numbers with routine CBC parameters. CVs of 10% or less can be achieved using automated analyzers. 24,25,49

use of coverslip preparations and spinner systems tends to minimize this

artifact of cell distribution. For wedge-pushed smears, it is recommended

that a battlement pattern of smear scanning be used in which one counts

fields in one direction, then changes direction and counts an equal number of fields before changing direction again to minimize distributional errors. 55

Differences in reticulocyte counts obtained from different analyzers

have been observed, which are likely related to instrument-specific technologies. 9 Current instruments also have the capability to report

In manual leukocyte counts, three main sources of error are found:

distribution of cells on the slide, cell recognition errors, and statistical

sampling errors. Poor blood smear preparation and staining are major

novel reticulocyte parameters such as immature reticulocyte fraction

contributors to cell recognition and cell distribution errors. Statistical

(IRF) and reticulocyte cellular indices such as cell volume and Hb

content. The IRF quantitates younger reticulocytes identified by more

errors are the main source of error inherent in manual counts because of

intense staining with RNA stains. However, the clinical utility of these

the small sample size in counts of 100 or 200 cells. The CV in manual

novel parameters is still being investigated. Potential clinical uses for

counts is between 5% and 10% and is also highly dependent on the skill

IRF include as an indicator of early marrow recovery in bone marrow

of the technician performing the differential. Ac uracy may be improved

transplant, an indicator of response to treatment with erythropoietic stimulating agents, and as an alternative to the manually calculated RPI. 46 Nucleated Red Blood Cell Counts

by increasing the numbers of cells counted, but for practical purposes, most laboratories will do a differential on 100 white cells. 11,57

Automated leukocyte differentials markedly decrease the time and

cost of performing routine examinations as well as improving precision

with CVs of approximately 3% for normal neutrophil and lymphocyte counts. 25,57,58 However, automated analysis is incapable of accurately

Circulating nucleated red blood cells (NRBCs) are abnormal in adults

and are seen in conditions such as acute hemolysis and hypoxic stress,

identifying and classifying all types of cells and is particularly insensi-

reflecting an increase in marrow erythropoietic activity, and can also be

tive to abnormal or immature cells, especially in small numbers. There

seen with bone marrow involvement by hematologic or other malignan-

have been some improvements in the ability of instrument to identify immature granulocytes, including blasts. 11 However, a comparison of five

cies. NRBCs are also normally seen in newborns, particularly premature

newborns, and young infants. Modern hematology analyzers provide

analyzers demonstrated that samples containing blasts may be missed,

enumeration of circulating NRBCs, with results expressed as number of

in particular with low WBC counts, and that blasts may sometimes be

NRBCs per volume of blood and as a percentage per 100 WBCs. Auto-

misclassified as other cell types, such as variant lymphocytes. Instrument

mated counts have been historically challenging because these cells have

blast flags may also be generated in samples where circulating blasts are not subsequently confirmed by microscopy. 9 For these reasons, instrument flags for possible abnormal white cell populations indicate the need for examination by a skilled morphologist. 15,58

a size and nucleus similar to mature lymphocytes and misclassification

because lymphocytes can lead to errors in the total leukocyte count and

differential. Correction of WBC counts may be necessary in the presence

of high numbers of NRBCs. Although analyzers have become more

Hematology analyzers identify cells based on the combinations of

sophisticated in the identification of NRBCs, a study of five common

cellular size, cell complexity, and staining characteristics, allowing for

hematology analyzers demonstrated poor concordance of NRBC counts

generation of a five-part differential count that enumerates neutrophils, monocytes, lymphocytes, eosinophils, and basophils. 24 Most analyzers use

between instruments and also between automated and manual counts, likely representing differences in instrument technologies. 9

flow cytometric techniques where the cells are suspended in diluent and

passed through an optical flow cell in a continuous stream so that single cells

LEUKOCYTE ANALYSIS White Blood Cell Counts

are analyzed (Figure 1.1). The differential data are plotted as a histogram

(Figure 1.2), which displays and classifies cell populations based on their

characteristics. Lymphocytes are characterized as small unstained cells (no

myeloperoxidase staining). Atypical/reactive lymphocytes, some blasts,

circulating plasma cells, or other abnormal cells are larger than mature

Leukocytes (WBCs) may also be enumerated by either manual methods

lymphocytes with low internal complexity and no myeloperoxidase activity

or automated hematology analyzers. WBCs are counted after dilution of

and are classified as large unstained cells. Neutrophils have higher internal

blood in a diluent that lyses the RBCs (usually acid or detergent). The

complexity (because of segmented nucleus and granules) and appear as

much lower numbers of leukocytes present require less dilution of the

larger cells. Eosinophils appear smaller than neutrophils because they tend

blood than is needed for RBC counts. As with red cell counts, manual

to absorb some of their own light scatter. Monocytes have lower levels of

leukocyte counts have more inherent error, with CVs ranging from 6.5%

complexity, are usually found between neutrophils and lymphocytes, and can be challenging to accurately classify. 11 To enumerate basophils, which are

in cases with normal or increased white cell counts to 15% in cases with decreased white cell counts. 50 Automated methods characteristically yield CVs in the 1% to 3% range for normal or elevated counts but also with increased CVs (approximately 6%) for lowWBC counts. 24,25 Automated leukocyte counts may be falsely elevated, with inaccurate differentials, in the presence of cryoglobulins or cryofibrinogen, 51 giant platelets or platelet clumps, 52 and nucleated RBCs, or when there is incomplete lysis counts have also been reported because of granulocyte agglutination secondary to surface immunoglobulin interactions. 53,54 Leukocyte Differentials of red cells, possibly requiring manual counting. Falsely low neutrophil

few in number and lack specific staining characteristics, a basophil-nuclear

lobularity channel may be utilized. For this determination, RBCs andWBCs

are differentially lysed, leaving bare leukocyte nuclei, with the exception

of basophils, which are resistant to lysis, and can then be counted based

on relatively large cell size because of the retained cytoplasm. Analysis

using this technique examines thousands of cells per sample, increasing

statistical accuracy, although the accuracy of automated basophil counts is still recognized as a challenge for all analyzers. 11,24,59

Copyright © 2019 Wolters Kluwer, Inc. Unauthorized reproduction of the content is prohibited. Hematology analyzers may have settings that allow for evaluation of red cell and white cell populations in very hypocellular specimens, such as body fluids. Because higher numbers of cells are evaluated, the

WBCs are analyzed to find the relative percentage of each cell type in a

accuracy of cell counts and differential counting is improved over manual counting methods. 60-63 However, manual techniques are still commonly used for cerebrospinal fluid and body fluid specimens. 11

differential leukocyte count. This information can be used to determine

absolute counts for each cell type by multiplying the percentage by the

total WBC count. Uniform standards for performing manual differential leukocyte counts on blood smears have been proposed by the CLSI 55 to

Automated digital image analysis is now used by some hematology

analyzers. For instance, CellaVision has an automated image analyzer

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Chapter 1: Examination of the Blood and Bone Marrow 5

that captures digital images of cells in a stained smear and classifies them

larger platelet volumes (secondary to new platelet production) seen in

thrombocytopenic patients in whom platelets are decreased because of peripheral destruction (as in immune thrombocytopenia). 76-78 MPVmay

to provide a differential that includes mature and immature WBCs and other cells, such as variant lymphocytes and plasma cells. 59 The images are reviewed by trained technologists to further refine the classifications if needed. RBC and platelet counts and morphology can also be analyzed. 64

also be increased in myeloproliferative disorders. However, it should be

noted that platelets tend to swell during the first 2 hours in EDTAantico- agulant, shrinking again with longer storage. 79 Decreased MPV has been associated with megakaryocytic hypoplasia and cytotoxic drug therapy. 80

The systems have the capacity to store images and are useful in training

technologists as well as providing an easily accessible means, whereby

smears obtained at different times from a single patient may be compared morphologically. 65 These systems perform well in normal blood specimens

Other platelet parameters may also be reported, depending on the

analyzer. The immature platelet fraction, or reticulated platelets, rep-

but have limitations in their ability to identify morphologically abnormal

resents newly released platelets that retain residual RNA, analogous to red cell reticulocytes. 64 Reticulated platelet counts are determined

cells, so specimens with dysplastic changes, unusual morphologic vari-

ants, or significant artifacts may not be evaluable or may provide false data. 11,59,66-69 Often, these systems will designate a certain percentage of

using RNA staining dyes, give an estimate of thrombopoiesis, and

may be useful in distinguishing platelet destruction syndromes from hypoplastic platelet production in bone marrow failure conditions. 64,81

cells as unclassifiable, requiring review by a technologist for definitive

identification of the cell type and completion of the differential.

Normal values vary between 3% and 20%, and 2.5- to 4.5-fold increases

in reticulated platelet counts are seen in the clinical setting of immune thrombocytopenia. 82 Increased reticulated platelets may herald the return of platelet production after chemotherapy. 83

PLATELET ANALYSIS

Platelets are anucleate cytoplasmic fragments that are 2 to 4 µm in

ADVANTAGES AND SOURCES OF ERROR WITH AUTOMATED HEMATOLOGY

diameter. As with the other blood components, they may be counted

by either manual or automated methods. Manual methods involve

dilution of blood samples and enumeration in a counting chamber or

hemocytometer using phase-contrast microscopy. Sources of error are

similar to other manual counting techniques and include dilution errors

Clearly, the use of automated hematology analyzers has reduced labo-

and low numbers of events counted. The CV of manual methods, es-

ratory costs and turnaround time while also improving the accuracy and

> 15%. 70

pecially in patients with thrombocytopenia, may be

Platelets

reproducibility of blood counts. Thorough verification of hematology

are counted in automated hematology analyzers after removal of red

analyzers prior to clinical use and adequate technical and quality control procedures are essential. 8,25,84 Despite the high level of accuracy and

cells by sedimentation or centrifugation, or using whole blood. Platelets

are identified by light scatter, impedance characteristics, and/or platelet antigen or platelet-specific cytoplasmic staining. 24,64 These give reliable

precision, automated hematology analyzers may generate a warning flag

in 10% to 25% of samples, requiring manual examination of the blood smear. 15-17,24,85 Blood smear examination still plays an important role

platelet counts with a CV of approximately 3% in the normal range.

in characterizing these samples. In addition, some cell types are only

However, achieving accurate counts in patients with thrombocytopenia

remains a challenge, and CVs in thrombocytopenic samples are closer to 5%. 25 Falsely low platelet counts may be caused by the presence of large platelets, platelet clumps/agglutinins, 52 or adsorption of platelets to leukocytes. 71 Fragments of RBCs or WBCs may falsely elevate the automated platelet count, but this usually gives rise to an abnormal histogram that identifies the spurious result. 72,73 Automated hematology analyzers also determine mean platelet vol- ume (MPV), which has been correlated with several disease states. 74-76

identified morphologically, such as Sézary cells, and red cell morphology is best analyzed by direct smear examination. 36

Laboratory Hematology

Certain disease states are associated with spuriously high or low results

from analyzers, although some of these are specific to a particular type of

instrumentation (summarized in Table 1.1 ). Therefore, values obtained

from the automated hematology analyzer must be interpreted in the context

of clinical findings. As previously mentioned, careful examination of the

stained blood film often imparts additional information that may not be

reflected in the average values reported by the automated CBC.

In general, MPV has an inverse relationship with platelet count, with

Table 1.1 Disorders and Conditions That May Reduce the Accuracy of Blood Cell Counting

Component Disorder/Condition

Effect on Cell Count

Rationale

Red cells

Microcytosis or schistocytes

May underestimate RBC

Lower threshold of RBC counting

window is greater than microcyte size

Howell–Jolly bodies

May spuriously elevate platelet count

Howell–Jolly bodies are similar in size

(in whole blood platelet counters only)

to platelets

Polycythemia

May underestimate RBC

Increased coincidence counting

White cells

Leukocytosis

Overestimate RBC

Increased coincidence counting

Acute leukemia and chronic lymphocytic

May spuriously lower WBC

Increased fragility of leukocytes,

leukemia, viral infections

including immature forms

Chemotherapy of acute leukemia

May artifactually increase platelet count

Leukemic cell nuclear or cytoplasmic

fragments identified as platelets

Platelets

Platelet agglutinins

May underestimate platelet count, sometimes

Platelet clumping

with spurious increase in WBC

Aggregates may be identified as

leukocytes

Copyright © 2019 Wolters Kluwer, Inc. Unauthorized reproduction of the content is prohibited. Plasma Cold agglutinins May underestimate RBC with spurious macrocytosis Red cell doublets, triplets, and so forth have increased volume

Cryoglobulins, cryofibrinogens

Variation in platelet count

Protein precipitates may be identified as

platelets

Some of these examples affect counts only when certain instruments are used. The effects depend on methodology, dilution, solutions used, and specimen temperatures.

Abbreviations: RBC, red blood cell count; WBC, white blood cell count.

Adapted from Koepke JA. Laboratory Hematology . New York, NY: Churchill Livingstone; 1984.

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6 Part 1: Laboratory Hematology— SECTION 1 MORPHOLOGIC ANALYSIS OF BLOOD CELLS

Preparation of Blood Smears

Blood films may be prepared on either glass slides or coverslips. Each method has specific advantages and disadvantages. 86 Blood smears are

Careful evaluation of a well-prepared blood smear is an important part

of the evaluation of hematologic disease. Although a specific diagnosis

often prepared from samples of anticoagulated blood remaining after

may be suggested by the data obtained from an automated hematology

automated hematologic analysis. However, artifacts in cell appearance and staining may be induced by anticoagulant. 7 Optimal morphology and

analyzer, many diseases may have normal blood counts but abnormal

cellular morphology. Examples of abnormal red cells that may be seen

staining are obtained from non–anticoagulated blood, most often from a

in the peripheral blood smear examination and which are associated

fingerstick procedure. Mechanical dragging of the cells across the glass

with specific disease states are found in Table 1.2 . Morphologic analysis

of the slide or coverslip and uneven distribution of blood may also distort

may be greatly hampered by poorly prepared or stained blood smears.

the cells; however, these artifacts are minimized with proper technique.

Table 1.2 Pathologic Red Cells in Blood Smears

Red Cell Type

Description

Underlying Change

Disease State Associations

Acanthocyte (spur cell)

Irregularly spiculated red cells with

Altered cell membrane lipids

Abetalipoproteinemia, parenchymal

projections of varying length and dense

liver disease, postsplenectomy

center

Basophilic stippling

Punctuate basophilic inclusions

Precipitated ribosomes (RNA)

Coarse stippling: Lead intoxication,

thalassemia

Fine stippling: A variety of anemias

Bite cell (degmacyte)

Smooth semicircle taken from one

Heinz body pitting by spleen

Glucose-6-phosphate dehydrogenase

edge

deficiency, drug-induced oxidant

hemolysis

Burr cell (echinocyte) or

Red cells with short, evenly spaced

May be associated with altered

Usually artifactual; seen in uremia,

crenated red cell

spicules and preserved central pallor

membrane lipids

bleeding ulcers, gastric carcinoma

Cabot rings

Circular, blue, threadlike inclusion

Nuclear remnant

Postsplenectomy, hemolytic anemia,

with dots

megaloblastic anemia

Ovalocyte (elliptocyte)

Elliptically shaped cell

Abnormal cytoskeletal proteins

Hereditary elliptocytosis

Howell–Jolly bodies

Small, discrete, basophilic, dense

Nuclear remnant (DNA)

Postsplenectomy, hemolytic anemia,

inclusions; usually single

megaloblastic anemia

Hypochromic red cell

Prominent central pallor

Diminished hemoglobin synthesis

Iron deficiency anemia, thalassemia,

sideroblastic anemia

Leptocyte

Flat, waferlike, thin, hypochromic cell

—

Obstructive liver disease, thalassemia

Macrocyte

Red cells larger than normal

Young red cells, abnormal red cell

Increased erythropoiesis; oval

( > 8.5 µm), well filled with hemoglobin

maturation

macrocytes in megaloblastic anemia;

round macrocytes in liver disease

Microcyte

Red cells smaller than normal

—

Hypochromic red cell

( < 7.0 µm)

Pappenheimer bodies

Small, dense, basophilic granules

Iron-containing siderosome or

Sideroblastic anemia, postsplenectomy

mitochondrial remnant

Polychromatophilia

Grayish or blue hue often seen in

Ribosomal material

Reticulocytosis, premature marrow

macrocytes

release of red cells

Rouleaux

Red cell aggregates resembling stack

Red cell clumping by circulating

Paraproteinemia

of coins

paraprotein

Schistocyte (helmet cell)

Distorted, fragmented cell; two or

Mechanical distortion in

Microangiopathic hemolytic

three pointed ends

microvasculature by fibrin strands,

anemia (disseminated intravascular

disruption by prosthetic heart valve

coagulation, thrombotic

thrombocytopenic purpura, hemolytic

uremic syndrome, prosthetic heart

valves, severe burns)

Sickle cell (drepanocyte)

Bipolar, spiculated forms, sickle-

Molecular aggregation of HbS

Sickle cell disorders, not including

shaped, pointed at both ends

S trait

Spherocyte

Spherical cell with dense appearance

Decreased membrane surface area

Hereditary spherocytosis,

and absent central pallor, usually

immunohemolytic anemia

decreased diameter

Stomatocyte

Mouth or cuplike deformity

Membrane defect with abnormal

Hereditary stomatocytosis,

Copyright © 2019 Wolters Kluwer, Inc. Unauthorized reproduction of the content is prohibited. cation permeability immunohemolytic anemia Target cell (codocyte) Targetlike appearance, often Increased redundancy of cell Liver disease, postsplenectomy,

hypochromic

membrane

thalassemia, hemoglobin C disease

Teardrop cell (dacryocyte)

Distorted, drop-shaped cell

—

Myelofibrosis, myelophthisic anemia

Adapted from Kjeldsberg C, Perkins SL, eds. Practical Diagnosis of Hematologic Disorders . 5th ed. Chicago, IL: ASCP Press; 2010.

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