Wintrobe's Clinical Hematology 14e SC

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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