Page 41 - Williams Hematology ( PDFDrive )

16 Part I: Clinical Evaluation of the Patient Chapter 2: Examination of Blood Cells 17

multiple parameters to identify and enumerate the five major morpho- count because of the small size, tendency to aggregate, and potential
logic leukocyte types in blood: neutrophils, basophils, eosinophils, lym- overlap of platelets with more numerous smaller red cells and cellular
phocytes, and monocytes, as well as indicate the possible presence of debris. Current instruments typically construct a platelet volume his-
immature or abnormal cell. Customarily, both absolute (cells per μL) togram based on platelet size within a reliably measured platelet vol-
and relative (percent of leukocytes) counts are reported in the leuko- ume window and mathematically extrapolate this histogram to account
cyte differential. It is the absolute values that relate to pathologic states, for platelets whose size overlaps with debris (smaller) or small red cells
and percentages are sometimes misleading (e.g., absolute neutropenia (larger). This works because platelet volumes in health or disease fol-
appearing as a relative lymphocytosis) if the absolute values are not care- low a log-normal distribution. Some analyzers compare platelet counts
fully examined. Some have proposed to eliminate the reporting of dif- determined by different methods (e.g., impedance, light scatter, or fluo-
ferential count percentages entirely for this reason. “Band” neutrophils rescence staining) to improve accuracy, especially useful for low platelet
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cannot be identified as such by automated analyzers, although they counts. Based on analysis of volume-distribution histograms of platelets
will usually trigger a manual review flag if present in increased num- and red cells and comparison of optical and impedance-based platelet
bers. Current high-throughput instruments can perform an accurate counts, suspect samples are flagged for microscopic review. Automated
automated “five-part” differential count with a false-positive rate (i.e., platelet counting by current instrumentation is accurate and far more
unnecessarily flagged for review) of 2 to 15 percent in samples from a precise than manual methods. At very low platelet counts (less than
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medical center patient population. Eosinophils are accurately counted 20 × 10 /L), results are less precise and there is a method-dependent
by current state-of-the-art instruments, but automated basophil counts tendency to overestimate platelet counts. Conversely, platelet activa-
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remain imprecise. Small numbers of abnormal cells can escape detec- tion in disorders such as disseminated intravascular coagulation (DIC)
tion by either automated or manual methods. The false-negative rate and acute leukemia may result in systematic slight undercounting of
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for detection of abnormal cells varies from 1 to 20 percent, depending platelets. Advances in instrumentation, such as fluorescent dyes to
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on the instrument, type of abnormal cell examined, and the detection more specifically identify platelets in thrombocytopenic and micro-
limit desired (1–5 percent abnormal cells). 62–64 Careful attention to use cytic samples, should improve accuracy. When reviewing the blood
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of flagging criteria designed to prompt manual review, which are linked film, platelet count may be roughly estimated as 2000 times the number
to instrument-specific methodology, is essential to insure that optimum of platelets in 10 consecutive oil immersion (1000×) fields. 75
workflow strategies are used to detect samples containing abnormal Falsely Decreased Platelet Counts Causes of falsely decreased
cells with a manageable rate of manual review. Many instruments have platelet counts include incomplete anticoagulation of the sample (some-
“blast” flags designed to pick up leukemic blasts, but the sensitivity of times accompanied by small clots in the specimen or fibrin strands on
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such flags alone varied from 65 to 94 percent in a recent study, and is the stained film) and platelet clumping (pseudothrombocytopenia) or
lower in leukopenic patients. One must rely not only on the specifi- “satellitism” (adherence of platelets to neutrophils), caused by aggrega-
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cally designed “blast” flags, but also on other abnormalities identified tion induced by nonpathogenic antibodies recognizing platelet adhesion
in the automated blood count, including other flags, to select samples molecule epitopes exposed as a result of chelation of divalent cations in
for manual morphologic smear review. Lymphoma cells and reactive the anticoagulated sample. Platelet clumping occurs in approximately
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lymphocytes are the most difficult for both automated instruments and 0.1 percent of hospitalized patients. The same phenomenon may occur
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the human observer to identify. If one needs to search for infrequent to a lesser degree in citrate, which is often used to obtain platelet count
abnormal cells or evaluate leukocyte morphology, there is still no sub- in such cases. Magnesium EDTA, as compared to sodium EDTA, anti-
stitute for microscopic examination of a properly stained blood film by a coagulant is reported to more effectively inhibit platelet aggregation in
trained observer. The variability of morphologic quantification of band these patients and provide an accurate platelet count. Classical causes
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neutrophils is so high that some have advocated ceasing quantitative of falsely elevated platelet count include severe microcytosis, cryoglob-
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reporting of band cells. In spite of instrumentation that permits auto- ulins, and leukocyte cytoplasmic fragmentation. Infrequently, it may
mated analysis of a majority of clinical samples, the leukocyte differen- be necessary to confirm automated results by a microscopic (phase con-
tial count is still labor intensive relative to other high-volume laboratory trast) platelet count or platelet estimate from the blood film, bearing in
tests, and its value as a cause-finding tool in screening of asymptomatic mind that these methods are imprecise.
patients has been questioned. 67 Mean Platelet Volume The mean platelet volume (MPV) has been
The normal differential leukocyte count varies with age. As proposed as a useful clinical tool in the differential diagnosis of throm-
described in Chap. 7, polymorphonuclear neutrophils are predominant bocytopenias, and is associated with cardiovascular risk, stroke, and
in the first few days after birth, but thereafter lymphocytes account for metabolic disease. Increased MPV may be related in a complex way to
the majority of leukocytes. This pattern persists up to approximately 4 to thrombopoietic stimuli that affect megakaryocyte ploidy, and not plate-
5 years of age, when the polymorphonuclear leukocyte again becomes let age per se. A platelet volume distribution width (PDW) can be calcu-
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the predominant cell and remains so throughout the rest of childhood lated just as the RDW, and is correlated with platelet count and MPV.
and adult life. Chapter 9 discusses the leukocyte count in older persons. However, platelet size parameters are difficult to accurately quantify and
The leukocyte count may decrease slightly in older subjects because of use diagnostically because of the wide physiologic variation of the MPV
a fall in the lymphocyte count with age. Neutrophil counts are lower in in normal subjects, lack of standardization of automated measurement
individuals of African descent, and in some Middle Eastern populations techniques and instability of platelet size parameters in the presence of
than in persons of European descent. 68 commonly used anticoagulants. 79
Newly Released (Reticulated) Platelets Newly released platelets
AUTOMATED ANALYSIS OF PLATELETS contain RNA, as do newly released red cells, and are functionally more
active, with enhanced expression of adhesion molecules and bound
Platelet Count coagulation factors. The number of platelets with high RNA content
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Platelets are usually counted electronically by enumerating particles in (sometimes termed reticulated platelets or immature platelet fraction,
the unlysed sample within a specified volume window (e.g., 2–20 fl), measured by flow cytometry with RNA-binding fluorescent dyes, or
where volume may be measured by electrical impedance or light scat- by certain automated analyzers ) is a marker of marrow megakary-
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ter. The platelet count was more difficult to automate than the red cell ocytopoiesis and is proposed as a way of differentiating decreased
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