Veterinary Hematology: A Diagnostic Guide and Color Atlas

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 C ha p ter 1 n Introduction to Veterinary Hematology 3

square root transformation) so that the frequency distribution conducting the test. Hematology indices such as the red
of the transformed data approximates a Gaussian distribution. cell distribution width (RDW) vary more between laborato-
The boundaries are determined as before and results are ries, making the use of published reference intervals less
retransformed to determine the reference interval. Alterna- acceptable.
tively, one can use percentiles to determine upper and lower The units used in reporting values can vary by laboratory
limits, especially if large numbers of healthy animals are evalu- and a conversion factor may be needed to compare a measured
ated. Values are listed in ascending order. The lower limit is value to a published reference interval. For example, blood
determined by the formula (n + 1) × 0.025, and the upper iron might be reported as 100 µg/dL or 18 µmol/L. Most U.S.
limit is determined by the formula (n + 1) × 0.975, where n laboratories continue to use conventional units, such as mg/
= the number of normal animals assayed.2 If 119 animals were dL; Canadian and European laboratories use the Interna-
used, the value for the 3rd lowest animal would be used as the tional System of Units (SI units), such as mmol/L. Where
lower limit and the value from the 117th animal (3rd from possible, moles are used rather than weight (e.g., mg) for SI
the top) would be used as the upper limit. units. This cannot be done for analytes, such as serum
protein concentration, where the molecular weight is variable
Interpretation of Test Results Relative to and/or unknown. For enzymes, an SI enzyme unit is defined
Reference Intervals as 1 µmol/min of substrate utilized or product formed. SI
The common usage of the 95% confidence interval to establish units are reported per liter.
reference intervals means that 5% of healthy animals will be For many wild animal species, reference intervals may not
reported as abnormal for a given test. When multiple tests are be published for some or all tests. The simultaneous measure-
done in laboratory medicine profiles, the probability of at least ment of a healthy “control” animal from the same species,
one test being abnormal increases with the number of tests preferably a cohort, can be used as a rough guideline reference
done. For example, there is a 64% chance that at least one value and therefore can aid interpretation of the patient’s
abnormal test result will be obtained when 20 analytes are results.
measured from a healthy animal.6 The degree to which a test
result is above or below the reference interval is generally
important in deciding whether a high or low value should be S EN S I T I V I T Y A N D S P E C I F I C I T Y
taken seriously. OF TESTS
Use of Published Reference Intervals Ideally analyte values obtained from a healthy animal popula-
Routine hematology test results are usually similar between tion would not overlap with values obtained form a diseased
laboratories; consequently published reference intervals for animal population. Unfortunately there is almost always some
values such as total leukocyte counts and hematocrits are often overlap in the distribution of individual analyte test results
used to interpret results from a species (e.g., wallaby) when between the two groups (Fig. 1-3). When the disease being
reference values have not been established in the laboratory considered has a major impact on an analyte, little overlap in

Mean
Healthy
Relative frequency
Relative frequency

Diabetes

0 0.2 0.4 0.6 0.8 1.0 1.2 0 50 100 150 200 250
Cells 103/L Glucose (mg/dL)

FIGURE 1-2 FIGURE 1 -3

Frequency diagram of hypothetical absolute blood cell counts with a Overlapping Gaussian distributions of a healthy dog population com-
skewed population. The central (tallest) vertical line denotes the mean. pared with a population of dogs with type 2 diabetes mellitus.
Each additional vertical line represents one standard deviation (SD) from
the adjacent vertical line. The use of mean ±2 SD to calculate the refer- The figure is redrawn from Farver TB. Concepts of normality in clinical bio-
ence interval is inappropriate, as demonstrated by the lower limit being chemistry. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical Biochemistry
an impossible negative value. of Domestic Animals. 6th ed. San Diego: Academic Press; 2008:1-25.
4 VETERINARY HEMATOLOGY

values will occur; however, extensive overlap occurs if the for which they are being tested. As can be seen in Figure 1-4,
analyte concentration is minimally altered by the disease being if one increases the reference interval of the healthy popula-
considered. True positives (TPs) are positive test results from tion in order to minimize the FPs, the number of FNs
animals with the disease for which they are being tested, false increases.
positives (FPs) are positive test results for animals without the A clinical test should be safe and practical, and should
disease for which they are being tested (Fig. 1-4), true nega- accurately indicate the presence or absence of a specific disease
tives (TNs) are negative test results from animals without the or pathology. Sensitivity, specificity, and predictive value con-
disease for which they are being tested, and false negatives stitute measures of a test’s utility for ruling in or ruling out a
(FNs) are negative test results from animals with the disease given disease.
Sensitivity is the likelihood of a positive or abnormal test
result occurring in animals with the disease being considered
(Box 1-1). For example, if 23 of 28 cats with feline infectious
peritonitis (FIP) are recognized to have a low absolute lym-
phocyte count in blood, the sensitivity of lymphopenia as a
Healthy diagnostic test for cats with FIP is calculated to be 82%
Relative frequency

(Tables 1-1 and 1-2).7

Specificity is the likelihood of obtaining a negative or
normal test result in nondiseased animals—that is, animals
FP without the particular disease under consideration. In other
TN
words, specificity represents the proportion of animals without
the disease in question that have normal tests. Specificity may
FN
TP be calculated in two distinctly different ways, either by assum-
Diabetes
ing that all of the nondiseased animals are healthy or by
A assuming that although nondiseased animals do not have the
particular disease for which the analysis is being performed,
they may have other diseases.
Determining the specificity of a test in a group of healthy
animals is of little value because reference intervals are gener-
ally established to include 95% of the total population of
Healthy healthy animals, with 2.5% of healthy animals having values
Relative frequency

above and 2.5% of healthy animals having values below the

reference interval. The specificity of a test is much more useful
when the population of animals typically evaluated in a vet-
FP erinary hospital setting is being used.1 In this approach, the
TN “nondiseased” group includes not only healthy animals pre-
sented for elective procedures but also animals with diseases
FN TP other than the disease being considered.

Diabetes
P R ED I C T I V E VA LU E S A N D
0 50 100 150 200 250 D I S E A S E P R EVA LEN C E
B Glucose (mg/dL)
Predictive values demonstrate how well a test performs in a
FIGURE 1-4 given population. In contrast to sensitivity determinations
Frequency diagrams of a healthy dog population compared with a popu- (which are made using only a population of animals with the
lation of dogs with type 2 diabetes mellitus. Graphs are redrawn from disease in question) and specificity determinations (which are
Figure 1-3 to demonstrate true-negative (TN), false-negative (FN), true- made using only a population of animals without the disease
positive (TP), and false-positive (FP) values used to calculate sensitivity,
specificity, and predictive values. The top graph (A) demonstrates the
under consideration), predictive value determinations are
effect of using the mean +2 standard deviations (SD) to set the upper made from populations that contain animal both with and
limit of the reference interval. The lower graph (B) demonstrates the without the disease in question.
effect of using the mean +3 SD to set the upper limit. The number of The predictive value of a positive test (PVPT) considers
FP tests are reduced but the number of FN tests are increased by using only animals in the population being studied that have a posi-
the higher reference limit.
tive test result and determines what percentage of animals
The figure is redrawn from Farver TB. Concepts of normality in clinical actually have the disease being considered (see Box 1-1). It
biochemistry. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical Biochemis- answers the question “How likely is it that an animal with a
try of Domestic Animals. 6th ed. San Diego: Academic Press; 2008:1-25. positive test will actually have the disease being considered?”
 C ha p ter 1 n Introduction to Veterinary Hematology 5

Table 1-1
Test Results from the Evaluation of 224 Cats with a History and Clinical Signs Consistent with Feline
Infectious Peritonitis (FIP) Resulting in the Inclusion of FIP in the List of Differential Diagnosesa
NUMBER OF CATS AFFECTED
Test Have FIP (N = 28) Do Not Have FIP (N = 196) Total Cats (N = 224)
Lymphopenia (<1.5 x 103 cells/µL) 23 43 66
Monocytosis (>0.9 x 103 cells/µL) 2 43 45
Hyperglobulinemia (>5.1 g/dL) 11 7 18
Coronavirus titer positive 22 84 106

N, Number of cats.
a
Data from Sparkes AH, Gruffydd-Jones TJ, Harbour DA. An appraisal of the value of laboratory tests in the diagnosis of feline infectious peritonitis. J Am Anim
Hosp Assoc. 1994;30:345-350.

Box 1-1 Table 1-2

Formulas for the Calculation of
Sensitivity, Specificity, Predictive Examination of Lymphopenia as a Diagnostic Test
Value of a Positive Test, for Feline Infectious Peritonitis (FIP)a,b
Predictive Value of a Negative NUMBER OF CATS AFFECTED
Test, and Prevalence
Have FIP Do Not Have FIP Total Cats
Test (N = 28) (N = 196) (N = 224)
TP × 100
Sensitivity (%) = Lymphopenia 23 43 66
TP + FN
True False positive Total
TN × 100
Specificity (%) = positive positive
TN + FP No lymphopenia 5 153 158
TP × 100 False True negative Total
Predictive value of a positive test (%) =
TP + FP negative negative
TN × 100
Predictive value of a negative test (%) =
TN + FN N, Number of cats.
a
( TP + FN) × 100 Cats with a history and clinical signs consistent with FIP were evaluated,
Prevalence (%) = resulting in FIP being included in the list of differential diagnoses. Lymphope-
TP + TN + FP + FN nia was defined as <1.5 x 103 lymphocytes per microliter of blood.
b
Data from Sparkes AH, Gruffydd-Jones TJ, Harbour DA. An appraisal of the
TP, true positive (the number of animals with the disease being tested for that
value of laboratory tests in the diagnosis of feline infectious peritonitis. J Am
have a positive test result); FP, false positive (the number of animals without
Anim Hosp Assoc. 1994;30:345-350.
the disease being tested for that have a positive test result); TN, true negative
(the number of animals without the disease being tested for that have a nega-
tive test result); and FN, false negative (the number of animals with the
disease being tested for that have a negative test result).
prevalence of a disease affects the predictive values of a test
used to diagnose the disease but not its sensitivity or specific-
Based on the selected population of cats presented in Tables ity. For most tests, the PVPT will be low and the PVNT will
1-1 and 1-2, there is a 23/66 or 35% chance that a cat with be high if the disease has a low prevalence in the population
lymphopenia in this population will have FIP.7 being studied. The PVPT will be low because low prevalence
The predictive value of a negative test (PVNT) considers magnifies the number of false-positive results—that is, most
only animals in the population being studied that have a nega- positive test results are false positives because few animals
tive or normal test result and determines what percentage of actually have the disease (see Box 1-1). The exception would
animals with negative test results do not have the disease be a test where false-positive results occur infrequently (e.g.,
being considered (see Box 1-1). It answers the question “How polymerase chain reaction tests for specific infectious agents
likely is that an animal with a negative or normal test result or inherited blood cell defects). The PVNT will be high
will be free of the disease being considered?” Based on the because few false-negative results are present in a population
selected population of cats presented in Table 1-2, there is a when the disease prevalence is low.
153/158 or 97% chance that a cat with a normal or increased To improve diagnostic accuracy, the prevalence (likelihood)
blood lymphocyte count will not have FIP. of the disease being considered can be increased by using the
The prevalence of a disease in a population is simply the history, physical examination, and adjunctive diagnostic tests
percentage of animals in a given population that have a certain to restrict the population, as described for cats in Table 1-1.
disease (see Box 1-1). The prevalence of FIP in the selected The prevalence of FIP in the general cat population is much
population presented in Table 1-1 is 28/224 or 12.5%. The lower than 12.5%. By ruling out one or more diseases that can
6 VETERINARY HEMATOLOGY

give the same positive test result as the disease being consid-
ered, a clinician decreases the size of the population being C U T O F F VA LU E S
studied, thereby increasing the prevalence of the disease in the
population and increasing the positive predictive value of the The PVPT may be increased by using a cutoff value above or
test for the disease being considered. below the standard reference interval, depending on whether
Laboratory tests are used to help rule in or rule out a spe- the disease under consideration results in an increase or a
cific disease. When significant hazards are associated with decrease in the analyte being measured. For example, low
treatment (e.g., amputation or high-risk chemotherapy) or mean cell volume (MCV) is a diagnostic test suggestive of
euthanasia is being considered, it is necessary to be as certain chronic iron deficiency in dogs. If we use 64 fL as the lower
as possible that the disease is actually present. Consequently limit of the reference interval to calculate its positive predic-
tests with high positive predictive values are needed for a tive value, the value would not be remarkably high because
rule-in strategy. When the penalty for missing a diagnosis is there are various other relatively common disorders that can
high, as with a disease for which therapy is effective if begun result in low MCVs in dogs, most notably inflammatory con-
quickly, tests with high negative predictive values are theoreti- ditions and portosystemic shunts. However, it is recognized
cally important as a rule-out strategy. A normal test result by that the other causes of microcytosis rarely result in MCV
virtue of its high negative predictive value would suggest that values below 52 fL. Consequently, if a dog has a MCV below
the disease is not present. Unfortunately many diseases have 52 fL, chronic iron deficiency anemia is highly likely and the
low prevalence, which by itself can result in a high negative PVPT using this cutoff value would approach 100%. However,
predictive value. The best evidence for ruling out a disease is 52 fL is not routinely used as a cutoff value for a positive test
finding a negative test result for an assay that has a high because many cases of chronic iron deficiency would be
sensitivity for recognizing the disease. Based on the selected missed. Nonetheless, it is important to realize that dogs with
population of cats presented in Tables 1-1 and 1-2, finding a especially low MCV values almost certainly have chronic iron
normal or increased blood lymphocyte count is more reliable deficiency anemia.
for ruling out FIP than is finding a low lymphocyte count for The effects of varying the cutoff value of a test on sensitiv-
making a diagnosis of FIP. ity, specificity, and predictive values are demonstrated in Table
Information is generally available concerning the sensitiv- 1-3, where plasma fibrinogen concentration was evaluated as
ity of routine tests for common diseases, but information is a diagnostic test for Rhodococcus equi pneumonia in 165 foals
often lacking concerning all diseases that may have a positive from a single farm.3 It is important to recognize that fibrino-
test result and the frequency of these diseases in the popula- gen is an acute-phase protein that often increases in associa-
tion being evaluated. Consequently, the specificity of a test can tion with various causes of inflammation in horses and that
vary when populations containing animals with other diseases the heat precipitation assay used to measure fibrinogen (while
are analyzed. PVPTs and PVNTs also vary considerably easily performed and clinically useful) is relatively imprecise.
depending on the population analyzed. Although accurate As the cutoff value for plasma fibrinogen concentration is
values are not usually available for PVPTs and PVNTs, clini- increased, the specificity and PVPT increase, but the sensitiv-
cians use their knowledge and experience, combined with the ity and PVNT decrease (see Table 1-3). Results from this
principles outlined above, to make informed judgments con- study also demonstrate that the PVPT increases and the
cerning the likelihood that a disease can be ruled in or ruled PVNT decreases as the prevalence of disease in a population
out of the differential diagnosis. These decisions are seldom increases. In choosing the most appropriate cutoff value for a
based on a single test result; instead, information in the history test, one must consider a number of factors including sensitiv-
is considered along with the clinical signs and results of the ity and specificity of the test, prevalence of disease in the
physical examination, diagnostic imaging, and other labora- population being tested, and consequences of false-positive
tory tests. The likelihood that a disease will be present increases and false-negative tests. In the example above, failure to iden-
if several findings are supportive of the diagnosis. For example, tify an infected foal (false-negative test) might result in the
in the FIP study discussed above, the PVPT was 35% for cats debilitation or death of the foal. Conversely, the treatment of
with lymphopenia, 77% for cats with lymphopenia and hyper- healthy foals based on false-positive test findings could result
globulinemia, and 89% for cats with lymphopenia, hyper- in unnecessary financial losses and potential injury to healthy
globulinemia, and a positive coronavirus titer. The PVNT foals as a result of the adverse side effects of antimicrobial
increased from 97% when lymphopenia alone was absent to therapy.
99% when all three findings were absent. Minimal change
occurs in the PVNT because the relatively low disease preva-
lence in the population is a major contributing factor to the ACC U R AC Y V ER S U S P R E C I S I O N
high negative PVNT. This contribution is most clearly dem-
onstrated by looking at blood monocyte data in the FIP study The accuracy of an analytical procedure is determined by
presented in Table 1-1. Only 7% of FIP cats have a monocy- how closely the result approaches the true value of the
tosis (sensitivity), and the PVPT for monocytosis is only 4%, analyte being measured. An accurate test is one where the
yet the PVNT for a cat lacking a monocytosis is 88%. average of several assay results is close to the true value (Fig.
 C ha p ter 1 n Introduction to Veterinary Hematology 7

Table 1-3
Sensitivity, Specificity, and Predictive Values of Plasma Fibrinogen Concentrations at Selected Cutoff Values
for the Early Identification of Foals with Rhodococcus equi Pneumonia, Assuming Two Different Prevalences
of Diseasea
PREDICTIVE VALUES
PREVALENCE 10% PREVALENCE 40%
Cutoff value (mg/dL) Sensitivity (%) Specificity (%) PVPT (%) PVNT (%) PVPT (%) PVNT (%)
300 100 6 11 100 42 100
400 91 51 17 98 55 89
500 71 68 20 96 60 78
600 38 96 51 93 86 70
700 29 97 51 92 86 67
800 12 100 100 91 100 63

PVPT, Predictive value of a positive test; PVNT, predictive value of a negative test.
a
Data from Giguère S, Hernandez J, Gaskin J, Miller C, Bowan JL. Evaluation of white blood cell concentration, plasma fibrinogen concentration, and an agar
gel immunodiffusion test for the early identification of foals with Rhodococcus equi pneumonia. J Am Vet Med Assoc. 2003;222:775-781.

Precise Accurate
40 accurate 40 imprecise

30 30
Test results (mmol/L)
Test results (mmol/L)

Precise Inaccurate
20 inaccurate 20
imprecise

10 10

0 10 20 30 40 0 10 20 30 40
Standard (mmol/L) Standard (mmol/L)

FIGURE 1-5 FIGURE 1 -6

Plots comparing test results of triplicate assays of three standards (y-axis) Plots comparing test results of four replicate assays of three standards
to the known values of the standards (x-axis). The top plot is accurate (y-axis) to the known values of the standards (x-axis). The top plot is
with good precision. The bottom plot has good precision but is accurate but imprecise. The bottom plot is inaccurate and imprecise.
inaccurate.

1-5). Analytic procedures with low accuracy are said to have sample. The CV is the standard deviation (SD) of the repeated
a negative bias if results are below the true value or a positive measurements expressed as a percent of the mean of the
bias if results are above the true value. repeated measurements (SD/mean × 100). The CV indicates
The precision of a test reflects how reproducible the test the amount of random error (imprecision) that is present in
results are when the assay is replicated. Precision is indepen- an assay. A high CV value (e.g., more than 10%) indicates that
dent of accuracy (Fig. 1-6); consequently test results can be an assay lacks precision. A low CV value (e.g., less than 5%)
highly reproducible but erroneous (see Fig. 1-5, lower plot). indicates that assay results are reproducible, varying little with
Precision or, more accurately, the amount of imprecision repeated measurement. The degree of imprecision of an assay
present in an assay, is determined by calculating the coefficient can also be measured over time intervals to assess within-run,
of variation (CV) for repeated measurements made on a single between-run, or between-day variation.
8 VETERINARY HEMATOLOGY

selected blood films (100 cells per differential). CVs were also
Automated versus Manual Methods calculated from results from 20 pairs of randomly selected
As can be seen in Figures 1-7 and 1-8, manual leukocyte and slides (200 cells per differential). Last, CVs were calculated
platelet counts are less precise (CV 15% and 13%, respectively) from results from 20 quads of randomly selected slides (400
than automated leukocyte and platelet counts (CV 2% and cells per differential). As expected, the CVs for leukocyte types
4%, respectively). These values do not indicate whether manual that were numerous (e.g., neutrophils) were much lower than
or automated methods are more accurate. In fact, the mean CVs for leukocyte types that were present in low numbers
manual platelet count is probably more accurate (more near (e.g., basophils), and CVs decreased as the total number of
the true platelet count) than the mean automated platelet cells counted in the differential increased (Figs. 1-9 and 1-10).
count because platelets in small platelet clumps can be visual- The CVs for each of the five leukocyte types from this dog
ized and counted separately in a hemacytometer chamber but were plotted versus the mean percentage of each leukocyte
would be counted as one platelet or not counted at all in an type for 100-, 200-, and 400-cell manual differential counts
automated cell counter. and compared with a like plot with data determined by per-
For manual differential leukocyte counts, the CV varies forming 20 automated differential counts on blood from a
with the percentage of a given leukocyte type present in the single dog using an Advia 120 (Siemens Healthcare Diagnos-
blood film and the total number of leukocytes included in the tics, Inc., Tarrytown, NY) hematology analyzer (Fig. 1-11).
differential leukocyte count. For example, 100 cell differential Automated hematology analyzers have lower CVs for each
counts were performed by a single technologist on each of 80 percentage of leukocyte type present because they examine
stained coverslip blood films from a dog with a mild baso- thousands of leukocytes (assuming a normal leukocyte
philia. CVs were calculated from results of 20 randomly count) in performing the differential leukocyte count.

10
300

200
Leukocytes (103/L)

Platelets (103/L)

100

0 0
Manual Automated Manual Automated

FIGURE 1-7 FIGURE 1 -8

Individual plots of total leukocyte counts performed 20 times each using Individual plots of platelet counts performed 20 times each using a
a manual method and an automated method on the same canine blood manual method and an automated method on the same canine blood
sample. The manual method utilized 20 separate dilutions (Unopette sample. The manual method utilized 20 separate dilutions (Unopette
365855, Becton Dickinson Co., Franklin Lakes, NJ), followed by the 365855, Becton Dickinson Co., Franklin Lakes, NJ), followed by the
counting of all leukocytes in 1 µL of 1/100 diluted blood in a hemacy- counting of all platelets in 1/25 µL of 1/100 diluted blood in a hema-
tometer chamber. A Cell-Dyn 3500 (Abbott Laboratories, North cytometer chamber. A Cell-Dyn 3500 (Abbott Laboratories, North
Chicago, IL) calibrated for canine blood was used to perform the auto- Chicago, IL) calibrated for canine blood was used to perform the auto-
mated cell counts. The mean and coefficient of variation (CV) for the mated cell counts. The mean and coefficient of variation (CV) for the
manual counts were 7.1 × 103/µL and 15% respectively. The mean and manual counts were 240 × 103/µL and 13% respectively. The mean and
CV for the automated counts were 6.7 × 103/µL and 2% respectively. CV for the automated counts were 219 × 103/µL and 4% respectively.
 C ha p ter 1 n Introduction to Veterinary Hematology 9

70 100
90 100 Cell manual
65 200 Cell manual
80

Coefficient of variation (%)

400 Cell manual
70 Advia 120
60
Neutrophils (%)

60

55 CV 6% 50
CV 5%
CV 9%
40
50 30
20
45
10
0
40 0 5 10 15 20 25 30 35 40 45 50 55 60
100 200 400
Leukocyte type (%)
Number of cells per differential count
FIGURE 1 -11
FIGURE 1-9
Mean coefficient of variation (CV) values for each of the five leukocyte
Box plots of neutrophil percentages and coefficients of variation (CVs) types from a single dog are plotted versus the mean differential counts
from manual differential counts from a single dog with 55.4% neutro- of each leukocyte type. Mean values were determined from 20 differential
phils. Values represent the results of 20 differential leukocyte counts each leukocyte counts each of 100, 200, and 400 nucleated cells. A like plot
of 100, 200, and 400 nucleated cells. A median line is shown. Boxes with data determined by performing 20 automated differential counts on
include 25th to 75th percentiles and error bars include 10th to 90th blood from a single dog using an Advia 120 hematology analyzer is
percentiles. included for comparison.

5
Critical Difference
4
The CV of an assay affects how the results are interpreted,
especially if an assay is being repeated to determine whether
3
a treatment is effective. For example, if the total leukocyte
Basophils (%)

count for a dog is 4600/µL before treatment and 5800/µL

after treatment, does this represent a real improvement or
2
might it reflect imprecision in the measurement of the total
CV 42% CV 33% leukocyte count? An additional confounding variable in this
1
example is the biological variability of the animal itself.
CV 94% Jensen et al.4 calculated the analytical CV for an automated
0
total leukocyte count in healthy laboratory beagles to be
3.7%, while the CV for repeated total leukocyte counts from
100 200 400
individual beagles (within dog CV) was 12.1%.4 From these
numbers, a critical difference of 35% was calculated. This
Number of cells per differential count
means that the total leukocyte count would have to increase
FIGURE 1-10 by more than 35% before the therapy could be assumed to
Box plots of neutrophil percentages and coefficients of variation (CVs) have an influence on this analyte. In the example above,
from manual differential counts from a single dog with 1.4% basophils. the automated total leukocyte count would have to exceed
Values represent the results of 20 differential leukocyte counts each of 4600/µL × 1.35, or 6200/µL, before a therapeutic effect might
100, 200, and 400 nucleated cells. A median line is shown. Boxes be assumed. A considerably greater difference would be
include 25th to 75th percentiles and error bars include 10th to 90th
percentiles.
required if total leukocyte counts were done using a manual
method because of its higher analytical CV. A greater critical
difference might also have been calculated in the above
example had client-owned animals been used for this study
However, they are not always more accurate. The inability to rather than laboratory animals, because it is likely that the
correctly identify certain cell types (especially basophils), biological variation would be higher in client-owned animals
abnormal cell morphology, or abnormal cell types can lead to that were not accustomed to the phlebotomy procedure, the
the misclassifications of cell types. For example, the Advia 120 individuals handling them, or the environment in which the
failed to identify any basophils in blood from a cat with 39% phlebotomy was done.
basophils or a dog with 14% basophils identified on manual Unfortunately, critical difference measurements have been
differential leukocyte counts. done for few analytes in veterinary medicine, and values will
10 VETERINARY HEMATOLOGY

vary depending on methods and instruments used and animal 3. Giguère S, Hernandez J, Gaskin J, et al. Evaluation of white blood cell concentration,
plasma fibrinogen concentration, and an agar gel immunodiffusion test for the early
populations evaluated. Nonetheless, clinicians develop knowl- identification of foal with Rhodococcus equi pneumonia. J Am Vet Med Assoc.
edge and intuition through study and experience that can help 2003;222:775-781.
them to make informed judgments concerning the impor- 4. Jensen AL, Iversen L, Petersen TK. Study on biologic variability of haematological
components in dogs. Comp Haematol Int. 1998;8:202-204.
tance of changes in laboratory data. 5. Lumsden JH. Reference values. In: Feldman BF, Zinkl JG, Jain NC, eds. Schalm’s
Veterinary Hematology. 5th ed. Philadelphia: Lippincott Williams & Wilkins;
2000:12-15.
R EF ER EN C E S 6. Marshall WJ. The interpretation of biochemical data. In: Marshall WJ, Bangert SK, eds.
Clinical Biochemistry. Metabolic and Clinical Aspects. 2nd ed. New York: Churchill
Livingstone Elsevier; 2008:17-27.
1. Braun JP, Concordet D, Lyazrhi M, et al. Overestimation of the predictive value of posi- 7. Sparkes AH, Gruffydd-Jones TJ, Harbour DA. An appraisal of the value of laboratory
tives by the usual calculations of the specificity of diagnostic tests. Vet Res Commun. tests in the diagnosis of feline infectious peritonitis. J Am Anim Hosp Assoc.
2000;24:17-24. 1994;30:345-350.
2. Farver TB. Concepts of normality in clinical biochemistry. In: Kaneko JJ, Harvey JW,
Bruss ML, eds. Clinical Biochemistry of Domestic Animals. 6th ed. San Diego: Academic
Press; 2008:1-25.
 C H A P T E R

2
Hematology Procedures

CO M P O S I T I O N O F B L O O D C A L C U L AT I O N O F
B L O O D VO LU M E
Blood is composed of cells (erythrocytes, leukocytes, and
platelets) circulating within fluid called plasma (Fig. 2-1). Total blood volume accounts for about 10% to 11% of body
Erythrocytes or red blood cells are most numerous, with weight in hot-blooded horses; 8% to 9% in dogs; 6% to 7%
several million erythrocytes per microliter of blood in in cats, ruminants, laboratory rodents, and cold-blooded
mammals (Appendix Table 1). Depending on the species, (draft) horses; and 5% to 6% in pigs. The total blood volume
erythrocytes typically account for one-fourth to one-half of of young growing animals often exceeds 10% of body weight.33
the total blood volume as measured by determining the hema- It may be desirable to calculate the total blood volume of an
tocrit. Platelets or thrombocytes are the next most numerous animal in determining the size of a needed blood transfusion,
cell type in blood, with platelet counts as low as 100 × 103/µL or the amount of blood that can safely be removed for a series
in healthy horses to several hundred thousand per microliter of diagnostic tests, or when an animal is to be used as a blood
in other mammalian species. Total leukocyte or white blood donor. For example, the total blood volume of a 4-kg cat is
cell counts are much lower than erythrocyte and platelet 0.07 × 4 kg = 0.28 kg = 280 mL, assuming that 7% of body
counts, with total leukocyte counts ranging from about 5 × weight is blood in cats and the specific gravity of blood is 1.0
103/µL to about 20 × 103/µL in mammals. The proportion of (1 mL weighs 1 g). Since one can safely remove 20% of the
leukocyte types present varies by species, with neutrophils blood volume from an animal, the calculated amount that can
being the most numerous leukocyte type present in the blood be removed from the cat in this example is 280 × 0.2 = 56 mL.
of carnivores and lymphocytes being the most numerous leu-
kocyte type present in the blood of ruminants and rodents.
Plasma consists primarily of water that contains about 6 to S A M P LE CO LLE C T I O N
8 g/dL of plasma proteins and 1.5 to 2.0 g/dL of inorganic A N D H A N D LI N G
salts, lipids, carbohydrates, hormones, and vitamins.19 Plasma
is prepared in the laboratory by collecting blood with an In monogastric animals, an overnight fast avoids postprandial
anticoagulant, followed by centrifugation to remove the blood lipemia, which can interfere with plasma protein, fibrinogen,
cells. If blood is collected without anticoagulant and allowed and hemoglobin determinations. Ethylenediaminetetraacetic
to clot, the fluid that is obtained following centrifugation is acid (EDTA) is the preferred anticoagulant for complete
called serum. The protein concentration in serum is usually blood count (CBC) determination in most species, but blood
about 0.2 to 0.5 g/dL lower than that in plasma, primarily from some birds and reptiles hemolyzes when collected into
owing to the absence of fibrinogen—which is consumed EDTA. In those species, heparin is often used as the anti­
during coagulation—in serum. Serum proteins may be sepa- coagulant. The disadvantage of heparin is that leukocytes
rated by electrophoresis into albumin, α-globulins, β-globulins, do not stain as well (presumably because heparin binds to
and γ-globulins (Fig. 2-2). Albumin is a single protein that leukocytes),24 and platelets generally clump more than
generally accounts for nearly half of the total plasma proteins they do in blood collected with EDTA. However, as discussed
present by weight. Each of the globulin classes is composed later, platelet aggregates and leukocyte aggregates may occur
of many different proteins.12 even in properly collected EDTA-anticoagulated blood

11
12 VETERINARY HEMATOLOGY

Whole
blood Formed Leukocytes
(volume) elements (differential)
(number per L)

Neutrophils
60-77%
Leukocytes
6,000-17,000
Formed
elements Erythrocytes
45% 5.5-8.5
million Eosinophils 2-10%
Platelets
Body 200,000-500,000 Basophils (rare)
weight Lymphocytes
12-30%
Blood 8%
Monocytes 3-10%
Proteins
(electrophoresis)
Plasma
Other weight
fluids
Proteins 7% Albumin
and Plasma

Serum proteins
tissues 55% 44%
92% Globulins
(electrophoresis)

Plasma proteins Alpha 14%

Water Beta 15%
91.5% Globulins
52% Gamma 23%

Fibrinogen 4%

Inorganic salts, lipids, hormones,

vitamins, carbohydrates 1.5%

FIGURE 2-1
Approximate composition of normal dog blood.

samples.10,29,41,49,53 In those cases, collection of blood using be made as soon as possible and rapidly dried to minimize
another anticoagulant (e.g., citrate) may prevent the forma- morphologic changes.
tion of cell aggregates. Cell aggregation tends to be more
pronounced as blood is cooled and stored; consequently
processing samples as rapidly as possible after collection G RO S S EXA M I N AT I O N O F
may minimize the formation of leukocyte and/or platelet B L O O D S A M P LE S
aggregates.
Collection of blood directly into a vacuum tube is preferred Samples are checked for clots and mixed well (gently inverted
to collection of blood by syringe and transfer to a vacuum tube. 20 times) immediately before removing aliquots for hematol-
This method reduces platelet clumping and clot formation in ogy procedures. Horse erythrocytes settle especially rapidly
samples for CBC determinations, as even small clots render because of rouleaux formation (adhesion of erythrocytes
a sample unusable. Platelet counts are markedly reduced, and together like a stack of coins). Blood should be examined
a significant reduction can sometimes occur in hematocrit grossly for color and evidence of erythrocyte agglutination.
(HCT) and leukocyte counts as well. Also, when the tube is The presence of marked lipemia may result in a blood sample
allowed to fill based on the vacuum within the tube, the proper with a milky red color resembling “tomato soup” when
sample-to-anticoagulant ratio will be present. Inadequate oxygenated.
sample size results in decreased HCT due to excessive EDTA
solution. Care should be taken to avoid iatrogenic hemolysis, Methemoglobinemia
which interferes with plasma protein, fibrinogen, and various Hemoglobin is a protein consisting of four polypeptide globin
erythrocyte measurements. Samples should be submitted to chains, each of which contains a heme prosthetic group within
the laboratory as rapidly as possible, and blood films should a hydrophobic pocket. Heme is composed of a tetrapyrrole
 C ha p ter 2 n Hematology Procedures 13

with a central iron molecule that must be maintained in the

ferrous (+2) state to reversibly bind oxygen. Methemoglobin
differs from hemoglobin only in that the iron molecule of the
heme group has been oxidized to the ferric (+3) state and is no
longer able to bind oxygen.28 The presence of large amounts of
A deoxyhemoglobin accounts for the dark bluish color of normal
venous blood samples. Methemoglobinemia may not be recog-
Albumin nized in venous blood samples because the brownish color of
methemoglobin is not readily apparent when methemoglobin
2
is mixed with deoxyhemoglobin (Fig. 2-3, A). When deoxyhe-
moglobin binds oxygen to form oxyhemoglobin, it becomes
bright red; consequently the brownish coloration of methemo-
globin becomes more apparent in the oxygenated samples (Fig.
2-3, B). A simple spot test provides a rapid way to oxygenate a
 venous blood sample and to determine whether clinically sig-

nificant levels of methemoglobin are present. One drop of
blood from the patient is placed on a piece of absorbent white
paper and a drop of normal control blood is placed next to it.
1 If the methemoglobin content is 10% or greater, the patient’s
blood will have a noticeably brown color compared with the
bright red color of control blood (Fig. 2-4).28 Accurate deter-
B mination of methemoglobin content requires that blood be
submitted to a laboratory that has this test available. Methe-
FIGURE 2-2 moglobinemia results from either increased production of
Serum protein electrophoresis from a dog with a polyclonal hyperglobu- methemoglobin by oxidants or decreased reduction of methe-
linemia, including increased α2-globulin and β-γ bridging. A, Agarose moglobin resulting from a hereditary deficiency in the eryth-
gel stained with Coomassie blue for protein. Albumin (band on the far
left) migrates more rapidly than other proteins. B, Densitometer tracing rocyte cytochrome-b5 reductase enzyme (see Chapter 4).27
of the agarose gel used to determine the contribution of each protein
type: total protein 7.7 g/dL, albumin 2.05 g/dL, α1 0.42 g/dL, α2 1.51 g/ Erythrocyte Agglutination
dL, β 1.86 g/dL, γ 1.86 g/dL. The appearance of red granules in a well-mixed blood sample
(Fig. 2-5) suggests the presence of erythrocyte autoagglutina-
tion. However, it is important to differentiate agglutination

A B

FIGURE 2-3
Gross appearance of mixtures of oxyhemoglobin, deoxyhemoglobin, and methemoglobin. A, Venous blood sample from a cat with 28% methemoglobin
(left sample) compared with blood from a normal cat with less than 1% methemoglobin (right sample). Both samples also contain a mixture of
oxyhemoglobin and deoxyhemoglobin. B, Oxygenated blood sample from a cat with 28% methemoglobin (left sample) compared with blood from a
normal cat with less than 1% methemoglobin (right sample). The sample on the left contains a mixture of oxyhemoglobin and methemoglobin; the
one on the right contains almost exclusively oxyhemoglobin.
14 VETERINARY HEMATOLOGY

FIGURE 2-4
Methemoglobin spot test. A drop of blood from a methemoglobin
reductase-deficient cat with 50% methemoglobin (left) is placed on
absorbent white paper next to a drop of blood from a normal cat with
less than 1% methemoglobin.

FIGURE 2 -6
Microscopic rouleaux in an unstained wet mount preparation of normal
cat blood.

FIGURE 2-5
Grossly visible agglutination in blood from a dog with immune-mediated
hemolytic anemia.

FIGURE 2 -7
(aggregation of erythrocytes together in clusters) from rou-
Microscopic agglutination in an unstained wet mount preparation of
leaux (adherence of erythrocytes together like a stack of coins), saline-washed erythrocytes from a foal with neonatal isoerythrolysis.
which can be seen in the blood from healthy horses and cats
(Fig. 2-6). Rouleaux formation is eliminated by washing
erythrocytes in physiologic saline, but agglutination is not. between negatively charged erythrocytes.67 In addition, there
This differentiation requires centrifugation of blood, removal are 10 antigen-binding sites per IgM molecule compared with
of plasma, and resuspension of erythrocytes in saline. A rapid 2 binding sites per IgG molecule (Fig. 2-8). A direct anti-
way to differentiate rouleaux from agglutination is to mix five globulin test is not needed to identify the presence of immu-
drops of physiologic saline with a drop of anticoagulated noglobulin bound to erythrocytes if agglutination is present
blood on a glass slide and examine it as a wet mount using a in saline solution-washed erythrocyte samples.
microscope. This dilution reduces rouleaux, but agglutination
is not affected. The microscopic appearance of agglutination
in a sample of washed erythrocytes is shown (Fig. 2-7). The M I C RO H EM AT O C R I T T U B E
presence of agglutination indicates that the erythrocytes have EVA LUAT I O N
increased surface-bound immunoglobulins. These immuno-
globulins are usually of the IgM type when agglutination is A microhematocrit tube is filled to about 90% of capacity with
present, because the greater distance between binding sites on well-mixed blood and sealed with clay at one end. The tube is
IgM molecules compared with IgG molecules makes it easier then placed in a microhematocrit centrifuge with the clay
for IgM molecules to overcome normal repulsive forces plug oriented to the periphery of the centrifuge head and
 C ha p ter 2 n Hematology Procedures 15

hyperbilirubinemia) in horses owing to reduced removal of

unconjugated bilirubin by the liver.13 Failure of the liver to
Erythrocytes
remove unconjugated bilirubin from blood has also been
reported to cause hyperbilirubinemia in one-fourth of the sick
(often anorectic) cattle in one study.40 In other species, yellow
plasma with a normal HCT suggests hyperbilirubinemia sec-
ondary to liver disease. Hyperbilirubinemia associated with a
marked decrease in the HCT suggests an increased destruc-
tion of erythrocytes; however, the concomitant occurrence of
liver disease and a nonhemolytic anemia could produce a
similar finding (Fig. 2-9, B).
Red discoloration of plasma indicates the presence of free
hemoglobin. This discoloration may represent either true
Anti-erythrocyte IgM hemoglobinemia, resulting from intravascular hemolysis, or
hemolysis occurring after sample collection due to such causes
as rough handling, fragile cells, lipemia, or prolonged storage
(Fig. 2-9, C,D). The HCT value may help differentiate between
FIGURE 2-8 these two possibilities, with red plasma and a normal HCT
Antierythrocyte IgM antibodies causing erythrocyte agglutination. suggesting in vitro hemolysis. The concomitant occurrence of
hemoglobinuria indicates that intravascular hemolysis has
occurred. Plasma will also appear red following treatment with
centrifuged for 5 minutes. After centrifugation, the blood cross-linked hemoglobin blood substitute solutions.
sample will be separated into three layers based on density, Lipemia is recognized as a white opaque appearance caused
with packed erythrocytes located at the bottom. A white by chylomicrons and very low density lipoproteins (VLDLs).
“buffy coat” is located above the erythrocyte layer, and the The presence of chylomicrons may also result in a white layer
acellular plasma is located above the buffy coat. at the top of the plasma column (Fig. 2-9, E). The presence
When blood is submitted for a CBC, most commercial of lipemia is frequently the result of a recent meal (postpran-
laboratories determine an electronic HCT rather than a dial lipemia), but diseases including diabetes mellitus, pancre-
packed cell volume (PCV) by centrifugation. This efficiency atitis, hypothyroidism, hyperadrenocorticism, protein-losing
negates the need to centrifuge a microhematocrit tube filled nephropathy, cholestasis, obesity, and starvation may also con-
with blood. Unfortunately useful information concerning the tribute to the development of lipemia in dogs and cats.23,72
appearance of plasma is missed unless a serum or plasma Transient lipemia and accompanying anemia has been
sample is also prepared for clinical chemistry tests. described as a syndrome in kittens, but a direct link between
hyper­lipidemia and anemia has not been clearly documented.23
Packed Cells Hereditary causes of lipemia include lipoprotein lipase defi-
The PCV is measured after centrifugation by determining the ciency in cats and idiopathic hyperlipidemia in miniature
fraction of total blood volume in a microhematocrit tube that schnauzer dogs.20,73
is occupied by erythrocytes. Leukocytes and platelets are pri- Equids of any age and either sex may develop lipemia, but
marily located within the buffy coat, although certain leuko- obese ponies, miniature horses, and miniature donkeys are
cyte types may be present in the top portion of the packed most susceptible. Lipemia has been associated with a broad
erythrocyte column in some species (e.g., neutrophils in range of diseases, but it is more prevalent in association with
cattle). The width of the buffy coat generally correlates directly pregnancy, lactation, and/or anorexia.65 Lipemia has also been
with the total leukocyte count. A large buffy coat suggests reported in llamas and alpacas with severe systemic diseases.
leukocytosis (Fig. 2-9, A) or thrombocytosis, and a small buffy It is not associated with age, sex, or reproductive status in these
coat suggests that low numbers of these cells may be present. camelids.64 The pathogenesis of marked secondary hyperlip-
The buffy coat may appear reddish owing to the presence of idemia is not always clear. It often appears to result from a
a marked reticulocytosis. mobilization of unesterified fatty acids from adipose tissue
and the subsequent overproduction of VLDLs by the liver,
The Appearance of Plasma but it may also result from ineffective clearance of VLDLs
Plasma is normally clear in all species. It is nearly colorless in by tissues or a combination of both, as occurs in diabetes
small animals, pigs, and sheep but light yellow in horses mellitus.
because they naturally have higher bilirubin concentrations.
Plasma varies from colorless to light yellow (carotenoid pig- Plasma Protein Determination
ments) in cattle, depending on their diet.62 Increased yellow After the PCV is measured and the appearance of the plasma
coloration usually indicates increased bilirubin concentration. and buffy coat is noted, the microhematocrit capillary tube is
This increase often occurs secondarily to anorexia (fasting broken just above the buffy coat and the plasma is placed in
16 VETERINARY HEMATOLOGY

A B C D E

FIGURE 2 -9
Gross appearance of microhematocrit tubes demonstrating leukocytosis, icterus, hemolysis, and lipemia.
A, Microhematocrit tube from a cat with a large buffy coat resulting from a chronic lymphocytic leukemia.
B, Microhematocrit tube from an anemic cat, with icteric plasma secondary to hepatic lipidosis. C, Micro-
hematocrit tube with evidence of hemolysis in plasma from a cat with acetaminophen-induced Heinz body
hemolytic anemia. Less dense erythrocyte ghosts can be seen above the packed intact erythrocytes. D, Micro-
hematocrit tube with evidence of hemolysis in plasma from a horse with intravascular hemolysis induced by
the inadvertent intravenous and intraperitoneal administration of hypotonic fluid. Less dense erythrocyte
ghosts can be seen above the buffy coat. E, Microhematocrit tube with marked lipemia in plasma from a dog
with hypothyroidism that was also being treated with prednisone for an allergic dermatitis. It was unclear
from the medical record if this was a fasting blood sample.

A, Courtesy of Heather Wamsley.

a refractometer for plasma protein determination. Plasma B L O O D C ELL CO U N T I N G

protein concentrations in newborn animals (approximately AND SIZING
4.5 to 5.5 g/dL) are lower than adult values and increase to
within the adult range by 3 to 4 months of age.33 The presence Total leukocyte counts, erythrocyte counts, and platelet counts
of lipemia or hemolysis will falsely increase the measured in mammals may be determined using manual or automated
plasma protein value. Maximum information can be gained techniques.
by interpretation of the HCT and plasma protein concentra-
tions simultaneously (see Chapter 4). Manual Cell Counting
Manual erythrocyte counts are not accurate enough to be
Fibrinogen Determination useful. Manual total leukocyte counts and platelet counts can
Fibrinogen can be measured in a hematocrit tube because it be performed using commercially available reservoirs and
readily precipitates from plasma when heated to 56°C to 58oC pipettes to dilute the sample and lyse erythrocytes prior to the
for 3 minutes. The difference between the total protein of the microscopic counting of leukocytes and platelets using a
plasma and the total protein of the defibrinogenated (heated) hemacytometer chamber. Manual leukocyte counts and plate-
plasma gives an estimate of the fibrinogen concentration in let counts are done when errors are suspected in cell counts
the plasma.32 This method is useful in identifying high fibrin- generated by automated cell counters. Manual cell counts may
ogen concentrations, but it is not accurate in identifying low also be done in an emergency situation when automated cell
fibrinogen concentrations.4,11 counts are not available.
 C ha p ter 2 n Hematology Procedures 17

All blood cell types in birds and reptiles are nucleated, must usually be corrected for the presence of nucleated eryth-
making accurate separation and counting difficult or impos- rocytes when present.
sible with automated cell counters. Consequently manual
leukocyte counts are generally required in nonmammalian Errors in Blood Cell Counting and Sizing
species. Thrombocytes can be estimated based on the number The accuracy of blood cell counting depends on the quality
present in stained blood films.47 and characteristics of the blood sample as well as the accuracy
If properly maintained, automated blood cell counters are of the analytic methods used. Storage of blood samples for
more precise and accurate than manual techniques in mam- more than a few hours can result in sample deterioration and
malian species. Various technologies—including quantitative inaccurate cell counts. The presence of even small clots in the
buffy coat analysis, impedance measurements, laser flow blood tube invalidates all cell counts. Even without clot for-
cytometry, and cytochemistry—are utilized to generate these mation, platelet aggregation may occur if platelets become
cell counts. activated during blood collection, as can happen when speci-
mens are collected with a syringe and then transferred to an
Automated Cell Counting anticoagulant tube. Heparin is generally not used as an anti-
Quantitative buffy coat analysis (QBC VetAutoread Hema- coagulant because it does not prevent platelet clumping, and
tology System, IDEXX, Inc., Westbrook, ME) depends on leukocyte staining is poor on blood films. EDTA is the pre-
variations in cell density to separate cell types. Cells are not ferred anticoagulant for CBC determinations in most species,
actually counted; instead the widths of the various layers of but EDTA may induce platelet, leukocyte, and erythrocyte
cell types that form are measured and the cell “count” is aggregate formation in some individuals with antibodies
generated assuming a standard cell size for the cell type in bound to the surfaces of these respective cell types.10,29,54,71,75
question.35 When this occurs, collection of blood into citrate may prevent
Impedance counters such as the Heska CBC-Diff (Heska the problem. Cell aggregation tends to be more pronounced
Corporation, Loveland, CO) and Abaxis VetScan HMII as blood is cooled and stored; consequently processing samples
(Abaxis, Union City, CA) depend on the principle that cells as rapidly as possible after collection may minimize the for-
are poor electrical conductors. Blood is diluted in an electri- mation of leukocyte and/or platelet aggregates. EDTA can
cally conductive solution and a precise volume of this diluted cause marked hemolysis in some species of birds (ostriches,
suspension is drawn through a small aperture between two emus, and jays),25,35 reptiles (turtles and tortoises),25,42 and fish
electrodes. Each cell produces a change in electrical imped- (carp and brown trout).43,66 Heparin is used as an anticoagu-
ance, resulting in a change in voltage that is proportional to lant in these species. Both lysis and clumping can result in
the size of the cell counted. Several thousand cells per second reduced cell counts of the cell types involved.
can be counted and sized. Erythrocytes and platelets can be Platelet clumping can not only result in an erroneously
differentiated by size alone in many species using impedance decreased platelet count59 but also in a falsely increased mean
counters, but not in cats, because their platelets are large and platelet volume (MPV) and occasionally a falsely increased
their erythrocytes relatively small. Leukocytes are counted as total leukocyte count.35,75 Autoagglutination of erythrocytes
free nuclei following lysis of erythrocytes and platelets. in the blood sample can result in a spuriously increased mean
The development of laser flow cytometry for use in cell volume (MCV) and mean cell hemoglobin concentration
instruments such as the CELL-DYN 3500R (Abbott Labo- (MCHC) and a decreased HCT and total erythrocyte count.48
ratories, Abbott Park, IL), the Advia 120 (Siemens Healthcare As indicated earlier, with the use of most hematology analyz-
Diagnostics, Inc., Tarrytown, NY), the Sysmex XT-2000iV ers, the presence of nucleated erythrocytes can result in a
(Sysmex Corporation, Kobe, Japan), and the LaserCyte falsely increased total leukocyte count. Residual erythrocyte
(IDEXX, Inc., Westbrook, ME) has allowed for cells to be stroma from inadequate lysing of erythrocytes may also result
characterized in greater detail. Individual cells pass through a in spuriously increased total leukocyte counts.35,76
laser beam, absorbing and scattering light. Interruptions in The presence of severe lipemia can result in spuriously
light are used to count cells and light scatter is used to deter- increased hemoglobin and MCHC values and possibly even
mine size and internal complexity or density. The Advia 120 increased platelet and total leukocyte counts.75 The presence
also utilizes a peroxidase channel to aid in determining spe- of in vitro or in vivo hemolysis in the sample or prior treat-
cific leukocyte types. When properly calibrated, laser flow ment with a cross-linked hemoglobin solution can result in
analysis allows for a more complete and accurate differential an erroneously increased MCHC value.38 The presence of
leukocyte count than can be done using cell size alone. Laser numerous Heinz bodies in erythrocytes can also result in
flow cytometry also permits the development of automated spuriously increased hemoglobin and MCHC values, increased
reticulocyte counts and reticulated platelet counts36,44 as well automated reticulocyte counts,14,52 and sometimes increased
as the development of new erythrocyte and reticulocyte total leukocyte counts.69 The precipitation of an IgM parapro-
parameters based on the simultaneous measurement of size tein in blood from a dog by the lysing reagent used in the
and hemoglobin concentration within individual cells.17 CELL-DYN 3500 falsely increased the spectrophotometric
Nucleated erythrocytes are counted as leukocytes in most measurement of hemoglobin (Hb), which falsely increased the
electronic cell counters; consequently total leukocyte counts calculated MCHC.8
18 VETERINARY HEMATOLOGY

Laboratory errors may occur as a result of mistakes made testing programs provide external quality control. Samples are
by operators. These operator errors include a lack of knowl- periodically sent to participating laboratories for analysis.
edge or the skills for the test being done, improper labeling Results are sent back to the agency supplying the samples and
of samples, dilution errors, use of outdated reagents, inade- these values are compared with those from reference labora-
quate quality control measures, or improper calibration of tories and other participating laboratories using the same
equipment. The instruments being used must be optimized methods. Proficiency testing programs provide valuable peer
and validated for the species being tested. review of instruments and methods used, but the expense is
Erythrocytes and platelets vary considerably in volume beyond the means of most private practices.
among animal species, and instruments must be adjustable to Manual methods are also used as quality control measures
accurately count and size these blood cells for the species for automated hematology instruments. A PCV can be mea-
being assayed. Cats naturally have large platelets and moder- sured following centrifugation of a blood sample for compari-
ately small erythrocytes. The resultant overlap of erythrocyte son to the HCT determined electronically. If values do not
and platelet size makes separation of cat platelets and eryth- match within a couple of percentage points after adequate
rocytes unreliable with the use of impedance counters.77 Con- sample mixing, it suggests that the MCV or the RBC count
sequently cat platelet counts are spuriously decreased when is probably incorrect, because these two values are used to
measured with impedance counters. This inclusion of platelets determine the HCT.
in the erythrocyte measurements can result in increased Stained blood films should be examined as a part of each
erythrocyte counts and HCT values and reduced MCV CBC. The blood film is scanned to verify that the automated
and MCHC values, but the ratio of platelets to erythrocytes total leukocyte count and the platelet count appear to be
is usually not large enough to have appreciable effects on correct. If the automated platelet count is low, it is especially
these parameters (exceptions include cats with severe anemia important to examine for platelet clumps that could result in
and/or marked thrombocytosis).76 Leukocytes are generally a spuriously low count. If an electronic differential leukocyte
included in erythrocyte measurements, but the ratio of leuko- count is to be reported, one must review the blood film to
cytes to erythrocytes is usually not large enough to have verify that the percentages of each leukocyte type recorded
appreciable effects on erythrocyte parameters (exceptions appear to be correct and that there are no other cell types
include animals with severe anemia and marked leukocytosis). present that are not identified (e.g., basophils are not identi-
In these instances the inclusion of leukocytes in erythrocyte fied by most electronic cell counters). More details concerning
measurements can result in increased erythrocyte counts, the estimation of cell counts and the proper examination of
HCT and MCV values, and reduced MCHC values because stained blood films are given later in this chapter.
leukocytes are larger than erythrocytes.76 These errors result-
ing from difficulties in separating cell type by size alone should
be minimized in automated cell counters that separate cells B L O O D - F I L M P R EPA R AT I O N
not only by size but also by internal complexity.
With the advent of new laser flow cytometry techniques, Blood films should be prepared within a couple of hours of
it is now possible to perform automated differential leukocyte blood sample collection to avoid artifactual changes that will
counts; however, most instruments cannot accurately count distort the morphology of blood cells. Blood films are pre-
basophils. For most instruments, a high basophil count is an pared in various ways including the slide (wedge) method,
error,35 as has been noted in old blood samples.63 These flow coverslip method, and automated slide spinner method. It is
cytometers must be specifically calibrated for each species, and essential that a monolayer of intact cells is present on the slide
they work best when leukocyte morphology is normal. More so that accurate examination and differential leukocyte counts
reliable flags are needed to identify the presence of left shifts can be performed. If blood films are too thick, cells will be
and neoplastic cells.35 shrunken and may be difficult to identify. If blood films are
The examination of a stained blood film is essential as a too thin, erythrocytes will be flattened and lose their central
quality control measure regardless of the technology used to pallor and some leukocytes (especially lymphocytes and blast
count blood cells. In addition to verifying the accuracy of cells) will be ruptured.30
leukocyte and platelet counts, a number of other evaluations
are made. Examples include determining whether erythrocyte Glass-Slide Blood-Film Method
polychromasia, erythrocyte shape abnormalities, neutrophilic A clean glass slide is placed on a flat surface and a small drop
left shifts, neutrophil toxicity, reactive lymphocytes, blast cells, of well-mixed blood is placed on one end of the slide (Fig.
mast cells, and/or blood parasites are present. 2-10). This slide is held in place with one hand and a second
glass slide (spreader slide) is placed on the first slide and held
Quality Control between the thumb and forefinger with the other hand at
Commercial control samples (preferably at two levels) about a 30- to 45-degree angle in front of the drop of blood.
approved by the instrument’s manufacturer should be run each The spreader slide is then backed into the drop of blood, and
day; the values obtained should fall within the confidence as soon as the blood flows along the back side of the spreader
intervals supplied with the control sample.37 Proficiency slide, the spreader slide is rapidly pushed forward. The
 C ha p ter 2 n Hematology Procedures 19

A B

A B C

C D

FIGURE 2-10
Slide blood-film preparation. A, A glass slide is placed on a flat surface D E
and a small drop of well-mixed blood is placed on one end of the slide
using a microhematocrit tube. B, A second glass slide (spreader slide) is FIGURE 2 -11
placed on the first slide at about a 30-degree angle in front of the drop Coverslip blood film preparation. A, B, One clean coverslip is held
of blood. The spreader slide is then backed into the drop of blood. C, As between the thumb and index finger of one hand and a small drop of
soon as the blood flows along the back side of the spreader slide, the blood is placed in the middle of it using a microhematocrit tube. C, A
spreader slide is rapidly pushed forward. D, The blood film produced is second clean coverslip is dropped on top of the first in a crosswise posi-
thick at the back of the slide, where the drop of blood was placed, and tion. D, Blood spreads evenly between the two coverslips and a feathered
thin at the front (feathered) edge of the slide. edge forms at the periphery. E, The coverslips are rapidly separated by
grasping an exposed corner of the top coverslip with the other hand and
pulling apart in a smooth, horizontal manner.
thickness of the smear is influenced by the size of the blood
drop, the viscosity of the sample (HCT), the angle of the
spreader slide, and the speed of spreading. The greater the One coverslip is held between the thumb and index finger of
angle (a more upright spreader slide) and faster the speed of one hand and a small drop of blood is placed in the middle
spreading, the thicker and shorter the blood film will be. Thus of the coverslip using a microhematocrit tube. The drop of
the spreader slide should be held more upright in preparing blood should be as perfectly round as possible to produce even
smears from anemic patients so as to create a thicker blood spreading between coverslips. The second coverslip is dropped
film.30 on top of the first in a crosswise position. After the blood
If the drop of blood is the proper size, all of the blood will spreads evenly between the two coverslips and a feathered
remain on the slide and a smear will be prepared that is thick edge forms at the periphery, the coverslips are rapidly sepa-
at the back of the slide, where the drop of blood was placed, rated by grasping an exposed corner of the top coverslip with
and thin at the front (feathered) edge of the slide. If the drop the other hand and pulling apart in a smooth, horizontal
of blood is too large, some of the blood will be pushed off the manner. Coverslips are immediately dried as described above
end of the slide, causing potential problems. Often these blood and then identified by marking on the thick end of the smears
films will be too thick for accurate evaluation. Second, clumps with a graphite pencil or a pen containing ink that is not
of cells tend to be pushed off the slide, making them unavail- removed by alcohol fixation.
able for examination. If the drop of blood used is too large, a feathered edge will
Once prepared, the slide is immediately dried by waving it not form and the blood film will be too thick. Multiple cov-
in the air or holding it in front of a hair dryer set on a slightly erslip blood films may be stained in small slotted coplin jars
warm-air setting. Holding the slide close to a dryer set on a or in ceramic staining baskets that are placed in beakers of
hot-air setting can result in fragmentation of cells. Slow fixative and stain.
drying can cause cells to contract, making them difficult to
identify.30 Slides are identified by writing on the thick end of
B L O O D - F I L M S TA I N I N G
the smear or the frosted end of the slide with a graphite pencil
P RO C ED U R E S
or a pen containing ink that is not removed by alcohol
fixation. Romanowsky-Type Stains
Blood films are routinely stained with a Romanowsky-type
Coverslip Blood-Film Method stain (e.g., Wright or Wright-Giemsa) either manually or
Two 22-mm square No. 1 1 2 coverslips are required to make using an automatic slide stainer. Romanowsky-type stains are
coverslip blood films (Fig. 2-11). A camel’s hair brush is used composed of a mixture of eosin and oxidized methylene blue
to remove particles from the surfaces that will contact blood. (azure) dyes. The azure dyes stain acids, resulting in blue to
20 VETERINARY HEMATOLOGY

A B

FIGURE 2-12
Appearance of blood films from a normal cat stained with Wright-Giemsa. A, Blood film was rinsed in distilled water. Five neutrophils, a basophil
( far right), and a lymphocyte (round nucleus) are present. Erythrocytes exhibit rouleaux, a normal finding in cats. B, Blood film was rinsed in tap
water. A neutrophil (left), monocyte (bottom right), and lymphocyte (top right) are present. The blue color of the erythrocytes results from using water
with inappropriate pH.

purple colors, and eosin stains bases, resulting in red color-

ation (Fig. 2-12, A). These staining characteristics depend on
the pH of the stains and the rinse water as well as the nature
of the cells present (Fig. 2-12, B). Low pH, inadequate stain-
ing time, degraded stains, or excessive washing can result in
excessively pink-staining blood films. High pH, prolonged
staining, or insufficient washing can result in excessively blue-
staining blood films.30
Blood films should be fixed in methanol within 4 hours
(preferably within 1 hour) of preparation. If the methanol
contains more than 3% water, morphologic artifacts including
loss of cellular detail and vacuolation may be present.30 Blood
films may have an overall blue tint if stored unfixed for long
periods before staining or if the unfixed blood films are
exposed to formalin vapors, as occurs when blood films are
shipped to the laboratory in a package that also contains
formalin-fixed tissue. Blood films prepared from blood col- FIGURE 2 -13
lected with heparin as the anticoagulant have an overall Refractile inclusions in erythrocytes from a horse are artifacts resulting
magenta tint owing to the mucopolysaccharides present.30 from drying or fixation problems. Erythrocytes exhibit rouleaux, a normal
finding in horses. Wright-Giemsa stain.
Drying or fixation problems can result in variably shaped
refractile inclusions in the erythrocytes; these may be confused
with erythrocyte parasites (Fig. 2-13). The presence of stain obtained by longer staining procedures, but staining quality is
precipitation can make identification of leukocytes and blood improved by allowing the blood film to remain in the fixative
parasites difficult (Fig. 2-14). Precipitated stain may be present for several minutes. The Diff-Quik stain is classified as an
because the stain or stains needed to be filtered, the staining aqueous stain, even though the fixation is done in methanol,
procedure was too long, or the washing was not sufficient. because the component stains were prepared in water. A sig-
Carboxymethylcellulose has been infused into the peritoneal nificant limitation of the Diff-Quik stain and other aqueous
cavity of horses and cattle in an attempt to prevent abdominal stains, such as Hema 3 (Fisher Scientific, Pittsburgh, PA) and
adhesions after surgery. This material can appear as a precipi- the stain used in the automated stainer Aerospray 7120
tate between cells in blood that resembles stain precipitation (Westcore, Inc, Logan, UT), is that they do not stain basophil,
(Fig. 2-15).6 mast cell, or large granular leukocyte granules well (Fig.
Various rapid stains, such as Diff-Quik (Dade Behring 2-16).1 However, these aqueous stains are superior to conven-
Inc., Newark, DE), are available. The quality of Diff-Quik- tional methanolic Wright or Wright-Giemsa stains in the
stained blood films is generally somewhat lower than that staining of distemper inclusions in canine blood cells.1,26
 C ha p ter 2 n Hematology Procedures 21

A B

FIGURE 2 -16
Band basophils in the blood of a horse. A, Granules stain well with
Wright-Giemsa. B, Most granules are not stained with Diff-Quik stain.

FIGURE 2-14
Stain precipitation in blood from a dog. The two neutrophils present
might be mistaken for basophils because of the adherent precipitated
stain. Wright-Giemsa stain.

FIGURE 2 -17
Reticulocytes in dog blood. Four reticulocytes (with blue-staining mate-
rial) and three mature erythrocytes in blood from a dog with a regenera-
tive anemia. New methylene blue reticulocyte stain.

reticulocytes. The use of a Miller’s disc in one of the microscope

FIGURE 2-15
oculars saves time in performing the reticulocyte count.
Carboxymethylcellulose precipitation. The blue-to-purple precipitate
present between erythrocytes in this horse blood results from treatment The blue-staining aggregates or “reticulum” seen in reticu-
with carboxymethylcellulose. Wright-Giemsa stain. locytes (Fig. 2-17) does not occur as such in living cells but
results from the precipitation of ribosomal ribonucleic acid
Photograph of a stained blood film from a 1994 ASVCP slide review case (RNA; the same RNA that causes the bluish color seen in
submitted by M.J. Burkhard, M.A. Thrall, and G. Weiser. polychromatophilic erythrocytes) in immature erythrocytes
during the staining process.33 As a reticulocyte matures, the
number of ribosomes decreases until only small punctate (dot-
Reticulocyte Stains like) inclusions are observed in erythrocytes (punctate reticu-
Reticulocyte stains are commercially available (N. Brecher, locytes) stained with the reticulocyte stain (Fig. 2-18). To
Harleco, EMD Chemicals). Those wishing to prepare their reduce the chance that a staining artifact would result in
own stain can do so by dissolving 0.5 g of new methylene blue misclassifying a mature erythrocyte as a punctate reticulocyte
and 1.6 g of potassium oxalate in 100 mL of distilled water. when using a reticulocyte stain, the cell in question should
Following filtration, equal volumes of blood and stain are have two or more discrete blue granules that are visible without
mixed together in a test tube and incubated at room tempera- requiring fine focus adjustment. These inclusions should be
ture for 10 to 20 minutes. After incubation, blood films away from the cell margin to avoid confusion with hemotro-
are made and reticulocyte counts performed by examining phic mycoplasmas (formerly Haemobartonella organisms) or
1000 erythrocytes and determining the percentage that are small Heinz bodies.
22 VETERINARY HEMATOLOGY

FIGURE 2-18 FIGURE 2 -19

Reticulocytes in cat blood. Three whole aggregate reticulocytes (contain- Reticulocytes in the blood of a dog. Five reticulocytes (with blue-staining
ing blue-staining aggregates of RNA) and half of an aggregate reticulo- material) in blood from a dog with a regenerative anemia in a new
cyte (far right) in blood from a cat with a markedly regenerative anemia. methylene blue-stained wet preparation. Note the difference in morphol-
A majority of the remaining cells are punctate reticulocytes containing ogy compared with reticulocytes stained with a standard reticulocyte
discrete dot-like inclusions. New methylene blue reticulocyte stain. stain (see Figure 2-17). New methylene blue-stained wet preparation.

In normal cats as well as in cats with a regenerative anemia,

the number of punctate reticulocytes is much greater than New Methylene Blue “Wet Mounts”
that seen in other species.2 This apparently occurs because A new methylene blue “wet mount” preparation can be used
the maturation (loss of ribosomes) of reticulocytes in cats is for rapid information concerning the number of reticulocytes,
slower than in other species. Consequently reticulocytes in platelets, and Heinz bodies present on a blood film. The stain
cats are classified as aggregate (if coarse clumping is observed) consists of 0.5% new methylene blue dissolved in 0.85% NaCl.
or punctate (if small individual inclusions are present). Per- One milliliter of formalin is added per 100 mL of stain as a
centages of both types should be reported. Based on compos- preservative. This stain is filtered after preparation and stored
ite results from several authors, normal cats generally have in dropper bottles. Alternatively, the stain may be stored in a
from 0% to 0.5% aggregate and 1% to 10% punctate reticu- plastic syringe with a 0.2-µm syringe filter attached so that
locytes as determined by manual means. Higher punctate the stain is filtered as it is used. Dry, unfixed blood films are
numbers of 2% to 17% have been reported using flow stained by placing a drop of stain between the coverslip and
cytometry.46 a glass slide. This preparation is not permanent and does not
The percentages of aggregate reticulocytes in cats directly stain mature erythrocytes or eosinophil granules. Punctate
correlate with the percentages of polychromatophilic erythro- reticulocytes are not demonstrated, but aggregate reticulocytes
cytes observed in blood films stained with Wright-Giemsa.2 appear as erythrocyte ghosts containing blue to purple granu-
Aggregate reticulocytes mature to punctate types in a day or lar material (Fig. 2-19). Platelets stain blue to purple, and
less. Several more days are required for maturation (total dis- Heinz bodies appear as refractile inclusions within erythro-
appearance of ribosomes) of punctate reticulocytes in cats.9,15 cyte ghosts. Although this staining method is not optimal for
In contrast to those of the cat, most reticulocytes in differential leukocyte counts, the number and type of leuko-
other species are of the aggregate type. Consequently no cytes present can be appreciated.
attempts are made to differentiate the stages of reticulocytes
in species other than the cat. In most species, the percentages Iron Stains
of reticulocytes directly correlate with the percentages of poly- An iron stain such as the Prussian blue stain is used to verify
chromatophilic erythrocytes observed on routinely stained the presence of iron-containing (siderotic) inclusions in blood
blood films. and bone marrow cells and to evaluate bone marrow iron
In examining a reticulocyte-stained blood film, one should stores. Smears may be sent to a commercial laboratory for this
also be careful not to confuse precipitated RNA with Heinz stain, or a stain kit can be purchased and applied in house
bodies. Heinz bodies are composed of denatured precipitated (Harleco Ferric Iron Histochemical Reaction Set, #6498693,
hemoglobin. They are spherical, stain pale blue with reticulo- EM Diagnostic Systems, Gibbstown, NJ). Iron-positive mate-
cyte stains, and are usually found at the periphery of the rial stains blue, in contrast to the pink color of the cells and
erythrocyte. the background when this stain is applied.
 C ha p ter 2 n Hematology Procedures 23

A B

FIGURE 2-20
Neutrophils containing hemosiderin (sideroleukocytes) in blood from a
dog with a hemolytic anemia. A, Wright-Giemsa stain. Hemosiderin
inclusions stain gray or brownish. B, Prussian blue stain. Hemosiderin
inclusions stain dark blue.
FIGURE 2 -21
The presence of focal areas of basophilic stippling within Autoagglutination of erythrocytes in blood from a dog with immune-
erythrocytes stained with Romanowsky-type blood stains sug- mediated hemolytic anemia and marked leukocytosis. Wright-Giemsa
stain.
gests that the stippling may contain iron. Iron-containing eryth-
rocytes are referred to as siderocytes. Neutrophils and monocytes
may also contain dark bluish-black or greenish iron-positive
particles within their cytoplasm when they are stained with
Romanowsky-type stains. Leukocytes containing iron-positive
inclusions have been called sideroleukocytes (Fig. 2-20).
Prussian blue stain applied to bone marrow aspirate smears
is a useful way to evaluate the amount of storage iron present
in the marrow. Minimal or no iron is expected in iron defi-
ciency anemia (although cats normally have no stainable iron
in the marrow), whereas normal or excess iron may be observed
in animals with hemolytic anemia and those with anemia
resulting from decreased erythrocyte production.

Cytochemical Stains
A variety of cytochemical stains—such as peroxidase,
chloroacetate esterase, alkaline phosphatase, and nonspecific
esterase—are utilized to classify cells in animals with acute
myeloid leukemias.22,33,50 Reactions vary not only by cell type
and stage of maturation but also by species.50 These stains are FIGURE 2 -22
done in a limited number of laboratories, and special training Leukocyte aggregate in blood from a dog. Leukocyte aggregates were
and/or experience is required to interpret the results. The present when EDTA was used as the anticoagulant but not when citrate
appearance of positive reactions also varies depending on the was used as the anticoagulant. Wright-Giemsa stain.
reagents used. Because of the complexities of the staining pro-
cedures and interpretation of results, minimal information on
cytochemical stains is presented in this book. As more antibod-
ies become available for immunophenotyping acute myeloid Blood films are generally examined following staining with
leukemias, the need for cytochemical stains will decrease.60 Romanowsky-type stains such as Wright or Wright-Giemsa.
These stains allow for the examination of erythrocyte, leuko-
cyte, and platelet morphology. Blood films should first be
EXA M I N AT I O N O F S TA I N ED scanned using a low power objective to estimate the total
BLOOD FILMS leukocyte count and to look for the presence of erythrocyte
autoagglutination (Fig. 2-21), leukocyte aggregates (Fig.
An overview and organized method of blood film examina- 2-22), platelet aggregates (Fig. 2-23), microfilaria (Fig. 2-24),
tion are presented here. Descriptions and photographs of and abnormal cells that might be missed during the differen-
normal and abnormal blood cell morphologies, inclusions, and tial leukocyte count. It is particularly important that the feath-
infectious agents are given in subsequent chapters. ered end of blood films made on glass slides be examined
24 VETERINARY HEMATOLOGY

FIGURE 2-23 FIGURE 2 -25

Platelet aggregate in blood from a cow. Wright-Giemsa stain. Leukocytes concentrated in the feathered edge of a blood film from a
dog. The blood film was prepared using glass slides. Wright-Giemsa
stain.

FIGURE 2-24 FIGURE 2 -26

Dirofilaria immitis microfilaria in blood from a cat with heartworm Platelet aggregate in the feathered edge of a blood film from a cat. The
disease. Wright-Giemsa stain. blood film was prepared using glass slides. Wright-Giemsa stain.

because leukocytes (Fig. 2-25) and platelet aggregates (Fig. Leukocyte Evaluation
2-26) may be concentrated in this area. Conversely, aggregates As a quality control measure, the number of leukocytes present
of cells tend to be in the center of blood films rather than at should be estimated to assure that the number present on the
the feathered edge when the coverslip blood-film method is slide is consistent with the total leukocyte count measured. If
used. 10× oculars and a 10× objective are used (100× magnification),
In examining a glass slide blood film, the blood film will the total leukocyte count in blood (cells per microliter) may
be too thick to evaluate blood cell morphology at the back of be estimated by determining the average number of leukocytes
the slide (Fig. 2-27, A) and too thin at the feathered edge, present per field and multiplying by 100 to 150. If a 20×
where cells are flattened (Fig. 2-27C). The optimal area for objective is used, the total leukocyte count may be estimated
evaluation is generally in the front half of the smear behind by multiplying the average number of leukocytes per field by
the feathered edge (Fig. 2-27, B). This area should appear as 400 to 600. The correction factor used may vary, depending
a well-stained monolayer (a field in which erythrocytes are on the microscope used. Consequently the appropriate cor-
close together with approximately half of the cells touching rection factors for the microscope being used should be deter-
each other). mined by performing estimates on a number of blood films