HOW-TO SESSION:
CLINICAL PATHOLOGY
How to Estimate White Blood Cell Mass and
Differential From a Peripheral Blood Smear
Kelsey A. Hart, DVM, PhD, DACVIM (LAIM)*; and
Melinda S. Camus, DVM, DACVP
Authors’ addresses: Departments of Large Animal Medicine (Hart) and Pathology (Camus),
University of Georgia, College of Veterinary Medicine, Athens, GA 30602; e-mail:
[email protected]. *Corresponding and presenting author. © 2015 AAEP.
1.
Introduction
Knowledge of the white blood cell (WBC) count
and differential distribution of the leukogram is
integral to correctly diagnosing and treating disease in any ill animal. In ambulatory equine
practice, obtaining this information typically requires submitting a complete blood count (CBC) to
a diagnostic laboratory, and is often associated
with a delay of a day or more until the results are
available. This can delay appropriate diagnosis
and initiation of therapy and ultimately can affect
the patient’s prognosis.
Although board-certified veterinary clinical pathologists are the most qualified to perform detailed microscopic analysis of blood and other
samples, general practitioners can use simple
techniques and inexpensive tools to make a peripheral blood smear from the CBC tube and use
this to rapidly estimate the WBC mass and differential. This information can help practitioners
make a tentative diagnosis and institute appropriate therapy without delay while awaiting confirmatory results from the laboratory. The same
tools can also be used for other cytologic applications in practice, ultimately improving patient
care and client satisfaction due to decreased delays in treatment while providing additional income through billable services.
Even in a referral institution with an in-house
clinical pathology service during routine hours,
evaluation of blood smears and cytologic samples
from out-of-hours cases can be very useful to expediently confirm presumptive diagnoses and initiate appropriate treatment. The goals of this
session are to demonstrate how to make and interpret a peripheral blood smear to estimate the
WBC mass and differential in equine samples, and
to illustrate common leukocyte abnormalities encountered in equine practice. The accuracy of
bench-top CBC analyzers at providing a differential cell count varies substantially among models:
some impedance counters are only able to provide
a total WBC count, whereas flow cytometer models
can provide an extremely accurate differential.
However, even with top-of-the-line equipment
with excellent cell type differentiation, a manual
differential and peripheral blood smear interpretation is considered standard of care in veterinary
clinical pathology laboratories to ensure that important cytologic findings such as nucleated eryth-
NOTES
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2.
3.
4.
5.
Fig. 1. Diagram of the regions of a peripheral blood smear. The
gray area at one end of the slide represents the frosted end that
is included for easier labeling on some slides; your slides may or
may not include a frosted end. The “butt end” is the area where
the drop of blood was initially placed before being smeared by the
pusher slide.
6.
7.
rocytes, band neutrophils, toxic changes in
leukocytes, or neoplastic cells are not missed.
2.
Materials and Methods
There are a number of different ways to prepare and
stain a peripheral blood smear,1 and as long as the
chosen method produces a “feathered edge” and a
region where the cells are spread in a monolayer
(Fig. 1), the particular method used is really a matter of personal preference. The method described
below has been compiled from best practices used by
clinical pathology and large-animal internal medicine personnel in our hospital who routinely prepare
and evaluate peripheral blood smears in routine and
emergent cases.
What you will need:
● A properly filled ethylenediaminetetraacetic
acid (EDTA) anti-coagulated blood sample
(purple top tube) from the patient
● Plain glass microhematocrit tubes (no heparin)
or two wooden-handled Q-tips
● Two glass microscope slides and coverslips
● Commercial hematology stain set (any stain in
the Romanowsky family of stains is acceptable.)
● A microscope, minimally with a 10⫻ objective
and a 40⫻ objective, and ideally with a 100⫻
(oil immersion) objective. There are a number of reasonably priced ($400 – 800), easy-touse microscopes suitable for general use in
clinical practice.
Preparation of the Blood Smear
1. Blood smears should be made within 2 hours
of collection to prevent artifacts that alter
cell morphology. Store the sample in a
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8.
refrigerator if you cannot make the smear
immediately. Make sure the sample is
brought back to room temperature prior to
making slides and that the slides themselves
are never refrigerated.
Mix the sample by repeated inversion for
30–60 seconds if recently collected, or for 3–5
minutes if it has been refrigerated.
Place one microscope slide on the table with a
long side facing you.
Partially fill the microhematocrit tube with
blood OR dip a wooden-handled applicator
stick into the well-mixed blood tube.
Place a small drop of blood on the slide approximately 0.5 cm from the short end of the slide
closest to your dominant hand.
Take a second slide (pusher slide), hold it on
the long sides fairly close to one short end,
and gently touch it at an approximately 30°
angle onto the first slide, adjacent to the
blood drop.
Slowly back the pusher slide into the blood
drop, wait while the blood spreads to the edge
of the slide, then gently and smoothly push the
pusher slide across the first slide in one smooth
motion to the end of the slide.
a. Do not push down: the weight of the slide
alone will spread the blood.
b. If you must make additional smears, you
may reuse the pusher slide as long as you do
not reuse the same edge that was used to
push the blood on the first attempt.
Dry the slide immediately by waving it in the
air to prevent crenation of the cells.
Staining of the Blood Smear
There are a number of different ways to stain blood
smears and cytologic preparations,1 but for general
applications a three-step Romanosky-type stain
such as Wright’s stain is simplest and most useful.
This generally will stain proteins pink, DNA/nuclei
purple, and bacteria and fungi blue. Follow the
manufacturer’s directions for specific stains. If you
plan to stain and analyze “dirty” samples (e.g., fecals, transtracheal washes, abscess aspirates, etc.),
it is helpful to keep two sets of stain: one for clean
samples such as blood smears, and one for the likely
contaminated samples listed above. In addition,
make sure to store and change out your stain properly according to the manufacturers’ protocols, to
avoid contamination with bacteria, fungus, or stain
precipitate that can impair your ability to analyze
your stained samples.
In our laboratory, we use a stain with the following basic protocol:
1.
Once slide is air dried, hold it by one short end
away from the sample and dip it in the fixative
(clear or light blue) for 10 –20 dips (⬃15 s),
HOW-TO SESSION:
CLINICAL PATHOLOGY
Fig. 3. Illustration of the pattern used for systematic evaluation
of the slide to perform a 100-cell differential cell count.
Fig. 2. Photomicrograph of the appearance of the monolayer at
40⫻ magnification.
then blot the short end opposite to the one you
are holding on a paper towel.
2. Dip the slide in the stain (pink), for 10 –20
dips (⬃15 s) and blot the short end again.
3. Dip the slide in the counterstain (purple) for
10 –20 dips (⬃15 s), and blot the short end,
then rinse gently with tap water.
4. Air dry again by waving in the air before viewing under the microscope, and label it with the
patient name or number on one edge away
from the smear.
Examination of the Blood Smear
1. Visually examine the slide to determine the
best area for microscopic examination (Fig.
1).
2. Place the slide on the microscope stage, and
focus on it using the lowest power objective (4⫻
or 10⫻). Find an edge of the smear near the
butt end and then quickly scan the whole
smear from butt end to feathered edge to note
cell clumps and thin areas.
3. Using the 10⫻ objective, examine the feathered edge. Big things such as platelet clumps
and large cells get dragged out to the feathered
edge. Equine platelets frequently clump in
EDTA anticoagulant, which can cause the automated platelet count to be falsely low. If
you see platelet clumping, do not panic over a
low platelet count on a CBC.
4. Now move three to four 10⫻ fields back into
the smear, to the monolayer, assuming that
the patient is not anemic. This is where
cellular morphology is best evaluated and
where the differential WBC count is best performed. You can tell you are at the right
part of the slide when erythrocytes are close
together but not touching (Fig. 2). If you
cannot find an area like this on the slide,
make another smear and try again.
5. Place a coverslip on the slide and change to the
40⫻ objective. The coverslip is important be-
cause it is required for the 40⫻ objective to
focus properly. Scan back and forth across
the short axis of the slide, noting the following:
a. Do
you
see
platelets? Are
they
clumped? You should see 15–20 platelets
per field at this magnification unless they
are severely clumped.2
b. Examine leukocyte morphology.
i. Get a general idea: are they all neutrophils or all lymphocytes? Horses
should have more neutrophils than lymphocytes (Based on equine reference
ranges established in our laboratory,
this is usually at least an approximately
2:1 to 3:1 neutrophil-to-lymphocyte
ratio.)
ii. Get a general number: usually you see
one to four leukocytes per field at this
magnification, so a lot fewer or a lot
more than this might be abnormal.
iii. Cell morphology specifics (see results
below for examples)
1. Mature vs immature neutrophils
2. Toxic changes in neutrophils
3. Reactive lymphocytes
4. Abnormal lymphocytes
5. Optional: are there any visible organisms (e.g., Babesia spp., Anaplasma
phagocytophilum)? Note: visualization of some organisms requires 100⫻
oil, so they could be missed if you are
only using a 40⫻ objective.
c. Perform a 100-cell differential cell count by
scanning back and forth across the short
axis of the slide (Fig. 3). This will require
either an actual differential counter, or a
differential worksheet where you make a
hash mark next to each cell type as you
scan, then tally up the number in each category when you reach 100 (see Appendix 1).
6. Optional: change to 100⫻ (oil) objective to
evaluate:
a. Erythrocyte morphology: size and shape,
and presence of any parasites or Heinz
bodies
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b. Estimated platelet concentration: take an
average over 10 fields (⬃7–22/100⫻ field is
adequate)
c. Note: if you elect to use an oil immersion
objective for higher magnification, take care
not to go back to the 40⫻ objective without
thoroughly cleaning the oil off the slide
bychanging to a new coverslip, or by wiping the coverslip with lens cleaner and
lens paper. If oil gets on the the 40⫻
objective, it will damage it and render it
unusably blurry without removal and
proper cleaning.
3.
Results
The above methodology is recommended for use in
the field because it is quick, inexpensive, and user
friendly. Below are three case examples that exemplify applications of this technique and commonly
encountered leukocyte abnormalities encountered in
equine practice.
●
●
●
Case 1: Peripheral blood smear from an 18year-old Quarter Horse gelding with colitis
(Fig. 4).
Case 2: Peripheral blood smear from a
5-year-old Warmblood mare with fever and
distal limb edema (Fig. 5).
Case 3: Peripheral blood smear from a
9-month-old Saddlebred foal with fever and
cough (Fig. 6).
Estimation of peripheral leukocyte counts from
analysis of blood smears can be inaccurate. When
seven experienced laboratory personnel in our laboratory analyzed the same 10 blood smears from different species in a prospective study, estimated
WBC count varied substantially among personnel
(coefficient of variation, 13– 43%), and less than half
the samples were acceptably consistent with WBCs
determined with the gold standard automated cell
counter.a Sources for error include different counting areas, counting broken cells, and variation in
microscope aperture size. As a result of our study,
WBC estimates are now interpreted qualitatively in
our laboratory (e.g., low/normal/high) and quantitative estimates are no longer given. Thus, implementation of this technique in your practice should
complement rather than replace automated complete blood counts.
4.
Discussion
Preliminary evaluation of a peripheral blood
smear is a simple procedure than can be performed quickly in a field setting and does not
require expensive equipment. Information regarding total leukocyte mass, leukocyte differential, platelet mass, and even detection of some
infectious organisms can be gleaned with this procedure, facilitating clinical decision making about
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2015 Ⲑ Vol. 61 Ⲑ AAEP PROCEEDINGS
Fig. 4. Peripheral blood smear images from a horse with colitis,
illustrating neutropenia and toxic changes (Döhle bodies, cytoplasmic basophilia, and cytoplasmic vacuolation). A, Many
fields in the monolayer region had no visible leukocytes in contrast with the expected one to four per field, due to the decreased
white blood cell mass. B, There is only one leukocyte (a toxic
band) in this field. C, On the left is a band neutrophil, with a
U-shaped nucleus, vs a mature, segmented neutrophil on the
right. Note that the band is also larger with more basophilic
cytoplasm. 100⫻ magnification, modified Wright’s stain.
the need for additional diagnostics, and permitting initiation of appropriate therapy without having to wait for the results of send-out tests. The
necessary microscope can also be used to perform
HOW-TO SESSION:
CLINICAL PATHOLOGY
Fig. 5. Peripheral blood smear from a horse with fever and
distal limb edema, illustrating a cytoplasmic inclusion in the
neutrophil (arrow) consistent with Anaplasma phagocytophilum
infection (formerly Ehrlichia equi, equine Erlichiosis). Neutropenia and thrombocytopenia are also frequently seen in this
disease. Although the diagnosis is confirmed with positive serology, recognition of the organisms on a blood smear can permit
earlier initiation of appropriate antimicrobial therapy with
oxytetracycline, which is generally quite effective when started
early. 100⫻ magnification, modified Wright’s stain.
in-house fecal flotation, skin cytology, and mass
aspirates, and if centrifugation is available, the
above slide preparation and staining procedures
can also be adapted for other cytologic
Fig. 7. A, Transtracheal wash cytology from the weanling with
fever and cough in Case 3, illustrating septic, purulent inflammation, and consistent with a diagnosis of bacterial pneumonia. Note the large number of degenerate neutrophils with
swollen, pale, puffy nuclei (black arrows) and intracellular bacterial cocci (white arrow). 100⫻ magnification, modified
Wright’s stain. B, Transtracheal wash cytology from a 12-yearold mare with cough and exercise intolerance, illustrating aseptic
purulent inflammation and increased mucus. In these two images, note the nondegenerate neutrophils (white arrows), absence
of bacteria, and preponderance of mucus (streaming blue/purple
background) with the presence of Curschmann’s spirals (black
arrow). This is consistent with a diagnosis of equine allergic/
inflammatory lower airway disease such as recurrent airway
obstruction (heaves) or inflammatory airway disease. Because
this horse was symptomatic at rest, she was ultimately diagnosed
with recurrent airway obstruction. 100⫻ magnification, modified Wright’s stain.
Fig. 6. Peripheral blood smear from a weanling with fever and
cough, illustrating a lymphocytosis. Note that all four leukocytes
visible in this field are lymphocytes, in contrast with the expected
finding of a 2–3:1 neutrophil-to-lymphocyte ratio in equine peripheral blood. The foal was ultimately diagnosed with bacterial pneumonia, and this blood smear was made 11 days into the course of
disease. Lymphocytosis is not an uncommon finding in chronic
inflammatory conditions in horses, particularly in younger animals. Other differential diagnoses for lymphocytosis would include
recent viral infection or hematologic neoplasia (lymphoma or lymphocytic leukemia). 100⫻ magnification, modified Wright’s stain.
specimens such as transtracheal wash fluid (Fig.
7), synovial fluid, and peritoneal fluid. Thus, although practitioners must understand the limitations of using this technique to estimate
peripheral leukocyte counts and morphology in
the field, investment in this equipment and the
time to perform the technique can improve efficiency and accuracy of patient care and increase
practice revenue in a number of ways.
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Appendix
Acknowledgments
Name(s)
Declaration of Ethics
The Authors declare that they have adhered to the
Principles of Veterinary Medical Ethics of the
AVMA.
Species
WBC estimate: NORMAL INCREASED DECREASED
#CELLS/100 WBC COUNTED
Conflict of Interest
The Authors declare no conflicts of interest.
Segs
References and Footnote
Bands
Lymphs
Monos
a
Camus M, Bush S. A method comparison study of two
routinely used methodologies for performing nonmammalian
complete blood counts. In: Proceedings of the Annual Meeting of the American College of Veterinary Pathology/American
College of Veterinary Clinical Pathology, Montreal, Canada,
2013.
Eos
Basos
114
1. Harvey J. Atlas of Veterinary Hematology. In: Blood and
Bone Marrow of Domestic Animals. Philadelphia: Saunders, 2001;3–18.
2. Stockham S, Scott M. Fundamentals of Veterinary Clinical
Pathology. 2nd ed. Ames: Blackwell, 2008;229.
2015 Ⲑ Vol. 61 Ⲑ AAEP PROCEEDINGS