Small Animal Hematology Lecture and Wet Lab
The Basics
Saundra E. Willis DVM DACVIM
Why Examine a Blood Smear?
To verify accuracy of bench-top analyzer
To evaluate smear for abnormalities the machine will not tell you about
• Hemoparasites
• WBC morphology – toxic change, left shift, unusual / unclassified cells
• RBC morphology, NRBCs
• Platelet morphology and clumping
Sample Handling
Take slides directly from blood draw
Fill lavender top tube
Label tubes, slides with animal first & last name
Completely dry slides before placing in plastic containers to avoid hemolysis
Test request form: Pertinent info, history, special requests
Hematology in the Clinic
Staining
Smear evaluation: 10X, 40X, 100X oil immersion
Staining Blood Smears
Change stain frequently
Hematology / cytology stain set-up should be separate from those for fecal, ear slides
Some quick stains may not stain mast cells, basophils, eosinophils well
If questions, send to lab
Basic Use of the Microscope
Adjust eye pieces
Set up binocular viewing
Kohler Illumination
Lower condenser to improve contrast
Right slide side up
Dirty and clean scopes
Kohler Illumination
Close light diaphragm
Center light
Move condenser so edges are clear
Open light diaphragm until just leaves field
On 10X
Scan the entire slide
Assess rbc/wbc distribution
Look for agglutination and/or rouleaux
Evaluate the feathered edge for platelet clumps, microfilaria, atypical cells including mast
cells
Estimate Leukocyte numbers
Count cells in 3 fields* of monolayer
Divide total # of cells by 3
Divide the average number of cells by 4
*At least 3 fields
Saline Test for Agglutination
Differentiates rouleaux & agglutination
Start with one drop blood, 10 drops buffered saline; mix gently
Examine under the microscope at 40X looking for clumps of rbc’s: Rouleaux will
disperse rbc’s and resolve clumping; clumping will remain with agglutination (clumps: 4
or more RBC’s)
Estimate Platelet Numbers
On 100x oil:
Count number of platelets in 10 fields of the monolayer
Calculate the average by dividing this number by 10
Multiply the average by 20 x 109/L
Example:
Total count is 100 in 10 fields
Average is 100/10 = 10
10 x (20 x 109/L) = 200 x 109/L
NOTE: PLATELET CLUMPING WILL FALSELY DECREASE THE PLATELET ESTIMATE
THUS SLIDE ESTIMATE OVERALL SHOULD INCLUDE AN ASSESSMENT FOR
CLUMPING.
Platelet Estimates from Smear that would indicate adequacy:
Dog
10 – 24 / 100x
Cat
15 – 40 / 100x
Horse 5 –17 / 100x
On 40x (50xoil)
Perform leukocyte (WBC) differential: neutrophils, lymphocytes, monocytes, eosinphils,
basophils, other
Assess leukocyte (WBC) morphology: color, shape, inclusions
Assess RBC morphology
Neutrophil morphology
Neutrophil morphology is important to assess as infection can present as neutropenia,
normal count, and neutrophilia
Presence of toxic change and left shift to immature forms can only be evaluated by a
blood smear evaluation. Hematology analyzers may indicate that neutrophil morphology
is abnormal but only on a smear evaluation can morphology be evaluated.
Toxic change within the neutrophil supports an active, perhaps septic source of the
inflammation. Left shift to immature forms including bands may also indicate severity
and systemic nature of the inflammation.
Neutropenia, particularly when toxic change and a left shift are present, supports an
overwhelming, potentially life threatening infection (such as sepsis) that is affecting
maturation of the neutrophils in the bone marrow.
Neutrophil Toxic Change
foamy vacuolation
blue granulation
dohle bodies (RER aggregates)
toxic/primary granules (red)
increased blue staining
Lymphocyte
Small (less than the size of a neutrophil)
Intermediate (the size of a neutrophil)
Large (larger than a neutrophil)
Cytoplasm - scant amounts of clear blue
Nucleus – round and occasionally indented/clefted
Granulated lymphocytes
Distinct pink granules within the cytoplasm
Reactive lymphocyte
Large lymphocytes with very blue cytoplasm
Lymphocytosis: Determining reactive vrs neoplastic cells
Flow cytometery
Antigen markers for different cell types
Clonality PCR on lymphocytes
Monocyte
Largest cell in peripheral blood 9-22um
Nucleus shape is extremely variable
Oval to bean, band like or segmented.
Cytoplasm is abundant blue-grey, sometimes vacuolated, grainy and coarse
Note: lymphocyte cytoplasm is often smooth
Eosinophil
Nucleus - band-formed or bilobed
Cytoplasm – orange- pink granules highly variable between species
dog - variable numbers and size (occasionally washes out to give a vacuolated
appearance -> greyhounds)
cat – numerous and rod-like
bovine – small, round and numerous
equine – large and bright orange-red like raspberries
Basophil
Nucleus lobulated
Cytoplasm - granules that vary in color and number with species:
dog – few dark violet granules
cat – pale orange-lavender granules
large animals – blue-black granules
Mast Cells vs. Basophils: A mast cells will have a round nucleus and a basophil will have a
segmented nucleus. Granules in mast cells are generally numerous and dark purple in color in
both species, nearly obliterating the nucleus. They are easily observed at 10X on scanning.
Basophil granules are small and lighter in color.
Atypical cells
Larger often round cells
Nuclei with fine chromatin
Nucleoli or nucleolar rings
Mitotic figures
Any questions, send CBC to the lab with a note for pathologist to review
WBC inclusions
Anaplasma phagocytophila
Differential
On 40x-50x, count 100 wbc’s in the monolayer area. If present, count nucleated rbc’s separately
as you go so as to determine how many nucleated rbc’s per 100 wbc’s there are. Determine
percentage, multiple by WBC count to determine absolute numbers of individual wbc’s.
Absolute Leukocyte Count
Total WBC count x differential % = Absolute number of leukocyte
Example: If WBC count is 10,000 /uL and 66/100(66%) are neutrophils
10, 000/uL x 66/100 = 6,600 /uL neutrophils
**This count is the one you want to assess on lab reports not the %
Corrected WBC Count
Use this formula if nRBCs >5 per 100 leukocytes in the differential count;
Corrected WBC count = WBC count x 100
(100 + nRBCs)
Example: If nRBCs are 15/100 WBC, WBC count is 30x109/L, the corrected WBC is
30,000/uLx100 = 3000000/uL = 26,100/uL WBC
100 + 15
115
Evaluate RBC Morphology
On 40x (50xoil):
Size
color (polychromasia/regeneration)
shape
inclusions
clumping
artifacts
Anemia
Regenerative: Hemorrhage, coagulopathy and immune-mediated hemolysis
Nonregenerative: Everything else and immune-mediated hemolysis
Nucleic Acid Remnants (seen mostly with a regenerative anemia)
Basophilic Stippling
Howell Jolly Bodies
Nucleated RBC’s
Poikilocytosis
Echinocytes: crenation, EDTA artifact
Spherocytes: immune-mediated anemia
Acanthocytes, schistocytes: red cell membrane changes, rbc fragmentation (liver, splenic
disease; DIC)
Microcytoses, hypochromasia: iron deficiency anemia from chronic blood loss
Eccentrocytes, Heinz bodies: oxidative damage (zinc pennies, onion, garlic, medications)
Oil Immersion
Estimate platelet count
Assess cell morphology
Size
Shape
Color
Parasites
Handouts will be provided at the lecture/laboratory session with examples of WBC and RBC
morphology and platelet estimates in color to complement these notes.