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.