THE BLOODY TRUTH ABOUT CATS AND DOGSHEMATOLOGY BASICS: DIFFERENTIALS

Sarah Ouellette, CVT

Why we run a CBC• Provides broad overview of overall health status

• Used for:• General health• Sick patients• Pre-anesthetic• Recheck abnormalities

Hematology Basics• Collection/handling

SAMPLE QUALITY IS A MAJOR CONTRIBUTOR TO ANALYTICAL ERRORS

• Poor collection/handling methods can lead to:• Inadequate cell counts• Morphologic artifacts

Hematology Basics• EDTA

• anticoagulant of choice• LTT

• Correct ratio• Low blood volume can cause

• False increase in plasma protein• False decrease in PCV/RBC count• Morphologic artifacts

Things to remember!• Make blood smears soon after collection

to reduce the risk of artifacts• Especially when sending out for pathology

review

• Make a good quality smear• unfixed

Why we do a differential• CBC analyzers do NOT give us any information about specific morphological changes or specific cell types

• CBC analyzers only “suspect” abnormalities such as nRBC/band cells, etc.

Blood Smear• How to make a good blood smear

Blood smear• Staining:

• Wright’s stain• New methylene blue

• IVG protocol: • 10 dips in each stain or 30 seconds/stain

Blood Smear Staining• Why its important? Overstaining

• Not reliable for accurate results

Reading a blood smear• NEVER NEVER NEVER go looking for something abnormal – unless indicated

• Examples:• Automated analyzer suspects an abnormality:

• Nucleated RBC• Band cells

• Abnormal (high/low) cell count

RED BLOOD CELLS:ERYTHROCYTES

Erythrocytes• Non-nucleated cells• Biconcave disk• Prominent central pallor• Stain pink/salmon color

• Function: transport oxygen between lungs and tissues• Hemoglobin is the O2 carrying component of the cell

Erythrocytes• Canine:

• Lifespan = 110-120 days• Slightly larger than cats• Mild polychromasia

• Abnormalities that can be seen in healthy dogs:• Occasional nucleated RBC • Occasional howell jolly bodies

Erythrocytes• Feline:

• Lifespan: 65-76 days• Smaller than canine RBC’s• More variable in size • Little to no central pallor

• Abnormalities that can be seen in healthy cats:• Occasional heinz bodies• Small # of howell jolly bodies

Abnormal Hemogram• Polychromasia

• Color variance with RBC• Indicative of immature RBC within the blood• MILD variance is accepted in cats and dogs

• Causes:• Anemia • Bone marrow disease

**importance of staining**

Abnormal Hemogram• Hypochromasia:

• Increased central pallor• Cells stain less overall• Low hemoglobin content

• Causes:• Iron insufficiency • Chronic blood loss• Anemia

Abnormal Hemogram• Poikilocytes or poikilocytosis:

• General term used to describe RBC’s with an abnormal shape

• Some abnormalities have specific significance relating to a particular disease while others may be non-specific

Poikilocytes• Acanthocytes (spur cells)

• 2-10 blunt/club shaped projections • Different lengths• Irregular intervals

• Causes:• Dogs with hemangiosarcoma• Increased blood cholesterol, iron deficiency anemia,

renal disease, and occasionally some liver diseases

Poikilocytes• Echinocytes (spiculated or crenated cells)

• Numerous sharp/blunt projections • Same length• Evenly spaced around the cell

• Causes:• Incidental

• excessive EDTA:Blood ratio causes dehydration of cells• Drugs

• salicylates, phenylbotazone, Lasix, and certain chemotherapeutic agents

Ecchinocytes• Shown at right are two

smears from the same sample, the upper one made while the blood was fresh

• The lower made after overnight storage of blood at refrigerator temperature

Poikilocytes• Eccentrocytes

• Ragged fringe of cytoplasm along one side of cell• Dense staining• May rupture (hemi-ghosts or “pseudo-spherocytes”)

• Causes:• Oxidative damage to cell

• Ex. onion toxicity

Poikilocytes• Keratocytes (helmet cells)

• Blister-like defect along the perimeter• May rupture (bite-shaped)

• Causes:• DIC• Vasculitis• Hemangiosarcoma• Iron deficiency anemia• Hepatic lipidosis in cats

Poikilocytes• Ovalocytes/elliptocytes

• Oval/elliptical shape

• Causes:• Non-clinical

• slide preparation

• Clinically in cats with• Hepatic lipidosis • Portosystemic shunt

Poikilocytes• Schistocytes

• Cell fragments • Irregular shape (triangular)• Indicates mechanical injury

• Causes:• Dogs: DIC, glomerular disease, certain inflammatory diseases, iron deficiency anemia, splenic disease, vasculitis, vascular cancer such as hemangiosarcoma

Schistocytes

Poikilocytes• Stomatocytes

• Elongated mouth-like area of central pallor• Due to the absence of hemoglobin

• Causes:• Regenerative anemias• Liver disease• Lead poisoning

• Non-clinical: blood smear that is too thick

Poikilocytes• Sphereocyte

• Smaller, more dense cell• Lack central pallor

• Causes:• Immune-mediated hemolytic anemia, fragmentation anemia, oxidative injuries, coral snake envenomation, pyruvate kinase deficiency

Spherocytes • Canine IMHA

• Nearly all the cells in this field are spherocytes

Poikilocytes• Codocytes (target cells)

• Cluster of hemoglobinized cytoplasm within the area of normal central pallor

• Only observed in dogs

• Causes:• Cholesterol/phospholipid abnormalities• Regenerative anemia• Liver disease• Non-clinical - Excessive amounts of EDTA

Target cells• Upper panel: numerous target cells in the blood of a dog with cholestasis.

• Lower panel: target cells in a dog with regenerative anemia

RBC Inclusions

• Red blood cell inclusions can arise from a variety of sources but is important in providing insights into metabolic, physiologic and pathogenic conditions

• Inclusion = nuclear or cytoplasmic aggregates (proteins) present within the cell

RBC inclusions• Howell-jolly bodies

• Small round basophilic inclusion• Deeply staining non-functioning nuclear remnant• Rare in normal dogs• Cats may have low #’s• Removed by the spleen

• Causes:• Regenerative anemias• Splenic disease

Howell-jolly bodies

• Howell-Jolly bodies in the blood of a (non-anemic) splenectomized dog

RBC Inclusions• Distemper

• Aggregates of viral particles • Found in RBC,WBC, epithelial cells• Rarely seen even with positive infection• Larger than Howell-jolly bodies

Abnormal Hemogram• Basophilic stippling• Spontaneous aggregation of ribosomal RNA in the

cytoplasm of young erythrocytes

• Causes:• Regenerative anemia• Bone marrow disorders• Lead poisoning (absence of anemia)

RBC inclusions• Heinz Bodies

• Small pink/clear short blunt projection• Precipitates of denatured or oxidized hemoglobin• Uncommon in dog/ common in cats• Confirm w/ New methylene blue stain

• Causes• Onion toxicity (dogs)• Endogenous HB in cats

• (diabetes, lymphoma, hyperthyroidism)

Heinz bodies• Blood from a cat with

Heinz body hemolytic anemia associated with acetaminophen toxicity. Upper panel: the large Heinz bodies are causing severe distortion of the cell outline. Lower panel: stained with New Methylene Blue.

Abnormal Hemogram• Metarubricytes (nucleated RBC)

• Late stage nucleated red blood cells• Chromatin is markedly clumped and the cytoplasm is

more abundant than that of a lymphocyte

• Causes:• Regenerative anemias• Acute lead toxicity• Bone marrow disease• Septicemia

Metarubricytes/Nucleated RBC

• Left panel: metarubricyte from a dog. • Middle panel: basophilic rubricyte from a dog.• Right panel: a megaloblastoid nRBC from a cat with

myelodysplastic syndrome.

Abnormal hemogram• Rouleaux formation

• Arranged in overlapping chains• Stacks of coins• Common in cats• Weak clumping (proteins)• Usually not significant

Abnormal hemogram• Agglutination

• Irregular disorganized clumps• Irreversible • Pathologically significant• Immune mediated disease• Saline agglutination test to confirm

Abnormal hemogram• Reticulocytes

• Young non-nucleated cells• Retained ribosomal DNA• Appear as small dark blue dots within the cell• Polychromatophils stained with New Methylene blue stain

• Causes:• Anemia • Bone marrow disease

Reticulocytes• Upper panel: Polychromatophilic RBCs in regenerative anemia in a cat.

• Lower panel: reticulocytes stained with NMB from the same cat

RBC Parasites• There are several organisms that can be detected from examination of a peripheral blood smear.

• Some organisms can cause significant disease in the host while others can be non-pathogenic.

RBC parasites• Babesia canis

• Protozoal parasite transmitted via brown dog tick• Hemolytic anemia seen in in mild dz• Intravascular hemolysis seen in severe dz

• B. canis appear usually as a single or multiple pear shaped organisms within the cell

• Babesia in endemic in greyhounds.

RBC parasites• Mycoplasma haemofelis

• Formally known as hemobartonella• Transmitted through infected fleas• Epicellular bacterial parasite • Observed as small blue cocci, rings, or rods on the edges or the surface of the red cells

• Can be mistaken for stain participate• Causes hemolytic anemia

RBC parasites• Mycoplasma haemocanis

• Formally known as hemobartonella• Transmitted through infected fleas• Epicellular bacterial parasite • Recognized as chains of cocci across the face of the red cell

• Can be mistaken for stain participate• Rarely causes anemia in dogs

Artifacts• Artifacts can significantly impair the examination of a blood smear

However,They can be easily avoided

• The most common cause of these artifacts is error in handling, collection, storage and preparation of the blood smear

Artifacts• Stain participate

• Can be mistaken for Inclusions or parasites

• Caused by:• Old stains• Bacterial growth

Artifacts• Water

• Caused by precipitation in fixative• Moth eaten appearance• Refractile

• As you focus up and down on the microscope the artifact “flashes”

** Always want to make sure stains covers are on tight**

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Questions?