Hematology basics pt 1 jpg
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THE BLOODY TRUTH ABOUT CATS AND DOGSHEMATOLOGY BASICS: DIFFERENTIALS
Sarah Ouellette, CVT
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Why we run a CBC• Provides broad overview of overall health status
• Used for:• General health• Sick patients• Pre-anesthetic• Recheck abnormalities
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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
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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
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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
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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.
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Blood Smear• How to make a good blood smear
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Blood smear• Staining:
• Wright’s stain• New methylene blue
• IVG protocol: • 10 dips in each stain or 30 seconds/stain
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Blood Smear Staining• Why its important? Overstaining
• Not reliable for accurate results
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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
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RED BLOOD CELLS:ERYTHROCYTES
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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
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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
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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
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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**
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Abnormal Hemogram• Hypochromasia:
• Increased central pallor• Cells stain less overall• Low hemoglobin content
• Causes:• Iron insufficiency • Chronic blood loss• Anemia
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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
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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
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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
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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
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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
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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
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Poikilocytes• Ovalocytes/elliptocytes
• Oval/elliptical shape
• Causes:• Non-clinical
• slide preparation
• Clinically in cats with• Hepatic lipidosis • Portosystemic shunt
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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
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Schistocytes
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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
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Poikilocytes• Sphereocyte
• Smaller, more dense cell• Lack central pallor
• Causes:• Immune-mediated hemolytic anemia, fragmentation anemia, oxidative injuries, coral snake envenomation, pyruvate kinase deficiency
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Spherocytes • Canine IMHA
• Nearly all the cells in this field are spherocytes
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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
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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
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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
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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
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Howell-jolly bodies
• Howell-Jolly bodies in the blood of a (non-anemic) splenectomized dog
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RBC Inclusions• Distemper
• Aggregates of viral particles • Found in RBC,WBC, epithelial cells• Rarely seen even with positive infection• Larger than Howell-jolly bodies
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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)
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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)
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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.
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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
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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.
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Abnormal hemogram• Rouleaux formation
• Arranged in overlapping chains• Stacks of coins• Common in cats• Weak clumping (proteins)• Usually not significant
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Abnormal hemogram• Agglutination
• Irregular disorganized clumps• Irreversible • Pathologically significant• Immune mediated disease• Saline agglutination test to confirm
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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
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Reticulocytes• Upper panel: Polychromatophilic RBCs in regenerative anemia in a cat.
• Lower panel: reticulocytes stained with NMB from the same cat
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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.
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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.
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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
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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
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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
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Artifacts• Stain participate
• Can be mistaken for Inclusions or parasites
• Caused by:• Old stains• Bacterial growth
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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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???
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Questions?