Focus on Heidi Ward, DVM, DACVIM Gulfcoast Veterinary Oncology and Internal Medicine Sarasota, FL

Mary Anna Thrall, DVM, MS, DACVP Ross University School of Veterinary Medicine St. Kitts, West Indies

Anemia is defined as the absolute decrease in the PCV, Hgb or RBC count. Anemia is a condition and not a diagnosis. The initial evaluation of anemia should include measurement of the PCV or HCT, quantification of the total protein, a CBC with examination of a and reticulocyte determination. The differential diagnosis of anemia is best classified based upon the severity of the anemia, bone marrow response (reticulocyte count) and size (MCV) and content (MCHC) of erythrocytes.

Packed cell volume/total protein Packed Cell Volume (PCV) is directly measured via centrifuging blood in a microhematocrit tube for 3 minutes. It is the quickest measure of the component of the blood and is affected by plasma trapping and how the red cells pack within the column. Examination of the HCT tube also yields valuable information about the color and clarity of the plasma (icterus, lipemia and hemolysis) as well as the size of the buffy coat (contains WBC and platelets). Total Protein should always be evaluated simultaneously with the PCV. The total protein concentration may help distinguish between blood loss (low PCV and TP) and hemolysis (low PCV with normal TP).

Complete blood count Hematocrit (HCT) is a calculated value obtained from the CBC analyzer. It is the product of the mean cell volume (MCV) and the red blood cell count (RBC). Anything that falsely increases or decreases the RBC or MCV will affect the HCT. Hemolysis will cause a decrease in the RBCs and thus falsely lower the HCT. Storage of blood will cause the RBCs to swell, increasing the MCV and falsely increasing the HCT.

MCV: Mean cell volume  Cats 40-50 fL Dogs 62-71 fL  MCV above reference interval: Macrocytic  MCV within the reference range: Normocytic  MCV below the reference range: Microcytic

MCHC: Mean cell hemoglobin concentration  Cats 32-36 g/dl Dogs 33.7-36.5 g/dl  MCHC above the reference interval: Hyperchromic  MCHC within the reference interval: Normochromic  MCHC below the reference interval: Hypochromic

RDW Index The red cell distribution width (RDW) is the variation in RBC volume and is the electronic equivalent of on a blood film; high RDW indicates more variation than normal. The blood film  Polychromatophils: Immature RBCs that stain blue-purple on blood smears (evidence of regeneration)  Spherocytes: Smaller and darker than normal RBCs. Moderate to marked numbers are highly suggestive for IHA, but can also be seen with anemia, zinc toxicity, vasculitis, post transfusion, neoplasia and ADIC  Autoagglutination: Grape like aggregation of RBCs (immune component to the anemia)  Heinz bodies: Clumps of denatured hemoglobin that appear as small circular projections off the RBC membrane; associated with oxidative damage (acetaminophen, garlic and onion, propofol, zinc).In cats they can also be seen with hyperthyroidism, lymphoma and diabetes  Howell Jolly bodies: Basophilic nuclear remnants; associated with regenerative anemia or hyposplenism  : Basophilic dots within cytoplasm; associated with regenerative anemia or : Fragmented RBCs; indicative of DIC, hemangiosarcoma, vasculitis, heartworm and liver disease  Erythroparasites: Mycoplasma spp., Cytauxzoon felis, Babesia gibsoni and canis  Nucleated RBCs: Metarubricytes, rubricytes and earlier stages of RBC development; appropriate/expected response to anemia. Inappropriately released with any bone marrow injury, lead toxicity; can be normal in Miniature Schnauzers  Other Cytopenias or Abnormal Cells

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Reticulocyte count Reticulocytes are immature red blood cells that are released from the bone marrow before they fully extrude their RNA and cellular organelles. New methylene blue is a supravital stain that precipitates the RNA and stains it blue. There are two types of reticulocytes including aggregate (clumps o f reticulum, larger and less mature) and punctate reticulocytes (single dots of reticulum). In cats, aggregate reticulocytes are considered evidence of current regeneration while punctate reticulocytes have a long half-life and their presence may indicate an insult that occurred days to weeks prior to testing. Both types are counted together in dogs. Reticulocytes are not seen for 2-4 days after an acute episode of blood loss or hemolysis and their response peaks between 4-7 days. Procedure for in-house reticulocyte count 1. Mix equal volumes (2-3 drops) of blood and NMB stain in a test tube and incubate the mixture for at least 10 minutes, then gently remix sample and remove a drop to make a blood film; air dry 2. Count number of reticulocytes per 1,000 RBCs 3. Report as a percentage by dividing the number of reticulocytes by 10 4. To get an absolute number, multiply the above percentage by analyzer generated RBC count Uncorrected reticulocyte percentage = Number of reticulocytes/10 Corrected reticulocyte percentage = Reticulocyte % X Patients HCT /normal HCT (45 for dogs or 35 for cats) Absolute reticulocyte& (/ul) = Reticulocyte% X RBC count (in million/uL)

%Retlculocvtes Retlculocytes/ul Degree of Dogs Cats Dogs Cats Regeneration• None <60,000 <15,000 Slight 1-4 0.5-2 60,000-15,000- 150,000 50,000

Moderate 5-20 150,000- 50,000 300,000 100,000 Marked >20 >4 >500,000 >200,000

Severity of anemia based on PCV Grade of anemia Dog Cat Reference normal 41-58 31-48 Mild 30-40 25-35 Moderate 20-30 15-25 Severe <20 <15

3 major mechanisms of anemia • Hemorrhage • Usually regenerative • Hemolysis • Usually regenerative • Decreased Production • Usually non-regenerative

Interpretation based on erythrocyte indices Macrocytic, hypochromic, mild to severe regenerative anemia Hemorrhage or hemolysis Macrocytic,normochromic, mild to severe non-regenerative anemia Vitamin B12, Folate or Cobalamin deficiency, FeLV, myelodysplasia, drugs, non-regenerative IHA, hereditary stomatocytosis Normocytic, normochromic, mild to moderate non-regenerative anemia Anemia of inflammatory disease, chronic renal disease, endocrine disease, infectious agents, drugs, lymphoma, histiocytic sarcoma, severe nutritional deficiencies Primary marrow disorder (non-regenerative IHA, hematopoietic or infiltrative neoplasia, bone marrow aplasia), drugs, Multifactorial anemia Microcytic, normochromic to hypochromic, mild to severe non-regenerative anemia Iron or copper deficiency, Portosystemic shunts

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Microcytic, hypochromic, mild to severe regenerative anemia

Regenerative vs. pre-regenerative vs. non-regenerative anemia Regenerative anemia The hallmark of regenerative anemia is the presence of a , which indicates an appropriate compensatory response to anemia. It is characterized by erythroid hyperplasia in the bone marrow and increased release of red cells into the circulation before they are fully mature (polychromatophilic red cells). Causes of regenerative include hemorrhage and hemolysis. Acute hemorrhage Common causes of acute hemorrhage include trauma, coagulopathies, platelet disorders, splenic rupture and gastrointestinal loss via neoplasia and ulcerative disease. The clinical signs of blood loss are dependent upon the amount of blood lost, time and site. Clinical signs of blood loss may be apparent before the onset of a decreased PCV due to the simultaneous loss of both plasma and RBCs as well as splenic contraction. Splenic contraction delivers a high PCV blood (80%) to the circulation. Polychromasia, anisocytosis, Howell Jolly bodies, increased nRBCs and reticulocytes become evident by 48-72 hours and reach a maximum about 5-7 days after hemorrhage. The hemogram returns to normal1-2 weeks following a single acute hemorrhagic episode. Hemolysis is often associated with a stronger regenerative response than hemorrhagic anemia. The total protein is usually normal. Hemolysis can be intravascular, extravascular or a combination of both. Both types can result in icterus. Intravascular hemolysis is the result of RBC destruction/rupture within the circulation and thus can result in hemoglobinemia, hemoglobinuria and bilirubinuria. Extravascular hemolysis occurs when RBCs are phagocytized by in the , liver and bone marrow; hemoglobinemia and hemoglobinuria are not present. Causes of hemolysis include: • Immune Mediated  Primary: Idiopathic IMHA, transfusion reaction  Secondary: Infectious, neoplasia, drugs, vaccine adverse reaction • Infectious  Hemotropic mycoplasmosis, ehrlichiosis, babesiosis, cytauxzoonosis, FeLV, septicemia • Fragmentation/physical damage to RBC membrane  DIC, caval syndrome, glomerulonephritis, hemolytic uremic syndrome, hemangiosarcoma, vasculitis, heatstroke, splenic torsion, hepatic disease, severe bums • Drugs, toxins, chemical damage  Zinc (pennies minted after 1983), snake envenomation, hypophosphatemia, bee stings, spider bites, oxidants  (onions, garlic, mothballs (naphthalene,) acetaminophen) • Congenital red cell abnormalities  Phosphofructokinase deficiency (English Springer Spaniels, Cocker Spaniels), Pyruvate Kinase deficiency  (Abyssinian, Somali and DSH, Beagles and Basenjis), hereditary stomatocytosis Pre-regenerative anemia An anemia with a regenerative response that is impending, but not yet apparent on the CBC, is termed pre-regenerative. It takes 3-4 days after an acute blood loss or hemolytic event until reticulocytes are evident on the CBC and several more days until the regenerative response peaks. Non-regenerative anemia The hallmark of non-regenerative anemia is the absence of reticulocytosis, which indicates erythropoiesis is being inhibited in some way. Most non-regenerative anemias are normocytic and normochromic other than secondary to FeLV (macrocytic) and iron deficiency (microcytic). Causes of non-regenerative anemia include anemia of inflammation, iron deficiency anemia, chronic renal failure, neoplasia, endocrinopathies, non-regenerative IMHA, liver disease, FeLV, toxic insults (chemotherapy, estrogen, etc.), space occupying disease in the bone marrow and nutritional deficiencies.

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