ERYTHROCYTES

  1. Overview: Erythrocytes (i.e. RBC) are blood cells that are primarily involved in transport of oxygen to body tissues and transport of carbon dioxide to the lungs. The major cytoplasmic protein is hemoglobin which transports both oxygen and CO2.

  2. RBC Metabolism
    1. Glycolysis (90% of glucose utilized by this route)
      1. Generates ATP needed to maintain ionic gradients across the cell membrane and maintain cell membrane flexibility.
      2. Generates NADH needed to reduce oxidized hemoglobin (i.e. methemoglobin) to oxyhemoglobin (i.e. the functional form of hemoglobin). Pathway involves methemoglobin reductase

    2. Hereditary Disorders of RBC Enzymes
      1. Pyruvate kinase deficiency - autosomal recessive disease seen in basenjis, west highland white terriers, caren terrieers and beagles. Lack of pyruvate kinase results in markedly reduced capacity to generate ATP through the glycolytic pathway
        1. see severe and persistent intravascular hemolysis
        2. dogs have severe hemolytic anemia and die within 4 years
      2. Phosphofructokinase Deficiency - seen in English springer spaniels
        1. clinically see hemoglobinuria associated with excitement or stress which induced respiratory alkalosis. Alkalosis inhibits the activity of what PFK is present.


    3. Pentose Phosphate Pathway
      1. Generates NADPH needed to reduce oxidized proteins including hemoglobin and membrane proteins and to reduce oxidized membrane phospholipids.
        oxidant
        Protein - SH----------> Protein - S
        SHS
        Disulfide bonds in proteins are reduced by glutathione (GSH).
        2GSH + Protein - S----------> GS-SG + Protein - SH
        SSH
        GSH becomes oxidized in the process and NADPH is needed to reduce GSH

        GSSG + NADPH   glutatione reductase   2GSH

      2. RBC hemoglobin is constantly being oxidized and therefore must be constantly reduced. Severe oxidation of hemoglobin results in aggregation of hemoglobin molecules which forms Heinz bodies.
      3. Other antioxidant pathways in RBC include superoxide dismutase, catalase, and vitamin E.


  3. Hemoglobin Synthesis
    1. Hemoglobin consists of 4 peptide chains (2 alpha and 2 beta chains) and 4 heme molecules.

    2. Heme synthesis occurs in the mitochrondria of developing RBC precursors.



  4. Hemoglobin disorders
    1. Mutations in the globin molecule may result in functional changes in the hemoglobin molecule such as altered affinity for oxygen.
    2. Congenital defects in the enzymes associated with heme synthesis result in accumulation of heme precursors in the body. These defects are termed congenital porphyria.


  5. RBC Circulation and Fate
    1. RBC lifespan varies with species
      Dog - 120 days    Cow - 160 days
      Cat - 60 days Pig - 60 days
      Horse - 145 days


    2. RBC destruction - old RBC undergo alteration in cell membrane antigens are recognized by macrophages primarily in the spleen. Macrophages bind and phagocytize old RBC. Iron is removed from heme and returned to the bone marrow for reuse. The heme rings are broken down to unconjugated bilirubin that is enters the plasma and is removed by the liver, conjugated, and excreted in bile.


  6. RBC MORPHOLOGY
    1. RBC Size - measured by determining mean cell volume (MCV)
      1. Anisocytosis - variation in RBC size
      2. Normocyte - normal sized RBC
        dog63-73 fl   horse39-53 fl   cow38-55 fl
        cat36-50 fl   pig50-68 fl   goat16-25 fl
      3. Microcyte - small RBC
        1. seen normally in Akita dogs and in iron deficiency anemia
      4. Macrocyte - large RBC
        1. seen with regenerative anemia (i.e. polychromasia), disorders of RBC production (i.e. feline leukemia virus infection, folate deficiency), and a congenital disorder in poodles (i.e. poodle macrocytosis).


    2. RBC color
      1. RBC color is dependent on hemoglobin concentration. Hemoglobin is measured as mean cell hemoglobin (MCH) of mean cell hemoglobin concentration (MCHC)
      2. Polychromasia - Immature RBC (i.e. reticulocytes) have not had time to produce all their hemoglobin so appear bluish rather than red. These cells show up as reticulocytes when stained with a vital stain like new methylene blue.
      3. Hypochromasia - normal size or small RBC with a large area of central pallor and a thin rim of hemoglobin.
        1. seen in iron deficiency anemia


    3. RBC shape
      1. Discocyte - normal RBC are flattened cell not round like a baloon. Dogs have a large enough RBC for the cell membrane to pinch in the center and form a biconcave disc that shows up on a blood smear as central pallor.
      2. Poikilocyte - a general term for misshapen RBC. All pigs and young goats normally have poikilocytosis in blood smears.
      3. Codocyte (target cell) - a cell that has an area of hemoglobin in the center surrounded by a clear area and an outer rim of hemoglobin
        1. seen in dogs with regenerative anemias and chronic liver disease
      4. Echinocyte - RBC with 4 to 15 regularly spaced sharp or blunt uniform projections
        1. Crenation - artifact with old blood samples, excess EDTA, slow drying of slides and high humidity
        2. Pathologic - seen in horses with electrolyte depletion and dogs and cats with a variety of disease conditions including chronic renal disease
      5. Acanthocyte - RBC with a few irregular blunt projections
        1. seen in fragmentation anemia (dogs only), chronic liver disease, and chronic renal disease
      6. Keratocyte - (helmet cell, bite cell) - RBC with a notch and projections on either side
        1. seen with fragmentation anemias
      7. Schizocyte (schistocyte) - irregularly shaped fragment of RBC
        1. seen with fragmentation anemias
      8. Spherocyte - a small, deeply stained, round, RBC that lack central pallor. Can only be seen with certainty in dogs.
        1. seen in large numbers (i.e. <50%) in immune-mediated hemolytic anemias. Low numbers can be seen normally, and in fragmentation anemias and Heinz body anemias


    4. RBC inclusions
      1. Heinz bodies - Denatured hemoglobin that precipitates as one or more round deposits in RBC. Cats normally have up to 10% Heinz bodies but other species should normally have none.
        1. Seen with oxidative injury to RBC
      2. Howell-Jolly Bodies - a single large round blue dot in RBC that represent a fragment of the nucleus.
        1. seen with regenerative anemias and splenic disease


  7. RBC PATHOLOGY: ANEMIA
    1. Anemia is defined as a decrease in hematocrit (i.e. PCV) hemoglobin concentration, or RBC count. Anemia occurs very frequently and has many causes. An organized approach to diagnosis is essential.

    2. Severity of anemia based on PCV
      Dog HorsePCV-Cat/Ruminant
      Mild30-37%29-32%20-26%
      Moderate20-29%20-29%14-19%
      Severe13-19%13-19%10-13%
      Real bad<13%<13%<10%


    3. Regenerative vs. nonregenerative anemias
      1. Regenerative anemia: anemia caused by blood loss (i.e. hemorrhage) or by RBC destruction (i.e. hemolytic anemia). In regenerative anemias, the bone marrow compensates by increasing RBC production. Increased RBC production is detected by observing increased numbers of young RBC (i.e reticulocytes or polychromasia) in the circulation.
      2. Dogs - release aggregate reticulocytes which mature rapidly to mature RBC (don't see punctate reticulocytes).
      3. Cats - release aggregate reticulocytes that mature rapidly to punctate reticulocytes. Punctate reticulocytes mature over 2 weeks. Therefore, cats have few aggregate reticulocytes and many punctate reticulocytes.
      4. Ruminants release aggregate reticulocytes but also have basophilic stippling of RBC. Both are considered indicators of RBC regeneration. Basophilic stippling looks much like punctate reticulocytes except that it is seen on Wright's stained blood smears where reticulocytes do not stain.
      5. Horses do not release reticulocytes even with marked RBC regeneration.

        Percent Reticulocyte Response
        Severity of
        anemia        		DogCat (aggregate) Cat (punctate)
        Normal1%0-0.4%1-10%
        Mild anemia1-4%0.5-2%10-20%
        Mod. anemia5-20%3-4%20-50%
        Severe anemia21-50%>5%>50%


    4. BLOOD LOSS ANEMIA
      1. Acute External Blood Loss
        1. Reticulocytes begin to increase on day 3
        2. Because plasma is lost with RBC, total plasma protein is usually decreased
      2. Chronic External Blood/Iron Deficiency Anemia
        1. Chronic blood loss eventually results in iron deficiency. As iron deficiency develops, the lack of iron limits RBC production and microcytosis and/or hypochromasia develop and anemia becomes poorly regenerative or nonregenerative.
        2. Causes of chronic blood loss: Intestinal parasites, fleas, sucking lice, bleeding gastrointestinal ulcers or neoplasia.
        3. See low total protein concentration


    5. HEMOLYTIC ANEMIAS
      1. Immune-mediated Hemolytic Anemia
        1. Caused by antibodies that bind to RBC and cause RBC hemolysis
        2. Frequently seen in dogs, less common in cats and horses
        3. Diagnosis: Spherocytosis (82% positive), Direct Coombs test (70% positive), autoagglutination (<20% positive)
      2. Heinz Body Anemia
        1. Acute intravascular hemolysis resulting from oxidative injury to RBC
        2. Canine Heinz body anemia
          • causes: ingestion of onions, methylene blue, and menadione
        3. Feline Heinz body anemia
          • healthy cats have up to 10% Heinz bodies
          • Causes: methylene blue, acetaminophen, phenazopyridine, cetacaine, propylene glycol, salmon- based diets, canned baby food diets containing onion salt, diabetes mellitus, hyperthyroidisn, and lymphosarcoma.
        4. Equine Heinz body anemia
          • causes: red maple ingestion, phenothiazine, onions
      3. Fragmentation Anemias
        1. Fragmentation of RBC results from direct physical trauma to RBC
        2. See acanthocytes (dogs only) keratocytes, and schizocytes
      4. Hereditary Hemolytic Anemias
      5. Blood Parasites
        1. Hemobartinella - causes acute extravascular hemolysis in cats and dogs. All are very small blue dots on RBC that look like stain precipitate. To differentiate the organism from stain precipitate, must identify specific forms of the organism.
          SpeciesOrganismMorphology for identification
          CatHemobartinellaRing form
          DogHemobartinellachains of cocci

        2. Eperythrozoon - causes acute intravascular hemolysis in pigs and ruminants. Organism similar in appearance to hemobartinella
        3. Anaplasma - protozoal organism larger than hemobartinella but smaller than Howell-Jolly body. Causes hemolytic anemia in cattle, sheep, and goats. Usually one to 3 organisms per RBC. Usually round but may have tails and loops.
        4. Babesia - a large pear-shaped organism that causes hemolytic anemia in cattle, horses, dogs, and cats.


    6. NONREGENERATIVE ANEMIAS
      1. Secondary Anemias - these are mild to moderate anemias that occur secondary to other disease processes.
        1. Causes: Inflammatory disease, neoplastic disease, chronic renal disease, chronic liver disease, hypothyroidism, and hypoadrenocorticism
      2. Pure Red Cell Aplasia - an immune-mediated disease, antibodies destroy developing RBC cells in bone marrow.
      3. Iron Deficiency Anemia
      4. Aplastic anemia (aplastic pancytopenia)
        1. See nonregenerative anemia and leukopenia and/or thrombocytopenia. Causes include drug toxicity, infectious agents, and toxins