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2013-10-26 21:32:26
Clin Path

Erythrocytes, clinical laboratory techniques lecture
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  1. Serum
    Liquid part of blood without clotting factors or fibrinogen (red top or tiger top), used for total proteins
  2. Plasma
    • Liquid part of blood with clotting factors and fibrinogen still inside.  
    • Lavender top (anticoagulant), used for total solids and CBC
  3. Separated blood (MHC) is made up of...
    • Plasma: 55% of blood.  91% water, 7% proteins, 2% nutrients, hormones and electrolytes.  
    • Cellular portion: 45% of blood.  Includes buffy coat (WBC, platelets) and RBCs.
  4. Considerations of internal vs external laboratories
    time, equipment, personnel, cost, quality control, stability of sample, hours of operation, veterinary commercial laboratory
  5. Packed Cell Volume (PCV)
    fraction of total blood volume in a microhematocrit tube that is occupied by red blood cells.  (WBC and platelets are located in the buffy coat).  40 is average(-ish)
  6. Plasma protein concentration
    determined on a refractometer.  Should be clear (can be icteric, hemolyzed, lipemic).
  7. icteric plasma
    • indicates excess bilirubin.  
    • With a normal hematocrit, can indicate liver disease.  
    • with low hematocrit, indicates an increased destruction of RBCs
  8. hemolyzed plasma
    • indicates presence of free hemoglobin.  
    • True hemoglobinemia - intravascular hemolysis, will not see RBCs under microscope
    • Secondary to sample collection - rough handling, fragile cells, lipemia, prolonged storage, spun too early
  9. lipemic blood
    • Indicates presence of chylomicrons and very low density lipoproteins (lipemia)
    • can be postprandial (after eating), caused by diseases (endocrinopathies like diabetes, protein-losing nephropathy, obesity, cholestasis, or starvation) or caused by heredity (idiopathic hyperlipidemia in Schnauzers, lipoprotein lipase deficiency in cats)
  10. Hgb
    hemoglobin concentration
  11. MCH
    mean cell hemoglobin, average hemoglobin per cell.
  12. MCHC
    mean cell hemoglobin concentration, amount of hemoglobin relative to other stuff
  13. HCT
    hematocrit--percent red blood cells
  14. MCV
    mean cell volume.  Average size.
  15. RBC
    erythrocyte concentration
  16. total and differential leukocyte concentrations
    total is how many WBC, overall.  Differential is how many types of each WBC.
  17. Reticulocytes
    • immature RBCs.  
    • Presence of too many indicates regenerative anemia
    • maturation begins in bone marrow, completed in blood and spleen.
  18. RP
    • Reticulocyte percentage (reticulocyte count)
    • Count 1000 RBC, categorize as reticulocytes or normal.  Reticulocytes/1000 x 100
    • 0-1.5% normal
    • RP>1.5% regenerative
    • RP wnl nonregenerative
  19. corrected and absolute reticulocyte count
    • Reticulocyte count must be corrected for degree of anemia present.  
    • HCT - corrected retic count = HCT/mean normal HCT for species x raw retic count in percent
    • RBC - absolute retic count (per microliter) = RBC count (per milliliter) x raw retic count (fraction).  Determined directly by flow cytometry
    • dogs and cats should have 0-60,000 cells/uL
    • cows should have none, horses don't even have reticulocytes.
  20. 0-10,000 cells per microliter
    nonregenerative anemia to very poor regeneration
  21. 10,000 - 60,000 cells per microliter
    nonregerative to poorly regenerative anemia
  22. 60,000 - 200,000 cells per microliter
    regenerative anemia to mild or moderate output
  23. 200,000 - 500,000 cells per microliter
    maximal regenerative response
  24. CBC
    complete blood count.  Summarizes and organizes test results.  Provides a framework for interpretation
  25. hematopoiesis
    production of blood cells
  26. Steps of RBC differentiation
    • Rubriblast (last nucleolus) > Prorubricyte > basophilic rubricyte > Polychromatophilic rubricyte > Metarubricyte (last nucleus) > reticulocyte > RBC
    • Get smaller as you go along
  27. Pluripotent stem cell
    • differentiates into all blood cells.
    • splits into lymphoid multipotential cell and myeloid multipotential cell in bone marrow.  
    • lymph goes to lymph organs and becomes lymphocyte colony cell, lymphoblast, B and T lymphocyte
    • myeloid splits into 6-erythrocyte colony, megakaryocyte forming, monocyte colony, granulocyte colony, eosinophil colony, basophil colony.
  28. RBCs life span
    3 months in d/c.  Up to 5 months.
  29. central pallor
    pale spot in center of biconcave RBC.  More in dogs than cats
  30. RBC shape and why
    biconcave.  Efficient for O2 exchange, deformable, larger surface area, minimal diffusion distance, greater osmotic swelling
  31. D/C/H/C reticulocyte concentration
    RBC concentration
    RBC life span
    RBC diameter
    RBC volume
    RBC central pallor
    • reticulocyte concentration: D>Ct.  H, Cw = 0
    • RBC concentration: H>Ct>/= D = Cw
    • RBC life span: H=Cw>D>Ct
    • RBC diameter: D>Ct>/= H=Cw
    • RBC volume: D>Cw>H=Ct
    • RBC central pallor: D>Cw>Ct>H
  32. Hemoglobin
    • Principal component of erythrocytes
    • transports O2 (50x more than plasma)
    • Carries more with more hemoglobin, higher partial pressure, higher hemoglobin affinity for O2.  
    • Transports CO2 from tissues to lungs, assists in acid/base.
  33. hemogram
    complete and detailed record of the findings in a thorough examination of the blood.  Includes numbers, proportions, morphology, hematocrit and total proteins
  34. HCT
    hematocrit.  Percentage of blood volume filled by erythrocytes.  Measure of O2 carrying capacity of blood.
  35. PCV
    packed cell volume.  Measure by centrifugation, measures % of RBC in blood.
  36. Hgb
    • blood hemoglobin content.  Grams hemoglobin per 100 ml (1 dL) of blood.  
    • more direct measure of O2 carrying capacity than HCT or RBC
  37. RBC
    red blood cell count.  Number of erythrocytes per unit volume of blood (cells per microliter).  Used to calculate MCH and MCV
  38. MCV definition
    • mean cell volume.  Volume per average erythrocyte expressed in fL (femtoliters) (femto = 1 quadrillionth, or 10-15)
    • Traditionally calculated or automated hematology analyzers.
  39. MCV formula
    MCV = (PCV/RBC) x 10
  40. microcytosis
    decreased MCV
  41. macrocytosis
    increased MCV
  42. Anisocytosis
    varying cell size
  43. Normocytosis
    size appropriate for the species (and age)
  44. MCH definition
    • mean cell hemoglobin.  Quantity of hemoglobin per average erythrocyte expressed in picograms (pg)
    • calculated from Hgb and RBC
  45. MCH formula
    MCH (pg) = (Hgb/RBC) x 10
  46. MCHC formula
    MCHC - (Hgb/HCT) x 100
  47. MCHC definition
    • mean cell hemoglobin concentration.  Cellular hemoglobin concentration per average erythrocyte expressed as grams per hemoglobin per 100 mL of erythrocytes (g/dL)
    • Calculated from Hgb and HCT
    • Increases with hemolyzsis, heinz bodies, cryoproteins or lipemia
    • decreases with regenerative anemia, hypernatremia or chronic iron deficiency.
  48. Hypochromasia
    decreased MCHC.  Increased central pallor
  49. Hyperchromasia
    increased MCHC, decreased central pallor
  50. Normochromasia
    MCHC wnl.  Appropriate level of central pallor for the species.
  51. RDW
    red cell distribution width.  Calculated value usually expressed as a percentage.  Reflects amount of variation in erythrocyte volume.  Measure of anisocytosis (increased number of reticulocytes will cause RDW).
  52. nRBC
    nucleated red blood cells.  Counted by hematology analyzers as WBCs, so WBC count must be corrected for nRBCs in circulation.
  53. Corrected WBC count
    (measured WBC count x 100) / (100 + nRBC)
  54. Reticulocytes
    Immature anucleated RBCs. Released to blood in increased numbers as a response to anemia caused by hemolysis or blood loss in most species (not in horses).
  55. Rouleaux
    • Aggregates of erythrocytes grouped like a stack of coins.  Depends on erythrocytes (more deformable with less surface charge) and plasma (high molecular weight proteins)
    • Normal in horse, sometimes in cat
  56. Agglutination
    • aggregation of erythrocytes in clusters.  Caused by immunoglobulins bound to erythrocyte surface
    • Happens in immune-mediated hemolytic anemia, tested for with saline test.
  57. Saline agglutination test
    • screens for immune-mediated hemolytic anemia.  Agglutination is a positive result (except a little in cat).  
    • Mix whole blood with saline 1:1 on a slide, look for agglutination.
  58. Polychromasia
    variable colors of RBC on a slide (reticulocytes)
  59. Ghost Cell
    erythrocytes after loss of hemoglobin.
  60. Polychromatophil
    immature RBC that stains bluish-red due to presence of both hemoglobin and RNA
  61. Anisocytosis
    variation in erythrocyte diameter.  Seen in regenerative anemia or post transfusion
  62. Microcytosis
    increased number of erythrocytes with small diameter.  Decreased iron or microcytic anemia
  63. macrocytosis
    increased number of erythrocytes with larger than normal diameter
  64. Poikilocytosis
    general term to describe presence of abnormally shaped erythrocytes.  Normal in goats and calves, can indicate liver disease, oxidative injury, chemo drug (doxorubracin), DIC (deceminated intravascular coagulation)
  65. Schistocytes
    • Erythrocyte fragments with pointed extremities (tiny bits).
    • Caval syndrome, when heartworms back up into vena cavae.  Can also be caused by DIC
  66. Prekaryatocyte
    • blister cell.  Erythrocytes containing one or more intact holes.  
    • Increase in fibrin
  67. Keratocyte
    Erythrocyte with ruptured holes.  "helmet cells".  Caused by trauma, endotoxemia, excess fibrin.
  68. Acanthocytes
    Erythrocytes with irregularly spaced and variably sized spicules.  "spur cells"
  69. Echinocytes
    Spiculated erythrocytes, evenly spaced and of similar size.  "burr cells".
  70. Spherocytes
    smaller diameter than normal erythrocytes, lacking central pallor.  Indicate immune-mediated hemolytic anemia.
  71. Elliptocytes
    elliptical or oval erythrocytes.  Generally flat.  Seen in nonmammals or camelidae family.
  72. Dacrocytes
    teardrop shaped erythrocytes with single elongated or pointed extremity.
  73. Drepanocyte
    spindle-shaped erythrocyte.  "sickle cell"
  74. Eccentrocytes
    bulk of erythrocyte is shifted to one side.  Hemoglobin is localized to one part of cell.  Visibly pale in hemoglobin-poor area.
  75. Leptocytes
    Thin, hypochromic erythrocytes with increased membrane-to-volume ratios (more border than inside).  Triconcave, central bar of hemoglobin.  Also called condocytes, or target cells.
  76. Stomatocyte
    cup shaped erythrocyte with oval or elongated areas of central pallor.
  77. Heinz bodies
    large aggregates of oxidized precipitated hgb that show primarily in methylene blue stain (Howell-Jolly are the blue in wright's)
  78. basophilic staining
    blue-staining punctate inclusions in reticulocytes (tiny blue dots).  Can indicate lead poisoning.
  79. Howell-Jolly bodies
    small spherical nuclear remnants that stain in Wright's stain (Heinz bodies do not)
  80. Siderotic granule
    iron positive inclusions in erythrocytes.  Also called siderocytes or "pappenheimer bodies"
  81. Infectious agents of RBC for feline, canine, bovine
    • feline: mycoplasma haemofelis (hemobartonella) and cytauxzoon felis
    • canine: mycoplasma haemocanis (hemobartonella canis), babesia canis, babesia gibsoni, distemper
    • bovine: anaplasma marginale (now also in canine) (tick-bourne)
  82. anemia
    Condition, not a diagnosis.  decrease in erythrocyte mass within the body.  HCT, hgb and RBC count decreased.  Reduced ability to transport O2.  Classifications assist in diagnosis.
  83. Classifications of anemia
    • bone marrow response: regenerative or nonregenerative
    • size and Hgb concentration: normocytic, macrocytic, microcytic, normochromic, hypochromic.  
    • etiology: loss or lysis.  (loss, destruction or decreased production
  84. regenerative anemia
    • anemia with reticulocytosis.  
    • Hemogram shows immature RBCs, Anisocytosis, Macrocytosis, Polychromasia.  Loss or lysis
    • Usually macrocytic hypochromic
    • can be visible or occult (internal bleeding)
    • takes 2-4 days to see regenerative response
    • Can be acute or chronic, caused by trauma, surgery, GI ulcers, parasites, poisonings (Warfarin, sweet clover, brackenfern), hemophilia, hematuria, thrombocytopenia, and vascular neoplasia
  85. Non-regenerative anemia
    • Anemia without reticulocytosis.  Hemogram is normocytic, normochromic with no poikilosis.  
    • Insufficient erythrocyte production in marrow.
  86. normocytic, normochromic
    disorders that reduce erythropoiesis.  Chronic inflammation, early regenerative and iron deficiency anemias
  87. macrocytic hypochromic
    regenerative anemia
  88. microcytic hypochromic
    iron deficiency
  89. Anemia develops when
    • loss or lysis.  
    • increased erythrocyte loss due to blood loss
    • accelerated erythrocyte destruction (pathological hemolysis)
    • decreased effective erythrocyte production
  90. clinical signs of anemia
    • reflect decreased O2 carrying capacity.  
    • decreased exercise tolerance, weakness, depression, rapid respiration, cold intolerance
  91. physical exam findings in anemia
    • pale mucous membranes
    • systolic heart murmur (due to watery blood)
    • tachycardia, tachypnea
    • icterus, bilirubinuria (dependant on mechanism)
    • fever
    • signs of shock if 1/3 of blood volume lost (move heat to core, so vasoconstriction, cold limbs, gingeva pale and cold, temperature abnormal.
  92. lab confirmation of anemia
    • decreased hematocrit.  Regenerative indicates look for hemorrhage or hemolysis.  Can take 2-4 days for regenerative response
    • non-regenerative look for bone marrow disease, either cancer or toxic drugs
  93. effects of external blood loss
    • can't re-use components.  
    • External includes alimentary tract and urinary tract
  94. effect of internal blood loss
    • erythrocytes move from intravascular space to extravascular space.  
    • Components can be reused.
  95. Acute blood loss
    • Animal presents with trauma, no time to adjust
    • initial PVC is normal due to splenic contraction
    • PVC and plasma protein decrease as blood is diluted by interstitial fluid as volume is restored over 2-3 days after clotting.  Reticulocytes are lower with hemorrhage than with hemolysis
  96. Length of time before maximal bone marrow response
    3-4 days
  97. Chronic blood loss anemia
    • Anemia develops slowly and animal adapts physiologically, so doesn't present with clinical signs
    • iron deficiency anemia may develop.  
    • microcytic, hypochromic.  
    • May become nonregenerative in prolonged cases due to lack of components (iron and proteins)
  98. Hemogram of hemolysis
    • decreased HCT
    • reticulocytosis
    • inflammatory leukogram (neutrophilia, monocytosis)
    • intravascular hemolysis (hemoglobinemia, hemoglobinuria, bilirubinuria, bilirubinemia
  99. causes of hemolysis
    • bacteria (leptospirosa, clostridia)
    • RBC parasite (Babesia, Mycoplasma, Anaplasma)
    • Chemicals (acetominophen, onions, garlic, propylene glycol)
    • Immune (IMHA, SLE(lupus), neonatal isoerythrolysis)
    • viral (Equine infectious anemia, FeLV)
  100. Diagnostic tests for hemolysis/regenerative anemia
    • Blood smear: parasites, spherocytes (IMHA), agglutination (vs rouleaux, IMHA)
    • Coombs: detects presents of antibody against RBCs.  Not definitive--only negative is definitive.  
    • Coggins: detects antibodies against EIA.  Definitive.
  101. Causes of nonregenerative anemia
    • 1.reduced erythropoiesis (kidneys)
    • 2.chronic diseases-most common in domestic animals, many reasons.  
    • 3. cytoxic bone marrow damage
    • 4. Myeloproliferative disorder
    • 5. infections
  102. chronic infection causing nonregenerative anemia
    most common in domestic animals.  Can be chronic infections, noninfectious disorders (neoplasia, chronic inflammatory disorders).  Lab=mild to moderate nonregnerative normocytic, chronic inflammatory leukogram, mature neutrophilia, lymphocytosis or monocytosis
  103. reduced erythropoeitin causing nonregenerative anemia
    due to lack of erythropoeitin from kidneys (chronic renal disease) or endocrine disease (hypothyroidism, hypoadrenocorticism
  104. cytoxic bone marrow damage causing nonregenerative anemia
    • caused by radiation
    • chemical (chemo like doxorubicin or vincristine)(drugs like phenylbutazone, griseofulvin, phenobarbitol, chloramphenicol, trimethoprim-sulfadiazine, estrogen toxicity
    • plants (bracken fern poisoning)
  105. myeloproliferative disorder as a cause of nonregenerative anemia
    • leukemia, multiple myeloma, metastatic lymphoma, metastatic mast cell tumor
    • neoplastic proliferation of hematopoietic cells in the bone marrow
  106. infections as a cause of nonregenerative anemia
    • FeLV: selectively damages erythroid cells or transform a cell into a neoplastic line
    • Panleukopenia virus and parvovirus: kills rapidly dividing cells
    • Ehrlichia: immune mediated destruction.  Caused by rhipicephalus sanguineous (american dog tick), loves WBC and causes anemia
  107. Polycythemia
    • Erythrocytosis (increased HCT, Hgb, RBC count), relative or absolute
    • Relative, pseudo or spurious (elevated HCT but RBC mass is normal), caused by dehydration (if proteins are elevated too) or by splenic contraction after excitement, fright or exercise.  Transient.
    • Absolute: Increased erythropoiesis, HCT and erythrocyte mass
  108. Causes of absolute polycythemia
    • Compensatory: body trying to compensate.  High altitude, chronic pulmonary disease, cardiac disease
    • Erythropoietin secreting tumors: 
    • Autonomous proliferation (primary).  Polycythemia vera (Bernese Mt. Dogs have such thick blood you have to bleed them).  Myeloproliferative neoplasm.