Hematology and Clinical Pathology Lecture Notes

Overview of Hematology and Clinical Chemistry

  • Hematology Defined: The study of blood, cellular fluid, and blood-forming organs. It encompasses the study of how blood is formed, stored, and circulated, as well as the study of blood diseases.

  • Diagnostic Utility: Hematology is used for the diagnosis and monitoring of health status and diseases, alongside history, physical exams, and other laboratory findings.

  • Clinical Chemistry: A subarea of clinical pathology involving the analysis of bodily fluids to assess organ function. A common example is a kidney function profile.

  • Serology: The study of blood serum and other bodily fluids, primarily focusing on the presence and activity of antibodies.

Blood Composition

  • Total Body Composition (Canine Example):

    • Whole Blood: 8%8\% of body weight.

    • Other fluids and tissues: 92%92\%

  • Whole Blood Components:

    • Formed Elements (45%45\%): Includes Leukocytes (6,0006,000 to 17,000/μL17,000/\mu L), Erythrocytes (5.55.5 to 8.5×106/μL8.5 \times 10^{6}/\mu L), and Thrombocytes (200,000200,000 to 500,000/μL500,000/\mu L).

    • Plasma (55%55\%—Note: The transcript mentions 55%55\% plasma whereas components sum to 100%100\% of the non-tissue portion): Includes Water (91.5%91.5\%), Proteins (7%7\%), and other substances (1.5%1.5\%—carbohydrates, lipids, vitamins, hormones, and inorganic salts).

  • Leukocyte Differential (Canine Example):

    • Neutrophils: 60−77%60-77\%

    • Lymphocytes: 12−30%12-30\%

    • Eosinophils: 2−10%2-10\%

    • Monocytes: 3−10%3-10\%

    • Basophils: Rare

  • Plasma Protein Components:

    • Albumin: 44%44\%

    • Globulins: 42%42\%

    • Fibrinogen: 4%4\%

Blood Volume and Clinical Removal

  • Percentage of Blood Volume in Relation to Body Weight (Adults):

    • Dogs: 8−9%8-9\%

    • Cats: 6−7%6-7\%

    • Lab Rodents: 6−7%6-7\%

    • Horse (Warm-blood): 10−11%10-11\%

    • Horse (Draft): 6−7%6-7\%

    • Ruminants: 6−7%6-7\%

    • Pigs: 5−6%5-6\%

  • Volume Calculation Example: For a 4 kg4\,kg cat:

    • Calculation: 0.07×4 kg=0.28 kg0.07 \times 4\,kg = 0.28\,kg of blood.

    • Conversion: Assuming 1 g=1 mL1\,g = 1\,mL, there is 0.28 kg×1000 mL/kg=280 mL0.28\,kg \times 1000\,mL/kg = 280\,mL of blood total.

  • Safe Removal Limit: One can safely remove up to 20%20\% of the total blood volume.

    • Example for the 4 kg4\,kg cat (280 mL280\,mL blood): Safe removal is 280 mL×0.20=56 mL280\,mL \times 0.20 = 56\,mL.

Sample Collection and Anticoagulants

  • Patient Preparation: Overnight fasting is preferred to prevent lipemia interference, which can affect measurements of plasma protein, fibrinogen, and hemoglobin.

  • Collection Tools: Vacutainers are preferred to ensure the correct ratio of anticoagulant to blood. This prevents clumping (platelets, fibrinogen) and trauma from manual transfer (leukocytes, erythrocytes).

  • Anticoagulants:

    • Purple Top (EDTA): Contains Ethylenediaminetetraacetic acid. It is the preferred anticoagulant for hematology. It works by forming calcium salts to remove calcium. Note: Causes hemolysis in some birds and reptiles.

    • Heparin: Often associated with Green (Ammonium heparin) or Red (Sodium heparin) tubes. It is less preferred for hematology because leukocytes stain poorly and platelets tend to clump.

    • Blue Top (Untreated): Used for specific tests or when no additive is required.

    • Capillary/Microhematocrit Tubes: Small tubes used for Packed Cell Volume (PCV). They are sealed with clay sealant.

Laboratory Analysis and Automated Hematology

  • Automated Hematology Methods:

    • Quantitative Buffy Coat Analysis: Measures variations in cell size and widths contrasted against a standard.

    • Laser Flow Cytometry: Uses a laser beam to assess light absorption and scattering to determine cell count, size, and density.

    • Impedance Counters: Measures changes in electrical currents. Cells are poor conductors; as they pass through, the change in impedance/voltage corresponds to cell size. WBCs are counted after RBCs are lysed.

    • Examples of Analyzers: Vetscan HM5, Abaxis, Hematrue, Heska.

  • Blood Film Examination (Manual Smear): This is essential for quality control to verify automated counts and to identify morphologic features that machines cannot read, such as:

    • Erythrocytes: Nucleated RBCs (nRBCs), polychromasia, target cells, Heinz bodies.

    • Leukocytes: Basophils, hypersegmentation, toxic granulation, left shift (increased immature WBCs).

    • Platelets: Megaplatelets and platelet clumps.

    • Other: Hemoparasites and bacteria.

Gross Examination of Blood

  • Color Meanings:

    • Bright red: Normal arterial sample or hemolysis.

    • Bluish red: Normal venous sample (deoxyhemoglobin).

    • Dark gray-blue: Low oxygen levels or an old sample.

    • Milky: Lipemic sample (postprandial or certain diseases).

  • Viscosity Meanings:

    • Watery: Hemolysis or over-hydration.

    • "Sludgy": Agglutination, clotting, or red granules indicating dehydration.

Complete Blood Count (CBC) and Packed Cell Volume (PCV)

  • PCV (also known as Hematocrit, Ht, or Erythrocyte Volume Fraction - EVF): The volume percentage of RBCs in blood.

  • Procedure for PCV:

    • Fill a heparinized microhematocrit tube to 75%75\%.

    • Seal one end with clay.

    • Centrifuge for 2−52-5 minutes (clay pointing outward).

  • Layers in a Hematocrit Tube:

    1. Plasma: Normally clear/transparent or pale yellow.

      • White/Milky: Lipemic (pregnancy, diabetes, pancreatitis, hypothyroidism).

      • Dark Yellow (Icterus): Liver disease, fasting, or diet of green plants.

      • Red: Hemolysis (improper handling or cell destruction).

    2. Buffy Coat: White blood cells and platelets (< 1\,mm wide). Red-tinged indicates immature RBCs.

    3. Packed cells: Red blood cells.

  • Calculating PCV Concentration:

    • PCV %=Length of RBC layerTotal length of sample (clay to plasma top)×100PCV\,\% = \frac{\text{Length of RBC layer}}{\text{Total length of sample (clay to plasma top)}} \times 100

  • Estimated Values from PCV:

    • Estimated RBC count: PCV6×106 cells/μL\frac{PCV}{6} \times 10^6\,cells/\mu L

    • Estimated Hemoglobin: PCV3 g/dL\frac{PCV}{3}\,g/dL

Erythrocyte Indices and Anemia Classification

  • Mean Corpuscular Volume (MCV): Average volume of a single RBC, measured in Femtoliters (fLfL).

    • Equation: MCV=PCVRBC concentration×10MCV = \frac{PCV}{RBC\,\text{concentration}} \times 10

  • Mean Corpuscular Hemoglobin Concentration (MCHC): Average concentration of hemoglobin in an average RBC (g/dLg/dL).

    • Equation: MCHC=HgbPCV×100MCHC = \frac{Hgb}{PCV} \times 100

  • Mean Corpuscular Hemoglobin (MCH): Average weight of hemoglobin per RBC (pgpg).

    • Equation: MCH=HgbRBC×10MCH = \frac{Hgb}{RBC} \times 10

  • Red Blood Cell Distribution (RDW): Measures variation in RBC volume (anisocytosis).

  • Anemia Classifications:

    • Regenerative: Bone marrow responds by releasing reticulocytes (takes 3−53-5 days). Causes: Hemorrhage or hemolysis.

    • Non-regenerative: No marrow response; implies marrow dysfunction.

    • Morphological Classifications:

      • Normocytic: Normal MCV (often chronic disease).

      • Microcytic: Low MCV (iron deficiency).

      • Macrocytic: High MCV (regenerative anemia).

      • Hypochromic: Low MCHC (iron deficiency or regenerative).

Blood Smear Preparation and Staining

  • Wedge Smear Technique:

    • Use a small drop of blood.

    • Hold a spreader slide at a 30∘30^{\circ} angle.

    • Pull back into the drop and then drag forward quickly and evenly.

    • Angle adjustment: Higher angle for high PCV (thick blood), lower angle for low PCV (thin blood).

  • Smear Zones: Application point, Body (thick), Monolayer (reading area/Zone of Morphology), and Feathered edge.

  • Staining (Romanowsky stains):

    • Fixative: 95%95\% methanol.

    • Eosin: Acidic pH; stains basic components (hemoglobin, eosinophilic granules).

    • Methylene blue: Alkaline pH; stains acidic components (nuclei).

    • Examples: Diff-Quik, Wright’s, Wright-Giemsa.

  • Differential Count:

    • Scan on Low Power (10×10\times) for large abnormalities and microfilaria.

    • Identify cells using Oil Immersion (100×100\times).

    • Count at least 100100 WBCs and categorize by type.

    • Absolute Count Calculation: Percentage of cell type×Total WBC count\text{Percentage of cell type} \times \text{Total WBC count}.

Erythrocyte Morphology

  • Color Variations:

    • Normochromia: Normal red.

    • Polychromia: Bluish tint (young cells with organelles).

    • Hypochromia: "Punched out" look; insufficient hemoglobin.

  • Size Variations:

    • Anisocytosis: General variation in size.

    • Macrocytes: Large (often young).

    • Microcytes: Small (iron deficiency).

  • Arrangement:

    • Rouleaux: Stacks of coins (normal in horses/cats; otherwise implies high fibrinogen/globulin).

    • Agglutination: Clumping due to antibodies; does not disperse with saline (unlike rouleaux).

  • Shape Abnormalities (Poikilocytes):

    • Schistocytes: RBC fragments (trauma like DIC).

    • Drepanocytes: Sickle cells (normal in deer/angora goats).

    • Keratocytes: "Bite" or "Blister" cells (trauma, hemangiosarcoma).

    • Spherocytes: Dark, small, no central pallor (immune mediated hemolytic anemia).

    • Acanthocytes (Spur cells): Uneven projections (liver disease).

    • Echinocytes (Burr cells): Even projections (renal disease, snake bites, or artifactual drying).

    • Codocytes (Target cells): Folded membrane (liver disease).

    • Dacrocytes: Teardrop shapes (myeloproliferative disease).

Cellular Parasites and Inclusions

  • Howell-Jolly bodies: Basophilic nuclear remnants; seen in regenerative anemia.

  • Heinz bodies: Denatured hemoglobin (oxidation/drugs); Blue with New Methylene Blue (5%5\% normal in cats).

  • Basophilic stippling: Residual RNA; common in lead poisoning in dogs.

  • Parasites:

    • Haemobartonella felis (Mycoplasma haemofelis): Feline infectious anemia; small cocci/rods on RBC surface.

    • Babesia: Teardrop-shaped pairs within RBCs.

    • Ehrlichia: Infects platelets (E.platysE. platys) or WBCs (E.canisE. canis); forms clusters called morulae (3−6 μm3-6\,\mu m).

    • Anaplasma marginale: Small cocci on the margins of ruminant RBCs.

    • Trypanosomes: Extracellular whiplike parasites with undulating membranes.

    • Dirofilaria immitis: Heartworm microfilariae; prelarval stage seen in smears.

Reference Normal Values (Summary Table)

  • Hematocrit (%): Canine 40−5940-59, Feline 29−5029-50

  • Hemoglobin (g/dL): Canine 14−2014-20, Feline 9−15.69-15.6

  • RBC (106/μL10^{6}/\mu L): Canine 5.6−8.75.6-8.7, Feline 6.1−11.96.1-11.9

  • WBC (/μL/\mu L): Canine 6,000−17,0006,000-17,000, Feline 4,900−20,0004,900-20,000

  • Platelets (103/μL10^{3}/\mu L): Canine 145−440145-440, Feline 190−800190-800