Practical Hematology Lab

Practical Hematology Lab

Introduction to RBC Morphology

• Understanding the morphology of red blood cells (RBCs) is a key component in hematology.

Abnormal RBC Morphology

• This includes abnormalities in size, shape, color, distribution, and inclusions of the RBCs, as well as other pathological states.

Recording RBC Morphology

• The procedure involves:
  • Scanning area using ×100 (oil immersion).
  • Observing 10 fields for red cell characteristics:
  • Size
  • Shape
  • Hemoglobin content
  • Inclusions
  • Assessment of abnormal morphology based on a grading system.
  • Key questions:
  • Is the morphology seen in every field?
  • Is the morphology pathologic, not artificially induced?

Types of Abnormal Erythrocyte Morphology

• Abnormalities observed can be categorized into:
  I. Distribution
  II. Size (anisocytosis)
  III. Hemoglobin content (color variation)
  IV. Shape (poikilocytosis)
  V. Presence of inclusion bodies

I. Erythrocyte Distribution Abnormalities

• Rouleaux Formation:

  • Description: Stacking of RBCs resembling coins due to increased plasma proteins.
  • Conditions Associated:
  • Hyperfibrinogenaemia
  • Hyperglobulinaemia

• Agglutination:

  • Description: Irregular clumping of RBCs, often antibody-mediated, and temperature-dependent.
  • Conditions Associated:
  • Cold agglutinins
  • Warm autoimmune hemolysis

II. Variation in Erythrocyte Size (Anisocytosis)

• Anisocytosis: Variations in RBC sizes.
  • Types:
  • Normocytic:
    • Normal size of RBC is approximately 8 μm, range: 7 - 9 μm (use the nucleus of a small lymphocyte as a size reference).
  • Microcytic:
    • Smaller than normal RBCs (< 7 μm) associated with decreased hemoglobin synthesis.
    • Conditions Associated:
    • Iron deficiency anemia
    • Thalassaemia
    • Sideroblastic anemia
    • Lead poisoning
    • Anemia of chronic disease.
  • Macrocytic:
    • Larger than normal RBCs (> 9 μm) due to defects in nuclear maturation or stimulated erythropoiesis.
    • Types by shape:
    • Oval (associated with folate and B12 deficiencies)
    • Round (associated with ethanol exposure, liver disease, reticulocytosis)

III. Variation in Erythrocyte Color

• Normal Erythrocyte Color: Pinkish-red with a central pallor when stained with Wright's stain. Indicates hemoglobin concentration.
• Normochromic: Normal color with proportional central pallor.
• Hypochromia:
  • Increased central pallor, indicating decreased hemoglobin concentration.
  • Central pallor occupies more than one-third of the RBC diameter.
  • Conditions Associated:
  • Iron deficiency
  • Thalassaemia
  • Any condition leading to microcytosis.
• Polychromasia:
  • RBCs appear blue-gray due to stained residual ribosomal RNA.
  • Found in:
  • Situations with reticulocytosis.

IV. Shape Abnormalities of Erythrocytes

• Poikilocytosis: General term for non-biconcave shaped mature erythrocytes, seen in various shapes.
  • Common types:
  • Target Cells:
    • Features: Central Hemoglobin with a target-shaped appearance.
    • Associated with: Liver disease, thalassaemia, severe iron deficiency.
  • Echinocyte:
    • Features: Short, equally spaced projections (burr cells).
    • Associated with: Uremia, hypokalemia.
  • Acanthocyte:
    • Features: Irregular projections, seen in liver diseases or after splenectomy.
  • Spherocyte:
    • Features: Spherical cells lacking central pallor.
    • Associated with: Hereditary spherocytosis, immune hemolytic anemia.
  • Schistocyte:
    • Features: Fragmented RBCs, seen in microangiopathic hemolytic anemia.
  • Ovalocyte:
    • Features: Elongated; result of membrane defect.
    • Associated with: Thalassaemia major, hereditary ovalocytosis.
  • Elliptocyte:
    • Features: Oval or elliptical shape, long axis twice the short.
    • Associated with: Hereditary elliptocytosis, megaloblastic anemia.

V. Erythrocyte Inclusions

• Types of Inclusions with Wright’s Stain:
  • Basophilic Stippling:
  • Composition: Precipitated ribosomes, appearing as fine or coarse granules.
  • Conditions: Lead poisoning, thalassaemia.
  • Howell-Jolly Bodies:
  • Composition: DNA remnants, appear as dense, round blue granules.
  • Conditions: Post-splenectomy.
  • Pappenheimer Bodies:
  • Composition: Iron-containing granules, appear as small clusters.
  • Conditions: Anemia.
  • Heinz Bodies:
  • Composition: Denatured hemoglobin, appear as round blue precipitates.
  • Significance: Indicates red cell injury, commonly associated with G6PD deficiency.
  • Cabot Rings:
  • Remnants of nuclear membranes, appear as thread-like rings.
  • Conditions: Severe anemias and lead poisoning.

Interpretation of Results

• The relevance of abnormal results helps to diagnose the cause of anemia:
  • Normocytic/Normochromic (NC/NC): Caused by blood loss, sepsis, long-term diseases.
  • Microcytic/Hypochromic: Caused by iron deficiency, lead poisoning.
  • Macrocytic/Normochromic: Caused by chemotherapy, vitamin deficiencies.

Reporting Results

• Recommendations for reporting:
  • Employ specific terminology: Use macrocytic and microcytic, as opposed to just anisocytosis.
  • When reporting abnormal cells, do not state normal if abnormalities are noted. For example, 7-10 microcytic RBCs should be reported as “2+ microcytosis.”

Quantitative and Qualitative Grading of RBC Morphology

• Table for Cell Count:
  • 1 to 5 cells/10 fields - slight abnormalities
  • 6 to 15 cells/10 fields - moderate abnormalities
  • >15 cells/10 fields - marked abnormalities

Examples of RBC Morphology in Conditions

• Spherocytes: Indicate autoimmune hemolytic anemia.
• Schistocytes: Indicate microangiopathic hemolytic anemia.
• Dacryocytes: Often seen in idiopathic myelofibrosis.
• Target Cells and Spur Cells: Morphological changes in liver disease.