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Hematology

Heinz Body

Overview of Heinz Body Staining

  • Definition: A histochemical staining technique used to visualize Heinz bodies within red blood cells (RBCs)
  • What are Heinz Bodies?
    • Inclusions within RBCs composed of denatured or precipitated hemoglobin
    • Formed due to oxidative damage to hemoglobin, causing the globin chains to unfold and aggregate
    • Not normally visible with Wright-Giemsa stain (the routine stain used for peripheral blood smears)
  • Clinical Significance: The presence of Heinz bodies indicates oxidative damage to red blood cells and is associated with several conditions, particularly G6PD deficiency and exposure to certain toxins
  • Key Principle: Heinz bodies can only be visualized with supravital stains, which stain living cells, allowing the denatured hemoglobin to precipitate and form visible inclusions

Principle of Heinz Body Staining

  • Supravital Staining:
    • The blood sample is stained while the cells are still living (in vitro) using a supravital dye
    • The supravital stain penetrates the RBCs and binds to the denatured hemoglobin, causing it to precipitate and form Heinz bodies
  • Staining Mechanism:
    • The supravital dye (typically crystal violet or brilliant cresyl blue) interacts with the denatured hemoglobin, causing it to aggregate and precipitate within the RBC
    • The precipitated hemoglobin forms small, round or irregular inclusions that are visible under a microscope

Materials and Reagents

  • Whole Blood: Fresh EDTA (purple-top) or heparin (green-top) whole blood sample
  • Supravital Stain:
    • Crystal Violet Stain:
      • Crystal violet
      • Ethanol
    • Brilliant Cresyl Blue Stain:
      • Brilliant cresyl blue
      • Sodium chloride
      • Distilled water
  • Glass Slides: Clean, grease-free microscope slides
  • Coverslips:
  • Test Tubes or Microcentrifuge Tubes:
  • Pipettes: To accurately measure reagents and samples
  • Incubator or Water Bath: To maintain a constant temperature during incubation
  • Microscope: With oil immersion objective (100x)

Procedure

  1. Prepare the Stain Solution:
    • Follow the manufacturer’s instructions or established laboratory protocols to prepare the crystal violet or brilliant cresyl blue stain solution
    • Filter the stain solution to remove any particulate matter
  2. Mix Blood and Stain:
    • In a small test tube or microcentrifuge tube, mix equal volumes of whole blood and the supravital stain solution (e.g., 0.5 mL of blood + 0.5 mL of stain)
  3. Incubate:
    • Incubate the mixture at 37°C (98.6°F) for 15-30 minutes. This allows the stain to penetrate the cells and react with the denatured hemoglobin
  4. Prepare Smears:
    • After incubation, gently mix the blood-stain mixture
    • Prepare thin wedge (push) smears on clean glass slides, similar to preparing a routine peripheral blood smear
  5. Air Dry:
    • Allow the smears to air dry completely at room temperature
    • Do not heat-fix the smears, as heat fixation can distort or destroy the Heinz bodies
  6. Microscopic Examination:
    • Examine the air-dried smears under oil immersion (100x objective)
    • Select a well-stained area of the smear where the RBCs are evenly distributed and not overlapping
  7. Identify and Count Heinz Bodies:
    • Look for small, round or irregular inclusions within the RBCs
    • Heinz bodies stain dark blue or purple with crystal violet and dark blue with brilliant cresyl blue
    • Count the number of Heinz bodies per 100 RBCs (or more, if necessary to obtain an accurate count)
    • Report the number of Heinz bodies per 100 RBCs (e.g., “5 Heinz bodies per 100 RBCs”)

Interpretation of Results

  • Normal: No Heinz bodies are seen in normal red blood cells

  • Heinz Bodies Present: Indicates oxidative damage to hemoglobin and suggests:

    • G6PD Deficiency: Exposure to oxidative stressors (e.g., drugs, infections, fava beans) in individuals with G6PD deficiency
    • Unstable Hemoglobin Variants: Hemoglobin variants that are prone to denaturation
    • Exposure to Oxidant Drugs or Chemicals: Certain drugs (e.g., dapsone, sulfonamides, phenazopyridine) and chemicals can cause oxidative damage
    • Splenectomy: The spleen normally removes Heinz bodies from RBCs, so they may be more readily seen in patients who have had a splenectomy

  • Important Considerations:

    • The number of Heinz bodies may vary depending on the severity of the oxidative stress and the time elapsed since the exposure
    • Heinz bodies are typically removed by the spleen, so they may not be seen in patients with an intact spleen, even if there is evidence of oxidative damage

Sources of Error and Troubleshooting

  • Poor Smear Preparation:
    • Ensure that the smears are thin, evenly distributed, and free of artifacts
  • Staining Artifacts:
    • Use fresh stain solutions and follow proper staining procedures
    • Avoid prolonged staining or washing times
  • Improper Microscope Settings:
    • Ensure that the microscope is properly aligned and the light source is adequate
    • Use the correct objective lens (100x oil immersion)
  • Misidentification of Heinz Bodies:
    • Distinguish Heinz bodies from other inclusions (e.g., Howell-Jolly bodies, Pappenheimer bodies) and staining artifacts
    • Consult a reference atlas or experienced hematologist if needed
  • Delay in Smear Preparation:
    • Prepare smears promptly after mixing the blood with the stain to prevent Heinz bodies from disappearing

Reporting Results

  • Report the presence or absence of Heinz bodies
  • Quantify the number of Heinz bodies per 100 RBCs (if present)
  • Indicate the type of stain used (e.g., crystal violet, brilliant cresyl blue)
  • Include a comment noting any factors that may have affected the results (e.g., recent transfusion, splenectomy)
  • Correlate the results with the patient’s clinical information

Key Terms

  • Heinz Bodies: Inclusions composed of denatured hemoglobin in RBCs
  • Supravital Stain: A dye used to stain living cells
  • Crystal Violet: A supravital stain used to visualize Heinz bodies
  • Brilliant Cresyl Blue: Another supravital stain used to visualize Heinz bodies
  • G6PD Deficiency: A genetic enzyme deficiency that predisposes to oxidative damage and Heinz body formation
  • Oxidative Stress: An imbalance between the production of reactive oxygen species and the body’s ability to detoxify them
  • Splenectomy: Surgical removal of the spleen