Blood Dilution Techniques in Hematology

Diluting the Blood

• The blood is diluted to facilitate counting and disperse red blood cells.
• A 1:200 dilution is commonly used for red blood cell counts.
• The dilution range for red blood cell counts using the Thoma pipette is 1:100 up to 1:1000.
• EDTA is used as an anticoagulant to preserve blood cells.
• A pipette should be fully immersed in the diluent to avoid air bubbles.
• The tip of the pipette should be wiped clean with a tissue.
• For anemic samples, a lower dilution (1:20, 1:100, or 1:10) may be required.

Diluting Fluids

• Hayem's solution can lead to rouleaux formation and autoagglutination of cells.
  • Composition:
    • Sodium chloride: 0.85 grams
    • Distilled water: 100.0 mL
• Gower's solution prevents rouleaux formation and precipitates protein in cases of hemoglobinemia and hyperglobulinemia.
  • Composition:
    • Sodium sulfate anhydrous: 12.5 grams
    • Sodium chloride: 2.0 grams
    • Distilled water: 100 mL
• 3.8% sodium citrate is isotonic and prevents cell shrinkage or bursting.
  • Composition:
    • Sodium chloride: 3.8 grams
• All diluting fluids should be isotonic to prevent cell shrinkage and bursting.

Charging the Counting Chamber

• RBC Thoma pipette: Used for red blood cell counts.
• WBC Thoma pipette: Used for white blood cell counts.
  • The color of the dot on the pipette distinguishes them - red for RBC and white for WBC.
• Charging procedure:
  • A representative sample of the diluted mixture is transferred to a counting chamber.
  • The pipette should be mixed by shaking, but not along the longitudinal axis.
  • Discard the first few drops (3-4) from the capillary system.
• WBC Thoma pipette dilution range: 1:10 - 1:100.

Counting Cells

• White blood cells:
  • Dilution: 1:20 for white blood cell counts.
  • Diluting fluid: Should be hypotonic to eliminate red blood cells and stain white blood cell nuclei.
    • Example diluents:
      • 2-3% acetic acid
      • 1% HCl with 1 drop of methyl violet or crystal violet
  • Counting procedure:
    • Familiarize yourself with the ruling of the chamber.
    • Let the cells settle for 1-2 minutes.
    • Use the low-power objective to locate and scan the ruled area.
    • Count the cells within the 4 corner squares (4 sq.mm.) which each have 16 medium squares.
• Differential count:
  • A 200-cell differential should be performed when the leukocyte count is in excess of 600,000-800,000/uL.
• Causes of precipitated stain:
  • Unclean slides
  • Drying during staining
  • Inadequate washing
  • Failure to hold the slide horizontally during initial washing
  • Inadequate filtration of the stain

Platelet Count

• Platelet count is the number of platelets per liter or milliliter of whole blood.
• Counting procedure:
  • Count platelets in the 25 small squares in the large center square (1 mm^2) of the hemacytometer using a phase contrast microscope.
• Platelet count formula:
  • Platelet count = Average platelet count x Depth factor x Dilution factor x Area correction factor
• Brecher-Cronkite method:
  • Most accurate method for platelet counting.
  • Formula:
    • Platelet/uL = Platelet x 5 x 10 x 100
• Dameshek's method (indirect):
  • Uses brilliant cresyl blue, sodium citrate, sucrose, and formalin.
  • Counterstained with Wright's stain.
• Reasons why platelets are hard to count:
  • Easily disintegrate.
  • Small size can lead to mistaking them for debris.
  • Easily clump.
  • Uneven distribution of blood.
• Sources of error in platelet counting:
  • Handling error.
  • Operator error.
  • Equipment and reagent error.
  • Inherent/field error.
• Causes of platelet clumping:
  • Initiation of platelet aggregation.
  • Clotting before blood reaches the anticoagulant.
  • Imperfect venipuncture.
  • Delay in sampling.
• Physiologic variation in platelet counts:
  • < 5 platelets/OIO field: Decrease in platelet number.
  • 5-20 platelets/OIO field: Adequate platelet supply.

  • 25 platelets/OIO field: Increase in platelet number.

Electronic Method for Platelet Counting

• Red blood cells must be removed from whole blood by sedimentation or centrifugation.

This quiz covers the critical techniques used in diluting blood samples for red blood cell counts, including the appropriate dilutions and anticoagulants. It also explores different diluting fluids and their compositions, such as Hayem's and Gower's solutions, highlighting their effects on cell behavior. Test your knowledge of these essential hematological practices.