Haematology: Microhaematocrit Method Study Notes

Aim

  • The objective of this practical exercise is to measure the proportion of red blood cells (RBCs) in a whole blood sample.

Principle

  • The measurement is based on centrifugation of a blood sample. Under the influence of centrifugal force (12,000 - 15,000 \text{ rpm}), cells with higher density (erythrocytes) are packed at the bottom of a narrow tube, while the less dense plasma remains at the top. This allows for the calculation of the packed cell volume (PCV).

Definition of Key Terms

  • Haematology: The study of blood and its components.

  • Microhaematocrit: A laboratory method using capillary tubes to measure the volume percentage of RBCs.

  • Erythrocytes: Red blood cells responsible for oxygen transport.

  • Anaemia: Condition where hemoglobin (Hb) or PCV levels are below reference ranges.

  • Polycythemia: Condition with abnormally high erythrocyte levels.

Materials Required

  • Microhaematocrit centrifuge

  • Microhaematocrit reader

  • Heparinized capillary tubes (for finger prick/capillary blood)

  • Plain capillary tubes (for EDTA anti-coagulated venous blood)

  • Sealing clay (Plasticine)

  • Cotton wool and alcohol swabs

Comparison of Methods

  1. Micro-method (Microhaematocrit):

    • Requires small volumes of blood (75 \mu\text{L}).

    • Centrifugation at 12,000 \text{ rpm} for 5 \text{ minutes}.

    • Advantages: Faster and requires less blood.

  2. Macro-method (Wintrobe method):

    • Requires approximately 1 \text{ mL} of blood.

    • Centrifugation at 3,000 \text{ rpm} for 30 \text{ minutes}.

    • Disadvantage: More time-consuming and increased risk of trapping plasma between cells.

Procedure

  1. Collect blood in an appropriate capillary tube (fill about 3/4 or 75\% of the tube).

  2. Seal the dry end of the tube with plasticine (seal depth should be approximately 2 \text{ mm}).

  3. Place the tube in the centrifuge with the sealed end facing outward against the rubber gasket.

  4. Centrifuge for 5 \text{ minutes} at 12,000 - 15,000 \text{ rpm}.

  5. Allow the centrifuge to stop naturally before opening the lid.

  6. Use a microhaematocrit reader to determine the result.

Calculation and Expression of Results

  • The Packed Cell Volume is expressed as either a percentage (\%) or a decimal fraction (\text{L/L}).

  • Formula:
    \text{PCV (\%)} = \frac{\text{Height of Packed RBC Column (mm)}}{\text{Total Height of Blood Column (mm)}} \times 100

  • Example: If the RBC column is 35 \text{ mm} and total column is 75 \text{ mm}, then:
    \text{PCV} = \frac{35}{75} \times 100 \approx 46.7\%

Results and Observations

  • Layers of Centrifuged Blood:

    1. Top layer: Clear, yellowish plasma.

    2. Middle layer (Buffy coat): A thin white/grey layer containing leukocytes (WBCs) and thrombocytes (platelets).

    3. Bottom layer: Packed erythrocytes (RBCs).

  • Normal reference ranges:

    • Males: 40\% - 54\%

    • Females: 36\% - 47\%

Precautions

  • Ensure the centrifuge is balanced by placing tubes symmetrically.

  • Ensure the sealed end faces the outer rim to prevent blood loss during spinning.

  • Read results immediately after centrifugation to prevent the cells from remixing with plasma.

  • Exclude the buffy coat layer when measuring the height of the RBC column.