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.