Standard Operating Procedure (SOP) for Total White Blood Cell (WBC) Count

Purpose

This SOP aims to outline the procedure for determining the Total White Blood Cell (WBC) count in a patient’s blood sample. The WBC count is essential for diagnosing infections, immune disorders, and hematological diseases.

Scope

This SOP applies to laboratory personnel performing the total WBC count as part of a full blood count (FBC) for patient diagnosis and monitoring.

Equipment and Materials

• EDTA anticoagulated blood sample (venous)
• Hemocytometer (Neubauer counting chamber)
• Micropipette (or pipette for manual dilution)
• WBC diluting fluid (e.g., Turk’s solution: 1% acetic acid with gentian violet)
• Cover slips
• Light microscope
• Hand tally counter (optional)
• PPE (gloves, lab coat)
• Biohazard waste container

Specimen Collection

Venous Blood:

• • Collect 2-5 mL of venous blood in an EDTA anticoagulated tube.
  • Mix the blood sample gently by inverting the tube 5-10 times to prevent clotting.

Preparation of the Hemocytometer

• Dilution of the Blood Sample:
  • Using a micropipette, pipette 20 µL of the blood sample into a clean test tube.
  • Add 380 µL of WBC diluting fluid (Turk’s solution) to the blood to make a 1:20 dilution.
  • Mix gently to avoid frothing, allowing the WBCs to be stained and red blood cells (RBCs) to lyse.

• Loading the Hemocytometer:
  • Clean the hemocytometer with alcohol and allow to dry.
  • Charge the counting chamber by placing a cover slip over the hemocytometer.
  • Use a micropipette or capillary pipette to transfer the diluted sample onto the hemocytometer. Ensure the chamber is fully charged without overfilling or air bubbles.

 

Microscopic Examination and Counting

• Let the Cells Settle:
  • Allow the hemocytometer to sit undisturbed for 2-3 minutes to allow the WBCs to settle evenly on the grid.

 

• Counting the Cells:
  • Place the hemocytometer on the microscope stage and examine under low power (10x objective).
  • Focus on the 4 large corner squares of the hemocytometer grid (the areas marked ‘W’ on the diagram) .
  • Count all WBCs present in four squares, using a hand tally counter if necessary to track the count.

• Exclusion Criteria:
  • Cells that touch the top and left borders of the grid squares are included in the count.
  • Cells that touch the bottom and right borders are excluded.

Calculating the Total WBC Count

The formula to calculate the total WBC count per liter

                              { Cells counted   X    Dilution factor (20)   X   10⁶} ÷ {Area counted (4mm²)   X    Depth of Chamber (0.1mm)}

In simplified term

• Divide the total number of cells counted by 2.
• Divide the number obtained by 10.
• The number obtained  X 10⁹is the white cell

 

Example:

• • Cells counted in 4 squares is 84
  •  84 /2 = 42
  • 42 /10 = 4.2
  • WBC count = 4.2 cells X 10⁹/L

Interpretation of Results

• Normal Reference Ranges:
  • Adults:………………..4.0-11.0 X 10⁹/L
  • Children at 1y):…. 6.0 – 18.0 X 10⁹/L
  • Children (4-7y):…. 5.0 – 15.0 X 10⁹/L

 

• Abnormal WBC Counts:
  • Leukocytosis: A total WBC count >11,000/µL, which may indicate an infection, inflammation, or malignancy (e.g., leukemia).
  • Leukopenia: A total WBC count <4,000/µL, which may indicate bone marrow suppression, viral infections, or autoimmune disorders.

Quality Control

• Equipment Calibration:
  • Ensure that the microscope, pipettes, and hemocytometer are regularly calibrated and maintained.

 

• Control Samples:
  • Use control blood samples with known WBC counts to validate the accuracy of the procedure.

 

• Reagent Quality:
  • Ensure that the WBC diluting fluid (Turk’s solution) is fresh and properly stored to ensure accurate lysing of RBCs and proper staining of WBCs.

Safety and Waste Disposal

• Personal Protective Equipment (PPE):
  • Wear gloves and a lab coat while handling blood samples to prevent exposure to potentially infectious materials.
• Disposal:
  • Dispose of all used pipettes, capillary tubes, and other contaminated materials in appropriate biohazard containers.
  • Sterilize reusable equipment, such as hemocytometers, before reuse.

References

• Cheesbrough, M. District Laboratory Practice in Tropical Countries, Part 2, 2nd edition. Cambridge University Press, 2005.