Enumeration of White Blood Cell (WBC) Count

Aim: Enumeration of White Blood Cell (WBC) Count

References

1. Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries. Cambridge University Press.
2. Henry, J. B. (2001). Clinical Diagnosis and Management by Laboratory Methods. W.B. Saunders Company.
3. Articles from Google Scholar/PubMed on manual WBC counting techniques.

Objective

To determine the total number of white blood cells (WBCs) per cubic millimeter of blood using a hemocytometer.

Introduction

White blood cells (WBCs), also known as leukocytes, are a critical component of the immune system, responsible for defending the body against infections, foreign invaders, and harmful substances. They are produced in the bone marrow and circulate through the bloodstream and lymphatic system.

Principle

White blood cells (WBCs) are vital components of the immune system, responsible for protecting the body against infections and foreign invaders. Enumeration of WBCs is essential for diagnosing various medical conditions such as infections, leukemia, and immune system disorders. A hemocytometer is used for manual WBC counting, where cells are visualized under a microscope and counted within a defined grid area.

Materials Required

1. Reagents
   1. Turk’s solution (acetic acid with gentian violet dye)
2. Apparatus

Hemocytometer (Neubauer chamber), Microscope, Capillary pipette or micropipette, Cover slip, Sterile lancet, Cotton and antiseptic, Test tubes

Procedure

1. Sample Preparation

• Prick the fingertip using a sterile lancet or collect venous blood in an anticoagulant-treated tube.
• Use a capillary pipette to draw blood to a mark indicating a specific volume.

2. Dilution of Blood Sample

• Add a known volume of blood (e.g., 0.02 mL) to Turk’s solution (e.g., 0.38 mL) in a test tube.
• Mix gently to lyse red blood cells (RBCs) and stain WBC nuclei for visibility.

3. Charging the Hemocytometer

• Clean the hemocytometer and cover slip to ensure no air bubbles.
• Place the cover slip over the Neubauer chamber.
• Using a micropipette, fill the chamber with the diluted blood sample. Ensure no overfilling or underfilling.

4. Microscopic Counting

• Place the hemocytometer under a microscope.
• Focus on the grid area for WBC counting, typically the four large corner squares.
• Count WBCs in each of the large squares under low power (10x objective).

Calculation

The total WBC count is calculated using the formula:

• Number of cells counted: Sum of WBCs in the four large squares.
• Dilution factor: As prepared (e.g., 20 if 0.02 mL blood + 0.38 mL Turk’s solution).
• Volume of one square: 0.1 mm³ for the large square in the Neubauer chamber.

Sample Data Table

Square No.	WBC Count (cells)
1	52
2	47
3	50
4	53
Total	202

Example Calculation

Interpretation

• Normal WBC count: 4,000–11,000 cells/mm³.
• Values outside this range indicate possible conditions:

Leukocytosis (high count): Infections, inflammation, leukemia.

Leukopenia (low count): Bone marrow disorders, autoimmune diseases.

Result: The WBC count of the given blood sample is 10,100 cells/mm³, which falls within the normal range (4,000–11,000 cells/mm³).

Precautions

1. Handle blood samples and reagents carefully to avoid contamination.
2. Ensure proper dilution to prevent cell clumping.
3. Use clean and calibrated equipment for accuracy.
4. Count systematically to avoid double-counting or missing cells.

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