Determination of Total Leukocyte Count

AIM: Determination of Total Leukocyte Count (TLC)

Principle

• The glacial acetic acid lyses the red cells while the gentian violet slightly stains the nuclei of the leukocytes.
• The blood specimen is diluted 1:20 in a WBC pipette with the diluting fluid, and the cells are counted under low power of the microscope by using a counting chamber.
• The number of cells in undiluted blood is reported per cu mm (pl) of whole blood.

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Requirements

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1. Blood Sample: EDTA blood sample 

2. Dilution Fluid: Turk’s solution: A mixture of acetic acid, gentian violet (a dye), and sometimes distilled water, which also helps to enhance the visibility of WBCs.

3. Hemocytometer

4. Microscope

5. Coverslip

6. WBC Pipettes

7. Timer

 

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Procedure

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1. Blood Sample Preparation:

   • Collect a small amount of venous blood up to 0.5 marks of WBC pipette.

   • Carefully wipe excess blood outside the pipette with cotton.

   • Mix the blood with a diluent (e.g., Turk’s solution) in a test tube. The typical ratio is 1 part blood to 20-100 parts of diluent, depending on the method used.

   • Draw diluting fluid up to 11 mark.

   • Allow the mixture to sit for 2-5 minutes to ensure complete lysis of the red blood cells, which are removed from the sample by the solution, while the white blood cells remain intact.

2. Loading the Hemocytometer:

   • After the red blood cells have lysed, mix the sample gently to ensure an even suspension of white blood cells.

   • Using a pipette, carefully place a drop of the diluted sample on the hemocytometer. Avoid overloading the chamber to prevent crowding of cells.

   • Place a coverslip over the hemocytometer, ensuring the fluid spreads evenly across the grid.

3. Microscopic Examination:

   • Place the hemocytometer under the microscope.

   • Focus on the grid lines at 400x magnification (or higher if needed).

   • WBCs will appear as larger, round cells with a prominent nucleus compared to RBCs. They are typically colorless, though staining solutions like Turk’s solution may make them easier to distinguish.

4. Counting the Cells:

   • In manual counting, count the number of white blood cells in the defined area of the hemocytometer. This area typically consists of 4 large squares in the grid.

   • Each square has a specific area, and by counting the WBCs in several squares, a representative count can be obtained.

5. Calculation of WBC Count:

   • The WBC count is determined by counting the cells in the grid squares and applying the dilution factor. The formula is:

     TLC (cells/mm³) = (Total WBC count in counted area/Area of counted grid)×Dilution Factor

   • Automated cell counters, if available, perform this calculation automatically after detecting and counting the white blood cells in the sample.

6. Record the Result:

   • The result is typically reported as the number of white blood cells per cubic millimeter (cells/mm³) of blood.

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Clinical Significance

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The total leukocyte count is an essential diagnostic tool in many clinical situations. Abnormal levels of WBCs can indicate various pathological conditions.

1. Leukocytosis (Elevated WBC Count):

   • A high TLC can be associated with:

     • Infections: Particularly bacterial infections, where the body increases WBC production to fight off pathogens.

     • Inflammation: Conditions like rheumatoid arthritis, allergies, and autoimmune disorders can cause elevated WBC counts.

     • Leukemia: A form of blood cancer that leads to the abnormal production of white blood cells.

     • Stress or trauma: Physical stressors or tissue damage can increase WBC count.

     • Drug-induced: Certain medications like corticosteroids may also lead to leukocytosis.

2. Leukopenia (Decreased WBC Count):

   • A low TLC can indicate:

     • Bone marrow disorders: Conditions like aplastic anemia or myelodysplastic syndromes can affect the production of WBCs.

     • Viral infections: Some viral infections (e.g., HIV, hepatitis) can suppress WBC production.

     • Autoimmune diseases: Conditions like lupus can cause a reduction in WBC counts.

     • Chemotherapy or radiation therapy: These treatments can lower the WBC count by affecting the bone marrow’s ability to produce new cells.

     • Nutritional deficiencies: Deficiencies in vitamin B12, folic acid, or copper can lead to low WBC counts.

3. Other Clinical Considerations:

   • Neutrophilia: A rise in neutrophils (a specific type of WBC) may occur due to bacterial infections, inflammation, or tissue damage.

   • Lymphocytosis: Increased lymphocytes can be seen in viral infections or lymphocytic leukemia.

   • Monocytosis: Elevated monocytes are often seen in chronic infections or inflammatory conditions.