Determination of Total Leukocyte Count

Mr. Topesh Patle

Principle

  • The Determination of Total Leukocyte Count (TLC) is a test used to measure the total number of white blood cells (WBCs) in a blood sample.
  • WBCs, or leukocytes, are a vital component of the immune system, and their count can indicate the presence of infection, inflammation, and other health conditions.

  • The principle behind the test involves counting the white blood cells under a microscope after diluting a blood sample with a reagent that lyses the red blood cells (RBCs), leaving the white blood cells intact.

  • The WBCs are then counted in a specified area of a hemocytometer or using an automated cell counter, and the total number of WBCs per unit volume of blood is calculated.

  • Typically, the WBC count is done on fresh blood to avoid changes in cell count over time.

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Requirements

  1. Blood Sample:

    • The blood should be freshly drawn, as prolonged storage can affect the WBC count.

    • A venous blood sample is preferred for accurate results.

  2. Dilution Fluid:

    • A diluent is used to dilute the blood sample and to lyse the red blood cells without affecting the WBCs. Commonly used diluents are:

      • Acetic acid: Helps to lyse RBCs while keeping WBCs intact.

      • 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.

      • Hayem’s solution: Another solution used to dilute blood for cell counting.

  3. Hemocytometer:

    • A specialized glass slide with a grid etched into it, used to count cells under a microscope. The hemocytometer chamber is designed to hold a known volume of liquid, which allows for calculating cell concentration.

    • The grid is divided into small squares where the cells are counted.

  4. Microscope:

    • A high-quality microscope (usually at least 400x magnification) is necessary to clearly visualize and count the WBCs in the hemocytometer.

  5. Coverslip:

    • A thin glass cover is placed over the hemocytometer to keep the sample in place and prevent the cells from moving during examination.

  6. Pipettes:

    • Precise pipettes transfer the blood and diluting fluid to the hemocytometer.

  7. Timer:

    • A timer is needed to maintain consistent timing when incubating the diluted sample, ensuring proper RBC lysis before counting.

 

Procedure

  1. Blood Sample Preparation:

    • Collect a small amount of venous blood using a sterile technique.

    • 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.

    • 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.

 

Clinical Significance

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.

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