Mostly white blood cells (WBC) or TLC are calculated by an automated hematology analyzer but WBC can be calculated by the formula for WBC count in the Neubauer chamber (TLC test).

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WBC / TLC count Principle

Anticoagulated blood mixed with diluting fluid in a WBC pipette. After mixing the charge in the Neubauer chamber. Count the four large squares of the Neubauer chamber.

Note: At least 100 WBC should be counted.

What are the Requirements for the WBC count test (TLC test) ?

Following are the requirements for manual white blood cell (WBC) count.

1. WBC pipette with bulb, capable of measuring 50 micro letter blood.
2. Improved Neubauer chamber
3. Coverslip.
4. White Blood cells (WBC) diluting fluid.
5. Microscope.

How to prepare WBC diluting fluid?

White blood cell diluting fluid is prepared by mixing 4 meli letter (ml) glacial acetic acid with a 3% aqueous solution of methylene blue.

Note: Methylene blue is used to color the nucleus of WBC, while acetic acid is used to destroy the Red blood cells (RBC) in the sample.

Total white blood cell counting procedure (TLC)

Here are the steps for calculating WBC by improved Neubauer chamber.

1. First, fill the WBC pipette up to the mark of 0.5. Clean the outside of the pipette.
2. In the second step draw the WBC diluting fluid up to the mark of 11. Gently mix by rotating the pipette between thumb and finger.
3. Draw 20 micro letter blood by automated pipette, and drop it in the test tube containing 0.38 ml diluting fluid.
4. Place the coverslip on the Neubauer chamber, and charge the chamber after discording 3 to 4 drops.
5. Wait for 4 to 5 minutes, so that cell may settle down.
6. Count the WBC at 4 large squares by using high dry (40x). At least 100 WBC should be counted.

WBC calculating formula (white blood cells)

• The large square area of the Neubauer chamber: 1 mm²
• Depth of Neubauer chamber: 0.1 mm
• Dilution of sample 1 in 20
• The volume of a large square area: 0.1 micro letter
• The mean number of cells counted: N
• WBC or Total Leucocyte Count Formula = N * 20 * 10⁶ / 0.1 = N * 200 * 10 ⁶ = N * 0.2 * 10⁹

Corrected WBC count calculator

Corrected WBC (white blood cells) count formula

If nucleated Red blood cells are seen in DLC (Differential leucocyte count), then Total leucocytes are corrected by the following formula.

Corrected TLC or WBC = 100 * observed TLC / 100 + NRBC / 100 WBC

What is the reference range of white blood cells for males and females?

The reference range for males and females: 4-11 *10⁹ /L

Note: NRBC stands for nucleated Red Blood Cells.

What are the 5 main precautions during the Total Leucocyte Count (TLC) OR WBC counting procedure?

1. WBC pipette should be dry and clean before filling.
2. Dilution should be correct.
3. Debris of RBC should not be confused with WBC.
4. Sometimes cells sticking to debris should be recognized and counted.
5. If nucleated RBC is found then correct the TLC by the CORRECT TLC formula. (As above mention)