Total Leukocyte Count (TLC): Methods and Reference Ranges

The total leukocyte count (TLC) measures the concentration of white blood cells (WBCs) in a given volume of blood, typically expressed as the number of cells per microliter (μl) or liter (L) of blood. Two main approaches are used to estimate TLC:

• Manual or microscopic method
• Automated method

A differential leukocyte count (DLC) should always accompany TLC, as it allows for the calculation of the absolute counts of specific types of leukocytes.

Purpose of TLC

The primary goal of performing a TLC is to detect abnormalities in the number of WBCs, indicating conditions such as leukocytosis (increased WBC count) or leukopenia (decreased WBC count). TLC is commonly used to investigate infections, fevers, hematologic disorders, malignancies, and for monitoring patients undergoing chemotherapy or radiotherapy.

Manual Method for Total Leukocyte Count

Principle

In the manual method, a blood sample is mixed with a diluent that lyses red blood cells and stains the nuclei of WBCs. The leukocytes are then counted using a hemocytometer under a microscope, with the results expressed as the total number of leukocytes per microliter or liter of blood.

Equipment

• Hemocytometer: A special counting chamber with an improved Neubauer ruling is used. It features a grid of 9 large squares for cell counting, with the four corner squares reserved for WBCs. The cover glass must be thick and optically flat to ensure a constant depth and volume throughout the chamber, with colored refraction rings (Newton's rings) indicating proper placement.
• Pipettes: Pipettes calibrated to deliver a precise volume of blood, such as the WBC bulb pipette, are used for dilution. However, these are not widely recommended due to mixing and calibration difficulties.
• Other equipment includes a graduated pipette, Pasteur pipette, and test tubes.

Reagent

WBC diluting fluid (Turk’s fluid) is composed of acetic acid, gentian violet, and distilled water. The acetic acid lyses red blood cells, while the gentian violet stains WBC nuclei, making the leukocytes more visible under the microscope. Its composition is as follows:

• Acetic acid, glacial 2 ml
• Gentian violet, 1% aqueous 1 ml
• Distilled water to make 100 ml

Specimen

Blood collected using EDTA as an anticoagulant is preferred. Capillary blood samples should be collected carefully to avoid dilution with tissue fluids.

Methodology for TLC

1. Dilution of blood: A small volume of blood is mixed with the diluent to create a 1:20 dilution.
2. Charging the counting chamber: The diluted blood is drawn into a Pasteur pipette and placed into the hemocytometer chamber, ensuring no air bubbles are trapped.
3. Counting the cells: Under a microscope, leukocytes are counted in four large squares. To improve accuracy, it is often advisable to count all nine squares.

Calculation of TLC

The total leukocyte count (TLC) per microliter is calculated using the following formula:

Where:

• N_w = number of WBCs counted
• C_d = correction for dilution (typically 20)
• C_v = correction for volume (10)
• N_LS = number of large squares counted

For example, if 200 WBCs are counted in 4 squares, the TLC would be 10,000/μl. In SI units, the count is converted to WBCs × 10⁶ per liter. If the TLC exceeds 50,000/μl, the dilution of blood should be adjusted to improve accuracy.

If TLC is more than 50,000/ml, then dilution of blood should be increased to 1:40 to increase the accuracy of the result.

If TLC is less than 2,000/ml then lesser dilution should be used.

Expression of Total Leukocyte Count (TLC)

TLC is typically presented in units of cells/μl, cells/cmm, or cells/mm³. In the International System of Units (SI), it is expressed as cells × 10⁹ per liter. To convert from conventional units to SI, a factor of 0.001 is applied, and to revert to conventional units from SI, the factor is 1000.

Correction of TLC for Nucleated Red Blood Cells (NRBC)

The diluting fluid used in counting does not break down nucleated red blood cells (erythroblasts), which can lead to their being mistakenly counted as leukocytes when using a hemocytometer. This miscalculation can significantly overestimate the total leukocyte count, particularly when there is a noticeable increase in erythroblasts in the blood sample. To correct this, if more than 10 nucleated red blood cells per 100 leukocytes are observed on a blood smear, the following formula should be used to adjust the TLC:

Here, CTLC represents the corrected total leukocyte count per microliter (μl), TLC is the total leukocyte count, and NRBC denotes the number of nucleated red blood cells per 100 white blood cells.

Reference Ranges for TLC

• Adults 4000-11,000/μl
• At birth 10,000-26000/μl
• 1 year 6,000-16,000/μl
• 6-12 year 5,000-13,000/μl
• Pregnancy up to 15,000/μl

Critical Values

TLC less than 2,000/μl or greater than 50,000/μl indicates a critical health condition that requires immediate attention.

Summary

Accurately determining the total leukocyte count provides critical information about a patient's immune status, especially when combined with a differential leukocyte count. Whether using manual or automated methods, ensuring precise results is vital for diagnosing and monitoring conditions such as infections, hematologic disorders, and responses to treatment.