Total Leucocyte Count (TLC) a.k.a Total WBC Count Using Hemocytometer

TOTAL WHITE BLOOD CELL (WBC) COUNT A.K.A TOTAL LEUCOCYTE COUNT (TLC) USING HEMOCYTOMETER / NEUBAUER’s CHAMBER

A BRIEF INTRODUCTION TO WHITE BLOOD CELL

White blood cells (WBCs) or Leucocytes are the round shaped, nucleated cells, present in the blood that provides defense against the foreign bodies. There are various types of White Blood Cells are present in blood and classified as Granulocytes (Neutrophils, Eosinophils & Basophils) and Agranulocytes (Monocytes & Lymphocytes). The size of the White Blood Cells varies from 10 – 20 µm (microns). Every type of White Blood Cell has a different function in our body….



The lifespan of White Blood Cells varies from few days to few years. The memory cells can live for many years and provide defense against various infectious agents.

Check out the functions of White Blood Cell

THE AIM / PURPOSE OF PERFORMING TOTAL WBC COUNT

The purpose of performing Total White Blood cell (WBC) count is to know whether or not you are suffering from Leucocytosis (i.e. the increase in the no. of White Blood Cells to more than 11000/mm3) or Leucopenia or (i.e. the Decrease in the no. of White Blood Cells to less than 1500 /mm3). The Total Leucocyte count (TLC) is also done to check the functioning of Bone marrow.

WHITE BLOOD CELLS - TOTAL LEUCOCYTE COUNT - TOTAL WBC COUNT - TOTAL WHITE BLOOD CELL COUNT - TLC - TWBC COUNT - NEUBAUER's CHAMBER - HEMOCYTOMETER
VARIOUS TYPES OF WHITE BLOOD CELLS

PRINCIPLE OF TOTAL LEUCOCYTE COUNT USING HEMOCYTOMETER

A Very large number of White Blood Cells or Leucocytes are present in the Blood Specimen. Practically, counting this much amount of White cells directly under the microscope is highly impossible. So, the Leucocytes are counted by using a special type of chamber, designed for the counting of blood cells in the specimen, known as Hemocytometer or Neubauer’s chamber.

For this, the blood specimen is diluted (usually in 1:20 ratio) with the help of WBC diluting fluid (commonly the Turk’s Fluid) which preserve, stains and fix the White blood cells and Lysis the Red Blood Cells. The Turk’s fluid is isotonic to the White blood cells and does not cause any damage to it.

After diluting the specimen, the content is charged on Hemocytometer / Neubauer’s chamber and the cells are counted in the areas specific for WBC count.

The composition of WBC diluting Fluid (Turk’s Fluid)

COMPONENTSQUANTITY
Glacial Acetic Acid2 ml
Gentian Violet (1% Aqueous)1 ml
NaCl0.9 gram
Distilled Water97 ml

The Final pH of the solution (at 25°C) varies from 2.0 – 2.4 which depends on the composition and companies who manufacture it.

The above Composition is based on HIMEDIA Turk’s diluting fluid protocol which you can check here.

Some Labs & Companies add the NaCl (Sodium Chloride) for isotonicity and Thymol crystals to prevent the Mould formation in the solution which may vary as per their Standard Operating Procedures (SOPs).

Hemocytometer / Neubauer’s Chamber

This is a special type of glass chamber that is used for the cell counting, especially for Blood cells. Nowadays, most commonly Improved Neubauer’s Chamber is used and in some laboratories, other types of chambers are also employed like Burkers chamber, Levy’s chamber and Fusch – Rosenthal chamber etc.

neubauer chamber - hemocytmeter - improved neubauer chamber - improved hemocytometer - improved markings in hemocytometer
HEMOCYTOMETER A.K.A NEUBAUER’s CHAMBER

The Neubauer’s Chamber has ruled the area of total 9 square mm and the depth is 0.1 mm as when the coverslip is placed on the surface of the counting chamber, the space between the bottom of the cover glass and the base of grooved area measures 0.1 mm in depth.

Out of the 9 Squares, the 4 corner squares are used for the White Blood Cell count. Every large corner square is further divided into the 16 small squares, so the total no. of the area to be counted for WBC Count –

rulings in neubauer chamber - neubauer chamber - rulings in hemocytometer - hemocytometer
R – RBC AREAS ; W – WBC AREAS

16 × 4 = 64 small squares of the 4 large corner squares

Two Method has been developed for the Manual Estimation of Total Leucocyte Count Using Hemocytometer / Neubauer’s chamber –

  • Microdilution Method
  • Macrodilution Method

Here, I’ll explain both the methods but the Microdilution method is not preferred nowadays due to the use of Mouth pipettes. So let’s start with Microdilution method and then we’ll move to Macrodilution method….



MICRODILUTION METHOD FOR THE ESTIMATION OF TOTAL LEUCOCYTE COUNT (TLC) USING HEMOCYTOMETER

Materials Required for the Total Leucocyte Count (TLC) by Microdilution Method –

  • Blood sample (Capillary blood or EDTA anticoagulated specimen)
  • WBC diluting fluid (preferably Turk’s fluid)
  • Gauze piece or Cotton
  • WBC pipette
  • Hemocytometer a.k.a. Neubauer’s Chamber
  • Coverslip
  • Microscope

A Brief Introduction to WBC Pipette….

WBC pipette is a graduated pipette that gives the dilution of 1:20. It has two markings at the bottom as 0.5 and 1 and the top of the pipette is marked 11. It has a round shape bulb which contains the White bead to mix the blood specimen and the diluting fluid. On the top, a rubber tube is attached to the pipette for sucking the blood specimen and diluting fluid.

When blood is sucked up to 0.5 mark and the diluting fluid up to 11 marks, gives the 1:20 dilution of Blood: Diluting fluid and When the Blood is sucked up to 1 mark and the diluting fluid up to 11, gives the 1:10 dilution of Blood: Diluting fluid which less commonly used. After sucking the Specimen & Diluting fluid, the content is gently mixed by rotating the pipette on its long axis to ensure thorough mixing of blood and diluting fluid.

Note: Nowadays Mouth pipetting is banned in most of the laboratories due to the high risk of getting infected with highly contaminated specimens of the patients. So instead of Microdilution method, the Macrodilution methods are employed in Laboratories…..

Procedure of the Total Leucocyte Count (TLC) Count by Microdilution Method

Fill the WBC pipette up to the 0.5 mark with the blood specimen and wipe out the pipette externally to avoid false high results.

Fill the same pipette with the WBC diluting fluid (preferably Turk’s Fluid) up to the mark 11.

Be cautious that there should be no air bubble in the pipette bulb.

Mix the Blood and Diluting fluid in the pipette by rotating the pipette (horizontally) between your palms.

Take out the Neubauer’s chamber / Hemocytometer from its case and clean it using a swab or gauze piece. Similarly, clean out the cover glass and place it over the grooved area of Hemocytometer.

Note: Here a special type of cover glass is used which is 0.4 mm thick with very smooth surface and even thickness so that the space between the grooved area of the chamber and cover glass is exactly 0.1 mm.

Now, put the WBC pipette, mix the solution present in it again and then discard 1-2 drops from the pipette before charging the chamber.

Gently press the rubber tube of the WBC pipette, so that the next drop of fluid is in hanging position.

Touch the Tip of the pipette with the hanging drop against the edge of the coverslip making an angle of 45° approximately.

Allow a small amount of fluid from the pipette to fill into the chamber which occurs by the Capillary action. Do not overcharge the chamber and there should be no air bubble in the Chamber.

After charging, wait for 3-5 min so that the cells settle down in the chamber & then focus the chamber under the microscope to calculate the White Blood Cells.



MACRODILUTION METHOD FOR THE ESTIMATION OF TOTAL LEUCOCYTES USING HEMOCYTOMETER

Materials Required for the Total Leucocyte Count (TLC) by Macrodilution Method

  • Blood sample (Capillary blood or EDTA anticoagulated specimen)
  • WBC diluting fluid (preferably Turk’s fluid)
  • Hb pipette or Micropipette (0.02 ml or 20 ml)
  • Hemocytometer / Neubauer’s Chamber
  • Gauze piece or Cotton swab
  • Graduated Pipette (5 ml)
  • Test tubes
  • Cover Slip

Procedure of the Total Leucocyte Count (TLC) Count by Macrodilution Method

Take 0.38 ml of WBC diluting fluid in a Clean, Dry and Grease free Test tube.

Note: If you don’t have variable pipette in the lab which can measure 0.38ml or 380 µl of Diluting fluid then take 400 µl of Diluting fluid with the help of micropipette in the test tube and discard 20 µl of fluid using a micropipette or Hb pipette.

Now add 0.02 ml or 20 µl of Blood Specimen to the tube containing diluting fluid with the help of micropipette or WBC pipette.

Alternatively, Take 1.9 ml of WBC Diluting Fluid in a Test tube & mix 0.1 ml of blood Specimen to it.

Mix well for few minutes and ready your Hemocytometer / Neubauer’s Chamber.

Take out the Neubauer’s chamber / Hemocytometer from its case and clean it using a swab or gauze piece. Similarly, clean out the cover glass and place it over the grooved area of Hemocytometer.

Note: Here a special type of cover glass is used which is 0.4 mm thick with very smooth surface and even thickness so that the space between the grooved area of the chamber and cover glass is exactly 0.1 mm.

Now, take out the WBC pipette and fill it with the Diluted Specimen, mix the solution well and then discard 1-2 drops from the pipette before charging the chamber.

Gently press the rubber tube of the WBC pipette, so that the next drop of fluid is in hanging position.

Touch the Tip of the pipette with the hanging drop against the edge of the coverslip making an angle of 45° approximately.

Allow a small amount of fluid from the pipette to fill into the chamber which occurs by the Capillary action. Do not overcharge the chamber and there should be no air bubble in the Chamber.

Using Micropipette instead of WBC pipette for charging the Hemocytometer

You can also use a micropipette instead of WBC pipette for charging the Hemocytometer. So, with a micropipette, carefully draw up around 20 µl of the diluted specimen. Press the knob of the pipette to make a hanging drop at the tip of the micropipette.

Now gently place the pipette tip against the edge of the cover glass and if required slowly expel the more liquid until the counting chamber is full. This process occurs by Capillary action, but care should be taken not to overfill the chamber. A volume of 10 µis sufficient to fill out the one counting chamber.

After charging, wait for 3-5 min so that the cells settle down in the chamber.

COUNTING THE WHITE BLOOD CELLS (WBC) UNDER MICROSCOPE

Focus the ruling using the 10x Objective lens and then Count the WBCs in 4 large corner squares as described above, using the 40x Objective lens.

Count the cells which are lying on the right and lower lines of the 4 Corner squares but not the opposite line. In case of marginal cells, count the cells on ‘L’ line that is either on Right and Lower lines or Left and Upper lines.

CALCULATIONS FOR THE TOTAL WBC COUNT USING HEMOCYTOMETER

After counting the cells under the microscope, we know the No. of WBC in 4 Corner squares. Let’s consider it as ‘N’ no. of cells.

Now, the volume of the fluid inside the chamber is the product of Area and depth of the Hemocytometer / Neubauer’s chamber.

4 × 1 × 1 × 1/10 = 2/5

The depth of the Hemocytometer is 0.1 or 1/10 mm as described above in a short description of Hemocytometer.

Now Apply the Following formula to get the Total White Blood Cell Count –

Thus, 2/5 mm3 Contains = N × Dilution

Then, 1 mm3 Contains = N × 20 × 5/2

Total WBC count = N × 50 / mm3

Using the Above formula we can calculate the Total No. of White Blood Cells present in the Blood Specimen.

NORMAL VALUES OF WHITE BLOOD CELLS

According to the Mayo Clinic, these are the normal ranges of WBCs per microlitre (or mm3) of blood by age:

AGE OF PERSONWBCs / Cu-mm OF BLOOD
immediately after birth9,000 to 30,000
1 to 7 days9,400 to 34,000
8 to 14 days5,000 to 21,000
15 days to 1 month5,000 to 20,000
2 to 5 months5,000 to 15,000
6 months to 1 year6,000 to 11,000
2 years5,000 to 12,000
3 to 5 years4,000 to 12,000
6 to 11 years3,400 to 10,000
12 to 15 years3,500 to 9,000
Adults3,500 to 10,500

The normal percentages of the types of WBCs in overall Leucocyte count are usually in these ranges:

TYPE OF WBCNORMAL PERCENTAGE IN BLOOD
Neutrophil45% to 75%
Eosinophil2% to 6%
Basophil0% to 1%
Monocytes2% to 10%
Lymphocytes20% to 40%

PRECAUTIONS TO BE TAKEN WHILE PERFORMING TOTAL LEUCOCYTE COUNT BY HEMOCYTOMETER

Use of Mouth pipettes (WBC pipette) is banned in many countries. However, in case you have to use it, be cautious that you should not intake the diluting fluid or Specimen.

Accurately measure the amount of specimen and Diluting Fluid to avoid any error in the results.

In case you are performing this test by Microdilution method, mix the specimen and diluting fluid appropriately by gently rotating the pipette in between your palms.

Carefully charge the Hemocytometer or Neubauer’s chamber that it should not be overcharged and do not contain any air bubble in it.

Check Out the Total RBC Count Using Hemocytometer


SAHIL BATRA

Hi, I'm the Founder and Developer of Paramedics World, a blog truly devoted to Paramedics. I am a Medical Lab Tech, a Web Developer and Bibliophiliac. My greatest hobby is to teach and motivate other peoples to do whatever they wanna do in life.

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