How is RBC Count Done? | Types | Blood Cells | Biology

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It is a quantitative measure of the population of blood cells in circulation. The counting of cells in circulation. The counting of cells can be done mainly with the help of a microscope after diluting the blood and making a wet mount. This technique is known as Hemocytometry.

Now a day’s automated system of blood cell counting by electrometric and photometric method are used. The hemocytometry technique has some drawbacks as they cannot differentiate between various type of WBC and Reticulocyte from the mature red blood cells.

Red Blood Cell Count:

Red Blood Cells (Erythrocytes):

(Erythrocytes)

Erythros – Red

Cyte – Cell

The cell which is red in appearance is generally known as Red blood cell. The colour of RBC is due to presence of haemoglobin in which haem is a colour pigment and globin is a protein.

The red blood cell count ranges between 4 to 5.5 million per cubic millimeter of blood.

In adult males it is 5 millions/cu mm of blood and in adult females it is 4.5 millions cu mm of blood.

Morphology or Red Blood Cells:

Normal Size

Diameter – 7.2 microns (µ) (6.9-7.4 m)

Thickness – At the peripheral it is thicker with 2.2 m % at the centre it is thinner with 1 micron

Normal Shape:

Normally the shape of R.B.C. is biconcave disc or dumb bell shaped.

Due to the biconcavity R.B.C. has some mechanical advantages which are following:

1. It helps in equal and rapid diffusion of oxygen and other substance into the interior of cell.

2. Large surface area is provided for absorption or removal of different substances.

3. Minimal tension is offered on the membrane when changes in volume of cell occur.

4. While passing through minute capillaries, these cells can squeeze through the capillaries very easily.

Total RBC Count by Hemocytometry:

Specimen:

i. Double oxalated or EDTA blood or

ii. Capillary blood.

Principle:

The blood specimen is diluted 1: 200 with the RBC diluting fluid cell are counted under high power (10 X objective) by using a counting chamber. The number of cells in undiluted blood calculated and reported as the number of red cells per cum (µl) of whole blood.

Requirement:

1. Microscope

2. Improved neubaeur chamber

3. RBC pipette

4. RBC diluting fluid preparation as follows:

a. Sodium Citrate: 3.09gm

b. Formalin: 1.0ml

c. Distilled water: 100ml.

This solutions is stable at room temperature (25°c ± 5°C) for at least one year.

Composition of RBC diluting fluid hayem’s fluid:

(1) Mercuric chloride — 0.5gm

(2) Sodium Chloride — 5gm

(3) Sodium Sulphate — 5gm

To prevent bacterial or fungal growth

(4) D.W. —200ml

Note:

1. RBC diluting fluid is isotonic with blood hence haemolysis does not take place. Normal saline also can be used but it causes slight crenation of red blood cells and allows rouleaux formation.

2. Formalin acts as a preservative and checks bacterial and fungal growth.

3. Sodium citrate prevents Coagulation of blood and provides correct osmotic pressure.

Procedure:

1. Mix the anti-coagulated blood carefully by swirling the bulb.

2. In the presence of capillary blood lancet stab should be sufficiently deep to allow free flow of blood; it is drawn quickly in the RBC pipette.

3. Draw blood up to 0.5 mark.

4. Carefully wipe the excess blood outside the pipette by using cotton or a gauze.

5. Draw diluting fluid up to 101 mark.

6. The pipette is rotated rapidly by keeping it horizontal during mixing.

7. After five minutes, by discarding few drops from the pipette and holding it slightly inclined small volume of the fluid is introduced under the cover slip which is placed on the counting chamber.

8. Allow the cell to settle for 2 to 3 minutes.

9. Place the counting chamber on the stage of the microscope.

10. Switch to lower power (10 x objective). Adjust light and locate the large square in the centre with 25 small squares.

11. Now switch to high power (40 x objective).

12. The red blood cells in the four corner squares and in the centre square are counted.

13. Use the following formula for the calculation of red blood cells.

Total red blood cell/cu mm (µl). = Number of red cells counted × dilution factor/Area counted × depth

Normal Value:

Male: 4.5 to 6.0 x 10⁶ cells/cu.mm (µl)

Female: 4.0 to 44.5 x 10⁶ cells/cu.mm (µl)

Clinical Significance:

An increase in total erythrocyte count is observed in conditions such as:

1. In burns, chlolera

2. Heart disease.

3. Polycythaemia

Decrease in erythrocyte count is observed in:

1. Old Age

2. Pregnancy.

3. Anaemia

Source of Error:

1. Diluting fluid should not be contaminated with RBC.

2. Keep the counting chamber and cover slip free from dust, lint and dried blood.

3. Use mild detergent (1% Sodium Bicarbonate) followed by washing with tap water and rinsing with deionzed water.

4. Some major source of technical error includes improper volume measurement (blood & diluent). Improper charging of the neubauer’s chamber, use of defective pipette improper counting, disturbance of the chamber during the switching of the objective, don’t allow the objective to touch the coverslip, failure to clean the blood sampling pipette, failure to clean the blood sampling pipette, wrong calculation and clinical mistake in recording.

Falsely High Count:

1. Inadequate wiping of the pipette.

2. Improper mixing.

3. Improper pipetting of blood as well as the fluid.

4. Error in calculations.

Falsely Low Count:

1. Blood dilution with tissues fluid

Deice’s Method:

Principle:

The number of red cell in a sample of diluted blood is counted in a hemolytic of known dimension. From the number of cell seen the total red cell count of undiluted sample is calculated.

Regents and Apparatus:

i. Diluting fluid

ii. Deice’s fluid

iii. Red cells pipette, small test tube and Hb pipette

iv. Hemocytometer

Method:

i. Anticoagulant or capillary blood is pipette to the 0.5 mark on the Thoma pipette and the external surface is wiped clean of blood.

ii. The diluting fluid is then taken to the 10% mark, care being taken to avoid the introduction of air bubble into the bulb.

iii. The pipette is shaken to mix and the first few drops of dilution are expelled.

iv. The hemocytometer is prepared by placing the covers Lipton transferees bars.

v. With the index finger controlling the end of pipette. Allow the drop of diluted blood to full one of side of the chamber by capillary attractions. Care should be taken that the drop is of sufficient size of completely fill the area. It shouldn’t over flow to surrounding area.

vi. The cells are allowed to settle in the ruled area for 2.3mins.

vii. With the 10x objective of microscope the cells seen in Fine Square out of 25 of central sequence of hemocytometer are counted.

Tube Dilution Techniques:

3.98ml diluting fluid

0.02ml blood using to Hb pipette

Rest of the steps is same as above.

Calculation:

Source of Errors:

1. Diluting fluid should not be contaminated with blood cells.

2. Keep the neubauer chamber and cover glass free from dust, lint and dried blood.

3. Some major source of technical error include improper volume measurement (specimen and diluents), improper charging of counting chamber, use of defective pipettes, improper counting, disturbance of chamber during the switching of the objective (Do not allow the objective to touch the cover slip) failure to clear the blood sampling pipette, failure wash the pipette properly, wrong calculation and clerical mistake in recording.