TOTAL RED BLOOD CELL COUNT

 INTRODUCTION 

• Total RBC count also known as erythrocyte-count.

• An RBC count is a blood test that is used to find out how many red blood cell person have.

PRINCIPLE

Very large number of red blood cell are present in the blood specimen, manually counting of red blood cell directly under the microscope is highly impossible, so the counting for this cell using a special type of chamber,known as hemocytometer.

For this, the blood specimen is diluted with the RBC diluting fluid which are isotonic to RBC , diluting fluid preserve and fix the RBC.

After diluting the specimen chamber gets filled with dilution, this is known as charging of the chamber.

After the charging we counted the cell easily under the microscope.

METHOD FOR TOTAL RBC COUNT

Two method are use for estimation of red blood cell count ,that is

1. Microdilution method
2. Macrodilution method

REQUIREMENTS FOR TEST 

Hemocytometer

This is a special type of glass chamber that is used for the blood cell counting.

Many types of hemocytometer is available in market such as

1. Neubauer's chamber.
2. Burkers chamber.
3. Levy's chamber.
4. Fusch Rosenthal chamber.

But mostly neubauer's chamber are used in medical laboratory.

Neubauer's chamber has ruled the area of total 9 square mm and the depth is 0.1 mm. The central 1 square is highly ruled which is divided into 25 square. Each square of the central square is further subdivided into 16small square.

For RBC count the cell are counted in the 5 squars of the central square. As 4 corner sub square of the central sub square and 1 central square of the larger central square .

RBC diluting fluid 

RBC diluting fluid is isotonic with blood hence hemolysis does not take place.

It is use for make dilution of RBC specimen.

Mainly Haym's fluid are use in laboratory for dilution of RBC.

Composition

• Sodium chloride - 0.5g
• Sodium sulfate - 2.5 g
• Mercuric chloride 0.25g
• Distilled water - 100 ml

RBC pipette

RBC pipette is graduated pipette that give dilution 1:200. It has bottom marked with 0.5 and 1 and tha top is marked with 101.

It has a round bulb containing red bead . A rubber tube attached to the top for sucking.

PROCEDURE FOR MICRODILUTION METHOD

1. Assemble all the equipment.
2. Drow the blood into the RBC pipette up to 0.5 mark.
3. Wipe off the top of pipette to remove extra blood.
4. Then immediately draw up the diluting fluid up to 101 mark.
5. Now rotate the pipette gently. So that the diluting fluid mixed properly. This will give dilution 1:200.
6. Place coverslip in position over the ruled area of chamber.
7. Discard 1-2 drop of blood.
8. Then apply slight pressure on the rubber tube and charged the chamber.
9. The care should be taken that , no air bubble is present inside the chamber.
10. Wait for 2 minute so that RBC settled down properly.
11. Place the chamber on the stage of microscope .adjust the light and ruled area.
12. Then count the RBC in central square, count four at each corner and one at center.

PROCEDURE OF MACRODILUTION METHOD

1. Take 3.98ml of RBC diluting fluid in a clean, dry and grease free test tube.
2. Now add 0.02ml (20ul) of blood specimen to the tube containing diluting fluid with the help of micropipette.
3. Mix well for fer minute
4. And place coverslip in posiyover the ruled area of chamber.
5. Carefully drow up around 20ul of the diluted specimen.
6. Press the knob of pipette to make a hanging drop at the top of the micropipette.
7. Touch the tip of pipette (hanging drop) against the edge of coverslip.
8. The angle between pipette and coverslip is 45°.
9. After charging wait for 3 minute so that the cells settle down in the chamber.
10. Place the chamber on the stage of microscope.
11. Now count the RBC on the central square, count four at each corner and one at center.

CALCULATION

Total RBC count =    N× Dilution

                                 Area × depth

Here

 N= number of cell counted

Dilution = 200

Area = 1/5 

Depth = 0.1

Then  total RBC count= N× 200

                                         1/5 ×0.1

Total RBC count = N × 10000.

NORMAL RANGE

• Male: 4.3-5.9 million/mm3
• Female: 3.5-5.5 million/mm3

CLINICAL SIGNIFICANCE

Erythrocytosis

Increase of RBC count from the normal range is called erythrocytosis. Condition:-

• Hypoxia
• Polycythemia

Erythrocytopenia

Decrease count of RBC is known as erythrocytopenia. Condition:-

• Anemia

SOURCE OF ERROR

• Inadequate whipping of the pipette.
• Improper mixing
• Improper pipetting of blood as well as fluid.
• Blood dilution with tissue fluid.

NOTE

• Nowadays Mouth pipetting is banned in most of the laboratories due to the high risk of getting infected with highly infected specimens of the patients. So instead of Microdilution method, the Macrodilution methods are employed in Laboratories.
• RBC diluting fluid is isotonic with blood hence haemolysis does not take place , normal saline also can be used but it cause slight creation of red blood cell and allow rouleaux formation.
• Keep the counting chamber and cover slip free from dust , lint and dried blood.