Determination of red blood cell (RBC) count

Determination of red blood cell (RBC) count in blood...pharmacyteach

A red blood cell (RBC) count test is done to determine average RBC counts in blood circulation(3). Before starting the practical we will see some details of red blood cell and their characteristic.

Characteristics of Red blood cells (RBCs)


The common name of RBC	Erythrocytes (1)
Shape of RBC	Biconcave discs (1)
Diameter of RBC	7.8 micrometer
Thickness of RBC	2.5 micrometers
The average volume of the RBC	90 – 95 cubic micrometers
A major function of RBCs	Transport hemoglobin
Lifetime of RBCs	120 days
Site of production of RBCs	In the fetus, the liver is majorly responsible for the production of RBCs but the spleen and lymph node are also somehow involved. After birth, Red blood cell production starts in the bone marrow.
The normal concentration of RBCs (2)	In males, 4.7—6.1 million/mm^3 In females, 4.2—5.4 million/mm^3 In children, 4.0—5.5milliom/mm^3

The process of making RBCs is known as erythropoiesis.

So, this will explain why the BRC count test is done and what happens when your RBC count is increase or decreases from the average range.

Clinical manifestation of RBC count

Alterations in RBCs count

Indication

Increased in RBC count (3)

Polycythemia

Dehydration

Chronic hypoxia conditions i.e. congenital disease and

Emphysema

Decrease in RBC count (3)

Anemia ( blood loss, aplastic, hemolytic, megaloblastic )

Kidney disease

malnutrition

Procedure for determination of RBCs count;

Apparatus :

Ø Microscope
Ø Hemocytometer (Neubauer counting chamber and RBC diluting pipette)
Ø Lancet
Ø Watch glass
Ø Coverslip

Chemicals required

Ø Hayem’s fluid ( sodium chloride, sodium sulfate, mercuric chloride, distilled water )

Step-by-step procedure of RBC count test :

Collection of blood samples:

Ø Wipe the finger or the tip of the subject with the help of an alcohol swab for pricking. Prick your finger with a sterile lancet. Wipe the first drop of blood with the sterile cotton gauze. Allow the blood to flow slowly and freely. Let a large drop of blood accumulate on a fingertip.

Dilution Of blood sample:

Ø Hold the pipette horizontally and dip its end into a drop of blood. Suck gently to draw blood to the required mark (0.5) on the stem of the pipette. Wipe the tip of the pipette to remove the extra amount of blood sticking to it.
Ø Maintain the blood level at the 0.5 mark and place the tip of the pipette in Hayem's fluid. Suck the diluting fluid into the pipette up to mark 101 above the bulb.
Ø Maintain the level of the mixture exactly to the mark by closing the pipette tip with an index finger Hold the pipette in the horizontal position.
Ø Mix the contents of the bulb thoroughly for 2-3 minutes by rotating the pipette while keeping the end closed with a finger Place the pipette on a horizontal surface

Charging Neubauer chamber

Ø Clean neubauer's chamber and coverslip. Place the coverslip on the central platform of the chamber Discard the first two drops of fluid from the pipette as it contains only diluting fluid
Ø Place the tip of the pipette on the surface of the chamber touching the edge of the coverslip at an angle of 45. Allow the diluted sample to flow under the coverslip by capillary action. Remove the pipette quickly from the edge of the coverslip as soon as the counting platform is filled with diluted blood. Take care to avoid the entry of air bubbles.
Ø Place the charged chamber on the microscope and observe under high power objective lens.
Ø Count the cells in five medium-sized squares (four comer and central medium-sized squares) which is a total of 80 small squares. Count the cells present in the square and on left and on lower lines

Calculations

Ø Area of 5 medium-sized squares = 1/25 x 5 = 1/5mm^3
Ø Volume of 5 medium-sized squares = 1/5 x 1/10 = 1/50mm^3
Ø Dilution factor = 1:200
Ø Cells in 1mm^3 of diluted blood = n x 50 (where n = number of cells)
Ø Cells in 1mm3 of undiluted blood = n x 50 x 200 = n x 10000 (where n is the total number of cells counted in 5 medium-sized RBC squares)