Determination of Total Red Cell Count

 Determination of Total Red Cell Count

 

AIM: To find out the total number of Red blood cell per Cu. mm.

 

Apparatus Required: Microscopic, Haemocytometer with counting Chamber (Neubauer ruling): Red cell pipette, RBC Diluting Fluid, Hayem’s fluid) Cover slip, Lancet, Cotton and Spirit.

 

A the Counting Chamber: The different parts of counting chamber the middle platform AAI which bears the ruled area, are exactly 0.1mm lower then the two wide platform C1 C2.

 

                The middle platform are separated from each other by the transverse groove ‘D’ and from the two side platform by the trenches Dl and D2 when the coverslip is placed upon the platform there is a space exactly 0.1mm. (1/10 mm) deep between it and the ruled areas on the middle platform. Upon each ruled area there are nine large fields each having an at of 1sqmm.

 

                The central field has an area of 1sq, mm and is used for R.B.C. count it is divided into 25 big squares, each having an area of 1/25 sq. mm each big square is further subdivided into 16 small square each having an area of 1/400 sp. mm. and there fore there are 400 small squares in the whole central field.

 

                Four large fields at the corners of the above central ruled area are used for counting the W.B.C. each field at the corner is divided into 16 big squares. The area of one large field is 1sq mm.

 

B.Red Cell Pipette: The capillary portation in the stem of the R.B.C. pipette is divided into  10 equal parts from tip of the pipette o the bottom of the bulb. The 5th and the 10^th marks are denoted by 0.5 and 1 marks respectively. There is another mark just above the bulb and is marked as 101 is given. To facilitate mixing a red bead is present in the bulb.

 

C Diluting Fluids:               1) Hayem’s Fluid: Composition given below

                                                    Sodium Chloride (acts is otonic) 1G

                                                    Sodium Sulphate Rouleax formation Hg Cl₂

                                                    (Acts as antiseptic) 0.5G

                                                    Dist Water …………………………….200 e.c

                                                2) 0 9 Persent Nacl, Solution can be also be used.

 

Method:

                With a dry R.B.C. pipette stick the blood upto mark 0.5. The blood column in the capillary stem should not be permitted to go beyond that mark. In case this happens gently touch the tip of the pipette to the filter paper Dip the tip of the pipette in the diluting fluid and suck to be exact 0-5 mark blood to be sucked mV the pipette. Stop sticking when the mark 101 is reached knot, to the top by using the extra rubber tube. Now rotate the pipette in between the palms of your hands to facilitate through mixing of blood with the diluting fluid. The dilution of blood in this case is 1 in 200.

Charging the Counting Chamber:

                Counting chamber and coverslip are cleaned and made dry put the coverslip over the counting chamber so that it covers the ruled areas fully, discard a few drops from the pipette and place the one drop or less than that over the counting chamber just near by margin of the overslip the drop will instantly flow under the coverslip by capillary action. The fluid should not flow into side chamber and should be just sufficient to fill up the ruled, area on the chamber.

 

Counting of the cells:

                Focus ruled area under low power and see whether the red cells are evenly placed or not if the distribution of the cells in whole field is uniform then see it under the high power count the total member of red cells present in five big squares (4 squares at the corners and one in the centre).

 

NB: The entire counting of R.B.C. completed only under high power.

 

Calculation:

                Let ‘X’ be the total number of cells present in 80 smallest squares.

                No. of cells present in smallest squares = x /80 cells.

                Area of the each smallest = L x B

                = l/20mm x l/20mm

                = 1/400sq.mm

Volume of one of smallest square L X B X H

                = 1/20mm x l/20mm x 1/l0mm

                = 1/4000cu. mm

i.e one smallest square having a volume of 1/4000 cu mm. contains

= X /80 cells 1 cu. mm. contains x/80 x 4/000 = X x 50 cells.

Dilution 1:200/cu. mm. of undiluted blood contains A X x 50 x 200 cells.

 

                                x 10.000 cells

                =              ---------------

                            millions/cu.mm

 

Normal Value: Total R.B.C. Count in male 5.5 to 6.5 millions / cu mm in female - 4.5 to 5.5

millions/cu. mm.

 

Precaution: While counting the red cells those cells lying on the upper and left hand boundary of square must be considered to insi4e that square and those cells lying on the right and lower boundary of square must be considered out side.

 

                A increase in number of red cells is called as polycythemia and occurs under the following condition.

 

Physiological Conditions: 1) New Born Infants. 2) During the working hours. 3) During muscular exercise. 4) Emotional states. 5) High turned environmental temperature. 6) High altitude A) Immediate Due to mobilisation from blood store house. B) Permanent Due to overreaction of red bone marrow 7) Injection of Aderenaline 8) Raised CO₂ tension. 9. Cells more in males then in females.