Estimation of Haemoglobin by Cyanmethemoglobin Method

Principle:Blood is diluted in a solution containing potassium cyanide potassium ferri-cyanide .Haemoglobin is oxidized to methaemoglobin by potassium ferri-cyanide, and methaemoglobin in turn combines with potassium cyanide to form cyanmethaemoglobin. The absorbance of the solution is measured in a spectrophotometer at wavelength 540 nm against Drabkin's solution as a blank. The result is (calculated from the formula provided below) expressed in gm/liter or mg/dl.

.Materials:

-         Spectrophotometer/Colorimeter

-         Automatic pipettes.

-         Racks.

-         Drabkin's solution.

-         Plastic or glass tubes.

-         Blood Collection set.

 Specimen: Venous blood or free-flowing capillary blood can be used.

Procedure:-

1.     Pipette 4 ml of Drab kin’s solution in a tube.

2.     Pipette exactly 0.02 ml (20µl) of well mixed blood using a pipette.

3.     Clean outside of the pipette and wash out the blood in the tube containing diluents (dilution=1: 200).

4.     Mix and leave for 5-10 minutes for reaction to be completed.

5.     Pour in a cuvette.

6.     The absorbance of the solution is measures in a spectrophotometer at wave length 540 nm against Drab kin’s solution as a blank. Hb is calculated by the following formula:

Calculation:

                                           Absorption of sample

Haemoglobin (g/dl) = ---------------------------------- X concentration of standard

                                            Absorption of standard