PRINCIPLE
When cyanide is added the absorption band at 630 nm disappears and the change in absorbance is proportional to the concentration of Hi. The total Hb in the sample is measured after complete conversion to HiCN by the addition of ferricyanide.
REAGENTS
Phosphate buffer 0.1 mol/l, pH 6.8; potassium cyanide 50 g/l, potassium ferricyanide 50 g/l.
PROCEDURE
0.2 ml of blood is lysed in a mixture of 4 ml buffer and 6 ml detergent and the lysate divided into equal parts (A and B). The absorbance of A is measured at 630 nm (D1). One drop of KCN solution is added and the absorbance is measured again (D2). One drop of potassium ferricyanide is added to B and after 5 minutes the absorbance is measured at the same wavelength (D3). One drop of potassium cyanide is then added to B and a final reading made (D4). All measurements are made against a blank of buffer/detergent in the same proportions as used in the test.2
CALCULATION
