Diagnostic Laboratory Section

CYANMETHEMOGLOBIN METHOD

• It is widely used technique for determining hemoglobin concentration. It must be done  on a photoelectric colorimeter at a wavelength of 540µm.

Advantages

• Solutions of cyanmethemoglobin are the most stable of the various hemoglobin pigments.
• These solutions can be accurately standardized.
• The absorbtion band of cyanmethemoglobin is broad in the region of 540 µm, and its solutions can be used both in filter photometers.
• All forms of hemoglobin likely to be found in blood, except sulfmethemoglobin, are quantitatively converted to cyanmethemoglobin by addition of this single reagent.

Procedure

• To perform this technique, place exactly 5 ml of diluent (cyanmethemoglobin reagent) into a clean cuvette and add exactly 0.02 ml of blood to the diluent.
• Care should be taken to preserve the  accuracy of this measurement by wiping off excess blood from the tip of the pipette. Since the quantity of the blood used is small, even a slight excess of blood will influence results.
• After the blood is expelled into a cuvette, the pipette is rinsed several times with diluent.
• After blood is added, the tube is stoppered and inverted two or three times. Allow the mixture of blood and reagent to stand for approximately 10 minutes for maximum conversion of hemoglobin to cyanmethemoglobin.
• The cuvette is wiped clean and placed into a spectrophotometer for reading.
• The percent transmisssion or optical density at 540 microns is recorded and compared with reading obtained using standard solution of cyanmethemoglobin or values on a previously prepared standard curve.
• This will convert the reading to grams of haemoglobulin per deciliter of blood.