Hemoglobin Determination Using Cyanmethemoglobin Method

Principle

In a reagent solution the ferrous ions (Fe2+) of hemoglobin are oxidized to the ferric (Fe3+) state by potassium ferricyanide to form methemoglobin. Methemoglobin subsequently reacts with the cyanide ions provided by potassium cyanide to form cyanmethemoglobin. The amount of cyanmethemoglobin can be measured spectrophotometrically at a wavelength of 540 nm on a spectrophotometer and compared to known hemoglobin standards in order to determine the hemoglobin concentration of the unknown sample. Hemoglobin, the main component of the RBC, transports oxygen to and CO2 from the body’s tissues. Hemoglobin in circulating blood is a mixture of hemoglobin, oxyhemoglobin, carboxyhemoglobin and minor amounts of other forms of this pigment. It is necessary to make a stable derivative involving all forms of hemoglobin in the blood in order to measure this compound accurately. The cyanmethemoglobin (HiCN) derivative can be conveniently and reproducibly prepared and is widely used for hemoglobin determinations. All forms of circulating hemoglobin are readily converted to HiCN except for sulfhemoglobin, which is rarely present in significant amounts. Cyanmethemoglobin can be measured accurately by its absorbance in a colorimeter.

Coulter Instrument Hemoglobin:

After the WBC solution is lysed, the system shines a beam of white light through the WBC aperture bath and then through an optical filter. This transmittance of light (525 nm wavelength) through a standard path length of Hgb solution is compared to the transmittance of such light in the same way through a reagent blank. The system converts this ratio to absorbance. It then converts absorbance to Hgb values in g/dL using a calibration factor.

Abott Cell-Dyne Hemoglobin:

This instrument uses a similar procedure, but is unique in that its reagent is cyanide-free.

Bayer Advia Hemoglobin:

Hemoglobin has dual readings - colorimetric or cyanmethemoglobin and corpuscular hemoglobin concentration mean.

Normal values:
Adult Male 14 - 17 g/dL
Adult Female 12.5 - 15 g/dL
Newborn 17 - 23 g/dL
3-month-old 9 - 14 g/dL
10-year-old 11.4 - 15.4 g/dL