Cyanmethemoglobin (Hemoglobin-Cyanide) Method for Estimation of Hemoglobin

When Blood is mixed with a solution of potassium cyanide, potassium ferricyanide, and Drabkin’s solution, the erythrocytes are lysed by producing evenly disturbed hemoglobin solution. Potassium ferricyanide transforms hemoglobin into methemoglobin, and methemoglobin combines with potassium cyanide to produce hemiglobincyanide (cyanmethemoglobin). This method is optional for the estimation of hemoglobin and this method is recommended by the International Committee for Standardization in hemotology. This is because in this method all type of hemoglobin is transformed to cyanmethemoglobin (except sulfhemoglobin), and a firm and trustworthy standard is available.

Principle

When Blood is mixed with a solution of potassium cyanide, potassium ferricyanide, and Drabkin’s solution, the erythrocytes are lysed by producing evenly disturbed hemoglobin solution. Potassium ferricyanide transforms hemoglobin into methemoglobin, and methemoglobin combines with potassium cyanide to produce hemiglobincyanide (cyanmethemoglobin). This way all types of hemoglobin present in the blood are entirely transformed into a single compound cyanmethemoglobin. When the reaction is entire, the absorbance of the solution is deliberate in a spectrophotometer at 540 nanometers. Hemoglobincyanide has a wide absorbance peak at this wavelength. The absorbance is compared with that of the standard hemiglobincyanide solution by using a formula to obtain the amount of hemoglobin.

Equipment

1. A spectrophotometer or photoelectric colorimeter
2. Pipette 5 ml
3. Sahli’s pipette

Reagents

1. Drabkin’s Solution
2. Cyanmethemoglobin standard solution with a known hemoglobin value

Specimen

Blood obtained from skin puncture or EDTA-anticoagulated venous blood.

Procedure

1. Take 5 ml of Drabkin’s solution in a test tube and add 20 μl of blood. This way, we will get the dilution of 1:25. Now mix the mixture and allow to stand for at least 5 minutes. This time is adequate for the transformation of hemoglobin to hemiglobincyanide.
2. Pour the test sample into a cuvette and read the absorbance of the test sample in a spectrophotometer at 540 nanometers or in a photoelectric colorimeter using a yellow-green filter. Also, read the absorbance of the standard solution. Absorbance must be read against Drabkin’s solution.
3. From the formula given below, the hemoglobin value is derived.

Hemoglobin in gm/dl = [Absorbance of test sample ÷ Absorbance of standard] x concentration of standard x [Dilution factor ÷ 100]

Preparation of table and graph

The result can be obtained quickly if the table of a graph is prepared which corresponds to absorbance with hemoglobin concentration. This is markedly acceptable when a huge number of samples are daily processed on the same instrument.

For the preparation of a calibration graph, adulterate cyanmethemoglobin standards are commercially available. As another option, a standard cyanmethemoglobin solution is diluted serially with Drabkin’s solution. The concentration of hemoglobin (horizontal axis) in each dilution is arranged against the absorbance (vertical axis) on a linear graph paper. A straight line connecting the points and passing through the origin is obtained. A table can be prepared relating absorbance to the concentration of hemoglobin from the help of this graph.

Notes:

1. The hemiglobincyanide solution is stable so that delay in getting the reading of absorbance does not influence the result.
2. High TLC (total leukocyte count) (> 25,000/μl), abnormal plasma proteins (e.g. in Waldenström’s macroglobulinemia, multiple myeloma), or lipemic blood (hypertriglyceridemia), can cause the error in results.