Methaemoglobin (MHb) Test

Methaemoglobin (metHb) is a form of protein haemoglobin in which iron is present in the form of Fe3+ instead of normal Fe2+ state. MetHb fail to bind oxygen with it. This causes oxygen deficiency in the tissues as they can’t get the oxygen and functional anaemia as well. If the metHb level in the blood is 1-2%, it is normal and one need not to worry about it. Generally, inorganic and organic nitrates and nitrites, aromatic amines and other toxic industrial chemicals make metHb by binding with Hb instead of O2. Apart from these, at times chromates, chlorobenzene, fertilisers and herbicides also bind with Hb to form metHb. In case of metHb reductase deficiency and G6PD (glucose-6-phosphate dehydrogenase) deficiency, the level of metHb rises in the blood.

There are a gamut of method through which level of metHb can be tested in the blood. All of the test are done by collecting the sample of blood through venipuncture. The blood sample is collected and tested in the laboratory to check the level of metHb and make the inference for the suspected defects and problems. Procedure: One of the method to check the level of metHb in the blood is Spectroscopic examination of blood for the methaemoglobin and sulfhemoglobin.

Method: The collected sample of blood is diluted with water in the ratio of either 1:5 or 1:10 and centrifuged. Using the hand spectroscope the sample is observed in daylight. A close and careful observation is needed in the especially in the red part of the absorption band of the spectrum at 620-630 nm. If the bands are observed in this area, add yellow ammonia sulfide to the sample. Bands with metHb will disappear after adding ammonia sulfide. The spectra of the unknown and known pigments might be observed in the reversion spectroscope. Measurement of methaemoglobin Method: 0.2 mL of the collected sample of blood is lysed in a solution of 4 mL of buffer and 6 mL detergent solution. The lysate solution is divided into two equal halves. Absorbance of first sample should be observed in a spectrophotometer at 630 nm. Now, add 1 drop of potassium cyanide to the solution and measure the absorbance again. To the second part, add a drop of potassium ferricyanide solution and observe the absorbance at the same wavelength after 5 mins.

To detect the methemoglobinemia due to the deficiency of methaemoglobin reductase.

One of the method to check the level of metHb in the blood is Spectroscopic examination of blood for the methaemoglobin and sulfhemoglobin.

Method: The collected sample of blood is diluted with water in the ratio of either 1:5 or 1:10 and centrifuged. Using the hand spectroscope the sample is observed in daylight. A close and careful observation is needed in the especially in the red part of the absorption band of the spectrum at 620-630 nm. If the bands are observed in this area, add yellow ammonia sulfide to the sample. Bands with metHb will disappear after adding ammonia sulfide. The spectra of the unknown and known pigments might be observed in the reversion spectroscope. Measurement of methaemoglobin

Method: 0.2 mL of the collected sample of blood is lysed in a solution of 4 mL of buffer and 6 mL detergent solution. The lysate solution is divided into two equal halves. Absorbance of first sample should be observed in a spectrophotometer at 630 nm. Now, add 1 drop of potassium cyanide to the solution and measure the absorbance again. To the second part, add a drop of potassium ferricyanide solution and observe the absorbance at the same wavelength after 5 mins.