IMPROVED PROCEDURES OF ALKALI-HEMOCHROME AND PYRIDINE HEMOCHROME METHODS FOR THE DETERMINATION OF HEMOGLOBINS (NORMAL AND ABNORMAL) AND THEIR DERIVATIVES
Pyridine hemochrome (PH) and alkali-hemochrome (AH) methods for the determination of blood hemoglobin concentration were improved in precision and accuracy through detailed examination of the original procedures. As to the PH method, an aliquot of 10.0 μll of blood or hemolysate is poured into 5.0 ml of 0.1 N-NaOH solution containing detergent (SDS in concentration of 3 g/dl), followed by addition of 1.0 ml of pyridine and a small amount of hydrosulfite (4-5 mg) to produce hemochrome. In alkali-hemochrome method, a volume of 10.0 μl blood or hemolysate is diluted with 4.0 ml of water to hemolyze the erythrocytes and subsequently 2.0 ml of 20.0 g/dl NaOH solution and a small amount of hydrosulfite (4-5 mg) are added. Both of these methods are suitable for the accurate determination of all derivatives and types of hemoglobins including Hb Ms, unstable Hbs, hemochromes, sulf-Hb, etc. The maximum discrepancy of Hb value of the same blood specimen between the AH and the standard cyanmethemoglobin (CMHb) methods is 1.46%, (Correlation coefficient r=+0.995, n = 109) and that between the PH and the CMHb method is 1.99% (r=+0,990, n = 77). Virtually Hb values identical with those by the standard method are obtained. The major points of improvement are: i) the use of 20.0 g/dl NaOH solution instead of the originally described 3 g/dl solution in the AH method and use of 0.1 N-NaOH solution containing 3 g/dl SDS in substitution of 0.1 N-NaOH solution in the original PH method and ii)specified time of alkaline incubation of blood for 10 minutes to attain strict accuracy of estimation. Both hemochrome methods are recomended for estimation of blood hemoglobin concentration as the alternate procedures of CMHb method, because they do not cause disastorousa water pollution. Heme concentration of blood in hemoglobinopathies is sasily detetmined by these methods.