Improvements in hemoglobin AI determination by rapid cation-exchange chromatography

The accuracy of many convenient methods for determining the proportion of hemoglobin AI to total hemoglobin by ion-exchange chromatography is in question because those methods have not been evaluated against a reference method. In addition, several if not all of these tests are influenced by laboratory temperature. We modified the chromatographic conditions usually used, to arrive at a method that gives results that agree well with those by the Trivelli method (N. Engl. J. Med. 284, 353, 1971) and is less influenced by ambient temperature than are results obtained with the commercially available Helena and Isolab kits. We accomplished this by lowering the pH of the fast-hemoglobin eluting buffer, with a corresponding adjustment in the ionic strength. We also use a larger elution volume, which assures more nearly complete elution of the hemoglobin A1 fraction. The final method (y) agreed being y = 0.98x + 0.63 r2 = 0.887. Average results from the Helena and Isolab methods were lower than with the reference method by 1.58 and 3.03% hemoglobin A1, respectively.