A comparison of the determination of glucosylated haemoglobin by isoelectric focusing and cation-exchange chromatography on minicolumns

Abstract

The labile intermediate, pre A1c, formed in the glycosylation of haemoglobin A is a potential contaminant in the measurement of glycosylated haemoglobin when this is determined as the amount of HbA1c present in the sample. By isoelectric focusing on polyacrylamide gel plates this contamination could be avoided either by excision of the HbA1c leaving the neighbouring pre A1c behind on the slab, or by converting the pre A1c to HbA in glucose-free medium before electrophoresis. In cation-exchange chromatography on minicolumns (from Bio-Rad) the pre A1c was removed in the haemolysis process by borate-induced transformation to the non-interfering HbA. The chromatographic method nevertheless gave about 10% higher values than isoelectric focusing. The linearity between paired results of the electrophoretic and chromatographic methods was not perfect (p less than 0.05). Both methods measured decreasing concentrations of HbA1c equally well and with the same precision at both high and low levels (CV less than 5%). All HbF was simultaneously determined in the chromatographic method, while HbF did not interfere in the electrophoretic method. The HbA1c in whole blood samples was stable at 4 degrees C for up to 1 week. Carbon monoxide treatment made the HbA1c in haemolysates stable for at least 3 months at -70 degrees C making possible long-term control by both methods.