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

Abstract

The labile intermediate, pre A_1c, 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 HbA_1c present in the sample. By isoelectric focusing on polyacrylamide gel plates this contamination could be avoided either by excision of the HbA,_c leaving the neighbouring pre A_lc behind on the slab, or by converting the pre A_ic to HbA in glucose-free medium before electrophoresis. In cation-exchange chromatography on minicolumns (from Bio-Rad) the pre A_lc 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>0.05). Both methods measured decreasing concentrations of HbA_lc equally well and with the same precision at both high and low levels (CV>5%). All HbF was simultaneously determined in the chromatographic method, while HbF did not interfere in the electrophoretic method. The HbA_lc in whole blood samples was stable at 4 °C for up to 1 week. Carbon monoxide treatment made the HbA_1c in haemolysates stable for at least 3 months at -70 °C making possible long-term control by both methods.

Key Words:

• Bio-Rad minicolumns
• CO-stabilization of HbA_lc
• HbA_lc (ketoamine)
• HbF
• LKB polyacrylamide gel plates
• pre A_1c (aldimine/Schiff base)
• precision
• quality control
• storage of samples