A comparative evaluation of glycosylated haemoglobin assays: feasibility of references and standards

Four assays; high pressure liquid chromatography, colorimetric with thiobarbituric acid, affinity columns, and microcolumn cation exchange were compared for (1) ability to discriminate between samples taken from diabetic and normal subjects; (2) correlation with each other; (3) stability over time at different temperatures; and (4) reproducibility between laboratories. The most discriminatory (10 samples from a diabetic and 10 samples from a normal group) was the microcolumn cation exchange method (t=5.25; p<0.001), but all were significantly different at p<0.005. The intra-assay coefficient of variation was 1%-6%, except for the affinity column method which was 13% in normal subjects. High pressure liquid chromatography was used as a reference and the other assays correlated well (r=0.93-0.99). Storage at-80 °C, -20 °C, 4 °C, and 24 °C showed marked differences. The thiobarbituric acid method results were stable except for 24 °C. Microcolumn cation exchange was labile under all conditions. Affinity column was stable for up to 15 days, only if samples were stored as whole blood. High pressure liquid chromatography showed an increase in haemoglobin A_1a+b and a decrease in the haemoglobin A_1c. Haemoglobin A_1c was reproducible for 4 days when stored at 4 °C and up to 11 days when stored at -80 °C. Samples exchanged between centres at 4 °C and performed within 5 days by high pressure liquid chromatography for haemoglobin A₁ and haemoglobin A_1c correlated well (r=0.98 and 0.99). Samples exchanged between centres after storage (up to 40 days -80°C) correlated (r=0.99) by the thiobarbituric acid method. Thus, standards can be prepared for the thiobarbituric acid method and this method with high pressure liquid chromatography could be used to establish references for clinical assays.