Critical Factors in the Chromatographic Measurement of Glycohemoglobin (HbA1)

Abstract

Measurement of glycohemoglobin has been proposed as a criterion for the management of diabetes mellitus. We evaluated various conditions critical to the accuracy and precision of the cation-exchange method. Tolerance limits for each variable were defined as follows: phosphate-eluding buffer (0.06 ± 0.005 mol/L ph 6.70−6.72), column temperature (19−21°C), and resin equilibration (to phosphate buffer, 0.07 mol/L, pH 6.70 ± 0.01). Hemoglobin absorbance measured in the Sorét region (approximately 416 nm) of the first chromatographic fraction divided by that of the total hemolysate provided the most accurate and precise result. Overall between-run precision expressed as coefficient of variation (CV, in percent) of normal and diabetic pools was 4.8% and 5.1%, respectively. When purified HbA1c was added to hemolysates, recovery was 90–95%. Results were linear to at least 18% glycohemoglobin. Hemoglobin F (HbF) interfered with the method, whereas HbC and HbS did not. Red cells could be stored frozen for at least 6 days, thus easing transport of outpatient samples. A reference range of 6.0−8.8% glycohemoglobin was established from 85 nondiabetic adults (ages 23–65 yr). In a clinical study, only 4 of 13 treated diabetic patients believed to be in good control showed glycohemoglobin results within the normal range. All of the 19 treated diabetics in fair or poor control showed glycohemoglobin results greater than 10% of total Hb, ie., well above the normal range.