Affinity binding assay of glycohemoglobin by two-dimensional centrifugation referenced to hemoglobin A1c

Abstract

We describe an automated assay of glycohemoglobin performed with the Abbott Vision analyzer. The assay is based on batch affinity-extraction with 3-aminophenylboronic acid-derivatized agarose beads. Reagents are packaged in a disposable test pack. Whole-blood specimens are hemolyzed with saponin within a glass capillary tube inserted into the test pack. The sample is automatically diluted with, mixed with, and separated from the solid-phase reagent. Bichromatic absorbance readings are used to calculate the percentage of hemoglobin bound. Based on the linear correlation between affinity-measured glycohemoglobin and HPLC-measured hemoglobin A1c, the percentage of hemoglobin bound is converted to a "standardized %HbA1c" result by use of regression parameters stored during a calibration run. The combination of affinity methodology with standardization by reference to HPLC produces values directly comparable with those obtained by methods specific for HbA1c. The method produces 10 results within 15 min. The assay operates with CVs < 5%, and the results correlate highly with those by ion-exchange and affinity minicolumn methods, and by ion-exchange HPLC.