Incomplete Removal of Labile Fraction When Measuring Hemoglobin A1c with Bio-Rad Variant® Analyzer

Abstract

When glucose reacts with hemoglobin (Hb), the first products are Schiff bases that slowly undergo Amadori rearrangement to produce stable ketoamines (glycohemoglobin). The relative fraction of the ketoamines to total Hb can be measured as hemoglobin A1c (Hb A1c), which constitutes ∼60% of the bound glucose (1). The Schiff base (or labile fraction) is unstable and is fairly readily hydrolyzed, especially at acid pH. Effective removal of the labile fraction is essential for the accurate determination of Hb A1c because the labile fraction cannot be separated from the true (ketoamine) fraction by simple ion-exchange procedures and because its concentration varies acutely with the plasma glucose concentration (2). Partial and variable removal of the labile fraction adds to assay imprecision and reduces the correlation (3) between measured Hb A1c and mean plasma glucose. The Bio-Rad Labs. Variant analyzer is a dedicated HPLC system designed to measure variant hemoglobins or Hb A1c. For Hb A1c the hemolyzing reagent is a pH 5.0 citrate buffer that also serves to remove the labile fraction. The Bio-Rad instrument manual advises users to “let samples stand at 18–28 °C for at least 10 min to allow Schiff base removal” (this was increased recently to 15 min). The manual also describes how to run the instrument essentially in batch mode. At start-up, or on restarting after running samples, the recommended protocol is an 8-min wash, a 3-min prime with hemolysate, followed by 3-min runs with two calibrators and two controls (total 23 min) before patients’ samples are processed. With this protocol, patients’ hemolysates are tested after variable periods of incubation at room temperature and at 8 °C (the temperature of the …