The effect of storage conditions on ion exchange and affinity chromatographic assays for glycated hemoglobin

Experiments were performed to evaluate the effect on glycated hemoglobin determinations when erythrocytes were stored under various conditions. We studied the influence of time, temperature, glucose concentration, and pH on results obtained by three commercially available column chromatographic procedures designed to quantify glycated hemoglobin. An affinity chromatographic procedure which measures total glycated hemoglobins (GHb) was compared with two ion-exchange column methods. One ion-exchange method measures total hemoglobin A1 and the other is purported to measure hemoglobin A1c. Stability studies were performed using specimens from nondiabetics and diabetics, supplemented in vitro with glucose at pH 7.4 and pH 5.0. Results obtained from the A1c and the GHb tests were unchanged over time for samples stored under the described conditions. Erythrocytes were incubated with glucose to produce labile glycated hemoglobin and assayed before and after overnight incubations in saline. These experiments showed that the A1c and GHb assays were unaffected by the presence of labile glycated hemoglobin. Column fractions were analyzed by isoelectric focusing and scanned with a laser densitometer for quantitative assessment of hemoglobin species found in each fraction. These experiments showed that the three column methods measure very different hemoglobin species. Focusing of eluates from the A1 and GHb tests revealed carryover of several hemoglobin species into the measured fractions, while the A1c test was found to be much more specific for HbA1c. Our data suggest that it may be possible to analyze glycated hemoglobins in samples transported to a reference laboratory without special treatment.