Hemoglobin A1C by isoelectric focusing.

We separated the hemoglobins in an unwashed erythrocyte hemolysate according to their isoelectric points on a thin-layer horizontal polyacrylamide gel containing ampholyte (40 g/L) over a pH gradient of 6-8. We scanned the fixed, unstained gels by microdensitometry and calculated the percentage of hemoglobin A1C. The overall CV (between-run imprecision) for a normal hemolysate (mean, 5.2% of total hemoglobin) stored at -70 degrees C was 12.6%. An above-normal pooled specimen (mean 8.5%) showed an overall CV of 8.4%. We confirmed that hemoglobins S, C, and F do not interfere; acetylated F co-migrates with A1C. The mean A1C percentage in non-diabetic adults was 4.9%, the reference interval 3.9-6.4%. The mean values for diabetics in various degrees of control were 7.6% in the group rated "good," 9.9% in the "fair" group, and 12.2% in the "poor" group. Results for patients' samples (y) were compared with results by cation-exchange chromatography (x). The slope was 1.0 and the intercept was 2.2%. The percentage of hemoglobin A1C in erythrocytes remains constant for seven days in samples stored at 30 degrees C.