Red cell life span heterogeneity in hematologically normal people is sufficient to alter HbA1c

Abstract

Although red blood cell (RBC) life span is a known determinant of percentage hemoglobin A1c (HbA1c), its variation has been considered insufficient to affect clinical decisions in hematologically normal persons. However, an unexplained discordance between HbA1c and other measures of glycemic control can be observed that could be, in part, the result of differences in RBC life span. To explore the hypothesis that variation in RBC life span could alter measured HbA1c sufficiently to explain some of this discordance, we determined RBC life span using a biotin label in 6 people with diabetes and 6 nondiabetic controls. Mean RBC age was calculated from the RBC survival curve for all circulating RBCs and for labeled RBCs at multiple time points as they aged. In addition, HbA1c in magnetically isolated labeled RBCs and in isolated transferrin receptor-positivereticulocytes was used to determine the in vivo synthetic rate of HbA1c. The mean age of circulating RBCs ranged from 39 to 56 days in diabetic subjects and 38 to 60 days in nondiabetic controls. HbA1c synthesis was linear and correlated with mean whole blood HbA1c (R(2) = 0.91). The observed variation in RBC survival was large enough to cause clinically important differences in HbA1c for a given mean blood glucose.

Figure 1

Scatter plot of TfR(+) reticulocyte HbA1c fraction and whole blood HbA1c fraction. There was a strong correlation of TfR(+) reticulocyte HbA1c fraction to whole blood HbA1c fraction obtained on the day of biotin labeling. (—) represents regression line; (), 95% confidence intervals.

Figure 2

The in vivo rate of formation of HbA1c. Autologous RBCs were isolated, labeled with biotin, and then reinfused into study subjects to track RBC survival and glycation over their life span. The HbA1c fraction over time after injection in the whole blood (□) and in the biotinylated RBCs (▿) is shown in 1 subject without (left panel) and 1 with (right panel) DM.

Figure 3

RBC disappearance in NDM and DM subjects. (Top) NDM subjects; (bottom) DM subjects. Because RBC disappearance curves were nonlinear, determination of RBC life span as the x-intercept is not the most meaningful approach to summarizing the exposure of the RBCs to plasma glucose. Mean cell age was therefore used, computed from the survival curves, fitted to cubic equations (“Analysis of RBC life span”).

Figure 4

Time course of measured HbA1c fraction versus mean age of biotinylated RBCs. HbA1c fraction was measured in autologous biotinylated RBCs, sampled at various times (triangles) after reinfusion in 6 NDM (left) and 6 DM (right) human subjects. Mean RBC age at sampling was calculated based on the time of sampling and the measured survival characteristics of each patient as described in “Analysis of RBC life span.” The initial mean cell age differs among both NDM and DM subjects resulting from differences in RBC survival. The slope of the biotinylated RBC HbA1c fraction vs time plot represents the Hb glycation rate. Slopes differ among subjects and are, in general, greater in the DM subjects. □ represent HbA1c fraction values in isolated TfR(+) cells; (—), regression line; (), 95% confidence intervals.

Figure 5

Relationship of Hb glycation rate to mean HbA1c fraction during the biotin label study. Glycation rates, determined as the slopes of the regression lines in Figure 4, were plotted versus the mean of HbA1c fraction values obtained for each subject over the course of the survival study.

Figure 6

Impact of differences in mean RBC age on measured HbA1c. Three hypothetical patients with identical glycation rates would be expected to have similar HbA1c values (in this case 0.086, the average of the 6 DM subjects in our study). However, variable mean RBC age results in a measured HbA1c that ranges from lower to higher than expected. This could lead to inappropriate clinical management.