Translating the A1C Assay

In the clinical management of diabetes, the A1C assay has become indispensable. Used worldwide to monitor chronic glycemia, the assay is an essential tool to determine whether a patient has achieved the core goal of therapy for diabetes: a marked and sustained reduction in plasma glucose to achieve as close to a normal level as can be safely attained. With the publication of the A1c-Derived Average Glucose (ADAG) study in this issue of Diabetes Care (1), the evolution of the A1C assay continues and an important milestone has been reached. To better appreciate this recent report, a brief and admittedly incomplete historical perspective may be useful. It was 60 years ago that Allen et al. (2) showed that hemoglobin A (which makes up about 97% of total hemoglobin) contains three minor components, designated HbA1a, HbA1b, and HbA1c (A1C). In the decades that followed, we learned that a hexose molecule is attached to these components (3) and that hemoglobin A actually has two more minor glycated derivatives. The five altogether comprise 5–7% of the HbA molecule (4). In the early course of the biochemical dissection of hemoglobin, Huisman and Dozy (5) noted, virtually in passing, that the level of glycated hemoglobin components was increased in a few individuals they studied who happened to have diabetes. It took 4 more years, however, for Rahbar and colleagues (6,7) to document that diabetes is clearly associated with an elevation in glycated hemoglobin. The Rahbar reports stimulated other investigators to confirm these initial findings and to seek an explanation for how glucose binds to hemoglobin. It was not for another few years, in 1972, that Bunn et al. (8) elegantly showed that the cause of the increased glycated hemoglobin in diabetes, which was predominantly the A1C component, was a result of excess nonenzymatic glycation that occurred throughout the lifespan of red cells and in an essentially irreversible process. The A1C-diabetes story then shifted from clinical chemistry to clinical medicine. Koenig et al. (9) were the first to show that A1C levels correlated well with fasting blood glucose, and they concluded that A1C levels “probably reflect . . . the mean daily blood glucose concentration . . . and may provide a better index of control of the diabetic patient.” Indeed, soon after their report, many other investigators confirmed a strong association between A1C and glycemic control and that the measurement had clinical utility (10–15), clearly surpassing in utility what was then the conventional assessment of metabolic control over time (e.g., signs, symptoms, urine, and blood glucose levels) (15). The thorough biochemical experiments performed in the 1970s and 1980s, most notably by Mortensen and Christophersen (16), demonstrated that the fraction of A1C in a sample depends on the glucose levels over a previous period, along with red cell turnover, reaching a steady state sometime between 4 and 12 weeks. Such kinetics were supported by many clinical studies in both type 1 and type 2 diabetic patients where the A1C level was found to correlate well with glucose regulation (17) or the mean blood glucose derived over time from multiple fingersticks (9,15,18–24). As the use of the A1C test gained traction, dozens of different analytical methods based on different assay principles (e.g., ion-exchange chromatography, affinity chromatography, immunoassay, and electrophoresis) were used to measure glycated hemoglobin. Without a common reference method and in the absence of a standardized assay, results varied considerably when the same sample was tested by different laboratories or methods or even when the same sample was tested repeatedly by one methodology. It was quite common, for example, to have values ranging from 4.0 to 8.1% on the same blood sample (25). In addition, the assays used then (and even now) in clinical medicine not only measured A1C itself but also more or lesser amounts of the other glycated hemoglobin components, and results were reported as A1C, HbA1, or total glycated hemoglobin. The results were also influenced by other interfering substances in the sample. The Diabetes Control and Complications Trial (DCCT) Study Group, recognizing these problems, centralized the measurement of A1C from the onset of the study so as to avoid confounding results if such a key analyte were to be measured at many sites (26). Also, in anticipation of the DCCT results, the American Association for Clinical Chemistry (AACC) established, in 1993, an A1C standardization workgroup to bring consistency to the measurement of A1C and to facilitate the traceability of results back to the DCCT such that these results could be directly related to the risk or progression of diabetes complications. After the standardization protocol was developed, the American Association for Clinical Chemistry group was dissolved and the National Glycohemoglobin Standardization Program (NGSP) began in 1996 (27). Briefly, in the NGSP, the reference method is the measurement of A1C by ion-exchange highperformance liquid chromatography, as was used in the DCCT. Manufacturers of testing equipment can receive NGSP certification if their instruments are calibrated to match the results obtained by the NGSP. Laboratories can also be certified by the same protocol and thereby document optimal performance in their setting. All this has led to a dramatic reduction in interlaboratory variability and a marked improvement in the precision and comparability of values (28). In 2007, 99% of all A1C test results in the U.S. were traceable to those obtained in the DCCT, with similar percentages in test results throughout the U.K. and in Canada (D. Sacks, personal communication). Although comparable data are not readily available from other countries, it appears that much of the world’s A1C testing is traceable to the DCCT numbers. Still, issues remain. First, the highperformance liquid chromatography reference method used by the NGSP is somewhat nonspecific in that the methodology, like many others, measures more than just A1C in a sample. Although this problem is obviated by the consistent use of one reference method, in the world of clinical chemistry, this situation is “metrologically unsound.” Second, although most methods used worldwide are NGSP certified, there are other standardization programs, most notably in Japan (29) and in Sweden (30). Thus, E d i t o r i a l s