Hemoglobin variants and hemoglobin A1c analysis: problem solved?

Measurement of glycohemoglobin (GHb) is an integral component of the management of patients with diabetes mellitus (1, 2). Glycation is the nonenzymatic addition of a sugar residue to amino groups of proteins. Numerous proteins in the body are glycated, but GHb in blood is the analyte most widely used clinically to monitor glycemic control. The concentration of GHb is directly proportional to the mean concentration of glucose in the blood and the lifespan of erythrocytes (mean, 120 days). Thus, the GHb concentration represents an integrated value for glucose over the preceding 2–3 months. GHb provides an index of glycemic control that is free of the wide diurnal glucose fluctuations and is unaffected by recent exercise or food ingestion. Two large, prospective, randomized clinical trials demonstrated a strong relationship between hyperglycemia and the development of microvascular complications of diabetes. In the Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS), which studied patients with type 1 and type 2 diabetes, respectively (3, 4), glycemic control was assessed by GHb. Both studies established a direct relationship between the GHb concentration and the risk of complications. These findings led the American Diabetes Association to recommend that a primary treatment goal in adults with diabetes should be near-normal glycemia with hemoglobin A1c (HbA1c) 7% when measured by the method used for the DCCT (2 ). The recommendation that HbA1c be measured at least every 6 months in patients with type 1 or type 2 diabetes (2 ) has had a dramatic impact on the use of this test. The number of laboratories participating in the GHb surveys offered by the College of American Pathologists increased from 707 in 1990 to 2008 in 2003. Clinical laboratories in the US currently perform 2 million GHb measurements each month (estimated from an unpublished survey by the College of American Pathologists). Analogous to all tests widely used in patient care, GHb analysis is subject to certain limitations. Conditions that affect red cell turnover, e.g., hemolysis or significant blood loss, will alter the amount of GHb that accumulates (5 ). In addition, interassay variability and hemoglobin variants have restricted the use of GHb testing. Considerable progress in the standardization of GHb assays (6, 7) and accurate GHb measurement in patients with hemoglobin variants, as exemplified by two recent reports in Clinical Chemistry (8, 9), lead to optimism that the latter problems may be approaching resolution. A brief overview of the principles of GHb analysis will facilitate comprehension of these issues. There are 30 different GHb assay methods, which are based on two general principles (10, 11). The first group of methods, which includes cation-exchange chromatography and electrophoresis, separates glycated from nonglycated hemoglobin components based on differences in their charges. The second group of assays exploit variations in structure related to the presence of the attached glucose; they include immunoassays and boronate affinity chromatography. The vast majority of GHb measurements in the US are performed by ion-exchange HPLC or immunoassay. These assays measure HbA1c (hemoglobin glycated at the N-terminal valine of the -chain) or total GHb (comprising HbA1c and hemoglobin glycated at other sites). Results reported on the same blood sample can vary substantially, depending on the analyte measured and the method used. For example, GHb values ranged from 4.0% to 8.1% in one study (12 ) and from 5.1% to 8.2% in another (13 ) on a single sample. To resolve this problem, committees were established independently under the auspices of the AACC and the IFCC to standardize GHb assays. In 1993, the AACC established a GHb standardization subcommittee, which recommended that the DCCT reference method be used as the designated comparison method for GHb standardization while reference methods and pure standards were being developed. The National Glycohemoglobin Standardization Program (now known as NGSP) was formed to harmonize GHb assays with the DCCT assay. Modeled on the Cholesterol Reference Method Laboratory Network program, the NGSP uses a network of reference laboratories and interacts with manufacturers of GHb methods to allow them to establish traceability to the Central Primary Reference Laboratory (which analyzes HbA1c by HPLC according to the method used in the DCCT) (6 ). Methods and laboratories can then be certified as traceable to the DCCT according to rigorous precision and bias criteria. A different approach was adopted by the IFCC working group, which developed a mixture of purified HbA0 and HbA1c as a primary reference material (7 ). A reference method was established in which hemoglobin is cleaved into peptides by endoproteinase Glu-C. The glycated and nonglycated N-terminal hexapeptides are separated and quantified by HPLC– electrospray ionization mass spectrometry (ESI-MS) or by HPLC–capillary electrophoresis (7 ). HbA1c is measured as the ratio between glycated and nonglycated N-terminal hexapeptides. The high specificity of this method yields values that are lower than those obtained with most commercial methods (7 ). The new reference method has been approved by the member societies of the IFCC and may ultimately provide the foundation for global standardization of GHb measurements. Hemoglobinopathies may interfere with GHb analysis, independent of their effects on erythrocyte survival [for a review, see Bry et al. (5 )]. Results may be falsely increased or decreased, depending on the particular method and the hemoglobinopathy. Hemoglobin variants that cannot be separated from HbA or HbA1c will produce spuriously increased or decreased results by ion-exchange HPLC [see Fig. 1 in Bry et al. (5 ) for details]. This is not a trivial Editorial