Background: Glycohemoglobin (GHB), reported as hemoglobin (Hb) A1c, is a marker of long-term glycemic control in patients with diabetes and is directly related to risk for diabetic complications. HbE and HbD are the second and fourth most common Hb variants worldwide. We investigated the accuracy of HbA1c measurement in the presence of HbE and/or HbD traits. Methods: We evaluated 23 HbA1c methods; 9 were immunoassay methods, 10 were ion-exchange HPLC methods, and 4 were capillary electrophoresis, affinity chromatography, or enzymatic methods. An overall test of coincidence of 2 least-squares linear regression lines was performed to determine whether the presence of HbE or HbD traits caused a statistically significant difference from HbAA results relative to the boronate affinity HPLC comparative method. Deming regression analysis was performed to determine whether the presence of these traits produced a clinically significant effect on HbA1c results with the use of ±10% relative bias at 6% and 9% HbA1c as evaluation limits. Results: Statistically significant differences were found in more than half of the methods tested. Only 22% and 13% showed clinically significant interference for HbE and HbD traits, respectively. Conclusions: Some current HbA1c methods show clinically significant interferences with samples containing HbE or HbD traits. To avoid reporting of inaccurate results, ion-exchange chromatograms must be carefully examined to identify possible interference from these Hb variants. For some methods, manufacturers’ instructions do not provide adequate information for making correct decisions about reporting results.

All methods either met or exceeded manufacturers' claims for stability. The CLC 385/330, Tosoh G7, and Bio-Rad Variant II high performance liquid chromatography methods showed better stability than the Tosoh 2.2 Plus and Bio-Rad Variant.

Background Carbamylated hemoglobin (carbHb) is reported to interfere with measurement and interpretation of HbA1c in diabetic patients with chronic renal failure (CRF). There is also concern that HbA1c may give low results in these patients due to shortened erythrocyte survival. Methods We evaluated the effect of carbHb on HbA1c measurements and compared HbA1c with glycated albumin (GA) in patients with and without renal disease to test if CRF causes clinically significant bias in HbA1c results using 11 assay methods. Subjects included those with and without renal failure and diabetes. Each subject’s estimated glomerular filtration rate (eGFR) was used to determine the presence and degree of renal disease. A multiple regression model was used to determine if the relationship between HbA1c results obtained from each test method and the comparative method were significantly (p<0.05) affected by eGFR. These methods were further evaluated for clinical significance using difference between the eGRF quartiles of >7% at 6 or 9% HbA1c. The relationship between HbA1c and glycated albumin (GA) in patients with and without renal failure was also compared. Results Some methods showed small but statistically significant effects of eGFR; none of these differences were clinically significant. If GA is assumed to better reflect glycemic control, then HbA1c was approximately 1.5% HbA1c lower in patients with renal failure. Conclusions Although most methods can measure HbA1c accurately in patients with renal failure, healthcare providers must interpret these test results cautiously in these patients due the propensity for shortened erythrocyte survival in renal failure.

An application of ion exchange chromatography for C-peptide analysis is described here. At the stage of C-peptide isolation, a strong cation exchanger (SP HP or MonoS) was used to purify the analyte from ballast proteins and peptides. The conditions of ion-exchange chromatographic separations were optimized using theoretical modeling of the net surface electric charge of the peptide as a function of pH. The purified and concentrated sample was further subjected to LC-MS/MS. In order to improve the reliability of analysis, two fragment ions were monitored simultaneously both for native C-peptide and internal standard, isotopically labeled C-peptides analogues (fragments with m/z of 927.7 and 147.2). Using ion-exchange chromatography, it became possible to process larger sample volumes, important for testing patients with very low C peptide levels, compared to currently used solid phase extraction methods.

Hemoglobin A1c (HbA1c) is an important indicator of risk for complications in patients with diabetes mellitus. Elevated fetal hemoglobin (HbF) levels have been reported to interfere with results of some HbA1c methods, but it has generally been assumed that HbA1c results from boronate-affinity methods are not affected by elevated HbF levels. None of the previous studies used the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method as the comparative HbA1c method. We, therefore, measured HbA1c in samples with normal and elevated HbF levels by several common assay methods and compared the results with those of the IFCC reference method.HbF levels of more than 20% artificially lowered HbA1c results from the Primus CLC 330/385 (Primus Diagnostics, Kansas City, MO), Siemens DCA2000 (Siemens Healthcare Diagnostics, Tarrytown, NY), and Tosoh 2.2+ (Tosoh Bioscience, South San Francisco, CA), but not the Bio-Rad Variant II (Bio-Rad Laboratories, Hercules, CA) and Tosoh G7. Physicians and laboratory professionals need to be aware of potential interference from elevated HbF levels that could affect HbA1c results, including those from boronate-affinity methods.