The National Glycohemoglobin Standardization Program: Over 20 Years of Improving Hemoglobin A1c Measurement.

BACKGROUND: Measurement of hemoglobin A1c (HbA1c) in the blood is integral to and essential for the treatment of patients with diabetes mellitus. HbA1c reflects the mean blood glucose concentration over the preceding 8 to 12 weeks. Although the clinical value of HbA1c was initially limited by large differences in results among various methods, the investment of considerable effort to implement standardization has brought about a marked improvement in analysis. CONTENT: The focus of this review is on the substantial progress that has been achieved in enhancing the accuracy and, therefore, the clinical value of HbA1c assays. SUMMARY: The interactions between the National Glycohemoglobin Standardization Program and manufacturers of HbA1c methods have been instrumental in standardizing HbA1c. Proficiency testing using whole blood has allowed accuracy-based assessment of methods in individual clinical laboratories that has made an important contribution to improving the HbA1c measurement in patient samples. These initiatives, supported by the efforts of the IFCC network, have led to a continuing enhancement of HbA1c methods.Many of the factors that previously influenced HbA1c results independently of blood glucose have been eliminated from most modern methods. These include carbamylation, labile intermediates, and common hemoglobin variants. Nevertheless, some factors (e.g., race and aging) may alter HbA1c interpretation, but whether these differences have clinical implications remains contentious. HbA1c has a fundamental role in the diagnosis and management of diabetes. Ongoing improvements in HbA1c measurement and quality will further enhance the clinical value of this analyte.

Two-step ion-exchange chromatographic purification combined with reversed-phase chromatography to isolate C-peptide for mass spectrometric analysis.

A liquid chromatography with mass spectrometry on-line platform that includes the orthogonal techniques of ion exchange and reversed phase chromatography is applied for C-peptide analysis. Additional improvement is achieved by the subsequent application of cation- and anion-exchange purification steps that allow for isolating components that have their isoelectric points in a narrow pH range before final reversed-phase mass spectrometry analysis. The utility of this approach for isolating fractions in the desired "pI window" for profiling complex mixtures is discussed.

Status of hemoglobin A1c measurement and goals for improvement: from chaos to order for improving diabetes care.

BACKGROUND: The Diabetes Control and Complications Trial (DCCT) and United Kingdom Prospective Diabetes Study (UKPDS) established the importance of hemoglobin A(1c) (Hb A(1c)) as a predictor of outcome in patients with diabetes mellitus. In 1994, the American Diabetes Association began recommending specific Hb A(1c) targets, but lack of comparability among assays limited the ability of clinicians to use these targets. The National Glycohemoglobin Standardization Program (NGSP) was implemented in 1996 to standardize Hb A(1c) results to those of the DCCT/UKPDS. CONTENT: The NGSP certifies manufacturers of Hb A(1c) methods as traceable to the DCCT. The certification criteria have been tightened over time and the NGSP has worked with the College of American Pathologists in tightening proficiency-testing requirements. As a result, variability of Hb A(1c) results among clinical laboratories has been considerably reduced. The IFCC has developed a reference system for Hb A(1c) that facilitates metrological traceability to a higher order. The NGSP maintains traceability to the IFCC network via ongoing sample comparisons. There has been controversy over whether to report Hb A(1c) results in IFCC or NGSP units, or as estimated average glucose. Individual countries are making this decision. SUMMARY: Variability among Hb A(1c) results has been greatly reduced. Not all countries will report Hb A(1c) in the same units, but there are established equations that enable conversion between different units. Hb A(1c) is now recommended for diagnosing diabetes, further accentuating the need for optimal assay performance. The NGSP will continue efforts to improve Hb A(1c) testing to ensure that clinical needs are met.

Evaluation of interference from hemoglobin C, D, E and S traits on measurements of hemoglobin A1c by fifteen methods.

BACKGROUND: Hemoglobin C, D Punjab, E or S trait can interfere with hemoglobin A1c (HbA1c) results. We assessed whether they affect results obtained with 15 current assay methods. METHODS: Hemoglobin AA (HbAA), HbAC, HbAD Punjab, HbAE and HbAS samples were analyzed on 2 enzymatic, 4 ion-exchange HPLC and 9 immunoassay methods. Trinity Premier Hb9210 boronate affinity HPLC was the comparative method. An overall test of coincidence of least-squared linear regression lines was performed to determine if HbA1c results were statistically significantly different from those of HbAA samples. Clinically significant interference was defined as >6% difference from HbAA at 6 or 9% HbA1c compared to Premier Hb9210 using Deming regression. RESULTS: All methods showed statistically significant effects for one or more variants. Clinically significant effects were observed for the Tosoh G11 variant mode (HbAD), Roche b 101 (HbAC and HbAE) and Siemens DCA Vantage (HbAE and HbAS). All other methods (Beckman Coulter B93009 and B00389 on DxC700AU, and Unicel DxC, Ortho Clinical Vitros 5.1, Roche cobas c 513, Siemens Dimension RxL and Vista, and Enzymatic on Advia and Atellica, Tosoh G8 5.24 and 5.28, and GX) showed no clinically significant differences. CONCLUSIONS: A few methods showed interference from one or more variants. Laboratories need to be aware of potential HbA1c assay interferences.

Effects of hemoglobin (Hb) E and HbD traits on measurements of glycated Hb (HbA1c) by 23 methods.

BACKGROUND: Glycohemoglobin (GHB), reported as hemoglobin (Hb) A(1c), 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 HbA(1c) measurement in the presence of HbE and/or HbD traits. METHODS: We evaluated 23 HbA(1c) 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 HbA(1c) results with the use of +/-10% relative bias at 6% and 9% HbA(1c) 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 HbA(1c) 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.

Effects of hemoglobin C and S traits on the results of 14 commercial glycated hemoglobin assays.

Glycated hemoglobin is widely used in the management of diabetes mellitus. At least 300,000 Americans with diabetes mellitus have the hemoglobin (Hb) C or S trait. The accuracy of HbA1c methods can be adversely affected by the presence of these traits. We evaluated the effects of HbC and HbS traits on the results of 14 commercial HbA1c methods that use boronate affinity, enzymatic, immunoassay, and ion exchange methods. Whole blood samples from people homozygous for HbA or heterozygous for HbC or HbS were analyzed for HbA1c. Results for each sample type were compared with those from the CLC 330 comparative method (Primus Diagnostics, Kansas City, MO). After correcting for calibration bias by comparing results from the homozygous HbA group, method bias attributable to the presence of HbC or HbS trait was evaluated with a clinically significant difference being more than 10% (ie, 0.6% at 6% HbA1c). One immunoassay method exhibited clinically significant differences owing to the presence of HbC and HbS traits.

The effect of elevated fetal hemoglobin on hemoglobin A1c results: five common hemoglobin A1c methods compared with the IFCC reference method.

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.

Effects of sample storage conditions on glycated hemoglobin measurement: evaluation of five different high performance liquid chromatography methods.

BACKGROUND: Glycated hemoglobin, reported as hemoglobin A1c (HbA1c), is widely used as a measure of long-term glycemic control in patients with diabetes. The accuracy of measurements depends in part on proper storage of the sample prior to analysis. METHODS: Three whole blood (WB) samples at three HbA1c levels were collected and stored at -70 degrees C, -20 degrees C, 4 degrees C, room temperature (17-23 degrees C), and 37 degrees C. One aliquot from each temperature was analyzed by each method on days 1, 2, 3, 6, 7, 10, 14, 21, 28, and 57. RESULTS: The Primus CLC (385 and 330) (Primus Corp., Kansas City, MO) showed stability of WB at -20 degrees C and 4 degrees C for 57 days, room temperature for 14 days, and 37 degrees C for 1 day. The Tosoh 2.2 Plus (Tosoh Bioscience, Inc., South San Francisco, CA) showed stability at -20 degrees C for 3 days, 4 degrees C for 14 days, room temperature for 3 days, and 37 degrees C for less than 24 h. With the Tosoh G7, results were acceptable at -20 degrees C for 10 days, 4 degrees C for 57 days, room temperature for 7 days, and 37 degrees C for less than 24 h. The Bio-Rad Variant (Bio-Rad Laboratories, Hercules, CA) showed stability at -20 degrees C for 6 days, 4 degrees C for 14 days, room temperature for 3 days, and 37 degrees C for less than 24 h. The Bio-Rad Variant II showed stability at -20 degrees C for 28 days, 4 degrees C for 57 days, room temperature for 7 days, and 37 degrees C for less than 24 h. CONCLUSIONS: 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.

Higher degree of glycation of hemoglobin S compared to hemoglobin A measured by mass spectrometry: Potential impact on HbA1c testing.

BACKGROUND: Glycated hemoglobin (GHb), reported as HbA1c, is used as marker of long-term glycemia for diabetic patients. HbA1c results from boronate affinity methods are generally considered to be unaffected by most hemoglobin variants; this assumes comparable glycation of variant and non-variant (HbAA) hemoglobins. In this report, glycation of HbA beta chain (ßA) and HbS beta chain (ßS) for the most common Hb variant trait (HbAS) are examined. METHODS: We analyzed 41 blood samples from subjects with HbAS, both with and without diabetes. Using LC-MS, ratios of glycated HbS to glycated HbA were determined by comparison of areas under the curves from extracted ion chromatograms. RESULTS: Glycation of ßS chains was significantly higher (p<0.001) than ßA chains; this difference was consistent across subjects. Total (α+ß) glycated HbAS was theoretically estimated to be ~5% higher than glycated HbAA. CONCLUSION: This novel mass-spectrometric approach described allows for relative quantification of glycated forms of ßS and ßA. Although ßS glycation was significantly higher than that of ßA, the difference in total glycation of HbAS versus HbAA was smaller and unlikely to impact clinical interpretation of boronate affinity HbA1c results. These data support the continued use of boronate affinity to measure HbA1c in patients with HbAS.

Effects of hemoglobin C, D, E and S traits on measurements of hemoglobin A1c by twelve methods.

BACKGROUND: Hemoglobin C, D Punjab, E or S trait can interfere with hemoglobin A1c (HbA1c) results. We assessed whether they affect results obtained with 12 current assay methods. METHODS: Hemoglobin AA (HbAA), HbAC, HbAD Punjab, HbAE and HbAS samples were analyzed on one enzymatic, nine ion-exchange HPLC and two Capillary Electrophoresis methods. Trinity ultra(2) boronate affinity HPLC was the comparative method. An overall test of coincidence of least-squared linear regression lines was performed to determine if HbA1c results were statistically significantly different from those of HbAA samples. Clinically significant interference was defined as >7% difference from HbAA at 6 or 9% HbA1c compared to ultra(2) using Deming regression. RESULTS: All methods showed statistically significant effects for one or more variants. Clinically significant effects were observed for the Tosoh G8 variant mode and GX (all variants), GX V1.22 (all but HbAE) and G11 variant mode (HbAC). All other methods (Abbott Architect c Enzymatic, Bio-Rad D-100, Variant II NU and Variant II Turbo 2.0, Menarini HA-8180T thalassemia mode and HA-8180V variant mode, Sebia Capillarys 2 and Capillarys 3) showed no clinically significant differences. CONCLUSIONS: Several methods showed clinically significant interference with HbA1c results from one or more variants which could adversely affect patient care.

Defining the relationship between plasma glucose and HbA(1c): analysis of glucose profiles and HbA(1c) in the Diabetes Control and Complications Trial.

OBJECTIVE: To define the relationship between HbA(1c) and plasma glucose (PG) levels in patients with type 1 diabetes using data from the Diabetes Control and Complications Trial (DCCT). RESEARCH DESIGN AND METHODS: The DCCT was a multicenter, randomized clinical trial designed to compare intensive and conventional therapies and their relative effects on the development and progression of diabetic complications in patients with type 1 diabetes. Quarterly HbA(1c) and corresponding seven-point capillary blood glucose profiles (premeal, postmeal, and bedtime) obtained in the DCCT were analyzed to define the relationship between HbA(1c) and PG. Only data from complete profiles with corresponding HbA(1c) were used (n = 26,056). Of the 1,441 subjects who participated in the study, 2 were excluded due to missing data. Mean plasma glucose (MPG) was estimated by multiplying capillary blood glucose by 1.11. Linear regression analysis weighted by the number of observations per subject was used to correlate MPG and HbA(1c). RESULTS: Linear regression analysis, using MPG and HbA(1c) summarized by patient (n = 1,439), produced a relationship of MPG (mmol/l) = (1.98 . HbA(1c)) - 4.29 or MPG (mg/dl) = (35.6 . HbA(1c)) - 77.3, r = 0.82). Among individual time points, afternoon and evening PG (postlunch, predinner, postdinner, and bedtime) showed higher correlations with HbA(1c) than the morning time points (prebreakfast, postbreakfast, and prelunch). CONCLUSIONS: We have defined the relationship between HbA(1c) and PG as assessed in the DCCT. Knowing this relationship can help patients with diabetes and their healthcare providers set day-to-day targets for PG to achieve specific HbA(1c) goals.

Effects of 49 Different Rare Hb Variants on HbA1c Measurement in Eight Methods.

BACKGROUND: Previous studies have shown interference with HbA1c measurement from the 4 most common heterozygous Hb variants (HbAS, HbAE, HbAC, and HbAD) with some assay methods. Here we examine analytical interference from 49 different less common variants with 7 different HbA1c methods using various method principles. METHODS: Hb variants were screened using the Bio-Rad Variant or Variant II beta thal short program, confirmed by alkaline and acid electrophoresis, and identified by sequence analysis. The Trinity ultra2 boronate affinity high-performance liquid chromatography (HPLC) method and Roche Tinaquant immunoassay were used as primary and secondary comparative methods, respectively, since these methods are least likely to show interference from Hb variants. Other methods included were the Tosoh G7 and G8, Bio-Rad D-10 and Variant II Turbo, Diazyme Enzymatic, and Sebia Capillarys 2 Flex Piercing. To eliminate any inherent calibration bias, results for each method were adjusted using regression verses the ultra2 with nonvariant samples. Each method's calibration-adjusted results were compared and judged to be acceptable if within the 99% prediction interval of the regression line for nonvariant samples. RESULTS: Almost all variant samples were recognized as such by the ion-exchange HPLC methods by the presence of abnormal peaks or results outside the reportable range. For most variants, interference was seen with 1 or more of the ion-exchange methods. Following manufacturer instructions for interpretation of chromatograms usually, but not always, prevented reporting of inaccurate results. RESULTS: Laboratories must be cautious about reporting results when the presence of a variant is suspected.

Effects of beta thalassemia minor on results of six glycated hemoglobin methods.

BACKGROUND: Beta-thalassemia minor (BTM) is a common benign condition that can be present in patients with diabetes mellitus. There are conflicting reports about the effect of BTM on glycated hemoglobin (gHb) measurements. We evaluated 6 gHb methods using samples from non-diabetic subjects with BTM. METHODS: Samples submitted for hemoglobin phenotype analysis were evaluated. A total of 57 samples (30 controls and 27 with BTM) from non-diabetic subjects were selected. GHb analysis was performed by Tosoh A1c 2.2+, Primus CLC 330, Bayer DCA 2000, Beckman Coulter, Synchron CX7 and LX20, and Roche Tina-quant II assays. RESULTS: The A1c 2.2+, CLC 330, DCA 2000 and Tina-quant II assays showed no statistically significant difference between the control and BTM groups. In contrast, BTM results were significantly higher than controls on the Synchron CX7 analyzer and borderline significant on the Synchron LX20 (p=0.051). Further investigation demonstrated an increase in Synchron %HbA(1c) results with decreasing hemoglobin concentrations. CONCLUSIONS: In this study using samples from subjects with normal or near-normal gHb, BTM does not affect gHb measurements per se. However, the Synchron methods yielded higher results for samples with lower hemoglobin concentrations, like those that can be seen in BTM. The Synchron method was improved at the end of 2003, which minimized this problem.

Comparing analytic performance criteria: evaluation of HbA1c certification criteria as an example.

BACKGROUND: Direct comparison of analytical performance criteria that utilize different statistical approaches can be problematic. We describe a mathematical approach to compare performance criteria for hemoglobin A1c (HbA1c) analysis used by the NGSP standardization program and the College of American Pathologists (CAP) to enhance consistency between the schemes. METHODS: The imprecision (CV) and bias combinations required to pass each criterion at probabilities of 0.95, 0.99 and 0.999 were calculated and used to construct contour plots to compare them. The CV/bias requirements were calculated mathematically for the 2011-2012 CAP (3/3 results within ±7% of the target) and different proposed NGSP (33/40 to 40/40 results within ±7% of the target) criteria, and using computer simulations for the existing NGSP criterion (95% confidence interval of the differences between the method and NGSP within ±0.75% HbA1c). RESULTS: Requiring 37 of 40 results to be within ±7% of the NGSP target best matched the CAP criterion at zero bias (95% chance of passing). CONCLUSIONS: The NGSP Steering Committee recommended a certification criterion of 37 of 40 results within ±7% of the NGSP (reduced to ±6% in 2014). The described evaluation approach may be useful in other situations where comparison of different performance criteria is desired.

The long and winding road to optimal HbA1c measurement.

The importance of hemoglobin A1c (HbA1c) as an indicator of mean glycemia and risks for complications in patients with diabetes mellitus was established by the results of long-term clinical trials, most notably the Diabetes Control and Complications Trial (DCCT) and United Kingdom Prospective Diabetes Study (UKPDS), published in 1993 and 1998 respectively. However, clinical application of recommended HbA1c targets that were based on these studies was difficult due to lack of comparability of HbA1c results among assay methods and laboratories. Thus, the National Glycohemoglobin Standardization Program (NGSP) was initiated in 1996 with the goal of standardizing HbA1c results to those of the DCCT/UKPDS. HbA1c standardization efforts have been highly successful; however, a number of issues have emerged on the "long and winding road" to better HbA1c, including the development of a higher-order HbA1c reference method by the International Federation of Clinical Chemistry (IFCC), recommendations to use HbA1c to diagnose as well as monitor diabetes, and point-of-care (POC) HbA1c testing. Here, we review the past, present and future of HbA1c standardization and describe the current status of HbA1c testing, including limitations that healthcare providers need to be aware of when interpreting HbA1c results.