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.

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.

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.

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.

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.

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.

Isotope dilution assay in peptide quantification: the challenge of microheterogeneity of internal standard.

Isotope dilution analysis allows quantitation of elements and different compounds in complex mixtures. The quantitation is based on a known amount of reference material (internal standard, IS) added to a sample that makes the result critically dependent on the value assigned to the standard. In the case of peptides, IS concentration is determined by nitrogen and amino acid analysis while purity is normally assessed by methods such as chromatography or electrophoresis that might not be able to detect many possible amino acid modifications, either naturally occurring or chemically induced. Microheterogeneity of the IS, if it is not accounted for when assigning a reference value to the standard, results in highly overestimated values in target analyte quantitation. In this viewpoint article, we illustrate the problem of internal standard microheterogeneity by analyzing synthetic human C-peptide labeled analogs.

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.

Measurement of Hba(1C) in patients with chronic renal failure.

BACKGROUND: Carbamylated hemoglobin (carbHb) is reported to interfere with measurement and interpretation of HbA(1c) 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 HbA(1c) measurements and compared HbA(1c) with glycated albumin (GA) in patients with and without renal disease to test if CRF causes clinically significant bias in HbA(1c) results by 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 the renal disease. A multiple regression model was used to determine if the relationship between HbA(1c) results obtained from each test method and the comparative method was significantly (p<0.05) affected by eGFR. These methods were further evaluated for clinical significance by using the difference between the eGRF quartiles of >7% at 6 or 9% HbA(1c). The relationship between HbA(1c) 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 HbA(1c) was approximately 1.5% HbA(1c) lower in patients with renal failure. CONCLUSIONS: Although most methods can measure HbA(1c) accurately in patients with renal failure, healthcare providers must interpret these test results cautiously in these patients due to the propensity for shortened erythrocyte survival in renal failure.

Human C-peptide Quantitation by LC-MS Isotope-Dilution Assay in Serum or Urine Samples.

In this communication we report a simple and efficient approach to C-peptide quantitation using isotope dilution mass-spectrometry analysis. The method facilitates quantitation of C-peptide levels at least one order of magnitude lower compared to concentration levels achieved with an IDA method reported previously. The improvement was due to more intensive sample preparation procedure that, in turn, makes it possible to increase the sample load without a corresponding increase in matrix effects. We also show the results of a comparison study with a second laboratory using a similar previously reported method for C-peptide quantitation.

Effects of whole blood storage on hemoglobin a1c measurements with five current assay methods.

BACKGROUND: Hemoglobin A1c (HbA1c) is an important index of average glycemia in patients with diabetes mellitus that is widely used in clinical trials and large-scale epidemiological studies. Previous studies have shown that adverse sample storage conditions can cause erroneous HbA1c results. We examined the effect of storage at different temperatures with five current HbA1c methods: Tosoh G7 and G8 (Tosoh Bioscience, Inc., South San Francisco, CA) and Bio-Rad Variant™ II (Bio-Rad Laboratories, Hercules, CA) (all ion-exchange high-performance liquid chromatography); Siemens DCA 2000+ (Siemens Healthcare Diagnostics, Deerfield, IL) (immunoassay); and Trinity Biotech (Kansas City, MO) ultra(2) (boronate-affinity high-performance liquid chromatography). METHODS: Five whole blood specimens with different HbA1c levels were analyzed by each assay method on Day 0 and then divided into aliquots that were stored at six different temperatures (-70°C, -20°C, 4°C, room temperature, 30°C, and 37°C) for analyses on subsequent days out to Day 84. Acceptance limits were defined as within ±3 SD of all -70°C results or ±0.2% HbA1c, whichever was wider, for each sample. Stability was considered acceptable for a given temperature only if results for all five specimens were acceptable on that day. RESULTS: The DCA 2000+ demonstrated the best stability at -20°C and room temperature, whereas the ultra(2) showed the best stability with specimens stored at 4°C. No methods demonstrated stability at 30°C or 37°C for more than 3 days. CONCLUSIONS: Exposure of specimens to high temperatures should be avoided regardless of assay methodology. For the ion-exchange methods tested 4°C storage is preferable to -20°C (stability 14-21 days vs. 4-10 days). For studies where long-term stability is required, samples should be stored at -70°C or colder.

Use of cation exchange chromatography for human C-peptide isotope dilution - mass spectrometric assay.

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.