C-peptide determination in the diagnosis of type of diabetes and its management: A clinical perspective.

Impaired beta-cell function is a recognized cornerstone of diabetes pathophysiology. Estimates of insulin secretory capacity are useful to inform clinical practice, helping to classify types of diabetes, complication risk stratification and to guide treatment decisions. Because C-peptide secretion mirrors beta-cell function, it has emerged as a valuable clinical biomarker, mainly in autoimmune diabetes and especially in adult-onset diabetes. Nonetheless, the lack of robust evidence about the clinical utility of C-peptide measurement in type 2 diabetes, where insulin resistance is a major confounder, limits its use in such cases. Furthermore, problems remain in the standardization of the assay for C-peptide, raising concerns about comparability of measurements between different laboratories. To approach the heterogeneity and complexity of diabetes, reliable, simple and inexpensive clinical markers are required that can inform clinicians about probable pathophysiology and disease progression, and so enable personalization of management and therapy. This review summarizes the current evidence base about the potential value of C-peptide in the management of the two most prevalent forms of diabetes (type 2 diabetes and autoimmune diabetes) to address how its measurement may assist daily clinical practice and to highlight current limitations and areas of uncertainties to be covered by future research.

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.

Implementing a Reference Measurement System for C-Peptide: Successes and Lessons Learned.

BACKGROUND: Assessment of endogenous insulin secretion by measuring C-peptide concentrations is widely accepted. Recent studies have shown that preservation of even small amounts of endogenous C-peptide production in patients with type 1 diabetes reduces risks for diabetic complications. Harmonization of C-peptide results will facilitate comparison of data from different research studies and later among clinical laboratory results at different sites using different assay methods. CONTENT: This review provides an overview of the general process of harmonization and standardization and the challenges encountered with implementing a reference measurement system for C-peptide. SUMMARY: Efforts to harmonize C-peptide results are described, including those by the National Institute of Diabetes and Digestive and Kidney Diseases-led C-peptide Standardization Committee in the US, activities in Japan, efforts by the National Institute for Biological Standards and Control in the UK, as well as activities led by the Bureau International des Poids et Mesures and the National Metrology Institute in China. A traceability scheme is proposed along with the next steps for implementation. Suggestions are made for better collaboration to optimize the harmonization process for other measurands.

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.

Investigation of 2 models to set and evaluate quality targets for hb a1c: biological variation and sigma-metrics.

BACKGROUND: A major objective of the IFCC Task Force on Implementation of HbA1c Standardization is to develop a model to define quality targets for glycated hemoglobin (Hb A1c). METHODS: Two generic models, biological variation and sigma-metrics, are investigated. We selected variables in the models for Hb A1c and used data of external quality assurance/proficiency testing programs to evaluate the suitability of the models to set and evaluate quality targets within and between laboratories. RESULTS: In the biological variation model, 48% of individual laboratories and none of the 26 instrument groups met the minimum performance criterion. In the sigma-metrics model, with a total allowable error (TAE) set at 5 mmol/mol (0.46% NGSP), 77% of the individual laboratories and 12 of 26 instrument groups met the 2σ criterion. CONCLUSIONS: The biological variation and sigma-metrics models were demonstrated to be suitable for setting and evaluating quality targets within and between laboratories. The sigma-metrics model is more flexible, as both the TAE and the risk of failure can be adjusted to the situation-for example, requirements related to diagnosis/monitoring or international authorities. With the aim of reaching (inter)national consensus on advice regarding quality targets for Hb A1c, the Task Force suggests the sigma-metrics model as the model of choice, with default values of 5 mmol/mol (0.46%) for TAE and risk levels of 2σ and 4σ for routine laboratories and laboratories performing clinical trials, respectively. These goals should serve as a starting point for discussion with international stakeholders in the field of diabetes.

Point-of-Care HbA1c in Clinical Practice: Caveats and Considerations for Optimal Use.

Hemoglobin A1c (A1C) is widely used for the diagnosis and management of diabetes. Accurate measurement of A1C is necessary for optimal clinical value. Assay standardization has markedly improved the accuracy and consistency of A1C testing. Devices to measure A1C at point of care (POC) are commercially available, allowing rapid results when the patient is seen. In this review, we describe how standardization of A1C testing was achieved, leading to high-quality results in clinical laboratories. We address the use of POC A1C testing in clinical situations and summarize the advantages and disadvantages of POC A1C testing. We emphasize the importance of considering the limitations of these devices and following correct testing procedures to ensure that accurate A1C results are obtained for optimal care of patients.

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.

Continuous Glucose Monitoring and Use of Alternative Markers To Assess Glycemia in Chronic Kidney Disease.

OBJECTIVE: In chronic kidney disease, glycated albumin and fructosamine have been postulated to be better biomarkers of glycemic control than HbA1c. We evaluated the accuracy, variability, and covariate bias of three biomarkers (HbA1c, glycated albumin, and fructosamine) compared with continuous glucose monitoring (CGM)-derived measurement of glycemia across estimated glomerular filtration rate (eGFR) in type 2 diabetes. RESEARCH DESIGN AND METHODS: A prospective cohort study was conducted of 104 participants with type 2 diabetes, 80 with eGFR <60 mL/min/1.73 m2 (not treated with dialysis) and 24 frequency-matched control subjects with eGFR ≥60 mL/min/1.73 m2. Participants wore a blinded CGM for two 6-day periods separated by 2 weeks, with blood and urine collected at the end of each CGM period. HbA1c, glycated albumin, and fructosamine were measured by high-performance liquid chromatographic, enzymatic, and colorimetric nitroblue tetrazolium methods, respectively. RESULTS: Within-person biomarker values were strongly correlated between the two CGM periods (r = 0.92-0.95), although no marker fully captured the within-person variability of mean CGM glucose. All markers were similarly correlated with mean CGM glucose (r = 0.71-77). Compared with mean CGM glucose, glycated albumin and fructosamine were significantly biased by age, BMI, serum iron concentration, transferrin saturation, and albuminuria; HbA1c was underestimated in those with albuminuria. CONCLUSIONS: Glycated albumin and fructosamine were not less variable than HbA1c at a given mean CGM glucose level, with several additional sources of bias. These results support measuring HbA1c to monitor trends in glycemia among patients with eGFR <60 mL/min/1.73 m2. Direct measurements of glucose are necessary to capture short-term variability.

Accuracy and Precision of a Point-of-Care HbA1c Test.

BACKGROUND: Point-of-care (POC) hemoglobin A1c (HbA1c) testing has advantages over laboratory testing, but some questions have remained regarding the accuracy and precision of these methods. The accuracy and the precision of the POC Afinion™ HbA1c Dx test were investigated. METHODS: Samples spanning the assay range were collected from prospectively enrolled subjects at three clinical sites. The accuracy of the POC test using fingerstick and venous whole blood samples was estimated via correlation and bias with respect to values obtained by an NGSP secondary reference laboratory (SRL). The precision of the POC test using fingerstick samples was estimated from duplicate results by calculating the coefficient of variation (CV) and standard deviation (SD), and separated into its components using analysis of variance (ANOVA). The precision of the POC test using venous blood was evaluated from samples run in four replicates on each of three test cartridge lots, twice per day for 10 consecutive days. The SD and CV by study site and overall were calculated. RESULTS: Across the assay range, POC test results from fingerstick and venous whole blood samples were highly correlated with results from the NGSP SRL (r = .99). The mean bias was -0.021% HbA1c (-0.346% relative) using fingerstick samples and -0.005% HbA1c (-0.093% relative) using venous samples. Imprecision ranged from 0.62% to 1.93% CV for fingerstick samples and 1.11% to 1.69% CV for venous samples. CONCLUSIONS: The results indicate that the POC test evaluated here is accurate and precise using both fingerstick and venous whole blood.

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.