A comparative analysis of current С-peptide assays compared to a reference method: can we overcome inertia to standardization?

Objectives: C-peptide is an equimolar by-product of insulin biosynthesis. It is used clinically to assess insulin secretion and differentiate types of diabetes. However, the lack of standardization across assays limits its broader application.

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.

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

Background: Measurement of hemoglobin A1c (HbA) in the blood is integral to and essential for the treatment of patients with diabetes mellitus. HbA reflects the mean blood glucose concentration over the preceding 8 to 12 weeks. Although the clinical value of HbA 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.

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 (βA) and HbS beta chain (βS) for the most common Hb variant trait (HbAS) are examined.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.