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International comparison of C-peptide measurements.

Human C-peptide provides an accurate assessment of residual beta-cell function and thus has been widely used as a marker of insulin secretion in patients with diabetes (1, 2). Some studies have also suggested that C-peptide is biologically active (3) and may play a role in preventing and possibly reversing some chronic complications of type 1 diabetes (4,5). C-peptide is also important in the diagnosis of insulinoma/endogenous hyperinsulinemia (6).

Despite the fact that measurement of C-peptide has become increasingly important, the accuracy and between-laboratory comparability of C-peptide results have not been thoroughly evaluated. In 2002, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) organized a C-peptide standardization committee and funded an international comparison study of C-peptide assays to assess the comparability of C-peptide results within and among laboratories involved in diabetes-related long-term research studies. The goal of the study was to assess the degree of comparability of C-peptide results among methods and laboratories and to determine whether C-peptide results could be normalized to make comparing and combining data from different laboratories and studies more feasible. The comparison study was coordinated by a central laboratory at the University of Missouri-Columbia and was overseen by the C-peptide Standardization Committee. The members of the Committee and the participating laboratories are listed in Appendix A.

The study was conducted by the Diabetes Diagnostic Laboratory (DDL) at the University of Missouri-Columbia. The protocols were approved by the University of Missouri Health Sciences Institutional Review Board and all participants gave written informed consent. Three laboratories used more than one assay for C-peptide analysis, which resulted in a total of 20 laboratory methods being evaluated. Five laboratories used the Linco RIA; 1 additional laboratory used Linco C-peptide antibody with in-house isotope and precipitating reagents. Four laboratories used the Diagnostic Product Corp. (DPC) Immulite chemiluminescence immunoassay system, and 3 laboratories used the DPC RIA kit. Two laboratories used Tosoh Bioscience AIA-60011, an automated immunoassay system. The other methods--Liaison Immunoluminometric assay (ILMA) (DiaSorin), Biochem Pharma RIA, Shionogi RIA, Dako ELISA, and PerkinElmer Time-resolved fluoroimmunoassay (FIA)--were each used by 1 laboratory.

Heparin plasma samples were collected from 8 nondiabetic volunteers after they had fasted overnight and 60 min after they had consumed a standard meal (Boost[TM], Novartis Medical Nutrition). The samples were shipped frozen on dry ice and kept at -70 [degrees]C in the laboratories before analysis. Laboratories were instructed to analyze the specimens on specified days, in the same manner as they would analyze clinical specimens, and to provide a single result for each specimen.

Along with patient specimens, the laboratories received a WHO C-peptide reference reagent (standard) to assess its commutability and to determine the feasibility of normalizing results to this standard (7). This standard was prepared in the central laboratory with a vial of C-peptide stock solution into which was dissolved 1 ampoule of WHO IRR 84/510 standard (10 /[micro]g) in 25 mL of distilled water (8,9). The resulting solution (400 ng/mL or 132.4 nmol/L) was shipped on dry ice along with the samples. Each laboratory serially diluted the standard solution with their own assay buffer to make 4 preparations that were then analyzed in each analytical run.

To assess the impact of the calibrator matrix on the comparability of results, 4 of the samples were used to normalize the results from the remaining 12 samples. The mean of all laboratories' results for each sample was used as the assigned value for each of these sample calibrators. To compare the effect of normalization with WHO standards and normalization with samples, normalization was applied to the same 12 samples for both analyses. The 4 samples used as sample matrix calibrators were therefore not included in the normalization with WHO standards.

We performed all data analyses with Excel and SAS. Most laboratories in this study reported C-peptide results in nanograms per milliliter, but a few reported results in SI units (nmol/L). All C-peptide results were converted to SI units (nmol/L) before data analyses. Because scatter plots of the data indicated increasing variability in responses with increasing C-peptide, the model assumed constant CV. Weighted regression methods were used with weights inversely proportional to the variance. We performed iterative re-weighting because the variance had to be estimated from the modeled regression results.

For each laboratory/method, 2nd-degree equations were fitted by use of laboratory /methods values for the WHO standards on y and the "true" values for the standards on x; weights based on constant CV were used in the weighted regression analyses. In all but one case the coefficient for the squared term was not significantly different from zero, and in that case the fitted quadratic curve visually showed minimal curvature. Therefore, we performed normalization with simple linear relationships. The weighted regression analyses were run again using the normalized values and the same analyses were performed when we used samples as calibrators.

Visual inspection of the data showed that one laboratory's results (using the DPC Immulite method) did not show consistent increases with increasing C-peptide concentrations and was therefore considered an outlier (Fig. 1a). This laboratory's results were therefore excluded from all further evaluation.

The assay survey of participating laboratories showed that 6 of the C-peptide assay methods were already calibrated to WHO standard IRR lot 84/510 (DPC RIA, DPC Immulite, Linco RIA, Tosoh AIA, DiaSorin ILMA, and in-house RIA). Two of the laboratories reported that their methods were not calibrated to WHO standards (PerkinElmer FIA, Shionogi RIA), and no information was available for 2 of the methods (Dako ELISA, Biochem Pharma RIA).

C-peptide results before and after normalization with WHO standard (Fig. 1A and 1B) showed that the 95% confidence interval (CI) estimate for the SD for the lab/ method effect (0.172-0.456) overlapped with the 95% CI estimated with the raw data (0.090-0.225). This result suggests that WHO normalization was ineffective in reducing the variability of C-peptide results within and among the lab/methods.

[FIGURE 1 OMITTED]

As with the WHO normalization, weighted linear regression analyses were used for each of the assay methods for normalization with the 4 patient samples. Results (Fig. 1C) showed that the 95% CI estimate for the SD for the lab/method effect (0.0-0.061) using normalized values did not overlap with the 95% CI estimated with the raw data (0.090-0.225). This result shows that normalization with sample calibrators was effective in reducing variability of C-peptide results.

The importance of measuring C-peptide has increased significantly in recent years with the evidence from the DCCT that higher C-peptide concentrations are associated with improved hemoglobin A1c concentrations, less hypoglycemia, and less retinopathy and nephropathy (10). Furthermore, stabilization of C-peptide concentrations is being used as a measurable endpoint for immunomodulatory trials in type 1 diabetes (11,12). Our data show that a wide variety of C-peptide assay methods are being used by laboratories worldwide, and that the C-peptide results generated by the different methods, and even in some cases by different laboratories using the same method, are not always comparable. This finding has important implications for studies in which C-peptide results from different laboratories involved in multicenter studies are compared or combined for data analyses and for clinicians following patients over time if samples are tested at different laboratories.

Normalization using specimens achieves a modest improvement in comparability but the harmonization may be inadequate to fulfill the requirements of clinical trials. Our data reaffirm the difficulty in achieving standardization of C-peptide measurements because, like many polypeptides, C-peptide does not appear to behave the same way in pure calibrators as it does in patient samples, i.e., the commutability of pure standards is poor (13,14). Recently, isotope-dilution liquid chromatography-mass spectrometry has been proposed as a reference measurement procedure for serum C-peptide (14,15). Further studies are needed to determine if this method may be useful in standardizing measurements of plasma/serum C-peptide.

It is important that comparability of C-peptide results between laboratories be addressed before the initiation of large-scale trials involving multiple laboratories performing C-peptide analyses. The data presented here support the concept of using a single core laboratory for such studies. Our data also illustrate the need for efforts to standardize C-peptide measurements by use of materials prepared in sample matrix.

Appendix

MEMBERS OF THE C-PEPTIDE STANDARDIZATION COMMITTEE AND PARTICIPATING LABORATORIES

NIDDK C-Peptide Standardization Committee. David Goldstein (University of Missouri), Gary Myers (Centers for Disease Control and Prevention), Jerry Palmer (University of Washington), Kenneth Polonsky (Washington University), Judith Fradkin (NIDDK), Lisa Spain (NIDDK), Randie Little (University of Missouri), Hsiao-Mei Wiedmeyer (University of Missouri).

Participating Laboratories. Anette Ziegler, Kerstin Koczwara, Diabetes Research Institute Munich (Germany); Paolo Pozzilli, University Campus Bio-Medico (Italy); Charlotte Becker, University Hospital Malmb (Sweden); Ezio Bonifacio, San Raffaele Hospital (Italy); Merete Frandsen, Thomas Mandrup-Poulsen, Steno Diabetes Center (Denmark); Anders Isaksson, Mona Landin-Olsson, Lund University (Sweden); Armando Mendez, Linda Jones, University of Miami (FL); Jean Bucksa, Vicky Makky, University of Minnesota Medical Center, Fairview (MN); Veronica Luzzi, Gene Sherrow, Washington University (MO); Liz Rinehart, Linco Diagnostic Services, Inc. (MO); Jon Nakamoto, Anne Caston-Balderrama, Quest Diagnostics Nichols Institute (CA); Vinod Gaur, Northwest Lipid Metabolism and Diabetes Research Laboratory, University of Washington (WA); Alethea Tennill, University of Missouri (MO); Akira Shimada, Taro Maruyama, Keio University (Japan); Spiros Fourlanos, Royal Melbourne Hospital (Australia).

Grant/funding support: This project was funded by the Centers for Disease Control and Prevention through a funding contract (No.200200409985), supported by the NIDDK Special Statutory Funding Program for Type 1 Diabetes Research.

Disclosures: None declared

Acknowledgments: We thank Judith Fradkin and Lisa Spain for their support. We also thank Alethea Tennill, Curt Rohlfing, and Donghua Huang of the Diabetes Diagnostic Laboratory, University of Missouri, for their technical assistance with this project, as well as Richard Madsen, University of Missouri, for statistical consultation.

Previously published online at DOI: 10.1373/clinchem.2006.081570

References

(1.) Polonsky, KS, Licinio-Paixao J, Given BD, Pugh W, Galloway RJ, Karrison T, et al. Use of biosynthetic human C-peptide in the measurement of insulin secretion rates in normal volunteers and type 1 diabetic patients. J Clin Invest 1986;77:98-105.

(2.) Kjems LL, Volund A, Madsbad S. Quantification of beta-cell function during IVGTT in Type II and non-diabetic subjects: assessment of insulin secretion by mathematical methods. Diabetologia 2001;44:1339-48.

(3.) Wahren J, Jornvall H. C-peptide makes a comeback. Diabetes Metab Res Rev 2003;19:345-7.

(4.) Johansson JW, Wallberg-Henriksson H, Linde B, Ferngvist-Forbes E, Zierath JR. C-peptide revisited-new physiological effects and therapeutic implications. J Intern Med 1996;240:115-24.

(5.) Johansson BL, Borg K, Ferngvist-Forbes E, Odergren T, Remahl S, Wahren J. C-peptide improves autonomic nerve function in IDDM patients. Diabetologia 1996;39:687-95.

(6.) Saddig C, Bender R, Starke A. A new classification plot for the C-peptide suppression test. JOP 2002;3:16-25.

(7.) Miller WG, Myers GL, Rey R. Why commutability matters. Clin Chem 2006;52:553-4.

(8.) WHO synthetic C-peptide reference material 84/510, National Institute for Biological Standards and Control (UK).

(9.) Bristow AF, Gaines Das RE. WHO International reference reagents for human proinsulin and human insulin C-peptide. J Biol Stand 1988;16:179-86.

(10.) Steffes MW, Sibley S, Jackson M, Thomas W. Beta-cell function and the development of diabetes-related complications in the diabetes control and complication trial. Diabetes Care 2003;26:832-6.

(11.) Diabetes Prevention Trial-Type 1 Diabetes Study Group. Effects of insulin in relatives of patients with type 1 diabetes mellitus. N Engl J Med 2002;346: 1685-91.

(12.) European Nicotinamide Diabetes Intervention Trial (ENDIT) Group. European Nicotinamide Diabetes Intervention Trial (ENDIT): a randomized controlled trial of intervention before the onset of type 1 diabetes. Lancet 2004;363: 925-31.

(13.) Stockl D, Franzini C, Kratochvilla J, Middle J, Ricos C, Thienpont LM. Current stage of standardization of measurements of specific polypeptides and proteins discussed in light of steps needed towards a comprehensive measurement system. Eur J Clin Chem Clin Biochem 1997;35:719-32.

(14.) Rodriguez-Cabaleiro D, St6ckl D, Kaufman JM, Fiers T, Thienpont LM. Feasibility of standardization of serum C-peptide immunoassays with isotope dilution-liquid chromatography-tandem mass spectrometry. Clin Chem 2006;52:1193-5.

(15.) Rogatsky E, Balent B, Goswami G, Tomuta V, Jayatillake H, Cruikshank G, et al. Sensitive quantitative analysis of C-peptide in human plasma by 2-dimensional liquid chromatography-mass spectrometry isotope-dilution assay. Clin Chem 2006;52:872-9.

Hsiao-Mei Wiedmeyer, [1] Kenneth S. Polonsky, [2] Gary L. Myers, [3] Randie R. Little, [1] * Carla J. Greenbaum, [4] David E. Goldstein, [1] Jerry P. Palmer, [5]

[1] Departments of Pathology & Anatomical Sciences and Child Health, University of Missouri-Columbia School of Medicine, Columbia, MO;

[2] Department of Medicine, Washington University School of Medicine, St. Louis, MO;

[3] Centers for Disease Control and Prevention, Division of Environmental Health Laboratory Sciences, Centers for Environmental Health (F25), Chamblee, GA;

[4] Benaroya Research Institute, Seattle, WA; 5 University of Washington and VA Medical Center, Seattle, WA;

* address correspondence to this author at: Diabetes Diagnostic Laboratory, M767, Departments of Pathology & Anatomical Sciences and Child Health, University of Missouri School of Medicine, 1 Hospital Dr. Columbia, MO 65212; fax 573-884-8823, e-mail: LittleR@health.missouri.edu
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Title Annotation:Technical Briefs
Author:Wiedmeyer, Hsiao-Mei; Polonsky, Kenneth S.; Myers, Gary L.; Little, Randie R.; Greenbaum, Carla J.;
Publication:Clinical Chemistry
Date:Apr 1, 2007
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