A comparative analysis of current С-peptide assays compared to a reference method: can we overcome inertia to standardization?
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Curt Rohlfing
Abstract
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. This study aimed to examine discrepancies between the leading C-peptide measurement methods used in clinical laboratories and propose a solution to reduce them based on a complete traceability chain.
Methods
Two sets of serum samples were distributed to 10 manufacturers of C-peptide assays. The first set (A, n=20) was analyzed independently by each manufacturer, who then returned their results to us. Subsequently, we sent out the second set (B, n=20) along with the reference values for set A. For set B, each manufacturer provided both non-calibrated and recalibrated values for each sample. The recalibration was performed according to each manufacturer’s internal standard protocols. We assessed how recalibration affected agreement between methods and alignment with the reference method. Non-parametric statistical approaches, including Passing-Bablok regression, level of agreement, and standard deviation analysis, were applied to compare data from multiple perspectives.
Results
Despite most manufacturers using the same WHO C-peptide calibrator material, significant disagreement was observed between methods prior to recalibration. Recalibration with matrix-appropriate serum samples reduced the discordance among assays, bringing them closer to the reference method. Overall, recalibration reduced both systematic bias and individual assay disagreement.
Conclusions
These findings underscore the importance of appropriate calibration schemes to improve agreement across C-peptide assays, enhancing the accuracy of C-peptide testing for clinical practice.
Funding source: NIH/NIDDK
Award Identifier / Grant number: U01DK096587
Acknowledgments
We would like to acknowledge the following manufacturers for their participation in our study: Abbott Laboratories, Beckman Coulter Inc., DiaSorin Deutschland GmbH, QuidelOrtho, Fujirebio Inc., Siemens Healthineers, Tosoh Corporation, ALPCO, Mercodia AB, and Roche Diagnostics GmbH.
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Research ethics: The protocols were approved by the University of Missouri Health Sciences Institutional Review Board (#2031722).
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Informed consent: Informed consent was obtained from all individuals included in this study.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Use of Large Language Models, AI and Machine Learning Tools: Not applicable.
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Conflict of interest: The authors state no conflict of interest.
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Research funding: This work was supported by NIH/NIDDK [Grant Number U01DK096587].
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Data availability: Not applicable.
References
1. Kruszynska, YT, Home, PD, Hanning, I, Alberti, KGMM. Basal and 24-h C-peptide and insulin secretion rate in normal man. Diabetologia 1987;30:16–21. https://doi.org/10.1007/bf01788901.Search in Google Scholar PubMed
2. Tillil, H. Assessment of insulin production and secretion dynamics in health and disease, including diabetes. Res Methodol Human Diabetes Part 2020;1:133–46.Search in Google Scholar
3. Bukmann Larsen, P, Linneberg, A, Hansen, T, Friis-Hansen, L. Reference intervals for C-peptide and insulin derived from a general adult Danish population. Clin Biochem 2017;50:408–13. https://doi.org/10.1016/j.clinbiochem.2016.12.004.Search in Google Scholar PubMed
4. Najjar, SM, Perdomo, G. Hepatic insulin clearance: mechanism and physiology. Physiology 2019;34:198–215. https://doi.org/10.1152/physiol.00048.2018.Search in Google Scholar PubMed PubMed Central
5. Bergman, RN, Piccinini, F, Kabir, M, Kolka, CM, Ader, M. Hypothesis: role of reduced hepatic insulin clearance in the pathogenesis of type 2 diabetes. Diabetes 2019;68:1709–16. https://doi.org/10.2337/db19-0098.Search in Google Scholar PubMed PubMed Central
6. Lachin, JM, McGee, P, Palmer, JP, For the DCCT/EDIC Research Group. Impact of C-peptide preservation on metabolic and clinical outcomes in the diabetes control and Complications trial. Diabetes 2014;63:739–48. https://doi.org/10.2337/db13-0881.Search in Google Scholar PubMed PubMed Central
7. Herold, KC, Gitelman, SE, Ehlers, MR, Gottlieb, PA, Greenbaum, CJ, Hagopian, W, et al.. Teplizumab (anti-CD3 mAb) treatment preserves C-peptide responses in patients with new-onset type 1 diabetes in a randomized controlled trial: metabolic and immunologic features at baseline identify a subgroup of responders. Diabetes 2013;62:3766–74. https://doi.org/10.2337/db13-0345.Search in Google Scholar PubMed PubMed Central
8. Taylor, PN, Collins, KS, Lam, A, Karpen, SR, Greeno, B, Walker, F, et al.. C-peptide and metabolic outcomes in trials of disease-modifying therapy in new-onset type 1 diabetes: an individual participant meta-analysis. Lancet Diabetes Endocrinol 2023;11:915–25. https://doi.org/10.1016/s2213-8587(23)00267-x.Search in Google Scholar PubMed
9. Latres, E, Greenbaum, CJ, Oyaski, ML, Dayan, CM, Colhoun, HM, Lachin, JM, et al.. Evidence for C-peptide as a validated surrogate to predict clinical benefits in trials of disease-modifying therapies for type 1 diabetes. Diabetes 2024;73:823–33. https://doi.org/10.2337/dbi23-0012.Search in Google Scholar PubMed
10. Leighton, E, Sainsbury, CA, Jones, GC. A practical review of C-peptide testing in diabetes. Diabetes Ther 2017;8:475–87. https://doi.org/10.1007/s13300-017-0265-4.Search in Google Scholar PubMed PubMed Central
11. Aleppo, G, Bannuru, RR, Bruemmer, D, Collins, BS, Ekhlaspour, L, et al., American Diabetes Association Professional Practice Committee. Diagnosis and classification of diabetes: standards of care in diabetes–2024. Diabetes Care 2024;47:S20–42. https://doi.org/10.2337/dc24-s002.Search in Google Scholar PubMed PubMed Central
12. Holt, RIG, DeVries, JH, Hess-Fischl, A, Hirsch, IB, Kirkman, MS, Klupa, T, et al.. The management of type 1 diabetes in adults: a consensus report by the American Diabetes Association and the European Association for the Study of Diabetes. Diabetes Care 2021;44:2589–625. https://doi.org/10.2337/dci21-0043.Search in Google Scholar PubMed
13. Wiedmeyer, HM, Polonsky, KS, Myers, GL, Little, RR, Greenbaum, CJ, Goldstein, DE, et al.. International comparison of C-peptide measurements. Clin Chem 2007;53:784–7. https://doi.org/10.1373/clinchem.2006.081570.Search in Google Scholar PubMed
14. Little, RR, Rohlfing, CL, Tennill, AL, Madsen, RW, Polonsky, KS, Myers, GL, et al.. Standardization of C-peptide measurements. Clin Chem 2008;54:1023–6. https://doi.org/10.1373/clinchem.2007.101287.Search in Google Scholar PubMed
15. Stoyanov, AV, Connolly, S, Rohlfing, CL, Rogatsky, E, Stein, D, Little, RR. Human C-peptide quantitation by LC-MS isotope-dilution assay in serum or urine samples. J Chromatogr Sep Technol 2013;4:172. https://doi.org/10.4172/2157-7064.1000172.Search in Google Scholar PubMed PubMed Central
16. Little, RR, Wielgosz, RI, Josephs, R, Kinumi, T, Takatsu, A, Li, H, et al.. Implementing a reference measurement system for C-peptide: successes and lessons learned. Clin Chem 2017;63:1447–56. https://doi.org/10.1373/clinchem.2016.269274.Search in Google Scholar PubMed PubMed Central
17. Little, RR, Kinumi, T, Connolly, S, Kabytaev, K. Implementing a reference measurement system for C-peptide: an addendum. Clin Chem 2017;63:1904–5. https://doi.org/10.1373/clinchem.2017.281170.Search in Google Scholar PubMed PubMed Central
18. Kolmogorov–Smirnov test. In: The Concise Encyclopedia of Statistics. New York, NY: Springer; 2008.Search in Google Scholar
19. Harrell, FE, Davis, CE. A new distribution-free quantile estimator. Biometrika 1982;69:635–40. https://doi.org/10.1093/biomet/69.3.635.Search in Google Scholar
20. 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 LC-MS isotope-dilution assay. Clin Chem 2006;52:872–9. https://doi.org/10.1373/clinchem.2005.063081.Search in Google Scholar PubMed
21. Ahlqvist, E, Storm, P, Käräjämäki, A, Martinell, M, Dorkhan, M, Carlsson, A, et al.. Novel subgroups of adult-onset diabetes and their association with outcomes: a data-driven cluster analysis of six variables. Lancet Diabetes Endocrinol 2018;6:361–9. https://doi.org/10.1016/s2213-8587(18)30051-2.Search in Google Scholar PubMed
22. Iqbal, S, Jayyab, AA, Alrashdi, AM, Reverté-Villarroya, S. The predictive ability of C-peptide in distinguishing type 1 diabetes from type 2 diabetes: a systematic review and meta-analysis. Endocr Pract 2023;29:379–87. https://doi.org/10.1016/j.eprac.2023.01.004.Search in Google Scholar PubMed
23. C-Peptide Standardization meetings. Available from: https://cpeptide.org/meetings.html.Search in Google Scholar
24. Florkowski, C, Crooke, M, Reed, M. Implementation of the HbA1c IFCC unit–from the laboratory to the consumer: the New Zealand experience. Clin Chim Acta 2014;432:157–61. https://doi.org/10.1016/j.cca.2013.10.009.Search in Google Scholar PubMed
25. Regulation (EU). 2017/746 of the European parliament and of the council. Off J Eur Union 2017. Available from: https://eur-lex.europa.eu/eli/reg/2017/746/oj.Search in Google Scholar
Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2024-1260).
© 2025 Walter de Gruyter GmbH, Berlin/Boston
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