The current status of serum insulin measurements and the need for standardization
-
Curt Rohlfing
Abstract
Objectives
Insulin assays are used to assess insulin resistance and to aid in the diagnosis of conditions such as insulinoma and various forms of hypoglycemia. However, discrepancies among commercial assays limit their clinical and research utility. This study evaluates the current comparability of the most widely used insulin assays.
Methods
Forty serum samples, including those from healthy individuals and patients with type 1 or type 2 diabetes, were analyzed by nine manufacturers using 12 commercial immunoassays. Results from each assay were compared both to the isotope dilution mass spectrometry (IDMS) method and across the different immunoassays.
Results
Intra-assay repeatability was excellent (ICCs>0.99), but substantial inter-assay variability was observed. Differences relative to LC-MS ranged from −298.2 to +302.6 pmol/L. Several assays overestimated insulin concentrations at low levels and underestimated them at higher levels. Only one assay method showed full agreement with the IDMS method.
Conclusions
Despite all methods claiming traceability to the WHO 66/304 standard, significant variability persists among insulin assays. These findings highlight the urgent need for insulin assay standardization using commutable certified reference materials.
Funding source: National Institute of Health
Award Identifier / Grant number: 5U01DK133106-03
Award Identifier / Grant number: U01 DK137097
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, Mercodia AB, and Roche Diagnostics GmbH.
-
Research ethics: The protocols were approved by the University of Missouri Health Sciences Institutional Review Board (#2031722).
-
Informed consent: Informed consent was obtained from all individuals included in this study.
-
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
-
Use of Large Language Models, AI and Machine Learning Tools: Not applicable.
-
Conflict of interest: The authors state no conflict of interest.
-
Research funding: This work was supported by NIH [Grant Number 5U01DK133106-03, U01 DK137097].
-
Data availability: Not applicable.
References
1. Proud, CG. Regulation of protein synthesis by insulin. Biochem Soc Trans 2006;34:213–6. https://doi.org/10.1042/bst0340213.Suche in Google Scholar
2. Carpentier, AC. 100th anniversary of the discovery of insulin perspective: insulin and adipose tissue fatty acid metabolism. Am J Physiol Endocrinol Metab 2021;320:E653–70. https://doi.org/10.1152/ajpendo.00620.2020.Suche in Google Scholar PubMed
3. Czech, MP. Insulin action and resistance in obesity and type 2 diabetes. Nat Med 2017;23:804–14. https://doi.org/10.1038/nm.4350.Suche in Google Scholar PubMed PubMed Central
4. Cantley, J, Ashcroft, FM. Q&A: insulin secretion and type 2 diabetes: why do β-cells fail? BMC Biol 2015;13:33. https://doi.org/10.1186/s12915-015-0140-6.Suche in Google Scholar PubMed PubMed Central
5. Park, SY, Gautier, JF, Chon, S. Assessment of insulin secretion and insulin resistance in human. Diabetes Metab J 2021;45:641–54. https://doi.org/10.4093/dmj.2021.0220.Suche in Google Scholar PubMed PubMed Central
6. Zhang, AMY, Wellberg, EA, Kopp, JL, Johnson, JD. Hyperinsulinemia in obesity, inflammation, and cancer. Diabetes Metab J 2021;45:285–311. https://doi.org/10.4093/dmj.2021.0131.Suche in Google Scholar PubMed PubMed Central
7. The Diabetes Prevention Program Research Group. Role of insulin secretion and sensitivity in the evolution of type 2 diabetes in the diabetes prevention program: effects of lifestyle intervention and metformin. Diabetes 2005;54:2404–14. https://doi.org/10.2337/diabetes.54.8.2404.Suche in Google Scholar PubMed PubMed Central
8. Kahn, SE, Lachin, JM, Zinman, B, Haffner, SM, Aftring, RP, Paul, G, et al.. Effects of rosiglitazone, glyburide, and metformin on β-cell function and insulin sensitivity in ADOPT. Diabetes 2011;60:1552–60. https://doi.org/10.2337/db10-1392.Suche in Google Scholar PubMed PubMed Central
9. Miller, WG, Thienpont, LM, Van Uytfanghe, K, Clark, PM, Lindstedt, P, Nilsson, G, et al.. Toward standardization of insulin immunoassays. Clin Chem. 2009;55:1011–8. https://doi.org/10.1373/clinchem.2008.118380.Suche in Google Scholar PubMed
10. Marcovina, S, Bowsher, RR, Miller, WG, Staten, M, Myers, G, Caudill, SP, et al.. Standardization of insulin immunoassays: report of the American Diabetes Association Workgroup. Clin Chem 2007;53:711–6. https://doi.org/10.1373/clinchem.2006.082214.Suche in Google Scholar PubMed
11. Staten, MA, Stern, MP, Miller, WG, Steffes, MW, Campbell, SE. Insulin assay standardization: leading to measures of insulin sensitivity and secretion for practical clinical care. Diabetes Care 2010;33:205–6. https://doi.org/10.2337/dc09-1206.Suche in Google Scholar PubMed PubMed Central
12. Foulon, N, Goonatilleke, E, MacCoss, MJ, Emrick, MA, Hoofnagle, AN. Multiplexed quantification of insulin and C-peptide by LC-MS/MS without the use of antibodies. J Mass Spectrom Adv Clin Lab 2022;25:19–26. https://doi.org/10.1016/j.jmsacl.2022.06.003.Suche in Google Scholar PubMed PubMed Central
13. Moradian, A, Goonatilleke, E, Lin, TT, Hatten-Beck, M, Emrick, M, Schepmoes, AA, et al.. Interlaboratory comparison of antibody-free LC-MS/MS measurements of C-peptide and insulin. Clin Chem 2024;70:855–64. https://doi.org/10.1093/clinchem/hvae034.Suche in Google Scholar PubMed PubMed Central
14. Li, J, Hatten-Beck, M, Lee, JKY, Hoofnagle, AN. Insights into insulin analog cross-reactivity: a comparative study of Siemens Atellica and LC-MS/MS. Endocrine 2025;87:79–84. https://doi.org/10.1007/s12020-024-03970-6.Suche in Google Scholar PubMed PubMed Central
15. Tu, S, Li, C, Zeng, D, Shepherd, BE. Rank intraclass correlation for clustered data. Stat Med 2023;42:4333–48. https://doi.org/10.1002/sim.9864.Suche in Google Scholar PubMed PubMed Central
16. Passing, H, Bablok, W. A new biometrical procedure for testing the equality of measurements from two different analytical methods. J Clin Chem Clin Biochem 1983;21:709–20. https://doi.org/10.1515/cclm.1983.21.11.709.Suche in Google Scholar PubMed
17. Potapov, S, Model, F, Schuetzenmeister, A, Manuilova, E, Dufey, F, Raymaekers, J, et al.. Mcr: method comparison regression [Internet]. Version 1.3.3.1; 2024. Available from: https://CRAN.R-project.org/package=mcr [cited 9 Jun 2025].Suche in Google Scholar
18. Miller, WG, Myers, GL, Rej, R. Why commutability matters. Clin Chem. 2006;52:553–4. https://doi.org/10.1373/clinchem.2005.063511.Suche in Google Scholar PubMed
19. Rohlfing, C, Petroski, G, Connolly, SM, Hanson, S, Little, RR, Kabytaev, K. A comparative analysis of current C-peptide assays compared to a reference method: can we overcome inertia to standardization? Clin Chem Lab Med 2025;63:1124–31. https://doi.org/10.1515/cclm-2024-1260.Suche in Google Scholar PubMed PubMed Central
20. World Health Organization. WHO reference reagent for human insulin [internet]. WHO/BS/2019.2366. Available from: https://www.who.int/publications/m/item/WHO-BS-2019.2366 [cited 9 Jun 2025].Suche in Google Scholar
21. Teoli, J, Chikh, K, Jouini-Bouhamri, R, Charriere, S, Fabien, N, Raverot, V. When discordant insulin and C-peptide levels lead to a medical diagnosis in a patient with transient hypoglycemia: varying degrees of interference of insulin-antibody complexes on three insulin immunoassays. Heliyon 2024;10:e34009. https://doi.org/10.1016/j.heliyon.2024.e34009.Suche in Google Scholar PubMed PubMed Central
22. Sims, EK, Bahnson, HT, Nyalwidhe, J, Haataja, L, Davis, AK, Speake, C, et al.. Proinsulin secretion is a persistent feature of type 1 diabetes. Diabetes Care 2019;42:258–64. https://doi.org/10.2337/dc17-2625.Suche in Google Scholar PubMed PubMed Central
23. Cotes, PM, Mussett, MV, Berryman, I, Ekins, R, Glover, S, Hales, N, et al.. Collaborative study of estimates by radioimmunoassay of insulin concentrations in plasma samples examined in groups of five or six laboratories. J Endocrinol 1969;45:557–69. https://doi.org/10.1677/joe.0.0450557.Suche in Google Scholar PubMed
24. Rosli, N, Kwon, HJ, Lim, J, Yoon, YA, Jeong, JS. Measurement comparability of insulin assays using conventional immunoassay kits. J Clin Lab Anal 2022;36:e24521. https://doi.org/10.1002/jcla.24521.Suche in Google Scholar PubMed PubMed Central
25. Tohidi, M, Arbab, P, Ghasemi, A. Assay-dependent variability of serum insulin concentrations: a comparison of eight assays. Scand J Clin Lab Invest 2017;77:122–9. https://doi.org/10.1080/00365513.2016.1278260.Suche in Google Scholar PubMed
26. Zhou, W, Deng, Y, Zhao, H, Zhang, C. Current status of serum insulin and C-peptide measurement in clinical laboratories: experience from 94 laboratories in China. Ann Lab Med 2022;42:428–37. https://doi.org/10.3343/alm.2022.42.4.428.Suche in Google Scholar PubMed PubMed Central
© 2025 Walter de Gruyter GmbH, Berlin/Boston
