Consistency of test results and applicability of reference intervals before and after 25-hydroxyvitamin D reagents upgrade_West China Medical Journal

Authors：

LI Xiaoling , ZHU Man , DING Fei , LIU Yin , CHEN Jie ,  GAN Wei

Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding?author：

GAN Wei, Email: 2004ganwei@scu.edu.cn

Keywords：

25-Hydroxyvitamin D; method comparison; reagent replacement; reference interval

DOI：

10.7507/1002-0179.202607110

Video：

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Objective To investigate the consistency of 25-hydroxyvitamin D (25-OH-VD) test results before and after a reagent generation change, assess the risk of misclassification associated with continued use of the previous reference interval after reagent replacement, and provide experimental evidence for the verification and revision of reference intervals following reagent changes in clinical laboratories. Methods The comparability of the two reagent generations was evaluated by simultaneously measuring samples using the pre-upgrade reagent (25-OH-VD Ⅰ) and the post-upgrade reagent (25-OH-VD Ⅲ). Healthy individuals who underwent physical examinations at the Health Management Center of West China Hospital, Sichuan University between March and June 2024 (25-OH-VD Ⅰ group, n=8203) and between March and June 2025 (25-OH-VD Ⅲ group, n=8203) were included. The distributions of results obtained using the two reagent generations across different concentration categories were compared. The consistency of the results was further evaluated through stratified analyses according to parathyroid hormone concentrations, and differences in the reference intervals between the two reagent generations were retrospectively analyzed. Results The relative differences between the results obtained using the 25-OH-VD Ⅲ and 25-OH-VD Ⅰ reagents ranged from –17% to +6%. In the 25-OH-VD deficiency category (<30 nmol/L), 428 individuals were identified using the 25-OH-VD Ⅰ reagent and 1195 using the 25-OH-VD Ⅲ reagent. In the insufficiency category (30-50 nmol/L), the corresponding numbers were 3324 and 4171, respectively. In the three parathyroid hormone strata of <1.6, 1.6-6.9, and >6.9 pmol/L, the median (lower quartile, upper quartile) of the 25-OH-VD concentrations measured using the 25-OH-VD Ⅲ reagent were 55.0 (41.9, 63.2), 44.9 (35.4, 57.6), and 39.1 (31.3, 49.4) nmol/L, respectively, all of which were lower than the corresponding values obtained using the 25-OH-VD Ⅰ reagent [70.8 (63.9, 79.1), 53.8 (42.6, 68.2), and 46.0 (37.1, 59.1) nmol/L, respectively; all P<0.05]. The P_2.5-P_97.5 reference interval for the 25-OH-VD Ⅲ reagent was 23.0-96.0 nmol/L, compared with 27.6-110.6 nmol/L for the 25-OH-VD Ⅰ reagent, representing decreases of 4.6 nmol/L in the lower limit and 14.6 nmol/L in the upper limit. Conclusions Significant differences were observed between the 25-OH-VD Ⅲ and 25-OH-VD Ⅰ reagents. Direct application of the reference interval established for the previous reagent may lead to overdiagnosis of vitamin D deficiency. Following a reagent generation change, laboratories should communicate the resulting differences to clinicians and, when necessary, establish a correction equation or revise the laboratory-specific reference interval.

Citation： LI Xiaoling, ZHU Man, DING Fei, LIU Yin, CHEN Jie, GAN Wei. Consistency of test results and applicability of reference intervals before and after 25-hydroxyvitamin D reagents upgrade. West China Medical Journal, 2026, 41(8): 1247-1252. doi: 10.7507/1002-0179.202607110 Copy

1.	Bouillon R, Manousaki D, Rosen C, et al. The health effects of vitamin D supplementation: evidence from human studies[J]. Nat Rev Endocrinol, 2022, 18(2): 96-110.
2.	Demay MB, Pittas AG, Bikle DD, et al. Vitamin D for the prevention of disease: an Endocrine Society clinical practice guideline[J]. J Clin Endocrinol Metab, 2024, 109(8): 1907-1947.
3.	Latic N, Erben RG. Interaction of vitamin D with peptide hormones with emphasis on parathyroid hormone, FGF23, and the renin-angiotensin-aldosterone system[J]. Nutrients, 2022, 14(23): 5186.
4.	Obo?ończyk ?, Berendt-Obo?ończyk M, Sworczak K, et al. Can vitamin D supplementation be dangerous? 25-Hydroxyvitamin D and 1,25-hydroxyvitamin D concentration in healthy population and primary hyperparathyroidism: a single-center experience and literature review[J]. Front Endocrinol (Lausanne), 2025, 16: 1700345.
5.	Mukhopadhyay P, Ghosh S, Bhattacharjee K, et al. Inverse relationship between 25 hydroxy vitamin D and parathormone: are there two inflection points?[J]. Indian J Endocrinol Metab, 2019, 23(4): 422-427.
6.	Alonso N, Zelzer S, Eibinger G, et al. Vitamin D metabolites: analytical challenges and clinical relevance[J]. Calcif Tissue Int, 2023, 112(2): 158-177.
7.	Zemp J, Erol C, Kaiser E, et al. A systematic review of evidence-based clinical guidelines for vitamin D screening and supplementation over the last decade[J]. Arch Public Health, 2025, 83(1): 221.
8.	Findeisen P, Leis M, Bendig G, et al. A multicenter performance evaluation of the new Elecsys Vitamin D total III assay versus reference isotope dilution liquid chromatography tandem mass spectrometry and commercially available comparators[J]. J Clin Lab Anal, 2022, 36(9): e24610.
9.	Wise SA, Camara JE, Sempos CT, et al. Vitamin D Standardization Program (VDSP) intralaboratory study for the assessment of 25-hydroxyvitamin D assay variability and bias[J]. J Steroid Biochem Mol Biol, 2021, 212: 105917.
10.	Zhou W, Gong M, Mao Y, et al. Analytical performance evaluation and optimization of serum 25(OH)D LC-MS/MS measurement[J]. Clin Chem Lab Med, 2025, 63(7): 1354-1365.
11.	Lee JH, Seo JD, Lee K, et al. Multicenter comparison of analytical interferences of 25-OH vitamin D immunoassay and mass spectrometry methods by endogenous interferents and cross-reactivity with 3-epi-25-OH-vitamin D₃[J]. Pract Lab Med, 2023, 38: e00347.
12.	Plebani M, Zaninotto M, Giannini S, et al. Vitamin D assay and supplementation: still debatable issues[J]. Diagnosis (Berl), 2025, 12(1): 35-44.
13.	WS/T 408-2024 定量檢驗程序分析性能驗證指南[S].
14.	WS/T 677-2020 人群維生素 D 缺乏篩查方法[S].
15.	CLSI. Defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline—third edition. CLSI document EP28-A3c[M]. Wayne, PA: Clinical and Laboratory Standards Institute, 2010.
16.	Favresse J, Schiettecatte J, Wolff F, et al. Two-site evaluation of the Roche Elecsys Vitamin D total III assay[J]. Clin Chem Lab Med, 2022, 60(10): 1598-1606.
17.	Scholze A, Shinkov A, Slavov GS, et al. 25-Hydroxyvitamin D₃ metabolism modulates the effect of variable UV exposure on 25-hydroxyvitamin D₃ plasma concentrations[J]. Commun Med (Lond), 2026, 6(1): 105.
18.	Geno KA, Alvarez YF, Hubbard JA, et al. Comparison of a reformulated automated vitamin D assay, its predecessor, and 2 contemporary formulations[J]. J Appl Lab Med, 2026, 11(3): 592-598.
19.	Ji Y, Bi R, Wang L. Research on vitamin D metabolic regulation in the pathogenesis of related diseases[J]. Front Endocrinol (Lausanne), 2026, 17: 1762302.
20.	Cavalier E, Bhattoa HP, Heijboer AC, et al. Assessment of measurement uncertainty of immunoassays and LC-MS/MS methods for serum 25-hydroxyvitamin D[J]. Clin Chem Lab Med, 2025, 63(10): 1981-1991.
21.	WS/T 402-2024 臨床實驗室定量檢驗項目參考區間的制定[S].
22.	趙怡, 韓昭昭. 體外診斷試劑參考區間審評要求及關鍵關注點[J]. 中國藥物評價, 2024, 41(6): 432-435.
23.	Perales-Afán JJ, Aparicio-Pelaz D, López-Triguero S, et al. Direct and indirect reference intervals of 25-hydroxyvitamin D: it is not a real vitamin D deficiency pandemic[J]. Biochem Med (Zagreb), 2024, 34(2): 020706.
24.	Black LJ, Anderson D, Clarke MW, et al. Analytical bias in the measurement of serum 25-hydroxyvitamin D concentrations impairs assessment of vitamin D status in clinical and research settings[J]. PLoS One, 2015, 10(8): e0135478.
25.	Fabregat-Bolufer AB, Escolà-Rodríguez A, Bedini-Chesa JL, et al. Redefining vitamin D status: establishing population-based indirect reference intervals through big data analysis[J]. Clin Chim Acta, 2025, 569: 120155.

1. Bouillon R, Manousaki D, Rosen C, et al. The health effects of vitamin D supplementation: evidence from human studies[J]. Nat Rev Endocrinol, 2022, 18(2): 96-110.
2. Demay MB, Pittas AG, Bikle DD, et al. Vitamin D for the prevention of disease: an Endocrine Society clinical practice guideline[J]. J Clin Endocrinol Metab, 2024, 109(8): 1907-1947.
3. Latic N, Erben RG. Interaction of vitamin D with peptide hormones with emphasis on parathyroid hormone, FGF23, and the renin-angiotensin-aldosterone system[J]. Nutrients, 2022, 14(23): 5186.
4. Obo?ończyk ?, Berendt-Obo?ończyk M, Sworczak K, et al. Can vitamin D supplementation be dangerous? 25-Hydroxyvitamin D and 1,25-hydroxyvitamin D concentration in healthy population and primary hyperparathyroidism: a single-center experience and literature review[J]. Front Endocrinol (Lausanne), 2025, 16: 1700345.
5. Mukhopadhyay P, Ghosh S, Bhattacharjee K, et al. Inverse relationship between 25 hydroxy vitamin D and parathormone: are there two inflection points?[J]. Indian J Endocrinol Metab, 2019, 23(4): 422-427.
6. Alonso N, Zelzer S, Eibinger G, et al. Vitamin D metabolites: analytical challenges and clinical relevance[J]. Calcif Tissue Int, 2023, 112(2): 158-177.
7. Zemp J, Erol C, Kaiser E, et al. A systematic review of evidence-based clinical guidelines for vitamin D screening and supplementation over the last decade[J]. Arch Public Health, 2025, 83(1): 221.
8. Findeisen P, Leis M, Bendig G, et al. A multicenter performance evaluation of the new Elecsys Vitamin D total III assay versus reference isotope dilution liquid chromatography tandem mass spectrometry and commercially available comparators[J]. J Clin Lab Anal, 2022, 36(9): e24610.
9. Wise SA, Camara JE, Sempos CT, et al. Vitamin D Standardization Program (VDSP) intralaboratory study for the assessment of 25-hydroxyvitamin D assay variability and bias[J]. J Steroid Biochem Mol Biol, 2021, 212: 105917.
10. Zhou W, Gong M, Mao Y, et al. Analytical performance evaluation and optimization of serum 25(OH)D LC-MS/MS measurement[J]. Clin Chem Lab Med, 2025, 63(7): 1354-1365.
11. Lee JH, Seo JD, Lee K, et al. Multicenter comparison of analytical interferences of 25-OH vitamin D immunoassay and mass spectrometry methods by endogenous interferents and cross-reactivity with 3-epi-25-OH-vitamin D₃[J]. Pract Lab Med, 2023, 38: e00347.
12. Plebani M, Zaninotto M, Giannini S, et al. Vitamin D assay and supplementation: still debatable issues[J]. Diagnosis (Berl), 2025, 12(1): 35-44.
13. WS/T 408-2024 定量檢驗程序分析性能驗證指南[S].
14. WS/T 677-2020 人群維生素 D 缺乏篩查方法[S].
15. CLSI. Defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline—third edition. CLSI document EP28-A3c[M]. Wayne, PA: Clinical and Laboratory Standards Institute, 2010.
16. Favresse J, Schiettecatte J, Wolff F, et al. Two-site evaluation of the Roche Elecsys Vitamin D total III assay[J]. Clin Chem Lab Med, 2022, 60(10): 1598-1606.
17. Scholze A, Shinkov A, Slavov GS, et al. 25-Hydroxyvitamin D₃ metabolism modulates the effect of variable UV exposure on 25-hydroxyvitamin D₃ plasma concentrations[J]. Commun Med (Lond), 2026, 6(1): 105.
18. Geno KA, Alvarez YF, Hubbard JA, et al. Comparison of a reformulated automated vitamin D assay, its predecessor, and 2 contemporary formulations[J]. J Appl Lab Med, 2026, 11(3): 592-598.
19. Ji Y, Bi R, Wang L. Research on vitamin D metabolic regulation in the pathogenesis of related diseases[J]. Front Endocrinol (Lausanne), 2026, 17: 1762302.
20. Cavalier E, Bhattoa HP, Heijboer AC, et al. Assessment of measurement uncertainty of immunoassays and LC-MS/MS methods for serum 25-hydroxyvitamin D[J]. Clin Chem Lab Med, 2025, 63(10): 1981-1991.
21. WS/T 402-2024 臨床實驗室定量檢驗項目參考區間的制定[S].
22. 趙怡, 韓昭昭. 體外診斷試劑參考區間審評要求及關鍵關注點[J]. 中國藥物評價, 2024, 41(6): 432-435.
23. Perales-Afán JJ, Aparicio-Pelaz D, López-Triguero S, et al. Direct and indirect reference intervals of 25-hydroxyvitamin D: it is not a real vitamin D deficiency pandemic[J]. Biochem Med (Zagreb), 2024, 34(2): 020706.
24. Black LJ, Anderson D, Clarke MW, et al. Analytical bias in the measurement of serum 25-hydroxyvitamin D concentrations impairs assessment of vitamin D status in clinical and research settings[J]. PLoS One, 2015, 10(8): e0135478.
25. Fabregat-Bolufer AB, Escolà-Rodríguez A, Bedini-Chesa JL, et al. Redefining vitamin D status: establishing population-based indirect reference intervals through big data analysis[J]. Clin Chim Acta, 2025, 569: 120155.

West China Medical Journal

Consistency of test results and applicability of reference intervals before and after 25-hydroxyvitamin D reagents upgrade

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