Measurement of 1,25-dihydroxyvitamin D in serum by LC-MS/MS compared to immunoassay reveals inconsistent agreement in paediatric samples.

Abstract

Objectives: Automated immunoassays for 1,25-dihydroxyvitamin D (1,25(OH)₂D) have increased the use of serum measurements in clinical and research settings, but disagreement with LC-MS/MS methods remains an issue.

Methods: In this study, we examined this problem using samples obtained from healthy young adults, n=80, mean age 21.7 (18-32) years, and a large cohort of paediatric samples, n=422, mean age 7.3 (0-17) years. We compared serum concentrations of 1,25(OH)₂D3/D2 produced by the DiaSorin LIAISON^® XL immunoassay against an LC-MS/MS method with immunoaffinity enrichment and DAPTAD derivation.

Results: Both assays showed intra/inter-assay imprecision of ≤9.4 % across their respective assay range. DEQAS between April 2020 to Jan 2024 (n=80) showed mean bias (SD, 95 %CI) for DiaSorin -0.6 % (6.2, -12.8 to 11.6) and LC-MS/MS of +1.3 % (7.4, -13.3 to 15.8) against their respective method group means. Comparison of measurements in the adult samples showed a strong correlation (r²=0.9331) and concordance (CCC=0.959) between the two methods. LC-MS/MS values were lower than DiaSorin by an overall mean (±SD, 95 %CI) of -1.6 (±14.3, -29.6 to 26.5) pmol/L with an increased negative bias at higher concentrations. In the paediatric samples, weaker correlation (r²=0.6536) and concordance (CCC=0.782) were observed, with greater bias mean (±SD, 95 %CI) of -9.8 (±23.4, -55.7 to 35.9) pmol/L. The variability in the paediatric samples was not associated with concentration or participant age. There was an increase in the correlation and concordance when 1,25(OH)₂D2 was included in the analysis.

Conclusions: It is likely that the metabolites of vitamin D present in the paediatric population contributed to the measurement of 1,25(OH)₂D. The inconsistent agreement highlights the need for better assay harmonisation and paediatric reference intervals using LC-MS/MS method.