Background/Objectives: Big data analysis has revolutionized medical research, making it possible to analyze vast amounts of data and gain valuable insights that were previously impossible to obtain. Our knowledge of the characteristics of vitamin D sufficiency is primarily based on data from a limited number of observations, generally spanning a few years at most. Methods: Here at the Medical Faculty of the University of Debrecen, the big data approach has allowed us to analyze trends in vitamin D status using nearly 60,000 25-hydroxyvitamin D (25(OH)D) concentration results from 2000 onwards. Results: Apart from analyzing the well-known phenomenon of seasonality in 25(OH)D concentration, we observed a trend in test requests, which increased from a few hundred in 2000 to almost 10,000 in 2020. Of particular interest is the change in the gender gap in test requests. In previous years, test requests were primarily from women, but by the end of the analysis period, a significant number of requests were from men as well. Since the data set includes all age groups, we analyzed 25(OH)D concentration for incremental age sets of five years, from a few months to 100 years old. The prevalence of vitamin D insufficiency (<75 nmol/L) was clearly demarcated among various years of observation, age groups, sexes, and seasons. Our data was particularly valuable for analyzing the effect of the methodology used for 25(OH)D determination. Three different methodologies were used during the study period, and clear, statistically significant bias was observed. Conclusions: Our results clearly demonstrate the effect of the methodology used to determine 25(OH)D concentrations on vitamin D status, explicitly highlighting the urgent need to standardize the various platforms used to measure this important analyte and its consequences for public health.

Background/Objective: Accurate measurement of serum 25-hydroxyvitamin D (25OHD) is essential for the diagnosis and management of vitamin D deficiency. Although automated immunoassays are widely used in routine clinical practice, analytical variability between platforms may lead to inconsistent patient classification around clinically relevant decision thresholds. This study comprehensively compared three automated immunoassays (DiaSorin Liaison XL, Beckman Coulter DxI 9000, and Roche cobas e602) with a Vitamin D Standardization Certification Program (VDSCP)-traceable liquid chromatography–tandem mass spectrometry (LC-MS/MS) reference method. Methods: A total of 400 anonymized adult outpatient serum samples were prospectively collected and equally distributed across four predefined vitamin D concentration categories (<25, 25–49, 50–74, and ≥75 nmol/L). Samples were stored at −70 °C before simultaneous analysis on the three automated immunoassays and the reference LC-MS/MS platform. Method comparison included Deming regression, Bland–Altman analysis, and evaluation of diagnostic performance at clinically relevant thresholds (<25, <50, and <75 nmol/L). The effects of sample storage, vitamin D metabolites (C3-epi-25OHD₃ and 24,25-dihydroxyvitamin D₃), and external quality assessment (DEQAS) performance were also investigated. Results: Significant analytical differences were observed between all immunoassays and LC-MS/MS. Roche cobas e602 consistently overestimated 25OHD concentrations, resulting in reduced sensitivity but excellent specificity and positive predictive value across all clinical thresholds. Beckman Coulter demonstrated the highest sensitivity for detecting vitamin D deficiency but generated more false-positive classifications, particularly at the <75 nmol/L threshold. DiaSorin Liaison XL achieved the best overall balance between sensitivity, specificity, accuracy, and agreement with LC-MS/MS, with Cohen’s κ values ranging from 0.818 to 0.895 across decision thresholds. Higher concentrations of C3-epi-25OHD₃ and 24,25-dihydroxyvitamin D₃ were associated with greater positive bias in Roche measurements, while Beckman Coulter showed no significant metabolite-related interference, although the present correlation analyses could not establish independent analytical interference. DEQAS performance did not consistently reflect agreement with LC-MS/MS in patient samples. Conclusions: Despite ongoing international standardization efforts, clinically important differences remain among automated 25OHD immunoassays. Assay-specific analytical characteristics significantly influence patient classification around established decision thresholds. DiaSorin Liaison XL demonstrated the closest overall agreement with the reference LC-MS/MS method, whereas Beckman Coulter prioritized sensitivity and Roche specificity. These findings highlight the importance of method-aware interpretation of vitamin D results and support continued assay harmonization to improve comparability in routine clinical practice.