Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2-Part 2 ligand binding assays - impact of 25-hydroxyvitamin D2 and 24R,25-dihydroxyvitamin D3 on assay performance

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of ligand binding assays (Part 2) for the determination of serum total 25-hydroxyvitamin D [25(OH)D]. Fifty single-donor samples were assigned target values for concentrations of 25-hydroxyvitamin D-2 [25(OH)D-2], 25-hydroxyvitamin D-3 [25(OH)D-3], 3-epi-25-hydroxyvitamin D-3 [3-epi-25(OH)D-3], and 24R,25-dihydroxyvitamin D-3 [24R,25(OH)(2)D-3] using isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 2 includes results from 17 laboratories using 32 ligand binding assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 50% of the ligand binding assays achieved the VDSP criterion of mean % bias <= |+/- 5%|. For the 13 unique ligand binding assays evaluated in this study, only 4 assays were consistently within +/- 5% mean bias and 4 assays were consistently outside +/- 5% mean bias regardless of the laboratory performing the assay. Based on multivariable regression analysis using the concentrations of individual vitamin D metabolites in the 50 single-donor samples, most assays underestimate 25(OH)D-2 and several assays (Abbott, bioMerieux, DiaSorin, IDS-EIA, and IDS-iSYS) may have cross-reactivity from 24R,25(OH)(2)D-3. The results of this interlaboratory study represent the most comprehensive comparison of 25(OH)D ligand binding assays published to date and is the only study to assess the impact of 24R,25(OH)(2)D-3 content using results from a reference measurement procedure.