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 D2 [25(OH)D2], 25-hydroxyvitamin D3 [25(OH)D3], 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] 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)D2 and several assays (Abbott, bioMérieux, DiaSorin, IDS-EIA, and IDS-iSYS) may have cross-reactivity from 24R,25(OH)2D3. 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)2D3 content using results from a reference measurement procedure.

Assessment of serum total 25-hydroxyvitamin D assays for Vitamin D External Quality Assessment Scheme (DEQAS) materials distributed at ambient and frozen conditions.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at -40 °C prior to distribution and the participants are instructed to store the samples frozen at -20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p < 0.05 for four assays (DiaSorin, DIAsource, Siemens, and SNIBE prototype). For all 14 LC-MS/MS assays, the differences in the results for the ambient- and frozen-shipped samples were not found to be significant at p < 0.05 indicating that these analytes were stable during shipment at ambient conditions. Even though assay results have been shown to vary considerably among different 25(OH)D assays in other studies, the results of this study also indicate that sample handling/transport conditions may influence 25(OH)D assay response for several assays.

Assessment of serum total 25-hydroxyvitamin D assay commutability of Standard Reference Materials and College of American Pathologists Accuracy-Based Vitamin D (ABVD) Scheme and Vitamin D External Quality Assessment Scheme (DEQAS) materials: Vitamin D Standardization Program (VDSP) Commutability Study 2.

An interlaboratory study was conducted through the Vitamin D Standardization Program (VDSP) to assess commutability of Standard Reference Materials® (SRMs) and proficiency testing/external quality assessment (PT/EQA) samples for determination of serum total 25-hydroxyvitamin D [25(OH)D] using ligand binding assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A set of 50 single-donor serum samples were assigned target values for 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] using reference measurement procedures (RMPs). SRM and PT/EQA samples evaluated included SRM 972a (four levels), SRM 2973, six College of American Pathologists (CAP) Accuracy-Based Vitamin D (ABVD) samples, and nine Vitamin D External Quality Assessment Scheme (DEQAS) samples. Results were received from 28 different laboratories using 20 ligand binding assays and 14 LC-MS/MS methods. Using the test assay results for total serum 25(OH)D (i.e., the sum of 25(OH)D2 and 25(OH)D3) determined for the single-donor samples and the RMP target values, the linear regression and 95% prediction intervals (PIs) were calculated. Using a subset of 42 samples that had concentrations of 25(OH)D2 below 30 nmol/L, one or more of the SRM and PT/EQA samples with high concentrations of 25(OH)D2 were deemed non-commutable using 5 of 11 unique ligand binding assays. SRM 972a (level 4), which has high exogenous concentration of 3-epi-25(OH)D3, was deemed non-commutable for 50% of the LC-MS/MS assays.

Assessment of an ultra-sensitive IFNγ immunoassay prototype for latent tuberculosis diagnosis.

Worldwide there are about 1.7 billion individuals with latent tuberculosis infection (LTBI) and only 5% to 15% will develop active tuberculosis (TB). It is recommended to treat only those most at risk of developing active TB to avoid problems of drug resistance. LTBI diagnosis involves reviewing the individual's medical history, physical examination, and biological tests. Interferon gamma release assays (IGRA) can yield "undeterminate" or "uncertain" results, which makes clinical management decisions difficult. We assessed an ultra-sensitive immunoassay prototype based on single molecule array (SiMoA) technology to evaluate its overall performance, and in particular, its performance for indeterminate and uncertain positive or negative samples, as classified by the results from the current ELISA technique used for IFNγ quantification. We analyzed samples from hospitalized or consulting patients and healthcare workers from three hospitals in Paris, previously classified as negative (n = 30), positive (n = 35), uncertain negative (n = 25), uncertain positive (n = 31), or indeterminate (n = 30). We observed that with the SiMoA assay 83.3% of the indeterminate samples became interpretable and could be classified as negative, whereas 74% of uncertain positive samples were classified as positive. Most uncertain negative samples (72%) were reclassified as uncertain positive (68%) or positive (4%). The results suggest that the ultra-sensitive SiMoA IFNγ assay could represent a useful tool for the identification of true positive and negative samples among those giving indeterminate or uncertain results with the TB IGRA assay currently used.