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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

The majority of circulating 25-hydroxyvitamin D (25(OH)D) is protein bound and perhaps less available than the free fraction of 25(OH)D; therefore, researchers have proposed that the measurement of free 25(OH)D in human serum may be a better indicator of vitamin D health status than total 25(OH)D. The availability of a new enzyme-linked immunosorbent assay (ELISA) for the determination of free 25(OH)D provides a method for direct measurement of the low levels of non-protein bound 25(OH)D. As an initial step towards harmonization of measurements of free 25(OH)D, the ELISA was used to measure free 25(OH)D in three existing Standard Reference Materials (SRMs): SRM 972a Vitamin D Metabolites in Frozen Human Serum, SRM 2973 Vitamin D Metabolites in Frozen Human Serum (High Level), and SRM 1949 Frozen Prenatal Human Serum. Target values for free 25(OH)D in the nine SRM serum pools, obtained by combining the results from two laboratories, ranged from 3.76 ± 0.36 to 10.0 ± 0.58 pg/mL. Of particular significance is the assignment of free 25(OH)D target values to SRM 1949, which consists of four serum pools from non-pregnant female donors of reproductive age and pregnant women in each of the three trimesters and which also has values assigned for vitamin D binding protein, which increases during pregnancy. The availability of target values for free 25(OH)D in these SRMs will allow researchers to validate new analytical methods and to compare their results with other researchers as an initial step towards harmonization of measurements among different studies and laboratories.

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays used for the determination of serum total 25-hydroxyvitamin D (25(OH)D), which is the sum of 25-hydroxyvitamin D2 (25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3). A set of 50 single-donor samples was assigned target values for concentrations of 25(OH)D2, 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 1 includes results from 14 laboratories using 14 custom LC-MS/MS 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 53% of the LC-MS/MS assays met the VDSP criterion of mean % bias ≤ |±5%|. For the LC-MS/MS assays not meeting the ≤ |±5%| criterion, four assays had mean % bias of between 12 and 21%. Based on multivariable regression analysis using the concentrations of the four individual vitamin D metabolites in the 50 single-donor samples, the performance of several LC-MS/MS assays was found to be influenced by the presence of 3-epi-25(OH)D3. The results of this interlaboratory study represent the most comprehensive comparison of LC-MS/MS assay performance for serum total 25(OH)D and document the significant impact of the lack of separation of 3-epi-25(OH)D3 and 25(OH)D3 on assay performance, particularly with regard to mean % bias.

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, bioMérieux, 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.

BACKGROUND Nutritional rickets is believed to result from the interaction of inadequate serum 25-hydroxyvitamin D [25(OH)D] concentration and dietary calcium intake, but this interaction has not been confirmed in children with rickets. Determining the vitamin D requirements to prevent nutritional rickets has been thwarted by inconsistent case definition, inadequate adjustment for calcium intake and other confounders, and 25(OH)D assay variability. OBJECTIVES To model the 25(OH)D concentration associated with nutritional rickets in calcium-deprived Nigerian children, adjusted for confounding factors, and develop a general approach to define vitamin D status while accounting for calcium intake. METHODS Logistic regression was used to model the association of serum 25(OH)D with having rickets adjusted for calcium intake in a reanalysis of a case-control study in Nigerian children. The matching variables age, sex, weight-for-age z score, and 4 additional significant variables were selected [religion, age began walking, phosphorus intake, and the 25(OH)D × calcium intake interaction] using a rigorous 7-step algorithm. RESULTS Cases had significantly (P

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 D(2) [25(OH)D(2)] and 25-hydroxyvitamin D(3) [25(OH)D(3)] 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)D(2) and 25(OH)D(3)] 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)D(2) below 30 nmol/L, one or more of the SRM and PT/EQA samples with high concentrations of 25(OH)D(2) 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)D(3) was deemed non-commutable for 50% of the LC-MS/MS assays.

Context Nutritional rickets results from the interaction of low vitamin D status and limited calcium intake. Serum alkaline phosphatase (AP) activity is a biomarker of impaired mineralization in rickets. Objective To assess the performance of serum AP activity in identifying nutritional rickets in calcium-deprived Nigerian children. Methods We reanalyzed data from a case-control study of children with active rickets and matched control subjects without rickets, using a multivariate logistic regression to assess the odds of rickets associated with AP activity, adjusting for age, sex, and weight-for-age z-score. Results A total of 122 children with rickets and 119 controls were included. Rachitic children had a mean (±SD) age of 54 ± 29 months, and 55 (45.1%) were male. Cases and controls had low dietary calcium intakes (216 ± 87 and 214 ± 96 mg/day, respectively). Serum AP activity levels in cases and controls were 812 ± 415 and 245 ± 78 U/L, respectively (P 350 U/L identified nutritional rickets in Nigerian children with sensitivity 0.93, specificity 0.92, positive likelihood ratio 11.3, and negative likelihood ratio 0.07. Conclusion An AP > 350 U/L effectively discriminated between Nigerian children with and without nutritional rickets. AP is a low-cost biochemical test that could be used to screen for nutritional rickets, but cutoff values require validation in other populations, and laboratory values need to be standardized for widespread population studies.

Testing representative populations to determine the prevalence or the percentage of the population with active severe acute respiratory syndrome coronavirus 2 infection and/or antibodies to infection is being recommended as essential for making public policy decisions to ease restrictions or to continue enforcing national, state, and local government rules to shelter in place. However, all laboratory tests are imperfect and have estimates of sensitivity and specificity less than 100%—in some cases, considerably less than 100%. That error will lead to biased prevalence estimates. If the true prevalence is low, possibly in the range of 1%–5%, then testing error will lead to a constant background of bias that most likely will be larger, and possibly much larger, than the true prevalence itself. As a result, what is needed is a method for adjusting prevalence estimates for testing error. Methods are outlined in this article for adjusting prevalence estimates for testing error both prospectively in studies being planned and retrospectively in studies that have been conducted. If used, these methods also would help harmonize study results within countries and worldwide. Adjustment can lead to more accurate prevalence estimates and to better policy decisions. However, adjustment will not improve the accuracy of an individual test.

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.

Context Supplementation with vitamin D has the potential to both reduce and increase risk of falling, and parathyroid hormone (PTH) may contribute to fall risk. Objective To assess the associations of intra-trial mean circulating levels of 25-hydroxyvitamin D [25(OH)D] and PTH on incident falls in healthy older adults. Design Observational within a clinical trial. Setting The Bone Metabolism Laboratory at the USDA Nutrition Center at Tufts University. Participants 410 men and women age ≥65 years who participated in the 3-year Boston STOP IT trial to determine the effect of supplementation with 700 IU of vitamin D3 plus calcium on incident falls (secondary endpoint). Intra-trial exposures of 25(OH)D and PTH were calculated as the mean of biannual measures up to and including the first fall. Main outcome measures Incidence of first fall. Results Intra-trial mean 25(OH)D was significantly associated with risk of falling in a U-shaped pattern; the range associated with minimal risk of falling was approximately 20 to 40 ng/mL. PTH was not significantly associated with risk of falling. Conclusions The findings highlight the importance of maintaining the circulating 25(OH)D level between 20 and 40 ng/mL, the range that is also recommended for bone health. At PTH levels within the normal range, there was no detectible independent association of PTH with fall risk.

It has been more than 80 years since the discovery of vitamin D and its ability to cure rickets in children. Vitamin D is a secosteroid and comes in two distinct forms, vitamin D2 and vitamin D3. During the last 40 years, the synthesis and metabolism of vitamin D were elucidated and more than 50 metabolites of vitamin D have been discovered, though commercial measurement procedures have been developed for only a few of them. The clinical significance of vitamin D in calcium and phosphorus homeostasis is well appreciated. However, recent epidemiological data have indicated that it has several extra-skeletal physiologic actions which are still a matter of scientific debate. Both research findings and the debate around the interpretation of the research results have created increased interest in more measurements of vitamin D. With the ever growing family of measurable vitamin D metabolites and the measuring techniques comes a question: What metabolic product will provide the right answers and which is the best way to measure it. The right choice of analytical technique is connected with the question of which metabolite we aim to measure, what is its serum concentration, and the purpose of the measurement. The aim of the first part of this review is to provide a brief overview of vitamin D metabolism and a more detailed analysis of the existing methods and the status of standardization for the measurement of 25-hydroxyvitamin D.

Background/aim:Cost-efficient methods are essential for successful participant recruitment in clinical trials. Patient portal messages are an emerging means of recruiting potentially eligible patients into trials. We assessed the response rate and complaint rate from direct-to-patient, targeted recruitment through patient portals of an electronic medical record for a clinical trial, and compared response rates by differences in message content.Methods:The Study to Understand Fall Reduction and Vitamin D in You (STURDY) trial is a National Institutes of Health–sponsored, community-based study of vitamin D supplementation for fall prevention in older adults conducted at Johns Hopkins. Potential participants were identified using the Epic electronic medical record at the Johns Hopkins Health System based on age (≥70 years), ZIP code (30-mile radius of study site), and prior activation of a patient portal account. We prepared a shorter message and a longer message. Both had basic information about study participation, but the longer message also contained information about the significance of the study and a personal invitation from the STURDY principal investigator. The Hopkins Institutional Review Board did not require prior consent from the patient or their providers. We calculated the response rate and tracked the number of complaints and requests for removal from future messages. We also determined response rate according to message content.Results:Of the 5.5 million individuals receiving care at the Johns Hopkins Health System, a sample of 6896 met our inclusion criteria and were sent one patient portal recruitment message between 6 April 2017 and 3 August 2017. Assessment of enrollment by this method ended on 1 December 2017. There were 116 patients who expressed interest in the study (response rate: 1.7%). Twelve (0.2%) recipients were randomized. There were two complaints (0.03%) and one request to unsubscribe from future recruitment messages (0.01%). Response rate was higher with the longer message than the shorter message (2.1% vs 1.2%; p = 0.005).Conclusion:Patient portal messages inviting seniors to participate in a randomized controlled trial resulted in a response rate similar to commercial email marketing and resulted in very few complaints or opt-out requests. Furthermore, a longer message with more content enhanced response rate. Recruitment through patient portals might be an effective strategy to enroll trial participants.

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays used for the determination of serum total 25-hydroxyvitamin D (25(OH)D), which is the sum of 25-hydroxyvitamin D

An intralaboratory study assessing assay variability and bias for determination of serum total 25-hydroxyvitamin D [25(OH)D] was conducted by the Vitamin D Standardization Program (VDSP). Thirteen assays for serum total 25(OH)D were evaluated in a single laboratory including 11 unique immunoassays and one liquid chromatography – tandem mass spectrometry (LC-MS/MS) assay. Fifty single-donor serum samples, including eight samples with high concentrations of 25(OH)D(2) (> 30 nmol/L), were assigned target values for 25(OH)D(2) and 25(OH)D(3) using reference measurement procedures (RMP). Using four replicate measurements for each sample, the mean total percent coefficient of variation (%CV) and mean % bias from the target values were determined for each assay using the 50 single-donor samples and a 42-sample subset, which excluded 8 high 25(OH)D(2) concentration samples, and compared with VDSP performance criteria of ≤ 10% CV and ≤ ±5% mean bias. All 12 assays achieved the performance criterion for %CV, and 9 of the 12 assays were within ≤ ±5 % mean bias. The Fujirebio Inc. assay exhibited the lowest %CV and highest percentage of individual measurements within ≤ ±5% mean bias. Ten immunoassays exhibited changes in response due to the high 25(OH)D(2) samples with Abbott, Biomérieux, DiaSorin, DIAsource, and IDS-iSYS assays having the largest deviations. The Fujirebio Inc. and Beckman Coulter assays were only minimally affected by the presence of the high 25(OH)D(2) samples. Samples with high concentrations of 25(OH)D(2) provided a critical performance test for immunoassays indicating that some assays may not have equal response or recovery for 25(OH)D(2) and 25(OH)D(3).

The Third International Conference on Controversies in Vitamin D was held in Gubbio, Italy, September 10-13, 2019. The conference was held as a follow-up to previous meetings held in 2017 and 2018 to address topics of controversy in vitamin D research. The specific topics were selected by the steering committee of the conference and based upon areas that remain controversial from the preceding conferences. Other topics were selected anew that reflect specific topics that have surfaced since the last international conference. Consensus was achieved after formal presentations and open discussions among experts. As will be detailed in this article, consensus was achieved with regard to the following: the importance and prevalence of nutritional rickets, amounts of vitamin D that are typically generated by sun exposure, worldwide prevalence of vitamin D deficiency, the importance of circulating concentrations of 25OHD as the best index of vitamin D stores, definitions and thresholds of vitamin D deficiency, and efficacy of vitamin D analogues in the treatment of psoriasis. Areas of uncertainly and controversy include the following: daily doses of vitamin D needed to maintain a normal level of 25OHD in the general population, recommendations for supplementation in patients with metabolic bone diseases, cutaneous production of vitamin D by UVB exposure, hepatic regulation of 25OHD metabolites, definition of vitamin D excess, vitamin D deficiency in acute illness, vitamin D requirements during reproduction, potential for a broad spectrum of cellular and organ activities under the influence of the vitamin D receptor, and potential links between vitamin D and major human diseases. With specific regard to the latter area, the proceedings of the conference led to recommendations for areas in need of further investigation through appropriately designed intervention trials. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research. ispartof: JBMR PLUS vol:4 issue:12 ispartof: location:England status: published

Background: Evidence from randomized controlled trials (RCTs) for the causal role of vitamin D on noncommunicable disease outcomes is inconclusive.Objective: The aim of this study was to investigate whether there are beneficial or harmful effects of cholecalciferol (vitamin D3) supplementation according to subgroups of remeasured serum 25-hydroxyvitamin D [25(OH)D] on cardiovascular and glucometabolic surrogate markers with the use of individual participant data (IPD) meta-analysis of RCTs.Design: Twelve RCTs (16 wk to 1 y of follow-up) were included. For standardization, 25(OH)D concentrations for all participants (n = 2994) at baseline and postintervention were re-measured in bio-banked serum samples with the use of a certified liquid chromatography-tandem mass spectrometry method traceable to a reference measurement procedure. IPD meta-analyses were performed according to subgroups of remeasured 25(OH)D. Main outcomes were blood pressure and glycated hemoglobin (HbA1c). Secondary outcomes were LDL, HDL, and total cholesterol and triglycerides; parathyroid hormone (PTH); fasting glucose, insulin, and C-peptide; and 2-h glucose. In secondary analyses, other potential effect modifiers were studied.Results: Remeasurement of 25(OH)D resulted in a lower mean 25(OH)D concentration in 10 of 12 RCTs. Vitamin D supplementation had no effect on the main outcomes of blood pressure and HbA1c. Supplementation resulted in 10-20% lower PTH concentrations, irrespective of the 25(OH)D subgroups. The subgroup analyses according to achieved 25(OH)D concentrations showed a significant decrease in LDL-cholesterol concentrations after vitamin D supplementation in 25(OH)D subgroups with Conclusions: For the main outcomes of blood pressure and HbA1c, the data support no benefit for vitamin D supplementation. For the secondary outcomes, in addition to its effect on PTH, we observed indications for a beneficial effect of vitamin D supplementation only on LDL cholesterol, which warrants further investigation. This trial was registered at www.clinicaltrials.gov as NCT02551835.

The First International Conference on Controversies in Vitamin D was held in Pisa, Italy, 14–16 June 2017. The meeting's purpose was to address controversies in vitamin D research, review the data available, to help resolve them, and suggest a research agenda to clarify areas of uncertainty. The serum 25‐hydroxyvitamin D [25(OH)D] concentration [i.e. the sum of 25(OH)D(3) and 25(OH)D(2)] remains the critical measurement for defining vitamin D status. Assay variation for 25(OH)D has contributed to the current chaos surrounding efforts to define hypovitaminosis D. An essential requirement to develop a consensus on vitamin D status is that measurement of 25(OH)D and, in the future, other potential vitamin D biomarkers [e.g. 1α,25(OH)(2)D(3), 3‐epi‐25(OH)D, 24,25(OH)(2)D(3,) vitamin D‐binding protein, free/bioavailable 25(OH)D and parathyroid hormone] be standardized/harmonized, to allow pooling of research data. Vitamin D Standardization Program tools are described and recommended for standardizing 25(OH)D measurement in research. In the future, similar methodology, based on National Institute for Standards and Technology standard reference materials, must be developed for other candidate markers of vitamin D status. Failure to standardize/harmonize vitamin D metabolite measurements is destined to promulgate continued chaos. At this time, 25(OH)D values below 12 ng ml(–1) (30 nmol l(–1)) should be considered to be associated with an increased risk of rickets/osteomalacia, whereas 25(OH)D concentrations between 20 ng ml(–1) and 50 ng ml(–1) (50–125 nmol l(–1)) appear to be safe and sufficient in the general population for skeletal health. In an effort to bridge knowledge gaps in defining hypovitaminosis D, an international study on rickets as a multifactorial disease is proposed.

Previous studies have assessed vitamin D status based on the 25-hydroxyvitamin D [25(OH)D] concentration measured in samples from dried blood spots (DBSs). In 40 individuals participating in a clinical study, we compared 25(OH)D levels measured from DBSs and in serum using an LC-MS/MS reference procedure in collaboration with the Vitamin D Standardization Program. The main objective was to simplify and optimize current methods to produce an assay that can be used as a screening tool for 25(OH)D concentration assessment without derivatization. The DBS 25(OH)D levels, compared to serum concentrations, were found to have 101% accuracy overall, and the correlation coefficient (r) was 0.83 (P < 0.0001), with a significant linear relationship. Free 25(OH)D and vitamin D binding protein (VDBP) were assessed in the serum samples for potential correlations to the DBS calculations: the levels of free 25(OH)D had moderate to strong correlation to DBS and serum concentrations, with r values of 0.67 (P < 0.0001) and 0.76 (P < 0.0001), respectively. VDBP and hematocrit had no significant correlation to either DBS or serum sample types, with r values

Assay variability has been cited as an obstacle to establishing optimal vitamin D exposure. As part of the Vitamin D Standardization Program (VDSP) effort to standardize the measurement of total 25-hydroxyvitamin D [25(OH)D], the value assignment of total 25(OH)D in 50 single-donor serum samples was performed using two isotope-dilution LC with tandem MS methods. Both methods are recognized as reference measurement procedures (RMPs) by the Joint Committee for Traceability in Laboratory Medicine. These samples and their assigned values serve as the foundation for several aspects of the VDSP. To our knowledge, this is the first time that two RMPs have been used to assign 25(OH)D values to such a large number of serum samples.

Six laboratories associated with the Vitamin D Standardization Program (VDSP) participated in an interlaboratory comparison of LC with tandem MS (MS/MS) methods for the determination of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] in human serum. The laboratories analyzed two different serum-based Standard Reference Materials (SRMs) intended for use in the determination of 25-hydroxyvitamin D and 30 samples from the Vitamin D External Quality Assessment Scheme (DEQAS). All laboratorymethods for 24,25(OH)2D3 were based on isotope dilution LC-MS/MS; three of the methods used derivatization of the vitamin D metabolites before LC-MS/MS. Laboratory results were compared to the National Institute of Standards and Technology (NIST) results, which were obtained using their newly developed candidate reference measurement procedure for 24,25(OH)2D3. Laboratory results for the SRM samples varied in comparability to the NIST results, with one laboratory in excellent agreement (−1.6% mean bias), three laboratories at 10–15% mean bias, and the remaining laboratory at 36% mean bias. For the 30 DEQAS samples, the mean bias for the five laboratories ranged from 6 to 15%; however, the SD of the bias ranged from 8 to 29%. As a result of this intercomparison study, one laboratory discovered and corrected a methodcalculation error and another laboratory modified and improved their LC-MS/MS method.

The National Institute of Standards and Technology (NIST) has developed Standard Reference Material (SRM) 972a Vitamin D Metabolites in Frozen Human Serum as a replacement for SRM 972, which is no longer available. SRM 972a was developed in collaboration with the National Institutes of Health’s Office of Dietary Supplements. In contrast to the previous reference material, three of the four levels of SRM 972a are comprised of unmodified human serum. This SRM has certified and reference values for the following 25-hydroxyvitamin D [25(OH)D] species: 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3. The value assignment and certification process included three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). The value assignment methods employed have been modified from those utilized for the previous SRM, and all three approaches now incorporate chromatographic resolution of the stereoisomers, 25(OH)D3 and 3-epi-25(OH)D3.

The 2nd International Conference on Controversies in Vitamin D was held in Monteriggioni (Siena), Italy, September 11-14, 2018. The aim of this meeting was to address ongoing controversies and timely topics in vitamin D research, to review available data related to these topics and controversies, to promote discussion to help resolve lingering issues and ultimately to suggest a research agenda to clarify areas of uncertainty. Several issues from the first conference, held in 2017, were revisited, such as assays used to determine serum 25-hydroxyvitamin D [25(OH)D] concentration, which remains a critical and controversial issue for defining vitamin D status. Definitions of vitamin D nutritional status (i.e. sufficiency, insufficiency and deficiency) were also revisited. New areas were reviewed, including vitamin D threshold values and how they should be defined in the context of specific diseases, sources of vitamin D and risk factors associated with vitamin D deficiency. Non-skeletal aspects related to vitamin D were also discussed, including the reproductive system, neurology, chronic kidney disease and falls. The therapeutic role of vitamin D and findings from recent clinical trials were also addressed. The topics were considered by 3 focus groups and divided into three main areas: 1) "Laboratory": assays and threshold values to define vitamin D status; 2) "Clinical": sources of vitamin D and risk factors and role of vitamin D in non-skeletal disease and 3) "Therapeutics": controversial issues on observational studies and recent randomized controlled trials. In this report, we present a summary of our findings. ispartof: Reviews In Endocrine & Metabolic Disorders vol:21 issue:1 pages:89-116 ispartof: location:Germany status: published

Today, the possibility exists to measure a number of different vitamin D metabolites with accurate and precise methods. The most abundant vitamin D metabolite, 25(OH)D, is considered the best marker for estimating vitamin D status and is therefore the most commonly measured in clinical practice. There is no consensus on the added value of measuring other metabolites beyond 25-hydroxyvitamin D, although, in some special clinical scenarios and complicated cases, these metabolites may provide just the information needed for an accurate diagnosis. The problem this review addresses is which metabolite to measure and when and how to measure it.

Although the association of vitamin D levels with cardiovascular risk profiles among Hispanics/Latinos has been studied, little is known about this association among Hispanics/Latinos with chronic conditions. This pilot study determined serum vitamin D and parathyroid hormone (PTH) levels in a sample of participants from the University of Illinois at the Chicago Cohort of Patients, Family and Friends (UIC Cohort) and examined their association with traditional cardiovascular disease risk factors. From July 2012 to June 2016, the UIC Cohort study enrolled and conducted clinical examinations on men and women ages 18 years and older, who had one or more diagnosed chronic diseases/conditions (excluding cancer). This pilot study sample included 40 participants from the six main Hispanic/Latino background groups in the United States, namely Dominican, Cuban, Puerto Rican, Mexican, Central American, and South American, and were grouped by Caribbean or mainland origin. No substantial differences were noted in the vitamin D-related measures by Hispanic/Latino background, but the PTH levels were somewhat higher in the Caribbean vs. mainland group (43.0 &plusmn, 4.6 vs. 38.6 &plusmn, 2.7 pg/mL). The associations between selected CVD risk factors (systolic and diastolic blood pressure (SBP, DBP), total cholesterol, glucose) and PTH and vitamin D-related analytes were investigated using interval-censored multivariate regression models adjusted for age, sex, percent body fat, serum albumin/calcium, and Hispanic/Latino background. A negative association between total 25[OH]D and blood pressure was corroborated (SBP: &beta, = &minus, 1.2, 95%CI = &minus, 2.0, &minus, 0.3, DBP: &beta, 0.7, 95% CI = &minus, 1.2, &minus, 0.1), whereas a positive association with total cholesterol was observed (&beta, = 1.9, 95% CI = 0.02, 3.7). Levels of 1, 25[OH]2D were not associated with CVD risk factors, whereas 24, 25[OH]2D3 was associated with blood pressure (SBP: &beta, 13.0, 95% CI = &minus, 20.7, &minus, 5.2, 6.3, 95% CI = &minus, 11.6, &minus, 1.0). Estimated free 25[OH]D was inversely associated with both SBP (&beta, 3.5, 95% CI = &minus, 6.1, &minus, 0.9) and DBP (&beta, 2.1, 95% CI = &minus, 3.8, &minus, 0.3). Similarly, calculated bioavailable 25[OH]D was inversely associated with both SBP (&beta, 9.2, 95% CI = &minus, 15.9, &minus, 2.4) and DBP(&beta, 5.3, 95% CI = &minus, 9.8, &minus, 0.8). In conclusion, a negative association between 25[OH]D with BP was observed and a positive association with lipids is suggested. Due to the small sample size, most associations did not reach statistical significance.

The vitamin D External Quality Assessment Scheme (DEQAS) for 25-hydroxyvitamin D (25-OHD) has approximately 1100 participants in 53 countries using 26 different methods or variants of methods (October 2014). In April 2015, the scheme was extended to cover 24,25-dihydroxyvitamin D (24,25(OH) 2 D). Since 2013, the 25-OHD scheme has been accuracy-based with values assigned by the NIST reference measurement procedure (RMP). DEQAS is uniquely placed to assess the accuracy (bias) and specificity of 25-OHD methods in a routine laboratory setting. Other vitamin D metabolites are known to interfere in 25-OHD assays and DEQAS has distributed samples spiked with 3- epi -25-OHD 3 (52.4 nmol/L), 24R,25(OH) 2 D 3 (14.4 nmol/L) and 24S,25(OH) 2 D 3 (57.9 nmol/L). The 3-epimer showed a cross reactivity of 56% in a competitive protein binding assay but was not detected in any antibody-based methods. Not all HPLC/UV or LC–MS/MS methods were able to resolve 3- epi -25-OHD 3 from 25-OHD 3 and thus overestimated total 25-OHD. The cross reactivity of 24R,25(OH) 2 D 3 (24S,25(OH) 2 D 3 ) ranged from

Unstandardized laboratory measurement of 25-hydroxyvitamin D (25(OH)D) confounds efforts to develop clinical and public health vitamin D guidelines. The Vitamin D Standardization Program (VDSP), an international collaborative effort, was founded in 2010 to correct this problem. Nearly all published vitamin D research is based on unstandardized laboratory 25(OH)D measurements. While it is impossible to standardize all old data, it may be possible to identify a small subset of prior studies critical to guidelines development. Once identified it may be possible to calibrate their 25(OH)D values to the NIST and Ghent University reference measurement procedures using VDSP methods thereby permitting future guidelines to be based on standardized results. We simulated the calibration of a small set of ten clinical trials of vitamin D supplementation on achieved 25(OH)D under minimal sun exposure. These studies were selected because they played a prominent role in setting the 2010 vitamin D dietary reference intakes (DRI). Using random-effects meta-regression analysis, Vitamin D External Quality Assessment (DEQAS) data on assay bias was used to simulate the potential bias due to the lack of assay standardization by calibrating the achieved 25(OH)D levels from those 10 studies to: (1) the largest negative, and (2) the largest positive bias from the DEQAS all laboratory trimmed mean (ALTM) for the appropriate assay and year of analysis. For a usual vitamin D intake of 600IU/day the difference in mean achieved 25(OH)D values for those two options was 20nmol/L. However, without re-calibration of 25(OH)D values it is impossible to know the degree to which any of the current guidelines may have been biased. This approach may help stimulate the search for and standardization of that small subset of key studies and, in the cases where standardization is impossible, to identify areas of urgently needed vitamin D research.

BACKGROUND Temporal trends in the US population's vitamin D status have been uncertain because of nonstandardized serum 25-hydroxyvitamin D [25(OH)D] measurements. OBJECTIVE To accurately assess vitamin D status trends among those aged ≥12 y, we used data from the cross-sectional NHANESs. DESIGN A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 25(OH)D (sum of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3), calibrated to standard reference materials, was used to predict LC-MS/MS-equivalent concentrations from radioimmunoassay data (1988-2006 surveys; n = 38,700) and to measure LC-MS/MS concentrations (2007-2010 surveys; n = 12,446). Weighted arithmetic means and the prevalence of 25(OH)D above or below cutoff concentrations were calculated to evaluate long-term trends. RESULTS Overall, mean predicted 25(OH)D showed no time trend from 1988 to 2006, but during 2007-2010 the mean measured 25(OH)D was 5-6 nmol/L higher. Those groups who showed the largest 25(OH)D increases (7-11 nmol/L) were older, female, non-Hispanic white, and vitamin D supplement users. During 1988-2010, the proportions of persons with 25(OH)D

BACKGROUND The 2007-2010 NHANES provides the first US nationally representative serum 25-hydroxyvitamin D [25(OH)D] concentrations measured by standardized liquid chromatography-tandem mass spectrometry. OBJECTIVE We describe patterns for total 25(OH)D and individual metabolites in persons aged ≥1 y stratified by race-ethnicity and grouped by demographic, intake, physiologic, and lifestyle variables. METHODS We measured 25-hydroxycholecalciferol [25(OH)D3], 25-hydroxyergocalciferol [25(OH)D2], and C3-epimer of 25(OH)D3 [C3-epi-25(OH)D3] in serum samples (n = 15,652) from the 2007-2010 cross-sectional NHANES [total 25(OH)D = 25(OH)D3 + 25(OH)D2]. RESULTS Concentrations (median, detection rate) of 25(OH)D3 (63.6 nmol/L, 100%) and C3-epi-25(OH)D3 (3.40 nmol/L, 86%) were generally detectable; 25(OH)D2 was detectable in 19% of the population. Total 25(OH)D, 25(OH)D3, and C3-epi-25(OH)D3 displayed similar demographic patterns and were strongly correlated (Spearman's r > 0.70). Concentrations of 25(OH)D2 (90th percentile) were much higher in persons aged ≥60 y (17.3 nmol/L) than in younger age groups (≤4.88 nmol/L). We noted significant race-ethnicity differences in mean total 25(OH)D [non-Hispanic blacks (NHBs), Hispanics, and non-Hispanic whites (NHWs): 46.6, 57.2, and 75.2 nmol/L, respectively] and in the prevalence of total 25(OH)D

ContextVitamin D is classically recognized as a regulator of calcium and phosphorus metabolism. Recent advances in the measurement of vitamin D metabolites, diagnosis of vitamin D deficiency, and clinical observations have led to an appreciation that along with its role in skeletal metabolism, vitamin D may well have an important role in nonclassical settings. Measurement of the circulating form of vitamin D that best describes total body stores, namely 25-hydroxyvitamin D, can be unreliable despite many sophisticated methodologies that have been proposed and implemented. Likewise, evidence from clinical studies showing a beneficial role of vitamin D in different disease states has been controversial and at times speculative. Moreover, the target concentrations of 25-hydroxyvitamin D to address a number of putative links between vitamin D inadequacy and nonskeletal diseases are further areas of uncertainty.SettingTo address these issues, an international conference on “Controversies in Vitamin D” was held in Pisa, Italy, in June 2017. Three main topics were addressed: (i) vitamin D assays and the definition of hypovitaminosis D; (ii) skeletal and extraskeletal effects of vitamin D; (iii) therapeutics of vitamin D.ResultsThis report provides a summary of the deliberations of the expert panels of the conference.ConclusionsDespite great advances in our appreciation of vitamin D metabolism, measurements, biological actions on classical and nonclassical tissues, and therapeutics, all of which this report summarizes, much more work remains to be done so that our knowledge base can become even more secure.

Background There is controversy regarding the potential influence of vitamin D deficiency, exposure to environmental tobacco smoke, BCG vaccination, season, and body habitus on susceptibility to Mycobacterium tuberculosis (MTB) infection. Methods We conducted a cross-sectional analysis to identify determinants of a positive QuantiFERON-TB Gold (QFT) assay result in children aged 6–13 years attending 18 schools in Ulaanbaatar, Mongolia. Data relating to potential risk factors for MTB infection were collected by questionnaire, physical examination, and determination of serum 25-hydroxyvitamin D (25[OH]D) concentrations. Risk ratios (RRs) were calculated with adjustment for potential confounders, and population attributable fractions (PAFs) were calculated for modifiable risk factors identified. Results Nine hundred forty-six of 9810 (9.6%) participants had a positive QFT result. QFT positivity was independently associated with household exposure to pulmonary tuberculosis (adjusted RR [aRR], 4.75 [95% confidence interval {CI}, 4.13–5.46, P < .001]; PAF, 13.1% [95% CI, 11.1%–15.0%]), vitamin D deficiency (aRR, 1.23 [95% CI, 1.08–1.40], P = .002; PAF, 5.7% [95% CI, 1.9%–9.3%]), exposure to environmental tobacco smoke (1 indoor smoker, aRR, 1.19 [95% CI, 1.04–1.35]; ≥2 indoor smokers, aRR, 1.30 [95% CI, 1.02–1.64]; P for trend = .006; PAF, 7.2% [95% CI, 2.2%–12.0%]), and increasing age (aRR per additional year, 1.14 [95% CI, 1.10–1.19], P < .001). No statistically significant independent association was seen for presence of a BCG scar, season of sampling, or body mass index. Conclusions Vitamin D deficiency and exposure to environmental tobacco smoke are potentially modifiable risk factors for MTB infection.

Background: The Canadian Health Measures Survey (CHMS) is an ongoing cross-sectional national survey that includes a measure of 25-hydroxyvitamin D [25(OH)D] by immunoassay. For cycles 1 and 2, the collection period occurred approximately every 2 y, with a new sample of w5600 individuals. Objective: The goal was to standardize the original 25(OH)D CHMS values in cycles 1 and 2 to the internationally recognized reference measurement procedures (RMPs) developed by the US National Institute for Standards and Technology (NIST) and Ghent University, Belgium. Design: Standardization was accomplished by using a 2-step procedure. First, serum samples corresponding to the original plasma samples were remeasured by using the currently available immunoassay method. Second, 50 serum samples with known 25(OH)D values assigned by the NIST and Ghent reference method laboratories were measured by using the currently available immunoassay method. The mathematical models for each step—i.e., 1) YCurrent = XOriginal and 2) YNIST-Ghent = XCurrent —were estimated by using Deming regression, and the 2 models were solved to obtain a single equation for converting the “original” values to NIST-Ghent RMP values. Results: After standardization (cycles 1 and 2 combined), the percentage of Canadians with 25(OH)D values ,40 nmol/L increased from 16.4% (original) to 19.4% (standardized), and values ,50 nmol/L increased from 29.0% (original) to 36.8% (standardized). The 25(OH)D standardized distributions (cycles 1 and 2 analyzed separately) were similar across age and sex groups; slightly higher values were associated with cycle 2 in the young and old. This finding contrasts with the original data, which indicated that cycle 2 values were lower for all age groups. Conclusion: The shifts in 25(OH)D distribution brought about by standardization indicate its importance in drawing correct conclusions about potential population deficiencies and insufficiencies and in permitting the comparison of distributions between national surveys. Am J Clin Nutr 2015;102:1044‐50.

Background: Multivitamin-mineral (MVM) products are the most commonly used supplements in the United States, followed by multivitamin (MV) products. Two randomized clinical trials (RCTs) did not show an effect of MVMs or MVs on cardiovascular disease (CVD) mortality; however, no clinical trial data are available for women with MVM supplement use and CVD mortality. Objective: The objective of this research was to examine the association between MVM and MV use and CVD-specific mortality among US adults without CVD. Methods: Anationallyrepresentativesample of adults fromthe restricted dataNHANESIII(1988‐1994;n= 8678; age$40y) were matched with mortality data reported by the National Death Index through 2011 to examine associations between MVM and MV use and CVD mortality by using Cox proportional hazards models, adjusting for multiple potential confounders. Results: We observed no significant association between CVD mortality and users of MVMs or MVs compared with nonusers; however, when users were classified by the reported length of time products were used, a significant association was found with MVM use of >3 y compared with nonusers (HR: 0.65; 95% CI: 0.49, 0.85). This finding was largely driven by the significant association among women (HR: 0.56; 95% CI: 0.37, 0.85) but not men (HR: 0.79; 95% CI: 0.44, 1.42). No significant association was observed for MV products and CVD mortality in fully adjusted models. Conclusions: In this nationally representative data set with detailed information on supplement use and CVD mortality data ;20 y later, we found an association between MVM use of >3 y and reduced CVD mortality risk for women when models controlled for age, race, education, body mass index, alcohol, aspirin use, serum lipids, blood pressure, and blood glucose/glycated hemoglobin. Our results are consistent with the 1 available RCT in men, indicating no relation with MVM use and CVD mortality. J Nutr doi: 10.3945/jn.114.204743.

Widespread variation in assay measurement of total 25-hydroxyvitamin D (25(OH)D) confounds international efforts to develop evidence-based clinical and public health guidelines. This chapter's subject is the Vitamin D Standardization Program (VDSP), its tools, and methods for measuring 25(OH)D in both commercially and laboratory developed assays worldwide. Two distinct VDSP protocols exist for standardizing 25(OH)D measurements: one for current/future measurements, i.e., Prospective Standardization and another for Retrospective Standardization of 25(OH)D measurements from past studies; both are described in detail. Special attention is given to the role that accuracy-based Performance Testing/External Quality Assessment schemes play in standardizing measurements in routine laboratories. The steps involved in retrospective standardization and its potential impact on interpretation of vitamin D research data are discussed in detail. Given the impact of assay standardization on research data interpretation, publication of meta-analyses based on unstandardized 25(OH)D data should be suspended. As discussed, both prospective and retrospective standardization are the way forward to developing consensus vitamin D guidelines.

Background: Comparability of 25-hydroxyvitamin D (25(OH)D) measurements is hampered by method-related differences in measurement values. International standardization of laboratory assays has been suggested to solve this problem. Methods: As part of the European Commission–funded project ‘Food-based solutions for optimal vitamin D nutrition and health through the life cycle’ (ODIN), original measurements of serum 25(OH)D of three German national health surveys conducted between 1998 and 2011 have been standardized retrospectively. In these representative population-based samples including persons aged between 1 and 79 years, the original 25(OH)D values were compared with those after standardization. Mean values and prevalences of vitamin D deficiency, insufficiency, and sufficiency (25(OH)D levels =50 nmol/l, respectively) were calculated by sex and age groups based on original and standardized 25(OH)D data. Results: In comparison to the original 25(OH)D levels, the standardized levels showed higher means overall and in age- and sex-specific analyses. After standardization, the prevalence of vitamin D deficiency was lower in all surveys while the prevalence of vitamin D sufficiency was higher. Nevertheless, even after standardization ~ 15% of adults and 12.5% of children had serum 25(OH)D levels

The science surrounding vitamin D presents both challenges and opportunities. Although many uncertainties are associated with the understandings concerning vitamin D, including its physiological function, the effects of excessive intake, and its role in health, it is at the same time a major interest in the research and health communities. The approach to evaluating and interpreting the available evidence about vitamin D should be founded on the quality of the data and on the conclusions that take into account the totality of the evidence. In addition, these activities can be used to identify critical data gaps and to help structure future research. The Office of Dietary Supplements (ODS) at the National Institutes of Health has as part of its mission the goal of supporting research and dialogues for topics with uncertain data, including vitamin D. This review considers vitamin D in the context of systematically addressing the uncertainty and in identifying research needs through the filter of the work of ODS. The focus includes the role of systematic reviews, activities that encompass considerations of the totality of the evidence, and collaborative activities to clarify unknowns or to fix methodological problems, as well as a case study using the relationship between cancer and vitamin D.

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health Office of Dietary Supplements and the Vitamin D Standardization Program, has recently issued a new serum-matrix Standard Reference Material (SRM): 2973 Vitamin D Metabolites in Frozen Human Serum (High Level). SRM 2973 was designed to provide a serum material with a total 25-hydroxyvitamin D [25(OH)D] concentration near 100 nmol/L to complement the existing serum-based SRMs with values assigned for total 25(OH)D between 20 and 80 nmol/L. Values were assigned for 25-hydroxyvitamin D2 [25(OH)D2], 25-hydroxyvitamin D3 [25(OH)D3], 3-epi-25(OH)D3, and total 25(OH)D [the sum of 25(OH)D2 + 25(OH)D3] using the NIST isotope dilution LC with tandem MS (MS/MS) reference measurement procedure (RMP) and related methods. SRM 2973 has a certified value of 98.4 ± 2.1 nmol/L for 25(OH)D3 and reference values of 1.59 ± 0.05 nmol/L for 25(OH)D2 and 5.23 ± 0.20 nmol/L for 3-epi-25(OH)D3. In addition, a candidate RMP for 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] based on LC-MS/MS was used to assign values to SRM 2973 and the existing SRM 972a Vitamin D Metabolites in Frozen Human Serum. Reference values for 24R,25(OH)2D3 were assigned to SRM 2973 (7.51 ± 0.26 nmol/L) and the four levels of SRM 972a: Level 1 (6.38 ± 0.23 nmol/L), Level 2 (3.39 ± 0.12 nmol/L), Level 3 (3.88 ± 0.013 nmol/L), and Level 4 (6.32 ± 0.22 nmol/L). The development of SRM 2973 [with a higher concentration of 25(OH)D3] and the addition of values for 24R,25(OH)2D3 assigned to both SRM 972a and SRM 2973 provide laboratories involved in vitamin D measurements with improved QA tools.

Since 2005, the National Institute of Standards and Technology (NIST) has collaborated with the National Institutes of Health (NIH), Office of Dietary Supplements (ODS) to improve the quality of measurements related to human nutritional markers of vitamin Dstatus. In support of the NIH-ODS Vitamin D Initiative, including the Vitamin D Standardization Program (VDSP), NIST efforts have focused on (1) development of validated analytical methods, including reference measurement procedures (RMPs); (2) development of Standard ReferenceMaterials (SRMs); (3) value assignment of critical study samples using NIST RMPs; and (4) development and coordination of laboratory measurement QA programs. As a result of this collaboration, NIST has developed RMPs for 25-hydroxyvitamin D2 [25(OH)D2], 25(OH)D3, and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3]; disseminated serum-based SRMs with values assigned for 25(OH)D2, 25(OH)D3, 3-epi-25(OH)D3, and 24R,25(OH)2D3; assigned values for critical samples for VDSP studies, including an extensive interlaboratory comparison and reference material commutability study; provided an accuracy basis for the Vitamin D External Quality Assurance Scheme; coordinated the first accuracy-based measurement QA program for the determination of 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3 in human serum/plasma; anddeveloped methods and SRMs for the determination of vitamin D and 25(OH)D in food and supplement matrix SRMs. The details of these activities and their benefit and impact to the NIH-ODS Vitamin D Initiative are described.

Until recently, the Vitamin D External Quality Assessment Scheme (DEQAS) assessed the performance of various assays for the determination of serum total 25-hydroxyvitamin D [25(OH)D] by using a consensus mean based on the all-laboratory trimmed mean (ALTM) of the approximately 1000 participants' results. Since October 2012, the National Institute of Standardsand Technology (NIST), as part of the Vitamin D Standardization Program, has participated in DEQAS by analyzing the quarterly serum sample sets using an isotope dilution LC-tandem MS (ID LC-MS/MS) reference measurement procedure to assign an accuracy-based target value for serum total 25(OH)D. NIST has analyzed90 DEQAS samples (18 exercises × 5 samples/exercise) to assign target values. The NIST-assigned values are compared with the ALTM and the biases assessed for various assays used by the participants, e.g., LC-MS/MS, HPLC, and several ligand-binding assays. The NIST-value assignment process and the resultsof the analyses of the 90 DEQAS samples are summarized. The absolute mean bias between the NIST-assignedvalues and the ALTM was 5.6%, with 10% of the samples having biases >10%. Benefits of the accuracy-based target values are presented, including for sample sets with high concentrations of 25(OH)D2 and 3-epi-25(OH)D3.

The Vitamin D Standardization Program (VDSP) coordinated an interlaboratory study to assess the comparability of measurements of total 25-hydroxyvitamin D [25(OH)D] in human serum, which is the primary marker of vitamin D status. A set of 50 individual donor samples were analyzed by 15 different laboratoriesrepresenting national nutrition surveys, assay manufacturers, and clinical and/or research laboratories to provide results for total 25(OH)D using both immunoassays (IAs) and LC tandem MS (MS/MS). The resultswere evaluated relative to bias compared with the target values assigned based on a combination of measurements at Ghent University (Belgium) and the U.S. National Institute of Standards and Technology using reference measurement procedures for the determination of 25(OH)D2 and 25(OH)D3. CV and mean bias for each laboratory and assay platform were assessed and compared with previously established VDSP performance criteria, namely CV ≤ 10% and mean bias ≤ 5%. Nearly all LC-MS/MS results achieved VDSP criteria, whereas only 50% of IAs met the criterion for a ≤10% CV and only three of eight IAs achieved the ≤5% bias. These results establish a benchmark for the evaluation of 25(OH)D assay performance and standardization activities in the future.

The Vitamin D Standardization Program (VDSP) coordinated a study in 2012 to assess the commutability of reference materials and proficiency testing/external quality assurance materials for total 25-hydroxyvitamin D [25(OH)D] in human serum, the primary indicator of vitamin D status. A set of 50 single-donor serum samples as well as 17 reference and proficiency testing/external quality assessment materials wereanalyzed by participating laboratories that used either immunoassay or LC-MS methods for total 25(OH)D. The commutability test materials included National Institute of Standards and Technology Standard Reference Material 972a Vitamin D Metabolites in Human Serum as well as materials from the College of AmericanPathologists and the Vitamin D External Quality Assessment Scheme. Study protocols and data analysis procedures were in accordance with Clinical and Laboratory Standards Institute guidelines. The majority of the test materials were found to be commutable with the methods used in this commutability study. These results provide guidance for laboratories needing tochoose appropriate reference materials and select proficiency or external quality assessment programs and will serve as a foundation for additional VDSP studies.

The Vitamin D Standardization Program (VDSP) has collaborated with numerous groups and agencies to assemble a set of tools, i.e., a reference measurementsystem, that can be used to establish the traceability of 25-hydroxyvitamin D [25(OH)D] assays to relevant reference measurement procedures and reference materials. This is done with the goal of verifying end-user laboratory performance using precise statistical criteria to determine whether a specific assay isstandardized. The purpose of this paper was to outline a set of steps that routine clinical and researchlaboratories can use to standardize their 25(OH)D assays using these tools. These steps apply to laboratories using commercially developed immunoassay measurement systems as well as in-house assays, usually based on high HPLC or LC tandem MS measurement systems. The steps are (1) initial calibration, (2) initial assessment of accuracy and bias, (3) assessment of total percent CV and mean bias, (4)use of trueness controls, and (5) participation in accuracy-based performance testing and/or external quality assessment schemes. The goal of each laboratory assay is to have a total CV of ≤10% and mean bias of ≤5%. Rigorous and less rigorous but low-cost options for meeting thesestatistical criteria are provided. Research laboratories who infrequently measure 25(OH)D are advised torepeat steps 1–4 for every measurement cycle.For users of commercial immunoassays who have relatively little control over standardization, we presentan option for using trueness controls to develop a master equation that can be used to standardize results to the reference methods.

Six laboratories associated with the Vitamin D Standardization Program (VDSP) participated in an interlaboratory comparison of LC with tandem MS (MS/MS) methods for the determination of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] in human serum. The laboratories analyzed two different serum-based Standard Reference Materials (SRMs) intended for use in the determination of 25-hydroxyvitamin D and 30 samples from the Vitamin D External Quality Assessment Scheme (DEQAS). All laboratory methods for 24,25(OH)2D3 were based on isotope dilution LC-MS/MS; three of the methods used derivatization of the vitamin D metabolites before LC-MS/MS. Laboratory results were compared to the National Institute of Standards and Technology (NIST) results, which were obtained using their newly developed candidate reference measurement procedure for 24,25(OH)2D3. Laboratory results for the SRM samples varied in comparability to the NIST results, with one laboratory in excellent agreement (-1.6% mean bias), three laboratories at 10-15% mean bias, and the remaining laboratory at 36% mean bias. For the 30 DEQAS samples, the mean bias for the five laboratories ranged from 6 to 15%; however, the SD of the bias ranged from 8 to 29%. As a result of this intercomparison study, one laboratory discovered and corrected a method calculation error and another laboratory modified and improved their LC-MS/MS method.

Low concentrations of total 25-hydroxyvitamin D [25(OH)D], the principal biological measure of vitamin D status, have been associated with clinical and public health outcomes. The determination of levels under which there is an increase in the risk of disease, as well as comparisons across populations, have been difficult to establish due the large assay variability in measuring 25(OH)D. Accordingly, the Vitamin D Standardization Program (VDSP) includes the retrospective standardization of existing 25(OH)D values collected by epidemiological and clinical studies,as well as clinical trials, as one of its main objectives. We introduce methodology developed by the VDSP that can be used to standardize the measurement oftime-stable analytes, including 25(OH)D, in samples that have been banked and maintained appropriately. Sample size estimation formulae are first applied tocalculate the required number of banked blood samples to be reanalyzed using either of two approaches. In the first approach, existing samples are remeasured using the current measurement procedure, and an equation relating “old” to “current” measurements is obtained. A second set ofsera, usually 40–50 single-donor serum samples, are measured with the current measurement procedure and an assay traceable to a reference measurementprocedure and/or certified reference materials, which yields a second calibration equation. These two equations are combined to produce standardized levels from the original old values. This approach is necessary when study restrictions prevent serum samples from being shipped to an external laboratory and is illustrated with samples from the Canadian Health Measures Survey. When serum samples are permitted to beshared with other laboratories, or the study investigators can carry out the measurements with a traceable assay, a single calibration equation methodis used. Existing samples are selected and remeasured using the available traceable assay. We outline the statistical theory supporting the VDSP protocol and provide implementation examples. The methods proposed are generalizable to any instance in which banked specimens have been properly prepared and stored and theanalyte of interest is stable under those conditions.

Recent years have seen a substantial increase in demand for 25-hydroxyvitamin D (25-OHD) assays. DEQAS (the Vitamin D External Quality Assessment Scheme) has been monitoring the performance of these assays since 1989. The first DEQAS distribution was in June 1989 and results were submitted by 13 laboratories in the UK, two of which used HPLC/UV; the rest used ligand binding assays with a tritium tracer. Inter-laboratory CVs (ALTM) ranged from 29.3% (42.7nmol/L) to 53.7% (20.0nmol/L). Currently the scheme has participants in 56 countries using 30 methods or variants of methods. In January 2017, 918 participants returned results and inter-laboratory CVs (ALTM) ranged from 10.3% (73.1nmol/L) to 15.3% (29.4nmol/L). Over the last 27 years, there have been a number of significant milestones in assay development. The first major advance was the development of an iodinated 25-OHD tracer by Hollis and Napoli in 1992, subsequently used in an RIA kit marketed by DiaSorin. This and other commercial radioimmunoassays that followed brought 25-OHD assays within reach of many more non-specialist routine laboratories. With the introduction of fully automated non-isotopic assays without solvent extraction, measurement of 25-OHD became available to any clinical chemistry laboratory with an appropriate analytical platform. However, as the limitations of these non-extraction assays became apparent more laboratories started using LC-MS/MS methodology. Meanwhile the variable accuracy of 25-OHD methods has been addressed by the Vitamin D Standardization Program (VDSP) which encourages manufacturers to produce methods traceable to the reference measurement procedures (RMPs) of NIST, University of Ghent and the Centers for Disease Control and Prevention (CDC). DEQAS changed to an accuracy-based scheme in 2013 and now assesses assay accuracy against the NIST RMP. This review will use DEQAS results and statistics to chart the historical development in 25-OHD assay technology and highlight some of the problems encountered in obtaining reliable results for this most challenging of analytes.