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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

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.

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.

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.

Mean serum total cholesterol levels appear to be higher in the Federal Republic of Germany (FRG) than in the United States (US) while coronary heart disease death rates are lower. The study examined possible factors for the difference including possible differences in laboratory methodology. Cross-sectional data from the first two waves of the German National Health Surveys (1984-1986 and 1987-1989; n = 9709) and from the Second National Health and Nutrition Examination Survey (1976-1980; n = 7832) were compared for men and women 25-69 years of age. The influence of age, body mass index, diet, cigarette smoking, education, income, use of oral contraceptives or antihypertensive agents, alcohol consumption and potential differences in laboratory measurement were explored using multiple regression techniques separately for men and women for ages 25-39, 40-59 and 60-69 years of age. Overall ages, unadjusted mean total cholesterol levels were higher in German than US men (6.02 vs. 5.64 mmol/l) and in German than US women (6.04 vs. 5.80 mmol/l) as were HDL cholesterol levels (men: 1.30 vs 1.14 mmol/l; women: 1.65 vs. 1.38 mmol/l). Adjusting for lifestyle factors explained, on the average, 40% of the differences in mean total cholesterol of which half or 20% was accounted for by adjusting for alcohol intake. Adjusting for possible laboratory differences explained, on the average, an additional 30% of the differences. Frequency of alcohol intake was the most important factor in explaining differences in mean HDL cholesterol levels. Adjustment for differences in alcohol intake had negligible effects on reducing the differences in mean non-HDL cholesterol.

Background Few data have been published on the validity of classification of overweight and obesity based on self-reported weight in representative samples of Hispanic as compared to other American populations despite the wide use of such data. Objective To test the null hypothesis that ethnicity is unrelated to bias of mean body mass index (BMI) and to sensitivity of overweight or obesity (BMI >= 25 kg/m2) derived from self-reported (SR) versus measured weight and height using measured BMI as the gold standard. Design Cross-sectional survey of a large national sample, the Third National Health and Nutrition Examination Survey (NHANES III) conducted in 1988–1994. Participants American men and women aged 20 years and over (n = 15,025). Measurements SR height, weight, cigarette smoking, health status, and socio-demographic variables from home interview and measured weight and height. Results In women and Mexican American (MA) men SR BMI underestimated true prevalence rates of overweight or obesity. For other men, no consistent difference was seen. Sensitivity of SR was similar in non-Hispanic European Americans (EA) and non-Hispanic African Americans (AA) but much lower in MA. Prevalence of obesity (BMI >= 30 kg/m2) is consistently underestimated by self-report, the gap being greater for MA than for other women, but similar for MA and other men. The mean difference between self-reported and measured BMI was greater in MA (men -0.37, women -0.76 kg/m2) than in non-Hispanic EA (men -0.22, women -0.62 kg/m2). In a regression model with the difference between self-reported and measured BMI as the dependent variable, MA ethnicity was a significant (p < 0.01) predictor of the difference in men and in women. The effect of MA ethnicity could not be explained by socio-demographic variables, smoking or health status. Conclusion Under-estimation of the prevalence of overweight or obesity based on height and weight self-reported at interview varied significantly among ethnic groups independent of other variables.