Vitamin D assays and the definition of hypovitaminosis D: results from the First International Conference on Controversies in Vitamin D

Abstract

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)D₃ and 25(OH)D₂ ] 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 D₃ , 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.

Keywords: 25-hydroxyvitamin D; Fibroblast Growth Factor (FGF23); Parathyroid Hormone (PTH); Vitamin D; Vitamin D Standardization Program (VDSP); Vitamin D-binding protein (DBP).

Figure 1

Vitamin D Standardization Program (VDSP): standardization phases. ABVD, accuracy‐based vitamin D survey; CAP, College of American Pathologists; CDC, Centers for Disease Control and Prevention; DEQAS, Vitamin D External Quality Assessment Scheme; NIST, National Institute for Standards and Technology; PT/EQA, performance testing/external quality assessment; RMP, reference method procedure; SRM, standard reference material; VDSCP, Vitamin D Standardization‐Certification Program

Figure 2

Vitamin D Standardization Program (VDSP): calibration traceability scheme. The VDSP calibration traceability scheme illustrates how the reference measurement system's tools are used to affect assay standardization (adapted from Myers 13). The goal of the complex set of calibration steps is to assure an unbroken chain between the reference method procedures, or National Institute for Standards and Technology standard reference material 2972 ethanolic calibration solutions and the routine laboratory. It emphasizes the central role that commercial assay manufacturers play in the standardization process and illustrates how accuracy‐based performance testing/external quality assessment (PT/EQA) are the only way to verify end‐user performance in routine laboratories

Figure 3

Vitamin D Standardization Program (VDSP): steps to the standardization of an individual laboratories assay to measure serum total 25‐hydroxyvitamin D. There are five steps to standardize an individual laboratory's serum total 25‐hydroxyvitamin D [25(OH)D] measurement (described in greater detail in the text and in Sempos et al. 48). Step 1, initial calibration, entails setting up the assay and calibrating it using the manufacturer's instructions. Step 2, an initial assessment of accuracy, is used to judge if the assay is performing correctly. This can be performed using NIST SRMs 972a and 2973, participating in the CDC's Vitamin D Standardization Certification Program – approaches potentially too costly for routine laboratories. A lower‐cost option in steps 2–4 is to use CAP ABVD and/or DEQAS serum samples, which have RMP‐assigned target values. Step 3 determines if the assay CV and mean bias meet the VDSP performance criteria – i.e. total CV ≤10% and mean bias ≤5%. In addition, a low‐cost method to estimate total CV and mean bias is to measure, for example, five CAP ABVD or DEQAS serum samples in duplicate on 2 days. If the assay does not meet those criteria, laboratory chemists should contact their commercial assay representative. If it is a laboratory‐developed assay, it may be necessary to start the process over. When patient/study participant samples are measured for 25(OH)D, it is recommended, in step 4, that trueness controls are used to assess the ongoing accuracy and precision. For commercial assay users, it is recommended that trueness controls be mixed in with patient/study samples. At the end of the laboratory analysis, these results should be used to develop a regression equation [i.e. RMP target values of the trueness controls (y) and routine laboratory assay results (x)]. If the laboratory does not meet VDSP performance criteria in step 3, then the regression equation may be needed to calibrate the results to RMP results [i.e. the best estimate of the true 25(OH)D concentration). All clinical and research laboratories should participate in an accuracy‐based performance testing programme (e.g. CAP ABVD and/or DEQAS) 61. ABVD, accuracy‐based vitamin D survey; CAP, College of American Pathologists; CDC, Centers for Disease Control and Prevention; CV, coefficient of variation; DEQAS, Vitamin D External Quality Assessment Scheme; NIST, National Institute for Standards and Technology; PT/EQA, performance testing/external quality assessment; RMP, reference method procedure; SRM, standard reference material; VDSCP, Vitamin D Standardization‐Certification Program