Table of Contents
The National Institutes of Health (NIH) Office of Dietary Supplements (ODS) established the Vitamin D Standardization Program (VDSP) in November 2010. Part of the ODS Vitamin D Initiative, VDSP is an international collaborative effort to standardize the laboratory measurement of vitamin D status. A standardized laboratory measurement is one that is accurate and comparable over time, location, and laboratory procedure.
- VDSP Goal
To improve the detection, evaluation, and treatment of vitamin D deficiency and insufficiency by promoting the standardized laboratory measurement of serum total 25-hydroxyvitamin D [25(OH)D]. VDSP promotes the standardized laboratory measurement of total 25(OH)D (a measure of vitamin D status) to improve clinical and public health practice worldwide.
- VDSP Objectives
- Standardize vitamin D measurement in national health surveys worldwide.
- Promote standardized 25(OH)D measurement by assay manufacturers and clinical and research laboratories.
- Conduct an international research program devoted to 25(OH)D and its laboratory measurement.
- VDSP Participants
All VDSP programs are open to assay manufacturers, clinical and commercial laboratories, national survey laboratories, and research laboratories. Current VDSP collaborators include:
- U.S. National Institutes of Health, Office of Dietary Supplements (ODS)
- U.S. National Institute of Standards and Technology (NIST)
- U.S. Centers for Disease Control and Prevention (CDC)
- Laboratory for Analytical Chemistry, Ghent University, Belgium
- Vitamin D External Quality Assessment Scheme, U.K. (DEQAS)
- College of American Pathologists (CAP)
- American Association of Clinical Chemists (AACC)
- International Federation of Clinical Chemistry (IFCC)
- The National Health Surveys of Australia, Canada, Germany, Ireland, Mexico, South Korea, United Kingdom, United States
Serum total 25-hydroxyvitamin D [25(OH)D] concentration is used to assess an individual’s vitamin D status. Research has consistently shown that there is a great deal of variation in 25(OH)D assays. This assay variation impedes pooling of 25(OH)D results from different studies in systematic reviews for the specific purpose of determining dose-response and/or clinical cutpoints. The Vitamin D Standardization Program (VDSP) was established to correct this problem.
- Definition of Serum Total 25(OH)D
Total 25(OH)D is the sum of the concentrations of 25(OH)D3 plus 25(OH)D2. It must be appreciated that this definition assumes that vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol) are of equal biological value, an assumption that requires further study. Total 25(OH)D concentration is commonly reported in the United States in units of nanograms per milliliter (ng/mL) and elsewhere in units of nanomoles per liter (nmol/L); ng/mL can be converted approximately into nmol/L using the formula: ng/ml x 2.5 ~ nmol/L.
Standardized Laboratory Measurement of Total 25-hydroxyvitamin D [25(OH)D]
A standardized laboratory measurement of 25(OH)D is one that is accurate and comparable to the NIST and Ghent Reference Measurement Procedures (RMPs) over time, location, and laboratory procedure. The effects of standardization are:
- Harmonization of laboratories and methods,
- Laboratories report "true" value – based on National Institute of Standards and Technology (NIST) and Ghent RMPs*
- Standardization leads to more informed decision making by physicians, policy makers and others.
* Tai SS-C, Bedner M, Phinney KW.Anal Chem 2010; 82:1942-8; Stepman HCM, Vanderroost A, Van Uytfanghe K, et al. Clin Chem 2011;57:441-8.
There are four basic steps necessary to achieve standardization.
Step 1: Develop Reference Measurement System (RMS)
Components: Reference Methods, Reference Materials,
Standardization Certification Program, Accuracy-based PT/EQA, and
Methods for Standardizing Old Measurements
Step 1: Develop an RMS. The VDSP RMS is a set of components that can be used to establish a calibration chain, i.e., traceability chain, from the true value as determined by an RMP to the routine clinical or research laboratory. This multi-component system includes:
- Definition of what is to be measured, i.e., serum total 25(OH)D in nmol/L or ng/mL;
- Development of RMPs and reference materials, e.g., the NIST Standard Reference Materials® (SRM);
- Development of a standardization-certification program;
- Development of accuracy-based performance-testing (PT) or external quality assessment (EQA) schemes, and, in the case of the VDSP,
- Development of procedures for standardizing research results measured in the past.
Step 2: Calibrate Commercial Assay Systems to Reference Methods
Tools: NIST Standard Reference Materials® (SRMs),
CDC Standardization Certification Program,
Single-Donor Serum Panels Standard
Step 2: Use the RMS to standardize commercial assay systems to the RMP. This can be accomplished using reference materials as well as single donor serum samples with concentrations assigned to them using the RMP, as part of a standardization-certification program.
Step 3: Calibrate Individual Clinical and Research Laboratory Assays to Reference Methods
Tools: NIST SRM®, CAP PT/EQA, DEQAS PT/EQA, and NIST-NIH VitDQAP PT/EQA
Step 3: Standardization of individual laboratories requires using multiple components of the VDSP RMS. NIST SRMs® are useful as trueness controls (SRM 972a) and calibrators (SRM 2972). Standardizing assays of individual laboratories can be achieved by two different pathways. The first and most rigorous way is by participation in the Centers for Disease Control and Prevention’s (CDC’s) Vitamin D Standardization Certification Program. That program is especially useful for commercial assay manufacturers and large clinical and commercial laboratories. As most vitamin D assays are commercially manufactured, this is the key link in the chain to standardizing 25(OH)D measurement by individual laboratories.
For routine clinical and research laboratories a second pathway is participation in accuracy-based PT or EQA schemes. Two such programs are provided by the College of American Pathologists’ (CAP) Accuracy-Based Vitamin D (ABVD) program and the Vitamin D External Quality Assessment Scheme (DEQAS). DEQAS materials have the true concentrations of 25(OH)D assigned to them by NIST using their RMP. CAP materials have true values assigned to them by the CDC laboratory, which is traceable to the NIST RMP. These first three steps of the VDSP RMS establish an unbroken chain of traceability between routine laboratory and the RMP. Routine assays are thereby calibrated or standardized to the RMP. As a result, they are measuring and reporting the true concentration of serum total 25(OH)D.
Step 4: Verify "End-User" Test Performance to Assure Consistency Across Assays
Tools: CDC Standardization Certification Program, CAP PT/EQA, DEQAS PT/EQA
Step 4: Verify "end-user" performance to assure consistency across different assay types:
- Commercial assay manufacturers and large commercial/clinical laboratories can verify end-user performance by obtaining certification through CDC’s Vitamin D Standardization Certification Program.
- Other clinical and research laboratories can establish end-use performance by participating in accuracy-based PT or EQA schemes provided by CAP and DEQAS.
Assessing "end-user" performance requires the setting of quantitative performance criteria*. The VDSP is advocating performance limits for both reference and routine laboratories. For routine laboratories these current performance limits are CV = 10% and bias ≤ 5%.
* Stöckl D, Sluss PM, Thienpont LM. Clin Chem Acta 2009;408:8-13.
VDSP Reference Measurement System (RMS) Specifics
VDSP developed an RMS to establish the world-wide standardization of 25-hydroxyvitamin D [25(OH)D] measurements.
- National Institute of Standards and Technology (NIST) and Ghent Reference Measurement Procedures (RMPs).
RMPs are the "gold-standard" laboratory procedures for measuring 25(OH)D; that is, the concentration values obtained using the NIST or the Ghent RMPs are considered to be the true concentrations.
- NIST Standard Reference Materials® (SRMs).
SRMs can be used as "trueness" controls, e.g., SRM 972a, and calibrators, e.g., SRM 2972.
- Centers for Disease Control and Prevention (CDC) Vitamin D Standardization Certification Program.
Most clinical and research laboratories measure total 25(OH)D using commercially developed assays. Laboratories using commercial assays must perform them rigorously as recommended by the manufacturer, but otherwise have very little control over how those assays function in comparison to RMPs. As such, commercially developed assays play a central role in the entire standardization effort. Moreover, as certification lasts for only one year, the maintenance of certification requires ongoing participation.
- Accuracy-Based Quality Assurance Programs.
The CDC program is not a practical solution for routine clinical and research laboratories. For such laboratories, accuracy-based performance testing or external quality assessment (EQA) schemes are available, i.e., College of American Pathologists Accuracy-Based Vitamin D (ABVD) program and Vitamin D External Quality Assessment Scheme (DEQAS). An additional resource for research and clinical laboratories is the NIST-NIH Vitamin D Metabolites Quality Assurance Program (VitDQAP). This program is conducted twice a year and it is free to all who wish to use it. NIST chemists provide technical support to help laboratories discover and correct problems with their assay. It is not a performance-testing program so there is no pass/fail assessment.
- Study designs for standardizing completed studies
Statistical procedures for standardizing 25(OH)D measurements from completed studies have been developed by the VDSP.* The procedures include methods for calculating sample size and for selecting samples to be re-measured. In the near future we will include links to manuals with instructions for using the VDSP statistical procedures.
* Cashman KD, Kiely M, Kinsella M, et al. Am J Clin Nutr 2013;97:1235-42.
An essential task in developing a reference measurement system is establishing that the Standardized Reference Materials® (SRMs), and the materials used in accuracy-based performance testing/external quality assessment programs are commutable. Commutability is defined as the equivalence of mathematical relationships between results obtained using different measurement procedures for representative samples. Demonstration of commutability is necessary in that materials used in PT/EQA programs are prepared from pooled serum samples. As a result, it is essential to document that such samples function in different assays just the way a sample from an individual patient would, i.e., that they are commutable.
Importantly, in 2013 the VDSP completed a commutability study from which preliminary data analyses suggest that SRM 972a, as well as College of American Pathologists (CAP) and Vitamin D External Quality Assessment Scheme (DEQAS) materials, are commutable. We are currently working to publish those results and to develop a new and larger study to test the commutability of SRM 972a and CAP and DEQAS materials in a wide range of currently used assay platforms.
A public meeting to showcase the VDSP’s progress was held on the National Institute of Standards and Technology campus on November 14, 2013.
Resources and References
Binkley N, Krueger D, Cowgill CS, et al. Assay variation confounds the diagnosis of hypovitaminosis D: A call for standardization. J Clin Endocrin Metab 2004;89:3152-3157.
Bunk DM. Reference materials and reference measurement procedures: an overview from a national metrology institute. Clin Biochem Rev 2007;28:131-137.
Carter GD. Accuracy of 25-hydroxyvitamin D assays: confronting the issues. Current Drug Targets 2011;12:19-28.
Cashman KD, Kiely M, Kinsella M, et al. Evaluation of Vitamin D Standardization Program protocols for standardizing serum 25-hydroxyvitamin D data: a case study of the program’s potential for National Nutrition/Health Surveys. Am J Clin Nutr 2013;97:1235-1242.
Miller WG, Jones GRD, Horowitz GL, et al. Proficiency testing/external quality assessment: current challenges and future directions. Clin Chem 2011;57:1670-1680.
Miller WG, Myers GL, Gantzer ML et al. Roadmap for harmonization of clinical laboratory methods. Clin Chem 2011;57:1108-1117.
Phinney KW, Bedner M, Tai S S-C, et al. Development and certification of a standard reference material for vitamin D metabolites in human serum. Anal Chem 2012;84:956-962.
Sempos CT, Vesper HW, Phinney KW, et al. Vitamin D status as an international issue: national surveys and the problem of standardization. Scand J Clin Lab Inves 2012;72(Suppl 243): 32-40.
Stepman HCM, Vanderroost A, Van Uytfanghe K, et al. Candidate reference measurement procedures for serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 by using isotope-dilution liquid chromatography-tandem mass spectrometry. Clin Chem 2011;57:441-448.
Stöckl D, Sluss PM, Thienpont LM. Specifications for trueness and precision of a reference measurement system for serum/plasma 25-hydroxyvitamin D analysis. Clin Chem Acta 2009;408:8-13.
Tai SS-C, Bedner M, Phinney KW. Development of a candidate reference measurement procedure for the determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry. Anal Chem 2010;82:1942-1948.
Thienpont LM, Stepman HCM, Vesper HW. Standardization of measurements of 25-Hydroxyvitamin D3 and D2. Scand J Clin Lab Inves 2012;72(Suppl 243):41-49.
Key Collaborating Organizations
National Institutes of Health, U.S.A.
Office of Dietary Supplements
Christopher T. Sempos, Ph.D.
Program Coordinator, Vitamin D Standardization Program
Web site: ods.od.nih.gov/vdsp
National Institute of Standards and Technology, U.S.A.
Stephen A. Wise, Ph.D.
Associate Chief, Chemical Sciences Division
Web site: www.nist.gov
Centers for Disease Control and Prevention, U.S.A.
Vitamin D Standardization-Certification Program
Hubert W. Vesper, Ph.D.
Director, CDC Standardization-Certification Program
Web site: www.cdc.gov/labstandards/hs.html
American Association for Clinical Chemistry
Gary Myers, Ph.D.
Vice President, Science and Practice Affairs
Web site: www.aacc.org
International Federation of Clinical Chemistry and Laboratory Medicine
Ian S. Young, M.D., Ph.D.
Professor of Medicine, Queen’s University Belfast
Web site: http://www.ifcc.org
Christopher T. Sempos, Ph.D.
Program Coordinator, Vitamin D Standardization Program
Phone: +1 301-435-2920