The VDSP is a collaborative venture that was organized in

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1 PERSPECTIVE JBMR Standardizing Vitamin D Assays: The Way Forward Neil Binkley 1 and Christopher T Sempos 2 for the Vitamin D Standardization Program (VDSP) 1 Osteoporosis Clinical Research Program and Institute on Aging, University of Wisconsin Madison, Madison, WI, USA 2 Office of Dietary Supplements (ODS), National Institutes of Health, Bethesda, MD, USA ABSTRACT For a number of years it has been widely assumed that measurement of serum 25 hydroxyvitamin D [25(OH)D] concentration is the best approach to assessing an individual s vitamin D status. (1,2) However, it has also been recognized that there is substantial within assay variation in 25(OH)D measurement and even greater between assay variability. (3 5) Such assay variation clearly confounds attempts to define what constitutes the diagnosis of hypovitaminosis D. Importantly, assay variability makes pooling of 25(OH)D results from different studies in systematic reviews for the specific purpose of determining dose response and/or clinical cut points at best problematic. Therefore, to develop and implement evidence based clinical guidelines, it is essential that 25(OH)D measurement be standardized in both clinical and research laboratories. In this Perspective we outline a way forward toward achieving this goal the Vitamin D Standardization Program (VDSP) American Society for Bone and Mineral Research KEY WORDS: VITAMIN D; STANDARDIZATION; VITAMIN D STANDARDIZATION PROGRAM; VDSP; 25 HYDROXYVITAMIN D The VDSP The VDSP is a collaborative venture that was organized in 2010 by the Office of Dietary Supplements (ODS) of the National Institutes of Health (NIH). (6) This collaboration involves the coordinated efforts of ODS, the National Institute for Standards and Technology (NIST), the Centers for Disease Control and Prevention (CDC), the Vitamin D External Quality Assessment Scheme (DEQAS), the College of American Pathologists (CAP), the American Association for Clinical Chemistry (AACC), the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), along with national surveys and collaborators around the world (Supporting Information). The VDSP goal is to promote standardized laboratory measurement of total 25(OH)D in order to improve decision making to inform clinical and public health practice worldwide. We describe the VDSP components, discuss how they can promote collection of standardized data now and in the future, and emphasize the role that individual researchers and clinicians can play to help standardize 25(OH)D measurement. The VDSP is a work in progress; we outline a research agenda currently in place to steadily improve standardization of vitamin D status assessment as the field moves forward. Finally, we make suggestions that we believe will further promote this effort. Initially, it is necessary to clarify what we mean by total 25(OH) D; this measurement consists of the summation of 25(OH)D 3 plus 25(OH)D 2. It must be appreciated that this definition assumes that vitamin D 3 (cholecalciferol) and vitamin D 2 (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 2.5 nmol/l. Next it is important to define standardization. A standardized laboratory measurement is one that is accurate and comparable over time, location, and laboratory procedure. (7,8) In the vitamin D context, a standardized laboratory measurement of total 25(OH)D is one that is accurate and comparable over time, location, and laboratory procedure to the values obtained using reference measurement procedures (RMPs) developed at the National Institute of Standards and Technology (NIST) (9) and Ghent University. (10) Reference measurement procedures are laboratory methods that have been judged to conform with the guidelines developed by the International Organization for Standardization (ISO) and are listed in the database of the Joint Committee for Traceability in Laboratory Medicine. (11) They are the gold standard laboratory procedures for measuring 25(OH) D; ie, the concentration values obtained using the NIST or the Ghent RMP are considered to be the true concentrations. Therefore, standardization will lead to all laboratories reporting the true concentration of total 25(OH)D as measured by the NIST and Ghent University RMPs. Received in original form January 31, 2014; revised form March 25, 2014; accepted April 2, Accepted manuscript online April 16, Address correspondence to: Christopher T Sempos, PhD, NIH Office of Dietary Supplements, 6100 Executive Blvd., Rm. 3B01, Bethesda, MD , USA. E mail: semposch@mail.nih.gov Additional Supporting Information may be found in the online version of this article. Journal of Bone and Mineral Research, Vol. 29, No. 8, August 2014, pp DOI: /jbmr American Society for Bone and Mineral Research 1709

2 VDSP Objectives The primary VDSP goal is to promote standardization of all 25 (OH)D laboratory procedures worldwide both commercial and laboratory developed in order to improve clinical and public health. Such standardization does not require a single analytic approach and, as such, the VDSP does not mandate or suggest a single analytic approach. The VDSP looks forward to collaborating with all assay manufacturers/developers, and clinical and research laboratories to work toward this goal. Given the VDSP goal, four main objectives have been developed for improving 25(OH)D measurement worldwide: 1. Promote standardized total 25(OH)D measurement by commercial assay manufacturers and clinical and research laboratories. 2. Standardize total 25(OH)D measurement in national health and nutrition surveys. 3. Conduct an international research program devoted to improving the laboratory measurement of total 25(OH)D. 4. Document and study differences in standardized total 25 (OH)D concentrations among national surveys. It is apparent from these objectives that a prominent focus is on national health and nutrition surveys around the world. These government conducted surveys collect data useful in assessing current status of, and monitoring trends in, population health over time. (12) Such data are used in the development of regulatory and health policy, conducting epidemiological research to facilitate determination of optimal vitamin D status, and in developing laboratory clinical reference ranges. As a result, all stakeholders, including commercial assay manufacturers and clinical/research laboratories, strive to produce results that are consistent with the laboratory methods used in the national surveys. In this way national surveys can help to catalyze the standardization effort and move the vitamin D field forward. How Can Standardization Be Achieved? There are four basic steps necessary to achieve standardization (Fig. 1). First, it is essential to develop a reference measurement system. (13) The VDSP reference measurement system is a set of components that can be used to establish a calibration chain, ie, traceability chain, from the true value as determined by a reference measurement procedure to the routine clinical or research laboratory. (14) This multicomponent system includes: a definition of what is to be measured, in this case serum total 25 (OH)D in nmol/l or ng/ml; development of reference measurement procedures (RMP); reference materials, eg, the NIST Standard Reference Materials (SRM); guidelines or limits for assessing assay performance; a standardization certification program; accuracybased performance testing (PT) or external quality assessment schemes (EQA); and, in the case of the VDSP, procedures for standardizing research results measured in the past (Table 1). Second, the reference measurement system, once developed, can be used to link commercial assay systems to the reference methods. 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 standardizationcertification program. Third, using SRMs along with participation in accuracy based PT or EQA schemes provided by CAP and DEQAS, individual assays are compared back to the reference methods (Fig. 2). Develop Reference Measurement System Calibrate Commercial Assay Systems to Reference Methods Calibrate Individual Clinical and Research Laboratory Assays to Reference Methods Verify End-User Test Performance Reference Methods, Reference Materials, Certification Program, Accuracy-based PT/EQA NIST Standard Reference Materials (SRM), CDC Standardization Certification Program, Single Donor Serum Panel NIST SRMs, CAP, DEQAS & NIST-NIH VitDQAP PT/EQA Consistency Across Assays Fig. 1. VDSP Steps to Standardization. The primary steps to standardization are as follows: (1) develop a reference system; (2) establish metrological traceability; and (3) verify end user test performance. (1) Reference system: the key components of a reference system are the development of reference measurement procedures, and the preparation of suitable materials that can be used to establish traceability. These can include assigning target values to single donor serum samples prepared using guidelines to assure commutability; development of standard reference materials (trademark of NIST and certified reference materials; and the development of accuracy based EQA/PT programs. (2) The materials can then be used to establish traceability between laboratories not using an RMP and those who are. (3) Verifying End User test performance is then essential to develop consistency across assays. This step can include a certification process as in the CDC s Standardization Certification Program or in a somewhat less expensive and less rigorous way, EAQ/PT (ie, DEQAS and CAP EQA/PT surveys) can be used to assess End User performance and consistency across surveys. Given the cost of participating in a program like the CDC s, which requires yearly certification participation in DEQAS and/or CAP is an essential step in the standardization effort. VDSP ¼ Vitamin D Standardization Program; NIST ¼ National Institute for Standards and Technology; EQA/PT ¼ external quality assessment performance testing; RMP ¼ reference measurement procedure; CDC ¼ Centers for Disease Control and Prevention; DEQAS ¼ Vitamin D External Quality Assessment Scheme; CAP ¼ College of American Pathologists. What this system accomplishes is establishing an unbroken chain of traceability between the RMP and the assays used in research and clinical laboratories to determine total 25(OH)D concentrations. (15) Traceability ensures that clinical and research laboratory 25(OH)D results are equivalent to the result that would be obtained using the RMP. (16) Routine assays are thereby calibrated or standardized to the RMP and they are measuring and reporting the true concentration of total 25(OH)D. Fourth, is the essential step of verifying end user performance to assure consistency across different assay types. Enduser performance can be established by obtaining certification through the CDC s Vitamin D Standardization Certification Program. (6,8) However, because of expense, the CDC program is primarily recommended for commercial assay manufacturers and large commercial/clinical laboratories. Other clinical and research laboratories can establish end use performance by participating in accuracy based PT such as that provided by CAP and DEQAS in the section Standardization Steps to Take Now Journal of Bone and Mineral Research

3 Table 1. VDSP Reference Measurement System Components NIST and Ghent University JCTLM approved Reference Measurement Procedures NIST SRMs. VDSP Assay Performance Limits CDC s Vitamin D Standardization Certification Program Accuracy Based Proficiency Testing/Quality Assessment Programs CAP ABVD DEQAS Accuracy Based Quality Assurance Programs: NIST NIH VitDQAP Study Designs for Standardizing Completed Studies VDSP ¼ Vitamin D Standardization Program; NIST ¼ National Institute for Standards and Technology; JCTLM ¼ Joint Committee for Traceability in Laboratory Medicine; SRM ¼ standard reference materials; CDC ¼ Centers for Disease Control and Prevention; CAP ¼ College of American Pathologists; ABVD ¼ Accuracy based Vitamin D Program; DEQAS ¼ Vitamin D External Quality Assessment Scheme; NIH ¼ National Institutes for Health; VitDQAP ¼ Vitamin D Metabolites Quality Assurance Program. Assessing end user performance requires the setting of quantitative performance criteria. The VDSP is advocating performance limits for both reference and routine laboratories estimated by Stöckl and colleagues (17) (Table 2). For routine laboratories these current performance limits are CV 10% and Bias 5%. VDSP Progress in Developing a Reference Measurement System Efforts to develop a reference measurement system began with a collaborative arrangement between ODS and NIST coordinated by the late Dr. Mary Frances Picciano and resulted in NIST developing a reference measurement procedure, (9) SRMs 2972 Table 2. VDSP Assay Performance Limits Based on Biological Variation (17) Measurements CV (%) Bias (%) Reference laboratories 5% 1.7% Routine laboratories 10% 5% VDSP ¼ Vitamin D Standardization Program; CV ¼ coefficient of variation. and 972, (18) and eventually SRM 972a to replenish stocks once SRM 972 was used up. Subsequently, another reference measurement procedure was developed by Ghent University. (10) Both reference methods use isotope dilution liquid chromatography with tandem mass spectrometry. In 2010, ODS collaborated with the CDC in setting up the CDC s Vitamin D Standardization Certification Program. In 2012, ODS provided support for NIST to assign values for total 25(OH)D to DEQAS materials, which commenced in April 2013, at which time DEQAS was converted into an accuracy based PT program. An essential task in developing a reference measurement system is establishing that the SRMs, and the materials used in accuracy based PT/EQA programs are commutable. (19) 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, ie, that they are commutable. (14 16,19) Importantly, in 2013, the VDSP completed a commutability study from which preliminary data analyses suggest that SRM 972a, as well as CAP and DEQAS materials are commutable. (20) True Value of 25(OH)D Established Using Reference Measurement Procedure (RMP) & Reference Materials (RM) Calibration Ca libration & Trueness Controls Accuracy-Based PT/EQA CDC Standardization- Certification Program NIST SRMs CAP, DEQAS & NIST-NIH VitDQAP Assay Manufacturer Clinical/Research Laboratory Fig. 2. VDSP Reference Measurement System and Traceability Scheme. This figure is useful because it emphasizes the central role of immunoassay manufacturers in the effort to standardize the measurement of vitamin D. All assay manufacturers should participate in the CDC s Standardization Certification Program. This is especially important for the manufacturers in house reference method and for assay measurement systems as they are being developed. This figure is also useful because it emphasizes the central role of accuracy based PT program; ie, ones using commutable materials. Individual serum donor samples, and especially SRMs or other certified materials can and especially SRMs and CRMs be used in clinical and research laboratories. VDSP ¼ Vitamin D Standardization Program; CDC ¼ Centers for Disease Control and Prevention; PT ¼ performance testing; SRM ¼ Standard Reference Materials; CRM ¼ Certified Reference Materials. Journal of Bone and Mineral Research 1711

4 Role of Assay Manufacturers in Standardization 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 (Fig. 2). All assay manufacturers are encouraged to work with the VDSP to standardize their assays; many are currently doing so. Specifically, a number of commercial assay manufacturers are using the NIST SRMs and are participating in the CDC s Standardization Certification Program in order to establish or certify that their assays both those currently available and those in development are standardized. However, those who are not participating should do so. It is anticipated that in the future the CDC will post on their website those assays which have been certified. In addition, many large commercial clinical laboratories also use NIST SRMs and participate in the CDC s program. Because certification lasts for only 1 year, the maintenance of certification requires continuous participation. Role of Clinical and Research Laboratories in Standardization The CDC program, as mentioned above, is expensive and it is not a practical solution for routine clinical and research laboratories. For such laboratories, accuracy based PT or EQA schemes are available. For example, PT programs offered by the College of American Pathologists (CAP) and the Vitamin D External Quality Assessment Scheme (DEQAS) exist, and should be used to standardize small clinical and research laboratories. Additionally, periodic use of the NIST SRMs can help verify assay performance. In the future, the VDSP will be publishing a detailed statement on how the performance criteria should be used. An additional resource for research and clinical laboratories is the NIST NIH Vitamin D Metabolites Quality Assurance Program (VitDQAP). (21) This program is conducted twice a year and it is free to all who wish to use it. It was designed so that NIST chemists could 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. (More information is given about the program at nist.gov/mml/csd/vitdqap.cfm.) If you are interested in joining the NIST NIH Vitamin D Quality Assurance Program please send an e mail to vitdqap@nist.gov. Progress in Standardizing National Health and Nutrition Surveys The VDSP is actively engaged in standardizing total 25(OH)D concentrations from national surveys in Australia, Canada, Germany, Ireland, Mexico, Korea, the United Kingdom, and the United States. (6) As this work progresses it will be possible to evaluate similarities and differences in the distribution of total 25 (OH)D concentrations among those nations. To this end, statistical study design algorithms were developed for standardizing measurements from previously completed surveys to the NIST and Ghent RMPs using stored sera in order to evaluate trends over time. (22) What Is the Future of Vitamin D Standardization? In the future, as the science of vitamin D advances, there may be changes to what vitamin D metabolites are considered biologically important and what constitutes the assessment of vitamin D status. Possible vitamin D metabolite candidates might include the 3 epimer of 25(OH)D 3, 24,25(OH) 2 D 3, cholecalciferol and ergocalciferol, free/bioavailable 25(OH)D (the assessment of which necessitates measurement of vitamin D binding protein (VDBP), or something not presently being considered. (23 26) As the science evolves, by necessity, the VDSP will need to evolve as well. For example, given concerns regarding VDBP measurement, an RMP is essential to accurate assessment of free/bioavailable 25(OH)D. (27,28) Similarly, it is plausible/likely that there may come a need to develop reference methods for free/bioavailable 25 (OH)D. (29) Currently, the VDSP, led by ODS and NIST, is engaged in an extensive research program to improve not only the laboratory measurement of total 25(OH)D but also other key vitamin D metabolites. NIST is leading the effort to conduct an expanded commutability study, which is scheduled to begin in Additionally, in response to requests, NIST in collaboration with ODS is developing a higher 25(OH)D concentration SRM and a new, faster RMP to meet an ever increasing demand to prepare materials with the true values assigned to them. This new reference method will include concentrations for not only for 25 (OH)D 3, and 25(OH)D 2, but also for the first time, concentrations for key vitamin D metabolites including 24,25(OH) 2 D and the 3 epimers of both 25(OH)D 3 and 25(OH)D 2. At NIST, there is currently an effort to develop an RMP for vitamin D binding protein. NIST and ODS are also evaluating the feasibility of developing an RMP and materials for blood spots and developing reference methods and SRMs for measuring 25 (OH)D in food. Moreover, the VDSP is sponsoring research to evaluate the biological importance of both 3 epimers of 25(OH) D. Finally, the VDSP is engaging in a detailed evaluation of the current performance guidelines; ie, CV 10% and Bias 5%. The objective is to evaluate their impact on clinical decision making and, depending on the results, it may be necessary to develop more stringent performance guidelines. Standardization Steps to Take Now Standardizing and certifying existing commercial and in house assays will not happen overnight, nor will every laboratory be able to meet the VDSP performance limits tomorrow. This effort will take time. However, steps can be taken now to move toward achieving this goal. Every commercial assay manufacturer and large clinical laboratory should participate in the CDC Standardization Certification Program. All commercial assay manufacturers and large commercial laboratories should enroll their existing assay platforms, their platforms in development, and their in house reference methods in this outstanding program. Users of commercial assays and/or commercial clinical laboratories should contact their representatives and find out if those assays and commercial laboratories are participating in the CDC program, and if not, why not. As a routine clinical or research laboratory, the next question to ask yourself is what is your laboratory s CV and Bias? Do you meet the 10% and 5% goals, respectively? If not, take steps to improve your laboratory s performance. Are you participating in 1712 Journal of Bone and Mineral Research

5 the CAP and DEQAS PT/EQA programs? If not, join one or both today. Are you using NIST SRM 972a as trueness controls and, in the case of in house methods, SRM 2972 to calibrate your assay? If not, please do so regularly or at a pace that is affordable. Moreover, we support establishing guidelines for conducting and reporting research. We suggest that Journal Editors consider requiring authors of research papers reporting total 25(OH)D to provide documentation of the performance of their assay at the time of the study and rejecting manuscripts using 25(OH)D methodology not meeting or exceeding VDSP performance guidelines. In addition, we suggest that members of granting agencies carefully consider what steps they can take to promote the collection of standardized vitamin D measurements in funded research. In this regard, should only one class of assay mass spectrometry assays be used in vitamin D research, as was recently suggested? (30) The choice of an assay is a complicated one often driven by cost and availability. Mass spectrometry assays, when calibrated and performed correctly, can be very accurate and precise and may give the researcher enhanced flexibility to simultaneously measure multiple metabolites of vitamin D in addition to total 25(OH)D to gain additional insights into vitamin D physiology. (31) Moreover, such assays engender less concern about commutability. Those advantages led to the recommendation by others that liquid chromatography with tandem mass spectrometry (LC MS/MS) assays be used in national surveys. (32) However, it must be recognized that mass spectrometry assays require a costly investment and experienced laboratorians to operate them; if not set up and run properly they can be both inaccurate and imprecise. Does this mean that research using non mass spectrometry methods such as immunoassays cannot make meaningful contributions to advance our knowledge of vitamin D biology? No; it is the policy of the VDSP that non mass spectrometry methods can be used if the assays meet the suggested performance guidelines (Table 2). Conclusion Substantial progress is being made to standardize total 25(OH)D measurement. Many commercial assay manufacturers and large commercial laboratories are participating in the CDC program. Many clinical and research laboratories participate in the accuracy based PT/EQA programs that CAP and DEQAS sponsor. Data from CAP and DEQAS programs can provide baseline information on the performance of participating assays and chart progress over time in standardizing total 25(OH)D measurement. However, much more work needs to be done. We all realize that large assay variation remains a critical barrier to standardizing clinical and research assay measurements and to developing clinical and public health guidelines based on standardized research results. Progress is being made. Please, join us in this effort. Disclosures All authors state that they have no conflicts of interest. Acknowledgments This work was supported in part by the NIH National Center on Minority Health and Health Disparities (NCMHD) Comprehensive Centers of Excellence (1P60/R01 MD ). The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the NIH, or the U.S. Department of Health and Human Services. Authors roles: Study design: NB and CS. Study conduct: NB and CS. Data collection: NB and CS. Data analysis: NB and CS. Data interpretation: NB and CS. Drafting manuscript: NB and CS. Revising manuscript content: NB and CS. Approving final version of manuscript: NB and CS. NB and CS take responsibility for the integrity of the data. References 1. IOM (Institute of Medicine). Dietary reference intakes for calcium and vitamin D. Washington, DC: The National Academies Press; Holick MF, Binkley NC, Bischoff Ferrari HA, et al. 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