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Vitamin D Standardization Program (VDSP) Standardized Laboratory Measurement

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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; 82:1942-8external link disclaimer; Stepman HCM, Vanderroost A, Van Uytfanghe K, et al. Clin Chem 2011;57:441-8external link disclaimer.

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.

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

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

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

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

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 Programexternal link disclaimer.
  • Other clinical and research laboratories can establish end-use performance by participating in accuracy-based PT or EQA schemes provided by CAPexternal link disclaimer and DEQASexternal link disclaimer.

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;408:8-13external link disclaimer.