Vitamin D - Laboratory Considerations

Vitamin D - Laboratory Considerations

Vitamin D has received increasing attention over the past few years. In fact, testing for Vitamin D deficiency is one of the fastest growing tests across the country, if not the fastest, with many clinical labs reporting doubling and tripling of requests year over year. Once many foods were fortified with Vitamin D and the incidence and prevalence of rickets in children plummeted, most clinicians thought the major health problems resulting from Vitamin D deficiency had been resolved. However, in recent years we have learned that Vitamin D deficiency is not uncommon. In adults it can precipitate or exacerbate osteopenia and osteoporosis, cause osteomalacia and muscle weakness, and increase the risk of fracture. The discovery that most tissues in the body have a Vitamin D receptor and a large variety of cells possess the enzymes necessary to convert the primary circulating form (25-hydroxyvitamin D) to the active form (1,25-dihydroxyvitamin D), has provided new insights into its function. Attention has turned to the role Vitamin D can play in reducing the risk of many illnesses, including common cancers, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, and cardiovascular disease.

Vitamin D is a prohormone, rather than a true vitamin, as one of its forms (Vitamin D3, cholecalciferol) is synthesized in the skin after solar UVB exposure. Aside from sunlight, the best nutritional sources of Vitamin D3 are oily fish, primarily salmon and mackerel. The other form of Vitamin D, Vitamin D2 (ergocalciferol), generally is a minor contributor to total Vitamin D and is derived only from the nutritional sources of some vegetables, yeast, and fungi. Some vegetarian diets, thus, are an exception to this usually small contribution. Both forms of Vitamin D are converted in the liver to 25-hydroxyvitamin D, which is the major circulating form. The accepted standard for clinical assessment of Vitamin D status is the measurement of total circulating 25-hydroxyvitamin D concentration.

Much of the ground breaking work on Vitamin D deficiency was based on RIA assays in which the total 25-hydroxyvitamin D was determined by the sum of 25-hydroxyvitamin D3 and D2. Our assay at John Muir Health and MuirLab is a newer version of this concept utilizing chemiluminescent immunoassay (CLIA) technology rather than radioactive isotopes, and achieves excellent accuracy and precision exhibited by within-assay variability <5% and between-assay variability <10% for total circulating 25-hydroxyvitamin D. Other newer assays, such as liquid chromatography mass spectroscopy (LC-MS), can measure the 2 forms separately. However, data from the College of American Pathologists and the UK-based DEQAS (external quality assessment surveys) have shown the LC-MS assays to have wide variability in the range of 20% between labs, thus supporting our decision to stick with our current assay. Much of the variability problem stems from the fact that there is no agreed upon reference standards, and the preparation of reagents for in-house LC-MS assays is conducted by individual labs per their own practices.

Another complexity follows as a result of the lack of agreed upon standardization, and that is the controversy on the optimum reference intervals for 25-hydroxyvitamin D to classify patients with moderate to severe deficiency. These controversies related to Vitamin D measurement are not unlike other issues in laboratory medicine that have been dealt with in the past. Such examples include the standardization of cholesterol measurement 20 years ago, the use of INRs related to prothrombin time and the anticoagulant effects of Warfarin, HbA1c traceability to the DCCT assay, and more recently creatinine to achieve standardization in eGFR calculations. Until such time as standardization is achieved, we will provide the following ranges for the classification of total 25-hydroxyvitamin D status as suggested in the literature:

Vitamin D status Total 25-hydroxyvitamin D

Deficiency < 10 ng/mL

Insufficiency 10-30 ng/mL

Optimum 30-100 ng/mL

Toxicity > 100 ng/mL

Finally, it is worth stressing the importance of the total 25-hydroxyvitamin D concentration rather than the individual D3 and D2 forms for clinical decision making. An interesting survey performed at the University of Wisconsin and the Medical University of South Carolina showed that reporting total 25(OH)D, 25(OH)D3 and 25(OH)D2 can confuse ordering clinicians, thereby leading to incorrect clinical decisions. For one of their scenarios of a hypothetical 82-year-old person with a hip fracture, and lab results of 25(OH)D3 <5 ng/mL and 25(OH)D2 = 40 ng/mL, 23% of the clinicians interpreted these results to indicate vitamin D or D3 deficiency requiring vitamin D treatment. Another 12% of clinicians interpreted the scenario of the same patient with 25(OH)D3 = 32 ng/mL and 25(OH)D2 < 5 ng/mL as being deficient in vitamin D or D2. In both scenarios, the total 25(OH)D was in the optimum range, thus neither warranted vitamin D treatment.

Should there be any questions or concerns about our total 25(OH) D assay, please do not hesitate to contact me (925.674.2508) or the supervisor of immunology at MuirLab, Cindy Liedstrand (925.692.5681). Thank you.


  1. Holick, MF. Vitamin D Deficiency. N Engl J Med 2007;357:266-81.
  2. Binkley, N et al. Laboratory Reporting of 25-Hydroxyvitamin D Results: Potential for Clinical Misinterpretation. Clin Chem 2006;52(11):2124-5.
  3. Singh, RJ. Are Clinical Laboratories Prepared for Accurate Testing of 25-Hydroxyvitamin D? Clin Chem 2008;54(1):221-3.