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In addition the literature contains reports that vitamin D insufficiency may play a role in the development of multiple sclerosis, rheumatoid arthritis, and asthma, and increases the risk of tuberculosis, pneumonia, poor cognitive function, periodontal disease, and reduced muscle tone and lower-extremity function. A further metaanalysis of second cancers after a diagnosis of nonmelanoma skin cancer concluded that solar ultraviolet-B (UVB) radiation reduces the risk of many internal cancers, including squamous cell carcinoma of the colon, stomach, and rectum (3).

THE IMPLICATIONS OF NEW ROLES FOR VITAMIN D FOR CLINICAL CHEMISTRY

The measurement of 25OHD in blood and other biological fluids is challenging. Early methods that relied on competitive protein binding or immunoassay to measure 250HD included a solvent extraction step to remove the sterol from vitamin D-binding protein. Manufacturers of 25OHD immunoassay methods have sought to replace this solvent-extraction step with blocking agents to facilitate the inclusion of 25OHD assays on automated platforms. Evidence in practice suggests that the success of this block-and-displace approach is limited. Clinical chemists are aware of spuriously high 25OHD results in individual patient samples and higher than expected imprecision. For example, the all-method mean CV for 20 recent specimens distributed through the Vitamin D External Quality Assessment Scheme was 18.7% (range 16.6%20.2%), and the CV for some individual immunoassay methods was greater than this all-method mean.

A second challenge to the measurement of 25OHD arises from the application of the assay to assess the adequacy of vitamin D replacement therapy. Vitamin D that is used for replacement purposes occurs in 2 major forms. Cholecalciferol (vitamin D3) is the natural animal form, and ergocalciferol (vitamin D2) is derived from plants. Increasing globalization of health and medicinal products means that both vitamin D3 and D2 are used by the public and by patients in all countries. Although vitamins D3 and D2 are structurally similar, the differences are sufficient to cause a variable response in the immunoassays used to measure the hydroxylated form of these preparations. Some immunoassays are claimed to be equipotent for the measurement of 25OHD3 and 25OHD2, whereas others preferentially detect one form. Some immunoassays in use may lead to serious clinical misclassification of individual patients.

To address the problems arising from immunoassay methods of vitamin D measurement, clinical chemists are establishing 25OHD measurement techniques based on mass spectrometry. Such methods typically involve sample pretreatment to separate the 25OHD from its binding protein followed by liquid chromatography and quantification using tandem mass spectrometry. Although these methods allow simultaneous measurement of both 25OHD3 and 25OHD2, method harmonization is limited, and there is no reference preparation or agreed calibrant for 25OHD. Clinical chemists also must clarify whether they are reporting 25OHD3, 25OHD2 or total 25OHD.

As we seek to understand the importance of newly discovered roles for vitamin D in health and disease, the accurate and precise measurement of 25OHD in blood is essential. Better agreement among methods is needed both to allow more meaningful comparison among research studies and to facilitate agreement on appropriate minimum and optimum replacement targets for protective treatment with vitamins D3 and D2. Clinical chemists and manufacturers have a responsibility to agree on quality standards and performance targets for the measurement of 250HD.