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Insight into Vitamin D Assays In Clinical Laboratory

Insight into Vitamin D Assays In Clinical Laboratory

 Shereen H. Atef, Consultant Clinical Pathologist, National Reference Laboratory

 16 January 2017

The status of vitamin D is usually assessed by measuring the serum concentration of 25-hydroxyvitamin D (25-OH-D). Over the recent years there has been a dramatic increase in 25-OH-D requests, prompting many laboratories to consider the use of automated immunoassays. In this article, the two major techniques that are used for measuring vitamin D will be discussed and compared (binding assay and chemical assay techniques).

Dramatic Increase in Vitamin D Testing

Vitamin D was first recognised as a very important component of the diet back in the late 1800s when rickets was initially described. Presently, rickets has been eradicated from most developed countries. However, it is still a very common problem in areas of the world where food is scarce.

The recent dramatic increase in vitamin D testing is primarily due to two causes. First, there has been a marked rise in vitamin D deficiency throughout the world. The second reason is that vitamin D has increasingly being used as general health marker and several diseases were linked to vitamin D deficiency.


What we commonly refer to as vitamin D actually comes in two different forms: vitamin D2 and vitamin D3. Vitamin D2  is also known as ergocalciferol, calciferol, or just vitamin D. Vitamin D3 is also known as cholecalciferol (it derives from cholesterol). There are two main ways via which vitamin D gets into the body: through the skin and through diet. In the intestine, either previtamin D or vitamin D is absorbed and trapped in the chylomicron molecules. In the skin, under the effect of UV rays of sunlight, 7-dehydrocholesterol is converted to cholecalciferol (Vitamin D3). Vitamin D from the two sources is subjected to hydroxylation in the liver to form 25-OH-D.The hydroxylated vitamin is then alpha hydroxylated in the kidney to form 1,25(OH)2 vitamin D, the active form of vitamin D. 1,25 (OH)2 vitamin D increases the absorption of calcium and phosphorous in the intestine. It also interacts with the parathyroid gland as feedback in the production of parathyroid hormone, therefore acting as a regulator of new bone formation. Vitamin D is also being recognised as a very important player in the signal transduction mechanisms in several organs like the brain, prostate, breast and colon tissue, as well as the immune cells. The cells in these organs have vitamin D receptors and respond to 1, 25 (OH)2 vitamin D.

In the circulation, vitamin D is transported by the vitamin D - binding protein, which belongs to the albumin and alpha-fetoprotein gene family. The concentration of vitamin D - binding protein in the plasma greatly exceeds that of 25-OH-D (9 nM versus 50 nM), with less than 5% of available binding sites being occupied.

Measurement of 25-OH-D

The analytical measurement of vitamin D is performed for two major reasons: to determine the nutritional status of vitamin D, and to monitor its therapeutic level. As mentioned before, there are two different types of vitamin D. To adequately monitor therapy, we need to be aware of which vitamin D entity is the one measured in the different assays. Specifically, if an immunoassay or protein-binding assay is to be used, is the antibody reacting equally with both types of vitamin D? The answer to this question is that  if the intention of measuring vitamin D is to monitor vitamin D2 therapy, then the assay must measure vitamin D2.

The assays currently available in the market (US and EU) can be classified as binding assays and chemical assays. Chemiluminescence immunoassays (CLIA), radioimmunoassay (RIA), and binding protein assay belong to the binding assays group, while chemical assays include high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The specificity and accuracy of these methods are very variable. Both RIA and CLIA are immunoassays in which the accuracy of the method will depend on the specificity of the antibody used (how well the antibody recognizes D2 and D3). The binding assays are affected by the matrix effects due to the tight binding of the vitamin D - binding protein to vitamin D. Currently, automated immunoassays are very popular and practical for the clinical laboratory.

The first automated vitamin D assay was based on Competitive-Protein Binding Assays (CPBA) for the Nicholis Advantage analyzer. It has the advantages of being inexpensive, can be performed on small sample size, and is co-specific for 25-OH-D2 and 25-OH-D3. This assay underestimated 25-OH-D at low levels and overestimated it at high levels. Immunoassay methods were first reported in the 1980s with a radioimmunoassay (RIA). This assay formed the basis for a subsequent chemiluminescent detection – based system. The Radioimmunoassay (RIA) requires a small sample size and the incorporation of Iodine – 125 as a tracer. It is not subjected to nonspecific interference, and in addition to being rapid it is inexpensive and accurate. Nevertheless, it still requires the use of radionuclides, and some RIA assays discriminate between 25-OH-D2 and 25-OH-D3.

Chemical assays have been originally more technically involved but are also now able to accommodate a large number of tests per day. Chemical methods (HPLC and LC-MS/MS) can report vitamin D2 and D3 independently. Ultraviolet quantitation following HPLC is a very stable, repeatable assay, and provides separate quantitation of 25-OH-D2 and 25-OH-D3. Nevertheless, it requires a larger sample size, needs a preparation step before chromatography and sometimes is subject to interferences with other compounds measured in the ultraviolet spectrum. Also, this assay requires a high level of technical expertise.

LC-MS/MS has been referred to as a “Gold Standard” technique for 25-OH-D3, although results can also be erroneous. This technique requires the skills of an experienced analyst. Another caveat with LC-MS/MS is the presence of the 25-OH-D2 and C3 epimers of vitamin D3 in pediatric specimens. If the assay is not optimised, vitamin D2/D3 result may be higher than expected in the pediatric population due to this epimer. Publications have shown that the C3 epimer may be present in adults as well.

Standardization and External Quality Control Assessment:

With the availability of many vitamin D assays, differences in the reported 25-OH-D values for the same samples were observed among different assays.

These differences could impact the classification of patients’ vitamin D status and therefore affect the clinical management of some patients. The question is if it is appropriate to have a clinical decision limit without assay standardization. To address this issue, the Vitamin D Standardization Program (VDSP), an initiative of the National Institutes of Health Office of Dietary Supplements (NIH ODS), was launched in 2010 in collaboration with the National Institutes of Health, the Center for Disease Control and Prevention (CDC), the National Institute for Standards and Technology (NIST), and Ghent University in Belgium.

Following, the CDC has introduced a Vitamin D Standardization-Certification Program to ensure reliable clinical measurement of vitamin D. It was recommended that all assay manufacturers should participate in the CDC’s Standardization-Certification Program. This is especially important for the in-house reference method of the manufacturers and for the assay measurement systems as they are being developed. The primary steps to standardization are as follows (fig.1):

(1) develop a reference system,

(2) establish metrological traceability and

(3) verify “end-user” test performance.

When participants pass four consecutive surveys, they are awarded a certification for one year, which can be renewed annually.

Controversies Regarding Vitamin D Testing                                                                                                                                        

Past: Over the past decade, a big number of studies linking low vitamin D levels to cancer, heart disease, diabetes, and other diseases haveled many doctors to routinely test vitamin D levels for their healthy patients. Consequently, laboratory professionals are confronted with the challenge of helping clinicians navigate the complexities of vitamin D assays.

The current evidence suggest that the main beneficial effects of vitamin D supplementation relate to musculoskeletal, rather than extraskeletal. Moreover, the exponential increase in vitamin D testing and supplements used in the past few years, have raised justifiable concerns if many vitamin D measurements are being undertaken without evidence-supported indications and if many individuals are being supplemented with little evidence of the benefit.

Present: In response to these concerns in 2013, the Royal College of Pathologists of Australasia (RCPA) published a position statement to clarify the role of vitamin D testing in the context of vitamin D deficiency, with guidelines about who should be tested and when repeat testing should be performed. Also, the U.S. Preventive Services Task Force (USPSTF) published a new recommendation in November 2014, which stated that there is no practical reason for most people to get a vitamin D test. Testing for vitamin D might be indicated in patients with osteoporosis or other bone-related problems, conditions that affect absorption of fat, including celiac disease or weight-loss surgery or who are taking medications that interfere with vitamin D activity, including anticonvulsants and glucocorticoids.


References are available on request.