25-Hydroxyvitamin D Testing: Immunoassays Versus Tandem Mass Spectrometry

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Key points

  • In addition to playing a vital role in bone health, vitamin D has been associated with many other health conditions, such as diabetes, cardiovascular diseases, cancer, multiple sclerosis, and immune system functions.

  • Several epidemiologic studies have shown that the prevalence of vitamin D deficiency is widespread in the general population.

  • Although, 1,25-dihydroxyvitamin D is the active form of vitamin D, 25-hydroxyvitamin D is the best marker of vitamin D status.

  • Immunoassays and HPLC-MS/MS are

25-Hydroxyvitamin D reference intervals

Currently, 25(OH)D is considered the best indicator of the vitamin D status because contrarily to 1,25(OH)2D, its level is not dependent on parathyroid harmone (PTH) concentration and it shows low intraindividual variability because of its longer half-life of approximately 3 weeks. Unlike other vitamins, population-based vitamin D reference ranges cannot be established. This is because several factors including season of the year, gender, ethnicity, sunlight exposure, diet, age, gender, body

Harmonization of 25-hydroxyvitamin D assays

Because medical decisions regarding vitamin D deficiency are based on the specific values of 25(OH)D, it is important that different methods for the measurement of 25(OH)D are comparable and harmonized. Various studies have shown that accurate measurement of 25(OH)D is challenging and there are clinically significant differences among various 25(OH)D assays. Many factors including the inconsistent release of 25(OH)D from VDBP and its similarity with many other endogenous compounds have

25-hydroxyvitamin D assays

There are two major categories of 25(OH)D assays: nonchromatographic methods involving 25(OH)D binding protein or antibody, and chromatographic methods.12 Before 1990, only a limited number of laboratories performed 25(OH)D assays. These laboratories used in-house CPBA or HPLC with UV detection. CPBA with and without chromatographic separation of 25(OH)D were developed. These assays were laborious and many suffered from inaccuracy, poor sensitivity, imprecision, and unequal cross-reactivity

Automated immunoassays and protein-binding assays for 25-hydroxyvitamin D

Several Food and Drug Administration (FDA) approved automated immunoassays and protein-binding assays are available for the determination of 25(OH)D. These assays are available on major chemistry platforms and provide rapid turnaround time. Unlike chromatographic methods, automated assays are simple to use, often require low sample volume, and do not need a large initial capital cost and specialized expertise. Most of these assays are competitive immunoassays or protein-binding assays based on

Chromatographic assays for 25-hydroxyvitamin D

Chromatographic methods have been widely used for the assay of 25(OH)D and other metabolites of vitamin D. These methods offer several advantages, the most obvious being the separation and simultaneous measurement of several clinically relevant vitamin D metabolites, such as 25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 1,25(OH)2D2, 24,25(OH)2D3, 24,25(OH)2D2, and 3-epi-25(OH)D. HPLC with UV detection, first described in 1978, was an earlier chromatographic method for the assay of vitamin D metabolites.

Comparison of immunoassays and protein-binding assays with chromatographic methods

Several studies have compared 25(OH)D immunoassays and protein-binding assays with chromatographic methods involving UV detection or mass spectrometry.15, 28, 29, 30, 31, 32 Until recently, immunoassays and protein-binding assays suffered from significant inaccuracies and imprecision. Several factors, including inconsistent release of 25(OH)D from VDBP, uneven cross-reactivity of antibody with 25(OH)D3 and 25(OH)D2, interferences from endogenous compounds, and lack of standard materials

Summary

Vitamin D deficiency has been associated with poor bone health and a myriad of other illnesses including diabetes, cardiovascular diseases, autoimmune disorders, and cancer. Because of these associations and the important role of vitamin D in health and disease, clinical laboratories have seen tremendous growth in 25(OH)D testing in recent years. To classify a patient into a right category of deficiency or sufficiency, accurate measurement of 25(OH)D is of the utmost importance. Although

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    Disclosure: The author has nothing to disclose.

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