Shining a light on vitamin D testing

Vitamin D is a pro-hormone that plays a key role in maintaining bone health.  It has also been linked with a vast number of health conditions from certain types of cancers to diabetes and overall mortality.¹ During the past decade, a dramatic increase in vitamin D-related research and ensuing media coverage has raised the awareness of vitamin D insufficiency/deficiency in the healthcare community as well as in the general public, which has resulted in a concurrent surge in vitamin D laboratory testing demand. The ever-accumulating information related to vitamin D, together with the rapid increase in both the testing volume and the number of available tests, has created some confusion regarding the health conditions that have been conclusively linked to vitamin D deficiency, the cutoffs defining vitamin D adequacy/insufficiency/deficiency, and the variance of the testing results among the types of testing platforms.² This article aims to explain the vitamin D testing methods currently available on the market, their clinical utilities, platform-specific attributes and limitations, as well as the approaches to ensure vitamin D tests provide clear, actionable results for patients and physicians.

Testing platforms

Available methods for vitamin D testing have evolved to the benefit of patients and laboratories over the past four decades, providing multiple options for labs seeking to offer the analyte as part of a broad test menu.³ A breakthrough at the time, the first measurements of vitamin D involved a manual competitive protein binding (CPB) assay that included solvent extraction, chromatography, and hand correction of each result to account for step-to-step analyte loss. A radioimmunoassay (RIA) that offered a more simplified assay procedure and a faster time to result was later developed, helping to make more routine vitamin D testing a reality.

The user-friendly enzyme immunoassay (EIA) was introduced more recently and, as most labs now seek to avoid using radio isotopic reagents, it has become the prevailing approach to vitamin D testing.⁴ Today, automated chemiluminescent vitamin D immunoassays have become the preferred method for measuring 25-OH vitamin D in sera.⁴ The reproducibility and ease-of-use attributes of these systems, including the ability to establish and follow standard workflows within the laboratory, perform random access sampling, automate calibration and QA/QC testing, include the test as part of a panel, and minimize hands-on time have made such systems extremely attractive for routine clinical labs.

Another highly regarded technique, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), is also regularly used clinically due to its high degree of sensitivity and precision.⁵ LC-MS/MS methods are typically “home-brew” and historically have not lent themselves to high throughput or automation, and samples are often batched to achieve economies of scale and throughput efficiency. In addition, the high cost of the instruments and advanced operational skills required to perform the test often push the LC-MS/MS methods beyond the means of mid- to small-size laboratories.

Testing for total vitamin D

Vitamin D exists in two forms. While vitamin D₂ (ergocalciferol) can be obtained from diet or supplement, vitamin D₃ (cholecalciferol) is mainly produced in the skin through sunlight exposure, though it can also be obtained from foods or supplements.¹ Today’s assays are required to measure both the vitamin D₂ and D₃ forms to accurately reflect total blood levels. This is particularly important for patients who are taking prescription supplements (which in some countries are in the form of vitamin D₂) because a partial 25-OH vitamin D measurement could erroneously underreport therapeutic or even toxic circulating vitamin D levels.⁶ Cases have been reported in the literature in which blood levels of 25-OH vitamin D appeared unchanged despite the patients taking large doses of supplements, which was due to the use of an assay that did not measure 25-OH vitamin D_2.⁷  This type of erroneous testing results can confuse physicians and affect patient care. In general, all LC-MS/MS methods measure both vitamin D₂ and D₃, but not all immunoassays recognize vitamin D₂. It is important to choose an assay that has equal detection of vitamin D₂ and D₃ for routine clinical lab use.⁶

Variability across platforms and assays

Despite good news about the variety of different vitamin D testing methods offered for clinical lab use, a number of issues and challenges have emerged. Recent studies seeking to compare assays reported differences in the 25-OH vitamin D values observed across platforms and manufacturers.^4,8,9 The observed differences could impact the classification of patients’ vitamin D status and could, in some extreme cases, affect the clinical management of some patients.

Variations among different platforms and assays may reflect inherent limitations of each technology and even the standards themselves, and must be taken into account when developing and calibrating assays to ensure reliable results. A recent report suggested that serum concentrations of vitamin D binding protein (VDBP) could cause bias of the reported vitamin D value by certain immunoassays.¹⁰ Similarly, certain immunoassays may be more sensitive to human anti-animal antibodies (HAAA).¹¹ In addition, the completeness of disassociation of vitamin D from VDBP may also add variations among immunoassays.

While LC-MS/MS is often used as the reference method, it is definitely not immune to inter-method variability. Most LC-MS/MS methods are developed in individual labs (home-brew) and can differ in assay conditions, such as sample preparation, instrument selection, ionization, instrument setting, and calibration.  Additionally, substances with the same molecular weight as vitamin D or its metabolites, as well as its C3-epimer, could also add bias unless accounted for by chromatography and detection conditions.²

The International Vitamin D External Quality Assessment Scheme (DEQAS) in the UK has been established for about 20 years with the goal of ensuring the analytical reliability of vitamin D assays. This organization distributes serum samples to participants on a quarterly basis and statistically analyzes submitted results to generate all lab trimmed means (ALTM), to which each participant can compare their own method.¹² Prior to 2009, no international standard reference materials (SRMs) existed, leaving developers to establish their own references and standardization curves. Since the introduction of the SRM 972 by the National Institute of Standard and Technology (NIST),² the inter-lab variations of the LC-MS/MS results submitted to DEQAS have decreased dramatically. However, since three of four SRM 972 reference materials are either spiked with exogenous metabolites or diluted with equine serum, the sample matrix is not suitable for many immunoassays. Thus, new reference materials applicable for both the LC-MS/MS method and immunoassays still need to be developed.

The future for vitamin D testing

Accumulating reports on vitamin D research have revealed the high global prevalence of vitamin D deficiency/insufficiency as well as its potential impact on public health.¹ The growing awareness of the potential impacts of low vitamin D and the dramatic upswing in vitamin D testing has driven the development of significantly improved assays, enabling far more labs to offer reliable, efficient testing than ever before.

Meanwhile, the need to standardize 25-OH vitamin D assays in order to provide accurate measurement and reliable patient care is now well recognized in the vitamin D measurement community. The Centers for Disease Control and Prevention (CDC) is expected to introduce a Vitamin D Standardization-Certification Program to ensure reliable clinical vitamin D measurement in October 2012.¹³ And as regulatory agencies and assay manufacturers continue to work toward these goals, test results are likely to achieve closer alignment across platforms and methodologies, improving consistency for clinical and research samples. This should create a clearer picture of vitamin D research conclusions that will better inform the establishment of standards of care as well as improve patient management.

References

1. Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357:266-281.
2. Carter DC. 25-hydroxyvitamin D: a difficult analyte. Clin Chem. 2012;58(3):486-488.
3. Wallace AM. Gibson S, de la Hunty A, Lamberg-Allardt C, Ashwell M. Measurement of 25-hydroxyvitamin D in the clinical laboratory: current procedure, performance characteristics and limitations. Steroids . 2010;75:477.
4. Schleicher RL. Vitamin D testing. Clin Lab News . 2009;12:10-12.
5. El-Khoury JM, Reineks EZ, Wang S. Progress of liquid chromatography-mass spectrometry in measurement of vitamin D metabolites and analogues. Clin Biochem . 2010;44:66.
6. Kleerekoper M. Clinical applications for vitamin D assays: what is known and what is wished for. Clin Chem . 2011;57:1227-1232.
7. Kolatkar NS, LeBoff MS. Analytic challenges in monitoring vitamin D therapy. Am J Clin Pathol. 2007;127(3):472-473.
8. Moon HW, Cho JH, Hur M. Comparison of four current 25-mydroxyvitamin D assays. Clin Biochem . 2012;45:326-330.
9. Tahsin-Swafir S, Blanco-Navarro I, Pérez-Sacristán B, Millán I, Granado-Lorencio F. The prevalence of vitamin D deficiency in clinical practice is assay-dependent. Clin Nutri . On Line 2012.
10. Heijboer AC. Accuracy of six routine 25-mydroxyvitamin D assays: influence of vitamin D binding protein concentration. Clin Chem . 2012;58:543-548.
11. Cavalier E, Carlisi I, Bekaert AC, Rousselle O, Chapelle, JP. Human anti-animal interference in DiaSorin Liason total 25(OH) vitamin D assay: towards the end of a strange story? Clin Chimica Acta . 2012;413:527.
12. DEQAS. http://www.deqas.org. Accessed July 30, 2012.
13. Laboratory Quality Assurance and Standardization Programs. Centers for Disease Control and Prevention. http://www.cdc.gov/labstandards. Accessed July 30, 2012.