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Are clinical laboratories prepared for accurate testing of 25-hydroxy vitamin D?

To the Editor:

Vitamin D deficiency is an important concern, but assays for serum 25-hydroxy vitamin D (25-OH-D), (1) the accepted marker for vitamin D nutritional status, are not standardized. A quick search of Medline yielded 6014 citations on vitamin D deficiency, 51 of which were published in the first 6 mo of 2007 (1). Recognition that vitamin D deficiency may be more prevalent in most patient populations than earlier assumed has resulted in an unexpected and marked increase in the volume of testing for 25-OH-D in clinical laboratories (1). Historically, not many tests in the clinical laboratories have increased at the rate of 80% to 90% per year, as is the case for 25-OH-D in our reference laboratory. Several million 25-OH-D tests are likely to be performed this year in the US by various reference and local hospital laboratories, raising questions about the adequacy of the methods used to make these measurements.

Various methods are available for measuring circulating concentrations of 25-OH-D (until recently, only RIA was available). Current methods include HPLC, RIA with low throughput to high throughput, automated chemiluminescence immunoassays, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). These new methods have already aroused controversy (2-4). Correlation and agreement studies between immunoassays and LCMS/MS methods for 25-OH-D have been reported by several investigators (2-4). These studies report reasonable correlations but with significant differences, the reasons for which are not transparent or well understood. Automated or manual competitive immunoassays are known to have less specificity for low-molecular-weight compounds, and immunoassays for 25-OH-D are no exception.

The College of American Pathologists (CAP) and the UK-based DEQAS (Vitamin D external quality assessment scheme) surveys provide independent approaches to monitor the performance of laboratories that use various methods for testing of 25-OH-D. The survey feedback does not assess the accuracy of 25-OH-D measurements by laboratories, but scores laboratories for agreement within the group using a particular method. Recent CAP data (CAP survey, 2006 Ligands Special) indicate that clinical laboratories using chemiluminescence immunoassays can report a result ranging from 41 to 96 [micro]g/L for a survey sample with a value of 31 [micro]g/L determined by LC-MS/MS (BGS-04 in Fig. 1). There could be many reasons for these variations, including drifts in the reagents being manufactured, but there is a clear and urgent need for harmonization and standardization.


NIST is developing additional quality control materials (human serum, SRM 972) that will contain 25-OH-D2, 25-OH-D3, and the metabolite 3-epi-25-OH-D at 4 different concentrations as characterized by LC-MS/MS. Although the biological significance of the metabolites remains to be elucidated, the preparation of this SRM is especially important for assays for which the cross-reactivity with these metabolites is not well defined (5).

LC-MS/MS is becoming the technique of choice for various reference laboratories. Laboratories that use in-house LC-MS/MS have responsibility for many steps of the assay. The LC-MS/MS technology for testing of human samples is not approved by the FDA, and manufacturers of LC-MS/MS instrumentation are not responsible for troubleshooting the assays. Laboratories performing 25-OH-D testing by LC-MS/MS technology have differences in their standard operating procedures, and thus interlaboratory CVs are in the range of 20%. The preparation of the reagents required for in-house LC-MS/MS assays is conducted by individual laboratories under their institutionally regulated standard procedures. The complexity of the LCMS/MS technology in its present form demands a robust, fully automated platform that can meet the need for throughput, precision, and accurate testing of vitamin D and metabolites. Multiplexed immunoassays may have the potential of achieving accuracy and precision for multiple vitamin D metabolites. For better patient care, the goal should be not only to have an accurate 25-OH-D value but also precision for 25-OH-D testing, with a CV < 1%.

Currently there are no guidelines or agreement among clinical laboratories on the optimum reference intervals for 25-OH-D to classify patients with moderate to severe vitamin D deficiency. Before it is too late, it is in the interest of clinical laboratories and diagnostic companies to work with each other to standardize the reagents and reference intervals to achieve the quality for 25-OH-D tests that our patients deserve.

Grant/funding Support: None declared.

Financial Disclosures: None declared.

Acknowledgments: The author thanks the staff of the Endocrine Laboratory at Mayo Clinic for sharing their expertise and experience in 25-OH-D testing.


(1.) Holick MF. Vitamin D deficiency. Engl J Med 2007;357:266-81.

(2.) Carter GD, Carter R, Jones J, Berry J. How accurate are assays for 25-hydroxyvitamin D? Data from the international vitamin D external quality assessment scheme. Clin Chem 2004;50: 2195-7.

(3.) Binkley N, Krueger D, Cowgill CS, Plum L, Lake E, Hansen KE, et al. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. J Clin Endocrinol Metab 2004;89: 3152-7.

(4.) Souberbielle JC, Fayol V, Sault C, Lawson-Body E, Kahan A, Cormier C. Assay-specific decision limits for two new automated parathyroid hormone and 25-hydroxyvitamin D assays. Clin Chem 2005;51:395- 400.

(5.) Singh RJ, Taylor RL, Reddy GS, Hollis BW, Grebe SK. C-3 epimers can account for a significant proportion of total circulating 25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. J Clin Endocrinol Metab 2006;91:3055-61.

(1) Nonstandard abbreviations: 25-OH-D, 25-hydroxy vitamin D; LC-MS/MS, liquid chromatography-tandem mass spectrometry; CAP, College of American Pathologists.

DOI: 10.1373/clinchem.2007.096156

Ravinder J. Singh *

Department of Laboratory Medicine and Pathology Mayo Clinic Rochester, MN, USA, 55905

* Address correspondence to the author at: Hilton 730 Department of Laboratory Medicine & Pathology Mayo Clinic and Foundation 200 First Street SW Rochester (MN) 55905 USA Fax (507) 284-9758 e-mail
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Title Annotation:Letters
Author:Singh, Ravinder J.
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Jan 1, 2008
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