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Technical Briefs |
1 Endocrine Laboratory, Clinical Chemistry Department, Charing Cross Hospital, London, United Kingdom;2 Sovereign Software Services, Haywards Heath, West Sussex, United Kingdom;3 Vitamin D Research Group, Medicine, Manchester Royal Infirmary, Manchester, United Kingdom;
aaddress correspondence to this author at: Endocrine Laboratory, Clinical Chemistry Department, Charing Cross Hospital, Fulham Palace Road, London, W6 8RF, United Kingdom; fax 44-208-846-7007, e-mail julia.jones{at}imperial.ac.uk
A recent report highlighted the interlaboratory variability in serum 25-hydroxyvitamin D (25OHD) results (1). Concerns have also been raised about the apparent inability of some 25OHD assays to reliably measure 25-hydroxyvitamin D2 (25OHD2) (2)(3)(4). The accurate measurement of this metabolite is essential for the monitoring of vitamin-D-deficient patients receiving ergocalciferol, which is the only supplement used in the United States (5) and widely used elsewhere.
The international Vitamin D Quality Assessment Scheme (DEQAS) has been monitoring the performance of 25OHD assays since 1989 and now has >100 registered participants in 18 countries. In essence, DEQAS is an ongoing, multicenter trial of the methods used by its participants and provides a unique opportunity to assess the accuracy and specificity of 25OHD methods as well as the analytical performance of a large number of their users.
The organization of DEQAS has been described elsewhere (6), and details are available on the DEQAS website (www.deqas.org). In brief, five samples of normal human sera are sent out at 3-month intervals. Participants are asked to measure the total 25OHD concentration in each and return their results within 6 weeks. After statistical analysis of results (7), participants receive a report giving an All-Laboratory Trimmed Mean (ALTM) and Standard Deviation (SD) for each sample. A small study conducted in 1997 (8) showed that the ALTM was a good surrogate for the "true" (target) value produced by gas chromatography–mass spectrometry. The accuracy of each result is defined by its percentage bias from the ALTM. Results for each sample are also grouped by method, and a method mean (MM) is produced. The overall accuracy of each method can be assessed from the percentage bias of the method mean from the ALTM: {[(MM – ALTM)/ALTM] x 100}.
The percentage bias of method means was calculated for each sample (n = 88) sent out between January 2000 and January 2004, except for the Nichols automated assay (n = 49) and the IDS enzyme immunoassay (EIA; n = 23); results for these methods were available only from 2001 and 2002, respectively. Samples known to contain 25OHD2 were excluded. The ALTMs (nmol/L) for the samples ranged from 12.1 to 118.5 (median, 39.3). The mean percentage bias over this period was calculated for each major method group. The results are presented in Fig. 1
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With the exception of the Nichols automated procedure, the mean bias of 25OHD methods over this period was within 7% of the ALTM. Plots of percentage bias vs ALTM (not shown) revealed that in the IDS EIA, bias increased with concentration, whereas bias was decreased slightly in the Nichols assay at higher concentrations. Since its inclusion in the scheme, the Nichols assay has, for most samples, shown a marked positive bias, averaging 
31%. Detection of the small amounts (<10 nmol/L) of 24,25-dihydroxyvitamin D found in human serum can lead to high results in nonchromatographic methods for 25OHD. However, this is unlikely to cause the positive bias in the Nichols assay because the stated cross-reactivity (100%) is the same as that quoted for the two other methods (DiaSorin and IDS RIA) used by the majority of DEQAS participants. The positive intercept (8.7 nmol/L) of the regression line method mean vs ALTM (see below) suggests that a sample matrix effect is a contributory factor. This would not be surprising. Early attempts to simplify competitive protein binding assays for 25OHD, by abandoning the preliminary chromatographic step, failed because interfering substances in the sample matrix led to high results (9)(10). The possibility that sodium azide, formerly used as a preservative in DEQAS samples, could have interfered in the chemiluminescence endpoint of the Nichols assay was considered but discounted because the bias persisted after the use of azide was abandoned in 2002. The continued use of the ALTM as the target value for 25OHD assays has also been questioned because the statistics could be distorted by the dominance of two methods (the DiaSorin and IDS RIAs), which currently account for >60% of the submitted results. However, the more rigorous chromatographic protein-binding assay and HPLC both continue to give results close to the ALTM. This supports the use of the ALTM as a target value, although its validity is under constant review.
Linear regression analysis was performed to define the relationship between method means and the ALTM (x); the results were as follows (correlation coefficients in parentheses): DiaSorin = 0.97x + 0.64 nmol/L (r = 0.99); IDS RIA = 0.98x – 1.13 nmol/L (r = 0.99); IDS EIA (all results) = 1.28x – 9.02 nmol/L (r = 0.95); IDS EIA (results <80 nmol/L) = 1.06x – 1.82 nmol/L (r = 0.96); HPLC = 0.98x + 2.15 nmol/L (r = 0.96); Nichols = 1.1x + 8.7 nmol/L (r = 0.90). P was <0.001 for all correlation coefficients.
Among the five samples distributed in January 2004 were two (samples 4 and 5) containing endogenous 25OHD2. A summary of the results is presented in Table 1
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The wide range of results submitted by users of the same method illustrates the degree to which 25OHD assays are operator-dependent. The one automated procedure (Nichols) produced more consistent values, but for the samples (1, 2, and 3) containing only 25OHD3, the results were again higher than those of other methods.
For samples 4 and 5, in which 25OHD2 is the predominant metabolite, method means for the IDS RIA and the Nichols automated assay were lower than those of other methods. The inference is that the IDS RIA and the Nichols automated procedure underestimate 25OHD2, the latter by a considerable margin. Indeed, the presence in these samples of 25OHD3, albeit in smaller amounts than 25OHD2, disguises the true extent of the problem. IDS acknowledge that their RIA has only a 75% cross-reactivity with 25OHD2 (11), but Nichols claims a 100% cross-reactivity and has presented data to support this (12). The reason for the disparity between the manufacturer’s claims and the DEQAS findings is unclear. Low recovery of 25OHD2 by the Nichols automated assay has also been observed in clinical samples (Dr. Carol Wagner, Department of Pediatrics, Medical University of South Carolina, Charleston, SC, personal communication). Vitamin-D-deficient neonates failed to show an increase in 25OHD, despite receiving large doses of ergocalciferol. When the samples were reanalyzed by the DiaSorin assay, the expected increase in 25OHD was observed. This suggests that the problem is not confined to DEQAS samples, an explanation originally proposed by the manufacturer to explain the overrecovery of 25OHD3. In the IDS RIA, the underrecovery of 25OHD2 can be attributed to differences in antibody specificity for the two forms of the metabolite. Interestingly, the same antibody is used in the nonisotopic version of the IDS assay, which despite the published cross-reactivity data of 75%, did not appear to underestimate 25OHD2 in the DEQAS samples.
In summary, an international quality assessment scheme has demonstrated that, for samples containing only 25OHD3, most commercial 25OHD methods are capable of giving results close to the target value, but the results are highly operator dependent. The Nichols automated assay gives more consistent results but generally produces higher values than other methods. For samples containing predominantly 25OHD2, the Nichols procedure and, to a lesser extent, the IDS RIA gave considerably lower results than other methods. The underrecovery of 25OHD2 by the Nichols assay, which has also been observed in clinical samples, occurred despite the manufacturer’s claim that the method is equally specific for both forms of 25OHD. The treatment of vitamin-D-deficient patients could be severely compromised by the use of assays that underestimate 25OHD2.
The validity of 25OHD results will, justifiably, continue to be questioned. The only way for laboratories to demonstrate the accuracy of their results is to participate in an external quality assessment scheme and to make details of their performance available to clinical colleagues.
Acknowledgments
Since this article was submitted, Nichols Institute Diagnostics has issued a technical note in which they acknowledge that some samples containing substantial quantities of 25OHD2 give low results in the Advantage automated assay.
References
The following articles in journals at HighWire Press have cited this article:
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  P. Leventis and S. Patel Clinical aspects of vitamin D in the management of rheumatoid arthritis Rheumatology, August 5, 2008; (2008) ken296v1. [Abstract] [Full Text] [PDF]
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A. Cranney, H. A Weiler, S. O'Donnell, and L. Puil Summary of evidence-based review on vitamin D efficacy and safety in relation to bone health Am. J. Clinical Nutrition, August 1, 2008; 88(2): 513S - 519S. [Abstract] [Full Text] [PDF]
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E. A Yetley Assessing the vitamin D status of the US population Am. J. Clinical Nutrition, August 1, 2008; 88(2): 558S - 564S. [Abstract] [Full Text] [PDF]
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K. D Cashman, T. R Hill, A. A Cotter, C. A Boreham, W. Dubitzky, L. Murray, J. Strain, A. Flynn, P. J Robson, J. M. Wallace, et al. Low vitamin D status adversely affects bone health parameters in adolescents Am. J. Clinical Nutrition, April 1, 2008; 87(4): 1039 - 1044. [Abstract] [Full Text] [PDF]
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 H. J. Roth, H. Schmidt-Gayk, H. Weber, and C. Niederau Accuracy and clinical implications of seven 25-hydroxyvitamin D methods compared with liquid chromatography-tandem mass spectrometry as a reference Ann Clin Biochem, March 1, 2008; 45(2): 153 - 159. [Abstract] [Full Text] [PDF]
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 S. Abbas, J. Linseisen, T. Slanger, S. Kropp, E. J. Mutschelknauss, D. Flesch-Janys, and J. Chang-Claude Serum 25-hydroxyvitamin D and risk of post-menopausal breast cancer--results of a large case-control study Carcinogenesis, January 1, 2008; 29(1): 93 - 99. [Abstract] [Full Text] [PDF]
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 E. Hypponen, S. Turner, P. Cumberland, C. Power, and I. Gibb Serum 25-Hydroxyvitamin D Measurement in a Large Population Survey with Statistical Harmonization of Assay Variation to an International Standard J. Clin. Endocrinol. Metab., December 1, 2007; 92(12): 4615 - 4622. [Abstract] [Full Text] [PDF]
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A. Burgaz, A. Akesson, A. Oster, K. Michaelsson, and A. Wolk Associations of diet, supplement use, and ultraviolet B radiation exposure with vitamin D status in Swedish women during winter Am. J. Clinical Nutrition, November 1, 2007; 86(5): 1399 - 1404. [Abstract] [Full Text] [PDF]
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I. Laaksi, J.-P. Ruohola, P. Tuohimaa, A. Auvinen, R. Haataja, H. Pihlajamaki, and T. Ylikomi An association of serum vitamin D concentrations < 40 nmol/L with acute respiratory tract infection in young Finnish men Am. J. Clinical Nutrition, September 1, 2007; 86(3): 714 - 717. [Abstract] [Full Text] [PDF]
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 C. C. Abnet, W. Chen, S. M. Dawsey, W.-Q. Wei, M. J. Roth, B. Liu, N. Lu, P. R. Taylor, and Y.-L. Qiao Serum 25(OH)-Vitamin D Concentration and Risk of Esophageal Squamous Dysplasia Cancer Epidemiol. Biomarkers Prev., September 1, 2007; 16(9): 1889 - 1893. [Abstract] [Full Text] [PDF]
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F. L Weng, J. Shults, M. B Leonard, V. A Stallings, and B. S Zemel Risk factors for low serum 25-hydroxyvitamin D concentrations in otherwise healthy children and adolescents Am. J. Clinical Nutrition, July 1, 2007; 86(1): 150 - 158. [Abstract] [Full Text] [PDF]
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N. Binkley, R. Novotny, D. Krueger, T. Kawahara, Y. G. Daida, G. Lensmeyer, B. W. Hollis, and M. K. Drezner Low Vitamin D Status despite Abundant Sun Exposure J. Clin. Endocrinol. Metab., June 1, 2007; 92(6): 2130 - 2135. [Abstract] [Full Text] [PDF]
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E. Hypponen and C. Power Hypovitaminosis D in British adults at age 45 y: nationwide cohort study of dietary and lifestyle predictors Am. J. Clinical Nutrition, March 1, 2007; 85(3): 860 - 868. [Abstract] [Full Text] [PDF]
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F. Ibrahim, C. Parmentier, and P. Boudou Divergence in Classification of 25-Hydroxyvitamin D Status with Respect to Immunoassays Clin. Chem., February 1, 2007; 53(2): 363 - 364. [Full Text] [PDF]
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J. A. Schmidt Measurement of 25-Hydroxyvitamin D Revisited Clin. Chem., December 1, 2006; 52(12): 2304 - 2305. [Full Text] [PDF]
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G. Lensmeyer, D. Wiebe, N. Binkley, and M. Drezner The authors of the article cited above respond: Clin. Chem., December 1, 2006; 52(12): 2305 - 2306. [Full Text] [PDF]
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G. L. Lensmeyer, D. A. Wiebe, N. Binkley, and M. K. Drezner HPLC Method for 25-Hydroxyvitamin D Measurement: Comparison with Contemporary Assays Clin. Chem., June 1, 2006; 52(6): 1120 - 1126. [Abstract] [Full Text] [PDF]
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Z. Maunsell, D. J. Wright, and S. J. Rainbow Routine Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Measurement of the 25-Hydroxy Metabolites of Vitamins D2 and D3 Clin. Chem., September 1, 2005; 51(9): 1683 - 1690. [Abstract] [Full Text] [PDF]
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P. Leventis, L. Garrison, M. Sibley, P. Peterson, M. Egerton, G. Levin, and P. Kiely Underestimation of Serum 25-Hydroxyvitamin D by the Nichols Advantage Assay in Patients Receiving Vitamin D Replacement Therapy Clin. Chem., June 1, 2005; 51(6): 1072 - 1074. [Full Text] [PDF]
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K. Ramakrishnan and M. F. Holick Underestimation of Serum 25-Hydroxyvitamin D by the Nichols Advantage Assay in Patients Receiving Vitamin D Replacement Therapy - Reply Clin. Chem., June 1, 2005; 51(6): 1074 - 1074. [Full Text] [PDF]
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G. T.Y. Chung, R. W.K. Chiu, K.C. A. Chan, T. K. Lau, T. N. Leung, L. W. Chan, and Y.M. D. Lo Detrimental Effect of Formaldehyde on Plasma RNA Detection Clin. Chem., June 1, 2005; 51(6): 1074 - 1076. [Full Text] [PDF]
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P. Glendenning and W. D. Fraser 25-OH-Vitamin D Assays J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 3129 - 3129. [Full Text] [PDF]
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