25-hydroxyvitamin D: a difficult analyte.

In their article in the current issue of Clinical Chemistry, Farrell et al. (1 ) describe the latest in a number of studies (2, 3 ) highlighting the method-related variability in serum 25-hydroxyvitamin D (25-OHD) results. There is common agreement that 25-OHD is a “difficult” analyte. That quality is generally ascribed to its hydrophobic nature, its existence in several different molecular forms, its tight binding to vitamin D– binding protein (VDBP) and, until recently, the absence of reference materials or a reference measurement procedure (RMP) against which assays could be standardized. This last problem was mitigated to some extent with the introduction in 2009 of the NIST standard reference materials (SRM 972 and SRM 2972) and the acceptance of the NIST and University of Ghent liquid chromatography–tandem mass spectrometry (LC-MS/MS) assays as RMPs (4 ). Unfortunately, 4 of the 5 NIST reference materials are either spiked with exogenous metabolites or diluted with equine serum, characteristics that render these reference materials unsuitable for many immunoassays (5, 6 ). The history of 25-OHD methodology could serve as a case study of the consequences of transferring a rigorous but labor-intensive method from the unhurried atmosphere of the research laboratory to the bustle of a routine clinical laboratory having to meet tight deadlines. The pioneering competitive proteinbinding (CPB) method of Haddad and Chyu (7 ) involved solvent extraction and chromatography, with the results of every sample corrected for procedural losses. Subsequent attempts at simplifying a CPB method by omitting the chromatography stage proved unsuccessful (8 ), and an automated version (Nichols Advantage) introduced in 2004 was withdrawn in 2006. The successive abandonment of sample extraction, chromatography, and correction for procedural losses in immunoassays has undoubtedly contributed to the inconsistencies reported by Farrell et al., as have differences in assay standardization. Nevertheless, results submitted to the international Vitamin D External Quality Assessment Scheme (DEQAS) have shown a gradual reduction in interlaboratory imprecision (CV) in recent years—from 30% in 1995 to 15% in 2011. This reduction may be due to the increasing dominance of automated immunoassays, in particular the DiaSorin LIAISON Total method and, more recently, greater use of the NIST SRMs in standardization. Whatever the explanation, it appears that there is currently reasonable agreement, with 1 or 2 notable exceptions, between most 25-OHD methods (9 ). Nevertheless, the fact that a change of method can affect the continuity of results has implications for the monitoring of patients on vitamin D supplements and for longterm epidemiologic studies. Even within the same method, the consistency of results can be compromised by subtle lot-to-lot changes in reagent kits or more overtly by a change of antibody or in a reformulation of reagents. With the introduction of the RMPs and the expected release in 2012 of a new generation of human serum– based SRMs, assay standardization should become less of an issue, and attention is likely to focus on the reliability of individual results, a far more intractable problem. The use of summary statistics in method comparisons, particularly the correlation coefficient, and linear regression equations can disguise a marked variance in the results given by 2 methods for the same sample. Method differences are best revealed by the use of Bland–Altman plots, which allow discrepancies between results for individual samples to be seen at a glance. These apparently random errors undermine the confidence in the veracity of all 25-OHD results and suggest that some methods are subject to interference from other components of the sample matrix. The study reported by Heijboer et al. (10 ) (also in the current issue) not only identifies a potential culprit (VDBP) but quantifies its effect. The automated 25OHD methods, which have necessarily abandoned solvent extraction or protein precipitation to free 25OHD from binding proteins, are likely to be more susceptible to variations in VDBP concentration. Man1 Imperial College Healthcare NHS Trust, Clinical Biochemistry Department, Charing Cross Hospital, London, UK. * Address correspondence to the author at: DEQAS Organizer, Imperial College Healthcare NHS Trust, Clinical Biochemistry Department, Charing Cross Hospital, Fulham Palace Rd., London, W6 8RF, UK. Fax 44-(0)20–8846-7007; e-mail [email protected]. Received December 29, 2011; accepted December 30, 2011. Previously published online at DOI: 10.1373/clinchem.2011.180562 2 Nonstandard abbreviations: 25-OHD, 25-hydroxyvitamin D; VDBP, vitamin D–binding protein; RMP, reference measurement procedure; SRM, standard reference material; LC-MS/MS, liquid chromatography–tandem mass spectrometry; CPB, competitive protein-binding (method); DEQAS, Vitamin D External Quality Assessment Scheme; 24,25-(OH)2D, 24,25-dihydroxyvitamin D; 3-epi25-OHD3, 3-epi-25-hydroxyvitamin D3. Clinical Chemistry 58:3 000 – 000 (2012) Editorials