Elsevier

Steroids

Volume 75, Issue 7, July 2010, Pages 477-488
Steroids

Review
Measurement of 25-hydroxyvitamin D in the clinical laboratory: Current procedures, performance characteristics and limitations

https://doi.org/10.1016/j.steroids.2010.02.012Get rights and content

Abstract

In this review we describe procedures, performance characteristics and limitations of methods available for the measurement of 25-hydroxyvitamin (25OHD) since the year 2000. The two main types of methods are competitive immunoassay and those based on chromatographic separation followed by non-immunological direct detection (HPLC, LC–MS/MS). Lack of a reference standard for 25OHD has, until recently, been a major issue resulting in poor between-method comparability. Fortunately this should soon improve due to the recent introduction of a standard reference material in human serum (SRM 972) from the National Institute of Standards and Technology (NIST). For immunoassay, specificity can be an issue especially in relation to the proportion of 25OHD2 that is quantified whereas HPLC and LC–MS/MS methods are able to measure the two major vitamin D metabolites 25OHD2 and 25OHD3 independently. HPLC and LC–MS/MS require more expensive equipment and expert staff but this can be offset against lower reagent costs. Increasingly procedures are being developed to semi-automate or automate HPLC and LC–MS/MS but run times remain considerably longer than for immunoassays especially if performed on automated platforms. For most HPLC and LC–MS/MS methods extraction and procedural losses are corrected for by the inclusion of an internal standard which, in part, may account for higher results compared to immunoassay. In general precision of immunoassay, HPLC and LC–MS/MS are comparable and all have the required sensitivity to identify severe vitamin D deficiency. Looking to the future it is hoped that the imminent introduction of a standard reference method (or methods) for 25OHD will further accelerate improvements in between method comparability.

Introduction

It is widely acknowledged that circulating 25-hydroxyvitamin D (25OHD) is the best indicator of vitamin D status [1]. There are two major vitamin D metabolites in the circulation, 25-hydroxyvitamin D3 (25OHD3) mainly derived from vitamin D3 produced by sunlight in the skin and 25-hydroxyvitamin D2 (25OHD2) derived from plants in the diet. In addition circulating 25OHD3 and 25OHD2 may be present due to supplementation with vitamin D3 or vitamin D2, respectively. Severe vitamin D deficiency (25OHD < 25 nmol/L) causes rickets in children and osteomalacia in adults [2]. Less severe deficiency, where the 25OHD concentration is between 25 and 50 nmol/L, causes secondary hyperparathyroidism and increases in bone turnover and bone loss [3], [4]. Furthermore vitamin D insufficiency has been implicated in an extremely wide range of clinical disorders. Some experts are of the opinion that for optimal health circulating 25OHD concentrations should be maintained above 75 nmol/L [5], [6].

The first methods for measuring 25OHD were described in the early 1970s being based on competitive protein binding after solvent extraction. The binding agent in these assays was vitamin D binding protein obtained from the serum of vitamin D-deficient rats. Later in the 1970s, methods based on high performance liquid chromatography (HPLC) became available. In 1985 the first radioimmunoassay (RIA) was developed which incorporate a specific 25OHD antibody. To avoid problems related to handling of radioactivity and the limited shelf-life of radioactive labels these have now been largely, but not completely, superseded in immunoassays by labels employing chemiluminescent substances (CLIA) or enzymes (EIA). Advances in tandem mass spectrometry towards the end of the last century enabled the introduction in 2004 of routine procedures based on LC–MS/MS for measuring vitamin D metabolites and the use of this methodology is increasing.

Current commercial immunoassays are supplied as kits which can be run manually or on platforms. With increasing clinical demand for 25OHD assays, fast automated platforms are attractive. The first automated procedure was a chemiluminescent competitive protein binding assay supplied by Nichols for their ‘Advantage’ platform. Unfortunately Nichols had to withdraw the assay at the end of 2005 as it overestimated total 25OHD concentrations and did not fully detect 25OHD2.

In 2004 Diasorin introduced a chemiluminescent immunoassay for use on their ‘Liaison’ automated immunoassay platform. In 2007 Diasorin updated and replaced this assay to improve sensitivity and precision and renamed it Liaison Total. All three Diasorin assays (RIA, Liaison and Liaison Total) use the same antibody. Recently Roche marketed an electrochemiluminescent immunoassay for the ‘Elecys’ and ‘Cobas E’ platforms which is specific for 25OHD3. The IDS EIA method is a manual assay which can also be performed on standard automated ELISA platforms (NEXgen, Triturus). IDS have recently released a new assay which uses a chemiluminescent label on an automated platform (iSYS). All three IDS methods (RIA, EIA and iSYS) use the same antibody. Detailed procedural and performance information on each immunoassay is described later.

Two methods employing non-immunological direct detection are currently available: HPLC and LC–MS/MS. Vitamin D metabolite measurement by high performance liquid chromatography (HPLC) incorporates a chromatographic separation followed by a variety of detection procedures. The term HPLC is usually restricted to procedures that have a ultra-violet (UV) or electrochemical detector. If HPLC is linked to mass detectors, the procedure is commonly termed LC–MS/MS or tandem mass spectrometry. Prior to chromatographic separation, an initial purification step is required. The simplest is extraction into an organic solvent (liquid/liquid extraction) or alternatively a simple protein crash procedure followed by solid phase extraction (SPE). Extraction and chromatographic separation will inevitably lead to loss of analyte which can be corrected by the inclusion of an internal standard. The internal standard should be indistinguishable from the analyte during the process of extraction and purification and choice is to some extent dictated by the quantitation procedure. For example, for procedures employing mass spectrometry deuterated internal standards are ideal, being chemically identical yet detectable by virtue of increased mass. This procedure generally compensates for any matrix related effects and is commonly termed isotope dilution mass spectrometry. When other types of detection systems such as light absorption and electrochemical properties are used, an internal standard is usually selected that has similar chemical properties to the analyte but is not present in biological samples. Detailed procedural and performance information on HPLC and LC–MS/MS methods is given later.

To summarise there are currently two main types of measurement used routinely for measuring the main circulating metabolites of vitamin D, 25OHD3 and 25OHD2. These are competitive immunoassay and methods based on chromatographic separation followed by non-immunological direct detection (HPLC, LC–MS/MS). Currently immunoassay is the most popular method with the majority using an automated platform. Recently there has been a steady increase in the use of LC–MS/MS.

Section snippets

Data source

A search of the literature was carried out on April 30th 2009 using the Pub Med database to identify all publications relating to a comparison of 25OHD measurement methods published since 2000, including letters and editorials. This date was chosen to ensure that the methods discussed were reasonably up-to-date. The search terms used and the number of papers identified were “25-hydroxyvitamin D and measurement (178 papers)”, “25-hydroxyvitamin D and measurement assay (149 papers)” and “25OHD

Method details

The methods detailed below are summarised in Table 1.

Assay procedure

The DiaSorin 25OHD RIA assay consists of a two-step procedure. The first step involves a rapid extraction of 25OHD and other hydroxylated metabolites from serum or plasma with acetonitrile. Following extraction, the treated sample is then assayed by competitive RIA using an antibody with specificity to 25OHD. The sample, antibody and tracer are incubated for 90 min at 20–25 °C. Phase separation is accomplished after 20-min incubation at 20–25 °C with a second antibody precipitating complex. To

Assay procedure

The IDS 25-hydroxy vitamin D EIA kit is an enzyme immunoassay for the quantitation of 25OHD and other hydroxylated metabolites in serum or plasma. Calibrators, controls and samples are diluted with 25OHD labelled with biotin. A propriety buffer reagent is used for dissociating 25OHD from its binding proteins. The diluted samples are incubated in microtitre wells which are coated with a highly specific sheep 25OHD antibody for 2 h at room temperature before aspiration and washing. Enzyme

Assay procedure

The method for quantitative determination of 25OHD is a direct, competitive chemiluminescence immunoassay (CLIA) on an automated platform. Specific antibody to vitamin D is used for coating magnetic particles (solid phase) and vitamin D is linked to an isoluminol derivative. During the incubation, 25OHD is dissociated from its binding protein, and competes with labelled vitamin D for binding sites on the antibody. After the incubation, the unbound material is removed with a wash cycle.

Assay procedure

The Liaison Total 25OHD assay is a direct competitive chemiluminescence immunoassay (CLIA) for quantitative determination of total 25OHD in serum or plasma on an automated platform. This is a reformulation of the Diasorin Liaison method. The same antibody is used but now in a two-step incubation procedure. During the first incubation, 25OHD is dissociated from its binding protein and binds to the specific antibody on the solid phase. After 10 min the tracer (vitamin D linked to an isoluminol

Assay procedure

A competitive immunoassay format is used based on the streptavidin–biotin technology. The assay employs a polyclonal sheep antibody against 25OHD3, which is ruthenium labelled. The vitamin D in the sample competes for binding with biotinylated 25OHD antigen which is bound to streptavidin coated microparticles. The test is intended for use on Elecsys and Cobas E automated immunoassay analysers. The literature search identified two papers that specifically examined the new Roche assay, Elecsys

Assay performance

This method was introduced early in 2009. The assay is based on chemiluminescence technology performed on an automated platform. Samples are subjected to a pre-treatment step to denature the vitamin D binding protein. The treated samples are then neutralised in assay buffer and a specific, anti-25OHD antibody labelled with acridinium is added. Following an incubation step, magnetic particles linked to 25OHD are added. After a further incubation step, the magnetic particles are “captured” using

Assay procedure

We identified seven publications (since 2000) with detailed information on the HPLC procedure [10], [13], [24], [25], [26]. Sample volumes ranged between 0.5 and 1 ml. Deproteinisation was achieved by acetonitrile [27], [10], ethanol [24], [26] an ethanol:acetonitrile mixture [25] or a methanol:isopropanol mixture [13]. Three [19], [25], [26] used an off-line solid phased extraction procedure and one used on automated on-line procedure [27]. Two procedures employed straight solvent extraction

Assay procedure

We identified seven publications (since 2000) containing detailed information on the LC–MS/MS method used [14], [15], [16], [22], [29], [30], [31]. Sample volumes were either 100 μl (n = 2) or 200 μl (n = 4). Deproteinisation was achieved by acetonitrile [16], acetonitrile plus sodium hydroxide [31], methanol [22], [30] or a methanol:propanol mixture [14]. Two used a liquid/liquid extraction with either n-heptane [15] or hexane [14]. The remaining five [16], [22], [30], [31] used SPE with either C8

Assay standardisation

There are a number of significant limitations of current methods for measuring 25OHD. Analysts have been aware of many of these problems for a number of years [7], [10], [35], [36] but it was a publication of Binkley et al. that first drew attention of the wider clinical community to the large variability in 25OHD results, both between methods and between laboratories [37]. They compared the values reported for samples from healthy individuals sent to six laboratories using different

Acknowledgements

We are extremely grateful to the Food Standards Agency (FSA) for support. This review formed part of an FSA International Workshop on 25OHD Methodology held at Gatwick Airport 2nd–4th November 2009 (Contract number NO8029).

We are also extremely grateful to the organizers of DEQAS, Graham Carter and Julia Jones, for sharing information on assay performance.

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