Measurement of Circulating 25-Hydroxy Vitamin D Using Three Commercial Enzyme-Linked Immunosorbent Assay Kits with Comparison to Liquid Chromatography: Tandem Mass Spectrometry Method

Aim. The purpose of this study was to compare the accuracy and clinical implications of three commercial enzyme-linked immunosorbent assay (ELISA) kits (Eagle Biosciences, Immundiagnostik, and MicroVue) with a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of serum 25(OH)D concentration. Methods. Blood samples were obtained from 225 healthy individuals who were recruited as subjects from Loughborough University, UK. Plasma samples were measured for 25(OH)D concentration by means of LC-MS/MS and ELISA kits from Eagle Biosciences, Immundiagnostik, and MicroVue. Results. The 25(OH)D concentration measured by the Eagle Biosciences, Immundiagnostik, and MicroVue ELISAs biased ？50.9 ± 79.1？nmol/L, ？14.2 ± 91.0？nmol/L, and ？7.2 ± 18.9？nmol/L (bias ± SD) from the LC-MS/MS method, respectively. We found that 52% (Eagle Biosciences), 48% (Immundiagnostik), and 38% (MicroVue) of participants were misclassified, and the results showed the poor agreement (Kappa: ？0.201~0.251) in classification of participants defined as vitamin D sufficiency and insufficiency between each method and LC-MS/MS. Conclusions. The present study demonstrated that there were negative biases and considerable misclassification of participants using the cut-off point (50？nmol/L) for vitamin D insufficiency and sufficiency using the Eagle Biosciences, Immundiagnostik, and MicroVue ELISAs compared with the LC-MS/MS assay. 1. Introduction Recently, there have been growing demands for measurement of vitamin D status because of the high prevalence of vitamin D insufficiency and the discovery of vitamin D nonclassical functions [1, 2]. The high prevalence of vitamin D insufficiency in the general population worldwide has been documented in a large number of studies [3]. Moreover, vitamin D insufficiency has also been reported to be common in athletes especially if exposure to natural sunlight is limited (e.g., when training in the winter months or when training mostly indoors) [4]. In addition, it has been recently recognised that vitamin D plays an important role in upregulating immunity. Several recent studies have found a negative association between vitamin D status and respiratory illness incidence in young and elderly adults [5, 6]. Measurement of plasma 25-hydroxy vitamin D (25(OH)D) concentration is widely used in clinical practice and research reports to assess vitamin D status. In humans, vitamin D can be obtained either from dietary sources or the epidermal layer of the skin via exposure to sunlight. Two forms of vitamin D can be obtained