The measurement of 25-hydroxy vitamin D – an analytical challenge

Abstract

Over the last decade requests for the measurement of 25-hydroxy vitamin D (25-OHD) have risen exponentially. For example, statistics from the Australian government provides evidence for a 100-fold increase between 2000 and 2010 (1) . This burst of vitamin D requests has several causes including an increased awareness of the medical community and the general population to the high prevalence of vitamin D defi ciency and its relevance for osteoporosis, cardiovascular disease, malignancies, infectious and autoimmune disease. The massively increased demand for 25-OHD testing has important implications for healthcare systems and clinical laboratories. Whereas healthcare systems worldwide face an explosion of costs for 25-OHD testing, laboratories have to adopt methods that can cope with the elevated workload. External quality control programs show that most laboratories use automated immunoassays. Diagnostic manufacturers have identifi ed 25-OHD as a highly profi table and strategic test, which has lead to the commercialization of new automated immunoassays during recent years. However, it has to be acknowledged that 25-OHD is a challenging analyte to accurately measure in human blood (2) . Analytical diffi culties are related to its lipohilic nature, strong affi nity to vitamin D binding protein (VDBP) and association with human serum albumin, as well as the presence of very low to high levels of 25-OH vitamin D 3 (25-OHD 3 ) and/or 25-OH vitamin D 2 (25-OHD 2 ), multiple 25-OHD metabolites (e.g., 24,25-dihydroxy vitamin D 3 ), C3-epimer of 25-OHD 3 and 25-OHD 2 (25-OHD 3 -epi and 25-OHD 2 -epi), and other sources of assay interference, such as heterophilic antibodies. Therefore, 25-OH vitamin D immunoassays are required to detect 25-OHD 2 and 25-OHD 3 in an equimolar fashion and report a total 25-OHD result.Previous automated immunoassay comparison studies have demonstrated signifi cant analytical limitations for some of these assays (2, 3) . External quality assurance programs, such as DEQAS, or the Quality Assurance Program of the Royal Australian College of Pathologists, also show a wide spread of reported results for the same sample, which in some cases vary from defi cient ( < 25 nmol/L) to suffi cient ( > 75 nmol/L). Analytical problems of 25-OHD immunoassays have also been noted by clinicians who often question the laboratory about results that do not correspond to the clinical picture of their patients. The apparent issues of automated immunoassays have resulted in the worldwide withdrawal of the Roche 25-OHD 3 assay in 2010 and to the modifi cation of the assay conditions of several other commercial immunoassays, such as SIEMENS Centaur, Abbott ARCHITECT and DiaSorin LIAISON. Regulatory bodies, such as the FDA have also become aware of analytical issues with some of the recently launched assays. For example, the FDA has imposed certain conditions on some of the recently cleared 510(k) assays, such as limiting the measuring range of the Abbott ARCHITECT assay from 13 ng/mL (32.5 nmol/ < L) to 96 ng/mL (240...

Automated Competitive Protein‐Binding Assay for Total 25‐OH Vitamin D, Multicenter Evaluation and Practical Performance

“…Other recent publications based on the Elecsys assay have confirmed that the assay has good precision and accuracy, and shows close agreement to other wellestablished methods for 25-OHD analysis, making it very suitable for routine assessment of vitamin D status (32,36,39,40). In conclusion, the Elecsys assay demonstrated low imprecision, high sensitivity, good lot-to-lot consistency, as well as good overall agreement with measurements obtained using LC-MS/MS and HPLC methods.…”

Automated measurement of 25-OH Vitamin D on the LUMIPULSE®G1200: analytical verification and method comparison

“…Characteristics of 25OH-D molecule that can interfere with assays include the lipophilic nature of Vitamin D, high levels of 25OH-D2, C3-epimers, heterophilic antibodies, strong affinity to VDBP and association with human serum albumin. [6] Furthermore, because of treatments with Vitamin D2 supplementation, it is also important that Vitamin D assays measures in an equimolar fashion both 25-OHD2 and 25-OHD3 and reports the total 25-OHD result. For these reasons, differences between assays are often remarkable and the latter studies about the performance of 25OH-D assays stressed the need of standardisation and harmonisation of 25OH-D measurements.…”

“…gov), specifically NIST SRM 972, and the application of reference measurement procedures (RMPs) validated by NIST and Ghent University. [6][7][8] Immunoassays are also a useful tool to measure 25OH-D, with the ability to detect both 25OH-D2 and 25OH-D3 in an equimolar manner, and so the opportunity to report total 25OH-D. [6] As a result of the increased Vitamin D testing, there has been a raised interest in automated Vitamin D immunoassays. Several manufacturers have launched automated 25OH-D immunoassays, such as Abbott (Chicago, IL, USA), Diasorin (Saluggia, Italy), IDS (Boldon, United Kingdom), Roche (Basel, Switzerland), Siemens (Munich, Germany) and, more recently, Fujirebio (Ghent, Belgium).…”

Evaluation of Vitamin D Assays in Relation to the Measurement Range and the Statistical Method

“…Despite huge number of studies, there is not a clear agreement on this subject due to several reasons: the study range and the number of participants, the cross-reactivity of heterophilic antibodies, the 25-OHD 2 -and 25-OHD 3 -epimer interferences with the methods used in the studies, and the influence of the concentration of vitamin D binding protein. 33,34 In addition to these factors, we hypothesized that the statistical procedures used to evaluate the performance of the methods can also contribute to the inconsistency observed between different studies. Therefore, in the present study, we attempted to investigate how the performances of three methods changed according to the study range chosen, and the statistical method used.…”