Hydroxyvitamin D assays: An historical perspective from DEQAS.

Recent years have seen a substantial increase in demand for 25-hydroxyvitamin D (25-OHD) assays. DEQAS (the Vitamin D External Quality Assessment Scheme) has been monitoring the performance of these assays since 1989. The first DEQAS distribution was in June 1989 and results were submitted by 13 laboratories in the UK, two of which used HPLC/UV; the rest used ligand binding assays with a tritium tracer. Inter-laboratory CVs (ALTM) ranged from 29.3% (42.7nmol/L) to 53.7% (20.0nmol/L). Currently the scheme has participants in 56 countries using 30 methods or variants of methods. In January 2017, 918 participants returned results and inter-laboratory CVs (ALTM) ranged from 10.3% (73.1nmol/L) to 15.3% (29.4nmol/L). Over the last 27 years, there have been a number of significant milestones in assay development. The first major advance was the development of an iodinated 25-OHD tracer by Hollis and Napoli in 1992, subsequently used in an RIA kit marketed by DiaSorin. This and other commercial radioimmunoassays that followed brought 25-OHD assays within reach of many more non-specialist routine laboratories. With the introduction of fully automated non-isotopic assays without solvent extraction, measurement of 25-OHD became available to any clinical chemistry laboratory with an appropriate analytical platform. However, as the limitations of these non-extraction assays became apparent more laboratories started using LC-MS/MS methodology. Meanwhile the variable accuracy of 25-OHD methods has been addressed by the Vitamin D Standardization Program (VDSP) which encourages manufacturers to produce methods traceable to the reference measurement procedures (RMPs) of NIST, University of Ghent and the Centers for Disease Control and Prevention (CDC). DEQAS changed to an accuracy-based scheme in 2013 and now assesses assay accuracy against the NIST RMP. This review will use DEQAS results and statistics to chart the historical development in 25-OHD assay technology and highlight some of the problems encountered in obtaining reliable results for this most challenging of analytes.

Quality assessment of vitamin D metabolite assays used by clinical and research laboratories.

The Vitamin D External Quality Assessment Scheme (DEQAS) was launched in 1989 and monitors the performance of 25-hydroxyvitamin D (25-OHD) and 1,25- dihydroxyvitamin D (1,25(OH)2D) assays. In April 2015 a pilot scheme for 24,25-dihydroxyvitamin D (24,25(OH)2D) was introduced. The 25-OHD scheme is accuracy - based with target values assigned by the NIST Reference Measurement Procedure (RMP) for 25-OHD2 and 25-OHD3. A similar method is used to assign values for 3-epi-25-OHD. Five samples of human serum are distributed quarterly to over 1000 participants in 58 countries (April 2016) and clinical laboratories are expected to submit results within approximately 5 weeks. Research laboratories with assays run less frequently are not given a deadline. Archived samples with NIST- assigned values are also available. Performance is assessed on the first four samples with the fifth reserved for investigations e.g. recovery experiments or to assess the influence of other serum constituents such as lipids. DEQAS provides rapid feedback, with an on-line preliminary report available immediately after a participant submits results and a comprehensive report soon after the results deadline. In 2015, DEQAS investigations revealed that several 25-OHD immunoassays under-recovered 25-OHD2 and 25-OHD results were falsely low on a sample with a modestly raised triglyceride concentration. An RMP for 1,25 (OH)2D is not yet available and results are judged against the Method Mean. Free advice is available from the DEQAS Advisory Panel which includes experts on methodology and biostatistics. DEQAS collaborates closely with the Vitamin D Standardization Program (VDSP) and both organizations have successfully worked with participants and manufacturers to improve the accuracy of vitamin D assays.

Proficiency testing of 25-hydroxyvitamin D (25-OHD) assays.

The Vitamin D External Quality Assessment Scheme (DEQAS) has been monitoring 25-OHD assay performance since 1989. The scheme has expanded rapidly in recent years and has 670 participants in 35 countries (July 2009). Five samples of human serum are distributed quarterly and the results analyzed to give an All-Laboratory Trimmed Mean (ALTM) and SD. Each participant has internet access to a preliminary report after submission of results and, following the results deadline, a final report is e-mailed to designated staff in each laboratory. The last 15 years has seen an improvement in mean inter-laboratory imprecision (CV), from 32.0% (1994) to 15.3% (2009) and most major methods are now giving results within plus or minus 7.4% of the ALTM (2009). DEQAS has regularly conducted and reported on a number of investigations into the performance of 25-OHD methods. A gas chromatography-mass spectrometry (GC-MS) reference method for 25-OHD is under development and will be used to assess whether the ALTM remains the most appropriate target for DEQAS samples.

Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation.

The International Quality Assessment Scheme for Vitamin D metabolites (DEQAS) was introduced in 1989. Initially, the aim was to improve the reliability of 25-hydroxyvitamin D (25-OHD) assays but the scheme was extended in 1997 to include 1,25-dihydroxyvitamin D (1,25(OH)(2)D). DEQAS has 95 members in 18 countries (January 2003). Five serum samples are distributed quarterly and participants are given up to 6 weeks to return their results for statistical analysis. The majority of participants use commercial kits for both analytes. A performance target was set by an advisory panel in 1997 and, at present, requires participants to get 80% or more of their results within +/-30% of the All-Laboratory Trimmed Mean (ALTM). The performance targets are under continual review. In 2003, 59% of participants met the target (cf. 52% in 2000). A questionnaire, distributed in January 2003, requested information on methods and the interpretation of results. Reference ranges varied but there was reasonable agreement on the 25-OHD concentrations below which Vitamin D supplementation was advised. A minority (22%) of respondents was unsure whether Vitamin D(3) or Vitamin D(2) was used to treat patients in their locality. The majority (52%) of assays for 1,25(OH)(2)D were done 'on demand' and others for apparently spurious reasons. Most respondents thought participation in DEQAS extremely important and the planned introduction of on-line reporting should enhance its value.