Impact of assay design on test performance: lessons learned from 25-hydroxyvitamin D.

BACKGROUND: Current automated immunoassays vary significantly in many aspects of their design. This study sought to establish if the theoretical advantages and disadvantages associated with different design formats of automated 25-hydroxyvitamin D (25-OHD) assays are translated into variations in assay performance in practice. METHODS: 25-OHD was measured in 1236 samples using automated assays from Abbott, DiaSorin, Roche and Siemens. A subset of 362 samples had up to three liquid chromatography-tandem mass spectrometry 25-OHD analyses performed. 25-OHD2 recovery, dilution recovery, human anti-animal antibody (HAAA) interference, 3-epi-25-OHD3 cross-reactivity and precision of the automated assays were evaluated. RESULTS: The assay that combined release of 25-OHD with analyte capture in a single step showed the most accurate 25-OHD2 recovery and the best dilution recovery. The use of vitamin D binding protein (DBP) as the capture moiety was associated with 25-OHD2 under-recovery, a trend consistent with 3-epi-25-OHD3 cross-reactivity and immunity to HAAA interference. Assays using animal-derived antibodies did not show 3-epi-25-OHD3 cross-reactivity but were variably susceptible to HAAA interference. Not combining 25-OHD release and capture in one step and use of biotin-streptavidin interaction for solid phase separation were features of the assays with inferior accuracy for diluted samples. The assays that used a backfill assay format showed the best precision at high concentrations but this design did not guarantee precision at low 25-OHD concentrations. CONCLUSIONS: Variations in design among automated 25-OHD assays influence their performance characteristics. Consideration of the details of assay design is therefore important when selecting and validating new assays.

Evaluation of two automated immunoassays for 25-OH vitamin D: comparison against LC-MS/MS.

BACKGROUND/METHODS: A total of 95 human serum specimens, and a 12 specimen precision panel, were measured by 2 automated immunoassays (investigation use only DiaSorin LIAISON(®) 25 OH Vitamin D TOTAL Assay [LSN], and Siemens ADVIA Centaur(®) Vitamin D Total (VitD) assay [Centaur]) and the results compared against LC-MS/MS [LCMS] used as the reference method (Esoterix Inc.). For functional sensitivity and precision, 12 serum specimens [range 1.2-148ng/mL] were run in six replicates [N=30] or four replicates [N=20], respectively, for 5 consecutive days. RESULTS: Passing-Bablok fit and Difference plot analysis [N=92] showed that although both immunoassays had comparable correlation coefficient [r] values to LCMS (0.936 and 0.933), the LSN assay results were statistically equivalent to those given by LCMS (slope 0.93, intercept -2.5), whereas the results of the Centaur assay showed overall significant assay bias compared to LCMS (slope 1.30, intercept -15.8) and this bias was more significant for doses <30ng/mL by LCMS [bias -30.4%; 95% limits of agreement -72.4% to 11.7%]. For specificity, based on 25-OHD2 and 25-OHD3 levels assessed by LCMS, we divided the specimens into 2 groups, one with detectable 25-OHD2 [Group 1, N=41] and the other with no detectible 25OHD2 [Group 2, N=51]. The 2 groups showed comparable correlation coefficient [r] values between the methods, but showed significant differences in slope: Centaur [1.48 with group 1 and 1.18 with group 2] compared to LSN [0.91 with Group 1 and 0.96 with Group 2]. LSN demonstrated better precision [total CV range 5.5-10.0%] compared to Centaur [total CV range 11.0-16.3%]. Functional sensitivity was calculated per EP-17A: 2.15ng/mL by LSN and 4.57ng/mL by Centaur. CONCLUSIONS: Though there was good overall correlation, substantial bias was present in Centaur. Although LSN had a slope and intercept that was not significantly different than LCMS, Centaur had a significantly higher slope in specimens containing measurable 25-OHD2 levels, a large negative intercept and a significant negative dose bias for doses <30ng/mL by LCMS, suggesting the Centaur assay would report a higher frequency of patients with apparent vitamin D insufficiency/deficiency at the low end and apparent vitamin D toxicity at the high end compared against LCMS. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

25-Hydroxyvitamin D testing: challenging the performance of current automated immunoassays.

BACKGROUND: Clinical laboratories require accurate and precise 25-hydroxyvitamin D (25-OHD) immunoassays to allow comparison of patient results with published decision limits. However, some variation in performance has been found with the previous generation of automated 25-OHD immunoassays. This study assessed the performance of four recently released automated 25-OHD immunoassays against a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. METHODS: A total of 983 samples from apparently healthy adults, plus 253 samples chosen to challenge the performance of the assays, were analyzed by the latest generation of immunoassays from Abbott, DiaSorin, Roche and Siemens. LC-MS/MS analysis was performed on a random subset of 264 samples. The precision of the immunoassays was assessed over 5 days with samples ranging between 3.0 and 370 nmol/L in concentration. RESULTS: Immunoassays showed significant differences in precision at 25-OHD concentrations of 3.0-86.5 nmol/L but all showed acceptable precision at higher concentrations. The DiaSorin assay agreed with LC-MS/MS across the measuring range of samples tested (7.7-425 nmol/L). The other assays showed generally good performance, but had some limitations when their performance was challenged with samples with low and high 25-OHD concentrations, heterophilic antibodies or high 25-OHD(2) concentrations. The C3-epimer of 25-OHD was identified in 40.4% of healthy adults tested and was a source of analytical variance in immunoassays. CONCLUSIONS: The latest generation of 25-OHD immunoassays has improved performance compared to previous assays. However, some immunoassays can still give discrepant results and this is most apparent when immunoassays are evaluated with a range of samples that challenge their analytical performance.