Evaluation of the Tosoh Bioscience 25OH vitamin D assay Wielders JPM1, te Stroet R1, Marivoet S2 1) Department of Clinical Chemistry, Meander Medical Center, Amersfoort, the Netherlands & 2) Tosoh Europe NV, Tessenderlo, Belgium Introduction Reliable 25(OH)vitamin D3 ( 25OHD3 ) assays are needed for the growing demand for measuring the vitamin D status. Tosoh introduced recently its ST AIA-PACK 25-OH Vitamin D assay which claims to measure both 25OHD3 and 25OHD2 in equimolar way. We tested precision and linearity, imprecision profile and LOQ, sample stability and the correlation with the Roche assay and the proposed LC-MSMS reference method. Methods We used an EP10 and EP5 protocol (EPevaluator) for basic precision and linearity testing. Method correlation with Roche Elecsys Vitamin Dtotal was performed using 75 left over serum samples. Sample stability was tested by 3 freezing – thawing cycles of 5 different sample pools and by storage of these 5 pools up to 4 days at room temperature and at 4 C. The Tosoh analyzer AIA-900 and the Tosoh 25OHD kit were used as prescribed by Tosoh. Results Imprecision and linearity: From the EP10 protocol we calculated a within run CV of 4.9% at 11.4 ng/ml and a CV of 2.2% at 41.9 ng/ml. The t values for non-linearity and drift are 1.2 and -0.2, respectively. Imprecision data calculated from the EP9 and EP5 protocol are given in table 1. Sample stability: At room temperature five left over samples (17.7 – 38.4 ng/ml) showed a increase up to 6.4%, probably due to evaporation. After 3 freeze/thawing cycles these 5 five samples showed an increase up to 4.3%. Method comparison: The Tosoh method correlated well with the Roche Dtotal assay (fig 3) n=75, especially in the normal range 20 – 40ng/ml. However, comparison with the LC-MSMS Figure 1: EP10 regression and Bland-Altman plots Tabel 1 Imprecision data EP5 protocol Level ( ng/ml ) 12.8 18.6 78.5 Inter run % CV 8.0 5.8 4.0 EP10 protocol Level ( ng/ml ) 11.4 26.7 41.9 Intra run %VC 4.9 2.4 2.2 Total % CV 5.9 3.8 2.3 Figure 3 Method comparison with Roche Dtotal Slope 0.87 (CI 0.83-0.91), intercept 3.3 (CI 1.3 – 4.7), R = 0.977 method of Thienpont (Ghent) using 29 samples containing both 25OHD3 and 25OHD2, showed significant bias (fig 4). Figure 2 below shows an LOQ < 4.6 ng/ml at CV 10%. titel Figure 4 Method comparison with proposed reference LC-MSMS Slope 1.35 (CI 1.12-1.57), intercept -5.3 (CI -14.5 – 4.0), R = 0.907 Declaration: Study design by Marivoet and Wielders. Equipment + reagents supplied by Tosoh. Results calculation, interpretation, conclusion by te Stroet and Wielders. Conclusion This basic evaluation shows that the imprecision and LOQ of the new Tosoh 25OHD kit are suitable for clinical use and comply with present standards for 25(OH)D3 analyses. However, the traceability to the proposed LC-MSMS reference method (Ghent) is at present unsatisfactory. Recalibration will probably solve this problem. Corresponding author JPM.Wielders@meandermc.nl