Comparison of assays to evaluate ADAMTS13 activity in patients’ plasma

BACKGROUND : several assays for measurement of ADAMTS-13 activity have been developed. Although the performance characteristics of these methods were recently evaluated (Tripodi A, et al. JTH 2004), little information is currently available on concordance of these assays in plasma. The main goal of this study was to investigate whether widely available assays concordantly measure the same amount of ADAMTS-13 activity in plasma METHODS : plasma samples of 72 healthy subjects and 124 patients affected with thrombotic microangiopathies were assayed for ADAMTS-13 activity using residual collagen binding activity (CBA) and FRET assays. The Spearman’s rho and the Kappa statistics were used to assess the correlation and agreement between assays after categorization of plasma samples into 5 subgroups with different levels of ADAMTS-13 activity RESULTS : the measurement of ADAMTS-13 activity by CBA and FRET assays showed a correlation of 0.85 (P<0.0001). The concordance between the two assays was confirmed in 81% of samples (Kappa=0.68, P<0.0001). The major discrepancies were observed in 10 patients where FRET assay measured a lower ADAMTS-13 activity compared to CBA (Table I, grey area). Only 30% of these discordant data could be ascribed to plasmatic hyperbilirubinemia (Meyer SC, et al. JTH 2007). Additionally, in other three patients with severe haemolysis, ADAMTS-13 activity was not measurable using the FRET assay due to a non-parallelism of serially diluted plasma samples. These samples were easily analysed by CBA CONCLUSIONS : ADAMTS-13 activity determined by CBA and FRET assays confirmed a good concordance. However, FRET assay measured almost a lower ADAMTS-13 activity compared to CBA. The major advantage of FRET assay compared with CBA is that the results are available within 1h. However, its downsides is related to the use of fluorescent probes, which could be influenced by plasma factors as bilirubin interfering to the fluorescence emission and consequently to the results of FRET assay