A microfluidic approach reveals ongoing disease activity in TTP patients despite clinical remission.

Background Thrombotic thrombocytopenic purpura (TTP) patients with reduced ADAMTS13 activity could benefit from a dynamic assay that comprehensively reflects disease activity and shows adequacy of treatment.Objective To use a microfluidic assay to monitor thrombogenicity in TTP patients.Methods Fluorescently-labelled whole blood was perfused through channels coated with collagen or an antibody against VWF A3 domain and monitored in real-time.Results Platelet coverage was significantly increased on both surfaces in samples from cTTP and, importantly, from iTTP patients with normal platelet counts, but ADAMTS13 activity below normal range (median activity 34.5IU/dl). There was heterogeneity in thrombogenicity in iTTP despite patients being in clinical remission. Surface coverage on anti-VWF A3 positively correlated with VWF antigen and activity, and with VWF:ADAMTS13 ratio. On collagen, samples from cTTP and iTTP patients with sub-normal ADAMTS13 activity formed extensive thrombi with increased area and length, positively correlated with VWF:ADAMTS13. In iTTP, these parameters normalised in complete remission (ADAMTS13 activity within normal range). In cTTP, there was a significant reduction in platelet coverage, thrombi area and length when patients were receiving treatment, particularly with recombinant ADAMTS13 prophylaxis compared to standard-of-care.Conclusions We successfully used a flow-based assay to investigate VWF-dependent platelet recruitment as a rapid and sensitive monitoring tool for TTP patients, and an important research tool to study thrombogenesis. Our results using this assay, as a marker of ongoing microvascular thrombi formation, confirm the need to normalise ADAMTS13 activity levels in iTTP patients in clinical remission and the benefit of using recombinant ADAMTS13 in cTTP.