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.