A microfluidic approach reveals ongoing disease activity in thrombotic thrombocytopenic purpura patients despite clinical remission

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) patients with reduced ADAMTS-13 activity could benefit from a dynamic assay that comprehensively reflects disease activity and shows adequacy of treatment. OBJECTIVES: To use a microfluidic assay to monitor thrombogenicity in TTP patients. METHODS: Fluorescently-labeled whole blood was perfused through channels coated with collagen or an antibody against von Willebrand factor (VWF) A3 domain and monitored in real-time. RESULTS: Platelet coverage was significantly increased on both surfaces in samples from congenital TTP (cTTP) and, importantly, immune TTP (iTTP) patients with normal platelet counts but ADAMTS-13 activity below normal range (median activity, 34.5 IU/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:ADAMTS-13 ratio. On collagen, samples from cTTP and iTTP patients with subnormal ADAMTS-13 activity formed extensive thrombi with increased area and length, positively correlating with VWF:ADAMTS-13. In iTTP, these parameters were normalized in complete remission (ADAMTS-13 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 ADAMTS-13 prophylaxis compared with standard o -care. CONCLUSION: We successfully used a flow-based assay to investigate VWF-dependent platelet recruitment as a rapid and sensitive monitoring tool for TTP patients, which is an important research tool to study thrombogenesis. Our results using this assay, as a marker of ongoing microvascular thrombi formation, confirm the need to normalize ADAMTS-13 activity levels in iTTP patients in clinical remission and the benefit of using recombinant ADAMTS-13 in cTTP.