OC 59.4 - A Microfluidic Approach in TTP Highlights the Need to Normalise ADAMTS13 Activity Levels in Clinical Remission in iTTP and the Effect of Different Treatments on Disease Activity in cTTP

Background: Thrombotic thrombocytopenic purpura (TTP) patients with sustained reduced ADAMTS13 activity could benefit from an assay that more comprehensively monitors disease activity and risk of relapse/response to treatment.

Aims: To use a dynamic microfluidic assay to monitor thrombogenicity in TTP patients in clinical remission.

Methods: Microfluidic channels were coated with collagen or an antibody against VWF-A3 domain (82D6A3). Blood was collected in citrate or lithium heparin from healthy volunteers (n=85), congenital TTP (cTTP) (n=47), or immune TTP (iTTP) (n=253) patients with normal platelet counts, following informed consent. Fluorescently-labelled whole blood was perfused at 1800s-1, monitored in real-time and analysed using software developed in-house. VWF and ADAMTS13 antigen and activity levels were also measured.

Results: Platelet coverage was significantly increased on both collagen and anti-VWF A3-coated microchannels in whole blood samples from cTTP and, importantly, also from iTTP patients with ADAMTS13 activity below the normal range (median activity 27.5IU/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, whole blood from TTP patients with sub-normal ADAMTS13 activity formed extensive thrombi with significantly increased area and length, positively correlated with VWF:ADAMTS13. In iTTP, these parameters normalised in matched complete remission samples. In cTTP, there was a significant reduction in platelet coverage, thrombi area and length when patients were receiving recombinant ADAMTS13 therapeutically compared to previous standard of care.

Conclusion(s): We successfully used a flow-based assay to investigate VWF-dependent platelet recruitment under flow as a rapid and sensitive monitoring tool for TTP patients, and an important research tool to study thrombogenesis. Our results using this assay suggest a need to normalise ADAMTS13 activity levels for iTTP patients in clinical remission and the benefit of using recombinant ADAMTS13 in cTTP.