OC 26.4 - A Microfluidic Approach in TTP Highlights the Need to Normalise ADAMTS13 Activity Levels in Clinical Remission

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

Aims: To develop a dynamic assay to monitor TTP patients and establish ADAMTS13 levels required for normalisation of platelet aggregation under flow.

Methods: Microfluidic channels were coated with collagen or an antibody against VWF-A3 domain (82D6A). Blood was collected in citrate or lithium heparin from healthy volunteers, congenital (cTTP), or immune (iTTP) TTP patients with normal platelet counts, following informed consent. Fluorescently-labelled 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: 150 patient samples (32 cTTP, 118 iTTP) and 54 healthy controls were analysed. Time-dependent platelet capture was observed in all conditions, forming thrombi on collagen, and small aggregates on anti-VWF A3, which were not forming in the presence of a GPIIb/IIIa inhibitor. On both collagen and anti-VWF A3, platelet coverage was increased, not only in cTTP but, importantly, also for iTTP samples with sub-normal ADAMTS13 activity. Thrombi forming on collagen presented different geometries in TTP patients with reduced ADAMTS13 activity, with significantly increased thrombi area mainly attributable to thrombi length. The ADAMTS13 levels that are required to normalise these parameters and reduce thrombotic risk are currently being established.

Conclusion(s): We present a method to investigate VWF-dependent platelet recruitment under flow using an antibody against the VWF A3 domain to capture plasma VWF. This assay provides a rapid and sensitive diagnostic tool to monitor TTP patients, and an important research tool to study thrombogenesis. Our results using this assay highlight the need to normalise ADAMTS13 activity levels for TTP patients in clinical remission.