Theodora J Steeghs, Nerissa M M Coolen, Elena Krivosheeva, Steven P L Kuijpers, Zixuan Li, Jona Merx, Thomas J Boltje, Nicole M A Blijlevens, Saskia E M Schols, Cornelis van 't Veer, Waander L van Heerde
Thrombotic thrombocytopenic purpura (TTP) is a severe type of thrombotic microangiopathy with a high mortality rate. Detection of plasma ADAMTS13 activity levels <0.1 IU/mL confirms the diagnosis of TTP, whereas detection of anti-ADAMTS13 antibodies allows differentiation between congenital TTP and immune-mediated TTP. Unfortunately, turnaround times for ADAMTS13 assays are often long, hampering appropriate treatment decisions. This study describes the design and validation of a microfluidic luminescence-based ADAMTS13 assay (ADAMTS13lum) to quantify ADAMTS13 activity levels and detect ADAMTS13 antibodies in plasma samples using a portable device. The platform consists of a reusable portable processor and a disposable microfluidic cartridge. The cartridge contains an immobilized chimera of the von Willebrand factor (VWF) A2 domain in tandem with luciferase (Nter-Nluc-VWFA2-Cys-Cter). Upon cleavage by ADAMTS13, the liberated luciferase of the chimera is transported to the detection area that contains furimazine, the substrate for luciferase. The portable processor contains miniaturized photon sensors to quantify the luminescent signals. The signal output of the ADAMTS13lum assay is linear with ADAMTS13 activity levels in plasma samples. ADAMTS13 levels measured with the ADAMTS13lum assay are in good agreement with commercially available ADAMTS13 activity assays. Finally, inhibition of recombinant ADAMTS13 printed in a parallel line detects ADAMTS13 antibodies, allowing simultaneous quantification of ADAMTS13 activity levels and detection of ADAMTS13 antibodies. This technology enables rapid quantification of ADAMTS13 activity levels and detection of ADAMTS13 antibodies using a portable device.