A chemiluminescent microfluidic thrombin generation assay for real-time monitoring in patient plasma.

Abstract

BACKGROUND: The thrombin generation assay (TGA) is a global coagulation test able to determine the amount of thrombin formed in plasma samples. TGA has been shown to be useful to monitor the effect of procoagulant and anticoagulation therapies in patients with bleeding diatheses or an increased thrombosis risk. OBJECTIVES: This study describes a new miniaturized platform to measure chemiluminescent-based microfluidic thrombin generation assay (TGA(lum)). METHODS: In TGA(lum), generated thrombin cleaves a β-Ala-Gly-Arg tripeptide from a caged aminoluciferin moiety, which almost instantaneously results in the generation of a photon by luciferase. The number of photons generated is proportional to the activity of thrombin in the microfluidic TGA(lum) system. By printing the assay components, a microfluidic cartridge was functionalized for TGA(lum). By only applying 20 μL of citrated plasma to the microfluidic cartridges, thrombin generation was monitored successfully with the characteristic peak profile in 110 nL detection chambers using a prototype readout device. RESULTS: The TGA(lum) was shown to be sensitive to therapeutic levels of anticoagulants (direct oral anticoagulants [DOACs]) to prevent thrombosis, as well as to the reversal effects of the antidotes thereof. Furthermore, TGA(lum) was able to detect the defect in the plasma of patients with hemophilia A (pwHA), as well as the hemostatic replacement effect of factor VIII infusion in pwHA. CONCLUSION: This study provides proof-of-concept that the present microfluidic TGA(lum) is applicable for monitoring therapy of pwHA at a global level and monitoring anticoagulant therapy with DOACs using a portable readout device.