Enzymatic functional assays of coagulation using small sample volumes

Abstract

Current laboratory methods for comprehensive thrombophilia status require large blood volumes and long turn-around times. We demonstrate the feasibility of performing thrombophilia panel testing of enzymatic functional assays on a microfluidic cartridge using low sample volume. Functional assays for Antithrombin III, Protein C, Factor VIII, and plasminogen were adapted on the digital microfluidic platform by developing novel fluorogenic substrates and establishing on-cartridge fluorescence (360/460 nm) detection. Cartridge vs. microtiter plate results were compared using samples obtained from pediatric patients. Linear regression and Bland-Altman plots were used to establish correlations. Results were not significantly different when performed on-cartridge compared to microtiter plates. Importantly, the sample volume required is significantly lower for all on-cartridge compared to microtiter plate assays (25 μL vs. 2 ml). This study demonstrates the feasibility of thrombophilia panel testing with high-fidelity using small plasma volume. The efficacy of this near-patient technology in clinical settings needs further investigation.