Enzymatic Functional Assays of Coagulation Using Small Sample Volumes

Area of Science:

• Biomedical Engineering
• Clinical Chemistry
• Hematology

Background:

• Current thrombophilia testing methods demand substantial blood volumes and extended processing times.
• Thrombophilia, a condition increasing clotting risk, requires accurate and timely diagnostic evaluation.

Purpose of the Study:

• To assess the feasibility of performing thrombophilia panel testing using enzymatic functional assays on a microfluidic cartridge.
• To evaluate the performance of microfluidic-based assays compared to traditional microtiter plate methods, focusing on sample volume reduction and accuracy.

Main Methods:

• Enzymatic functional assays for Antithrombin III, Protein C, Factor VIII, and plasminogen were adapted to a digital microfluidic platform.
• Novel fluorogenic substrates were developed, and on-cartridge fluorescence detection was established.
• Assay results from the microfluidic cartridge were compared against microtiter plate results using pediatric patient samples.

Main Results:

• Functional assays for key thrombophilia markers were successfully adapted to the microfluidic platform.
• On-cartridge assay results showed no significant difference compared to conventional microtiter plate methods.
• The microfluidic cartridge assays required significantly less plasma volume (25 μL) compared to microtiter plate assays (2 mL).

Conclusions:

• Microfluidic cartridge technology enables high-fidelity thrombophilia panel testing with substantially reduced sample volume.
• This approach offers a promising alternative to current laboratory methods, potentially enabling faster, near-patient diagnostics.
• Further clinical validation is necessary to establish the efficacy of this near-patient technology in real-world settings.