Scalable manufacture of a disposable, storage-stable eight-channel microfluidic device for rapid testing of platelet, coagulation, and drug function under whole blood flow

Abstract

Custom polydimethylsiloxane (PDMS) microfluidic devices allow for small-volume human blood research under hemodynamic conditions of bleeding and clotting. However, issues of PDMS molding/assembly, bio-coating, and sample preparation often limit their point-of-care use. We aim to develop a microfluidic device that has the same utility as previously established PDMS devices but which is more usable in point-of-care operation. We designed an injection-molded 1 × 3 in.² device with eight flow paths crossing a bio-printed surface of a collagen/tissue factor. The device is rapidly primed and compatible with multi-channel pipetting (<0.5 ml blood) and operates under venous or arterial shear rates using constant flow rate or constant pressure modes. Platelet and fibrin deposition were monitored dynamically by the imaging of immunofluorescence. For whole blood clotting at a wall shear rate of 200 s^-1, the intrachip CV at 400 s for platelet and fibrin deposition was 10% and the interdonor CV at 400 s was 30% for platelet and 22% for fibrin deposition (across 10 healthy donors). No significant difference was detected for samples tested on a new chip vs a chip stored for 6 months at 4 °C. Using the fibrin signal, dose-response testing of whole blood revealed IC₅₀'s of 120 nM for rivaroxaban and apixaban, and 60 nM for dabigatran. A complete reversal of apixaban inhibition was observed for an equimolar addition of Xa DOAC reversal agent Andexanet Alfa. We demonstrate the ability to manufacture single-use, storage-stable eight-channel chips. In clinical settings, such chips may help evaluate patient bleeding risk, therapy choice, drug activity, or reversal.

FIG. 1.

(a) Schematic of injection molded chip with eight channels designed to have equal flow when applying a negative pressure at outlet. (b) Visual demonstration of priming, where priming fluid (green) injected in the dedicated priming inlet arrives at each inlet port and the outlet port simultaneously. (c) Blood added to inlet wells displaces priming fluid when a negative pressure applied at the outlet.

FIG. 2.

(a) 2D side view of the focal region of a single channel from a 3D model of chip. (b) Volumetric flow rate as a function of occlusion fraction (h/60), for a constant outlet vacuum pressure. (c) Pressure drop across the clot as a function of occlusion fraction. (D) Shear rate at the top surface of the clot as a function of clot height.

FIG. 3.

Average platelet intensity (a) and fibrin intensity (b) vs time for N = 10 donors, and n = 8 clots per donor for a constant outlet pressure yielding an initial wall shear rate of 200 s⁻¹. (c) Fluorescence microscopy images for platelets (AF488 CD-61) and fibrin (AF594-conjugated human fibrinogen) at t = 400 s.

FIG. 4.

Comparison of a chip used immediately after preparation (day 1) and a chip used 6 months after preparation (stored at 4 degrees Celsius in a desiccated chamber) for platelet (a) and fibrin (b) intensity. Data for N = 1 donor, and n = 8 clots per chip, where blood is perfused with constant outlet pressure yielding an initial wall shear rate of 200 s⁻¹. No significance was detected in the fresh vs stored chips for either platelets or fibrin (p > 0.05).

FIG. 5.

Fluorescence data for whole blood treated with Factor Xa inhibitors rivaroxaban (a)–(c) and apixaban (d)–(f), showing limited platelet response (a) but a strong dose-dependent response for fibrin signal (b). (c) and (f) Log[inhibitor] vs fibrin intensity indicating an IC50 of 120 nM for both inhibitors in this assay. A statistically significant difference in fibrin signal was observed at 500 s for both drugs (_*p < 0.01).

FIG. 6.

Fluorescence data for whole blood treated with Factor IIa inhibitor Dabigatran, showing limited platelet response (a) but a strong dose-dependent response for fibrin signal (b), and micrographs of the same at t = 400 s (d). (c) Log[inhibitor] vs fibrin intensity indicating an IC50 of 60 nM in this assay. A statistically significant difference in fibrin signal was observed at 500 s (_*p < 0.01).

FIG. 7.

Fluorescence data for whole blood treated with 200 nM Factor Xa inhibitor apixaban with and without mutant inactive factor Xa molecule Andexanet Alfa (Andexxa) as a reversal agent. Platelets (a) show no dependence on either drug or reversal agent, whereas fibrin (b) is partially inhibited by apixaban, but is recovered completely by an equimolar ratio of Andexanet Alfa, and partially recovered by 1:5 deficit of Andexanet Alfa.