Rapid on-chip recalcification and drug dosing of citrated whole blood using microfluidic buffer sheath flow

Abstract

Millions of clotting tests each year require recalcification of blood treated with sodium citrate, a calcium chelator that prevents prothrombinase assembly. We validated a converging trifurcated microfluidic device to measure platelet and fibrin accumulation following on-chip recalcification of citrated whole blood. Recalcification was accomplished by sheathing the blood with Ca²⁺ buffer. Fluorescein rapidly diffused across the buffer-blood interface (achieving 62.5% of maximum centerline concentration within ∼4 cm of flow), while albumin remained relatively unchanged in blood due to its lower diffusivity (<20% decrease). Since Ca²⁺ diffuses faster than fluorescein, full recalcification of whole blood was achieved within ∼1 cm of flow prior to encountering a collagen/tissue surface. Platelet and fibrin were reduced by 87.3% and 99.0%, respectively, when the sheath buffer was Ca²⁺-free. A 30-min preincubation of citrated whole blood prior to on-chip recalcification increased platelet (159%) and fibrin (86.6%) deposition, compared to 5-min preincubation, likely due to factor XIIa generation in citrated blood. The P2Y₁ inhibitor, MRS-2179, was delivered by diffusion into flowing blood and inhibited platelet deposition on collagen with a calculated IC₅₀ of 0.155 μM. On-chip recalcification and drug dosing of citrated blood allows for assays of platelet function in a whole blood milieu under flow.