title: 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.
authors.affiliation: Institute for Medicine and Engineering, Department of Chemical and Biomolecular Engineering, 1024 Vagelos Research Laboratory, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Ryan W
authors.initials: RW
authors.lastname: Muthard
authors.affiliation: Institute for Medicine and Engineering, Department of Chemical and Biomolecular Engineering, 1024 Vagelos Research Laboratory, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Scott L
authors.initials: SL
authors.lastname: Diamond
mesh_terms: D054316:Biomarkers, Pharmacological; D001777:Blood Coagulation / Q000502:physiology*; D002951:Citrates / Q000378:metabolism*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D044085:Microfluidics / Q000379:methods*; D010973:Platelet Adhesiveness; D000077559:Sodium Citrate
chemical_list: D054316:Biomarkers, Pharmacological; D002951:Citrates; D000077559:Sodium Citrate
keywords: Thrombosis; citrate; fibrin; platelet