title: A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time.
abstract: Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+and Mg2+levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.
authors.affiliation: Transfusion Research Center, Belgian Red Cross-Flanders; Faculty of Medicine and Health Sciences, Ghent University.
authors.forename: Katrijn R
authors.initials: KR
authors.lastname: Six
authors.affiliation: Transfusion Research Center, Belgian Red Cross-Flanders.
authors.forename: Rosalie
authors.initials: R
authors.lastname: Devloo
authors.affiliation: Transfusion Research Center, Belgian Red Cross-Flanders.
authors.forename: Britt
authors.initials: B
authors.lastname: Van Aelst
authors.affiliation: Faculty of Medicine and Health Sciences, Ghent University; Blood Service, Belgian Red Cross-Flanders; Department of Public Health and Primary Care, KULeuven - University of Leuven.
authors.forename: Philippe
authors.initials: P
authors.lastname: Vandekerckhove
authors.affiliation: Transfusion Research Center, Belgian Red Cross-Flanders; hendrik.feys@rodekruis.be.
authors.forename: Hendrik B
authors.initials: HB
authors.lastname: Feys
authors.affiliation: Transfusion Research Center, Belgian Red Cross-Flanders; Faculty of Medicine and Health Sciences, Ghent University; Blood Service, Belgian Red Cross-Flanders.
authors.forename: Veerle
authors.initials: V
authors.lastname: Compernolle
mesh_terms: D000925:Anticoagulants / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology*; D001780:Blood Coagulation Tests / Q000379:methods*; D001792:Blood Platelets / Q000378:metabolism / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D005174:Factor XII / Q000494:pharmacology; D005337:Fibrin / Q000378:metabolism*; D006487:Hemostasis / Q000502:physiology; D006801:Humans; D044085:Microfluidics / Q000379:methods*; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology*; D017713:Platelet Transfusion / Q000379:methods*
chemical_list: D000925:Anticoagulants; D005174:Factor XII; D005337:Fibrin; D003094:Collagen; D002118:Calcium