Perfusion chamber with static mixer: new possibilities to investigate the interplay between platelet aggregation and coagulation under flow S.W.J. de Vet (BSc) BMTE 12.01 Supervisors: Frans van de Vosse* Arjen Bogaerds* Attie Tuinenburg** Committee: Frans van de Vosse* Arjen Bogaerds* Mark Roest** Han Meijer*** Peter Neerincx*** * Eindhoven University of Technology Department of Biomedical Engineering ∗∗ University Medical Center Utrecht Department of Clinical Chemistry and Hematology ∗∗∗ Eindhoven University of Technology Department of Mechanical Engineering Eindhoven, January 18, 2012 Abstract In this study a perfusion chamber has been developed in which both platelet aggregation and fibrin formation can be visualized in real-time during flow over a coated coverslip. This has been reached by including a static mixer in the perfusion chamber. In this way, a CaCl2 solution and sodium-citrated blood can be properly mixed within the order of ten seconds before the blood flows over the coverslip. By re-adding calcium to citrated blood, clotting factors can be activated again. Due to reactivation of clotting factors, not only the primary hemostasis has been visualized but also the coagulation. By means of injection molding production process the perfusion chamber has been made as a disposable. By using a disposable perfusion chamber, it is assured that no clotting factors which will disturb the measurements are present in the chamber. To approach physiological conditions an aluminium frame that can be attached to the perfusion chamber has been equiped with a temperature controller. The operation of the disposable perfusion chamber has been proved by labeling platelets with DiOC6 and by adding fluorescent fibrinogen to citrated blood. By using a microscope with dual camera, the aggregation of platelets as well as the development of the fibrin network at the coverslip was recorded simultaneously. Samenvatting In dit onderzoek is een perfusiekamer ontwikkeld waarmee zowel de aggre- gatie van bloedplaatjes als de ontwikkeling van het fibrine netwerk realtime gevisualiseerd kan worden tijdens stroming over een dekglaasje gecoat met collageen. Dit is gerealiseerd door een statische mixer in de perfusiekamer in te bouwen. Op die manier kan een CaCl2-oplossing en gecitreerd blood binnen een tiental seconde homogeen mixen voordat het mengsel over het dekglaasje stroomt. Door calcium opnieuw toe te voegen aan gecitreerd bloed, worden stollingsfactoren opnieuw geactiveerd. Door de reactivatie van de stollingsfactoren, kan behalve de primaire hemostase ook de coagu- latie gevisualiseerd worden. Om massaproductie mogelijk te maken worden de perfusiekamers gespuitgiet, wat resulteert in wegwerpbare perfusiekamers. Door gebruik te maken van wegwerpbare perfusiekamers heb je geen last van stollingsfactoren die vanuit het vorige experiment zijn achtergebleven in de perfusiekamer, wat resulteert in de vermindering van de ruis op de volgende metingen. Om fysiologische thermische omstandigheden te bereiken wordt het aluminium frame, dat om de perfusiekamer zit, opgewarmd met behulp van een temperatuurregelaar. Om de werking van de wegwerpbare perfusiekamer te testen zijn de bloed- plaatjes gelabeld met DiOC6 en is er gefluoresceerd fibrinogeen aan gecitreerd bloed toegevoegd. Door gebruik te maken van een microscoop met duale camera, kan zowel de aggregatie van bloedplaatjes als de ontwikkeling van het fibrine netwerk tegelijkertijd opgenomen worden. Contents 1 Introduction 3 1.1 Hemostasis . 4 1.2 Perfusion experiments . 7 1.3 Problem description . 10 1.4 Requirements perfusion chamber . 12 2 Design process 14 2.1 Prototype 1 perfusion chamber . 14 2.1.1 Introduction . 14 2.1.2 Design . 16 2.1.3 Results . 18 2.1.4 Conclusion . 18 2.2 Prototype 2 perfusion chamber . 18 2.2.1 Introduction . 18 2.2.2 Design . 18 2.2.3 Results . 20 2.2.4 Conclusion . 20 2.3 Final model perfusion chamber . 21 2.3.1 Introduction . 21 2.3.2 Design . 21 2.3.3 Results . 24 2.3.4 Conclusion . 25 3 Pilot studies with whole blood 26 3.1 Materials and methods . 26 3.1.1 Blood donation . 26 3.1.2 Materials and Reagents . 26 3.1.3 Coating coverslips . 27 3.1.4 Labeling platelets . 27 3.1.5 Perfusion experiments . 28 3.2 Results . 30 1 4 Discussion 35 5 Future research 40 5.1 Coating coverslips . 40 5.2 Contact activation test . 40 5.3 Hemolysis test . 42 5.4 Platelet activation test . 43 6 Conclusion 44 A Homogeneity shear rate 49 2 Chapter 1 Introduction Hemostasis is the process of blood clot formation at the site of vessel injury. When a blood vessel wall is disrupted, the hemostatic response must be fast, localized, and carefully regulated. When specific elements of this process are missing or dysfunctional, abnormal bleeding or nonphysiologic thrombosis may occur. At the Department of Clinical Chemistry and Hematology of the University Medical Center Utrecht (UMCU) hemostasis is being studied to get more insight into bleeding and thrombotic disorders. To study these disorders and hemostasis in vitro, perfusion chambers (also named flow cham- bers) have been developed. The process of hemostasis is dependent on the interplay between platelets, coagulation, the vessel wall and blood rheology. Perfusion chambers enable experiments with blood under well-controlled con- ditions of exposure time, thrombogenic surface and reproducible flow, thereby approaching physiological conditions. When blood is drawn from a donor it is collected in a tube with an anticoagulant buffer. Without this anticoagulant buffer the blood will start clotting soon after it leaves the vein, and it will not be usable for in vitro experiments. Therefore, the effects of thrombin genera- tion are missing in the perfusion experiments. Sodium-citrate is a frequently used anticoagulant and inhibits coagulation by chelating calcium ions in the blood. By re-adding calcium to the citrated blood, clotting factors can be activated again. However, a proper mix between anticoagulated blood and a calcium solution can not be obtained in the standard perfusion chamber. The goal of this study is to design a perfusion chamber with a static mixer to achieve a proper and reproducible mix between citrated blood and CaCl2 solution in a short time, and thus to visualize the whole hemostasis. To pre- vent after each experiment to clean the remaining clots and clotting factors in the perfusion chamber, the chamber will be made disposable by the use of mass production. To enable mass production of the perfusion chamber, the final perfusion chamber has been designed such that it can be manufac- 3 tured by means of injection molding. By adding fluorescent fibrinogen to the blood sample which is perfused, the performance of the new perfusion chamber will be tested. The microfluidic device with static mixer gives us a more accurate in vitro model of hemostasis. It gives us new possibilities to study the interplay between platelets and coagulation under conditions of flow. By involving the coagulation to the perfusion experiments it is possible to: 1) study the effects of thrombin generation to the hemostasis, 2) diagnose the case at patients with bleeding disorders 3) study the effects of a stenose on the forming thrombi thereby reproducing patients with atherosclerosis, 4) replace in vivo animal testing by these perfusion experiments, and 5) study the effects of different anticoagulants to the hemostasis. 1.1 Hemostasis Hemostasis is a complex process which aims to stop bleeding by sealing a defect in the vessel wall. After injury, the vessel wall constricts, thereby reducing blood loss. Due to the injury, the subendothelial matrix becomes exposed to the circulating blood. The subendothelial matrix consists of adhe- sive proteins, of which collagen is an important one. Platelets adhere to these proteins, become activated and form unstable platelet aggregates. This pro- cess is called primary hemostasis. Simultaneously with the process of primary hemostasis, secondary hemostasis (coagulation) is triggered due to the ex- posed tissue factor. The end product of coagulation is a fibrin network which stabilizes the unstable platelet plug. The interplay between platelets, coagu- lation and endothelium or subendothelial matrix is important in hemostasis. In addition, also the flow and rheology of blood has a significant impact on hemostasis [1]. In the following paragraphs, a detailed description of in vivo platelet aggregation and coagulation will be given. After vessel wall injury, platelets are exposed to the subendothelial ma- trix. The plasma protein von Willebrand factor (vWF) binds to the collagen fibers of the subendothelium. From that moment vWF becomes able to interact with glycoprotein (GP) Ib [2], [3]. This unstable vWF-GPIb inter- action facilitates tethering and rolling of platelets over the subendothelium. By doing so, platelets are slowed down. A continuous loss of vWF-GPIb interactions at one site of the platelet and the formation of new interactions on the other side of the platelet support the rolling process [4], [5]. The rolling of the platelets will finally result in firm adhesion to collagen via the platelet GPIa/IIa (integrin α2β1) [6], [7], followed by activation of intracellu- lar signaling pathways by platelet GPVI [7], [8]. Alternative interactions to get firm adhesion are: 1) GPIIb/IIIa (integrin αIIbβ3) via vWF, fibrinogen, 4 fibronectin and vitronectin [2], [3], 2) integrin α5β1 via fibronectin [2], 3) in- tegrin α6β1 via laminin and 4) integrin αV β3 via vitronectin [2]. Due to these adhesive proteins platelets are now activated. It results in an increase of the cytosolic calcium concentration [9], exposure of phosphatidylserine (PS) and P-selectin, secretion of α-granules (e.g. with vWF, fibrinogen and activated factor V), secretion of dense bodies (e.g. with calcium, ADP and throm- boxane A2) and conformational changes of specific platelet receptors [2], [3], [10], [11]. Subsequently the activated platelets along with PS, P-selectin and activated factor V (Va) support the coagulation cascade [10], [12]. The co- agulation will run parallel to the primary hemostasis and will be discussed in the next paragraph.