M. R. Helder1, B. Tefft2, R. Hennessy2, C. D. Koch4, D. B. Spoon2, R. D. Simari3, A. Lerman2 1Mayo Clinic,Cardiovascular Surgery,Rochester, MN, USA 2Mayo Clinic,Cardiovascular Diseases,Rochester, MN, USA 3University Of Kansas,School Of Medicine,Kansas City, KS, USA 4Mayo Clinic,Clinical Core Laboratory Services,Rochester, MN, USA
Introduction: One goal of tissue engineering is to develop heart valves for implantation with reduced thrombogenicity. Current in-vitro thrombogenicity tests are static and do not reflect the true thrombogenicity potential of valves. We present a novel in-vitro method to dynamically test the thrombogenic potential of heart valves and present our experiments for optimizing test conditions.
Methods: The Haemobile (Haemoscan, Inc) was modified for in-vitro thrombogenicity testing of heart valves with intact roots. The Haemobile is comprised of a polycarbonate fluid circuit that contains the valve, which is mounted onto a motor that creates pulsatile fluid flow without a pump system. Mechanical valves with aortic conduits were utilized in this study to validate the model and establish a positive control as well as optimal conditions for future studies. Various conditions were tested including temperature (4ºC, 25ºC, 37ºC), run time (3 hr, 5 hr), and blood:saline dilution (100% blood, 50% blood, 40% blood, 35% blood, 30% blood). Blood was obtained in an aseptic manner from sheep and immediately used for testing. Kaolin activated thromboelastography (TEG® 5000, Haemonetics,) was performed in duplicate to measure the clotting potential of blood samples collected prior to valve exposure and at specified time points following valve exposure. Thromboelastography showed clot potential at every dilution prior to starting the run. Any clots produced in the circuit were weighed.
Results: The optimal temperature for testing was determined by exposing undiluted blood to a mechanical valve for 5 hr. A macroscopic clot was produced only at 25ºC, and thus, this temperature was used in subsequent experiments. The shortest test time that formed a thrombus was 3 hr and this was the time point utilized in further studies. Blood dilutions were tested in order to create more stringent criteria to differentiate valve thrombogenicity. Dilutions of ≥ 40% blood created a clot that decreased linearly by mass with increasing dilution of blood samples. Correspondingly, TEG readings of the blood sample were more depressed with larger clots indicating that increasing amounts of coagulation factors and platelets were utilized (Figure).
Conclusions: The Haemobile provides a dynamic method to test the thrombogenicity of heart valves. A clot formed on a mechanical valve with dilutions ≥ 40% blood following 3 hr of exposure at 25ºC. In this model, there was excellent agreement between blood sample dilution, mass of thrombus and TEG readings allowing for small differences in valve thrombogenicity to be detected. Future studies will test the thrombogenicity of decellularized and other tissue engineered heart valves.
