The Evaluation of Leukocytes in Response to the In Vitro Testing of Ventricular Assist Devices *†Chris H.H. Chan, †Andrew Hilton, †Graham Foster, *Karl M. Hawkins, *Nafiseh Badiei, and *Catherine A. Thornton *Institute of Life Science, College of Medicine, Swansea University; and †Calon Cardio-Technology Ltd, Swansea, UK Abstract: Infection is a clinically relevant adverse event in patients with ventricular assist device (VAD) support.The risk of infection could be linked to a reduced immune response resulting from damage to leukocytes during VAD support.The purpose of this study was to develop an under- standing of leukocyte responses during the in vitro testing of VADs by analyzing the changes to their morphology and biochemistry. The VentrAssist implantable rotary blood pump (IRBP) and RotaFlow centrifugal pump (CP) were tested in vitro under constant hemodynamic conditions. Automated hematology analysis of samples collected regu- larly over 25-h tests was undertaken. A new flow cytometric assay was employed to measure biochemical alteration, necrosis (7-AAD) and morphological alteration (CD45 expression) of the circulating leukocytes during the pumping process. The results of hematology analysis show the total leukocyte number and subset counts decreased over the period of in vitro tests dependent on different blood pumps. The percentage of leukocytes damaged during 6-h tests was 40.8 ± 5.7% for the VentrAssist IRBP, 17.6 ± 5.4% for the RotaFlow CP, and 2.7 ± 1.8% for the static control (all n = 5). Flow cytometric monitoring of CD45 expression and forward/side scatter characteristics revealed leukocytes that were fragmented into smaller pieces (microparticles). Scanning electron microscopy and imaging flow cytometry were used to confirm this. Device developers could use these robust cellular assays to gain a better understanding of leukocyte-specific VAD per- formance. Key Words: Leukocyte damage—Ventricular assist devices—Flow cytometric assay—Leukocyte-derived microparticles. The availability of organs for transplantation has declined over recent years. Innovations in ventricular assist device (VAD) technology have provided an alternative therapeutic option for patients with advanced heart failure (1). The mechanisms underly- ing problems with cardiovascular devices such as bleeding, thromboembolism, infections, and systemic inflammation are not well understood, but damage to the cellular components of blood have been impli- cated (2–7). Infection is a clinically relevant adverse event in patients with VAD support (8). Previously, platelet– leukocyte aggregates were suggested to be the root cause of activation of the thrombotic and inflam- matory cascade in patients with VAD support (9,10). Shive et al. suggested a mechanism through which shear stress plays an important role in the persistence of bacterial infections at sites of cardiovascular device implantation under flow conditions (11). Fur- thermore, Diehl et al. recently noted significantly elevated platelet, leukocyte, and endothelial cell- derived microparticles in response to high shear stress (12). The artificial surface of mechanical assist devices and nonphysiological flow regimes could promote vascular procoagulation, thromboembolic complications, and inflammation. Additional insights into the role that microparticles play in patients with VAD are eagerly anticipated. In vitro testing remains a common part of cardio- vascular device development due to its being less complicated than surgery and of low cost. Minimizing blood trauma is a fundamental requirement for all blood pumps and most VAD research focuses on hemolysis (damage to red blood cells) but not on damage to leukocytes. In this article, we have doi:10.1111/aor.12161 Received January 2013; revised May 2013. Address correspondence and reprint requests to Dr. Chris Hoi Houng Chan, Swansea University, College of Medicine, Singleton Park, Swansea SA2 8PP, UK. E-mail: chris_houng@caloncardio .com; h.h.chan@swansea.ac.uk Presented in part at the 20th Congress of the International Society for Rotary Blood Pumps held September 20–22, 2012 in Istanbul, Turkey. © 2013 Wiley Periodicals, Inc. and International Center for Artificial Organs and Transplantation Artificial Organs 2013, 37(9):793–801