Detecting Erythrocyte-Derived Extracellular Vesicles Generated from Blood Pump Flow and the Challenges Encountered

Utilization of a blood pump to aid in circulating a patient's blood, otherwise known as mechanical circulatory support, is an effective and often life-saving treatment for cardiac/pulmonary failure patients, yet adverse events remain a common complication often attributed to mechanical trauma inflicted on blood components. This work specifically focuses on erythrocyte-derived extracellular vesicles (ErEVs) as a marker of this mechanical trauma as they are elevated in patients with blood pumps and have been tied to adverse events. Despite this, ErEVs are typically neglected during device development which usually includes testing with animal blood, most commonly porcine and bovine. Flow cytometry was employed to monitor ErEVs generated during a 6 h perfusion of porcine or bovine red blood cells (RBCs) in a blood circulatory loop with the CentriMag blood pump. Successful measurement meant overcoming limitations in suitable stains for the RBCs and ErEVs of the two species. Between the two species, 12 different antibodies and dyes were evaluated, including multiple glycophorin A clones, the typical human erythrocyte antigen. Only CD46 and carboxyfluorescein succinimidyl ester (CFSE) were found to successfully and reliably label porcine and bovine RBCs, respectively. With these stains, statistically significant increases for both porcine and bovine ErEVs with perfusion time were observed. Bovine erythrocytes produced significantly more ErEVs than porcine, indicating they are more sensitive to mechanical trauma and could be useful in early-stage device development. The utility of CD46 and CFSE used for porcine and bovine ErEV detection was demonstrated for in vitro pump testing with implications for physiological and pathological research with these animals.