Poster U7 - Erythrocyte Extracellular Vesicle Production at a Flow Contraction

Introduction: Mechanical circulatory support (MCS) is a treatment for cardiac/pulmonary failure patients, and while it can be lifesaving, it still has complications often attributed to hemocompatibility issues. MCS device development has historically focused on minimizing hemolysis and shear stress magnitudes to improve patient issues and outcomes, and while this has improved MCS treatment, adverse events, such as thrombosis and bleeding, persist. Two areas that have received little attention in MCS development are extensional stresses and sublethal trauma to blood components. The effect of extensional stresses was thought to be negligible due to their small magnitude and prevalence in devices compared to shear, but recent studies indicate that extensional stresses might be more damaging to erythrocytes than shear stresses of equal magnitude. Additionally, other recent studies indicate that elevated levels of phosphatidylserine positive (PS+) EVs in MCS patients, could contribute to the development of common adverse events, such as thrombosis and bleeding. Further study is warranted to better understand the effect of both types of fluid stresses on blood components and the resulting sublethal damage. In this preliminary study, we examined the production of erythrocyte EVs (ErEVs) in either extensional or shear stress dominated environments. Exposure of erythrocytes to extensional or shear stress occurred during flow through a nozzle in a mock flow loop with either a sharp (3:1) or gradual contraction, respectively. The nozzle was inspired by the FDA Critical Path Initiative nozzle, which was designed to emulate stresses in MCS.

Materials and

Methods: Isolated porcine red blood cells (RBCs), from abattoir blood, at 37% hematocrit were perfused through a mock loop (180 mL) using an MCS device (CentriMag) at 1.5 L/min for 5 hours. Two loops were run in an alternating order on each experimental day, one with a flow contraction (sharp or gradual) and one with no contraction as a control. A total of 5 experiments were performed for each contraction type (one outlier removed for the gradual runs) and10 experiments for the control loop. Additionally, a 50 mL aliquot of RBCs was used as a stagnant control for each loop to account for EV production due to aging of the RBCs (n=20). For each loop and stagnant control, samples were collected from bulk RBCs (t=-1hr), the start of perfusion, and after every hour. ErEVs were labeled with anti-porcine CD46-AlexaFluor 488 (labels RBCs) and annexin V-AlexaFluor 647 (labels PS) and analyzed for concomitant (CD46-PS+) ErEVs over the size range 0.5-1 μm using flow cytometry. Hemolysis was measured by the Harboe assay. Statistical analysis was performed using a one-way ANOVA at each time point with Tukey’s HSD post-hoc testing, with all data reported as mean ± standard error. Significance was defined as p < 0.05.

Results, Conclusions, and Discussions: The concentration of CD46-PS+ ErEVs increased from 1062±288 ErEV/μL at baseline to 3537±881 ErEV/μL at t=5 hours for the sharp contraction and from 413±77 to 1416±136 ErEV/μL for the gradual contraction, while the concentration remained constant for both controls (Figure 1). The sharp contraction was significantly different from the controls starting at t=1 hr (p < 0.05) and from the gradual contraction at t=5 hr (p < 0.01). Trends observed for hemolysis with the sharp and gradual contractions were similar having a change in free hemoglobin at t=5 hr from baseline of 67.1±7.7 and 87.2±5.2 mg/dL respectively, with both controls showing no to minimal hemolysis (Figure 2). Both the sharp and gradual contractions were significantly different from controls starting at t=1 hr (p < 0.001) and significantly different from each other at t=3 and t=5 hr only (p < 0.05). The normalized index of hemolysis was 0.017±0.002, 0.023±0.001, and 0.002±0.001 g/100L for the sharp, gradual, and control respectively, with the sharp and gradual contraction significantly different from only the control (p < 0.001).

While both ErEV concentration and hemolysis increased for both the sharp and gradual contraction, the difference in hemolysis is minimal between contraction type. In contrast, the sharp contraction produced significantly more ErEVs compared to the gradual contraction. This observation shows that extensional stresses were more damaging to RBCs than shear stresses when it comes to EV production. It also suggests that evaluating EV presence in MCS patients could indicate potential problems/adverse events prior to clinically significant levels of hemolysis being observed. EV production might be a more sensitive measure of blood cell damage than the current benchmark standard, hemolysis, with improved capability to assess device performance. This information could be valuable when it comes to improving hemocompatibility in MCS.

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