Shear Stress‐Induced Total Blood Trauma in Multiple Species

Abstract

The common complications in heart failure patients with implanted ventricular assist devices (VADs) include hemolysis, thrombosis, and bleeding. These are linked to shear stress-induced trauma to erythrocytes, platelets, and von Willebrand factor (vWF). Novel device designs are being developed to reduce the blood trauma, which will need to undergo in vitro and in vivo preclinical testing in large animal models such as cattle, sheep, and pig. To fully understand the impact of device design and enable translation of preclinical results, it is important to identify any potential species-specific differences in the VAD-associated common complications. Therefore, the purpose of this study was to evaluate the effects of shear stress on cells and proteins in bovine, ovine, and porcine blood compared to human. Blood from different species was subjected to various shear rates (0-8000/s) using a rheometer. It was then analyzed for complete blood counts, hemolysis by the Harboe assay, platelet activation by flow cytometry, vWF structure by immunoblotting, and function by collagen binding activity ELISA (vWF : CBA). Overall, increasing shear rate caused increased total blood trauma in all tested species. This analysis revealed species-specific differences in shear-induced hemolysis, platelet activation, and vWF structure and function. Compared to human blood, porcine blood was the most resilient and showed less hemolysis, similar blood counts, but less platelet activation and less vWF damage in response to shear. Compared to human blood, sheared bovine blood showed less hemolysis, similar blood cell counts, greater platelet activation, and similar degradation of vWF structure, but less impact on its activity in response to shear. The shear-induced effect on ovine blood depended on whether the blood was collected via gravity at the abattoir or by venepuncture from live sheep. Overall, ovine abattoir blood was the least resilient in response to shear and bovine blood was the most similar to human blood. These results lay the foundations for developing blood trauma evaluation standards to enable the extrapolation of in vitro and in vivo animal data to predict safety and biocompatibility of blood-handling medical devices in humans. We advise using ovine venepuncture blood instead of ovine abattoir blood due to the greater overall damage in the latter. We propose using bovine blood for total blood damage in vitro device evaluation but multiple species could be used to create a full understanding of the complication risk profile of new devices. Further, this study highlights that choice of antibody clone for evaluating platelet activation in bovine blood can influence the interpretation of results from different studies.

Artificial shear stress effects on leukocytes at a biomaterial interface

“…Decrease in platelet counts under shear has been noted previously as a sign of platelet activation. (8,42) This cell type was not studied further here but is the focus of ongoing work. Decreases in leukocyte counts under shear have been noted in the past in a model wherein whole blood was sheared for 10 minutes and there was a 25% decrease in leukocyte count at 60 Pa and membrane disruption at 15 Pa. (14) This is not only a longer time but a higher shear stress than in our study (~10 Pa).…”

Ovine Leukocyte Microparticles Generated by the CentriMag Ventricular Assist Device In Vitro

“…Levels of pfHb obtained with ovine blood were one quarter of those obtained in bovine blood after 360 min pumping in the CentriMag at the standard condition (2.5 6 1.0 vs. 10 6 1.5 mg/dL), with mg NIH roughly one-third in ovine compared to bovine blood (0.38 6 0.15 compared to 1.10 6 0.5 mg/100 L) (13). In our previous multispecies comparison study, in which we evaluated the response to shear caused by a rheometer, we also observed greater hemolysis in bovine abattoir compared to ovine venepuncture blood at high shear (75 vs. 50 mg/dL at 8000/s, respectively), although this difference was not statistically significant (34).…”

In Vitro Hemocompatibility Evaluation of Ventricular Assist Devices in Pediatric Flow Conditions: A Benchmark Study

“…Our results showed that bovine blood was the most similar to human blood overall, therefore bovine blood was chosen for this study (20). Our results showed that bovine blood was the most similar to human blood overall, therefore bovine blood was chosen for this study (20).…”

“…A 20 lL aliquot of the every 120 min blood samples, along with a positive control of blood treated with 5 lM phorbol 12-myristate 13-acetate (PMA) for 60 min at room temperature, were stained with 5 lL of CAPP2A (1.0 mg/dL, an antiruminant CD41/61 antibody) (from Monoclonal Antibody Center, Washington State University), as staining and analyzing methods were explained in detail previously (12,20). Samples were acquired and analyzed using BD LSR II Flow Cytometry Cell Analyzer with FACSDiva 6.1.3 software (BD Bioscience, Oxford, UK).…”

“…The centrifuged PPP was subjected to electrophoresis, protein blotting transfer, vWF specific staining as methods were explained in detail previously (20,23). Platelet-poor plasma (PPP) was prepared by further centrifuging PRP for 5 min at 15 000 3 g to remove debris.…”