Implantable ventricular assist devices (VADs) have proven efficient in advanced heart failure patients as a bridge-to-transplant or destination therapy. However, VAD usage often leads to infection, bleeding, and thrombosis, side effects attributable to the damage to blood cells and plasma proteins. Measuring hemolysis alone does not provide sufficient information to understand total blood damage, and research exploring the impact of currently available pumps on a wider range of blood cell types and plasma proteins such as von Willebrand factor (vWF) is required to further our understanding of safer pump design. The extracorporeal CentriMag (Thoratec Corporation, Pleasanton, CA, USA) has a hemolysis profile within published standards of normalized index of hemolysis levels of less than 0.01 g/100 L at 100 mm Hg but the effect on leukocytes, vWF multimers, and platelets is unknown. Here, the CentriMag was tested using bovine blood (n = 15) under constant hemodynamic conditions in comparison with a static control for total blood cell counts, hemolysis, leukocyte death, vWF multimers, microparticles, platelet activation, and apoptosis. The CentriMag decreased the levels of healthy leukocytes (P < 0.006), induced leukocyte microparticles (P < 10−5), and the level of high molecular weight of vWF multimers was significantly reduced in the CentriMag (P < 10−5) all compared with the static treatment after 6 h in vitro testing. Despite the leukocyte damage, microparticle formation, and cleavage of vWF multimers, these results show that the CentriMag is a hemocompatible pump which could be used as a standard in blood damage assays to inform the design of new implantable blood pumps.

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.

The therapeutic use of ventricular assist devices (VADs) for end-stage heart failure (HF) patients who are ineligible for transplant has increased steadily in the last decade. In parallel, improvements in VAD design have reduced device size, cost, and device-related complications. These complications include infection and thrombosis which share underpinning contribution from the inflammatory response and remain common risks from VAD implantation. An added and underappreciated difficulty in designing a VAD that supports heart function and aids the repair of damaged myocardium is that different types of HF are accompanied by different inflammatory profiles that can affect the response to the implanted device. Circulating inflammatory markers and changes in leukocyte phenotypes receive much attention as biomarkers for mortality and disease progression. However, they are seldom used to monitor progress during and outcomes from VAD therapy or during the design phase for new devices. Even the partial reversal of heart damage associated with heart failure is a desirable outcome from VAD use. Therefore, improved understanding of the interplay between VADs and the recipient's inflammatory response would potentially increase their uptake, improve patient lives, and fuel research related to other blood-contacting medical devices. Here we provide a review of what is currently known about inflammation in heart failure and how this inflammatory profile is altered in heart failure patients receiving VAD therapy.

Clinical outcomes from ventricular assist devices (VADs) have improved significantly during recent decades, but bleeding episodes remain a common complication of long-term VAD usage. Greater understanding of the effect of the shear stress in the VAD on platelet aggregation, which is influenced by the functional activity of high molecular weight (HMW) von Willebrand factor (vWF), could provide insight into these bleeding complications. However, because VAD shear rates are difficult to assess, there is a need for a model that enables controlled shear rates to first establish the relationship between shear rates and vWF damage. Secondly, if such a dependency exists, then it is relevant to establish a rapid and quantitative assay that can be used routinely for the safety assessment of new VADs in development. Therefore, the purpose of this study was to exert vWF to controlled levels of shear using a rheometer, and flow cytometry was used to investigate the shear-dependent effect on the functional activity of vWF. Human platelet-poor plasma (PPP) was subjected to different shear rate levels ranging from 0 to 8000/s for a period of 6 h using a rheometer. A simple and rapid flow cytometric assay was used to determine platelet aggregation in the presence of ristocetin cofactor as a readout for vWF activity. Platelet aggregates were visualized by confocal microscopy. Multimers of vWF were detected using gel electrophoresis and immunoblotting. The longer PPP was exposed to high shear, the greater the loss of HMW vWF multimers, and the lower the functional activity of vWF for platelet aggregation. Confocal microscopy revealed for the first time that platelet aggregates were smaller and more dispersed in postsheared PPP compared with nonsheared PPP. The loss of HMW vWF in postsheared PPP was demonstrated by immunoblotting. Smaller vWF platelet aggregates formed in response to shear stress might be a cause of bleeding in patients implanted with VADs. The methodological approaches used herein could be useful in the design of safer VADs and other blood handling devices. In particular, we have demonstrated a correlation between the loss of HMW vWF, analyzed by immunoblotting, with platelet aggregation, assessed by flow cytometry. This suggests that flow cytometry could replace conventional immunoblotting as a simple and rapid routine test for HMW vWF loss during in vitro testing of devices.

Ventricular assist devices (VADs) are an effective bridging or destination therapy for patients with advanced stage heart failure. These devices remain susceptible to adverse events including infection, bleeding, and thrombus; events linked to the foreign body response. Therefore, the biocompatibility of all biomaterials used is crucial to the success of medical devices. Biomaterials common in VADs-DLC: diamond-like carbon coated stainless steel; Sap: single-crystal sapphire; SiN: silicon nitride; Ti: titanium alloy; and ZTA: zirconia-toughened alumina-were tested for their biocompatibility through incubation with whole human blood for 2 h with mild agitation. Blood was then removed and used for: complete cell counts; leukocyte activation and death, and the production of key inflammatory cytokines. All were compared to time 0 and an un-exposed 2 h sample. Monocyte numbers were lower after exposure to DLC, SiN, and ZTA and monocytes showed evidence of activation with DLC, Sap, and SiN. Neutrophils and lymphocytes were unaffected. This approach allows comprehensive analysis of the potential blood damaging effects of biomaterials. Monocyte activation by DLC, Sap, ZTA, and SiN warrants further investigation linking effects on this cell type to unfavorable inflammatory/thrombogenic responses to VADs and other blood handling devices. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1730-1738, 2018.

Medical devices, such as ventricular assist devices (VADs), introduce both foreign materials and artificial shear stress to the circulatory system. The effects these have on leukocytes and the immune response are not well understood. Understanding how these two elements combine to affect leukocytes may reveal why some patients are susceptible to recurrent device‐related infections and provide insight into the development of pump thrombosis. Biomaterials—DLC: diamond‐like carbon‐coated stainless steel; Sap: single‐crystal sapphire; and Ti: titanium alloy (Ti6Al4V) were attached to the parallel plates of a rheometer. Whole human blood was left between the two discs for 5 minutes at +37°C with or without the application of shear stress (0 s–1 or 1000 s–1). Blood was removed and used for complete blood cell counts, flow cytometry (leukocyte activation, cell death, microparticle generation, phagocytic ability, and reactive oxygen species [ROS] production), and the production of pro‐inflammatory cytokines. L‐selectin expression on monocytes was decreased when blood was exposed to the biomaterials both with and without shear. Applying shear stress to blood on a Sap and Ti surface led to activation of neutrophils shown as decreased L‐selectin expression. Sap and Ti blunted the LPS‐stimulated macrophage migration inhibitory factor (MIF) production, most notably when sheared on Ti. The biomaterials used here have been shown to activate leukocytes in a static environment. The introduction of shear appears to exacerbate this activation. Interestingly, a widely accepted biocompatible material (Ti) utilized in many different types of devices has the capacity for immune cell activation and inhibition of MIF secretion when combined with shear stress. These findings contribute to our understanding of the contribution of biomaterials and shear stress to recurrent infections and vulnerability to sepsis in some VAD patients as well as pump thrombosis.

Ventricular assist devices (VADs) are a life-saving form of mechanical circulatory support in heart failure patients. However, VADs have not yet reached their full potential due to the associated side effects (thrombosis, bleeding, infection) related to the activation and damage of blood cells and proteins caused by mechanical stress and foreign materials. Studies of the effects of VADs on leukocytes are limited, yet leukocyte activation and damage including microparticle generation can influence both thrombosis and infection rates. Therefore, the aim was to develop a multicolor flow cytometry assessment of leukocyte microparticles (LMPs) using ovine blood and the CentriMag VAD as a model for shear stress. Ovine blood was pumped for 6 h in the CentriMag and regular samples analyzed for hemolysis, complete blood counts and LMP by flow cytometry during three different pump operating conditions (low flow, standard, high speed). The high speed condition caused significant increases in plasma-free hemoglobin; decreases in total leukocytes, granulocytes, monocytes, and platelets; increases in CD45 LMPs as well as two novel LMP populations: CD11b /HLA-DR and CD11b /HLA-DR , both of which were CD14 /CD21 . CD11b /HLA-DR LMPs appeared to respond to an increase in shear magnitude whereas the CD11b /HLA-DR LMPs significantly increased in all pumping conditions. We propose that these two populations are released from granulocytes and T cells, respectively, but further research is needed to better characterize these two populations.

Elevation of Hemoglobin F ameliorates symptoms of β-thalassemia, a common autosomal recessive disorder. The transcription factor SOX6 plays a key role in the γ to β-globin gene switching. In the current investigation, a mutation was induced using the CRISPR/Cas9 technology in the binding domain region of SOX6 to reactivate γ-globin expression. Three CRISPR/Cas9 cassettes were provided, whose single-guide RNAs targeted different regions in the SOX6 gene-binding domain. After transfection of K562 cells with CRISPR a, b and c, and subsequent erythroid differentiation, the indel percentage of the cells was about 30%, 25%, and 24%, respectively. Relative quantification showed that the γ-globin mRNA level increased to 1.3-, 2.1-, and 1.1-fold in the cells treated with CRISPR/Cas9 a, b, and c, respectively, compared with untreated cells. Our results show that mutation induction in the binding site of the SOX6 gene leads to γ-globin reactivation. These findings support the idea that CRISPR interrupts the SOX6 binding site, and, as a result, SOX6 is incapable of binding the γ-globin promoter. In conclusion, SOX6 disruption could be considered as a therapeutic approach for β-thalassemia treatment. CRISPR/Cas9 was selected for this purpose as it is the most rapidly evolving technology.