Production of erythrocyte microparticles in a sub-hemolytic environment.

Microparticles are produced by various cells due to a number of different stimuli in the circulatory system. Shear stress has been shown to injure red blood cells resulting in hemolysis or non-reversible sub-hemolytic damage. We hypothesized that, in the sub-hemolytic shear range, there exist sufficient mechanical stimuli for red blood cells to respond with production of microparticles. Red blood cells isolated from blood of healthy volunteers were exposed to high shear stress in a microfluidic channel to mimic mechanical trauma similar to that occurring in ventricular assist devices. Utilizing flow cytometry techniques, both an increase of shear rate and exposure time showed higher concentrations of red blood cell microparticles. Controlled shear rate exposure shows that red blood cell microparticle concentration may be indicative of sub-hemolytic damage to red blood cells. In addition, properties of these red blood cell microparticles produced by shear suggest that mechanical trauma may underlie some complications for cardiovascular patients.

Assessment of Thrombelastography and Platelet Life Span in Ovines.

Ovines are a common animal model for the study of cardiovascular devices, where consideration of blood biocompatibility is an essential design criterion. In the ovine model, tools to assess blood biocompatibility are limited and continued investigation to identify and apply additional assays is merited. Toward this end, the thrombelastograph, clinically utilized to assess hemostasis, was used to characterize normal ovine parameters. In addition, platelet labeling with biotin was evaluated for its potential applicability to quantify ovine platelet life span. Mean ovine thrombelastograph values were reaction-time: 4.9 min, K-time: 2 min, angle: 64.1°, maximum amplitude: 68.6mm, actual clot strength: 11.9 kd/s, and coagulation index: 1.5. Reaction time was significantly shorter and maximum amplitude, actual clot strength, and coagulation index were all significantly higher when compared to normal human thrombelastograph values suggesting some hypercoagulability of sheep blood. Biotinylation and reinfusion of ovine platelets allowed temporal tracking of the labeled platelet cohort with flow cytometry. These data indicated a mean ovine platelet life span of 188h with a half-life of 84h. The collection of these parameters for normal ovines demonstrates the applicability of these techniques for subsequent studies where cardiovascular devices may be evaluated and provides an indication of normal ovine values for comparison purposes.

Flow-Field Simulations and Hemolysis Estimates for the Food and Drug Administration Critical Path Initiative Centrifugal Blood Pump.

The design of blood pumps for use in ventricular assist devices, which provide life-saving circulatory support in patients with heart failure, require remarkable precision and attention to detail to replicate the functionality of the native heart. The United States Food and Drug Administration (FDA) initiated a Critical Path Initiative to standardize and facilitate the use of computational fluid dynamics in the study and development of these devices. As a part of the study, a simplified centrifugal blood pump model generated by computer-aided design was released to universities and laboratories nationwide. The effects of changes in fluid rheology due to temperature, hematocrit, and turbulent flow on key metrics of the FDA pump were examined in depth using results from a finite volume-based commercial computational fluid dynamics code. Differences in blood damage indices obtained using Eulerian and Lagrangian formulations were considered. These results are presented and discussed awaiting future validation using experimental results, which will be released by the FDA at a future date.

Preclinical Device Thrombogenicity Assessments: Key Messages From the 2018 FDA, Industry, and Academia Forum.

Device-related thrombosis and thromboembolic complications remain a major clinical concern and often impact patient morbidity and mortality. Thus, improved preclinical thrombogenicity assessment methods that better predict clinical outcomes and enhance patient safety are needed. However, there are several challenges and limitations associated with developing and performing preclinical thrombogenicity assessments on the bench and in animals (e.g., the clinical relevance of most in vitro tests has not been established, animal studies may not accurately predict clinical thrombotic events). To facilitate a discussion on how to overcome some of these challenges and to promote collaboration between the Food and Drug Administration (FDA), industry, and academia for the development of more reliable test methods, a scientific forum was organized by FDA and held in Washington, DC, on June 15, 2018 at the ASAIO 64th Annual Conference. Three subject matter experts from the medical device industry and FDA presented their perspectives at this forum, and several audience experts provided input during the open dialogue session. This article summarizes the key messages from the forum regarding the current status and challenges of preclinical thrombogenicity testing, important areas of needed research, and mechanisms for working with FDA to further improve thrombogenicity evaluations of medical devices.

A biohybrid artificial lung prototype with active mixing of endothelialized microporous hollow fibers.

Acute respiratory distress syndrome (ARDS) affects nearly 150,000 patients per year in the US, and is associated with high mortality ( approximately 40%) and suboptimal options for patient care. Mechanical ventilation and extracorporeal membrane oxygenation are limited to short-term use due to ventilator-induced lung injury and poor biocompatibility, respectively. In this report, we describe the development of a biohybrid lung prototype, employing a rotating endothelialized microporous hollow fiber (MHF) bundle to improve blood biocompatibility while MHF mixing could contribute to gas transfer efficiency. MHFs were surface modified with radio frequency glow discharge (RFGD) and protein adsorption to promote endothelial cell (EC) attachment and growth. The MHF bundles were placed in the biohybrid lung prototype and rotated up to 1,500 revolutions per minute (rpm) using speed ramping protocols to condition ECs to remain adherent on the fibers. Oxygen transfer, thrombotic deposition, and EC p-selectin expression were evaluated as indicators of biohybrid lung functionality and biocompatibility. A fixed aliquot of blood in contact with MHF bundles rotated at either 250 or 750 rpm reached saturating pO(2) levels more quickly with increased rpm, supporting the concept that fiber rotation would positively contribute to oxygen transfer. The presence of ECs had no effect on the rate of oxygen transfer at lower fiber rpm, but did provide some resistance with increased rpm when the overall rate of mass transfer was higher due to active mixing. RFGD followed by fibronectin adsorption on MHFs facilitated near confluent EC coverage with minimal p-selectin expression under both normoxic and hyperoxic conditions. Indeed, even subconfluent EC coverage on MHFs significantly reduced thrombotic deposition adding further support that endothelialization enhances, blood biocompatibility. Overall these findings demonstrate a proof-of-concept that a rotating endothelialized MHF bundle enhances gas transfer and biocompatibility, potentially producing safer, more efficient artificial lungs.

A Flow Induced Autoimmune Response and Accelerated Senescence of Red Blood Cells in Cardiovascular Devices.

Red blood cells (RBCs) passing through heart pumps, prosthetic heart valves and other cardiovascular devices undergo early senescence attributed to non-physiologic forces. We hypothesized that mechanical trauma accelerates aging by deformation of membrane proteins to cause binding of naturally occurring IgG. RBCs isolated from blood of healthy volunteers were exposed to high shear stress in a viscometer or microfluidics channel to mimic mechanical trauma and then incubated with autologous plasma. Increased binding of IgG was observed indicating forces caused conformational changes in a membrane protein exposing an epitope(s), probably the senescent cell antigen of band 3. The binding of immunoglobulin suggests it plays a role in the premature sequestration and phagocytosis of RBCs in the spleen. Measurement of IgG holds promise as a marker foreshadowing complications in cardiovascular patients and as a means to improve the design of medical devices in which RBCs are susceptible to sublethal trauma.

Benchtop von Willebrand Factor Testing: Comparison of Commercially Available Ventricular Assist Devices and Evaluation of Variables for a Standardized Test Method.

Gastrointestinal bleeding occurs in 20-30% of patients receiving ventricular assist devices (VADs) due, in part, to acquired von Willebrand syndrome. We examined factors to optimize a benchtop method to quantify changes in von Willebrand Factor (VWF) multimer distribution and function in VADs, then applied them to evaluate commercially available devices. Human plasma was circulated through flow loops with VADs. Several experimental conditions were examined, including temperature, viscosity, and enzyme inhibition. Samples were analyzed for VWF collagen-binding activity (VWF:CB) and VWF antigen level. von Willebrand Factor multimer profiles were quantified using gel electrophoresis, near-infrared in-gel visualization, and densitometric analysis. The VWF:CB/antigen ratio in the HeartMate II, CentriMag, and HVAD exhibited average decreases of 46%, 44%, and 36% from baseline after 360 minutes of operation. High molecular weight (hVWF) multimer loss occurred within 30 minutes, although the Levacor and control loop profiles were unchanged. Varying temperature and viscosity altered hVWF degradation rate, but not the final results. Inhibition of a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13 (ADAMTS13) can potentially distinguish mechanoenzymatic cleavage of VWF from mechanical degradation. We developed a repeatable benchtop method to evaluate VWF compatibility of VADs similar to hemolysis testing that can be adopted for preclinical VAD evaluation.

Flow cytometric assays for quantifying activated ovine platelets.

Ovines are a common animal model for preclinical evaluation of cardiovascular devices including heart valves, endovascular grafts, and ventricular assist devices. Biocompatibility is essential to the success of these devices; however, tools to assess biocompatibility in ovines are limited. To address this need, antibodies that bind to activated human and bovine platelets and annexin V protein were evaluated for potential cross-reactivity to activated ovine platelets. These candidate markers were incubated with stimulated and quiescent ovine whole blood, and binding to platelets was quantified by flow cytometry. Several antihuman CD62P antibodies including one polyclonal antibody, three monoclonal antibodies, and annexin V selectively bound to activated ovine platelets. An assay to quantify platelet microaggregates was also developed. The availability of assays to quantify ovine platelet activation can increase the quality of biocompatibility data obtainable during preclinical development of artificial organs in the ovine model, potentially aiding in the evaluation of design refinements to enhance device biocompatibility.

Effects of Transient Exposure to High Shear on Neutrophil Rolling Behavior.

INTRODUCTION: Neutrophils display an array of behaviors ranging from rolling and migration to phagocytosis and granule secretion. Several of these behaviors are modulated by the local shear conditions. In the normal circulation, neutrophils experience shear rates from approximately 10-2,000 s-1. However, neutrophils are also exposed to pathological shear levels in natural conditions such as severe stenosis and arteriosclerosis, as well as in blood-contacting devices such as ventricular assist devices (VADs) and hemodialysis machines. The effects of transiently (< 1 sec) exposing neutrophils to abnormally high shear rates (>3,000 s-1) are not well understood. METHODS: We developed a set of microfluidic devices capable of exposing neutrophils to high shear rates for short durations (100-400 msec). Suspensions of isolated neutrophils were perfused through the devices and their rolling velocities on P-selectin were analyzed before and after shear exposure. RESULTS: We observed a significant increase in neutrophil rolling velocities on P-selectin coated regions following transient high shear exposure. The magnitude of the rolling velocity increase was dependent upon the duration of high shear exposure and became statistically significant for exposure times of 310 msec or longer. When polystyrene beads coated with a glycosulfopeptide that mimics the binding region of P-selectin glycoprotein ligand-1 (PSGL-1) were perfused through the devices, no change between the pre-shear and post-shear rolling velocities was observed. CONCLUSIONS: These results suggest that high shear levels alter normal neutrophil rolling behavior and are important for understanding neutrophil biology in high shear conditions, as well as for improving medical device performance.

Preclinical biocompatibility assessment of the EVAHEART ventricular assist device: coating comparison and platelet activation.

Thromboembolism and bleeding remain significant complications of ventricular assist device (VAD) support. Increasing the amount of biocompatibility data collected during preclinical studies can provide additional criteria to evaluate device refinements, while design changes may be implemented before entering clinical use. Twenty bovines were implanted with the EVAHEART centrifugal VAD for durations from 30 to 196 days. Titanium alloy pumps were coated with either diamond-like carbon or 2-methoxyethyloylphosphoryl choline (MPC). Activated platelets and platelet microaggregates were quantified by flow cytometry, including two new assays to quantify bovine platelets expressing CD62P and CD63. Temporally, all assays were low preoperatively, then significantly increased following VAD implantation, before declining to a lower, but still elevated level over 2-3 weeks. MPC-coated VADs produced significantly fewer activated platelets after implant trauma effects diminished. Three animals receiving no postoperative anticoagulation had similar amounts of circulating activated platelets and platelet microaggregates as animals receiving warfarin anticoagulation. Two new methods to quantify bovine activated platelets using antibodies to CD62P and CD63 were characterized and applied. These measures, along with previously described assays, were able to differentiate between two biocompatible coatings and assess effects of anticoagulation regimen in VAD preclinical testing.

Constricted microfluidic devices to study the effects of transient high shear exposure on platelets.

Due to the critical roles that platelets play in thrombosis during many biological and pathological events, altered platelet function may be a key contributor to altered hemostasis, leading to both thrombotic and hemorrhagic complications. Platelet adhesion at arterial shear rates occurs through binding to von Willebrand Factor via the glycoprotein (GP) GPIb receptor. GPIb binding can induce platelet activation distinguishable by P-selectin (CD62P) surface expression and αIIbß3 activation, resulting in platelet aggregation and formation of the primary hemostatic plug to stop bleeding. Previous studies have used cone and plate viscometers to examine pathologic blood flow conditions, applied shear rates that are relatively low, and examined exposure times that are orders of magnitude longer compared to conditions present in ventricular assist devices, mechanical heart valves, or pathologic states such as stenotic arteries. Here, we evaluate the effect of short exposure to high shear on granule release and receptor shedding utilizing a constricted microfluidic device in conjunction with flow cytometry and enzyme-linked immunosorbent assay. In this study, platelets were first perfused through microfluidic channels capable of producing shear rates of 80 000-100 000 s-1 for exposure times of 0-73 ms. We investigated platelet activation by measuring the expression level of CD62P (soluble and surface expressed), platelet factor 4 (PF4), and beta-thromboglobulin (ßTG). In addition, we measured potential platelet receptor shedding of GPVI and GPIb using flow cytometry. The results showed that a single pass to high shear with short exposure times (milliseconds) had no effect on the levels of CD62P, GPVI and GPIb, or on the release of alpha granule content (PF4, ßTG, and sP-selectin).

Towards the development of a pediatric ventricular assist device.

The very limited options available to treat ventricular failure in children with congenital and acquired heart diseases have motivated the development of a pediatric ventricular assist device at the University of Pittsburgh (UoP) and University of Pittsburgh Medical Center (UPMC). Our effort involves a consortium consisting of UoP, Children's Hospital of Pittsburgh (CHP), Carnegie Mellon University, World Heart Corporation, and LaunchPoint Technologies, Inc. The overall aim of our program is to develop a highly reliable, biocompatible ventricular assist device (VAD) for chronic support (6 months) of the unique and high-risk population of children between 3 and 15 kg (patients from birth to 2 years of age). The innovative pediatric ventricular assist device we are developing is based on a miniature mixed flow turbodynamic pump featuring magnetic levitation, to assure minimal blood trauma and risk of thrombosis. This review article discusses the limitations of current pediatric cardiac assist treatment options and the work to date by our consortium toward the development of a pediatric VAD.

The PediaFlow pediatric ventricular assist device.

The very limited options available to treat ventricular failure in patients with congenital and acquired heart diseases have motivated the development of a pediatric ventricular assist device (VAD). Our effort involves a consortium consisting of the University of Pittsburgh, Carnegie Mellon University, Children's Hospital of Pittsburgh, World Heart Corporation, and LaunchPoint Technologies, LLC. The overall aim of our program is to develop a highly reliable, biocompatible VAD for chronic support (6 months) of the unique and high-risk population of children between 3 kg and 15 kg (patients from birth to 2 years of age). The innovative pediatric VAD we are developing (PediaFlow) is based on a miniature mixed-flow turbodynamic pump featuring magnetic levitation, with the design goal being to assure minimal blood trauma and risk of thrombosis. This article discusses the limitations of current pediatric cardiac assist treatment options and the work to date by our consortium toward the development of a pediatric VAD.

Development of standard tests to examine viscoelastic properties of blood of experimental animals for pediatric mechanical support device evaluation.

We investigated the applicability of measuring the viscoelasticity of bovine, ovine, and porcine whole blood for the evaluation of sublethal damage to red blood cells (RBCs). An increase in blood viscosity and elasticity without changes in hematocrit and plasma viscosity would signify a decrease in RBC deformability. Blood viscoelasticity was assessed using a Vilastic Scientific viscoelastometer. Due to the natural absence of RBC aggregation and small RBC size in normal bovine and ovine blood, viscoelastic properties are less readily detected. However, we found that adjustment of blood hematocrit to a standard level of 40-50% allows for sensitive assessment of viscoelasticity in these blood types demonstrating a marked non-Newtonian behavior mostly related to RBC deformability. Porcine blood showed a pronounced non-Newtonian behavior at all tested hematocrit values, which makes it rheologically comparable to human blood. Both viscosity and elasticity were elevated after blood exposure to a uniform mechanical stress. RBCs rigidified by heat exposure demonstrated a loss of viscoelasticity dependence on shear rate. Measurements of blood viscoelasticity can be meaningful in bovine, ovine, and, especially, porcine blood, and can be used for evaluation of sublethal blood damage during in vitro and animal trials of heart-assist devices.

Elimination of adverse leakage flow in a miniature pediatric centrifugal blood pump by computational fluid dynamics-based design optimization.

We investigated a miniature magnetically levitated centrifugal blood pump intended to deliver 0.3-1.5 l/min of support to neonates and infants. The back clearance gap between the housing and large volume of the rotor, where the suspension and motor bearings are located, forms a continuous leakage flow path. Within the gap, flow demonstrates a very complex three-dimensional structure: the fluid adjacent to the rotating disk tends to accelerate by centrifugal force to flow radially outwards toward the outlet of the impeller against an unfavorable pressure gradient, which in turn forces blood to return along the stationary housing surfaces. Consequently, one or multiple vortices may be generated in the gap to block blood flow and cause the formation of a retrograde and antegrade leakage flow phenomenon at the gap outlet using an optimization process including extensive computational fluid dynamics (CFD) analysis of impeller refinements, we found that secondary blades located along the back or extended to the side surfaces of the rotor have the capacity to reduce and eliminate the retrograde flow in the back clearance gap. Flow visualization confirmed the CFD-predicted flow patterns. This work demonstrates the utility of CFD-based design optimization to optimize the fluid path of a miniature centrifugal pump.

Platelet activation, aggregation, and life span in calves implanted with axial flow ventricular assist devices.

BACKGROUND: A variety of rotary blood pumps are under development worldwide to serve as chronic ventricular assist devices (VADs). Historically VADs have been associated with thrombotic and thromboembolic complications, yet the ability to evaluate the thrombotic process in preclinical device testing has been limited. METHODS: We have developed and applied flow cytometric assays for activated platelets, platelet microaggregates, and platelet life span and consumption to calves implanted with an axial flow VAD and calves undergoing a sham surgical procedure. RESULTS: Surgical sham calves had significant increases in circulating activated platelets (p < 0.05) that resolved within 17 days, and no increases in circulating platelet microaggregates. Calves with uneventful VAD implant periods had early transient elevations in platelet microaggregates and prolonged elevations in activated platelets that did not recover to preoperative values during the study. Daily platelet consumption in VAD implanted calves was increased by 20% +/- 3%. Calves with thrombotic deposition within the VAD and elevated thromboembolism observed at autopsy experienced increases in circulating activated platelets and microaggregates at the end of the implant period when VAD flow decreased. CONCLUSIONS: This study demonstrates the ability of flow cytometry-based platelet assays to differentiate VAD implant operations from VAD support, and suggests differences that exist between uneventful VAD support and support with complications. These techniques should have value in evaluating other cardiovascular devices undergoing preclinical testing and provide insight into the temporal impact of these devices on the hemostatic system.

Postimplant left ventricular assist device fit analysis using three-dimensional reconstruction.

Left ventricular assist devices (LVADs) are blood pumps that augment the function of the failing heart to improve perfusion, resulting in improved survival. For LVADs to effectively unload the left ventricle, the inflow cannula (IC) should be unobstructed and ideally aligned with the heart's mitral valve (MV). We examined IC orientation deviation from a hypothesized conventional angle (45° right-posterior) and the approximate angle for direct IC-MV alignment in many patients. Three-dimensional anatomic models were created from computed tomography scans for 24 LVAD-implanted patients, and angles were measured between the IC and the apical z-axis in both the coronal and the sagittal planes. Common surgical IC angulation was found to be 22 ± 15° rightward and 21 ± 12° posterior from the apical z-axis; 38% (n = 9) of patients fell in this range. Direct IC-MV angulation was found to be 34 ± 8° rightward and 15 ± 7° posterior; only 8% (n = 2) of patients fell in this range. Rightward deviation toward ventricular septal wall and anterior deviation toward LV anterior freewall are associated with mortalities more so than leftward and posterior deviation. In conclusion, anatomic reconstruction may be a useful preoperative tool to obtain general population and patient-specific alignment for optimal LVAD implantation.

Effects of gamma radiation on a ventricular assist device and its percutaneous lead components.

A patient supported by a left ventricular assist device (LVAD) presented with an abdominal tumor requiring consolidative radiation therapy. To assess the effects of radiation therapy on the operation of the ventricular assist device (VAD) system and assure that the treatment would be safe for the patient with regard to the operation of the VAD system, sample equipment was irradiated and then tested for functionality. Changes in the mechanical properties of components of the percutaneous lead were measured. After testing, it was concluded that radiation therapy would not impede the operation of the pump or produce deleterious alterations of mechanical properties of the various system components. The patient underwent radiation therapy with a total dose of 2,000 cGy without evident complications. There were no observed effects on the LVAD operation nor any indication of alarms or malfunctions. Subsequently, the patient recovered adequate cardiac function for explant of the LVAD and the recovered components were also analyzed confirming the absence of alterations in material properties that would endanger patient safety.

The role of fibrinogen spacing and patch size on platelet adhesion under flow.

Platelet adhesion to the vessel wall during vascular injury is mediated by platelet glycoproteins binding to their respective ligands on the vascular wall. In this study we investigated the roles that ligand patch spacing and size play in regulating platelet interactions with fibrinogen under hemodynamic flow conditions. To regulate the size and distance between patches of fibrinogen we developed a photolithography-based technique to fabricate patterns of proteins surrounded by a protein-repellant layer of poly(ethylene glycol). We demonstrate that when mepacrine labeled whole blood is perfused at a shear rate of 100 s ⁻¹ over substrates patterned with micron-sized wide lines of fibrinogen, platelets selectively adhere to the areas of patterned fibrinogen. Using fluorescent and scanning electron microscopy we demonstrate that the degree of platelet coverage (3-35%) and the ability of platelet aggregates to grow laterally are dependent upon the distance (6-30 µm) between parallel lines of fibrinogen. We also report on the effects of fibrinogen patch size on platelet adhesion by varying the size of the protein patch (2-20 µm) available for adhesion, demonstrating that the downstream length of the ligand patch is a critical parameter in platelet adhesion under flow. We expect that these results and protein patterning surfaces will be useful in understanding the spatial and temporal dynamics of platelet adhesion under physiologic flow, and in the development of novel platelet adhesion assays.