In Vitro models for thrombogenicity testing of blood-recirculating medical devices

ASAIO Journal

To develop and standardize a reliable in vitro dynamic thrombogenicity test protocol, the key test parameters that could impact thrombus formation need to be investigated and understood. In this study, we evaluated the effect of temperature on the thrombogenic responses (thrombus surface coverage, thrombus weight, and platelet count reduction) of various materials using an in vitro blood flow loop test system. Whole blood from live sheep and cow donors was used to assess four materials with varying thrombogenic potentials: negative-control polytetrafluoroethylene (PTFE), positive-control latex, silicone, and high-density polyethylene (HDPE). Blood, heparinized to a donor-specific concentration, was recirculated through a polyvinyl chloride tubing loop containing the test material at room temperature (22–24°C) for 1 hour, or at 37°C for 1 or 2 hours. The flow loop system could effectively differentiate a thrombogenic material (latex) from the other materials for both test temperature...

Artificial organs, 2016

We designed an experimental setup to characterize the thrombogenic potential associated with blood recirculating devices (BRDs) used in extracorporeal circulation (ECC). Our methodology relies on in vitro flow loop platelet recirculation experiments combined with the modified-prothrombinase platelet activity state (PAS) assay to quantify the bulk thrombin production rate of circulated platelets, which correlates to the platelet activation (PA) level. The method was applied to a commercial neonatal hollow fiber membrane oxygenator. In analogous hemodynamic environment, we compared the PA level resulting from multiple passes of platelets within devices provided with phosphorylcholine (PC)-coated and noncoated (NC) fibers to account for flow-related mechanical factors (i.e., fluid-induced shear stress) together with surface contact activation phenomena. We report for the first time that PAS assay is not significantly sensitive to the effect of material coating under clinically pertinen...

PLOS ONE, 2012

Mechanical circulatory support (MCS) devices provide both short and long term hemodynamic support for advanced heart failure patients. Unfortunately these devices remain plagued by thromboembolic complications associated with chronic platelet activation-mandating complex, lifelong anticoagulation therapy. To address the unmet need for enhancing the thromboresistance of these devices to extend their long term use, we developed a universal predictive methodology entitled Device Thrombogenicity Emulation (DTE) that facilitates optimizing the thrombogenic performance of any MCS device-ideally to a level that may obviate the need for mandatory anticoagulation. DTE combines in silico numerical simulations with in vitro measurements by correlating device hemodynamics with platelet activity coagulation markersbefore and after iterative design modifications aimed at achieving optimized thrombogenic performance. DTE proof-ofconcept is demonstrated by comparing two rotary Left Ventricular Assist Devices (LVADs) (DeBakey vs HeartAssist 5, Micromed Houston, TX), the latter a version of the former following optimization of geometrical features implicated in device thrombogenicity. Cumulative stresses that may drive platelets beyond their activation threshold were calculated along multiple flow trajectories and collapsed into probability density functions (PDFs) representing the device 'thrombogenic footprint', indicating significantly reduced thrombogenicity for the optimized design. Platelet activity measurements performed in the actual pump prototypes operating under clinical conditions in circulation flow loopsbefore and after the optimization with the DTE methodology, show an order of magnitude lower platelet activity rate for the optimized device. The robust capability of this predictive technology-demonstrated here for attaining safe and costeffective pre-clinical MCS thrombo-optimization-indicates its potential for reducing device thrombogenicity to a level that may significantly limit the extent of concomitant antithrombotic pharmacotherapy needed for safe clinical device use.

Journal of materials science. Materials in medicine, 1997

The increasing number of patients requiring prosthetic substitution of segments of the vascular system strongly supports the need to optimize a relevant, standardized testing panel for new materials designed for synthetic vascular prostheses. The ISO gives the standard requirements for testing biomaterials provided for implantation. Our primary interest was the establishment of a reliable in vitro panel as a useful and relevant screening system for vascular implant devices to evaluate blood/device interactions under flow conditions. The aim of the present study was to evaluate influences of different flow conditions on blood cell-biomaterial interactions with special emphasis on the interactions of human granulocytes (PMN) and polymeric surfaces. PMN were isolated and vital cells were quantified by flow cytometrical analysis directly before, as well as immediately after the experiments. The viscosity of the final cellular suspension was analysed by using a computerized cone-plate rh...

Journal of Visualized Experiments, 2020

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 °C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces

Journal of Biomedicine and Biotechnology, 2007

Extended use of cardiopulmonary bypass (CPB) systems is often hampered by thrombus formation and infection. Part of these problems relates to imperfect hemocompatibility of the CPB circuitry. The engineering of biomaterial surfaces with genuine longterm hemocompatibility is essentially virgin territory in biomaterials science. For example, most experiments with the well-known Chandler loop model, for evaluation of blood-biomaterial interactions under flow, have been described for a maximum duration of 2 hours only. This study reports a systematic evaluation of two commercial CPB tubings, each with a hemocompatible coating, and one uncoated control. The experiments comprised (i) testing over 5 hours under flow, with human whole blood from 4 different donors; (ii) measurement of essential blood parameters of hemocompatibility; (iii) analysis of the luminal surfaces by scanning electron microscopy and thrombin generation time measurements. The dataset indicated differences in hemocompatibility of the tubings. Furthermore, it appeared that discrimination between biomaterial coatings can be made only after several hours of bloodbiomaterial contact. Platelet counting, myeloperoxidase quantification, and scanning electron microscopy proved to be the most useful methods. These findings are believed to be relevant with respect to the bioengineering of extracorporeal devices that should function in contact with blood for extended time.

Biomedical Instrumentation & Technology, 2011

Preclinical hemolysis testing is a critical requirement toward demonstrating device safety for U.S. Food and Drug Administration (FDA) 510(k) approval of mechanical circulatory support devices (MCSD). FDA and ASTM (formerly known as the American Society for Testing and Materials) have published guidelines to assist industry with developing study protocols. However, there can be significant variability in experimental procedures, study design, and reporting of data that makes comparison of test and predicate devices a challenge. To overcome these limitations, we present a hemolysis testing protocol developed to enable standardization of hemolysis testing while adhering to FDA and ASTM guidelines. Static mock flow loops primed with fresh bovine blood (600 mL, Hematocrit = 27±5%, heparin titrated for ACT >300 sec) from a single-source donor were created as a platform for investigating test and predicate devices. MCSD differential pressure and temperature were maintained at 80 mmHg a...

2006

Bleeding and thromboembolism remain major complications of ventricular assist device (VAD) support. The amount of biocompatibility information that may be collected during preclinical studies is limited due to a lack of available assays, leaving the evaluation of investigational devices incomplete. To address these issues, flow cytometric assays were developed to quantify bovine circulating activated platelets, platelet microaggregates, platelet-leukocyte aggregates, and monocytes expressing tissue factor. Platelet lifespan was determined using an ex vivo biotinylation technique. These assays were applied in 50 animals receiving the Nimbus/Heartmate II axial flow VAD, 29 receiving the SunMedical EVAHEART centrifugal VAD, over 20 animals receiving a variety of other cardiovascular devices, and eight animals that underwent a sham VAD implantation procedure. The results demonstrated significantly increased circulating activated platelets and leukocytes, and cell aggregates following VAD implantation, which then usually declined to a lower but still significantly elevated level. Deviations from this pattern were observed in several pumps with obstructive thrombi in the blood flow path. Platelet life span decreased and platelet consumption correspondingly increased. The sham studies demonstrated that the effects of the implant procedure abated within three weeks. Thus, the ongoing platelet and leukocyte activation and aggregation, and decreased platelet life span could be attributed to the VADs, even while accounting for surgical effects. To identify the potential causes of the observed cellular activation, VAD surface modifications, revolutions per min increases, and anticoagulant regimen changes were evaluated in vivo. Two blood-shearing devices were constructed to investigate the effects of the supraphysiologic shear field within rotary VADs, although heat generation and sealing issues v limited their effectiveness. Flow visualization of the Heartmate II VAD revealed vortices developed at low flow rates, frequently encountered in vivo. In conclusion, the propensity of cardiovascular devices to activate platelets and leukocytes was quantified, while accounting for the effects of the implant procedure. Through in vivo and in vitro investigations, it was demonstrated that the blood-contacting surface and adverse flow effects each contributed to the observed cellular activation. Thus, applying novel biocompatibility assays to preclinical studies, including those evaluating design enhancements and refinements, may be used to develop safer cardiovascular devices. vi TABLE OF CONTENTS

Biomechanics and Modeling in Mechanobiology, 2018

Thrombosis is a major concern in bloodcoated medical devices. Contact activation, which is the initial part of the coagulation cascade in device-related thrombosis, is not considered in current thrombus formation models. In the present study, pro-coagulant reactions including the contact activation system are coupled with a fluid solver in order to evaluate the potential of the contact system to initiate thrombin production. The biochemical/fluid model is applied to a backward facing step configuration, a flow configuration that frequently appears in medical devices. In contrast to the in vivo thrombosis models in which a specific thrombotic zone (injury region) is set a priori by the user to initiate the coagulation reaction, a reactive surface boundary condition is applied to the whole device wall. Simulation results show large thrombin concentration in regions related to recirculation zones without the need of an a priori knowledge of the thrombus location. The numerical results align well with the regions prone to thrombosis observed in experimental results reported in the literature. This approach could complement thrombus formation models that take into account platelet activity and thrombus growth to optimize a wide range of medical devices.

Artificial Organs, 2019

Mechanical circulatory support (MCS) devices continue to be hampered by thrombotic adverse events, a consequence of device-imparted supraphysiologic shear stresses, leading to shearmediated platelet activation (SMPA). In advancing MCS devices from design to clinical use, invitro circulatory loops containing the device under development and testing are utilized as a means of assessing device thrombogenicity. Physical characteristics of these test circulatory loops may too contribute to inadvertent platelet activation through imparted shear stress, adding inadvertent error in evaluating MCS device thrombogenicity. While investigators normally control for the effect of a loop, inadvertent addition of what are considered innocuous connectors may impact test results. Here we tested the effect of common, additive components of in-vitro circulatory test loops, i.e. connectors and loop geometry, as to their additive contribution to shear stress via both in-silico and in-vitro models. A series of test circulatory loops containing a ventricular assist device (VAD) with differing constituent components, were established in-silico including: loops with 0 ~ 5 Luer connectors, a loop with a T-connector creating 90° angulation, and a loop with 90° angulation. Computational fluid dynamics (CFD) simulations were performed using a kω shear stress transport (SST) turbulence model to platelet activation index based on a power law model. VAD-operated loops replicating in-silico designs were assembled in-vitro and gel-filtered human platelets were recirculated within (1 h) and SMPA was determined. Results: CFD simulations demonstrated high shear being introduced at non-smooth regions such as edge-connector boundaries, tubing, and at Luer holes. Noticeable peaks' shifts of sss distributions towards high shear-region existed with increasing loop complexity. Platelet activation also increased with increasing shear exposure time, being statistically higher when platelets were exposed to connector-employed loop designs. The extent of platelet activation in-vitro could be