In Vitro Thrombogenicity Testing of Biomaterials in a Dynamic Flow Loop: Effects of Length and Quantity of Test Samples.

The results of in vitro dynamic thrombogenicity testing of biomaterials and medical devices can be significantly impacted by test conditions. To develop and standardize a robust dynamic in vitro thrombogenicity tool, the key test parameters need to be appropriately evaluated and optimized. We used a flow loop test system previously developed in our laboratory to investigate the effects of sample length and the number of samples per test loop on the thrombogenicity results. Porcine blood heparinized to a donor-specific target concentration was recirculated at room temperature through polyvinyl chloride (PVC) tubing loops containing test materials for 1 h at 200 mL/min. Four test materials (polytetrafluoroethylene (PTFE), latex, PVC, and silicone) with various thrombotic potentials in two sample lengths (12 and 18 cm) were examined. For the 12-cm long materials, two different test configurations (one and two samples per loop) were compared. Thrombogenicity was assessed through percent thrombus surface coverage, thrombus weight, and platelet count reduction in the blood. The test system was able to effectively differentiate the thrombogenicity profile of the materials (latex > silicone > PVC ≥ PTFE) at all test configurations. Increasing test sample length by 50% did not significantly impact the test results as both 12 and 18 cm sample lengths were shown to equally differentiate thrombotic potentials between the materials. The addition of a second test sample to each loop did not increase the test sensitivity and may produce confounding results, and thus a single test sample per loop is recommended.

Effect of Temperature on Thrombogenicity Testing of Biomaterials in an In Vitro Dynamic Flow Loop System.

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 temperatures and blood species ( p < 0.05). However, compared with 37°C, testing at room temperature appeared to have slightly better sensitivity in differentiating silicone (intermediate thrombogenic potential) from the relatively thromboresistant materials (PTFE and HDPE, p < 0.05). These data suggest that testing at room temperature may be a viable option for dynamic thrombogenicity assessment of biomaterials and medical devices.

Comparison of animal and human blood for in vitro dynamic thrombogenicity testing of biomaterials.

BACKGROUND: To determine suitable alternatives to human blood for in vitro dynamic thrombogenicity testing of biomaterials, four different animal blood sources (ovine, bovine, and porcine blood from live donors, and abattoir porcine blood) were compared to fresh human blood. METHODS: To account for blood coagulability differences between individual donors and species, each blood pool was heparinized to a donor-specific concentration immediately before testing in a dynamic flow loop system. The target heparin level was established using a static thrombosis pre-test. For dynamic testing, whole blood was recirculated at room temperature for 1 h at 200 ml/min through a flow loop containing a single test material. Four materials with varying thrombotic potentials were investigated: latex (positive control), polytetrafluoroethylene (PTFE) (negative control), silicone (intermediate thrombotic potential), and high-density polyethylene (HDPE) (historically thromboresistant). Thrombus weight and surface area coverage on the test materials were quantified, along with platelet count reduction in the blood. RESULTS: While donor-specific heparin levels varied substantially from 0.6 U/ml to 7.0 U/ml among the different blood sources, each source was able to differentiate between the thrombogenic latex and the thromboresistant PTFE and HDPE materials (p < 0.05). However, only donor ovine and bovine blood were sensitive enough to differentiate an increased response for the intermediate thrombotic silicone material compared to PTFE and HDPE. CONCLUSIONS: These results demonstrated that multiple animal blood sources (particularly donor ovine and bovine blood) may be suitable alternatives to fresh human blood for dynamic thrombogenicity testing when appropriate control materials and donor-specific anticoagulation levels are used.

Platelet and leukocyte count assay for thrombogenicity screening of biomaterials and medical devices: Evaluation and improvement of ASTM F2888 test standard.

An appropriate preclinical thrombogenicity evaluation of a blood-contacting device is important to reduce thrombosis and thromboembolism risks to patients. The in vitro platelet and leukocyte count assay, as described in the ASTM F2888 test standard, aims to assess thrombogenic potentials of blood-contacting materials. The goals of this study were to evaluate whether this standardized test method can effectively differentiate materials with different thrombogenic potentials and to investigate the impact of anticoagulation conditions on test sensitivity. Using human blood with various anticoagulation conditions, we performed the platelet and leukocyte count assays on four biomaterials and three positive control materials. We found that the use of sodium citrate anticoagulation as stipulated in the 2013 version of the ASTM F2888 standard cannot differentiate materials with different thrombogenic potentials. The modification to use low-concentration heparin, either with recalcified citrated blood or with direct heparinization, substantially improved the test sensitivity and enabled the assay to distinguish platelet count reduction between the positive controls and commonly used biomaterials. Leukocyte count was shown to be a much less sensitive indicator than platelet count for thrombogenicity evaluations of biomaterials. The findings from this study have been incorporated in the recent 2019 version of the ASTM F2888 standard.

Influence of shear rate and surface chemistry on thrombus formation in micro-crevice.

Thromboembolic complications remain a central issue in management of patients on mechanical circulatory support. Despite the best practices employed in design and manufacturing of modern ventricular assist devices, complexity and modular nature of these systems often introduces internal steps and crevices in the flow path which can serve as nidus for thrombus formation. Thrombotic potential is influenced by multiple factors including the characteristics of the flow and surface chemistry of the biomaterial. This study explored these elements in the setting of blood flow over a micro-crevice using a multi-constituent numerical model of thrombosis. The simulations reproduced the platelet deposition patterns observed experimentally and elucidated the role of flow, shear rate, and surface chemistry in shaping the deposition. The results offer insights for design and operation of blood-contacting devices.

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.

An In Vitro Blood Flow Loop System for Evaluating the Thrombogenicity of Medical Devices and Biomaterials.

A reliable in vitro dynamic test method to evaluate device thrombogenicity is very important for the improvement of the design and safety of blood-contacting medical devices, while reducing the use of animal studies. In this study, a recirculating flow loop system was developed for thrombogenicity testing, using donor sheep blood anticoagulated with Anticoagulant Citrate Dextrose Solution A (ACDA) and used within 24-36 hr postdraw. Immediately before testing, the blood was recalcified and heparinized to a donor-specific target concentration. The heparinization level was based on a static pretest, in which latex tubes were incubated at room temperature for 30 min in blood with a series of heparin concentrations and evaluated for thrombus deposition. For dynamic testing, blood was recirculated at room temperature through a polyvinyl chloride (PVC) tubing loop containing a test material for 1 hr at 200 ml/min using a roller pump. Nine materials were investigated: a negative control (polytetrafluoroethylene [PTFE]), a positive control (latex), and seven commonly used biomaterials including PVC, two silicones with different formulations (Q-Sil and V-Sil), nylon, polyurethane (PU), high-density polyethylene (HDPE), and polyether block amide (PEBAX). The results showed that latex was significantly more thrombogenic than all the other materials (p < 0.05), PVC and Q-Sil exhibited intermediate thrombogenicity with significantly more thrombus surface coverage and thrombus weight than PTFE (p < 0.05), whereas PTFE and the rest of the biomaterials had little to no thrombus deposition. In summary, the test loop system was able to effectively differentiate materials with different thrombogenic potentials.

Multi-Constituent Simulation of Thrombus Deposition.

In this paper, we present a spatio-temporal mathematical model for simulating the formation and growth of a thrombus. Blood is treated as a multi-constituent mixture comprised of a linear fluid phase and a thrombus (solid) phase. The transport and reactions of 10 chemical and biological species are incorporated using a system of coupled convection-reaction-diffusion (CRD) equations to represent three processes in thrombus formation: initiation, propagation and stabilization. Computational fluid dynamic (CFD) simulations using the libraries of OpenFOAM were performed for two illustrative benchmark problems: in vivo thrombus growth in an injured blood vessel and in vitro thrombus deposition in micro-channels (1.5 mm × 1.6 mm × 0.1 mm) with small crevices (125 µm × 75 µm and 125 µm × 137 µm). For both problems, the simulated thrombus deposition agreed very well with experimental observations, both spatially and temporally. Based on the success with these two benchmark problems, which have very different flow conditions and biological environments, we believe that the current model will provide useful insight into the genesis of thrombosis in blood-wetted devices, and provide a tool for the design of less thrombogenic devices.

Visualization and analysis of biomaterial-centered thrombus formation within a defined crevice under flow.

The blood flow pathway within a device, together with the biomaterial surfaces and status of the patient's blood, are well-recognized factors in the development of thrombotic deposition and subsequent embolization. Blood flow patterns are of particular concern for devices such as blood pumps (i.e. ventricular assist devices, VADs) where shearing forces can be high, volumes are relatively large, and the flow fields can be complex. However, few studies have examined the effect of geometric irregularities on thrombus formation on clinically relevant opaque materials under flow. The objective of this study was to quantify human platelet deposition onto Ti6Al4V alloys, as well as positive and negative control surfaces, in the region of defined crevices (∼50-150 µm in width) that might be encountered in many VADs or other cardiovascular devices. To achieve this, reconstituted fresh human blood with hemoglobin-depleted red blood cells (to achieve optical clarity while maintaining relevant rheology), long working optics, and a custom designed parallel plate flow chamber were employed. The results showed that the least amount of platelet deposition occurred in the largest crevice size examined, which was counterintuitive. The greatest levels of deposition occurred in the 90 µm and 53 µm crevices at the lower wall shear rate. The results suggest that while crevices may be unavoidable in device manufacturing, the crevice size might be tailored, depending on the flow conditions, to reduce the risk of thromboembolic events. Further, these data might be used to improve the accuracy of predictive models of thrombotic deposition in cardiovascular devices to help optimize the blood flow path and reduce device thrombogenicity.

Real time visualization and characterization of platelet deposition under flow onto clinically relevant opaque surfaces.

Although the thrombogenic nature of the surfaces of cardiovascular devices is an important aspect of blood biocompatibility, few studies have examined platelet deposition onto opaque materials used for these devices in real time. This is particularly true for the metallic surfaces used in current ventricular assist devices (VADs). Using hemoglobin depleted red blood cells (RBC ghosts) and long working distance optics to visualize platelet deposition, we sought to perform such an evaluation. Fluorescently labeled platelets mixed with human RBC ghosts were perfused across six opaque materials (a titanium alloy (Ti6Al4V), silicon carbide (SiC), alumina (Al2O3, 2-methacryloyloxyethyl phosphorylcholine polymer coated Ti6Al4V (MPC-Ti6Al4V), yttria partially stabilized zirconia (YZTP), and zirconia toughened alumina (ZTA)) for 5 min at wall shear rates of 400 and 1000 s(-1). Ti6Al4V had significantly increased platelet deposition relative to MPC-Ti6Al4V, Al2 O3 , YZTP, and ZTA at both wall shear rates (p < 0.01). For all test surfaces, increasing the wall shear rate produced a trend of decreased platelet adhesion. The described system can be a utilized as a tool for comparative analysis of candidate blood-contacting materials with acute blood contact.