BIO4: Investigation of Two Molecular Biomarkers for In Vitro Thrombogenicity Assessment of Device Materials

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Purpose: The goal of this study was to investigate two molecular biomarkers, beta-thromboglobulin (β-TG) and Thrombin-Antithrombin (TAT), for in vitro thrombogenicity testing of materials and to assess the impact of heparin concentration on test sensitivity. Methods: Four commonly used medical device materials: polytetrafluoroethylene (PTFE), high–density polypropylene (HDPE), 316 L stainless steel (SS), and silicone (Si), and two thrombogenic materials: Buna-N rubber (Buna) and glass beads, were investigated. Acid-Citrate-Dextrose Solution A (ACDA) anticoagulated human blood, drawn from healthy adult donors (n=6 and tested separately), was used within 4 hrs post blood draw. Immediately before starting each test, the blood was recalcified with calcium chloride and heparinized with a concentration range of 0.5- 1.5 U/mL heparin. Test materials were incubated in the heparinized blood at an exposure ratio of 6 cm2/ml in a polypropylene tube (Figure 1A) for 1 hr in a shaking water bath (37 °C, 60 rpm). After the incubation, additional ACDA was added to inhibit further blood reactions and the tube was placed on ice. Platelet (PLT) and leukocyte (WBC) counts were measured using a hematology analyzer (Hemavet 950 FS). Platelet free plasma was prepared by centrifuging the blood at 4 °C (in two steps: 1500g for 10min, then 10000g for 10 min) and then stored at −80 °C until enzyme-linked immunosorbent assay (ELISA) analysis for molecular biomarkers β-TG and TAT. Results: The results show that test sensitivity was improved when donor-specific heparin concentration (based on the heparin concentration that yielded Activated Clotting Time (ACT) between 200-235 sec) was used in the test for each donor blood compared to a fixed heparin concentration. As shown in Figure 1B-D, similar to PLT and WBC count based thrombogenicity markers, both molecular biomarkers (β-TG and TAT) could differentiate between the thrombogenic materials and commonly used biomaterials under the test conditions of the current study, which used 6 cm2/mL material surface area to blood volume ratio (lower than ASTM F2888-19 standard recommended ratio of 12 cm2/mL). In addition, compared to PLT and WBC count markers, the molecular biomarkers showed slightly better consistency in differentiating materials with intermediate thrombogenicity potential (e.g., Si and SS) from thrombogenic materials. Summary: Both molecular biomarkers (β-TG and TAT) can distinguish thrombogenic potentials of different materials at a surface area to volume ratio of 6 cm2/mL. The test sensitivity can be improved by using a donor-specific heparin concentration, which can be determined by ACT measurement for each donor blood. Additional testing is needed to further validate these results and to determine their regulatory and clinical relevance. Different molecular biomarkers will also be investigated in the future.Figure 1. (A) Experimental setup showing the test material segments were incubated in recalcified whole blood with a series of heparin concentrations for 1 hr at 37°C. For positive control: Test was run with 20 µM ADP in recalcified blood. For negative control: Test was run without any test material. (B-D) Test results for Platelet and WBC counts, Mean TAT and β-TG concentrations after 1 hour incubation of materials at ACT-based donor-specific heparin concentrations, respectively. Data are shown as Mean ± SD (n=6).