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

Abstract

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.

Keywords: animal blood sources; blood flow loop; human blood; in vitro testing; platelets; thrombogenicity; thrombosis.

Figure 1:

A) Representative image of the test material tubing segment showing how a test material is introduced into the flow loop. B) Experimental set-up of the dynamic flow dual-loop system.

Figure 2:

Static latex pre-test performed to estimate donor-specific heparin concentrations. A) Uniform latex tubing segments were incubated in re-calcified blood with a series of species-dependent heparin concentrations for 15 minutes at 37°C (ovine blood was incubated for 30 minutes at room temperature). Example images of the pre-test results: B) Donor porcine blood, C) Abattoir porcine blood, D) Donor bovine blood, E) Donor ovine blood, and F) Fresh human blood. Threshold Concentration is defined as the minimum heparin concentration that resulted in a thrombus surface coverage ≤ 10% on the latex tubing. The difference between the Initial Concentration for the dynamic flow loop test and the Threshold Concentration was blood species dependent, as indicated by the arrows. The initial heparin concentration was 1.5 U/mL lower than the threshold concentration for donor porcine blood, 2.5 U/mL lower for abattoir porcine blood, 0.4 U/mL lower for bovine blood, 0.2 U/mL lower for ovine blood, but 0.4 U/mL higher for fresh human blood.

Figure 3:

Images of the resulting thrombus deposition on the positive (latex) and negative (PTFE) control materials after 1 hr of circulation with ovine blood from two separate donors anticoagulated to the same heparin concentration (1.2 U/mL).

Figure 4:

Representative images of typically seen thrombus formation on the test materials after 1 hour of circulation for the different blood species. Blood flow direction was from right to left.

Figure 5:

Effect of blood source on thrombus surface coverage. N = 6 for human blood, N = 5 for all other blood sources.

Figure 6:

Effect of blood source on normalized thrombus weight. N = 6 for human blood, N = 5 for all other blood sources.

Figure 7:

Effect of blood source on platelet count reduction. N = 6 for human blood, N = 5 for all other blood sources.