2.6. In Vitro Blood Compatibility Testing.

The hemolytic activity of the optimized GSNO–PVC material was determined in vitro based on ASTM F756, Standard Practice for Assessment of Hemolytic Properties of Materials. Whole porcine blood was drawn immediately prior to testing using 3.8% sodium citrate at a 9:1 blood to citrate ratio. Prior to hemolysis testing, the plasma hemoglobin levels were determined by combining plasma (750 μL, collected through centrifugation) with Drabkin’s reagent (750 μL) for 15 min before measuring the absorbance at 540 nm via the plate reader. The hemoglobin concentration was determined using a standard curve composed of known concentrations, and its level in the drawn blood must be below 2 mg mL⁻¹ to proceed.

To begin testing the hemolytic activity of the samples, well-mixed whole blood (20 μL) was combined with Drabkin’s reagent (5 mL), allowed to stand for 15 min, and measured at 540 nm to quantify the total hemoglobin concentration present in the whole blood. The whole blood was then diluted with CMF-PBS to achieve a hemoglobin concentration of 10 mg mL⁻¹. To test the samples, diluted whole blood (1 mL) and CMF-PBS (7 mL) were pipetted into falcon tubes, and the samples were individually placed in the tubes for incubation for 3 h at 37 °C. For blank and positive controls, falcon tubes containing only CMF-PBS with whole blood and sterile DI water with whole blood, respectively, were also incubated. The falcon tubes were gently inverted every 30 min throughout the incubation time. After 3 h, the samples were removed, and the falcon tubes were centrifuged at 700 rpm for 15 min. After centrifugation, supernatant (1 mL) from each sample tube was collected combined with Drabkin’s reagent (1 mL). After 15 min, absorbance was measured at 540 nm and the concentration of free hemoglobin was determined (eq 1).

where WB means whole blood and Abs means absorbance.

To evaluate the thrombotic activity in vitro, unmodified PVC and optimized GSNO–PVC samples were exposed to whole blood for 60 s and 5 min. SEM images of samples were taken after being fixed in 2% glutaraldehyde, serially exposed to increasing ethanol concentrations, and chemically dried using HMDS.