This protocol presents a model for the hemocompatibility investigation of blood-contacting devices according to International Standard Organization guidelines. The model is ideal for mimicking the physiological conditions that correspond to the implant since the low background for thrombotic events and the low concentration of anticoagulants enable a broad analysis of blood parameter. This method is ideal for biomaterial research since it provides a simple way to evaluate hemocompatibility.
Moreover, it can be used for preclinical studies. For heparin loading, first mix 5, 000 international units of undiluted heparin with 0.9%sodium chloride to obtain a 15 international units per milliliter of heparin concentration. Next, add 900 microliters of the diluted heparin solution to three neutral monovettes and three reserved monovettes per donor and store the heparin-loaded monovettes at four degrees Celsius.
30 minutes before the blood sample collection, place the monovettes at room temperature. When the monovettes have warmed, collect nine milliliters of blood into each of three monovettes per donor before pooling all 27 milliliters of blood from each donor into a single plastic container. To prepare the flow loop, load at least one tube per condition with the neurovascular laser cut implant of interest and place one end of each tube in a reservoir filled with 0.9%sodium chloride.
Place an unloaded tube into the reservoir and connect all of the tubes to the pump head. Then insert the other end of each tube into a measuring cylinder and adjust the settings of the peristaltic pump to a flow rate of 150 milliliters per minute using a timer while checking the fill level in the measuring cylinder. For hemocompatibility testing, use a 12 milliliter syringe to fill each tube with six milliliters of blood.
After forming a circuit, use a 0.5 centimeter length of silicone connecting tubing to close the tubes tightly and place the tubes in a 37 degree Celsius water bath. Then start the 60 minute tube. For whole blood count analysis, add 1.2 milliliters of nonperfused blood or 1.2 milliliters of blood from each tube after the perfusion into individual monovettes containing EDTA.
Carefully invert the monovettes five times and load the vials into a blood analyzer for a blood count analysis of each sample. At the end of the analysis, place the monovettes on ice for 15-60 minutes. For citrate plasma collection, fill citrate-containing monovettes with 1.4 milliliters of nonperfused or perfused blood and carefully invert each monovette five times.
Separate the plasma by centrifugation and transfer three 250 microliter aliquots of the plasma fraction into individual 1.5 milliliter reaction tubes. Then freeze the plasma samples in liquid nitrogen and store them at minus 20 degrees Celsius until their analysis. For EDTA plasma collection, after the four degrees Celsius incubation after the blood count measurement, separate the plasma by centrifugation and transfer three 250 microliter aliquots of the plasma fraction into individual 1.5 milliliter reaction tubes.
Then freeze the plasma samples in liquid nitrogen for minus 20 degrees Celsius storage. For CTAD plasma collection, fill CTAD-containing monovettes with 2.7 milliliters of freshly drawn or perfused blood and carefully invert the tubes five times. Place the monovettes on ice for 15-60 minutes before collecting the plasma by centrifugation.
Transfer 700 microliters of each middle plasma fraction into individual 1.5 milliliter reaction tubes and centrifuge the filled reaction tubes again. Then transfer two 100 microliter aliquots of the middle fraction into individual 1.5 milliliter reaction tubes and freeze the tubes in liquid nitrogen for their storage at minus 20 degrees Celsius. To measure the TAT levels in the citrate plasma samples, add 50 microliters of ELISA sample buffer into each well of a 96-well flat bottom plate and 50 microliters of plasma standard, plasma control, and undiluted plasma samples in duplicates to the appropriate wells of the plate.
After sealing the plate, incubate the samples at 37 degrees Celsius for 15 minutes with gentle shaking. At the end of the incubation, wash the plate three times with 300 microliters of washing solution per well before adding 100 microliters of peroxidase conjugated anti-human TAT antibody to each well. After a 15-minute incubation at 37 degrees Celsius with shaking, wash the plate three times with 300 microliters of fresh washing solution per well.
After the last wash, add 100 microliters of freshly prepared chromogen solution per well for a 30-minute incubation at room temperature. At the end of the incubation, add 100 microliters of stop solution to each well and read the optical density on a photometer at 490 to 500 nanometers using the standard curve data as a trend line for calculating the TAT concentration of each sample. To prepare the implants for scanning electron microscopy, use forceps to remove the implants from the tubes and briefly rinse each implant three times in fresh 0.9%sodium chloride per wash.
After the last wash, store the implants in individual containers of 2%glutaraldehyde in PBS without calcium and magnesium overnight at four degrees Celsius. The next morning, incubate the implants in individual containers of PBS for 10 minutes before dehydrating the implants in ascending concentrations of ethanol for 10 minutes per concentration as indicated. Directly after blood collection and perfusion, no changes are detected in the number of white or red blood cells or in the hematocrit values.
However, a decrease in hemoglobin levels is detected after perfusion within the flow loop system. A decrease in platelet numbers is also observed, that is increased when an uncoated stent is present within the tubing. Notably, this loss is reduced when the blood is incubated with a fibrin-heparin-coated stent.
The TAT complex concentration is mildly increased in response to the perfusion. When a bare metal stent is added, however, a significant increase in the TAT is detected indicating a profound activation of the coagulation system. The use of a fibrin-heparin-coated stent prevents this activation.
Perfusion leads to an increased activation of the compliment cascade that is not affected by the presence of uncoated or fibrin-heparin-coated stents. Similarly, neutrophil granulocytes are activated as evidenced by the quantification of polymorphonuclear neutrophil elastase levels. Visualization by scanning electron microscopy reveals that the presence of a dense network of blood cells and proteins on the surface of the uncoated stent after perfusion that is not observed on the fibrin-heparin-coated stents.
It is of utmost importance to confirm that the blood fulfill the inclusion criteria for obtaining samples and to use freshly drawn blood as quick as possible. Human blood may contain bloodborne viruses and other agents and carries the risk of infection so be sure to always wear appropriate PPE when handling the samples.
