Thrombosis is associated with numerous medical conditions and current treatments risk severe bleeding. Circulatory system flow significantly impacts blood clot structure. Incorporating flow into in vitro models may improve their physiological relevance, enhancing the selection of better candidates for in vivo testing.
The assays for the study of fibrinolytic predominantly utilize static clot or microfluidic systems to assess properties such as lysis time, clot structure, or viscoelasticity. Static clot lysis assays typically rely on spectrophotometric measurements or clot mass determination as endpoints to evaluate fibrinolysis efficiency. Current thrombolytic testing is a statically formed clots, which differs structurally from those formed under flow conditions.
Microfluidic systems consider flow dynamics, but their smaller scales create unique hemodynamics and clot structures. The currently developed RT-FluFF essay provides ease in monitoring for lysis in real-time under physiological flow condition. This means we now have a platform that allows control over multiple parameters to mimic condition closer to study models for better translation when screening new thrombolytics.
To begin, add fluorescently-tagged fibrinogen to three milliliters of citrated human whole blood to achieve a final concentration of 60 micrograms per milliliter. Set up the Chandler loop with a 120 millimeter diameter drum in a 37 degree Celsius water bath. Cut a standard 200 microliter PCR tube and connect the ends of the tubing.
To initiate blood clotting, add 176 microliters of 200 millimolar calcium chloride into the three milliliters of whole blood. After properly mixing with gentle inversion, using a three milliliter syringe, load the blood into the tubing, immediately place the tubing onto the Chandler loop drum connecting the ends. Under low light conditions, rotate the drum partially submerged in the water bath at 40 rpm.
After 40 to 60 minutes, remove the clots from the tubing. Gross Chandler loop FITC fibrinogen-tagged whole blood characteristics showed uniform dispersion of fluorescence under ultraviolet light. Further, varying the degree of FITC fibrinogen within the whole blood does not alter clot weights.
To begin, connect the flow loop apparatus and ensure that all connections are secure. Turn on the pressure monitor. Ensure the monitor reads zero millimeters of mercury for both the inlet and outlet sensors.
Turn on the water bath and adjust the temperature to 37 degrees Celsius to mimic physiologic human body temperature. Start the pump at the desired flow rate to check for any leaks, and confirm the proper function of the pressure sensors. Turn off the pump to facilitate the plasma mobile phase and clot loading.
To measure previously prepared a human whole blood clot mass, gently blot clots on laboratory wipes until no significant amount of liquid is released. Submerge the clot in type matched pulled plasma in a weighing disk. Remove the central section of the flow loop tubing between the inlet and outlet sensors and attach a 10 milliliter syringe to one end of the tubing.
Through the free end, prime the tubing with a small volume of plasma. After identifying the head and tail of the clot, position the head toward the inlet pressure sensor. Place the tubing at the tail end of the clot and aspirate into the tubing with the syringe.
Attach the tubing to the flow loop apparatus, positioning the clot closest to the outlet pressure sensor with the head facing away. To secure the clot in position, puncture the tubing and clot head with two 30-gauge needles in an X pattern. Transfer the remaining mobile phase solution into a 50 milliliter tube as the reservoir.
Place the reservoir in the water bath. Then insert inlet and outlet tubing in the reservoir. Turn on the fluorometer and verify that the initial value is appropriate by comparing it to a mobile phase-only system baseline.
Add 500 microliters of tissue plasminogen activator directly to the reservoir. Start the pump and adjust the flow rate to 160 milliliters per minute. The system then fills with fluid and bubbles.
Once the bubbles are gone, turn off the light and start the data acquisition. Record until the clot is significantly degraded. While the plasma is flowing, monitor the pre and post-clot system pressures, which will change over the course of the clot digestion process as the clot mass decreases.
After the clot digestion, reduce the volumetric flow rate and remove the inlet tubing while the pump is still running to push most of the fluid into the reservoir. Disconnect the clot containing tubing section at the outlet pressure sensor side, and lower it into a weigh boat. Remove the needles to collect the remaining fluid and clot fragments.
Weigh the remaining clot. Between samples, load warm water into the reservoir and run through the system for two minutes at 150 rpm. After all samples have been run for the day, remove and discard the tubing.
Scrub the T-junctions with hot water and a bristle brush until clean and leave them to dry. Rinse the syringe dampers with hot water and wipe with 70%ethanol to maintain sterility.
