We aim to develop a practical and accessible benchtop protocol to identify potential thrombogenic hotspots in ventricular assist devices, bridging the critical gap between early prototyping and animal testing. Animal testing has persisted as the primary method for evaluating thrombosis in ventricular assist devices. While computational fluid dynamics has become an invaluable tool for design verification, it cannot supplant in vitro and in vivo validation yet.
The complex nature of blood coagulation presence and numerous confounding factors, making it difficult to distinguish between actual device thrombogenicity and experimental artifacts. In vitro methods for assessing thrombosis in VADs remain underutilized, and no standardized method exists to-date. By offering a comprehensive protocol and video guide, we hope to facilitate broader adoption of benchtop methods.
This method addresses both a technical and ethical challenge in medical device development, reducing reliance on animal testing, while improving the ability to detect thrombosis risks early in the development process. To begin, fill the test loop with 150 milliliters of blood. Eliminate all air trapped in the loop.
If using an axial flow blood pump, rotate it into a vertical position to release air via buoyancy. If using a centrifugal pump, flip it upside down and rotate it to ensure no air is trapped in secondary flow paths. Gently tap the tubing and reservoir surfaces and squeeze any air bubbles to dislodge them.
Closely inspect horizontal sections of tubing and the junctions between tubing and connectors to ensure no air bubbles are present. Squeeze the intravenous bag to bring the fluid level close to the narrow top portion. Then clamp the bag with a hemostat across the fluid line to eliminate the fluid-air interface, and close the air vent stopcock.
Next, close the stopcock at the end of the pressure lines. Remove the manometer tubing and attach a three-milliliter syringe. Open the stopcock and draw one to two milliliters of fluid into the extension line, approximately four centimeters.
Close the stopcock, detach the syringe, and reconnect the manometer tubing. Open the stopcock to enable pressure readings. Finally, prime the sampling port using a syringe.
Next, cut or tear a lint-free wipe into three equal fragments. Twist the corner of one fragment to form a tip and insert it into the injection port to absorb residual blood. Twist the second fragment.
Moisten it with saline, and insert the wet tip into the port to remove all remaining blood. Use the third fragment to absorb any residual saline in the port. Now start the ventricular assist device, or VAD, at a low speed and operate it for about five seconds to dislodge any trapped air bubbles within the pump.
Stop the pump. If any bubbles appear in the bag, repeat de-airing. Restart the pump at a low speed to circulate the blood in the loop.
Transfer one milliliter of heparin sodium, two milliliters of the prepared calcium chloride solution, and 1.5 milliliters of EDTA into separate tubes. To add heparin to the blood in the loop, aspirate 75 units of heparin into a micropipette, and dispense it into a three-milliliter syringe by simultaneously dispensing the pipette and drawing the syringe plunger without spilling. Use the transverse port of the three-way stopcock to administer substances into the loop with a syringe.
Rotate the male lure lock on the stopcock so the injection port faces upward to trap air bubbles at the top. Now attach the syringe to the injection port via the lure lock with the port facing up and the syringe pointing vertically down. Draw the syringe plunger to fill it with blood and mix the heparin with the blood while aspirating any air into the syringe.
Allow the air bubbles to rise to the top of the syringe. Then inject the mixture into the loop, ensuring no air enters. Shuttle blood in and out of the syringe four to five times to ensure that the heparinized blood does not stay confined to the space in the port.
To titrate the activated clotting time, or ACT, of blood in the loop, first add 750 microliters of one-molar calcium chloride solution to 150 milliliters of blood in the loop. To prevent blood coagulation, swiftly inject the fluid after removing residual air. Alternatively, dilute the calcium chloride in the syringe with one milliliter of tris-buffered saline to reduce premature coagulation.
After letting the injected calcium chloride circulate for at least two minutes, attach a one-milliliter syringe to the sampling port. Draw and discard 0.5 milliliters of waste blood to clear stagnant blood from the port. Next, attach a new one-milliliter syringe to the sampling port and draw 0.5 milliliters of blood for analysis.
Measure the activated clotting time using a point-of-care whole blood coagulation system. Clean the sampling port using lint-free wipes as demonstrated before. Refer to the titration values to inform the target calcium chloride concentration.
Incrementally increase the calcium concentration to achieve an activated clotting time of 300 seconds. Commence the in vitro thrombosis test once the ACT of 300 seconds is achieved in the loop. Adjust the pump to the desired flow rate and pressure by modifying the rotor speed and regulating the resistance using the Hoffman clamp.
Measure the ACT every 15 minutes by drawing a blood sample from the loop and placing a drop of blood into the ACT instrument, as demonstrated earlier. If the ACT drops below 200 seconds, inject an additional 25 units of heparin sodium into the loop. After one hour, inject 1.5 milliliters of 0.5-molar EDTA into the loop to inhibit further coagulation.
Let the EDTA circulate and mix for two minutes, then stop the pump. Using hemostats, clamp the tubing connected to the pump inlet and outlet, positioning the clamps three to four centimeters away from the inlet and outlet barbs. Carefully disconnect the tubing to release the pump.
Then drain the blood from the pump and the flow loop into a container. Finally, pipette saline through the pump inlet and outlet to wash any residual blood. Platelet deposition was consistently observed along the roots of the blades in the pump prototypes.
Electro-polishing the titanium components eliminated the thrombus formation on these areas. The prototype also had imperfect cooptation in the fore and aft housing component junction, resulting in a crevice where blood seeped in and coagulated. Lapping and polishing improved the fitting of the components and reduced thrombus formation.
Procedural errors and incomplete de-airing of the loop can introduce artifacts. In this instance, an experimental error during calcium chloride injection triggered localized coagulation, and the resulting thrombus entered the centrifugal Maglev pump, occluding the flow path. Spherical, loosely adhered clots observed on surfaces are often the result of air bubbles encapsulated within clots.
Foreign material and debris circulating in the loop were also encapsulated in thrombi and adhered to pump surfaces. Ring thrombi at tubing connector junctions were indicative of optimal ACT range during the test.
