An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

This protocol provides a benchtop method for assessing the efficacy and mechanisms of action of histotripsy aided thrombolytic therapy or lysotripsy, an alternative non-invasive approach for treating critical deep vein thrombosis. This technique uses bubble activity to have a two-way effect through an increased fibrinolysis due to an enhanced lytic delivery and the hemolysis of red blood cells within the clot. This benchtop approach to treating deep vein thrombosis facilitates the modeling of blood clots, treatment with lysotripsy, and simultaneous imaging during treatment.

Bubble cloud generation, treatment planning, and image guidance can be further used to investigate in vitro histotripsy-based treatments of other diseases, such as kidney and liver tumors. To set up the histotripsy source, mount the source onto a motorized positioning system. Cover the imaging array with a probe cover and fix the array coaxially in the aperture of the histotripsy source.

Connect the imaging array to an ultrasound scanning system and completely submerge the histotripsy source and imaging array in the tank. Use a syringe to gently remove any air bubbles and use the imaging array and scanner to acquire degassed B-mode water images at 20 frames per second. While acquiring the images, adjust the position of the imaging array inside the confocal transducer opening until the bubble cloud is located approximately at the center of the image window and record the detected focal location in the imaging window.

Then discontinue the insonation and set the voltage applied to the histotripsy source to zero. To prepare a clot for analysis, use pliers to cut the sealed end of a pipette containing a clot and let the clot and serum slide into a petri dish. Use a scalpel to cut the clot to a one centimeter length and use a cleaning wipe to gently blot the cut section to remove any excess fluid.

Use tweezers to carefully place the section of clot onto a scale and record the weight. Next, manually raise the flow channel out of a degassed reverse osmosis water tank and remove the model vessel from the channel. Use tweezers to carefully place the clot into the model vessel without damaging the clot and attach the vessel to the flow channel.

Lower the flow channel into the tank such that the proximal end of the stage relative to the reservoir is low compared to the distal side, and use a pipette to add 30 microliters of 37 degrees Celsius warmed plasma to the reservoir. Monitor the temperature of the water tank until it reaches at least 36 degrees Celsius, and use a pipette to add 80.4 micrograms of rt-PA into the plasma reservoir. To prime the flow channel, use a syringe pump to draw plasma from the reservoir into the channel until the model vessel has been filled.

Then manually level the model vessel to ensure that no air bubbles are present in the model vessel, as seen within the imaging window. To plan a path for the histotripsy source and imaging array for a uniform histotripsy exposure along the clot length, use the imaging script to use the motorized positioners to align the imaging array parallel to the length of the clot and confirm that no air bubbles are present in the vessel. Use the motorized positioners to align the imaging array such that the imaging plane is parallel to the cross section of the clot.

Using the imaging window as a guide, move the histotripsy source to the proximal end of the clot, relative to the reservoir. To determine the insonation path along the clot length, set waypoints along the length of the clot in five millimeter increments. Prior to finalizing each waypoint, fire test pulses from the histotripsy source with the same insonation parameters as demonstrated, but with the overall exposure reduced to 10 total pulses.

At each waypoint, save the motor positions using the commands designated by the manufacturer. To treat the clot along its length according to the path defined in the pre-treatment step, set the syringe pump to 0.65 milliliters per minute, and wait for the meniscus of the plasma to move. Interpolate the treatment path with intermediate steps between the established waypoints with a fixed step size, and use the positioners to move the histotripsy source at each path location, using the originally set insonation parameters.

Next, create a script to set the imaging array to acquire a B-mode image of the clot and model vessel a few seconds before the application of histotripsy pulse at each location. Then apply the histotripsy pulse at each waypoint, acquiring the acoustic emissions in the script to form passive cavitation images after the analysis. Use the imaging window to image the bubble activity during the application of the histotripsy pulse at each path location.

After the analysis, manually raise the model vessel out of the water tank to drain the perfusate via gravity, and use the syringe pump to draw the plasma solution from the flow channel to allow collection of the entire perfusate in a small beaker. Disconnect the model vessel and remove the clot. Then wipe the clot with lab tissues and weigh the clot to assess the clot mass loss.

Upon application of sufficient voltage to the histotripsy source, a bubble cloud is generated in the focal region of the transducer and visualized via ultrasound imaging. The focal position is defined as the center of the bubble cloud. This control perfusate of a clot exposed to plasma alone, and this perfusate of a lysotripsy treated clot were used to assess the hemoglobin and D-dimer content as demonstrated.

The variability in hemoglobin concentration can be quantified by optical absorbance. Here, a clot can be visualized within a model vessel via B-mode imaging prior to histotripsy exposure to determine the clot position for segmentation of the passive cavitation image. As this passive cavitation image co-registered with the B-mode image of the clot confirms, the acoustic energy is contained primarily within the clot during the histotripsy exposure.

In samples exposed to histotripsy, disruption is primarily restricted to the clot center, consistent with the observed locations of bubble activity tracked via passive cavitation imaging. With the addition of lytic, mass loss also occurs in regions closer to the periphery of the clot. Alternatively, ecogenic liposomes containing rt-PA can be used in place of systemic rt-PA delivery to allow the assessment of targeted drug delivery to the clot.

Lysotripsy provides an alternative non-invasive approach to the treating of many diseases. This in vitro protocol enables assessment of the efficacy of the treatment and its potential success in in vivo application.