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

Summary

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.

Abstract

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.

Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Introduction

Thrombosis is the condition of clot formation in an otherwise healthy blood vessel that obstructs circulation^1,2. Venous thromboembolism has an annual healthcare cost of $7-10 billion, with 375,000-425,000 cases in the United States³. Pulmonary embolism is the obstruction of the pulmonary artery and is the most serious consequence of venous thromboembolism. The primary source of pulmonary obstruction is deep vein thrombi, primarily from iliofemoral venous segments^4,5,6. Deep vein thrombosis (DVT) has inherent sequela besides pulmonary obstructions, with long term complications that result in pain, swelling, leg ulcerations, and limb amputations^7,8,9. For critical obstructions, catheter directed thrombolytics (CDT) are the frontline approach for vessel recanalization¹⁰. The outcome of CDT depends on a number of factors, including thrombus age, location, size, composition, etiology, and patient risk category¹¹. Moreover, CDT is associated with vascular damage, infections, bleeding complications, and long treatment time¹⁰. Next generation devices aim to combine mechanical thrombectomy with thrombolytics (i.e., pharmacomechanical thrombectomy)^12,13. Use of these devices lower the lytic dosage leading to reduced bleeding complications, and shortened treatment time^12,13,14 as compared to CDT. These devices still retain issues of hemorrhagic side-effects and incomplete removal of chronic thrombi¹⁵. An adjuvant strategy is thus needed that can remove the thrombus completely with lower bleeding complications.

One potential approach is histotripsy-aided thrombolytic treatment, referred to as lysotripsy. Histotripsy is a non-invasive treatment modality that uses focused ultrasound to nucleate bubble clouds in tissues¹⁶. Bubble activity is generated not via exogenous nuclei, but by the application of ultrasound pulses with sufficient tension to activate nuclei intrinsic to tissues, including clot^17,18. The mechanical oscillation of the bubble cloud imparts strain to the clot, disintegrating the structure into acellular debris¹⁹. Histotripsy bubble activity provides effective degradation of retracted and unretracted blood clots both in vivo and in vitro^20,21,22. Prior studies have^23,24 demonstrated that the combination of histotripsy and the lytic recombinant tissue-type plasminogen activator (rt-PA) significantly increases treatment efficacy compared to lytic alone or histotripsy alone. It is hypothesized that two primary mechanisms associated with histotripsy bubble activity are responsible for the improved treatment efficacy: 1) increased fibrinolysis due to enhanced lytic delivery, and 2) hemolysis of red blood cells within the clot. The bulk of the clot mass is comprised of red blood cells²⁴, and, therefore, tracking erythrocyte degradation is a good surrogate for ablation of the sample. Other formed clot elements are also likely disintegrated under histotripsy bubble activity but are not considered in this protocol.

Here, a benchtop approach to treat DVT in vitro with lysotripsy is outlined. The protocol describes critical operating parameters of the histotripsy source, assessment of treatment efficacy, and image guidance. The protocol includes designing a flow channel to mimic an iliofemoral venous segment and manufacturing human whole blood clots. The experimental procedure outlines the positioning of the histotripsy source and imaging array to achieve histotripsy exposure along the clot placed in the flow channel. Relevant insonation parameters to attain clot disruption and minimize off-target bubble activity are defined. The use of ultrasound imaging for guidance and assessment of bubble activity is illustrated²⁴. Metrics to quantify treatment efficacy such as clot mass loss, D-dimer (fibrinolysis), and hemoglobin (hemolysis) are outlined^{23,24,25,26,27}. Overall, the study provides an effective means for executing and assessing the efficacy of lysotripsy to treat DVT.

Protocol

For the results presented here, venous human blood was drawn to form clots after approval from the local internal review board (IRB #19-1300) and written informed consent provided by volunteer donors²⁴. This section outlines a design protocol to assess lysotripsy efficacy. The protocol is based on a previous work by Bollen et al.²⁴.

1. Clot modeling

NOTE: Prepare the clots within 2 weeks but more than 3 days prior to the day of the experiment to ensure clot stability and maximize retraction²⁸. Prepare the clot following the approval from local institutional review board.

1. Prepare borosilicate Pasteur pipette for storing the blood (see Table of Materials for specifications of the pipette). Borosilicate tubes are used because of the hydrophilic nature of the material which promotes platelet activation and clot retraction²⁹. Seal the tip of the pipette via heating over a Bunsen burner.
2. Draw fresh human venous blood. Aliquot the total blood drawn in 2 mL increments per desired clot. Transfer each 2 mL aliquot to one Pasteur pipette.
   NOTE: Execute step 1.2 within approximately 3 min of blood draw so that blood does not clot before transferring to pipettes. Also, ensure the volunteer donor is not on any medications that may alter the clotting cascade (e.g. blood thinners or platelet inhibitors).
3. Incubate the blood aliquots within the pipettes (equal to the number of clots required) in a water bath for 3 h at 37 °C.
4. Store the pipettes for a minimum of 3 days at 4 °C to allow for retraction of clots²⁸. As the clots retract, serum will be observed to accumulate at the top of the clot within the pipette. The rt-PA response of clots remains stable for 2 weeks following retraction²⁸.

2. Water tank preparation

1. Fill the water tank with reverse osmosis water. Line the bottom surface of the tank with an acoustic absorbing material to reduce reflection of the therapy or imaging ultrasound pulses. Use a water handling system to degas and filter the water to minimize bubble nuclei.
   NOTE: One way to filter the water is using an inline filter. The specifications of the bag used for generating the representative results is given in the Table of Materials.
2. Place two heating elements at the bottom surface of the tank. Heat the water to 37.3 ˚C to achieve the maximum lytic enzymatic activity³⁰.
3. Setup a flow channel as shown in Figure 1A. The flow channel consists of tubing, a model vessel with material and geometric properties representative of an iliofemoral vein, a reservoir for plasma, and a syringe on the distal most end of the reservoir (Table of Materials). The syringe is connected to a pump to regulate a flow through the channel during the experiment.
4. Manually submerge the flow channel in the water tank to bring the channel to physiologic temperature during the degassing/filtering/heating stage (steps 2.1 and 2.2).

3. Preparation of plasma and rt-PA mixture

1. Prior to experiment day
   ​NOTE: When stored at -80 °C, plasma is stable for at least 2.5 years³¹ and rt-PA is stable for at least 7 years³². Therefore, execute step 3.1 anytime within these periods to ensure stability of the two components.
   1. Dilute the rt-PA obtained from a manufacturer in powdered form to 1 mg/mL in sterile water.
   2. Aliquot 100 µL of diluted rt-PA into 0.5 mL centrifuge tubes and store them at -80 °C.
   3. Aliquot 35 mL of human fresh-frozen type O plasma in 50 mL centrifuge tubes. Store the tubes at -80 °C.
2. On the experiment day
   1. Retrieve plasma aliquots from the freezer. Retrieve as many aliquots as the number of clots to be tested that day. Immerse the frozen aliquots in a water bath at 37 °C to thaw (~10 min).
   2. Once plasma has thawed, pour it into a beaker that is triply rinsed with ultrapure water. Lightly cover the mouth of the beaker with aluminum foil to prevent contamination and place the beaker in the water bath. Allow the foil to be loose enough to allow air to contact the plasma.
   3. Let plasma equilibrate at 37 °C to atmospheric pressure for at least 2 h.
   4. Take out the frozen rt-PA vials and place on ice until needed, with one vial for each experiment run.
   5. Make low gelling agarose (2%) in a 50 mL flask, by dissolving agarose in ultrapure water. Choose the total amount of agarose solution such that approximately 2 mL is available for each specimen to be analyzed. Heat the solution in flask in a microwave until bubbly. Secure the flask with a waterproof screw lid on it. Submerge the flask in the water bath alongside the plasma.
      ​NOTE: This step ensures agarose is available to secure exposed clot segments for histology analysis following histotripsy insonation.

4. Setting up histotripsy source and imaging array

1. Ensure the motorized positioners can be controlled from the runtime environment of a programming platform, using directions and commands provided by the manufacturer. Check that the motors of the system are connected to the appropriate port of the computer with the runtime environment.
2. Mount the histotripsy source on the motorized positioning system as shown in Figure 1B.
3. Connect the histotripsy source to its driving electronics (e.g., power amplifier and function generator) via the appropriate connectors (e.g., BNC cables) as specified by the manufacturer.
   NOTE: Ensure that the driving electronics of the histotripsy source can be controlled via the runtime environment used in step 4.1.
4. Cover the imaging array with a probe cover and affix the array coaxially in the aperture of the histotripsy source as shown in Figure 2. Be sure to understand the orientation of the imaging plane relative to the orientation of the histotripsy source.
5. Connect the imaging array to an ultrasound scanning system. Ensure that this system can control the operation and triggering of the imaging array, and collect imaging data, as per the commands provided by the manufacturer of the scanner.
6. Submerge the histotripsy source/imaging array into the tank while degassing as shown in Figure 1A. Gently remove any air bubbles using a syringe from the surface of the histotripsy source or imaging array.
   NOTE: Immerse the histotripsy source and imaging array completely in water before operation. Avoid touching the surface of the histotripsy source.
7. Acquire B-mode images at a rate of 20 frames per second using the imaging array and the built-in commands of the scanner. Adjust the imaging window to ensure visualization of the focus of the histotripsy source in these real-time images.
   NOTE: It is assumed that the dimensions of the focal region of the therapeutic source are known.
8. Set the operating parameters of the histotripsy source, including fundamental frequency (e.g., 1.5 MHz), pulse repetition frequency (e.g., 20-100 Hz), pulse duration (e.g., 1-20 cycles per pulse), and total number of pulses per location (e.g., 100-2,000)^18,23,24,33. Modify these parameters if sufficient clot lysis is not achieved or if the bubble activity extends beyond the lumen of the model vessel. To set these parameters, use the protocol provided by the manufacturer of the source or use a programming platform that can communicate with the source (Step 4.3).
9. Using the manufacturer's protocol or programming platform used in step 4.8, run the histotripsy source at the set parameters in degassed water only, without any obstruction in the surrounding environment. Increase the voltage applied to the histotripsy source until a bubble cloud is formed.
10. Using the real-time imaging of step 4.7, 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. The bubble cloud is the region of hyperechoic pixels in the imaging plane (Figure 3). Tighten the screws to hold the imaging array firmly in the transducer opening.
    NOTE: If the array is aligned properly, the azimuthal position of the bubble cloud should be approximately at 0 mm in the imaging plane. The imaging array may project slightly from the inner surface of the therapy source, and therefore the range position of the bubble cloud may differ from the focal length of the source.
11. Identify the bubble cloud location in the imaging plane. Assign the focus of the histotripsy source as the center of the bubble cloud (Figure 3).
12. Record the detected focal location (step 4.11) in the imaging window (Figure 3). A possible way to mark the focal position is placing a cursor to note the location in the imaging window, if available with the imaging platform.
13. Discontinue insonation and set the voltage applied to the histotripsy source to 0 V.

5. Clot preparation

1. Remove the clot from the pipette by cutting the sealed end with pliers. Let the clot slide into a Petri dish along with the serum. If the clot does not dislodge, gently apply pressure from the other end of the pipette via saline flush to remove the clot.
2. Cut the clot to 1 cm length using a scalpel, aiming for a uniform piece from the center (i.e., away from sections of the clot formed at the top or bottom of the pipette).
3. Use a cleaning wipe to blot the cut section of the clot gently to remove excess fluid.
4. Using tweezers, place the clot section gently on a weighing scale and record the weight.
5. Manually raise the flow channel out of the water tank and remove the model vessel from the flow channel.
6. Place the clot in the model vessel using tweezers and attach the model vessel again to the flow channel.
   NOTE: A nylon rod can be placed within the model vessel to prevent the clot from moving downstream due to the flow.
7. Lower the flow channel into the tank in such a way that the proximal end of the stage relative to the reservoir is low compared to the distal side. The angling of the stage in this manner prevents trapping of bubbles in the model vessel when plasma is drawn through the flow channel in step 6.1.
8. Add 30 mL of plasma to the reservoir using a pipette and monitor the temperature until it reaches at least 36 °C.
9. Use a pipette to dispense the rt-PA (80.4 µg in 30 mL of plasma, 2.68 µg/mL) into the plasma reservoir. Stir the plasma with the pipette to ensure a uniform rt-PA distribution within the reservoir.

6. Priming the flow channel

1. Draw plasma into the flow channel from the reservoir via the syringe pump until the plasma fills the model vessel.
   NOTE: If the clot is not flush with the nylon rod, use short pump draws at 60 mL/min to try to force the plasma downstream the clot or manually draw through the syringe. Limit the amount of plasma drawn in this process or refill the reservoir using additional plasma/rt-PA to ensure 30 mL of solution in the flow channel.
2. Using the motorized positioners, align the imaging array parallel to the length of the clot using the imaging script (step 4.7). The parallel alignment enables the user to visually ensure proper placement of the clot and absence of bubbles inside the model vessel.
3. Level the model vessel manually and visually ensure that no air bubbles are present using the imaging window (step 4.7).

7. Experiment procedure

1. Pre-treatment
   ​NOTE: This step is to plan a path for the histotripsy source/imaging array for uniform histotripsy exposure along clot length.
   1. Align the imaging array using the motorized positioners such that the imaging plane is parallel to the cross-section of the clot (i.e., perpendicular to the orientation described in step 6.2).
   2. Under guidance via the imaging window (step 4.7), move the histotripsy source to the proximal end of the clot relative to the reservoir using the motorized positioners. At this point, adjust the histotripsy source position such that the marked focal point in step 4.12 aligns with the center of the clot.
   3. Determine the insonation path along the clot length. To define this path, set three waypoints along the length of the clot (i.e., positions of the motors where histotripsy bubble activity is contained within the clot) in 5 mm increments. Align the waypoints such that the overall motion of the histotripsy source along the path is antegrade with flow in the system (i.e., the first waypoint is at the most proximal end of the clot relative to the reservoir, and the third waypoint is in the distal position relative to the reservoir).
   4. Prior to finalizing each waypoint, fire test pulses from the histotripsy source with the same insonation parameters as step 4.8 but reduce the overall exposure to 10 total pulses. Adjust the position of the histotripsy source using the motorized positioners if necessary, to contain bubble activity within the clot.
   5. At each waypoint, save the motor positions using the commands provided by the manufacturer, similar to the step 4.1.
2. Treatment
   ​NOTE: This step defines the procedure to treat the clot along its length according to the path defined in the pre-treatment step.
   1. Run the syringe pump at 0.65 mL/min and wait for meniscus of the plasma to move. This flow rate mimics a near total occlusion of the iliofemoral vasculature^24,34.
   2. Interpolate the path created in step 7.1.3 with intermediate steps between the established waypoints with a fixed step size (e.g., 0.5 mm). The step size is chosen to be smaller than half the width of the focal region as measured along the clot length (elevational direction of the imaging array). Move the histotripsy source using motorized positioners at each path location with insonation parameters defined in step 4.8.
   3. Monitor/image bubble activity during application of the histotripsy pulse at each path location using the imaging window (step 4.7). Center the image on the histotripsy focus with the image dimensions covering 15 mm in the azimuth and range. Prior to the application of histotripsy pulse at each location, acquire a B-mode image to provide visualization of the clot and model vessel, by creating a script in a programming platform. Ensure the script establishes communication with the scanner using the manufacturer's commands.
   4. During the application of the histotripsy pulse, implement the acquisition of acoustic emissions in the script in step 7.2.3 to form passive cavitation images post hoc³⁵. Acquire one passive cavitation image after every 10 treatment pulses. Apply a flat time gain compensation of 50 at 8 incremental depths till the end of the imaging depth. Choose an appropriate acquisition window size such that the entire signal from the clot is captured with minimum loss of energy due to windowing³⁵.
   5. If off-target bubble clouds are present, adjust the transducer position in situ with the motorized positioners.
      ​NOTE: Monitor for missed triggers of the imaging array. Adjust the number of acquired imaging data sets to ensure the storage of data is completed before subsequent triggering.

8. Post experiment procedure

1. Manually raise the model vessel out of the water tank to drain the perfusate via gravity. Be sure to keep the flow channel levelled to prevent the clot from moving downstream and out of the model vessel during draining.
2. Collect the entire perfusate for further analysis in a small beaker (Figure 4A) by drawing plasma solution from the flow channel through the syringe pump at a very low flow rate.
3. Disconnect the model vessel and remove the clot. If necessary, inject a small amount of saline into the model vessel to gently dislodge the clot.
4. Wipe the clot similar to step 1.2.3. Weigh the clot on the weighing scale for assessing the clot mass loss.
5. To analyze the D-dimer content, add 100 mg of aminocaproic acid to a microcentrifuge tube followed by 1 mL of perfusate, and mix well using a pipette. Perform a latex immune-turbidimetric assay to quantify the D-dimer within the sample³⁶.
6. To assess hemolysis, add 1 mL of perfusate to centrifuge tubes and spin at 610 x g (3,500 rpm) for 10 min. Combine 0.5 mL of supernatant (concentrate) with 0.5 mL of Drabkins solution and let the mixture rest at room temperature for 15 min. Transfer 200 µL to well plates, as shown in Figure 4B. Use a plate reader to read absorbance at 540 nm, Figure 4C (Drabkins assay³⁷).
7. Histology assessment
   1. Cut a section from the center of the clot of about 2-3 mm in length with a scalpel.
   2. Add the section to a cassette. Maintain orientation of the section relative to the direction of histotripsy pulse propagation.
   3. Add 2 mL of low gelling agarose solution prepared in step 3.2.5 into the cassette to fix the clot in place.
   4. Fix the sample in 10% formalin for 24 h. Place the sample in 70% alcohol after 24 h and perform standard hemotoxylin-eosin staining³⁸.

9. Passive cavitation image analysis

1. Process the signals acquired from the imaging array during the histotripsy excitation (step 7.2.4) using an algorithm based on the robust Capon beamformer³⁹ to create an image of acoustic emissions generated by the bubble cloud at each treatment location.
   NOTE: To generate quantitative images, follow the steps described in Haworth et al.³⁵. Otherwise, each pixel value in the image should represent the relative bubble cloud acoustic energy (units of V²) at each corresponding location.
2. Segment the B-mode image aquired in steps 7.2.3 to distinguish between the pixels representing clot and the model vessel.
3. Co-register the passive cavitation image with the B-mode image as shown in Figure 5B. Sum up the acoustic energy within the clot over the exposure period³⁵.

Results

The protocol outlined in this study highlights the details of venous clot modeling, lysotripsy for clot disruption, and ultrasound imaging in an in vitro setup of DVT. The adopted procedure demonstrates the steps necessary to assess clot disruption due to the combined effects of rt-PA and histotripsy bubble cloud activity. The benchtop setup was designed to mimic the characteristics of a venous iliofemoral vein. Figure 1A shows a model vessel that has the acoustic, mechanical, and geometrica...

Discussion

The proposed protocol presents a model to quantify treatment efficacy of lysotripsy. While the key details have been discussed, there are certain critical aspects to consider for the success of this protocol. The enzymatic activity of rt-PA has an Arrhenius temperature dependence³⁰. Temperature is also a contributing factor to the speed of sound in water and tissue, and variations in temperature can cause minor alterations of the focal zone geometry. Thus, the water temperature should be carefully...

Materials

Name	Company	Catalog Number	Comments
Absorbing sheets	Precision acoustics	F28-SMALL-M	300mm x 300 mm x 10 mm
Borosilicate Pasteur pippettes	Fisher Scientific	1367820A	14.6 cm length, 2 mL capacity
Centrifuge tubes	Eppendorf	22364111	1.5 mL capacity
Drabkin's assay	Sigma Aldrich	D5941-6VL
Draw syringe	Cole-Parmer	EW-07945-43	60 mL capacity
Filter bags	McMaster-Carr	5162K111	Remove particle size upto 1 microns
Flow channel tubing	McMaster-Carr	5154K25	Polyethylene-lined EVA plastic tubing (Outer diameter: 3/8", Inner diameter: 1/4"
Heating elements	Won Brothers	HT 300 Titanium	Titanium rods placed at the bottom of tank
Imaging array	Verasonics	L11-5v	128 element with sensitivity from -55 to -49 dB
Low gelling agarose	Millipore Sigma	A9414
Model vessel	McMaster-Carr	5234K98	6.6 cm length, 0.6 cm inner diameter, 1 mm thickness
Nanopure water	Barnstead	Nanopure Diamond	ASTM type I, 18 Mohm-cm resistivity
Plasma	Vitalant	4PF000	Plasma frozen within 24 hours
Plate reader	Biotek	Synergy Neo HST Plate Reader	For haemoglobin quantification
Probe cover	Civco	610-362
Programming platform	MATLAB (the Mathworks, Natick, MA, USA)
Recombinant tissue-type plasminogen activator (rt-PA)	Genentech	Activase
Reservoir	Cole-Parmer	EW-07945-43	60 mL capacity
Syringe pump	Cole-Parmer	EW-74900-20	pump attached to the syringe to draw the flow in the flow channel at a pre-determined fized rate
Transducer	In-house customized		Eight-element, elliptically-focused transducer (9 cm major axis, 7 cm minor axis and 6 cm focal length), powered by custom designed and built class D amplifier and matching network
Ultrasound scaning system	Verasonics		Vantage Research Ultrasound System
Water tank	Advanced acrylics	C133	14 x 14 x 12, 1/2"