This work was funded by the National Institutes of Health, Grant R01HL13334. The authors would like to thank Dr. Kevin Haworth for assisting with Drabkin&#39;s assay and Dr. Viktor Bollen for his support in designing the protocol. The authors are also thankful to Dr. Adam Maxwell for his guidance on designing the histotripsy source.

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&#160;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 t...

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...

Thrombosis is the condition of clot formation in an otherwise healthy blood vessel that obstructs circulation1,2. 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 segments4,5,6. Deep vein thrombosis (DVT) has inherent sequela besides pulmonary obstructions, with long term complications that result in pain, swelling, leg ulcerations, and limb amputations7,8,9. 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 time12,13,14 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 clot17,18. 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 vitro20,21,22. Prior studies have23,24 demonstrated that the combination of histotripsy and the lytic&#160;recombinant tissue-type plasminogen activator (rt-PA)&#160;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 outlined23,24,25,26,27. Overall, the study provides an effective means for executing and assessing the efficacy of lysotripsy to treat DVT.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absorbing sheets</td>
			<td>Precision acoustics</td>
			<td>F28-SMALL-M</td>
			<td>300mm x 300 mm x 10 mm</td>
		</tr>
		<tr>
			<td>Borosilicate Pasteur pippettes</td>
			<td>Fisher Scientific</td>
			<td>1367820A</td>
			<td>14.6 cm length, 2 mL capacity</td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Eppendorf</td>
			<td>22364111</td>
			<td>1.5 mL capacity</td>
		</tr>
		<tr>
			<td>Drabkin&#39;s assay</td>
			<td>Sigma Aldrich</td>
			<td>D5941-6VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Draw syringe</td>
			<td>Cole-Parmer</td>
			<td>EW-07945-43</td>
			<td>60 mL capacity</td>
		</tr>
		<tr>
			<td>Filter bags</td>
			<td>McMaster-Carr</td>
			<td>5162K111</td>
			<td>Remove particle size upto 1 microns</td>
		</tr>
		<tr>
			<td>Flow channel tubing</td>
			<td>McMaster-Carr</td>
			<td>5154K25</td>
			<td>Polyethylene-lined EVA plastic tubing (Outer diameter: 3/8&#34;, Inner diameter: 1/4&#34;</td>
		</tr>
		<tr>
			<td>Heating elements</td>
			<td>Won Brothers</td>
			<td>HT 300 Titanium</td>
			<td>Titanium rods placed at the bottom of tank</td>
		</tr>
		<tr>
			<td>Imaging array</td>
			<td>Verasonics</td>
			<td>L11-5v</td>
			<td>128 element with sensitivity from -55 to -49 dB</td>
		</tr>
		<tr>
			<td>Low gelling agarose</td>
			<td>Millipore Sigma</td>
			<td>A9414</td>
			<td></td>
		</tr>
		<tr>
			<td>Model vessel</td>
			<td>McMaster-Carr</td>
			<td>5234K98</td>
			<td>6.6 cm length, 0.6 cm inner diameter, 1 mm thickness</td>
		</tr>
		<tr>
			<td>Nanopure water</td>
			<td>Barnstead</td>
			<td>Nanopure Diamond</td>
			<td>ASTM type I, 18 Mohm-cm resistivity</td>
		</tr>
		<tr>
			<td>Plasma</td>
			<td>Vitalant</td>
			<td>4PF000</td>
			<td>Plasma frozen within 24 hours</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Biotek</td>
			<td>Synergy Neo HST Plate Reader</td>
			<td>For haemoglobin quantification</td>
		</tr>
		<tr>
			<td>Probe cover</td>
			<td>Civco</td>
			<td>610-362</td>
			<td></td>
		</tr>
		<tr>
			<td>Programming platform</td>
			<td>MATLAB (the Mathworks, Natick, MA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant tissue-type plasminogen activator (rt-PA)</td>
			<td>Genentech</td>
			<td>Activase</td>
			<td></td>
		</tr>
		<tr>
			<td>Reservoir</td>
			<td>Cole-Parmer</td>
			<td>EW-07945-43</td>
			<td>60 mL capacity</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Cole-Parmer</td>
			<td>EW-74900-20</td>
			<td>pump attached to the syringe to draw the flow in the flow channel at a pre-determined fized rate</td>
		</tr>
		<tr>
			<td>Transducer</td>
			<td>In-house customized</td>
			<td></td>
			<td>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</td>
		</tr>
		<tr>
			<td>Ultrasound scaning system</td>
			<td>Verasonics</td>
			<td></td>
			<td>Vantage Research Ultrasound System</td>
		</tr>
		<tr>
			<td>Water tank</td>
			<td>Advanced acrylics</td>
			<td>C133</td>
			<td>14 x 14 x 12, 1/2&#34;</td>
		</tr>
	</tbody>
</table>

an in vitro system to gauge the thrombolytic efficacy of histotripsy and a lytic drug

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&#160;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.

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...

Watch this Scientific Journal Video about An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug at JoVE.com

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

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.