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

Published: June 4th, 2021



1Department of Radiology, University of Chicago, 2Graduate Program in Medical Physics, University of Chicago

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.

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.

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 (DV....

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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 t.......

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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.......

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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.......

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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'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.


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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"

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