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 (DV....

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 l....

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"