Quantification of Hepatocellular Carcinoma Vascular Dynamics With Contrast-Enhanced Ultrasound for LI-RADS Implementation.

OBJECTIVE: The aim of this study is to describe a comprehensive contrast-enhanced ultrasound (CEUS) imaging protocol and analysis method to implement CEUS LI-RADS (Liver Imaging Reporting and Data System) in a quantifiable manner. The methods that are validated with a prospective single-center study aim to simplify CEUS LI-RADS evaluation, remove observer bias, and potentially improve the sensitivity of CEUS LI-RADS. MATERIALS AND METHODS: This prospective single-center study enrolled patients with hepatocellular carcinoma (April 2021-June 2022; N = 31; mean age ± SD, 67 ± 6 years; 24 men/7 women). For each patient, at least 2 CEUS loops spanning over 5 minutes were collected for different lesion scan planes using an articulated arm to hold the transducer. Automatic respiratory gating and motion compensation algorithms removed errors due to breathing motion. The long axis of the lesion was measured in the contrast and fundamental images to capture nodule size. Parametric processing of time-intensity curve analysis on linearized data provided quantifiable information of the wash-in and washout dynamics via rise time ( RT ) and degree of washout ( DW ) parameters extracted from the time-intensity curve, respectively. A Welch t test was performed between lesion and parenchyma RT for each lesion to confirm statistically significant differences. P values for bootstrapped 95% confidence intervals of the relative degree of washout ( rDW ), ratio of DW between the lesion and surrounding parenchyma, were computed to quantify lesion washout. Coefficient of variation (COV) of RT , DW , and rDW was calculated for each patient between injections for both the lesion and surrounding parenchyma to gauge reproducibility of these metrics. Spearman rank correlation tests were performed among size, RT , DW , and rDW values to evaluate statistical dependence between the variables. RESULTS: The mean ± SD lesion diameter was 23 ± 8 mm. The RT for all lesions, capturing arterial phase hyperenhancement, was shorter than that of surrounding liver parenchyma ( P < 0.05). All lesions also demonstrated significant ( P < 0.05) but variable levels of washout at both 2-minute and 5-minute time points, quantified in rDW . The COV of RT for the lesion and surrounding parenchyma were both 11%, and the COV of DW and rDW at 2 and 5 minutes ranged from 22% to 31%. Statistically significant relationships between lesion and parenchyma RT and between lesion RT and lesion DW at the 2- and 5-minute time points were found ( P < 0.05). CONCLUSIONS: The imaging protocol and analysis method presented provide robust, quantitative metrics that describe the dynamic vascular patterns of LI-RADS 5 lesions classified as hepatocellular carcinomas. The RT of the bolus transit quantifies the arterial phase hyperenhancement, and the DW and rDW parameters quantify the washout from linearized CEUS intensity data. This unique methodology is able to implement the CEUS-LIRADS scheme in a quantifiable manner for the first time and remove its existing issues of currently being qualitative and suffering from subjective evaluations.

Ultrasound stiffness and perfusion markers correlate with tumor volume responses to immunotherapy.

Immunotherapy has revolutionized the treatment of dozens of cancers and became a standard of care for some tumor types. However, the majority of patients do not benefit from current immunotherapeutics and many develop severe toxicities. Therefore, the identification of biomarkers to classify patients as likely responders or non-responders to immunotherapy is a timely task. Here, we test ultrasound imaging markers of tumor stiffness and perfusion. Ultrasound imaging is non-invasive and clinically available and can be used both for stiffness and perfusion evaluation. In this study, we employed syngeneic orthotopic models of two breast cancers, a fibrosarcoma and a melanoma, to demonstrate that ultrasound-derived measures of tumor stiffness and perfusion (i.e., blood volume) correlate with the efficacy of immune checkpoint inhibition (ICI) in terms of changes in primary tumor volume. To modulate tumor stiffness and perfusion and thus, get a range of therapeutic outcomes, we employed the mechanotherapeutic tranilast. Mechanotherapeutics combined with ICI are advancing through clinical trials, but biomarkers of response have not been tested until now. We found the existence of linear correlations between tumor stiffness and perfusion imaging biomarkers as well as strong linear correlations between the stiffness and perfusion markers with ICI efficacy on primary tumor growth rates. Our findings set the basis for ultrasound biomarkers predictive of ICI therapy in combination with mechanotherapeutics. STATEMENT OF SIGNIFICANCE: Hypothesis: Monitoring Tumor Microenvironment (TME) mechanical abnormalities can predict the efficacy of immune checkpoint inhibition and provide biomarkers predictive of response. Tumor stiffening and solid stress elevation are hallmarks of tumor patho-physiology in desmoplastic tumors. They induce hypo-perfusion and hypoxia by compressing tumor vessels, posing major barriers to immunotherapy. Mechanotherapeutics is a new class of drugs that target the TME to reduce stiffness and improve perfusion and oxygenation. In this study, we show that measures of stiffness and perfusion derived from ultrasound shear wave elastography and contrast enhanced ultrasound can provide biomarkers of tumor response.

Controlled Hyperthermia With High-Intensity Focused Ultrasound and Ultrasound Contrast Agent Microbubbles in Porcine Liver.

OBJECTIVE: The objective of this work was to study microbubble-enhanced temperature elevation with high-intensity focused ultrasound (HIFU) at different acoustic pressures and under image guidance. The microbubbles were administered with either local or vascular injections (that mimic systemic injections) in perfused and non-perfused ex vivo porcine liver under ultrasound image guidance. METHODS: Porcine liver was insonified for 30 s with a single-element HIFU transducer (0.9 MHz, 0.413 ms, 82% duty cycle, focal pressures of 0.6-3.5 MPa). Contrast microbubbles were injected either locally or through the vasculature. A needle thermocouple at the focus measured temperature elevation. Diagnostic ultrasound (Philips iU22, C5-1 probe) guided placement of the thermocouple and delivery of microbubbles and monitored the procedure in real time. RESULTS: At lower acoustic pressures (0.6 and 1.2 MPa) in non-perfused liver, inertial cavitation of the injected microbubbles led to greater temperatures at the focus compared with HIFU-only treatments. At higher pressures (2.4 and 3.5 MPa) native inertial cavitation in the tissue (without injecting microbubbles) resulted in temperature elevations similar to those after injecting microbubbles. The heated area was larger when using microbubbles at all pressures. In the presence of perfusion, only local injections provided a sufficiently high concentration of microbubbles necessary for significant temperature enhancement. CONCLUSION: Local injections of microbubbles provide a higher concentration of microbubbles in a smaller area, avoiding acoustic shadowing, and can lead to higher temperature elevation at lower pressures and increase the size of the heated area at all pressures.

Contrast-Enhanced Ultrasound with Optimized Aperture Patterns and Bubble Segmentation Based on Echo Phase.

Amplitude modulation (AM) suppresses tissue signals and detects microbubble signals in contrast-enhanced ultrasound (CEUS) and is often implemented with checkerboard apertures. However, possible crosstalk between transmitting and non-transmitting array elements may compromise tissue suppression in AM. Using AM aperture patterns other than the conventional checkerboard approach (one on, one off) may reduce the degree of crosstalk and increase the contrast-to-tissue-ratio (CTR) compared with conventional AM. Furthermore, previous studies have reported that the phase difference between the echoes in AM pulsing sequences may be used to segment tissue and microbubbles and improve tissue signal suppression and the CTR of CEUS images. However, the CTR of the image produced by alternative AM aperture patterns and the effect of segmentation approach on these alternative apertures have not been investigated. We evaluated a number of AM aperture patterns to find an optimal AM aperture pattern that provides the highest CTR. We found that the aperture that uses alternating groups of two elements, AM2, had the highest CTR for the probe evaluated. In addition, a segmentation technique based on echo phase differences (between the full and half-pulses, ΔΦAM, between the complementary half-pulses, ΔΦhalf, and the maximum of the two ΔΦmax) was also considered in the AM aperture optimization process. The segmentation approach increases the CTR by about 25 dB for all apertures. Finally, AM2 segmented with ΔΦmax had a 7-dB higher CTR in a flow phantom and a 6-dB higher contrast in a perfused pig liver than conventional AM segmented with ΔΦAM, and it is the optimal transmit aperture design.

Investigation of the Phase of Nonlinear Echoes From Microbubbles During Amplitude Modulation.

Contrast-enhanced ultrasound (CEUS) imaging relies on distinguishing between microbubble and tissue echoes. Amplitude modulation (AM), a nonlinear pulsing scheme, has been developed to take advantage of the amplitude-dependent nonlinearity of microbubble echoes. However, with AM, tissue nonlinear propagation can also degrade the image contrast. Segmentation of CEUS images based on amplitude-dependent phase difference in the echoes, defined in this article as [Formula: see text], has been proposed as an additional method of enhancing contrast-to-tissue ratio as tissue is not expected to create the same degree of [Formula: see text]; however, this has not been robustly investigated. In this work, we evaluate the source of [Formula: see text] through simulations of unshelled versus shelled microbubble oscillation and simulations of nonlinear propagation in tissue. We then validate the simulated [Formula: see text] results with experimental [Formula: see text] measurements during in vitro scattering and imaging in a flow phantom. We show that shelled and unshelled microbubbles resulted in a [Formula: see text] with similar overall magnitude with some differences in trends, and that tissue echoes have a small yet detectable degree of [Formula: see text] due to nonlinear propagation. The results from this work can help inform optimal parameter selection for phase segmentation and implementation on a clinical scanner.

The Role of Ultrasound in Modulating Interstitial Fluid Pressure in Solid Tumors for Improved Drug Delivery.

The unique microenvironment of solid tumors, including desmoplasia within the extracellular matrix, enhanced vascular permeability, and poor lymphatic drainage, leads to an elevated interstitial fluid pressure which is a major barrier to drug delivery. Reducing tumor interstitial fluid pressure is one proposed method of increasing drug delivery to the tumor. The goal of this topical review is to describe recent work using focused ultrasound with or without microbubbles to modulate tumor interstitial fluid pressure, through either thermal or mechanical effects on the extracellular matrix and the vasculature. Furthermore, we provide a review on techniques in which ultrasound imaging may be used to diagnose elevated interstitial fluid pressure within solid tumors. Ultrasound-based techniques show high promise in diagnosing and treating elevated interstitial pressure to enhance drug delivery.

Safety of Image-Guided Treatment of the Liver with Ultrasound and Microbubbles in an in Vivo Porcine Model.

Ultrasound and microbubbles are useful for both diagnostic imaging and targeted drug delivery, making them ideal conduits for theranostic interventions. Recent reports have indicated the preclinical success of microbubble cavitation for enhancement of chemotherapy in abdominal tumors; however, there have been limited studies and variable efficacy in clinical implementation of this technique. This is likely because in contrast to the high pressures and long cycle lengths seen in successful preclinical work, current clinical implementation of microbubble cavitation for drug delivery generally involves low acoustic pressures and short cycle lengths to fit within clinical guidelines. To translate the preclinical parameter space to clinical adoption, a relevant safety study in a healthy large animal is required. Therefore, the purpose of this work was to evaluate the safety of ultrasound cavitation treatment (USCTx) in a healthy porcine model using a modified Philips EPIQ with S5-1 as the focused source. We performed USCTx on eight healthy pigs and monitored health over the course of 1 wk. We then performed an acute study of USCTx to evaluate immediate tissue damage. Contrast-enhanced ultrasound exams were performed before and after each treatment to investigate perfusion changes within the treated areas, and blood and urine were evaluated for liver damage biomarkers. We illustrate, through quantitative analysis of contrast-enhanced ultrasound data, blood and urine analyses and histology, that this technique and the parameter space considered are safe within the time frame evaluated. With its safety confirmed using a clinical-grade ultrasound scanner and contrast agent, USCTx could be easily translated into clinical trials for improvement of chemotherapy delivery. This represents the first safety study assessing the bio-effects of microbubble cavitation from relevant ultrasound parameters in a large animal model.

Microbubble-Enhanced Heating: Exploring the Effect of Microbubble Concentration and Pressure Amplitude on High-Intensity Focused Ultrasound Treatments.

High-intensity focused ultrasound (HIFU) is a non-invasive tool that can be used for targeted thermal ablation treatments. Currently, HIFU is clinically approved for treatment of uterine fibroids, various cancers, and certain brain applications. However, for brain applications such as essential tremors, HIFU can only be used to treat limited areas confined to the center of the brain because of geometrical limitations (shape of the transducer and skull). A major obstacle to advancing this technology is the inability to treat non-central brain locations without causing damage to the skin and/or skull. Previous research has indicated that cavitation-induced bubbles or microbubble contrast agents can be used to enhance HIFU treatments by increasing ablation regions and shortening acoustic exposures at lower acoustic pressures. However, little research has been done to explore the interplay between microbubble concentration and pressure amplitude on HIFU treatments. We developed an in vitro experimental setup to study lesion formation at three different acoustic pressures and three microbubble concentrations. Real-time ultrasound imaging was integrated to monitor initial microbubble concentration and subsequent behavior during the HIFU treatments. Depending on the pressure used for the HIFU treatment, there was an optimal concentration of microbubbles that led to enhanced heating in the focal area. If the concentration of microbubbles was too high, the treatment was detrimentally affected because of non-linear attenuation by the pre-focal microbubbles. Additionally, the real-time ultrasound imaging provided a reliable method to monitor microbubble activity during the HIFU treatments, which is important for translation to in vivo HIFU applications with microbubbles.

Linear Signal Cancellation of Nonlinear Pulsing Schemes in a Verasonics Research Scanner.

Contrast-enhanced ultrasound (CEUS) is a real-time imaging technique that allows the visualization of organ and tumor microcirculation by utilizing the nonlinear response of microbubbles. Nonlinear pulsing schemes are used exclusively in CEUS imaging modes in modern scanners. One important aspect of nonlinear pulsing schemes is the near-complete elimination of the linear signals that originate from tissue. Up until now, no study has investigated the performance of Verasonics scanners in eliminating the linear signals during CEUS and, by extension, the optimal pulsing sequences for performing CEUS. The aim of this article was to investigate linear signal cancellation of the Verasonics scanner performing nonlinear pulsing schemes with two different probes (L7-4 linear array and C5-2 convex array). We have considered two pulsing schemes: pulse inversion (PI) and amplitude modulation (AM). We have also compared our results from the Verasonics scanner with a clinical scanner (Philips iU22). We found that the linear signal cancellation of the transmitted pulse by Verasonics scanner was ~40 dB in AM mode and ~30 dB in PI mode when operated at 0.06 MI. The linear signal cancellation performance of Verasonics scanner was comparable with Philips iU22 scanner in focused AM mode and on average 3 dB better than Philips iU22 scanner in focused PI mode.

Cavitation Therapy Monitoring of Commercial Microbubbles With a Clinical Scanner.

The ability to monitor cavitation activity during ultrasound and microbubble-mediated procedures is of high clinical value. However, there has been little reported literature comparing the cavitation characteristics of different clinical microbubbles, nor have current clinical scanners been used to perform passive cavitation detection in real time. The goal of this work was to investigate and characterize standard microbubble formulations (Optison, Sonovue, Sonazoid, and a custom microbubble made with similar components as Definity) with a custom passive cavitation detector (two confocal single-element focused transducers) and with a Philips EPIQ scanner with a C5-1 curvilinear probe passively listening. We evaluated three different methods for investigating cavitation thresholds, two from previously reported work and one developed in this work. For all three techniques, it was observed that the inertial cavitation thresholds were between 0.1 and 0.3 MPa for all agents when detected with both systems. Notably, we found that most microbubble formulations in bulk solution behaved generally similarly, with some differences. We show that these characteristics and thresholds are maintained when using a diagnostic ultrasound system for detecting cavitation activity. We believe that a systematic evaluation of the frequency response of the cavitation activity of different microbubbles in order to inform real-time therapy monitoring using a clinical ultrasound device could make an immediate clinical impact.

Image-Guided Treatment of Primary Liver Cancer in Mice Leads to Vascular Disruption and Increased Drug Penetration.

Despite advances in interventional procedures and chemotherapeutic drug development, hepatocellular carcinoma (HCC) is still the fourth leading cause of cancer-related deaths worldwide with a <30% 5-year survival rate. This poor prognosis can be attributed to the fact that HCC most commonly occurs in patients with pre-existing liver conditions, rendering many treatment options too aggressive. Patient survival rates could be improved by a more targeted approach. Ultrasound-induced cavitation can provide a means for overcoming traditional barriers defining drug uptake. The goal of this work was to evaluate preclinical efficacy of image-guided, cavitation-enabled drug delivery with a clinical ultrasound scanner. To this end, ultrasound conditions (unique from those used in imaging) were designed and implemented on a Philips EPIQ and S5-1 phased array probe to produced focused ultrasound for cavitation treatment. Sonovue® microbubbles which are clinically approved as an ultrasound contrast agent were used for both imaging and cavitation treatment. A genetically engineered mouse model was bred and used as a physiologically relevant preclinical analog to human HCC. It was observed that image-guided and targeted microbubble cavitation resulted in selective disruption of the tumor blood flow and enhanced doxorubicin uptake and penetration. Histology results indicate that no gross morphological damage occurred as a result of this process. The combination of these effects may be exploited to treat HCC and other challenging malignancies and could be implemented with currently available ultrasound scanners and reagents.

Evaluation of the Reproducibility of Bolus Transit Quantification With Contrast-Enhanced Ultrasound Across Multiple Scanners and Analysis Software Packages-A Quantitative Imaging Biomarker Alliance Study.

OBJECTIVES: Contrast enhanced ultrasound (CEUS) is now broadly used clinically for liver lesion detection and characterization. Obstacles to the efforts to quantify perfusion with CEUS have been the lack of a standardized approach and undocumented reproducibility. The use of multiple scanners and different analysis software packages compounds the degree of variability. Our objectives were to standardize a CEUS-based approach for quantification of perfusion-related parameters of liver lesions and to evaluate the variability of bolus transit parameters (rise time [RT], mean transit time [MTT], peak intensity, and area under the curve) obtained from various clinical ultrasound scanners and analysis software. MATERIALS AND METHODS: Bolus transit as a way of evaluating perfusion has been investigated both in vivo and in vitro in the past but without establishing its reproducibility. We developed a tissue flow phantom that produces time-intensity curves very similar to those extracted from clinical cine loops of liver lesions. We evaluated the variability of the bolus transit parameters with 4 commercial scanners (Philips iU22, Philips EPIQ, GE LOGIQ E9, and Siemens Acuson Sequoia) and 3 different analysis software packages in multiple trials (15 per scanner). RESULTS: The variability (coefficient of variation) from repeated trials and while using a single scanner and software was less than 8% for RT, less than 12% for MTT, less than 49% for peak intensity, and less than 50% for area under the curve. Currently, it is not possible to directly compare amplitude values from different scanners and analysis software packages owing to the arbitrary linearization algorithm used among manufacturers; however, it is possible for time parameters (RT and MTT). The variability when using a different scanner with the same analysis software package was less than 9% for RT and less than 21% for MTT. The variability when using a different analysis software with the same scanner was less than 9% for RT and less than 15% for MTT. In all the evaluations we have performed, RT is the least variable parameter, while MTT is only slightly more variable. CONCLUSIONS: The present study will lay the groundwork for multicenter patient evaluations with CEUS quantification of perfusion-related parameters with the bolus transit technique. When using the protocol and method developed here, it is possible to perform perfusion quantification on different scanners and analysis software and be able to compare the results. The current work is the first study that presents a comparison of bolus transit parameters derived from multiple systems and software packages.

Contrast-enhanced US for the Interventional Radiologist: Current and Emerging Applications.

US is a powerful and nearly ubiquitous tool in the practice of interventional radiology. Use of contrast-enhanced US (CEUS) has gained traction in diagnostic imaging given the recent approval by the U.S. Food and Drug Administration (FDA) of microbubble contrast agents for use in the liver, such as sulfur hexafluoride lipid-type A microspheres. Adoption of CEUS by interventional radiologists can enhance not only procedure guidance but also preprocedure patient evaluation and assessment of treatment response across a wide spectrum of oncologic, vascular, and nonvascular procedures. In addition, the unique physical properties of microbubble contrast agents make them amenable as therapeutic vehicles in themselves, which can lay a foundation for future therapeutic innovations in the field in drug delivery, thrombolysis, and vascular flow augmentation. The purpose of this article is to provide an introduction to and overview of CEUS aimed at the interventional radiologist, highlighting its role before, during, and after frequently practiced oncologic and vascular interventions such as biopsy, ablation, transarterial chemoembolization, detection and control of hemorrhage, evaluation of transjugular intrahepatic portosystemic shunts (TIPS), detection of aortic endograft endoleak, thrombus detection and evaluation, evaluation of vascular malformations, lymphangiography, and percutaneous drain placement. Basic physical principles of CEUS, injection and scanning protocols, and logistics for practice implementation are also discussed. Early adoption of CEUS by the interventional radiology community will ensure rapid innovation of the field and development of future novel procedures. Online supplemental material is available for this article. ©RSNA, 2020.

Imaging Methods for Ultrasound Contrast Agents.

Microbubble contrast agents were introduced more than 25 years ago with the objective of enhancing blood echoes and enabling diagnostic ultrasound to image the microcirculation. Cardiology and oncology waited anxiously for the fulfillment of that objective with one clinical application each: myocardial perfusion, tumor perfusion and angiogenesis imaging. What was necessary though at first was the scientific understanding of microbubble behavior in vivo and the development of imaging technology to deliver the original objective. And indeed, for more than 25 years bubble science and imaging technology have evolved methodically to deliver contrast-enhanced ultrasound. Realization of the basic bubbles properties, non-linear response and ultrasound-induced destruction, has led to a plethora of methods; algorithms and techniques for contrast-enhanced ultrasound (CEUS) and imaging modes such as harmonic imaging, harmonic power Doppler, pulse inversion, amplitude modulation, maximum intensity projection and many others were invented, developed and validated. Today, CEUS is used everywhere in the world with clinical indications both in cardiology and in radiology, and it continues to mature and evolve and has become a basic clinical tool that transforms diagnostic ultrasound into a functional imaging modality. In this review article, we present and explain in detail bubble imaging methods and associated artifacts, perfusion quantification approaches, and implementation considerations and regulatory aspects.

Differentiation of Vascular Characteristics Using Contrast-Enhanced Ultrasound Imaging.

Ultrasound contrast imaging has been used to assess tumour growth and regression by assessing the flow through the macro- and micro-vasculature. Our aim was to differentiate the blood kinetics of vessels such as veins, arteries and microvasculature within the limits of the spatial resolution of contrast-enhanced ultrasound imaging. The highly vascularised ovine ovary was used as a biological model. Perfusion of the ovary with SonoVue was recorded with a Philips iU22 scanner in contrast imaging mode. One ewe was treated with prostaglandin to induce vascular regression. Time-intensity curves (TIC) for different regions of interest were obtained, a lognormal model was fitted and flow parameters calculated. Parametric maps of the whole imaging plane were generated for 2 × 2 pixel regions of interest. Further analysis of TICs from selected locations helped specify parameters associated with differentiation into four categories of vessels (arteries, veins, medium-sized vessels and micro-vessels). Time-dependent parameters were associated with large veins, whereas intensity-dependent parameters were associated with large arteries. Further development may enable automation of the technique as an efficient way of monitoring vessel distributions in a clinical setting using currently available scanners.

Super-Resolution Contrast-Enhanced Ultrasound Methodology for the Identification of In Vivo Vascular Dynamics in 2D.

OBJECTIVES: The aim of this study was to provide an ultrasound-based super-resolution methodology that can be implemented using clinical 2-dimensional ultrasound equipment and standard contrast-enhanced ultrasound modes. In addition, the aim is to achieve this for true-to-life patient imaging conditions, including realistic examination times of a few minutes and adequate image penetration depths that can be used to scan entire organs without sacrificing current super-resolution ultrasound imaging performance. METHODS: Standard contrast-enhanced ultrasound was used along with bolus or infusion injections of SonoVue (Bracco, Geneva, Switzerland) microbubble (MB) suspensions. An image analysis methodology, translated from light microscopy algorithms, was developed for use with ultrasound contrast imaging video data. New features that are tailored for ultrasound contrast image data were developed for MB detection and segmentation, so that the algorithm can deal with single and overlapping MBs. The method was tested initially on synthetic data, then with a simple microvessel phantom, and then with in vivo ultrasound contrast video loops from sheep ovaries. Tracks detailing the vascular structure and corresponding velocity map of the sheep ovary were reconstructed. Images acquired from light microscopy, optical projection tomography, and optical coherence tomography were compared with the vasculature network that was revealed in the ultrasound contrast data. The final method was applied to clinical prostate data as a proof of principle. RESULTS: Features of the ovary identified in optical modalities mentioned previously were also identified in the ultrasound super-resolution density maps. Follicular areas, follicle wall, vessel diameter, and tissue dimensions were very similar. An approximately 8.5-fold resolution gain was demonstrated in vessel width, as vessels of width down to 60 µm were detected and verified (λ = 514 µm). Best agreement was found between ultrasound measurements and optical coherence tomography with 10% difference in the measured vessel widths, whereas ex vivo microscopy measurements were significantly lower by 43% on average. The results were mostly achieved using video loops of under 2-minute duration that included respiratory motion. A feasibility study on a human prostate showed good agreement between density and velocity ultrasound maps with the histological evaluation of the location of a tumor. CONCLUSIONS: The feasibility of a 2-dimensional contrast-enhanced ultrasound-based super-resolution method was demonstrated using in vitro, synthetic and in vivo animal data. The method reduces the examination times to a few minutes using state-of-the-art ultrasound equipment and can provide super-resolution maps for an entire prostate with similar resolution to that achieved in other studies.

Modeling of the Acoustic Field Produced by Diagnostic Ultrasound Arrays in Plane and Diverging Wave Modes.

Recent advances in ultrafast contrast imaging have facilitated innovations, such as superresolution imaging and ultrafast contrast-enhanced Doppler imaging. Combining plane and diverging wave imaging (PWI/DWI) with tissue harmonic imaging (THI) may offer improvements in image quality in applications such as 3-D THI and harmonic color flow. However, no studies have reported simulations of the nonlinear acoustic fields produced by diagnostic arrays in either plane or diverging wave mode. The aim of this study is to model three typical diagnostic arrays that are used in clinical practice and research, Verasonics L11-4v linear array, C5-2v convex array, and P4-2v phased array with the Khokhlov-Zabolotskaya-Kuznetsov (KZK) equation. We have two specific objectives: first, to investigate whether there is increased bubble destruction due to the nature of the plane and diverging fields in contrast imaging; and second, to investigate the feasibility of combining PWI/DWI and THI by quantifying the second harmonic generated by these fields. We showed in linear simulations that using such arrays for ultrafast contrast imaging produced pressures that are greater in the near field and lower in the far field than those of focused beams and thus may induce more near-field bubble destruction. In nonlinear simulations, the second harmonic produced by ultrafast THI was found to be 2-16 dB lower than that of focused beams for all arrays considered when operated at the same MI. This moderate difference of the second harmonic between PWI/DWI and focused ultrasound suggests that it is feasible to combine PWI/DWI and THI.

Contrast-enhanced ultrasound to ultrasound fusion during microwave ablation: feasibility study in a perfused porcine liver model.

PURPOSE: To obtain a complete response with thermal ablation, the margin and entire tumor volume must be treated. Real-time ultrasound visualization is limited during ablation due to gas production. This study assesses the feasibility of fusing volumetric contrast-enhanced ultrasound (CEUS), obtained immediately prior to microwave ablation, with real-time CEUS during and following ablation in a machine-perfused porcine liver. METHODS: Ten, 3-4 cm microwave ablations were performed in five explanted perfused livers. Prior to ablation, microbubbles were injected into the vasculature while an ultrasound sweep across the liver captured a volumetric image during maximum enhancement. This volumetric image was then fused to overlay the real-time ultrasound imaging. Since the perfused livers did not have tumors, a spherical marker circumscribing a target volume was placed on the images. Approximatively, 75% of the total intended circumscribed spherical volume was ablated. Following ablation, a second bolus injection of ultrasound contrast was administered demonstrating continued enhancement of the intentionally non-ablated 25%. A second volumetric image of the post-ablation CEUS was then fused to overlay the real-time ultrasound images for guidance during ablation of the remaining enhancing volume. RESULTS: Technical success was achieved in 100% of the cases. The pre- and then the post-ablation CEUS volume was fused with real-time imaging during antenna placement for initial and subsequent ablation. CONCLUSION: CEUS-CEUS fusion during thermal ablation is feasible and greatly improves the workflow. The approach may augment the use of dynamic CEUS for guidance, improving antenna placement, and aiding in the identification and ablation of initial and residual enhancing tissue.

Ultrasound Imaging of Microbubble Activity during Sonoporation Pulse Sequences.

Ultrasound-mediated drug delivery using the mechanical action of oscillating and/or collapsing microbubbles has been studied on many different experimental platforms, both in vitro and in vivo; however, the mechanisms remain to be elucidated. Many groups use sterile, enclosed chambers, such as Opticells and Clinicells, to optimize acoustic parameters in vitro needed for effective drug delivery in vivo, as well as for mechanistic investigation of sonoporation or the use of sound to permeate cell membranes. In these containers, cell monolayers are seeded on one side, and the remainder of the volume is filled with a solution containing microbubbles and a model drug. Ultrasound is then applied to study the effect of different parameters on model drug uptake in cell monolayers. Despite the simplicity of this system, the field has been unable to appropriately address what parameters and microbubble concentrations are most effective at enhancing drug uptake and minimizing cellular toxicity. In this work, a common in vitro sonoporation experimental setup was characterized through quantitative analysis of microbubble-dependent acoustic attenuation in combination with high-frame-rate and high-resolution imaging of bubble activity during sonoporation pulse sequences. The goal was to visualize the effect that ultrasound parameters have on microbubble activity. It was observed that under literature-derived sonoporation conditions (0.1-1 MPa, 20-1000 cycles and 10,000 to 10,000,000 microbubbles/mL), there is strong and non-linear acoustic attenuation, as well as bubble destruction, gas diffusion and bubble motion resulting in spatiotemporal pressure and concentration gradients. Ultimately, it was found that the acoustic conditions in common in vitro sonoporation setups are much more complex and confounding than often assumed.

Engineered 3D Microvascular Networks for the Study of Ultrasound-Microbubble-Mediated Drug Delivery.

Localized and targeted drug delivery can be achieved by the combined action of ultrasound and microbubbles on the tumor microenvironment, likely through sonoporation and other therapeutic mechanisms that are not well understood. Here, we present a perfusable in vitro model with a realistic 3D geometry to study the interactions between microbubbles and the vascular endothelium in the presence of ultrasound. Specifically, a three-dimensional, endothelial-cell-seeded in vitro microvascular model was perfused with cell culture medium and microbubbles while being sonicated by a single-element 1 MHz focused transducer. This setup mimics the in vivo scenario in which ultrasound induces a therapeutic effect in the tumor vasculature in the presence of flow. Fluorescence and bright-field microscopy were employed to assess the microbubble-vessel interactions and the extent of drug delivery and cell death both in real time during treatment as well as after treatment. Propidium iodide was used as the model drug while calcein AM was used to evaluate cell viability. There were two acoustic parameter sets chosen for this work: (1) acoustic pressure: 1.4 MPa, pulse length: 500 cycles, duty cycle: 5% and (2) acoustic pressure: 0.4 MPa, pulse length: 1000 cycles, duty cycle: 20%. Enhanced drug delivery and cell death were observed in both cases while the higher pressure setting had a more pronounced effect. By introducing physiological flow to the in vitro microvascular model and examining the PECAM-1 expression of the endothelial cells within it, we demonstrated that our model is a good mimic of the in vivo vasculature and is therefore a viable platform to provide mechanistic insights into ultrasound-mediated drug delivery.