Assessment of histotripsy-induced liquefaction with diagnostic ultrasound and magnetic resonance imaging in vitro and ex vivo

Abstract

Histotripsy is a therapeutic ultrasound modality under development to liquefy tissue mechanically via bubble clouds. Image guidance of histotripsy requires both quantification of the bubble cloud activity and accurate delineation of the treatment zone. In this study, magnetic resonance (MR) and diagnostic ultrasound imaging were combined to assess histotripsy treatment in vitro and ex vivo. Mechanically ablative histotripsy pulses were applied to agarose phantoms or porcine livers. Bubble cloud emissions were monitored with passive cavitation imaging (PCI), and hyperechogenicity via plane wave imaging. Changes in the medium structure due to bubble activity were assessed with diagnostic ultrasound using conventional B-mode imaging and T ₁-, T ₂-, and diffusion-weighted MR images acquired at 3 Tesla. Liquefaction zones were correlated with diagnostic ultrasound and MR imaging via receiver operating characteristic (ROC) analysis and Dice similarity coefficient (DSC) analysis. Diagnostic ultrasound indicated strong bubble activity for all samples. Histotripsy-induced changes in sample structure were evident on conventional B-mode and T ₂-weighted images for all samples, and were dependent on the sample type for T ₁- and diffusion-weighted imaging. The greatest changes observed on conventional B-mode or MR imaging relative to baseline in the samples did not necessarily indicate the regions of strongest bubble activity. Areas under the ROC curve for predicting phantom or liver liquefaction were significantly greater than 0.5 for PCI power, plane wave and conventional B-mode grayscale, T ₁, T ₂, and ADC. The acoustic power mapped via PCI provided a better prediction of liquefaction than assessment of the liquefaction zone via conventional B-mode or MR imaging for all samples. The DSC values for T ₂-weighted images were greater than those derived from conventional B-mode images. These results indicate diagnostic ultrasound and MR imaging provide complimentary sets of information, demonstrating that multimodal imaging is useful for assessment of histotripsy liquefaction.

Figure 1.

Top-down view of experimental setup for phantoms containing red blood cell layers (left) and liver samples (right). Three locations were targeted with the therapy source (shown in gray on the left) in each phantom, separated by 1.5 cm. In red blood cell phantoms, agarose was poured in layers, with thin blood cell layers (red lines) applied via pipette on top of each solidified agarose layer. Additional fiducial markers (indicated by blue dotted lines) were used to orient the diagnostic ultrasound images, magnetic resonance (MR) images, and collected histologic samples parallel to the direction of ultrasound propagation.

Figure 2.

(a) Side view of experimental setup for red blood cell (RBC) phantom insonation with a therapeutic ultrasound transducer. RBC layers of the phantoms were oriented parallel to the direction of ultrasound propagation from the histotripsy source. Fiducial markers embedded within the agarose were used to register diagnostic ultrasound and magnetic resonance (MR) images to digital photographs of phantoms post-insonation. The imaging plane of the L11–4v imaging array was registered to the RBC layers and visualized bubble activity along the acoustic axis of the histotripsy source. (b) Timeline of all image data acquisition. Passive and plane wave acquisitions were acquired every tenth histotripsy pulse due to data transfer rate limitations. Post hoc conventional B-mode images were acquired within 1 min of the histotripsy insonation. Samples were transferred to the MR scanner within 10 min, and MR images were acquired over the course of 1 h.

Figure 3.

Demonstration of Otsu thresholding to generate binary mask of liquefaction zone. Red-green-blue images (a) are converted to grayscale and a global threshold is determined based on the gray level histogram to generate a binary image (b).

Figure 4.

Gross observations (top row) and post hoc visualizations of liquefaction zones with conventional B-mode images (middle row) and T₂-weighted images (bottom row). Insonations consisted of 13 (left) 17 (middle-left), 21 (middle-right), or 25 (right) MPa peak negative pressure pulses of 5 μs pulse duration and 1 MHz fundamental frequency in red blood cell phantoms. Therapeutic ultrasound pulses propagated from left to right in all images. In post-insonation conventional B-mode images, fiducial markers have been removed to allow better grayscale windowing for visualization of hypoechoic liquefaction zones.

Figure 5.

Plane wave ultrasound images (top row) and passive cavitation images (bottom row) acquired during histotripsy insonation with 13 (left) 17 (middle-left), 21 (middle-right), and 25 (right) MPa peak negative pressure pulses of 5 μs pulse duration and 1 MHz fundamental frequency in red blood cell phantoms. Therapeutic ultrasound pulses propagated from left to right in all images.

Figure 6.

Registration of imaging with gross observation of red blood cell phantom liquefaction. (a) Gross observation of liquefaction generated by histotripsy in a red blood cell phantom with the liquefaction zone outlined in green, (b) coregistration of passive cavitation imaging (PCI) acoustic power and red blood cell liquefaction, (c) coregistration of plane wave grayscale and red blood cell liquefaction, (d) parametric T₂ map of red blood cell liquefaction, (e) coregistration of PCI acoustic power and T₂ map, and (f) coregistration of plane wave grayscale and T₂ map. The liquefaction zone outline is shown in panels (a)-(c). The histotripsy pulse (1 MHz fundamental frequency, 5 μs pulse duration, 17 MPa peak negative pressure) propagated from left to right in the image. The azimuth/range dimensions of the diagnostic ultrasound imaging plane are indicated in the panel (a).

Figure 7.

Registration of imaging with histologic observation of liver sample liquefaction. (a) Hematoxylin and eosin (H&E) stain of liver sample exposed to histotripsy with the ablation zone indicated by the dotted black outline, (b) coregistration of passive cavitation imaging (PCI) acoustic power and H&E-stained liver sample, (c) coregistration of plane wave grayscale and H&E-stained liver sample, (d) parametric T₂ map of treated liver sample, (e) coregistration of PCI acoustic power and T₂ map, (f) coregistration of plane wave grayscale and T₂ map, (g) parametric apparent diffusion coefficient (ADC) map of treated liver sample, (h) coregistration of PCI acoustic power and ADC map, and (i) coregistration of plane wave grayscale and ADC map. The histotripsy pulse (1 MHz fundamental frequency, 5 μs pulse duration, 25 MPa peak negative pressure) propagated from left to right in the image. The azimuth/range dimensions of the diagnostic ultrasound imaging plane are indicated in the panel (a). Reflections from connective tissue in (c), (f), and (i) have been removed for better windowing and visualization of the histotripsy bubble cloud.

Figure 8.

Hematoxylin and eosin (H&E) stain, (b) post hoc conventional B-mode image, and (c) T₂-weighted image of liquefaction in the same liver sample. The liquefaction zone (dotted black dotted outline in (a), red arrows) can be distinguished as hypoechoic in (b) and hyperintense in (c). Regions of more thorough liquefaction exhibit minimal H&E staining, (yellow arrow in (a)). Small hyperechoic structures can be seen in the post hoc conventional B-mode image (yellow arrows in (b)), possibly indicating residual bubbles or accumulations of cellular debris. The histotripsy pulse (1-MHz fundamental frequency, 5-μs pulse duration, 25-MPa peak negative pressure) propagated from left to right in each image.

Figure 9.

Examples of parametric (a) T₁, (b) T₂, and (c) apparent diffusion coefficient (ADC) maps for red blood cell phantoms. The liquefaction zone is indicated by a red arrow in (a) and (b). The red blood cell layer (dark area) is indicated by yellow arrows in (a) and (b). The histotripsy pulse (1 MHz fundamental frequency, 5 μs pulse duration, 17 MPa peak negative pressure) propagated from left to right in each image.

Figure 10.

Normalized amplitudes of passive cavitation imaging (PCI) acoustic power, plane wave grayscale, and change in T₂ from background along the central axis of the liquefaction zone (dashed blue line in top images) for (a) a red blood cell (RBC) phantom, and (b) a liver sample. The locations of liquefaction are binarized for the bottom plots, with values of 1 indicating liquefaction and 0 indicating intact media. The areas of greatest change in T₂ in the RBC phantom (black arrows) correspond to areas of relatively low PCI power and plane wave grayscale intensity (red arrow) within the liquefaction zone. In the liver sample, locations of T₂ and apparent diffusion coefficient maxima (black dashed arrows) correspond with more thorough liquefaction as indicated by Hematoxylin and eosin staining (yellow dashed arrow). Two maxima are present for PCI power (red dashed arrows) and plane wave grayscale (gray dashed arrows). The proximal (leftmost) plane wave grayscale peak is small compared with the distal (rightmost) peak, while the larger proximal PCI peak more accurately reflects the increased liquefaction and magnetic resonance parameter changes in this location. The histotripsy pulse (1 MHz fundamental frequency, 5 μs pulse duration, 17 MPa peak negative pressure for RBC phantom, 25 MPa peak negative pressure for liver sample) propagated from left to right in each image.

Figure 11.

Azimuthal position of proximal edge of liquefaction zone (solid black bars), maximum passive cavitation imaging (PCI) acoustic power (solid red bars), plane wave grayscale (checkered blue bars), and maximum (striped orange bars) and minimum (cross-hatched green bars) T₂ for all pressures applied to red blood cell phantoms. Colored bars cover 25% to 75% quantiles, and error bars extend to maximum and minimum azimuthal positions recorded for each parameter. 90 mm corresponds to the geometric focus of the histotripsy source. Locations of maximum PCI acoustic power and T₂ shift proximally with increasing pressure, while plane wave maxima and T₂ minima shift distally.

Figure 12.

Receiver Operating Characteristic (ROC) curves obtained from (a) red blood cell phantoms and (b) liver samples. Quantitative plane wave grayscale (gray dotted line), passive cavitation imaging acoustic power (red solid line), apparent diffusion coefficient (black dashed line), T₁, (gray solid line), T₂, (brown dash-dot line), and post hoc conventional B-mode grayscale (blue dotted line) were used to predict presence of liquefaction/necrosis along azimuth dimension of histotripsy lesions across all peak negative pressures of the histotripsy pulse (only 1 pressure level was employed in the liver studies). The black dotted line indicates the resulting ROC curve from random guessing (area under the curve = 0.5).