In Vitro Thrombolytic Efficacy of Single- and Five-Cycle Histotripsy Pulses and rt-PA

Abstract

Although primarily known as an ablative modality, histotripsy can increase the efficacy of lytic therapy in a retracted venous clot model. Bubble cloud oscillations are the primary mechanism of action for histotripsy, and the type of bubble activity is dependent on the pulse duration. A retracted human venous clot model was perfused with and without the thrombolytic recombinant tissue plasminogen activator (rt-PA). The clot was exposed to histotripsy pulses of single- or five-cycle duration and peak negative pressures of 0-30 MPa. Bubble activity within the clot was monitored via passive cavitation imaging. The combination of histotripsy and rt-PA was more efficacious than rt-PA alone for single- and five-cycle pulses with peak negative pressures of 25 and 20 MPa, respectively. For both excitation schemes, the detected acoustic emissions correlated with the degree of thrombolytic efficacy. These results indicate that rt-PA and single- or multicycle histotripsy pulses enhance thrombolytic therapy.

Keywords: Deep vein thrombosis; Histotripsy; Intrinsic threshold; Microtripsy; Shock scattering; Thrombolysis; Thrombotripsy.

Figure 1:

Measured waveforms for (a) nominally single-cycle and (b) five-cycle excitation pulses at the focus of the transducer. The peak negative pressures were approximately 18 and 21 MPa, respectively. The geometric focus of the transducer is at 75 mm, or 50 μs. The associated normalized spectra of the (c) single-cycle and (d) five-cycles excitations. The spectra for both pulse durations were multiplied by a Hamming window to reduce side lobes. The peak positive pressure (PPP) and peak negative pressure (PNP) at a given voltage applied to the class-D amplifier for (e) single cycle, and (f) five cycle excitation pulses, respectively. The legend in (e) is the same for (f). The arrowhead in (f) notes the approximate inflection point of the peak positive pressure curve, indicative of the onset of shock formation.

Figure 2:

Experimental setup. A) shows the flow phantom setup. The flow was from right to left in this diagram. The lateral dimension for the imaging array is into the page. B) is the timing diagram for histotripsy insonation and data acquisition. An a priori scanline B-Mode image was taken at each location. Each location is then treated with 2000 pulses at a 40 Hz pulse repetition frequency. Every tenth pulse passive cavitation imaging (PCI) waveforms are acquired for offline analysis. Due to time of flight propagation of the histotripsy pulse to the focal zone (Histotripsy Focal Insonation in diagram), the acquisition of cavitation emissions was delayed 64 μs after the trigger of the pulse.

Figure 3:

Duplex B-mode/passive cavitation images for (a) single-cycle and (b) five-cycle pulse duration. For both panels, the peak negative pressure was 15 MPa. Note that the color bar has different limits in each subfigure. B-mode images are acquired prior to histotripsy exposure (Figure 2b).

Figure 4:

(a) Mass loss for each experimental arm as a function of the peak negative pressure of the histotripsy pulse. (b) Mass loss grouped by treatment arm. The asterisk (*) denotes a significant difference with respect to rt-PA alone. The triangle (Δ) denotes a significant difference to all other arms at a given peak negative pressure.

Figure 5:

Average emission spectrum of (a) single-cycle pulses of 15 MPa peak negative pressure, (c) five-cycle pulses of 15 MPa peak negative pressure, (b) single-cycle pulses of 25 MPa peak negative pressure, and (d) five-cycle pulses of 25 MPa peak negative pressure. The acoustic emissions were mapped using passive cavitation imaging (PCI). The spectra were taken from the pixel with the maximum amplitude, normalized by the sensitivity of the imaging array and the maximum amplitude.

Figure 6:

(A) Acoustic power, P_clot, from the passive cavitation images for each peak negative pressure and treatment arm. (B) Normalization of P_clot by the excitation pulse duration (1 or 5 μs).

Figure 7:

Scatter plot comparing the clot mass loss and bubble activity-induced acoustic power, P_clot, for clots treated with (A) histotripsy alone, or (B) histotripsy and rt-PA. The Pearson correlation coefficients for each treatment arm indicated a significant correlation between thrombolytic efficacy and the strength of the bubble cloud activity for each arm (p < 0.05). Instances where the clot mass loss and P_clot coincided for the single and five cycle pulse durations were observed (arrows), indicating that the thrombolytic efficacy was independent of pulse duration.

Figure 8:

Flow cytometry particle size measurements. The distribution is split into three size ranges with respect to the diameter of a red blood cell: (a) subcellular (between 4 and 6 μm), (b) cellular (between 6 and 8 μm), and (c) supercellular (between 8 and 38 μm).

Figure 9:

Histological sample of an untreated clot (i.e. no exposure to plasma, rt-PA, or histotripsy). (a) Masson’s Trichrome stain of clot section, (b) magnified Masson’s Trichrome stain of clot section. The fibrin network is faintly visible on the edge of the clot and marked with a black arrowhead. (c) CD61 stain of clot section. Platelets are stained brown. Blue coloring is a counterstain. (d) CD61 stain of a magnified clot section. All slides were scanned at 20× magnification.

Figure 10:

Masson’s trichrome and CD61 stains of clot samples subject to the flow channel. The numerical suffix 1 denotes Masson’s trichrome stains, and 2 denotes CD61 stains. (a) Plasma exposure alone. (b) Plasma and rt-PA exposure. (c) Exposure to histotripsy alone: single-cycle pulses, 25 MPa peak negative pressure. (d) Histotripsy and rt-PA: single-cycle pulses, 25 MPa peak negative pressure. (e) Exposure to histotripsy alone: five-cycle pulses, 25 MPa peak negative pressure. (f) Histotripsy and rt-PA: five-cycle pulses, 25 MPa peak negative pressure. For clots in the second and third rows (c-f), the histotripsy pulse propagated from top to bottom in the image. The orientation in sample d was lost. The mass losses were 5, 24, 21, 55, 29 and 62% for samples a through f, respectively. Black arrowheads denote areas of ablation. The red arrowhead in (f.1) marks deformation of the anterior surface of the clot. The scale bar in panel a.1 applies to all panels. All slides were scanned at 20× magnification. The −6 dB width of the histotripsy focal zone ran 5 mm from top to bottom in the image, and 1 mm along the left to right axis of the image.