PONE-D-21-29418(More Than) Doubling Down: Effective fibrinolysis at a reduced rt-PA dose for catheter-directed thrombolysis combined with histotripsyPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This paper reports an interesting sonothrombolysis by using catheter infusion and histotripsy. The findings on reduced tPA is promising. The following comments may be addressed to strengthen the manuscript:

1. Abstract

Page 2. Line 29. “… rt-PA at a one-twentieth dose reduction (0.23 μg/mL) when combined with histotripsy.”. This should be changed to “… rt-PA at a reduction to one-twentieth of the common clinic dose (0.23 μg/mL) when combined with histotripsy.”.

Page 2. Line 36. “Overall, the data collected in this study indicate the rt-PA dose can be reduce by…”. This should be changed to “Overall, the data collected in this study indicate the rt-PA dose can be reduced by…”. (use past tense for “reduce”)

2. Introduction

Page 3. Line 49. ” This approach is not without…”. This could be changed to “This approach has…” to be more concise.

Page 3 Line 55. Authors may add a paragraph here to introduce the status of sonothrombolysis including the ultrasound catheter based sonothrombolysis (e.g. EKOS, forward-viewing catheter based sonothrombolysis), focused ultrasound based sonothrombolysis, etc. , and their limitations, and then come to the uniqueness of the histotripsy based sonothrombolysis that is reported in this paper.

3. Methods and materials

Page 5 Line 74-81. Authors may comment on the clot retraction. Are they retracted clots or unretracted ones?

Page 6. Line 109. “Bubble activity was initiate in the clot…”. This should be changed to “Bubble activity was initiated in the clot…” so that “was” and “initiated” are both past tense.

Page 7. Line 129. “Motor positions that contained bubble activity within the clot were assigned in 5 mm increments along the length…”. I’m not sure if it is correct to say that a motor position can contain bubble activity. How about this: “Motor positions that correspond with bubble activity within the clot were assigned in 5 mm increments along the length…”.

Page 7. Line 139. “…scanned the histotripsy source scanned along the length…”. Remove the second “scanned” so that this phrase is written as “…scanned the histotripsy source along the length...”.

Page 8. Line 156. “A total of eight clots were tested for each arm, and a total of 72 total clots used in the study.”. This should be changed to: “Eight clots were tested for each arm, with a total of 72 clots used in the study.” to avoid repeating “total” 3 times.

Page 8. Line 158. “…the residual clot was removed from the flow model and was weighed…”. The second “was” can be removed to yield: “…the residual clot was removed from the flow model and weighed…”.

Page 8. Line 161. “The instrument was examination with brightfield microscopy…”. For this sentence, “examined” should be used. The sentence becomes: “The instrument was examined with brightfield microscopy…”.

4. Results

Page 11. Line 232. “Hemoglobin, intact erythrocytes, and intact platelets shed from the clot are reported for all treatment arms in”. This sentence/section needs to be completed.

Page 12. Line 241. There was already a “Figure 4” mentioned previously.

Page 12. Line 248. “Figure Error! No text of specified style in document.”. Add the figure number.

Page 12. Line 253. “…histotripsy significantly increased D imer…”. Change “D imer” to “D-dimer” so that the phrase becomes “…histotripsy significantly increased D-dimer…”.

Page 13. Line 257. “Figure Error! No text of specified style in document.:”. Add the figure number.

Page 13. Line 268. “The multi-path scheme was similarly skewed FDP production to higher concentrations compared to lytic alone.”. Change this to: “Similarly, the multi-path scheme skewed FDP production to higher concentrations compared to lytic alone.”.

Page 15. Line 299. “The average Euclidean distance between acoustic emissions maxima and catheter location…”. Change “emissions” to “emission’s” so that it is possessive. The phrase becomes: “The average Euclidean distance between the acoustic emission’s maxima and catheter location…”.

5. Discussion

Page 18. Line 357. “The histotripsy pulse will be attenuated as it propagated through the clots…”. Change “propagated” to “propagates” so that the tenses in the sentence correspond chronologically. The phrase becomes: “The histotripsy pulse will be attenuated as it propagates through the clots…”.

Page 20. Line 415. “…the release of non-specific fibrin degradation products was increased for histotripsy arms at two highest lytic dose…”. This should be changed to “…the release of non-specific fibrin degradation products was increased for histotripsy arms at the two highest lytic doses…” such that “dose” is switched to plural.

Page 21. Line 436. “The in vitro clots used in this study were homogenous, and which may not be representative…”. This should be changed to “The in vitro clots used in this study were homogenous, which may not be representative…” because the “and” after the comma is unnecessary.

Reviewer #2: This manuscript describes the effects of rt-PA dose on histotripsy-combined, catheter-directed thrombolytic efficacy in vitro.

1. The designed test schemes and procedures seem technically sound and clearly described. Sufficient data support the conclusion that histotripsy can reduce the dose of rt-PA infusion via catheter by a factor of twenty compared to the standard dose for the equivalent thrombolytic efficacy.

2. Statistical analyses conducted in this study seem appropriate. Data interpretation seems unbiased and reasonable without misleading explanation.

3. All data underlying the findings in the manuscript are available as an excel file and jpg figure files on the journal website.

4. The manuscript contains organized contents written in standard English.

5. (Additional comments) Some minor revisions seem required before publication. The itemized comments are below:

Page 3-4, introduction:

The research motivation can be more specific for catheterized conditions. Many previous works already demonstrated that ‘rt-PA + histotripsy’ improves sonothrombolytic efficacy and further reduces the rt-PA dose for the equivalent clot mass reduction. Although the novelty of this work is catheter-directed rt-PA delivery instead of systematic venous administration, the given hypothesis ‘histotripsy may reduce the required rt-PA dose’ seems not significant to justify the further experimental study since the overall trend of the results can easily be anticipated based on the previously published works. The authors may emphasize 1) what is the specific benefit of combining histotripsy to catheter-directed lytic infusion in comparison with standard venous administration and 2) what kinds of obstacles or advantages can uniquely involve in the catheter-directed environment, e.g. if catheter tip may provide an impedance boundary that is different from free field cavitation, if ~200 ul/min infusion-induced local streaming and associated local agitation may help or hinder nucleation, etc. Some of these points are briefly discussed in the Discussion, but it would be more helpful to follow the research motivation by clarifying them in the Introduction.

Page 5, lines 92-93:

The frequency of the used histotripsy pulses should be provided.

Page 7, lines 136-137:

Please add the information of duty-cycle and corresponding time-averaged acoustic intensity.

Does ‘13.3 us for 20 cycle’ burst mean that the frequency is 1.5 MHz (1/(13.3e-6/20))? Please clarify the frequency so the readers easily catch the specifications and estimate the 1) frequency-dependent focal volume, 2) scattering, attenuation through the propagation, and 3) cavitation cloud dynamics.

Reviewer #3: The authors describe use of histotripsy and rt-PA for treatment of human blood clots, with the goal of utilizing histotripsy while reducing rt-PA dose to increase safety. This work addresses a significant problem with potential for significant impact. The work is well performed and described, limitations are clearly explained. The discussion is well considered, especially catheter effects and methods of clot dissolution, and was interesting to read. I have only a few minor comments.

Specific comments:

1. “Clots were stored at 4 ℃ for 3 days to allow for full retraction [22], and were used within 2 weeks of formation.”

Alternatively, if aged clots were used, what would be the expected effect on experiments?

2. “A multi-side port, 5F catheter (Cragg-McNamara, Medtronic, Minneapolis, Minnesota, USA) was introduced into the model vessel 104 via a hemostasis valve and positioned approximately through the central axis of the clot. No guidewires or sheaths were used to advance the catheter through the clot.”

Would there be any risk of downstream embolization due to mechanical contact with the clot?

3. What factors must be considered before using one of these strategies for in vivo treatment? For example, how will the transducer be coupled to the patient and focused at the correct location? Is 21 min an acceptable treatment time considering both the urgency of recanalization and potential for motion/registration errors during this time? Are there concerns regarding heating at the skin?

4. Some internal reference/bookmark errors need to be corrected: “The generation of D-dimer is reported in Figure Error! No text of specified style in document”. The figure before this is Fig. 4 and the figure after this is Fig. 5, so it’s unclear if figures are numbered correctly.

5. “Regardless, these data indicate that histotripsy bubble clouds can be initiated in conjunction with catheter-directed thrombolysis.”

In this location, can the authors remind the reader of the significance of why it is important that these can be done in conjunction?

6. “The data in this study indicate fibrinolysis is equivalent between the standard catheter-directed thrombolysis dosing scheme and a twenty-fold reduction in rt-PA when combined with histotripsy (Figure 5). A reduction in lytic dose may improve the treatment safety profile [9,21].”

This is true that reducing t-PA dose could improve one component of safety, but what are the safety considerations of histotripsy at peak acoustic pressures of 35 MPa?

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Reviewer #1: No

Reviewer #2: Yes: Jinwook Kim

Reviewer #3: No

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(More Than) Doubling Down: Effective fibrinolysis at a reduced rt-PA dose for catheter-directed thrombolysis combined with histotripsy

PONE-D-21-29418R1

Dear Dr. Bader,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Virginie Papadopoulou, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear authors,

Thank you for your careful revision. All comments raised have been addressed. It is my pleasure to accept the manuscript for publication.

Sincerely,

Virginie Papadopoulou, PhD

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The responses with a revised manuscript have addressed the previous comments. I do not have any other questions or comments.

Reviewer #3: The authors have addressed the previous comments adequately.

Suggest changing "...transverse several centimeters of tissue..." to "..traverse several centimeters of tissue..." in the revised manuscript.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No