Leukocyte differentiation in bronchoalveolar lavage fluids using higher harmonic generation microscopy and deep learning

Laura M. G. van Huizen; Max Blokker; Yael Rip; Mitko Veta; Kirsten A. Mooij Kalverda; Peter I. Bonta; Jan Willem Duitman; Marie Louise Groot

doi:10.1371/journal.pone.0279525

Peer Review History

Original SubmissionDecember 9, 2022
14 Feb 2023 Decision Letter - Rahul Gomes, Editor PONE-D-22-33842Differentiation of leukocytes in bronchoalveolar lavage fluid samples using higher harmonic generation microscopy and deep learningPLOS ONE Dear Dr. van Huizen, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please address the reviews as mentioned. There are some issues with image resolution. Provide training and loss graphs. Show a comparative analysis of how the model performs with and without adding weights to address class imbalance. Please submit your revised manuscript by Mar 31 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2.Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information 3. Thank you for stating the following in the Competing Interests section: "I have read the journal's policy and the authors of this manuscript have the following competing interests: M.G. declares to have financial and non-financial interest in Flash Pathology B.V. However, Flash Pathology B.V. was not involved in the design of the study or analysis of the data." Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ****** 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 ****** 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: No ****** 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: -The motivation and background need to be improve and describe contribution and organization clearly in seprate paragraph in introduction section. "A Numerical Study of Heat Transfer and Fluid Flow in a Channel with an Array of Pin Fins in Aligned and Staggered Configurations" "Spectral control of high order harmonics through non-linear propagation effects" -Author should uniform border in all tables. -where is conclusion part? Author should add conclusion section and make sure not repeat phrases from the Introduction!. -future work should more focused and clearly mention at the end of conclusion. -please give a proofread check to the paper. Reviewer #2: The title is very long, you can summare it the inrorudcrion paer in the abstract section is long you can highlight the main work in the intorudction section The stucture of this paper can be added into the end of the intorudctions section the mathmatical notation in this paper is missing some related works are needed to be metioned in this paper Artocarpus Classification Technique Using Deep Learning Based Convolutional Neural Network Mango Varieties Classification-Based Optimization with Transfer Learning and Deep Learning ApproachesClassification Applications with Deep Learning and Machine Learning Technologies ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0279525.r001
Revision 1
12 May 2023 Author Response Dr. Rahul Gomes PLOS ONE ATS Peer Review Office 1265 Battery Street, Suite 200 San Francisco, CA 94111 April 20, 2023 Subject: Submission of revised manuscript PONE-D-22-33842 Dear Dr. Rahul Gomes, We thank the editors and reviewers for their time and constructive comments that have helped to improve our manuscript. We have carefully reviewed the comments and have revised the manuscript accordingly. Below, we provide a point-by-point response explaining how we have addressed each of the reviewers’ comments, corresponding changes to the text of the manuscript are marked in red. Below you will also find the updated financial disclosure and competing interests statements: Financial Disclosure statement This publication is part of the project InstantPathology (with project number 15825) of the research program Applied and Engineering Sciences which is (partly) financed by the Dutch Research Council (NWO), awarded to M.G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests statement I have read the journal's policy and the authors of this manuscript have the following competing interests: M.G. declares to have financial and non-financial interest in Flash Pathology B.V. However, Flash Pathology B.V. was not involved in the design of the study or analysis of the data. This does not alter our adherence to PLOS ONE policies on sharing data and materials. We hope the revised version is now suitable for publication and look forward to hearing from you in due course. Yours sincerely, On behalf of the co-authors, PhD candidate Laura van Huizen, PhD candidate Max Blokker and Prof. Dr. Marloes Groot Response to editor: C1: “There are some issues with image resolution”. R1: Thank you for pointing this out. We checked the resolution of our images, and our images are fulfilling the image requirements of the journal (format, dimensions, size, resolution). We agree that in the submitted PDF file the images have lower resolution. However, the single downloadable images have the correct quality. We hope you can appreciate the quality of these images, as the image resolution is important for this study. C2: “Provide training and loss graphs.” R2: We added a new figure, Figure 5, which displays the training and validation mean absolute error for the top-performing model during optimization. C3: “Show a comparative analysis of how the model performs with and without adding weights to address class imbalance.” R3: Thank you for raising this point. In the newly added Figure 5, this has been included. In short, the figure highlights the penalizing effect of the class weights on the raw training mean absolute error. Response to Journal requirements: C4: “Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.” R4: We made some small adjustments to the manuscript to meet all PLOS ONE’s style requirements, including placing the table legends at the bottom of the tables and adjusting the title page. C5: “Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information” R5: Thank you for pointing this out. We added the type of participant consent (written) to the ethics statement in Methods and online submission information, resulting in the full ethical statement (p. 6 line 124-127): “All patients provided written informed consent as part of studies approved by a Medical Research Board under protocol number NTR NL7634 or FP02C-18-001. The research was conducted in accordance with the Netherlands Code of Conduct for Research Integrity and the Declaration of Helsinki.” C6: “Thank you for stating the following in the Competing Interests section: "I have read the journal's policy and the authors of this manuscript have the following competing interests: M.G. declares to have financial and non-financial interest in Flash Pathology B.V. However, Flash Pathology B.V. was not involved in the design of the study or analysis of the data." Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.” R6: We adjusted the conflict-of-interest statement accordingly, resulting in the full Competing Interests statement: “I have read the journal's policy and the authors of this manuscript have the following competing interests: M.G. declares to have financial and non-financial interest in Flash Pathology B.V. However, Flash Pathology B.V. was not involved in the design of the study or analysis of the data. This does not alter our adherence to PLOS ONE policies on sharing data and materials.” Response to Reviewer #1: Thank you for your review of our manuscript. We have answered each of your points below. C7: “The motivation and background need to be improve and describe contribution and organization clearly in seprate paragraph in introduction section.” R7: Thank you for your comments on the introduction. We improved the motivation and background of our study by simplifying and making them more concrete in the first paragraph (p. 4 line 63-75). Additionally, we added our contribution and the structure of the paper in the last paragraph of the introduction (p. 5-6 line 106-116). C8: “ "A Numerical Study of Heat Transfer and Fluid Flow in a Channel with an Array of Pin Fins in Aligned and Staggered Configurations" "Spectral control of high order harmonics through non-linear propagation effects" “ R8: Thank you for the references. We assume the reviewer suggested these papers as references for in our manuscript. To our understanding the first paper describes the investigation of different configurations of array of pin fins for heat transfer and fluid flow experiments. We do not see how this paper connects to our manuscript, and therefore we did not include it as a reference. In the second mentioned paper the spectral shifts in generated harmonics in silicon and zinc oxide were investigated. Although this paper utilizes higher harmonic generation, similar as we do, we do not see any other overlap with our research, and therefore decided to not use it as a reference. C9: “Author should uniform border in all tables.” R9: Thank you for pointing this out. We adjusted all tables such that they have uniform borders and are fulfilling the requirements of the journal. C10: “where is conclusion part? Author should add conclusion section and make sure not repeat phrases from the Introduction!.” R10: We agree with the reviewer that the conclusion part was not directly visible. We added the section “Conclusions” at the end of the manuscript, where we summarize the results (p. 23 line 530-540). C11: “future work should more focused and clearly mention at the end of conclusion.” R11: Thank you for raising this point. In the new section “Conclusions” we added concrete suggestions for future work (p. 23 line 530-540). C12: “please give a proofread check to the paper.” R12: Thank you for pointing this out. All authors have read the paper again carefully and we revised it to the best of our knowledge. Response to Reviewer #2: Thank you for reviewing and providing comments on our manuscript. We have answered each of your points below. C13: “The title is very long, you can summare it” R13: Thank you for pointing this out. We shorted the title to be: “Leukocyte differentiation in bronchoalveolar lavage fluids using higher harmonic generation microscopy and deep learning”. We believe that the components “leukocyte differentiation”, “BAL fluids”, “higher harmonic generation microscopy” and “deep learning” are important to be stated in the title, as together they form the focus of the manuscript. We are welcoming suggestions that also cover the content of the manuscript. C14: “the inrorudcrion paer in the abstract section is long.” R14: Thank you for raising this point. We shorted the introduction part of the abstract and provided more structure by adding headings (“background”, “objective”, “methods”, “results”, and “conclusions”). C15: “you can highlight the main work in the intorudction section” R15: We agree with the reviewer that the main work can be highlighted more in the introduction section. We added, especially in the last paragraph of the introduction (p. 5-6 line 106-116), more concrete our contribution. C16: “The stucture of this paper can be added into the end of the intorudctions section” R16: We agree with the reviewer that the structure of the paper at the end of the introduction section is useful. Therefore, we outlined the structure of the manuscript in the last paragraph of the introduction (p. 5-6 line 106-116). C17: “the mathmatical notation in this paper is missing” R17: We assume that the reviewer is referring to “1e-05” in line 228 and 360 (old version). We replaced this by “1×10^(-5)” in line 228 and 358, respectively (revised version). C18: “some related works are needed to be metioned in this paper Artocarpus Classification Technique Using Deep Learning Based Convolutional Neural Network Mango Varieties Classification-Based Optimization with Transfer Learning and Deep Learning Approaches Classification Applications with Deep Learning and Machine Learning Technologies” R18: Thank you for pointing out these references. We agree with the reviewer that the book containing these references is relevant given the similar models utilized in our work. Therefore, we have added a reference at p. 11 line 223. R19: Reviewer #2 answered “No” to the following question: “Is the manuscript presented in an intelligible fashion and written in standard English?”. To the best of our knowledge, our manuscript is written in standard English in an intelligible fashion. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0279525.r002
12 Jun 2023 Decision Letter - Rahul Gomes, Editor Leukocyte differentiation in bronchoalveolar lavage fluids using higher harmonic generation microscopy and deep learning PONE-D-22-33842R1 Dear Dr. van Huizen, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Rahul Gomes, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): 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: (No Response) Reviewer #2: All comments have been addressed ******** 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: (No Response) Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) Reviewer #2: 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: (No Response) Reviewer #2: Yes ****** 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: (No Response) Reviewer #2: 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: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0279525.r003
Formally Accepted
19 Jun 2023 Acceptance Letter - Rahul Gomes, Editor PONE-D-22-33842R1 Leukocyte differentiation in bronchoalveolar lavage fluids using higher harmonic generation microscopy and deep learning Dear Dr. van Huizen: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Rahul Gomes Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0279525.r004

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