Intra-individual variation of particles in exhaled air and of the contents of Surfactant protein A and albumin

Spela Kokelj; Jeong-Lim Kim; Marianne Andersson; Gunilla Runström Eden; Björn Bake; Anna-Carin Olin

doi:10.1371/journal.pone.0227980

Peer Review History

Original SubmissionNovember 4, 2019
25 Nov 2019 Decision Letter - Ajaya Bhattarai, Editor PONE-D-19-30683 Intra-individual variation of particles in exhaled air and of the contents of Surfactant protein A and albumin PLOS ONE Dear Professor Spela Kokelj, 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. We would appreciate receiving your revised manuscript by Jan 09 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Please include the updated Competing Interests Statement and Funding Statement in your cover letter. We will change the online submission form on your behalf. Additional Editor Comments (if provided): Major revision is necessary. After getting the revised manuscript, the further decision will be given. [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes ****** 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: Yes ****** 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 findings from a small-sample size study. Given the novelty of the exhaled particle technique and the scarcity of the data on this topic, the results are interesting and important enough to be published despite that the results may be considered preliminary. The findings support the need for a more in-depth study with larger sample size to generate more generalizable results. Specific comments are as follows. Abstract: CRs should be defined. As written, the units of CRs for different analytes are not normalized to mean values. e.g., reporting a CR as 0.85 kn L-1 gives the reader no idea on variability. Table 1: Since this is not a randomly selected cohort of subjects, normal distributions would not be expected for any of the anthropocentric (and perhaps spirometric) parameters shown in Table 1. It is more informative to provide range, median, etc, in addition to mean and SD. Methods: The method of exhaled particle collection should be described in more detail perhaps with a diagram of the sampling set up. How was the sampling air flow controlled? What sampling medium was used to collect particles? How long did it take to collect 120 ng particles? How was this monitored? Statistical analyses section: the sentence starting "particle mean mass differences..." (Line 141-143) is unclear. Next sentence - what's the rationale to define CR as SD multiplied by 1.96? ANOVA tests presented in the paper are okay; but given the data structure, statistical testing for intra-day and between-day differences can be done more appropriately using mix-effects models, allowing control for subject as a random effect. Table 3: the heading should be "mean(95% CI) or median (interquartile range)". Discussion section is very well done. Reviewer #2: This paper reports on measurements of exhaled breath from 16 subjects. It seems to be partly an advertisement for the PExA instrument (the authors state a conflict) and partly an evaluation of some of the contents of exhaled breath. The paper has some major flaws, discussed below, but the bottom line is that the hypothesis is not clearly stated. The hypothesis that I find is that intra-individual variation is small as measured by CR so that larger variations might indicate onset or resolution of disease. The paper should be restructured around this hypothesis or another one if my interpretation is incorrect. In addition, the authors present the context of this work in terms of that of their prior co-authors. There is an extensive and growing literature on exhaled breath analysis. The introduction needs to be restructured to address the goals of this work in the context of other work in the field. Major Comments Line 47-48: this statement needs references. Line 50-51: what is a PExA instrument? The introduction should describe the context for the work not the instrumentation used to perform the work. If the point of this work is to highlight the PExA instrument, it should be compared to other instruments that produce similar results. Line 36: Define lnPEx. Is it the log of PEx? Line 200-201: BAL contains information on cellular content of for instance neutrophils and eosinophils, which are important health markers. It would be very interesting if exhaled breath particles also contained cellular information, but this was not tested in this study. This sentence is more of a sales pitch for the PExA instrument and not a balanced evaluation of the pros and cons of exhaled breath analysis vs. BAL. Lines 202-211: No analysis was performed of SP-D or CC-16 in this study. Why is it discussed here? Line 218: this study did not include any subjects with asthma – why is this included here? Line 223-228: speculation. It could be that gravity plays a role. At night the subjects were horizontal while in the day vertical, which may alter mucociliary transport and may explain some of the observations. This is also speculation. Table 4: wt% might vary with relative humidity – drier conditions might lead to higher concentrations. Was RH in the clinic measured? If not, are these results valid? ****** 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: Yes: Junfeng (Jim) Zhang 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 to be viewed.] 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. 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Revision 1
10 Dec 2019 Author Response Dear Ajaya Bhattarai, Thank you for giving us the opportunity to submit a revised manuscript entitled “Intra-individual variation of particles in exhaled air and of the contents of Surfactant protein A and albumin”. We appreciate the time and effort that PLOS ONE and the reviewers have dedicated providing valuable feedback on our manuscript and we have incorporated changes in the manuscript to reflect most of the suggestions provided by the reviewers. These changes are highlighted in a separate file. We consider this revision, incorporating reviewers’ insightful comments, to have substantially improved the manuscript. Looking forward to hearing from you regarding our revision and to respond to any further questions and comments you may have that would improve the manuscript. Sincerely, Spela Kokelj (on the behalf of the co-authors) Journal Requirements: 1. Updated Competing Interests statement is provided in the cover letter and the patent number has been included. 2. The Funding Statement was incorrect and it has been revised. The funding related text has been removed from the manuscript and the Acknowledgements section has been changed (line 310-313). Response to the Reviewer #1: Comment: This paper reports findings from a small-sample size study. Given the novelty of the exhaled particle technique and the scarcity of the data on this topic, the results are interesting and important enough to be published despite that the results may be considered preliminary. The findings support the need for a more in-depth study with larger sample size to generate more generalizable results. Specific comments are as follows. Reply: We appreciate the positive feedback from the reviewer. Comment: Abstract: CRs should be defined. As written, the units of CRs for different analytes are not normalized to mean values. e.g., reporting a CR as 0.85 kn L-1 gives the reader no idea on variability. Reply: We agree with this comment. The abstract is rewritten to highlight diurnal variation and effect of time between the measurements on variation. CR is defined in the method section and CRs are instead presented only in Table 4 and Table 3 gives the reader an overview of mean values for all variables. Comment: Table 1: Since this is not a randomly selected cohort of subjects, normal distributions would not be expected for any of the anthropocentric (and perhaps spirometric) parameters shown in Table 1. It is more informative to provide range, median, etc, in addition to mean and SD. Reply: We agree and have therefore revised Table 1 where we present the mean value and range for all variables. Comment: Methods: The method of exhaled particle collection should be described in more detail perhaps with a diagram of the sampling set up. How was the sampling air flow controlled? What sampling medium was used to collect particles? How long did it take to collect 120 ng particles? How was this monitored? Reply: A figure of the PExA instrument and the sampling set-up has been added as a supplement (S2 Fig.) Exhalation flow was measured using an ultrasonic flow meter (OEM flow sensor; Spiroson-AS, Medical Technologies, Zürich, Switzerland), enabling visualization of the expiratory flow and volume. The sampling air flow was, however, not controlled, since the flow through the impactor is controlled and constant and the exhalations exceeding the flow rate are buffered in the reservoir. The sampling medium used to collect particles was a hydrophilic silicon wafer inside the impactor. We have included this in the Methods section under Exhaled particles (line 104). The median number of breathing maneuvers required to collect 120 ng was 7, with a range from 4 to 53. This has been added to the Methods section under Exhaled particles (line 114-115). Comment: Statistical analyses section: the sentence starting "particle mean mass differences..." (Line 141-143) is unclear. Reply: Regarding the sentence about particle mean mass differences (line 141 -142) – the text has been revised. The sentence (line 143-146) now reads: “Particle mean mass differences were expressed as the percentage of the mean of corresponding measurements (calculated by subtracting a later from an earlier measurement and dividing it by the mean of the two measurements).” Comment: Next sentence - what's the rationale to define CR as SD multiplied by 1.96? Reply: The rationale behind CR = ±1.96SD is that CR then defines the maximum difference which is likely to occur between measurements with 95% probability, assuming normal distribution. Comment: ANOVA tests presented in the paper are okay; but given the data structure, statistical testing for intra-day and between-day differences can be done more appropriately using mix-effects models, allowing control for subject as a random effect. Reply: As the reviewer mentioned above, we could apply mix-effects models allowing control for subjects as a random effect. Then, we can obtain the coefficients of predictor variables, which are adjusted for subjects as random. However, we are not only interested in coefficients of predictors but also of the effects of actual day- and daytime variance. Therefore, we treat them as fixed variables to yield the value of coefficients. Comment: Table 3: the heading should be "mean (95% CI) or median (interquartile range)". Reply: The suggested correction in Table 3 has been made. Comment: Discussion section is very well done. Reply: The positive feedback is appreciated. Response to the Reviewer #2: Comment: This paper reports on measurements of exhaled breath from 16 subjects. It seems to be partly an advertisement for the PExA instrument (the authors state a conflict) and partly an evaluation of some of the contents of exhaled breath. The paper has some major flaws, discussed below, but the bottom line is that the hypothesis is not clearly stated. The hypothesis that I find is that intra-individual variation is small as measured by CR so that larger variations might indicate onset or resolution of disease. The paper should be restructured around this hypothesis or another one if my interpretation is incorrect. In addition, the authors present the context of this work in terms of that of their prior co-authors. There is an extensive and growing literature on exhaled breath analysis. The introduction needs to be restructured to address the goals of this work in the context of other work in the field. Reply: We thank the reviewer for the keen concern. However, we claim that the authors do not want to promote any commercial advertisement of a specific instrument and are only interested in evaluating the method of exhaled particles itself. Therefore, we have now revised the manuscript and the PExA® instrument is mentioned in the method section only, which is necessary in this context. We have also clearly stated a hypothesis in the introduction, as suggested (line 66-69). Comment: Line 47-48: this statement needs references. Reply: The reference has been added. Comment: Line 50-51: what is a PExA instrument? The introduction should describe the context for the work not the instrumentation used to perform the work. If the point of this work is to highlight the PExA instrument, it should be compared to other instruments that produce similar results. Reply: We have restructured the introduction to shift focus away from the PExA instrument and instead focus on the context of the work. We have also mentioned the work of the others (Schwarz et al.) that are using a similar method to sample exhaled particles. However, the results and the methods are hard to compare since there is nobody using this breathing maneuver that is used with our method. The results would be different if tidal breathing would be used like with other similar methods. On top of that, there is lack of methods that allow a similar chemical analysis of the exhaled particles to our method. Comment: Line 36: Define lnPEx. Is it the log of PEx? Reply: lnPEx is a natural logarithm of PEx. The abstract has been restructured and does therefore not include lnPEx. The definition of lnPEx has been added to the manuscript (line 143). Comment: Line 200-201: BAL contains information on cellular content of for instance neutrophils and eosinophils, which are important health markers. It would be very interesting if exhaled breath particles also contained cellular information, but this was not tested in this study. This sentence is more of a sales pitch for the PExA instrument and not a balanced evaluation of the pros and cons of exhaled breath analysis vs. BAL. Reply: You have raised an important point here, however, we do not agree with the comment on “a sales pitch”. The authors would like to emphasize that the aim of the present study was to evaluate the PEx method and not to promote the PExA instrument. To the best of our knowledge, there is no comparable method sampling exhaled particles. The exhaled particles obtained with our instrument unfortunately do not contain cellular information such as for example BAL. We have changed the sentence and it now reads (line 207-210): “The PEx method samples RTLF from small airways while avoiding invasive techniques and unknown dilutions, as in, for example, bronchoalveolar lavage (BAL), induced sputum, and exhaled breath condensate. However, the method has some drawbacks, namely the sample not containing any cellular material that can be obtained will BAL.” Comment: Lines 202-211: No analysis was performed of SP-D or CC-16 in this study. Why is it discussed here? Reply: They are other known components of respiratory tract lining fluid. We know nothing about variability of SP-A or albumin, since it has not been studied previously, we wanted to compare it to other known components of respiratory tract lining fluid that have been studied before, especially since they also exhibit diurnal variation and are secreted by the same cells as SP-A (for SP-D). Comment: Line 218: this study did not include any subjects with asthma – why is this included here? Reply: The suggestion has been noted and asthma has been omitted (line 228-229). Comment: Line 223-228: speculation. It could be that gravity plays a role. At night the subjects were horizontal while in the day vertical, which may alter mucociliary transport and may explain some of the observations. This is also speculation. Reply: Thank you for bringing this interesting point up. We agree with the comment and have incorporated it in the discussion section (line 225-228). Comment: Table 4: wt% might vary with relative humidity – drier conditions might lead to higher concentrations. Was RH in the clinic measured? If not, are these results valid? Reply: The humidity in the instrument is similar to the humidity of the air we are exhaling. The humidity in the instrument is kept constant throughout the measurement by the exhaled air that is saturated at 36 °C circling around. We assume the humidity of exhaled air is similar between subjects. Furthermore, the exhaled particles are not at any point in contact with room air, so they are not under the influence of the relative humidity in the room. Attachments Attachment Submitted filename: Response to Reviewers.docx* https://doi.org/10.1371/journal.pone.0227980.r002
6 Jan 2020 Decision Letter - Ajaya Bhattarai, Editor Intra-individual variation of particles in exhaled air and of the contents of Surfactant protein A and albumin PONE-D-19-30683R1 Dear Dr. Kokelj, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Ajaya Bhattarai 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: 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) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) ****** 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) ****** 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) ****** 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) ****** 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: Yes: Junfeng (Jim) Zhang https://doi.org/10.1371/journal.pone.0227980.r003
Formally Accepted
14 Jan 2020 Acceptance Letter - Ajaya Bhattarai, Editor PONE-D-19-30683R1 Intra-individual variation of particles in exhaled air and of the contents of Surfactant protein A and albumin Dear Dr. Kokelj: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ajaya Bhattarai Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0227980.r004

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