An experimental erythrocyte rigidity index (Ri) and its correlations with Transcranial Doppler velocities (TAMMV), Gosling Pulsatility Index PI, hematocrit, hemoglobin concentration and red cell distribution width (RDW)

Antonio Valadão Cardoso

doi:10.1371/journal.pone.0229105

Peer Review History

Original SubmissionOctober 15, 2019
4 Dec 2019 Decision Letter - Jeffrey Chalmers, Editor PONE-D-19-28834 An experimental erythrocyte rigidity index (Ri) and its correlations with Transcranial Doppler velocities (TAMMV), Gosling Pulsatility Index PI, hematocrit, hemoglobin concentration and red cell distribution width (RDW) PLOS ONE Dear Professor Valadão Cardoso, 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. Normally I require more than one review; however, based on quality of the single review, and the desire of promptness, I made my decision on one review. Please address all raised questions; also, I believe the reviewer meant, if you are NOT including EM pictures, do not describe protocol in Materials and Methods. 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The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: To CEMIG S / A for the financial support and the Undergraduate Grant to Gabriel Leon de Queiroz Sousa We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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: 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: This paper analyzes the Red Blood Cell (RBC) rigidity index from healthy donors and unhealthy young patients (2-17 years). The author employs a straightforward photometric technique to measure the rigidity of the samples. The main goal of this research was to verify if the overall rigidity of RBCs is higher in unhealthy (sicke) cells obtained from young donors. The samples were obtained from 71 donors and 98 patients. The technique employed to measure the rigidity index is simple and the required equipment is available in low-resource areas, which makes it very interesting. Furthermore, the author tried to correlate this value with other parameters (Hct, Hb, velocity, Gosling's pulsatility index and Red Cell Distribution Width) to improve understanding of flow in cerebral arteries. Nevertheless, a poor and inconclusive correlation between the Ri and the rest of the parameters was found. Moreover, the manuscript is long, it contains multiple grammar mistakes and some figures are not important and could be moved to the supplemental materials. For the previous reasons, I do not recommend its publication unless the following issues are addressed: - The patients and blood donors have different range of ages (from 2-17 and 20-65, respectively). Why did the author compare blood samples from underage patients against adult donors? Can we expect any difference in the measured quantities based on the age of the donors? - If electron microscopy images are going to be presented in future publications, the author should remove the explanation regarding the procedure for taking these images from materials and methods section. The same applies to the procedure for recording the sedimentation process by optical microscopy. - Author stated that “The values of Hct, Hb, and RDW obtained from the archive files are from the date of sample collection for the test of the rigidity index Ri or the time closest to that day”. Regarding the values that were obtained in a different day, I recommend the author to include the time that had passed between the Ri measurement and the Hct, Hb or RDW measurements, and also, to take into account that Hct and Hb could have changed if several days had passed between measurements. - In Figure 3, the author explained that the difference between the initial and final absorbance values for the donors is higher because the deformable red cells form rouleaux. What does it mean? Do the RBCs form aggregates and different structures when they are sickle? Also, in Figure 3, I wonder if the slope of the curve at times between 100-200 s could give more information, as it looks like the slope is more pronounced for donors in comparison to unhealthy cells. - HbS (sickle) and HbF (fetal) hemoglobin should be explained and introduced before their abbreviations. Also, the use of acronyms and abbreviations of other terms should be checked. What is the meaning of SCC, Sβ0thal, SD, MCA, ACA, ICA, PCA, BA? - In Figure 4, the difference in the Ri value for the SCA patient is attributed to the change in Hct, however, this value does not change with Hct for healthy donors. Since the Hct may vary from donor to donor and also at different times for the same donor, I wonder if the value of Hct should be fixed before running the experiments or should be kept constant at the native value for all the donors. - Table 2.A and Table 5 are too wide and are not correctly visualized, and Tables 3 and 4 are very difficult to read, I believe these could be better understood if presented as a Figure. - There are several grammatical mistakes in the manuscript. I suggest the author to thoroughly review the whole manuscript before submission. ****** 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 [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
17 Jan 2020 Author Response Belo Horizonte January 16, 2020 PONE-D-19-28834 An experimental erythrocyte rigidity index (Ri) and its correlations with Transcranial Doppler velocities (TAMMV), Gosling Pulsatility Index PI, hematocrit, hemoglobin concentration and red cell distribution width (RDW) – PLOS ONE Dear Professor Jeffrey Chalmers, I separated into items each of the criticisms made by you and the Reviewer and tried to answer them as best as possible below. Grateful for your attention and Cordially, Antonio Valadão Cardoso, Professor at Minas Gerais State University – Brazil 1- Questions raised by Academic Editor (in italics in the file "Response to Reviewers"): 1.1- "…I believe the reviewer meant, if you are NOT including EM pictures, do not describe protocol in Materials and Methods." Answer: EM (Electron Microscopy) protocol mention removed from the manuscript. 1.2- "Please ensure that your manuscript meets PLOS ONE's style." Answer: Manuscript meets PLOS ONE's style. 1.3- "Please removes any funding-related text from the manuscript." Answer: Funding-related text removed. 1.4- "And let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript." Answer: Funding Statement maintained. 1.5- "While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool." Answer: All figure files approved by PACE 2- Questions raised by Reviewer (in italics in the original file "Response to Reviewers"): 2.1. "Moreover, the manuscript is long, it contains multiple grammar mistakes…" Answer: 496 words of the text were cut (~ 5% of the total). Additionally, two tables were cut, and two figures transferred to supporting material at the suggestion of the reviewer. The text has been completely revised, and the grammatical errors corrected. To check for grammatical errors, I paid online grammar correction services. Also, I asked a colleague (Ph.D. in the UK) to do a final text check and correct any remaining grammatical errors. 2.2- "…and some figures are not important and could be moved to the supplemental materials." Answer: I cut two figures (figures 2 and 10) and transferred them to the supplementary material section. 2.3- "The patients and blood donors have different range of ages (from 2-17 and 20-65, respectively). Why did the author compare blood samples from underage patients against adult donors? Can we expect any difference in the measured quantities based on the age of the donors?" Answer: Sickle cell stiffness is the result of gelatinization (partial or total) of the hemoglobin suspension, containing mutant S hemoglobin. Hemoglobin S polymerizes into fibers when this S concentration reaches a particular value. The deformability of healthy red blood cells is mainly due to hemoglobin remaining in a stable suspension. The difference in age ranges, in this case, does not affect the outcome when the S concentration is high enough to initiate polymerization, regardless of age (adult or child). Also, Hemominas Foundation (acronym HBH, the blood center that provided the samples) only accepts blood from donors older than 18 years. 2.4- "If electron microscopy images are going to be presented in future publications, the author should remove the explanation regarding the procedure for taking these images from materials and methods section." Answer: Removed. 2.5- The same applies to the procedure for recording the sedimentation process by optical microscopy. Answer: Removed. 2.6- "Table 2.A and Table 5 are too wide and are not correctly visualized…" Answer: Tables have been reformatted, and visualization is correct now. 2.7- "… and Tables 3 and 4 are very difficult to read, I believe these could be better understood if presented as a Figure." Answer: Tables 3 and 4 are, now, presented as Figures 9 and 10. 2.8- Author stated that "The values of Hct, Hb, and RDW obtained from the archive files are from the date of sample collection for the test of the rigidity index Ri or the time closest to that day". Regarding the values that were obtained in a different day, I recommend the author to include the time that had passed between the Ri measurement and the Hct, Hb or RDW measurements, and also, to take into account that Hct and Hb could have changed if several days had passed between measurements. Answer: This part of sub-item 2.5.1 changed to: ………………………………………………………………………………………………… Data collection in the HBH files was performed directly in the patient's record. In possession of the date of blood collection to measure the Ri, we searched the patient's folder for the blood count performed on the date closest to the date of the project collection. HBH has not permitted reproduction by any means of these patient medical records. It only allowed copying the blood count data. Since errors can be made in the process of collecting and transcribing data manually, the coincidence of dates between Ri measurement and blood count is not guaranteed. On the other hand, as it is a blood donation procedure, it is ensured that the donor hemoglobin concentration data (g / l) are from the same collection date to measure Ri from that donor. 2.9- In Figure 3, the author explained that the difference between the initial and final absorbance values for the donors is higher because the deformable red cells form rouleaux. What does it mean? Do the RBCs form aggregates and different structures when they are sickle? Answer: Microstructure differences, between healthy and SCA patient blood, are easily identifiable under the optical microscope. To observe under the optical microscope, we placed a drop of blood, with EDTA anticoagulant only, on the glass slide and gently deposited a coverslip over this drop. Deformable donor red blood cells form very large, stable, and interconnected rouleaux when the blood is at rest. Like large stacks of coins, fitted precisely on top of each other. SCA RBC forms smaller, poorly structured, disconnected rouleaux. Irreversibly polymerized SS erythrocytes generally do not form rouleaux and are generally separate, individualized. 2.10- Also, in Figure 3, I wonder if the slope of the curve at times between 100-200 s could give more information, as it looks like the slope is more pronounced for donors in comparison to unhealthy cells. Answer: This Reviewer remark has been added to the text (line number 323). In the case of donors, possibly between 100-200s, the already settled and connected rouleaux form 3-D structures and contract, increasing absorbance. In the case of deformable donor red blood cells, this contraction (increased absorbance) is faster because there is a 3D connection between the rouleaux. In the case of patients, the connection between rouleaux is much smaller, and the increase in absorbance occurs more slowly. 2.11- HbS (sickle) and HbF (fetal) hemoglobin should be explained and introduced before their abbreviations. Answer: HbS introduced and explained starting at line 61, and HbF at line 93. 2.12 - Also, the use of acronyms and abbreviations of other terms should be checked. What is the meaning of SCC, Sβ0thal, SD, MCA, ACA, ICA, PCA, BA? Answer: All full name acronyms and abbreviations added to the text. 2.13- In Figure 4, the difference in the Ri value for the SCA patient is attributed to the change in Hct, however, this value does not change with Hct for healthy donors. Since the Hct may vary from donor to donor and also at different times for the same donor, I wonder if the value of Hct should be fixed before running the experiments or should be kept constant at the native value for all the donors. Answer: This Reviewer remark has been added to the text (line number 368). 2.15- There are several grammatical mistakes in the manuscript. I suggest the author to thoroughly review the whole manuscript before submission. Answer: A thorough revision of the entire manuscript was made. The grammatical errors found were corrected, as can be seen in the file "Revised manuscript with track changes." Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0229105.r002
30 Jan 2020 Decision Letter - Jeffrey Chalmers, Editor An experimental erythrocyte rigidity index (Ri) and its correlations with Transcranial Doppler velocities (TAMMV), Gosling Pulsatility Index PI, hematocrit, hemoglobin concentration and red cell distribution width (RDW) PONE-D-19-28834R1 Dear Dr. Valadão Cardoso, 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, Jeffrey Chalmers, 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: 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: No https://doi.org/10.1371/journal.pone.0229105.r003
Formally Accepted
6 Feb 2020 Acceptance Letter - Jeffrey Chalmers, Editor PONE-D-19-28834R1 An experimental erythrocyte rigidity index (Ri) and its correlations with Transcranial Doppler velocities (TAMMV), Gosling Pulsatility Index PI, hematocrit, hemoglobin concentration and red cell distribution width (RDW) Dear Dr. Valadão Cardoso: 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. Jeffrey Chalmers Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0229105.r004

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