Differentiating iron-loading anemias using a newly developed and analytically validated ELISA for human serum erythroferrone

Laura Diepeveen; Rian Roelofs; Nicolai Grebenchtchikov; Rachel van Swelm; Leon Kautz; Dorine Swinkels

doi:10.1371/journal.pone.0254851

Peer Review History

Original SubmissionMay 4, 2021
8 Jun 2021 Decision Letter - Kostas Pantopoulos, Editor PONE-D-21-13559 Differentiating iron-loading anemias using a newly developed and analytically validated ELISA for human serum erythroferrone PLOS ONE Dear Dr. Diepeveen, 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. Both reviewers appreciated the importance in developing and validating a new ELISA assay for ERFE. However, they also raised issues about the cohort of patients used in this work, the comparison of the new assay with another available ERFE ELISA kit, and the interpretation of some data. These issues should be addressed in a revision. Please submit your revised manuscript by Jul 23 2021 11:59PM. 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Kind regards, Kostas Pantopoulos, PhD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. 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 Additional Editor Comments (if provided): Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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: 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: 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 manuscript describes a new ERFE Elisa assay, which is thoroughly validated for use with human plasma. The laboratory has extensive previous experience with hepcidin assays, and it is expected that the described erythroferrone assay will contribute to the ongoing studies regarding the role and mode of action of erythroferrone. The steps taken to validate of the assay should result in increased confidence in obtained data. There are some minor points which should be addressed: 1) The authors repeatedly point out that their assay could be useful in clinical practice - as an example, they demonstrate the ability of the assay to distinguish between thalassemia major and X-linked sideroblastic anemia. In this respect, they probably should point out that the determination of plasma ERFE provides only supplementary information for the clinician. Differential diagnosis of thalassemia major vs. XLSA will always be based on overall hematologic parameters and the presence/absence of ring sideroblasts. In the case of blood donors, the main criteria will always be hemoglobin and ferritin. 2) The authors repeatedly point out the association between ERFE and ineffective erythropoiesis. However, for the testing of their assay, they use transfused thalassemia major patients. The cited reference (7) by Camaschella and Nai explicitly mentions non-transfusion-dependent thalassemia as a typical example of a disease associated with ineffective erythropoiesis. Generally, the text (for example lines 66-71) gives the impression that ERFE is the main factor influencing iron loading in thalassemia major. This is certainly not the case, given the need for repeated transfusions in thalassemia major. The need for transfusions, as well as the consequences of transfusions in thalassemia major patients should be more emphasized in the text. Did the authors consider the possibility of testing thalassemia intermedia patients? 3) Line 305: The authors probably mean "ERFE" rather than "hepcidin" (?). 4) Table 4: The difference in values for plasma and serum is interesting. Did the authors have the opportunity to check this effect with the other erythroferrone assays? 5) Table 5: The reader could benefit from adding the normal range values. Overall, the validation of the assay appears to be competently done, and the assay itself has the potential to provide interesting data regarding the role of erythroferrone in iron metabolism. Reviewer #2: It will be interessting to compare with human ERFE ELISA described in Ramirez Cuevas K et al, Drug Test Anal. 2020. Also, optimal samples to test are samples from volunteers injected with rhEPO. It should be added as other experiment. ****** 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.0254851.r001
Revision 1
15 Jun 2021 Author Response Additional Journal Requirements 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. 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 Authors: The revised files were checked to meet PLOS ONE's style requirements. 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Authors: We checked the reference list. Although no references were retracted, we noticed reference 15 and 29 were incorrect. Therefore, reference 15 was replaced and reference 29 was textually changed. Reviewer #1 1) The authors repeatedly point out that their assay could be useful in clinical practice - as an example, they demonstrate the ability of the assay to distinguish between thalassemia major and X-linked sideroblastic anemia. In this respect, they probably should point out that the determination of plasma ERFE provides only supplementary information for the clinician. Differential diagnosis of thalassemia major vs. XLSA will always be based on overall hematologic parameters and the presence/absence of ring sideroblasts. In the case of blood donors, the main criteria will always be hemoglobin and ferritin. Authors: The reviewer is correct that ERFE measurement would only provide supplementary information to clinicians regarding the diagnosis and prognosis of XLSA and b-thalassemia major. Therefore, we propose the assay can be used as indicator of severity of the phenotypes and we use the subsequent experiment, in which we aim to differentiate between the two pathologies, as biological validation of functionality of the assay. We clarified both the future utility of ERFE measurement as the goal of our experiment (line 73-76, 284, 346). 2) The authors repeatedly point out the association between ERFE and ineffective erythropoiesis. However, for the testing of their assay, they use transfused thalassemia major patients. The cited reference (7) by Camaschella and Nai explicitly mentions non-transfusion-dependent thalassemia as a typical example of a disease associated with ineffective erythropoiesis. Generally, the text (for example lines 66-71) gives the impression that ERFE is the main factor influencing iron loading in thalassemia major. This is certainly not the case, given the need for repeated transfusions in thalassemia major. The need for transfusions, as well as the consequences of transfusions in thalassemia major patients should be more emphasized in the text. Did the authors consider the possibility of testing thalassemia intermedia patients? Authors: We agree with the reviewer that we should provide more information on both the need as well as the consequences of transfusions in b-thalassemia major patients. We included this in the manuscript (line 71-73). Regarding the thalassemia intermedia patients, indeed it would be interesting to use this patient population. Yet, the Radboudumc biobank, that includes patient samples of the Dutch Center for Iron Disorders, could only provide us with thalassemia major patient samples. 3) Line 305: The authors probably mean "ERFE" rather than "hepcidin" (?). Authors: The referee is correct. Thank you, we changed it. 4) Table 4: The difference in values for plasma and serum is interesting. Did the authors have the opportunity to check this effect with the other erythroferrone assays? Authors: In this current study, we did not have the opportunity to check with the ILS kit and the other in-house assay. However, this characteristic has been tested before in a study by Appleby et al. (reference 40) who did use the ILS kit and interestingly observed the same effect. 5) Table 5: The reader could benefit from adding the normal range values. Authors: We agree with the referee and added this information to Table 5. Reviewer #2 1) It will be interesting to compare with human ERFE ELISA described in Ramirez Cuevas K et al, Drug Test Anal. 2020. Also, optimal samples to test are samples from volunteers injected with rhEPO. It should be added as other experiment. Authors: Thank you for your suggestions. It would indeed be interesting to compare our results with the AdipoGen Life Sciences ERFE kit, as used by Cuevas et al. Besides this kit, more commercially ERFE ELISAs are currently increasingly available. Most of these assays are not published and data on extensive analytical validation is lacking. To increase applicability and reliability of these methods, eventually future studies should focus on harmonization or standardization of these assay, for which overall comparison of all assays is needed to ensure good analytical performance since this a prerequisite for reducing inter-assay variation. Yet, this is out of the scope of our manuscript. For the current study, we performed the comparison experiment to provide extra data on the reliability of our assay and therefore chose to solely compare our ELISA with two existing methods. Moreover, we agree that testing our ELISA with samples from volunteers injected with rhEPO would increase the biological plausibility of our assay and will keep this in mind for future experiments. However, since the most important message of our paper is to extensively analytically validate an assay in order to use the assay correctly, we believe one biological validation experiment is sufficient. In addition, the suggested experiment would require approval according to the Dutch Medical Research Involving Human Subjects acts, which is currently not feasible for this manuscript. https://doi.org/10.1371/journal.pone.0254851.r002
5 Jul 2021 Decision Letter - Kostas Pantopoulos, Editor Differentiating iron-loading anemias using a newly developed and analytically validated ELISA for human serum erythroferrone PONE-D-21-13559R1 Dear Dr. Diepeveen, 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, Kostas Pantopoulos, PhD 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.0254851.r003
Formally Accepted
9 Jul 2021 Acceptance Letter - Kostas Pantopoulos, Editor PONE-D-21-13559R1 Differentiating iron-loading anemias using a newly developed and analytically validated ELISA for human serum erythroferrone Dear Dr. Diepeveen: 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. Kostas Pantopoulos Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0254851.r004

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