Automated dry thawing of cryopreserved haematopoietic cells is not adversely influenced by cryostorage time, patient age or gender

Peter Kilbride; Julie Meneghel; Giovanna Creasey; Fatemeh Masoudzadeh; Tina Drew; Hannah Creasey; David Bloxham; G. John Morris; Kevin Jestice

doi:10.1371/journal.pone.0240310

Peer Review History

Original SubmissionJune 10, 2020
11 Aug 2020 Decision Letter - Jeffrey Chalmers, Editor PONE-D-20-17327 Automated dry thawing of cryopreserved haematopoietic cells is not adversely influenced by cryostorage time, patient age or gender PLOS ONE Dear Dr. Kilbride, 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. There is obviously a large differences of opinion between the two reviews. Please try to address the critical issues raised by the one reviewer. Please submit your revised manuscript by Sep 25 2020 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. When you're 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Jeffrey Chalmers, Ph.D. 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 2. Thank you for including your competing interests statement; "I have read the journal's policy and the authors of this manuscript have the following competing interests: Authors PK, JM, GC, and GJM are employees of Cytiva." We note that one or more of the authors are employed by a commercial company: Cytiva, Danaher Corporation, Cambridge, UK Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form. Please also include the following statement within your amended Funding Statement. “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement. 2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Within your Competing Interests Statement, please confirm that this commercial affiliation 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 this adherence statement is not accurate and 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 both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [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: No Reviewer #2: Yes ******** 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: In this study cryopreserved cell viability after dry and wet thawing is compared. No differences between both methods are observed in a comparison of obtained viabilities by t-test/Wilcoxon SR test. Furthermore, no correlation between viabilities and storage time, age, or sex is observed. The authors interpret these observations as being supportive for the use of dry thawing. General: The choice of statistical analysis in this study is not convincing. Instead of a t-test or Wilcoxon SR test, other methods (e.g., linear regression, Bland-Altman analysis) should have been applied. CD34+ viability ranged between ~10% and ~90% for both methods with a quite homogenous distribution and a possible range of 0-100%. It is obvious that P would not be <.05. The current way of presentation does not allow pairwise comparison of dry and wet thaw results on an individual basis. The attentive reader, however, can see in Fig. 2,3 that viability differs by up to 20-30 percent points in some cases (e.g., the ~64-year-old individual whose cells were stored for ~3 years). This suggests poor agreement between both methods of thawing, which in turn would impair assessment of the subgroup analyses. Abstract: The abstract should adhere to the conventional objectives-methods-results-conclusions structure in compliance with the journal author guidelines. Currently, it does not include information on background/objectives. Introduction: - I understand that all analyzed samples were cryopreserved PBMCs from stimulated donors (although this is never explicitly stated). Therefore, the introduction should be shortened, and it should focus on these products. While being the hot topic of the time, CAR-T cells have not much to do with this study. - In the last paragraph the authors state that the studied samples were from “donors treated for myeloma and now in remission”, and that 2x10^6 viable cells/kg BW already had been used for therapy prior to the study. In case of the products with ~10% CD34+ viability there must have been a unplausibly high number of cryobags available to achieve this therapeutic dose. I am assuming that the viability of the cells used for infusion was as low as the viability observed in this study, as the authors state that cryogenic storage time does not affect post-thaw viability. Materials and Methods: - It should be explicitly stated what products are studied, e.g., PBMCs from autologous donors after stimulation (GCSF only? GCSF plus plerixafor?). It should be explicitly stated, if the cryobags used for comparison were identical pairs (Same total volume? Same content?) - The dry thawing device should be described in more detail, as the reader might not be familiar with it. Results: - Viability is unusually low in a quite high number of products. Pre-freeze viability usually is near 100%. In the literature recovery rates of viable CD34+ cells are usually described to lay between 80% and 90%. In real life recovery rates might be as low as 50-60% in some cases. But viabilities of 10-20% implicate recovery rates of <50%, and it is not clear to me, how one could successfully collect enough CD34+ cells for therapeutic use with such low viability. - In the last paragraph the authors state that trypan blue viability was higher than CD34+ and CD45+ viability, but no statistical comparison is provided. Discussion: - First paragraph: The absence of a significant difference in this case does not in turn implicate that the compared methods are equivalent (see general comment). - Second paragraph discusses importance of thaw time without much concrete reference to the performed analyses and observed results. - Third paragraph: See above (second comment regarding Results section). - Fourth paragraph: Again, too much general information. - Sixth paragraph: “Red blood cell transplant effectiveness” and ref. 43 refer to common allogenic RBC transfusions (without cryopreservation). These cannot be compared with autologous PBMC infusions. Author contributions: - The comment used for internal communication between the authors should be removed from the word document before submission. Reviewer #2: This study compared two methods for thawing hematopoietic stem cell grafts cryopreserved using DMSO, thawing using the traditional water bath and thawing using an automated water-free thawing device. The study also evaluated the effect of storage duration, patient age and patient gender on the post-thaw recovery of leukocytes and CD34+ cells. While all of the data is important to laboratories involved with processing hematopoietic stem cells, the most relevant data involves the finding that automated water-free thawing device yielded similar results as a water bath. These finding are of particular importance to laboratories thawing genetically engineered T-cells such as chimeric antigen receptor (CAR) T-cells. The processing of CAR T-cells is, in general, expected to meet strict Good Manufacturing Practice requirements. Thawing cells using the automated water-free thawing device prevents variations in the thawing process due to technique differences among lab staff which allows for a more consistent thawing process. In addition, maintaining a water bath in a processing laboratory presents a risk for microbial contamination of the laboratory and the product. The study did not directly evaluate thawing of genetically engineered T cells, but the results of thawing with the automated water-free thaw device would likely be similar. While the manuscript mentions the advantages of the automated water-free thaw device in the introduction section, it may be worthwhile to include some mention of these advantages in the discussion section. ****** 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.0240310.r001
Revision 1
4 Sep 2020 Author Response Dear Editor, Please note that these have also been uploaded as a word document. Thank you for giving us the opportunity to revise our work for PLOS. We’d also like to thank the reviewers for their helpful comments, and we hope that you find the revised text suitable for publication. In the below document, we first answer the journal requests regarding competing interests and the financial statement, before going on to answer the reviewer comments. We have kept the reviewer comments in black, with our responses in blue. Best Wishes, Peter and the authors, 1. We have prepared a Financial Statement as requested: The funder Cytiva provided support in the form of salaries only for authors PK, GC, JM, and GJM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. And an updated Competing Interests Statement as requested: Authors PK, JM, GC, and GJM are employees of Cytiva, which provided salaries for these authors. No consultancy, patents, products in development, or marketed products were derived from this study. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Responses to reviewers: Reviewer #1: In this study cryopreserved cell viability after dry and wet thawing is compared. No differences between both methods are observed in a comparison of obtained viabilities by t-test/Wilcoxon SR test. Furthermore, no correlation between viabilities and storage time, age, or sex is observed. The authors interpret these observations as being supportive for the use of dry thawing. General: The choice of statistical analysis in this study is not convincing. Instead of a t-test or Wilcoxon SR test, other methods (e.g., linear regression, Bland-Altman analysis) should have been applied. CD34+ viability ranged between ~10% and ~90% for both methods with a quite homogenous distribution and a possible range of 0-100%. It is obvious that P would not be <.05. The current way of presentation does not allow pairwise comparison of dry and wet thaw results on an individual basis. The attentive reader, however, can see in Fig. 2,3 that viability differs by up to 20-30 percent points in some cases (e.g., the ~64-year-old individual whose cells were stored for ~3 years). This suggests poor agreement between both methods of thawing, which in turn would impair assessment of the subgroup analyses. We agree that for these age and storage time tests a linear regression is appropriate for population-wide tests i.e. for when we plot say storage time or patient age against post-thaw viability. We have in fact done this for these datasets shown in figures 2 and 3 (now numbered figures 3 and 4 in the revised manuscript), using a Pearson correlation coefficient to a linear regression model. We have changed the text to be more explicit about the method used here (p. 8, l. 216-217 in the revised manuscript with track changes). We agree that Bland-Altman analyses, as suggested by the reviewer, on CD45+ and CD34+ post-thaw cell viabilities relative to wet and dry thawing is appropriate as the differences between both methods for each cellular parameter were normally distributed (test performed: Shapiro-Wilk normality test, p-values = 0.3 and 0.2, respectively). We have carried out these analyses using Bland-Altman as suggested by the reviewer, and the plot of both thawing methods with line of equality for CD45+ and CD34+ post-thaw cell viabilities are shown in the updated Figure 1. Overall, agreement between both thawing methods is considered substantial for CD45+ and moderate for CD34+ (Altman, 1991; McBride, 2005), which was confirmed by concordance correlation coefficients (Lin et al. 1989), with values of 0.95 and 0.80, respectively. We have updated the methods (p. 8, l. 210-214), figure numbering (throughout the results and discussion sections), and results section accordingly (p. 9, l. 234-238). Abstract: The abstract should adhere to the conventional objectives-methods-results-conclusions structure in compliance with the journal author guidelines. Currently, it does not include information on background/objectives. We agree and have added some new text to the start of the abstract to better put the work and aims in context (p. 2, l. 28-34). Introduction: - I understand that all analyzed samples were cryopreserved PBMCs from stimulated donors (although this is never explicitly stated). Therefore, the introduction should be shortened, and it should focus on these products. While being the hot topic of the time, CAR-T cells have not much to do with this study. We’ve updated the methods section to make the mobilization of the cells, and this general process, more detailed and reproducible (also based on another comment below; p. 5, l. 125-128). We have removed some extra text in the introduction where possible (p. 3, l. 72-76). We have also removed some references to T cells too (p. 3, l. 61-65) to streamline the introduction, although we think it is relevant to keep some mention of the CAR-T area. This is because CAR-T is perhaps the fastest growing area of research in apheresis samples, and so we think that there is relevance and usefulness from this work to researchers in that area. Specifically, in CAR-T processing the initial step usually involves taking an apheresis sample from a patient, cryopreserving and shipping it, before thawing at a manufacturing site, a process for which this work is directly relevant (except perhaps the extended storage times part). While this paper focuses on apheresis samples which were taken for use in myeloma samples, this doesn’t preclude the relevance for the data for apheresis samples used in other areas. - In the last paragraph the authors state that the studied samples were from “donors treated for myeloma and now in remission”, and that 2x10^6 viable cells/kg BW already had been used for therapy prior to the study. In case of the products with ~10% CD34+ viability there must have been a unplausibly high number of cryobags available to achieve this therapeutic dose. I am assuming that the viability of the cells used for infusion was as low as the viability observed in this study, as the authors state that cryogenic storage time does not affect post-thaw viability. We have rephrased this statement in the text as it was unacceptably ambiguous (p. 5, l. 112-113). What we intended to state in this paragraph was that the myeloma samples had been taken from patients who in the past had been successfully treated and so were in remission at point of apheresis. The 2x10^6 viable cells/kg is actually the pre-freeze value target (this was reworded in p. 5, l. 115-117). In practice we thaw and transfuse cells immediately, as the cells start to die due to the DMSO toxicity as soon as they are warmed so we cannot wait for flow work. In the case of very low viabilities (trypan blue as flow takes too long), we advise the clinical who can decide the best course of action (such as give a larger number of cryobags for example). The work shows that cryogenic storage duration does not significantly affect post-thaw viability, however the freeze-thaw process itself does, to an extent that is minimal to considerable (with for instance CD34+ cell viability ranging from 8% to 87%), due to patient sample variability at apheresis. Materials and Methods: - It should be explicitly stated what products are studied, e.g., PBMCs from autologous donors after stimulation (GCSF only? GCSF plus plerixafor?). It should be explicitly stated, if the cryobags used for comparison were identical pairs (Same total volume? Same content?) - The dry thawing device should be described in more detail, as the reader might not be familiar with it. We have edited the methods section to give more detail about the mobilization process of the patients (p. 5, l. 125-128), as well as making clear that all bags were identical (p. 6, l. 143-144). We have added another reference about dry thawing devices and explained further how they work in the introduction (p.4, l. 96-98 and 103-104) and in the methods (p.6, l. 162-163). One thing we want to avoid in this paper is making it too specifically focused on a single thawing device – the main outcome of this part of the work is that water-free thawing has the same outcome as a water bath, even though the thaw takes a little longer (which would be true for any dry thawing system as thermal transfer relies on conduction rather than on convection) – we believe these results aren’t limited to the system tested here so don’t want to limit the readers’ interest. The details of the system are mentioned in the text, so a reader should find out more detailed specifics if required. Results: - Viability is unusually low in a quite high number of products. Pre-freeze viability usually is near 100%. In the literature recovery rates of viable CD34+ cells are usually described to lay between 80% and 90%. In real life recovery rates might be as low as 50-60% in some cases. But viabilities of 10-20% implicate recovery rates of <50%, and it is not clear to me, how one could successfully collect enough CD34+ cells for therapeutic use with such low viability. These are taken from a patient in the expectation of having enough cells, with a target of 2X10^6 cells/kg body weight minimum taken from each patient. However with some patients, either because they’re very ill, have a poor response to mobilization, or have a compromised immune system etc. have cells which freeze poorly (the exact mechanism by which one patient has ‘better’ cells in terms of freezing related to another is poorly understood), but we’ve highlighted this variation in results (p. 9, l. 239-240) and in the first section of the discussion (p. 13, l. 337-341). We didn’t want to remove these very poor results as this might artificially boost the suggested numbers, however we agree that these patients would have a low chance of engraftment – in practice we usually advise the clinician in the cases of very low viability, however infusing lower than 2x10^6 cell/kg doesn’t necessarily mean there won’t be engraftment. - In the last paragraph the authors state that trypan blue viability was higher than CD34+ and CD45+ viability, but no statistical comparison is provided. We have moved this text to the first section of the results where we think this sits better, and also added in the statistical comparison (p. 9, l. 239-240). Discussion: - First paragraph: The absence of a significant difference in this case does not in turn implicate that the compared methods are equivalent (see general comment). See response to first comment. - Second paragraph discusses importance of thaw time without much concrete reference to the performed analyses and observed results. We have now added the thawing data to the results section to make the discussion better placed (p. 9, l. 240-241). - Third paragraph: See above (second comment regarding Results section). Answer same as previous. - Fourth paragraph: Again, too much general information. We have removed the general information to shorten the paragraph (p. 13, l. 330-335). - Sixth paragraph: “Red blood cell transplant effectiveness” and ref. 43 refer to common allogenic RBC transfusions (without cryopreservation). These cannot be compared with autologous PBMC infusions. This reference has been removed, and the final paragraph of this section re-written (p. 14, l. 363-371). Author contributions: - The comment used for internal communication between the authors should be removed from the word document before submission. This has been removed. Reviewer #2: This study compared two methods for thawing hematopoietic stem cell grafts cryopreserved using DMSO, thawing using the traditional water bath and thawing using an automated water-free thawing device. The study also evaluated the effect of storage duration, patient age and patient gender on the post-thaw recovery of leukocytes and CD34+ cells. While all of the data is important to laboratories involved with processing hematopoietic stem cells, the most relevant data involves the finding that automated water-free thawing device yielded similar results as a water bath. These finding are of particular importance to laboratories thawing genetically engineered T-cells such as chimeric antigen receptor (CAR) T-cells. The processing of CAR T-cells is, in general, expected to meet strict Good Manufacturing Practice requirements. Thawing cells using the automated water-free thawing device prevents variations in the thawing process due to technique differences among lab staff which allows for a more consistent thawing process. In addition, maintaining a water bath in a processing laboratory presents a risk for microbial contamination of the laboratory and the product. The study did not directly evaluate thawing of genetically engineered T cells, but the results of thawing with the automated water-free thaw device would likely be similar. While the manuscript mentions the advantages of the automated water-free thaw device in the introduction section, it may be worthwhile to include some mention of these advantages in the discussion section. We have added some more detail in the discussion (p. 12, l. 313-318). To avoid this being a repeat of the introduction, we have mentioned some of the GMP practical considerations related to dry thawing, which is likely useful to many clinical readers. Attachments Attachment Submitted filename: 2020-08-28 Reviewer Comments Response Final.docx https://doi.org/10.1371/journal.pone.0240310.r002
24 Sep 2020 Decision Letter - Jeffrey Chalmers, Editor Automated dry thawing of cryopreserved haematopoietic cells is not adversely influenced by cryostorage time, patient age or gender PONE-D-20-17327R1 Dear Dr. Kilbride, 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, 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 #2: (No Response) ******** 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: I Don't Know ****** 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 #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 #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 #2: The authors have addressed all of the comments made by this reviewer. I have no further concerns about the manuscript. ****** 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 #2: No https://doi.org/10.1371/journal.pone.0240310.r003
Formally Accepted
15 Oct 2020 Acceptance Letter - Jeffrey Chalmers, Editor PONE-D-20-17327R1 Automated dry thawing of cryopreserved haematopoietic cells is not adversely influenced by cryostorage time, patient age or gender Dear Dr. Kilbride: 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. Jeffrey Chalmers Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0240310.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .