PONE-D-21-00625

Intensity of de novo DSA detected by Immucor Lifecodes Assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation.

PLOS ONE

Dear Dr. Bertrand,

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.

The study examined the role of de novo subclinical DSA and their potential C3-fxing antibody. Based on over 100 patients, the authors conclude that no correlation was observed between subclinical antibody mediated rejection (sAMR) and the detection of C3-fixing antibody. The reviewer # 1 requested that the authors make mor conclusive statement based on their analysis. Indeed, the statement that more study should be performed is not satisfactory as almost every study needs a continuation. The Reviewer # 2 indicated at several editorial changes which need to be addressed. 

The authors need to address points raised by the reviewers 1 and 2:

Reviewer # 1:

Bertrand and colleagues conducted a retrospective, multicenter, study to test potential correlation between the C3D-fixing antibody and IgG SAB by Imucor in a cohort of patients previously tested by the IgG SAB assay by One Lambda (MFI > 1000).

The authors confirmed previous report showing some differences between data obtained by the two vendors (Immucor and One Lambda), looking either at immunedominant DSA or sum of DSAs. They further attempted comparison between histological diagnosis on the biopsy and antibody characteristics and show no correlation

Biopsies were obtained with significant time difference from sera test (within 6 months for some samples) and the purpose for obtaining them is not clear – does not look like the same time point from transplant for all patients – the only thing in common is that they did not correlate with graft dysfunction.

Overall, this is a relatively small study cohort, utilizing patients from 9 different centers, with no systematic approach to obtaining either biopsies or serum samples. With that – it is not clear what the clinical value of their finding is. 

Reviewer # 2:

Review Comments to the Authors Manuscript title: Intensity of de novo DSA detected by Immucor Lifecodes Assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation. 

The rationale and objective of this study is clear. Moreover, authors successfully describe the contribution of this work to the field, while recognizing its limitations and noting outstanding questions. However, the manuscript requires much improvement to the writing quality and data presentation and interpretation. The manuscript lacks organization and focus due to run-on and awkwardly structured sentences. In addition, figures are missing crucial details required to interpret the data. 

Several results are stated without a clear connection to the supporting data. Authors should address the following points: 

Readers could benefit from a more detailed description of HLA donor specific antibodies and de novo DSA in the introduction, and how this is relevant to transplant pathology. Please explain rationale for accepting positive results for the IM Lifecodes LSAB after 2 of 3 criteria were met, instead of all requiring all criteria to be met. Please indicate negative control beads for the IM Lifecodes LSAB. 

Authors should specify concentration of beads, phycoerythrin conjugated goat antihuman IgG, anti-human C3d, instead of, or in addition to the volume used, as volume is arbitrary without knowing this information. 

Please describe methods for light microscopy and immunofluorescence staining of kidney tissue. In statistical analysis, authors should indicate a Student t-test was used. Please describe the advantages/disadvantages of the iDSA and DSA parameters. For example, why were both used, and is one superior to the other? 

Please indicate all abbreviations used in Table 1. Authors should consider reformatting the methods and results sections from bullet points to paragraphs. For reader’s ease, authors should consider labeling Figure 1 panels A-C to indicate the correlation described (as indicated in the Figure legend). For Figure 2, please define the descriptive statistics reflected in the box and whisker plot. Please label y-axes in Figures 2, 3, 4, 5, and 6. Please specific what the numbers refer to on the top of each bar in Figure 3. In addition, please explain in more detail the significance of Figure 3. Figure 4 is not clearly described and thus, difficult to interpret. To address this, please indicate the abbreviations used (g+cpt, i+t, ci+ct, cv, and cg) in the figure legend. Please define the numbers indicated on top of the bar graphs in the figure legend. 

Authors should indicate the Figure showing the data supporting the following results: “The proportions of patients with positive C3d-fixing DSA were not statistically different in patients with active sABMR, chronic active sABMR or without sABMR (56.7% vs 47.1 % vs 57.1%, p=NS).” 

Please indicate the figure showing the data supporting the following results that begin with the sentence: “The intensity (BCM) of iDSA and sDSA were statistically higher in the C3dfixing DSA group…” 

Please be consistent with reporting non-significant results as either “NS” (without a p-value) or reporting a p-value greater than 0.05. Preference is to report p-values. Authors should also consider reporting actual p-values for significant data (instead of indicating p 

The discussion should be condensed to be more concise and direct because it lacks clear focus. The discussion should also better delineate between the findings from the group’s previous work and the findings form the current study. Finally, authors should be cautious about describing data as “perfectly comparable” to previous studies. 

“Finally” is spelled incorrectly in the discussion.

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We look forward to receiving your revised manuscript.

Kind regards,

Stanislaw Stepkowski

Academic Editor

PLOS ONE

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6.Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found some minor occurrences of overlapping text with the following previous publication(s), some of which you are an author, which needs to be addressed:

- https://journals.lww.com/transplantjournal/Abstract/2021/03000/Complex_Linkage_Disequilibrium_Effects_in_HLA_DPB1.27.aspx?context=FeaturedArticles&collectionId=23

- https://onlinelibrary.wiley.com/doi/full/10.1111/ajt.15414

- https://www.nejm.org/doi/10.1056/NEJMoa1302506

- https://journals.lww.com/transplantjournal/Fulltext/2019/03000/Comparison_of_Two_Luminex_Single_antigen_Bead_Flow.28.aspx

We would like to make you aware that copying extracts from previous publications word-for-word, especially outside the methods section, is unacceptable. In addition, the reproduction of text from published reports has implications for the copyright that may apply to the publications.

Please revise the manuscript to quote or rephrase the duplicated text and cite your sources for text outside the methods section. Please note that further consideration is dependent on the submission of a manuscript that addresses these concerns about the overlap in text with published work.

Additional Editor Comments:

The study examined the role of de novo subclinical DSA and their potential C3-fxing antibody. Based on over 100 patients, the authors conclude that no correlation was observed between subclinical antibody mediated rejection (sAMR) and the detection of C3-fixing antibody. The reviewer # 1 requested that the authors make mor conclusive statement based on their analysis. Indeed, the statement that more study should be performed is not satisfactory as almost every study needs a continuation. The Reviewer # 2 indicated at several editorial changes which need to be addressed.

The authors need to address points raised by the reviewers 1 and 2:

Reviewer # 1:

Bertrand and colleagues conducted a retrospective, multicenter, study to test potential correlation between the C3D-fixing antibody and IgG SAB by Imucor in a cohort of patients previously tested by the IgG SAB assay by One Lambda (MFI > 1000).

The authors confirmed previous report showing some differences between data obtained by the two vendors (Immucor and One Lambda), looking either at immunedominant DSA or sum of DSAs. They further attempted comparison between histological diagnosis on the biopsy and antibody characteristics and show no correlation

Biopsies were obtained with significant time difference from sera test (within 6 months for some samples) and the purpose for obtaining them is not clear – does not look like the same time point from transplant for all patients – the only thing in common is that they did not correlate with graft dysfunction.

Overall, this is a relatively small study cohort, utilizing patients from 9 different centers, with no systematic approach to obtaining either biopsies or serum samples. With that – it is not clear what the clinical value of their finding is.

Reviewer # 2:

Review Comments to the Authors Manuscript title: Intensity of de novo DSA detected by Immucor Lifecodes Assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation.

The rationale and objective of this study is clear. Moreover, authors successfully describe the contribution of this work to the field, while recognizing its limitations and noting outstanding questions. However, the manuscript requires much improvement to the writing quality and data presentation and interpretation. The manuscript lacks organization and focus due to run-on and awkwardly structured sentences. In addition, figures are missing crucial details required to interpret the data.

Several results are stated without a clear connection to the supporting data. Authors should address the following points:

Readers could benefit from a more detailed description of HLA donor specific antibodies and de novo DSA in the introduction, and how this is relevant to transplant pathology. Please explain rationale for accepting positive results for the IM Lifecodes LSAB after 2 of 3 criteria were met, instead of all requiring all criteria to be met. Please indicate negative control beads for the IM Lifecodes LSAB.

Authors should specify concentration of beads, phycoerythrin conjugated goat antihuman IgG, anti-human C3d, instead of, or in addition to the volume used, as volume is arbitrary without knowing this information.

Please describe methods for light microscopy and immunofluorescence staining of kidney tissue. In statistical analysis, authors should indicate a Student t-test was used. Please describe the advantages/disadvantages of the iDSA and DSA parameters. For example, why were both used, and is one superior to the other?

Please indicate all abbreviations used in Table 1. Authors should consider reformatting the methods and results sections from bullet points to paragraphs. For reader’s ease, authors should consider labeling Figure 1 panels A-C to indicate the correlation described (as indicated in the Figure legend). For Figure 2, please define the descriptive statistics reflected in the box and whisker plot. Please label y-axes in Figures 2, 3, 4, 5, and 6. Please specific what the numbers refer to on the top of each bar in Figure 3. In addition, please explain in more detail the significance of Figure 3. Figure 4 is not clearly described and thus, difficult to interpret. To address this, please indicate the abbreviations used (g+cpt, i+t, ci+ct, cv, and cg) in the figure legend. Please define the numbers indicated on top of the bar graphs in the figure legend.

Authors should indicate the Figure showing the data supporting the following results: “The proportions of patients with positive C3d-fixing DSA were not statistically different in patients with active sABMR, chronic active sABMR or without sABMR (56.7% vs 47.1 % vs 57.1%, p=NS).”

Please indicate the figure showing the data supporting the following results that begin with the sentence: “The intensity (BCM) of iDSA and sDSA were statistically higher in the C3dfixing DSA group…”

Please be consistent with reporting non-significant results as either “NS” (without a p-value) or reporting a p-value greater than 0.05. Preference is to report p-values. Authors should also consider reporting actual p-values for significant data (instead of indicating p

The discussion should be condensed to be more concise and direct because it lacks clear focus. The discussion should also better delineate between the findings from the group’s previous work and the findings form the current study. Finally, authors should be cautious about describing data as “perfectly comparable” to previous studies.

“Finally” is spelled incorrectly in the discussion.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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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: No

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: The rationale and objective of this study is clear. Moreover, authors successfully describe the contribution of this work to the field, while recognizing its limitations and noting outstanding questions. However, the manuscript requires much improvement to the writing quality and data presentation and interpretation. The manuscript lacks organization and focus due to run-on and awkwardly structured sentences. In addition, figures are missing crucial details required to interpret the data. Several results are stated without a clear connection to the supporting data.

Please see attached document for remaining comments.

Reviewer #2: Bertrand and colleagues conducted a retrospective, multicenter, study to test potential correlation between the C3D-fixing antibody and IgG SAB by Imucor in a cohort of patients previously tested by the IgG SAB assay by One Lambda (MFI > 1000).

The authors confirmed previous report showing some differences between data obtained by the two vendors (Immucor and One Lambda), looking either at immunedominant DSA or sum of DSAs. They further attempted comparison between histological diagnosis on the biopsy and antibody characteristics and show no correlation

Biopsies were obtained with significant time difference from sera test (within 6 months for some samples) and the purpose for obtaining them is not clear – does not look like the same time point from transplant for all patients – the only thing in common is that they did not correlate with graft dysfunction.

Overall, this is a relatively small study cohort, utilizing patients from 9 different centers, with no systematic approach to obtaining either biopsies or serum samples. With that – it is not clear what the clinical value of their finding is.

**********

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Review Comments to the Author_Intensity of de novo DSA detected by Immucor Lifecodes Assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation.pdf

Intensity of de novo DSA detected by Immucor Lifecodes Assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation.

PONE-D-21-00625R1

Dear Dr. Bertrand,

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.

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Stanislaw Stepkowski

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: