Reviewer’ s report
Title: Low mean fluorescence intensity donor-specific class II antibodies can also cause
complications after liver transplant
Version: 1Date: 24 May 2020
Reviewer's report:
This manuscript attempts to associate HLA donor-specific antibodies (DSA) identified in Luminex
single allele bead assays and with low mean fluorescence intensity (MFI) values are associatedwith complications post liver transplantation.A large cohort of 430 liver transplants are included, however the study is incomplete lacking alot of information required to address the issue of MFI associations with post-transplantcomplications. I have several general comments.
First, the title of the manuscript is misleading. Very little data is provided to describe DSA with
low MFI values and no information is given to describe the types of complications present inpatients with low MFI DSA. Rather, the authors emphasize the presence of HLA class II withcomplications, especially with cirrhosis post-transplant. The Conclusion of the paper does noteven mention low MFI DSAs.
ABSTRACT: At the end of the Results section of the Abstract, the authors write that "All patients
with cirrhosis of the graft showed HLA class II antibodies", but this is incorrect. As described laterin the paper, all DSA-positive patients with cirrhosis had antibody to HLA class II.
METHODS - PATIENTS: The authors state that "The screening for DSA was performed
independently at the time of LT…", which is very confusing. This same sentence is repeated laterin the Discussion, with an explanation that it is unknown if the DSAs were preformed or de novo.Hence, there is no information about DSAs at the time of transplant. I would suggest the sentencein quotations be removed from the Methods and a statement included that DSA was determinedonly post-transplantation and no information about pre-transplant DSA was available.
METHODS - ANTIBODY DETECTION: It is stated that DSA were detected with a Luminex
screening assay "… and were subsequently specified with a Luminex single antigen bead assay." It
should be stated that only sera that were positive in the screening assay were tested with the singleantigen bead assay. This is then a limitation of the study, in that some DSAs may have been missedby using only the screening assay. The timing of DSA testing is not clear- was it in associationwith liver complication? Also was any patient tested more than once?How were results handled when multiple DSAs were detected? The authors identified somepatients with both class I and class II DSA, or HLA-DR + -DQ, so were the results combined andMFI values added together? Or was only a single DSA and a single MFI value used in theanalysis? A description is needed to explain the wide ranges of MFI values in the figures.
Regarding the assignment of the DQ specific DSA , were the authors considering for the
specificity both DQA and DQB chains- were the donors typed for DQA and DQB, When multiplebeads positive for a specificity how they determine the DSA MFI?
RESULTS - BASELINE CHARACTERISITCS: The first sentence should state that the results
are "… outlined in Table 1", not "the Table". Table 1 should include definitions of all the
abbreviations.RESULTS - MFI LEVEL IS NOT AN INDICATOR OF FUTURE COMPLICATIONS: Theterms "HLA-1" and "HLA-2" should not be used to describe "class I" and "class II" antibodies. In
the second sentence, it is stated that the range of class I DSA MFI values is 530-41,300, but
Figures 3B and 3C do not show any values greater than 5000. What is the correct range?In this same section, the authors state that there is no significant difference for MFI values in DSA-positive patients with vs. without complications. No data is shown on the types of complicationsassociation with different levels of MFI values. This seems to be a very incomplete analysis ofMFI values.
RESULTS - CIRRHOSIS OF THE GRAFT .. : The third paragraph of this section describes
differences in DSA MFI values among DSA-positive patients with cirrhosis. It appears that thereis a very wide range of MFI values, but no other information is shown to explain the differences.What is the point of adding this information? Out of curiosity, were the very high MFI values dueto patients having multiple DSAs?
RESULTS - DONOR DATA: Similarly, what is the point of describing gender disparities among
the donors? This information could easily be added to Table 1.DISCUSSION: the Discussion does not address the title of the paper, that DSA with low MFIvalues cause post-liver transplant complications. The Discussion is too long and wanders,describing many studies of liver and kidney transplants that looked at HLA DSA associated with
complications. However, the authors do not adequately address how their study compares to the
others. There seems to be no purpose to most aspects of this section.There was a very wide range of post-transplant time for DSA testing (from 1 month to 29 years),and no information was provided as to any differences in results that may be associated with thosetime differences.The last paragraph of the Discussion brings up the study's monitoring of patients'immunosuppression trough levels, but no data was shown. While they may "regularly monitor"levels, were changes seen for the time that DSA were detected? Did DSA detection correlate withany changes in immunosuppression trough levels for DSA-positive patients? I'm not sure thisparagraph should be included in the paper.
CONCLUSION: As stated above, the conclusion does not address the title of the manuscript. The
last few sentences of the Conclusion pertain to immunosuppression, specific regimens and troughlevels. Neither of these aspects are addressed in this manuscript.
FIGURE 3: What do the boxes represent? This should be specified.
Comment on wording: The authors used the term "HLA subspecies" throughout the paper todifferentiate between HLA antibodies in general and those against one or a few HLA loci. This is
not an appropriate term. Depending on the context, this could be changed to terms such as "HLA
locus-specific" or "HLA class-specific" or the specific loci described.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
No
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
YesAre the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
NoAre you able to assess any statistics in the manuscript or would you recommend an
additional statistical review?If an additional statistical review is recommended, please specify what aspects require furtherassessment in your comments to the editors.
I am able to assess the statistics
Quality of written English
Please indicate the quality of language in the manuscript:
Needs some language corrections before being publishedDeclaration of competing interests
Please complete a declaration of competing interests, considering the following questions:
1. Have you in the past five years received reimbursements, fees, funding, or salary from an
organisation that may in any way gain or lose financially from the publication of thismanuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose
financially from the publication of this manuscript, either now or in the future?
3. Do you hold or are you currently applying for any patents relating to the content of the
manuscript?
4. Have you received reimbursements, fees, funding, or salary from an organization that holds
or has applied for patents relating to the content of the manuscript?
5. Do you have any other financial competing interests?6. Do you have any non-financial competing interests in relation to this paper?
If you can answer no to all of the above, write 'I declare that I have no competing interests' below.
If your reply is yes to any, please give details below.
I declare that I have no competing interest
Reviewer Publication Consent. I agree for my report to be made available under an Open Access
Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manu-script is accepted for publication. Any comments that I do not wish to be included in the publishedreport have been included as confidential comments to the editor, which will not be published.Ifyou are not happy for us to publish this report, please contact the editorial office before completingthe review.If you wish, you can include your name in your published report. Please note you mustdecide whether to include your name at the start of the process and confirm this decision wheneverprompted. If you change your decision later, we will not be able to publish your name.
I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report.
(default)
Reviewer’ s report
Title: Low mean fluorescence intensity donor-specific class II antibodies can also cause
complications after liver transplant
Version: 1Date: 24 May 2020
Reviewer's report:
This manuscript attempts to associate HLA donor-specific antibodies (DSA) identified in Luminex
single allele bead assays and with low mean fluorescence intensity (MFI) values are associatedwith complications post liver transplantation.A large cohort of 430 liver transplants are included, however the study is incomplete lacking alot of information required to address the issue of MFI associations with post-transplantcomplications. I have several general comments.
First, the title of the manuscript is misleading. Very little data is provided to describe DSA with
low MFI values and no information is given to describe the types of complications present inpatients with low MFI DSA. Rather, the authors emphasize the presence of HLA class II withcomplications, especially with cirrhosis post-transplant. The Conclusion of the paper does noteven mention low MFI DSAs.
ABSTRACT: At the end of the Results section of the Abstract, the authors write that "All patients
with cirrhosis of the graft showed HLA class II antibodies", but this is incorrect. As described laterin the paper, all DSA-positive patients with cirrhosis had antibody to HLA class II.
METHODS - PATIENTS: The authors state that "The screening for DSA was performed
independently at the time of LT…", which is very confusing. This same sentence is repeated laterin the Discussion, with an explanation that it is unknown if the DSAs were preformed or de novo.Hence, there is no information about DSAs at the time of transplant. I would suggest the sentencein quotations be removed from the Methods and a statement included that DSA was determinedonly post-transplantation and no information about pre-transplant DSA was available.
METHODS - ANTIBODY DETECTION: It is stated that DSA were detected with a Luminex
screening assay "… and were subsequently specified with a Luminex single antigen bead assay." It
should be stated that only sera that were positive in the screening assay were tested with the singleantigen bead assay. This is then a limitation of the study, in that some DSAs may have been missedby using only the screening assay. The timing of DSA testing is not clear- was it in associationwith liver complication? Also was any patient tested more than once?How were results handled when multiple DSAs were detected? The authors identified somepatients with both class I and class II DSA, or HLA-DR + -DQ, so were the results combined andMFI values added together? Or was only a single DSA and a single MFI value used in theanalysis? A description is needed to explain the wide ranges of MFI values in the figures.
Regarding the assignment of the DQ specific DSA , were the authors considering for the
specificity both DQA and DQB chains- were the donors typed for DQA and DQB, When multiplebeads positive for a specificity how they determine the DSA MFI?
RESULTS - BASELINE CHARACTERISITCS: The first sentence should state that the results
are "… outlined in Table 1", not "the Table". Table 1 should include definitions of all the
abbreviations.RESULTS - MFI LEVEL IS NOT AN INDICATOR OF FUTURE COMPLICATIONS: Theterms "HLA-1" and "HLA-2" should not be used to describe "class I" and "class II" antibodies. In
the second sentence, it is stated that the range of class I DSA MFI values is 530-41,300, but
Figures 3B and 3C do not show any values greater than 5000. What is the correct range?In this same section, the authors state that there is no significant difference for MFI values in DSA-positive patients with vs. without complications. No data is shown on the types of complicationsassociation with different levels of MFI values. This seems to be a very incomplete analysis ofMFI values.
RESULTS - CIRRHOSIS OF THE GRAFT .. : The third paragraph of this section describes
differences in DSA MFI values among DSA-positive patients with cirrhosis. It appears that thereis a very wide range of MFI values, but no other information is shown to explain the differences.What is the point of adding this information? Out of curiosity, were the very high MFI values dueto patients having multiple DSAs?
RESULTS - DONOR DATA: Similarly, what is the point of describing gender disparities among
the donors? This information could easily be added to Table 1.DISCUSSION: the Discussion does not address the title of the paper, that DSA with low MFIvalues cause post-liver transplant complications. The Discussion is too long and wanders,describing many studies of liver and kidney transplants that looked at HLA DSA associated with
complications. However, the authors do not adequately address how their study compares to the
others. There seems to be no purpose to most aspects of this section.There was a very wide range of post-transplant time for DSA testing (from 1 month to 29 years),and no information was provided as to any differences in results that may be associated with thosetime differences.The last paragraph of the Discussion brings up the study's monitoring of patients'immunosuppression trough levels, but no data was shown. While they may "regularly monitor"levels, were changes seen for the time that DSA were detected? Did DSA detection correlate withany changes in immunosuppression trough levels for DSA-positive patients? I'm not sure thisparagraph should be included in the paper.
CONCLUSION: As stated above, the conclusion does not address the title of the manuscript. The
last few sentences of the Conclusion pertain to immunosuppression, specific regimens and troughlevels. Neither of these aspects are addressed in this manuscript.
FIGURE 3: What do the boxes represent? This should be specified.
Comment on wording: The authors used the term "HLA subspecies" throughout the paper todifferentiate between HLA antibodies in general and those against one or a few HLA loci. This is
not an appropriate term. Depending on the context, this could be changed to terms such as "HLA
locus-specific" or "HLA class-specific" or the specific loci described.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
No
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
YesAre the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
NoAre you able to assess any statistics in the manuscript or would you recommend an
additional statistical review?If an additional statistical review is recommended, please specify what aspects require furtherassessment in your comments to the editors.
I am able to assess the statistics
Quality of written English
Please indicate the quality of language in the manuscript:
Needs some language corrections before being publishedDeclaration of competing interests
Please complete a declaration of competing interests, considering the following questions:
1. Have you in the past five years received reimbursements, fees, funding, or salary from an
organisation that may in any way gain or lose financially from the publication of thismanuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose
financially from the publication of this manuscript, either now or in the future?
3. Do you hold or are you currently applying for any patents relating to the content of the
manuscript?
4. Have you received reimbursements, fees, funding, or salary from an organization that holds
or has applied for patents relating to the content of the manuscript?
5. Do you have any other financial competing interests?6. Do you have any non-financial competing interests in relation to this paper?
If you can answer no to all of the above, write 'I declare that I have no competing interests' below.
If your reply is yes to any, please give details below.
I declare that I have no competing interest
Reviewer Publication Consent. I agree for my report to be made available under an Open Access
Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manu-script is accepted for publication. Any comments that I do not wish to be included in the publishedreport have been included as confidential comments to the editor, which will not be published.Ifyou are not happy for us to publish this report, please contact the editorial office before completingthe review.If you wish, you can include your name in your published report. Please note you mustdecide whether to include your name at the start of the process and confirm this decision wheneverprompted. If you change your decision later, we will not be able to publish your name.
I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report.
(default)
Please review this paper.
user
Reviewer’ s report
Title: Low mean fluorescence intensity donor-specific class II antibodies can also cause
complications after liver transplant
Version: 1Date: 24 May 2020
Reviewer's report:
This manuscript attempts to associate HLA donor-specific antibodies (DSA) identified in Luminex
single allele bead assays and with low mean fluorescence intensity (MFI) values are associatedwith complications post liver transplantation.A large cohort of 430 liver transplants are included, however the study is incomplete lacking alot of information required to address the issue of MFI associations with post-transplantcomplications. I have several general comments.
First, the title of the manuscript is misleading. Very little data is provided to describe DSA with
low MFI values and no information is given to describe the types of complications present inpatients with low MFI DSA. Rather, the authors emphasize the presence of HLA class II withcomplications, especially with cirrhosis post-transplant. The Conclusion of the paper does noteven mention low MFI DSAs.
ABSTRACT: At the end of the Results section of the Abstract, the authors write that "All patients
with cirrhosis of the graft showed HLA class II antibodies", but this is incorrect. As described laterin the paper, all DSA-positive patients with cirrhosis had antibody to HLA class II.
METHODS - PATIENTS: The authors state that "The screening for DSA was performed
independently at the time of LT…", which is very confusing. This same sentence is repeated laterin the Discussion, with an explanation that it is unknown if the DSAs were preformed or de novo.Hence, there is no information about DSAs at the time of transplant. I would suggest the sentencein quotations be removed from the Methods and a statement included that DSA was determinedonly post-transplantation and no information about pre-transplant DSA was available.
METHODS - ANTIBODY DETECTION: It is stated that DSA were detected with a Luminex
screening assay "… and were subsequently specified with a Luminex single antigen bead assay." It
should be stated that only sera that were positive in the screening assay were tested with the singleantigen bead assay. This is then a limitation of the study, in that some DSAs may have been missedby using only the screening assay. The timing of DSA testing is not clear- was it in associationwith liver complication? Also was any patient tested more than once?How were results handled when multiple DSAs were detected? The authors identified somepatients with both class I and class II DSA, or HLA-DR + -DQ, so were the results combined andMFI values added together? Or was only a single DSA and a single MFI value used in theanalysis? A description is needed to explain the wide ranges of MFI values in the figures.
Regarding the assignment of the DQ specific DSA , were the authors considering for the
specificity both DQA and DQB chains- were the donors typed for DQA and DQB, When multiplebeads positive for a specificity how they determine the DSA MFI?
RESULTS - BASELINE CHARACTERISITCS: The first sentence should state that the results
are "… outlined in Table 1", not "the Table". Table 1 should include definitions of all the
abbreviations.RESULTS - MFI LEVEL IS NOT AN INDICATOR OF FUTURE COMPLICATIONS: Theterms "HLA-1" and "HLA-2" should not be used to describe "class I" and "class II" antibodies. In
the second sentence, it is stated that the range of class I DSA MFI values is 530-41,300, but
Figures 3B and 3C do not show any values greater than 5000. What is the correct range?In this same section, the authors state that there is no significant difference for MFI values in DSA-positive patients with vs. without complications. No data is shown on the types of complicationsassociation with different levels of MFI values. This seems to be a very incomplete analysis ofMFI values.
RESULTS - CIRRHOSIS OF THE GRAFT .. : The third paragraph of this section describes
differences in DSA MFI values among DSA-positive patients with cirrhosis. It appears that thereis a very wide range of MFI values, but no other information is shown to explain the differences.What is the point of adding this information? Out of curiosity, were the very high MFI values dueto patients having multiple DSAs?
RESULTS - DONOR DATA: Similarly, what is the point of describing gender disparities among
the donors? This information could easily be added to Table 1.DISCUSSION: the Discussion does not address the title of the paper, that DSA with low MFIvalues cause post-liver transplant complications. The Discussion is too long and wanders,describing many studies of liver and kidney transplants that looked at HLA DSA associated with
complications. However, the authors do not adequately address how their study compares to the
others. There seems to be no purpose to most aspects of this section.There was a very wide range of post-transplant time for DSA testing (from 1 month to 29 years),and no information was provided as to any differences in results that may be associated with thosetime differences.The last paragraph of the Discussion brings up the study's monitoring of patients'immunosuppression trough levels, but no data was shown. While they may "regularly monitor"levels, were changes seen for the time that DSA were detected? Did DSA detection correlate withany changes in immunosuppression trough levels for DSA-positive patients? I'm not sure thisparagraph should be included in the paper.
CONCLUSION: As stated above, the conclusion does not address the title of the manuscript. The
last few sentences of the Conclusion pertain to immunosuppression, specific regimens and troughlevels. Neither of these aspects are addressed in this manuscript.
FIGURE 3: What do the boxes represent? This should be specified.
Comment on wording: The authors used the term "HLA subspecies" throughout the paper todifferentiate between HLA antibodies in general and those against one or a few HLA loci. This is
not an appropriate term. Depending on the context, this could be changed to terms such as "HLA
locus-specific" or "HLA class-specific" or the specific loci described.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
No
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
YesAre the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
NoAre you able to assess any statistics in the manuscript or would you recommend an
additional statistical review?If an additional statistical review is recommended, please specify what aspects require furtherassessment in your comments to the editors.
I am able to assess the statistics
Quality of written English
Please indicate the quality of language in the manuscript:
Needs some language corrections before being publishedDeclaration of competing interests
Please complete a declaration of competing interests, considering the following questions:
1. Have you in the past five years received reimbursements, fees, funding, or salary from an
organisation that may in any way gain or lose financially from the publication of thismanuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose
financially from the publication of this manuscript, either now or in the future?
3. Do you hold or are you currently applying for any patents relating to the content of the
manuscript?
4. Have you received reimbursements, fees, funding, or salary from an organization that holds
or has applied for patents relating to the content of the manuscript?
5. Do you have any other financial competing interests?6. Do you have any non-financial competing interests in relation to this paper?
If you can answer no to all of the above, write 'I declare that I have no competing interests' below.
If your reply is yes to any, please give details below.
I declare that I have no competing interest
Reviewer Publication Consent. I agree for my report to be made available under an Open Access
Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manu-script is accepted for publication. Any comments that I do not wish to be included in the publishedreport have been included as confidential comments to the editor, which will not be published.Ifyou are not happy for us to publish this report, please contact the editorial office before completingthe review.If you wish, you can include your name in your published report. Please note you mustdecide whether to include your name at the start of the process and confirm this decision wheneverprompted. If you change your decision later, we will not be able to publish your name.
I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report.
(default)
assistant