PONE-D-20-04529

Development of an assay of plasma neurofilament light chain utilizing immunomagnetic reduction technology

PLOS ONE

Dear Dr. Yang,

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Academic Editor

PLOS ONE

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"Huei-Chun Liu and Shieh-Yueh Yang are employees at MagQu Co., Ltd. Shieh-Yueh Yang is also an employee of MagQu LLC. Shieh-Yueh Yang owns stock in MagQu Co., Ltd. There are no conflicts of interest for other authors. The other authors have no competing interests."

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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

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

Reviewer #1: No

Reviewer #2: Yes

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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

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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

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This paper details the validation of a novel assay for neurofilament light (NfL), a commonly-used biomarker of neurodegeneration derived from CSF or blood. The authors do a good job of describing the background and importance of this biomarker as it relates to AD research and diagnosis. They also provide a rigorous and comprehensive description of their assay which is helpful in interpreting their results. There are several statistical issues that need to be addressed in this manuscript which are outlined below.

Statistical Methods

1. The authors give a brief description of the statistical analyses that were employed, but they did not describe the analyses used to ascertain the diagnostic accuracy of their NfL assay. They report sensitivity, specificity, and AUC values, but it is unclear what statistical tests were used to derive these values.

2. The authors also state that t-tests were used to compare continuous variables, but it is unclear what comparisons they are referring to. This statement is very vague as it is unclear if the authors are referring to paired t-tests for the reproducibility studies or whether they used a series a two-sample t-tests for the clinical group comparisons. If two-sample t-tests were used for the clinical group comparisons, this would be an incorrect approach. When comparing three or more groups, a one-way ANOVA should be used so that the groups are compared simultaneously and so that the p-values of groupwise comparisons that are statistically significant are adjusted for multiple comparisons.

3. Did the authors check to ensure that the distribution of NfL values met the normality assumption. If they are not normally distributed, then non-parametric tests should be used to test group differences.

4. Why did the authors report the diagnostic accuracy results in the Discussion section? This information should be included in the “Plasma NFL concentrations in patients with dementia” section. In addition, I emphasize the need to describe the statistical methods that were used to derive the AUC values, sensitivity, and specificity. Were these from a logistic regression model or a specific ROC function. Which of the stated statistical software packages were used for these analyses?

5. For AUC, sensitivity, and specificity, please report 95% confidence intervals with these values.

Reviewer #2: An ultrasensitive assay for measuring plasma NFL utilizing IMR technology has been developed. The NC group showed a plasma NFL level of 7.70 ± 4.00 pg/ml, which is significantly lower than that of the PD/PDD (15.85 ± 7.82 pg/ml, p < 0.001) and AD (19.24 ± 8.99 pg/ml, p < 0.001) groups . A significant difference in plasma NFL levels was determined between the PD and AD groups (p < 0.01). Through ROC curve analysis, the cut-off value of the plasma NFL concentration for differentiating NCs from dementia patients (AD and PD/PDD) was found to be 12.71 pg/ml, with a clinical sensitivity and specificity of 73.5% and 90.3% , respectively . The AUC was 0.868. Furthermore, the cut-off value of the plasma NFL concentration for discriminating AD from PD/PDD was found to be 18.02 pg/ml, with a clinical sensitivity and specificity of 61.3% and 65.4%, respectively. The AUC was 0.630. Although clear differences in plasma NFL concentrations were observed among NCs and PD and AD patients, these diagnostic cut-off values should be validated in another group of more patients in further studies.

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Reviewer #1: Yes: Michael Malek-Ahmadi

Reviewer #2: No

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Development of an assay of plasma neurofilament light chain utilizing immunomagnetic reduction technology

PONE-D-20-04529R1

Dear Dr. Yang,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Kewei Chen, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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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: Yes

**********

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

Reviewer #1: Yes

**********

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: Yes

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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: 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 #1: The authors have addressed all of my comments sufficiently and no further revisions are required for this manuscript.

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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: Yes: Michael Malek-Ahmadi