Dear Dr. Fucharoen,

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.

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Kind regards,

J Francis Borgio, Ph.D.,

Academic Editor

PLOS One

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“This study was financially supported by Khon Kaen University, Thailand, to SF (Contract ID: RP68-Research Center KKU), and Genomics Thailand, the Health System Research Institute (Contract ID: HSRI 68-049) to KrS.”

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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?

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: No

Reviewer #2: N/A

Reviewer #3: 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.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

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: The work presented is technically sound. However, the phenomenon of Hemoglobin variants interfering with HbA2 quantification (leading to false-positive beta-thalassemia diagnosis) is well-documented in the literatures.

Major comments:

1. It is not quite clear how 372 were selected. Were these based on availability of the samples related to problematic reports of the hemoglobin variants? Or were these randomly selected from a larger pool of samples? Please clarify. The selection criteria could impact the generalizability of the findings.

2. The manuscript as currently presented does not provide sufficient novelty to warrant publication. The interference of hemoglobin variants on HbA2 quantification has been reported in multiple prior studies. The authors should better highlight what new information this study provides that is not already known in the field.

Minor comments:

1. The title which emphasize 12-year experience may not be necessary as the data chosen for analysis did not show how the trends evolved over the years. Consider revising the title to better reflect the content of the manuscript.

2. When presenting the prevalence of different hemoglobin variants, consider including the years the samples were collected to provide context on whether certain variants were more common in specific time periods. This should help strengthen the epidemiological aspect of the study.

3. The discussion section could benefit from a more in-depth comparison with prior studies on the same topic. Highlighting similarities and differences in findings would help situate this work within the broader literature.

4. The images included in the draft manuscript at present could be improved to have a better resolution when published, e.g. Fig 1 texts within the pictures are not legible.

5. The authors mentioned that the all data have been presented in the paper. However, as the aggregate data with mean, median, sd only. Individual levels data without PII could also be beneficials for future interests such as correlating specific type of mutation with the percentage of hemogobin electrophoresis results.

Reviewer #2: The authors emphasize that in their publications, the use of two different methods to measure Hb A2 and F values in the diagnosis of hemoglobinopathies, together with molecular analysis, highlights an important distinction in the interpretation of beta thalassemia. In this sense, they provide valuable data.

In some places in the text, the spelling of “Hb A2” varies. Consistency in spelling throughout the text may be preferable.

Reviewer #3: The manuscript addresses a well-chosen topic that offers a practical solution to a genuine laboratory challenge. However, I have a concern regarding the study population. In Thailand, Hb E is the most common hemoglobin variant and is known to be associated with elevated Hb A2 levels. Given the stated objectives of the study, it would be reasonable to include Hb E in the analysis. Nevertheless, this variant appears to have been excluded, and the authors did not provide a clear rationale for its omission. I would kindly request the authors to clarify why Hb E was not considered in the present study.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Common hemoglobin variants affecting the diagnosis of β-thalassemia: a large cohort data at a single center

PONE-D-25-66507R1

Dear Dr. Fucharoen,

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.

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Kind regards,

J Francis Borgio, Ph.D.,

Academic Editor

PLOS One

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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.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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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: I have reviewed the revised manuscript (PONE-D-25-66507R1) and the authors' corresponding rebuttal letter. The authors have made sufficient efforts to address the primary concerns raised during the initial review, particularly regarding the clarification of their inclusion criteria and the commendable provision of the underlying dataset. While the manuscript remains predominantly descriptive and misses an opportunity to employ more advanced statistical modeling—which could have substantially elevated its diagnostic impact—the data presented are technically sound and offer practical value to the field of hematological screening. The current revisions meet the threshold for publication, and I therefore recommend that the manuscript be accepted in its current form.

Reviewer #2: The authors have been done all the comments from the reviewers. I think this is sufficient for publish.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #1: No

Reviewer #2: No

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