-->PONE-D-25-68655-->-->Association between results of component-resolved diagnostics and basophil activation in Hymenoptera venom allergy: a registry-based cross-sectional study in adults.-->-->PLOS One

Dear Dr. Piwowarek,

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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PLOS One

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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?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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-->5. Review Comments to the Author

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Reviewer #1: The manuscript presents a registry-based cross-sectional study on Hymenoptera venom allergy (HVA) diagnosis in 154 adults from Poland (pp. 1, 8). The authors investigated the association between component-resolved diagnostics (CRD) and the basophil activation test (BAT) to improve the identification of the culprit venom and qualification for specific immunotherapy (SCIT). Key findings indicate a moderate correlation between BAT results and sIgE to major components Api m 1 (honeybee) and Ves v 5 (wasp) (pp. 1, 9, 20). The study concludes that combining CRD and BAT enhances diagnostic accuracy, particularly in complex cases like polysensitization (pp. 9, 26). Notably, the severity of a patient's historical sting reaction did not correlate with either of the measured laboratory parameters. The manuscript is well-structured, the methodology is sound, and the results are clearly presented. I have no comments.

Reviewer #2: The author has carried out a well-designed, registry-based, cross-sectional study evaluating the association between component-resolved diagnostics (CRD) and basophil activation testing (BAT) in adults with Hymenoptera venom allergy. It is clinically relevant and addresses a clear diagnostic challenge in allergy and immunology. The use of clearly defined patient groups, and integration of CRD with functional testing are notable strengths of this study. This study addresses an important diagnostic issue in venom allergy and presents clinically meaningful findings, but the author needs to address some minor clarifications in methodology, and interpretation of results.

1.Lines 45–46, 317–320: Statements that CRD and BAT ‘enhance accuracy’ or ‘enhance sensitivity and specificity’ imply improved diagnostic performance. Given the retrospective, cross-sectional design of the study, I recommend rephrasing these statements to emphasize association or supportive clinical use rather than diagnostic enhancement.

2.Lines 68–72, 79–82: The discussion of limitations of conventional diagnostics and the potential benefits of CRD and BAT overlaps conceptually and leaves some speculations regarding improved subcutaneous immunotherapy qualification. I suggest condensing these sections and clarifying that the statements reflect hypotheses or emerging evidence rather than established clinical practice.

3.Lines 104–107: Patients in groups B and C were subdivided by reaction severity without a clear explanation of the rationale. I recommend to briefly clarify how this stratification aligns with the study objectives.

4.Lines 120–127, 321–326: BAT was conducted using a single venom concentration without dose–response testing. I recommend acknowledging this limitation explicitly in the limitations section and discussing its potential impact on interpretation of basophil sensitivity.

5.Lines 150–151: No correction for multiple testing was applied despite numerous correlation analyses. I recommend providing a clearer justification for this decision, even within an exploratory framework.

6.Lines 169–172: Missing data are reported, but their potential impact on bias or statistical power is not discussed. I suggest briefly stating whether the missing data was assumed to be at random.

Reviewer #3: This registry-based cross-sectional study analyzes the utility of the Basophil Activation Test (BAT) and Component-Resolved Diagnostics (CRD) in 154 patients evaluated for Hymenoptera venom allergy. The topic is clinically relevant, particularly regarding the specific sensitization profiles in Central Europe. (In addition, when considering the therapeutic potential of venom extracts, it can also give an insight in clinical context.) The manuscript presents interesting data regarding the correlation between specific IgE (sIgE) components and BAT reactivity.

However, the manuscript requires revision before it can be accepted. There are gaps in the reporting of methodology (specifically the BAT protocol and patient grouping criteria), discrepancies in the results (missing data in tables, conflicting statistical significance), and issues with data availability statements. Furthermore, the figures and tables require reorganization to improve readability and standard scientific formatting.

Below are my specific comments arranged by section.

#Major Revisions

1. There is a contradiction regarding data access. The metadata states "No - some restrictions will apply", yet the text under Data Availability states "All relevant data have been included within the paper and its Supporting Information files". If the data is fully available in S1 Table, the metadata should be updated to "Yes." If restrictions apply, the text must explain them.

2. The Methods section states that participants were divided into three groups (A, B, C) "according to the final diagnosis and qualification for treatment". However, the specific clinical criteria used to make these assignments are missing.

- The definition for Group A is currently located in the Introduction ("absence of confirmed venom allergy") rather than the Methods. Please provide the definition or criteria of group A, B and C to the Methods. Please define what constitutes "absence of confirmed allergy" (e.g., negative skin tests? negative sIgE?) and state the criteria (e.g., skin test reaction size, sIgE thresholds) used to categorize a patient into the Bee group vs. the Wasp group, also shown in Table 6 which separates patients based on qualification for Bee vs. Wasp immunotherapy.

3. Missing Methodological Details (BAT Protocol)** The "Laboratory procedures" section can be strengthened by adding the detail required for replication.

- In line 122 and line 128, the duration of the incubation is not stated.

- In line 128, it is unclear if staining occurred simultaneously with stimulation or as a subsequent step. Please specify the time and temperature conditions for the antibody staining.

- Was a stopping solution (e.g., EDTA) used to halt the reaction?

4. Please add the row for rVes v 5 to Table 3, which is omitted despite the text identifying it as a major component with significant correlations.

5.Figure 3 identifies a patient with double-positive reactivity. Please clarify in the Methods or Results section which group (B or C) this patient was assigned to, or if they were treated as a separate entity, as this affects the specificity analysis of the respective groups.

6. The Results section states that "nonspecific reactivity assessed by the positive control did not differ significantly" among groups. However, Table 3 reports a p-value of 0.046 for "BAT control pos.", which is statistically significant (<0.05). Please clarify this discrepancy.

7. The Conclusion section need to be improved by addressing the study objectives mentioned in the Introduction section.

- The conclusion focuses on specific molecules (Api m 1, Ves v 5) but does not explicitly relate the findings back to the differentiation of the three study groups (A, B, C) as proposed in the primary objective.

- In line 87-88, "The secondary objective was to analyze the association between the investigated parameters and the severity of anaphylactic reactions in the medical history of participants." I suggest to state even the negative finding, no association, in the Conclusion.

#Minor Revisions

1.In Methods Section,

- In "Setting and study participants", please replace "included all adult patients" with the specific number (n=154) to provide immediate context on the study power.

- There is potential confusion regarding consent. It stated "individuals unable to provide informed consent were not referred", but later says consent was "waived". Please clarify that patients lacking legal capacity for _clinical_ consent were excluded, which is distinct from the _research_ consent waiver.

- Please mention the approximate volume of blood collected to indicate the sample requirement for this battery of tests.

2.In Result Section

- I suggest clarifying the sentence ""BAT results were significantly higher for the venom of culprit insect" for general readers.

3. In Discussion Section

- In Discussion section, please remove the bulleted list of limitations and rewrite this as a continuous narrative paragraph.

- In the Discussion, the statement "few previous reports have examined..." requires citations (e.g., referencing Eberlein et al. or Korošec et al.).

4.In Figures

- I suggest to merge figure 1 and 2. These figures show the same groups responding to different stimuli. It would be much clearer to merge them into a single figure with two panels ((A): Wasp stimulation, (B) Honeybee stimulation). Ensure Y-axes have identical scales and titles.

- In figure 3, please add row labels (A, B, C, D) directly to the image and update the caption to match. The current description ("Following lines from top to bottom") is difficult to follow.

- In figure 4, please add clear axis titles (e.g., "sIgE Components", "Basophil Activation") to make the heatmap self-explanatory.

5.In tables.

- The URL citation in the title of Table 1 should be converted to a formal reference. I also suggest to consider to move this table to a more proper location not to disrupts the Introduction or I'd like to additional explanation why only eight allergen picked in this study.

- I suggest to reorganize table 2 and 3 into one with patient demographics(combining age and sex) and the other with laboratory findings(Tryptase, cells, slgE, BAT).

- 'Table 4. Distribution of missing data' disrupts the flow of the results. I suggest to merge it to the new table with laboratory findings or move it to Supplementary Information.

- The readability of Table 5 can be improved. Please regroup the columns so that **rho** and **p-value** are listed side-by-side under their respective headers (BAT Wasp / BAT Honeybee).

- All abbreviations used in tables (e.g., SD, Med, NEUT) must be defined in the table footnotes.

6. Style

- Please unify the use of abbreviation throughout the manuscript. The full name (e.g. Apis mellifera for Api m) should be defined upon first use in the body text and the abbreviation should be use consistently after it is defined (e.g. sIgE)

Reviewer #4: The manuscript entitled "Association between results of component-resolved diagnostics and basophil activation in Hymenoptera venom allergy" reports on a retrospective cross-sectional study that evaluates the association between component-resolved diagnostics (CRD) and the basophil activation test (BAT) in 154 adults with Hymenoptera venom allergy. Patients were divided into three groups: not qualified for subcutaneous immunotherapy (SCIT), qualified for bee venom SCIT, and qualified for wasp venom SCIT.

The key findings include moderate correlations between BAT results and sIgE levels to Api m 1 and Ves v 5, no association between test parameters (BAT, sIgE) and the severity of prior sting reactions (Müller grades), and that CRD and BAT may provide complementary diagnostic information, particularly in cases of polysensitization or ambiguous conventional test results.

The study is well-designed, with appropriate statistical methods and a balanced discussion of limitations. However, minor revisions are needed to clarify the study rationale and clinical implications, ensuring accessibility for PLOS ONE's broad readership.

Minor Revisions Required

1.Abstract Issue: The problem statement is underdeveloped. The rationale for comparing CRD and BAT is not explicitly stated. Please explain the interest to compare these two methods in the diagnostic and therapeutic management of HVA patients.

Refine the clinical implications: I do not think one can assert "enhances diagnostic accuracy" in the conclusion since the study objectives were not focused on diagnostic accuracy (sensitivity, specificity etc.). Instead, I would suggest replacing it with something like: CRD and BAT provide complementary information, particularly in cases of polysensitization or ambiguous conventional test results, potentially improving patient selection for SCIT.

2.Introduction Issue: Briefly explain SCIT indications before this sentence: "The combined use of BAT and CRD may potentially improve the accuracy of qualification for subcutaneous allergen immunotherapy (SCIT), etc."

The problem statement is underdeveloped. The rationale for comparing CRD and BAT is not explicitly stated. Please explain the interest to compare these two methods in the diagnostic and therapeutic management of HVA patients.

**********

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Reviewer #1: Yes: Dr Daniel Elbirt

Reviewer #2: Yes: Mary Olorunkosebi

Reviewer #3: Yes: Heejin Jo

Reviewer #4: No

**********

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-->PONE-D-25-68655R1-->-->Association between results of component-resolved diagnostics and basophil activation in Hymenoptera venom allergy: a registry-based cross-sectional study in adults.-->-->PLOS One

Dear Dr. Piwowarek,

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.

Please submit your revised manuscript by Apr 26 2026 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 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'.
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-->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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Petr Heneberg

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

In my view, the manuscript still attributes more certainty to the results than the design permits. A retrospective cross-sectional study can show that certain markers are associated with BAT results in this sample, but it cannot establish that one marker exerts the strongest impact on diagnosis or that it has a decisive biological role. Expressions such as influence, impact, and decisive role suggest causal hierarchy or clinical superiority. The data presented support a more limited statement, namely that Api m 1 and Ves v 5 were the markers most strongly correlated with BAT among the variables examined.

The way the groups are defined leaves unresolved circularity. Group A is characterized in part by negative venom-specific IgE and negative BAT, yet the manuscript does not specify in operational terms how patients were assigned to the bee and wasp immunotherapy groups. If clinical history, skin testing, extract-specific IgE, BAT, or component-resolved diagnostics contributed to assignment, and those same variables are then compared across groups as if they were independent outcomes, the observed differences may largely reproduce the initial classification logic. The manuscript should therefore provide a stepwise diagnostic algorithm showing which criteria determined group membership and which measures were reserved for comparative analysis.

The ethics wording remains difficult to reconcile. The text states that individuals unable to provide informed consent were excluded, but also states that written informed consent was waived because the study was retrospective and anonymized. As written, it is not clear whether the exclusion referred to inability to consent to routine clinical procedures, inability to consent to research participation, or something else. There is also a chronology issue that should be addressed explicitly, since the patient evaluation period ended before the stated ethics approval date. If the approval covered retrospective analysis of already collected data, the manuscript should say that directly and unambiguously.

The statistical approach is not sufficiently guarded in relation to the number of analyses performed. The manuscript labels the work exploratory, but it still interprets multiple nominal p values below 0.05 as positive findings across a broad set of tests and correlations. In that setting, isolated borderline results can arise from multiplicity alone. Several of the reported associations fall very close to the conventional threshold, which limits confidence in their stability. A stricter presentation would either apply a multiple-testing correction or state clearly that these p values are descriptive and hypothesis-generating rather than confirmatory.

Some reported summary statistics appear inconsistent enough to require a full audit of the tables. The neutrophil data are one example, where the means are presented in the thousands but the standard deviations are around 1.26 and 1.27, which suggests a unit mismatch, formatting error, or incorrect transcription. Another problematic entry is the severe bee subgroup value for rApi m 3, where the reported mean, standard deviation, and median do not appear to form a plausible combination. These inconsistencies should be checked against the raw dataset and the statistical output, with corrections made wherever necessary.

The mechanistic explanation offered for Api m 1 and Ves v 5 is not well grounded in the underlying biology. The manuscript suggests that their relatively low molecular weight may increase accessibility to FcεRI receptors, but FcεRI binds IgE already attached to the basophil surface, not the allergen itself. Basophil activation depends on allergen-mediated cross-linking of receptor-bound IgE, and that process is shaped by factors such as sensitization profile, epitope architecture, and functional binding characteristics. The proposed explanation is therefore speculative and should not be presented as if it were a supported interpretation of the current results.

The severity analysis should be stated more carefully. The absence of a statistically significant association in these subgroups does not justify a firm conclusion that severity is unrelated to the measured markers. The bee subgroup is especially limited because the mild-reaction category contains very few patients compared with the severe category, and that imbalance reduces the ability to detect anything other than large differences. The manuscript should therefore report that no association with severity was detected in this dataset, rather than presenting the result as evidence against any relationship.

In my view, the manuscript was improved when compared to its first version, but the above-mentioned issues need to be addressed in order to avoid data overinterpretation and misinterpretation.

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-->

-->PONE-D-25-68655R2-->-->Association between results of component-resolved diagnostics and basophil activation in Hymenoptera venom allergy: a registry-based cross-sectional study in adults.-->-->PLOS One

Dear Dr. Piwowarek,

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.-->-->

Please submit your revised manuscript by May 15 2026 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 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

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We look forward to receiving your revised manuscript.

Kind regards,

Petr Heneberg

Academic Editor

PLOS One

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If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

The manuscript was improved and the revisions were made in a correct direction, but several of them were incomplete. In my view, the main remaining concerns are the ones that still limit interpretation even after revision, together with practical fixes that could be implemented without additional wet lab work.

The severity analysis remains a key weakness. The authors now acknowledge that some subgroups were small, especially the honeybee mild-reaction subgroup, and that the findings therefore do not justify a firm conclusion. That is an improvement, but the analysis is still underpowered for a confident statement about severity-related associations. I would therefore encourage the authors to tone this down even further in the abstract, discussion, and conclusions, and to present the severity results explicitly as inconclusive subgroup analyses rather than negative findings. It would also strengthen the manuscript if the authors reported effect sizes and confidence intervals, not only p values, so that readers can better judge the uncertainty around these estimates.

Circularity remains a substantive methodological limitation. The authors added a clearer diagnostic algorithm and now acknowledge that some circularity cannot be excluded because component-resolved diagnostics contributed to routine clinical decision-making. This improves transparency, but it does not remove the issue. I would encourage the authors to distinguish more clearly which variables were used for clinical classification and which were treated as analytic outcomes, and then to frame the between-group comparisons as descriptive only. If possible, the authors could also add a sensitivity analysis excluding variables most directly involved in treatment assignment, or at least provide a table that identifies classification-related versus evaluation-only variables.

The statistical analysis part is still not very strong. The revision presents the findings more cautiously, but the manuscript still relies on multiple comparisons and nominal significance testing. A practical improvement, without requiring new experiments, would be to restructure the Results around effect sizes, confidence intervals, and a smaller set of prespecified primary comparisons, while moving more exploratory correlations to the Supplement. Alternatively, if the authors prefer to retain the present structure, the manuscript should state even more explicitly that these analyses are hypothesis-generating rather than confirmatory.

The retrospective single-center design remains an important limitation for generalizability. The authors now acknowledge this more clearly, which is appropriate, but the claims should remain tightly aligned with what this dataset can actually support. I would therefore suggest that the authors narrow the wording where needed and avoid implying broader diagnostic applicability beyond similar referral populations.

The absence of skin test data and the use of only a single BAT concentration still weaken the diagnostic interpretation. Since this cannot be remedied experimentally at this stage, I would encourage the authors to address it more explicitly in the manuscript by clarifying how these limitations constrain interpretation, and by avoiding any implication that BAT performance or thresholds were comprehensively characterized here. A brief supplementary flowchart or summary table showing which diagnostic modalities were available for how many patients would also improve transparency.

Missing data remain a lesser but still relevant concern. The explanation provided by the authors is reasonable, but the manuscript would be stronger if the authors added a compact supplementary table showing missingness by variable and group, together with a brief statement on whether complete-case analyses differed materially from the full analytic sample.

Some conclusions also still extend slightly beyond what the design can firmly support. I would encourage the authors to make the final take-home message as precise as possible, emphasizing association and complementary diagnostic information rather than clinical utility in a stronger sense. Statements about decision support would be better framed as requiring prospective validation.

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-->

Association between results of component-resolved diagnostics and basophil activation in Hymenoptera venom allergy: a registry-based cross-sectional study in adults.

PONE-D-25-68655R3

Dear Dr. Piwowarek,

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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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,

Petr Heneberg

Academic Editor

PLOS One

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