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Thank you for your thorough and thoughtful revision. Both reviewers have evaluated the updated manuscript and confirmed that all previously raised concerns have been fully addressed. The scientific rationale, methodological clarifications, statistical transparency, and overall presentation have significantly improved, and the revised version now reads as a solid and clinically relevant validation study.
I have also reviewed the revision myself, and I am satisfied that the manuscript meets PeerJ’s criteria for clarity, methodological rigor, and transparency.
Your manuscript is now ready for acceptance.
Thank you for your careful work throughout the revision process.
[# PeerJ Staff Note - this decision was reviewed and approved by Vladimir Uversky, a PeerJ Section Editor covering this Section #]
The revised manuscript is written in fluent, professional language and is easy to follow. The abstract accurately reflects the content of the study, and the structure of the paper is consistent with the format. The introduction now provides sufficient background and appropriately situates the study within the existing body of work on high-fluorescent cells and CEA in malignant effusions. The authors have expanded the literature review to include key studies, such as those by Wu et al., Favrese et al., and Cho et al., which helps the readers undertand the context and relevance of this large validation cohort. The terminology has been corrected throughout, and the previously overstated claim of HFC as a "novel biomarker" has been replaced by the more accurate term "rapid screening tool"
The figures and tables are clear and well presented, and the removal of the three-variable model has improved readability. The statistical results are reported in detail with confidence intervals and p-values.
The expanded figure legends are now informative and properly describe the used data.
The study design is appropriate and clinically meaningful. The inclusion of 978 effusion samples provides excellent statistical power and enhances generalizability across effusion types. The authors have clarified essential methodological points that were previously missing, including the blinding of cytology findings to HFC data, explicit exclusion criteria for hemolyzed or insufficient samples, adherence to ISO calibration standards for the Sysmex analyzer, and consistent handling of samples within two hours of collection. These clarifications strengthen the reproducibility and transparency of the work.
The use of logistic regression and ROC analysis is correct and well justified. The results are internally consistent, and the choice of cutoff values appears logical. The paper now describes a reproducible and clinically applicable approach that can easily be adopted in similar laboratory settings.
The data are statistically clear and well interpreted. The revised manuscript emphasizes that the best diagnostic accuracy is achieved when HFC values are combined with CEA levels, rather than using HFC alone. This correction aligns with the conclusions of evidence-based medicine. The authors have also added a thoughtful discussion on false positive and false negative results, noting that reactive mesothelial cells and macrophages can contribute to false positives, while low tumor cellularity may lead to false negatives.
These observations reflect the realities of diagnostic cytology and demonstrate that the authors understand the limitations of automated fluorescence-based methods.
The conclusion is now balanced and correctly describe the role of HFC as an adjunct to cytology and biochemical testing, rather than as a replacement. The limitations section appropriately acknowledges the dependency on specific analyzer models, the need for standardization across laboratories, and the inability of the Sysmex platform to distinguish between mesothelial and tumor cells.
This revision has transformed the paper into a coherent, mature, and clinically relevant validation study. The results are meaningful, the design is meaningful and the conclusions are credible. The work provides a practical and low-cost screening parameter that could help laboratories triage effusion samples more efficiently.
The authors efforts to improve the manuscript are evident throughout, and the language is now polished and professional. As someone who has worked on similar biomarker and cytological correlation studies, I find the data convincing and the clinical implications valuable.
I would only recommend final proofreading for punctuation consistency and formatting in tables, but these are purely editorial refinements and do not affect the scientific integrity of the work.
After revision, they improved the text and provided more detail and references.
The methods are described with sufficient detail, and overall, the manuscript provides valuable insight into the potential clinical utility of HFCs for diagnosing malignant effusions.
The conclusion is clear and is supported by the results. All underlying data are robust, statistically correct, and well controlled.
Thank you for submitting your manuscript on the diagnostic value of high-fluorescent cells (HFCs) in malignant effusions. While the reviewers appreciated the clinical relevance and the large dataset, they also highlighted important areas requiring substantial revision before the paper can be considered further.
Specifically:
1) Novelty claims should be toned down. HFCs should not be described as a “novel biomarker”; instead, please position your work as a valuable validation in a larger cohort.
2) Contextualisation: Expand the introduction and discussion with appropriate references, including prior studies using HFCs and HFC+CEA (e.g., Wu et al. 2019). Clarify how your work differs from earlier studies.
3) CEA vs HFC: Ensure the results and discussion accurately reflect that the best diagnostic performance arises from combining CEA and HFC, not HFC alone. Revise the title and conclusions accordingly.
4) Methodological clarity: Please indicate blinding of cytology vs HFC assessment, describe exclusion criteria more explicitly, and address false positive/negative findings.
5) Limitations: Expand this section to acknowledge that mesothelial cells and macrophages may also be classified as HFCs, and discuss the impact on diagnostic accuracy.
6) Figures and text: Improve figure structure and legends for clarity, simplify overlapping models, and perform thorough language editing for readability.
Taken together, these revisions are essential to improve clarity, ensure accurate interpretation, and place your findings in the context of the existing literature. We are therefore issuing a decision of Revision, and we invite you to carefully address all reviewer and editorial comments in a resubmission.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
The manuscript is generally well written in clear, professional English and adheres to PeerJ’ s standards. The title is concise and informative. The abstract is appropriately structured and summarizes the objective, methods, key findings, and conclusions.
The introduction provides adequate background and contextualizes the rationale behind evaluating high-fluorescent cells (HFCs) as a diagnostic marker for malignant effusions. Relevant literature is cited, though additional discussion on current limitations of cytology and competing methods like cell-block IHC or next-gen sequencing could strengthen the context. The figures and tables are clearly labeled, relevant to the data presented. The structure conforms to the IMRAD format. The raw data are reported transparently in tables, with cut-off values and AUCs detailed for multiple biomarkers.
Suggestion:
Replace "nonmalignant" with "benign".
The study describes original, hypothesis-driven thorough clinical research. The research question is well-defined. The design is appropriate, involving a large cohort (n = 978) with clinically validated classification of effusions (malignant vs. benign) based on cytology, pathology, and imaging. The inclusion of multiple types of effusions (pleural, pericardial, ascites) improves generalizability.
Methodology is precise and reproducible. The use of Sysmex XN-10 in BF mode is adequately described, including the fluorescence signal basis. Biomarker assays are conducted on validated platforms and follow SOP. Statistical analysis using ROC and logistic regression is clearly presented.
Suggestions for improvement:
Please clarify whether cytological findings were blinded to HFC results to reduce confirmation bias.
Describe the exclusion criteria in more way.
The findings are statistically clear, well controlled, and supported by the raw data. The use of multivariate logistic regression strengthens the conclusions. The sample size is appropriate, and ROC analyses were thoroughly executed with proper AUC reporting and confidence intervals. The conclusions are in line with the results.
This is a well-structured, clinically relevant, and technically organized study that addresses a significant diagnostic challenge. It introduces a potentially low-cost, high-throughput screening method for malignant effusions using available hematological platforms.
Minor language polishing would be good.
Title (lines 1-2): The effectiveness of HFC detection as a marker for malignant effusions is best when combined with CEA levels, and this should be reflected in the title.
Line 51: please add reference for “In particular, exfoliative examination is the gold standard for the diagnosis of malignant effusion.”
Line 51: please add reference for “However, this method is time-consuming, has low sensitivity, and is easily affected by the quality of material collected and the ability of the technician to identify cells.”
Line 59: please add reference for “The absolute number of HFCs (HFC#) and the percentage of HFCs (HFC%) have received attention for their diagnostic value in malignant effusions.”
Table 3: Cuto-ff value should be cutoff-value
Line 238: please cite other similar studies and contextualize these results.
The paper is concise and the results well-presented, however, this has been studied previously and these findings need to be better contextualized with previously published works (PMID 31018036, for instance). If the point is that this study is performed on effusions in general and other studies are performed on pleural effusion specifically, then data from the different effusion sites should be separately presented in the current paper. Additionally, CEA levels still appear to have a superior diagnostic performance to HFC detection, and this should be clarified at some points throughout the paper to more accurately present the significance of these findings. Please see specific comments.
Line 231: It would be helpful to also add to the results section the reasons behind false negative/ false positive HFC results in this study
Line 148 and Table 2: “These results further showed that HFC has an advantage in identifying malignant effusion and can serve as a valuable biomarker.” This sentence should more accurately reflect the multivariate analysis results or at least provide more of a qualifier. As presented, the odds ratios in Table 2 are likely not clinically significant even though they are statistically significant, because they are very close to 1. This should be addressed at some point in the paper.
Line 175: eliminate “and HFC could be used as a valuable biomarker for the identification of malignant effusions,” as it is really the CEA and HFC combination that has the best diagnostic performance.
Discussion:
Line 196: “When conventional cytology examination is not sensitive enough, HFC, as a novel biomarker, shows high potential for clinical application.” Please include a brief discussion of published sensitivities of cytology for malignant effusions. Ideally, a more specific suggested role for HFC should be presented, such as screening, or diagnostic adjunct.
Line 223: “However, compared with CEA, HFC may have special significance in the diagnosis of malignant effusion because of its higher sensitivity and faster detection speed,” though this may be true, the trade-off with lower specificity of HFCs compared to CEA should also be mentioned. The proposed role for HFC in clinical or research settings should be clarified, ie, in conjunction with CEA levels and conventional cytology.
Line 245: “Therefore, HFC has high potential as a novel biomarker for rapidly identifying malignant effusions.” Rephrase, as it is not clear that there is an improvement over CEA levels. This study shows that the combination of CEA and HFC values has the highest diagnostic accuracy.
Conclusions:
Line 253: Rephrase, as it is not clear that HFC alone provides much of an improvement over current methods
Line 256: “HFC can provide a reliable basis for rapid clinical screening of malignant effusions.” Rephrase, as this is not entirely supported. More accurately, “In combination with CEA levels, HFC may provide a valuable adjunct to cytomorphologic evaluation of pleural fluids”
The study addresses an important clinical problem on detection of malignancy in serous effusions; however, the manuscript requires substantial improvement in terms of background and justification with literature, data interpretation, and language. At present, the introduction and discussion do not provide sufficient context. English language and coherence of the text need major revision to ensure that the findings are accessible to an international audience.
Please consider following instructions for improvement:
1. Your most important issue is incorrect use of the term novel biomarker which reflects overstating of your finding and is not justified by the presented evidence. Not only can HFCs not be considered a biomarker, as they are also expressed in non-tumor cells in effusions, but the definition of a novel biomarker requires extensive investigations, which are not present in your work. The outcome of this study is not a novel biomarker, and this claim should be avoided in whole manuscript.
2. The next most important item is lacking an appropriate citation to prior works. For example, Wu et al. (Int J Lab Hematol. 2019. 41(4):509-512), applied a similar strategy using combination of CEA and HFCs for diagnostic purposes. Please explain how your work is different.
3. Your introduction needs more detail and references:
3.1. Please clarify “other conditions” and provide references in Line 47.
3.2. You are highlighting the problems with exfoliative cytology and advantages of Sysmex XN-10 as a solution. However, this section lacks detailed comparison based on the literature. Readers need more detail on specificity and sensitivity of Sysmex XN-10 compared to exfoliative cytology. In this context, please specify most relevant parameters to malignant effusion detection.
3.3. In Line 50, you state that exfoliative cytology is affected by the quality of collected material, but you do not explain how this differs from Sysmex XN-10. Both methods use effusion/liquid biopsy material. Please clarify this distinction.
3.4. Please indicate how low sensitivity improved by Sysmex XN-10.
3.5. Please provide more explanation and background on how following markers: WBC, RBC, CEA, LDH, and ADA are related to malignant effusion diagnosis.
3.6. Please provide rationale for focusing specifically on lung cancer as one of your major results.
3.7. Please provide more explanation and background on CEA.
3.8. Please provide systematic comparisons and cite prior studies regarding conventional cytology examination in Line 196.
3.9. In Lines 199–201, I suggest that, rather than comparing various translational research methods, compare Sysmex XN-10 with clinical diagnostic methods currently used in liquid biopsy. This will establish stronger clinical relevance.
3.10. “Faster detection speed “in Line 225, needs elaboration and supporting evidence.
3.11. In Line 250, the limitations section is incomplete. Using Sysmex XN-10, mesothelial cells and macrophages may be counted as high‐fluorescent cells, affecting the accuracy of the cell count results. You only investigated the values of HFC% and HFC# detected by the instrument, without accounting for this inaccuracy. This impacts the diagnostic efficiency for distinguishing benign and malignant pleural effusions.
3.12. In line 256, you must precise that HFC in combination with CEA provide a reliable basis for rapid screening.
4. The manuscript requires significant editing for clarity and coherence. Some sentences, as well as titles of results, and figure legends are unclear and are difficult to understand. I recommend professional editing.
5. Figures:
Thank you for providing the raw data files. However, figures need improvement based on the following remarks:
5.1. I recommend restructuring Figure 1 to interpret easier. Specifically, merge panels A and B Include following markets: HFC#, HFC%, CEA, LDH, HFC#+CEA, HFC%+CEA.
5.2. Avoid the three-variable (HFC# + HFC% + CEA) model in both figure 1 and figure 2. HFC# and HFC% are strongly collinear. Presenting that model in the main figure is confusing.
5.3. Improve figure legends for both figure 1 and figure 2. This part needs clearer wording and explanation.
The methods are described with sufficient detail, and overall, the manuscript provides valuable insight into the potential clinical utility of HFCs for diagnosing malignant effusions.
The conclusions are not appropriately stated and are not fully supported by the results. Claims of a novel biomarker are overstated; establishing novel biomarkers requires an extensive experimental validation, which is not present in this manuscript. Moreover, the strategy presented here is not new, as both HFCs and the combination of CEA with HFCs have already been reported in the literature.
That said, this study makes meaningful contribution by validating HFCs as an indicator of malignancy in a larger patient dataset than previously published. All underlying data are robust, statistically correct, and well controlled.
In conclusion, there is a weakness in written and interpreting data (noted above) which should be improved before acceptance.
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