Virtual Evaluation of Hematoxylin & Eosin via Digital Pathology Survey (VEED) Project: Results from a Non-Inferiority Study of a Tabs-Based Staining Method

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Resp sir/madam,

The topic feel timely and potentially valuable,

Study is significant for standardization in anatomic pathology departments,

but the current version remain conceptually fragmented and require substantial restructuring before it can reach publication standard.

--structural reorganization, deeper mechanistic integration, and more deliberate translational framing, the manuscript could become a cohesive and impactful.

 

--add note that----The image shows high resolution virtual staining that maintains morphological detail equivalent to traditional chemical staining methods.-

--Note to add of tech---This technology enables rapid tissue assessment, reduces processing time, and facilitates telepathology applications. The virtual H&E approach represents an important advancement in pathological diagnosis, particularly relevant for intraoperative consultation and resource-limited settings.

 

---data are limited to test the proposed tech through satisfying the approved outcome-neutral conditions (such as through quality checks and positive controls);

also requires to study on different organ/tissue representative samples for ex-adipose tissue,neural,lung,renal,bone etc.

 

---mention the suitability and feasibility of the analytical methodology and  advantage over others.

requires minor revision.

 

----intra- and inter-laboratory variability----checking is required.

 

Author Response

We sincerely thank the reviewer for the valuable and insightful comments. We appreciate the time and effort devoted to evaluating our manuscript. Below, we provide a detailed point-by-point response to each comment:

1) Structural reorganization, deeper mechanistic integration, and more deliberate translational framing, the manuscript could become a cohesive and impactful.

We thank the Reviewer for this insightful comment and for the overall positive assessment of the manuscript’s potential. We agree that stronger mechanistic integration and translational framing can often enhance the clarity and impact of a study.

However, we would like to clarify that the formulation of the Stain Tabs used in this work is proprietary, and therefore detailed mechanistic disclosure of their internal composition and modes of action cannot be further expanded within the scope of this manuscript. For this reason, we are not able to provide additional molecular or mechanistic breakdown beyond what is already reported.

2) Add note that----The image shows high resolution virtual staining that maintains morphological detail equivalent to traditional chemical staining methods.

We thank the reviewer for this suggestion. As our study investigated a histochemical rather than a virtual staining method, we have added the following statement to the Discussion: "Our findings demonstrate that TABS-based staining provides high-quality histological images while maintaining morphological detail comparable to that achieved with conventional pre-diluted H&E staining methods."

To avoid any misunderstanding, we have emphasized this concept in the Abstract and Introduction by explicitly stating that we evaluated a histochemical staining method.

3) Note to add of tech---This technology enables rapid tissue assessment, reduces processing time, and facilitates telepathology applications. The virtual H&E approach represents an important advancement in pathological diagnosis, particularly relevant for intraoperative consultation and resource-limited settings.

In the present study, we evaluated a TABS-based histochemical staining method (see the Methods and Discussion sections) and assessed its non-inferiority using a customized digital pathology survey. Therefore, the potential advantages of this histochemical approach are not related to rapid assessment, but rather to the reduction of shipping and storage costs, as well as the potential improvement of staining reproducibility, although further studies are required to investigate this aspect.

As we mentioned in point n. 2, to avoid any misunderstanding, we have emphasized this concept in the Abstract and Introduction by explicitly stating that we evaluated a histochemical staining method.

4) Data are limited to test the proposed tech through satisfying the approved outcome-neutral conditions (such as through quality checks and positive controls); also requires to study on different organ/tissue representative samples for ex-adipose tissue,neural,lung,renal,bone etc.

We thank the reviewer for this insightful comment. We agree that the current dataset does not include all possible kind of tissue. We also acknowledge that further studies on a broader range of representative tissue types will be necessary to fully assess the generalizability of the method. Importantly, these aspects have already been addressed in the limitations section of the Discussion, where we explicitly acknowledge the need for expanded validation across multiple tissue types in future studies.

Following the reviewer suggestion, we expanded the Limitation section as follows: "The limitations of our study lie primarily in the incomplete representation of tissues; for example, the survey did not include tissues from the lung, bladder, lymph nodes, bone, and central nervous system. Additionally, all specimens were collected and stained at a single institution, precluding assessment of interlaboratory variability; future studies will include a multicenter evaluation involving independent laboratories, different staining platforms, different tissue samples and diverse workflow settings to further validate the present findings and investigate both intra- and inter-laboratory variability. Nevertheless, these findings provide a pivotal foundation for future investigations using the same described approach, with broader inclusion of tissue types and samples from multiple centers"

5) Mention the suitability and feasibility of the analytical methodology and advantage over others.

We thank the reviewer for this valuable suggestion. We have revised the manuscript to better emphasize the suitability and feasibility of the proposed analytical methodology in the Discussion section.

Here the sentence: "The tabs-based histochemical approach appears to be compatible with routine digital pathology workflows, allowing straightforward implementation without the need for substantial changes to standard laboratory procedures. It only requires the preparation of tabs dilution and hematoxylin filtration as additional steps, offering substantial advantages in terms of storage and shipping, as well as potential benefits in standardization".

Reviewer 2 Report

Comments and Suggestions for Authors

The paper presents a practical reagent-standardization approach rather than a fundamentally new staining technology. Its main contribution lies in demonstrating that tablet-based H&E staining can achieve diagnostic quality comparable to conventional staining while potentially improving reagent stability and staining consistency for digital pathology workflows. I have some observations regarding the reproducibility/standardization claims and objective quantitative analysis which are listed below:

1. The manuscript repeatedly suggests that the proposed tablet based staining approach may improve intra/inter-laboratory reproducibility and standardization. Whereas all staining procedures were performed at a single institution using a single staining platform. Therefore, the study does not directly evaluate inter-laboratory reproducibility, representing one of the principal motivations for the proposed method. The authors should either provide additional evidence supporting this claim or substantially moderate the corresponding statements throughout the manuscript.
2. The authors should explicitly acknowledge that the current study demonstrates diagnostic adequacy and non-inferiority within a single-center setting only. A dedicated discussion should be included outlining the need for future multicenter studies involving independent laboratories, different staining platforms, and diverse workflows to validate the proposed benefits regarding standardization and reproducibility.
3. The study relies predominantly on subjective pathologist evaluations. The manuscript would be significantly strengthened by including objective quantitative analyses such as colorimetric measurements, optical-density analysis, stain variability assessment, or other digital image analysis metrics. Given the manuscript's emphasis on digital pathology and AI applications; such analyses would provide more robust evidence regarding the potential benefits of the proposed staining method for computational pathology workflows.

Author Response

We sincerely thank the reviewer for the valuable and insightful comments. We appreciate the time and effort devoted to evaluating our manuscript. Below, we provide a detailed point-by-point response to each comment:

1) "The manuscript repeatedly suggests that the proposed tablet based staining approach may improve intra/inter-laboratory reproducibility and standardization. Whereas all staining procedures were performed at a single institution using a single staining platform. Therefore, the study does not directly evaluate inter-laboratory reproducibility, representing one of the principal motivations for the proposed method. The authors should either provide additional evidence supporting this claim or substantially moderate the corresponding statements throughout the manuscript"

We agree with the reviewer that the assessment of inter-laboratory variability is an important aspect of method validation. However, the present study was not designed to evaluate inter-laboratory reproducibility. Our primary objective was to demonstrate the non-inferiority of the tablet-based H&E staining method compared with the conventional pre-diluted H&E approach. This represents a necessary first step before investigating its potential impact on reducing staining variability.

Notably, the tablet-based method is expected to be less susceptible to factors such as temperature fluctuations and reagent storage time, which may affect the performance of pre-diluted H&E solutions. Nevertheless, the effect of this approach on inter- and intra-laboratory variability cannot be inferred from the current dataset and requires dedicated investigation.

To state this limitation, we had just included the following statement in the manuscript: “Additionally, all specimens were collected and stained at a single institution, precluding assessment of inter-laboratory variability.”

Furthermore, following the reviewer's suggestion, we have expanded the Conclusions section by adding the following statement: “Once non-inferiority has been established, further studies are needed to evaluate the impact of the tablet-based H&E staining method on staining variability, including both intra- and inter-laboratory reproducibility, supported by quantitative analytical assessments.”

2) The authors should explicitly acknowledge that the current study demonstrates diagnostic adequacy and non-inferiority within a single-center setting only. A dedicated discussion should be included outlining the need for future multicenter studies involving independent laboratories, different staining platforms, and diverse workflows to validate the proposed benefits regarding standardization and reproducibility.

We thank the reviewer for this valuable suggestion, which allowed us to better outline the future directions of our research. In response, we have added the following statement to the Limitations section:

“Future studies will include a multicenter evaluation involving independent laboratories, different staining platforms, different tissue samples and diverse workflow settings to further validate the present findings and investigate both intra- and inter-laboratory variability. To this end, quantitative image analysis may be incorporated in subsequent investigations. .”

3) The study relies predominantly on subjective pathologist evaluations. The manuscript would be significantly strengthened by including objective quantitative analyses such as colorimetric measurements, optical-density analysis, stain variability assessment, or other digital image analysis metrics. Given the manuscript's emphasis on digital pathology and AI applications; such analyses would provide more robust evidence regarding the potential benefits of the proposed staining method for computational pathology workflows.

We agree with the reviewer regarding the potential value of quantitative analyses, and it is planned for the second phase of the project, with different aims regarding inter-laboratory variability: quantitative analyses performed on blocks stained in different centers could help to assess the variability associated with both standard and TABS-based H&E staining.

However, the aim of the present project is to investigate the non-inferiority of the described method in terms of pathologist's qualitative evaluation.

Following the reviewer suggestion, we added this consideration in the limitation paragraph in the Discussion section (see point 2). Moreover, as we reported in point 1, we expanded the Conclusion section adding these considerations.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have adequately addressed the major concerns through clearer acknowledgment of the single-center nature of the study and the need for future multicenter validation of reproducibility and standardization.

The absence of objective quantitative image-based analyses remains a limitation, but this has also been appropriately recognized and discussed as future work.