Enzyme-linked immunosorbent assay and immunohistochemical analysis of mast cell related biochemicals in oral submucous fibrosis

Harshkant Gharote; Rahul Bhowate; Suwarna Dangore-Khasbage

doi:10.12688/f1000research.141179.3

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Study Protocol

Revised

Enzyme-linked immunosorbent assay and immunohistochemical analysis of mast cell related biochemicals in oral submucous fibrosis

[version 3; peer review: 2 approved]

Harshkant Gharote ¹, Rahul Bhowate¹, Suwarna Dangore-Khasbage¹

PUBLISHED 20 May 2024

Author details Author details

¹ Oral Medicine and Radiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Higher Education and Research, Sawangi (Meghe), Wardha, Maharashtra, 442004, India

Harshkant Gharote
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation

Rahul Bhowate
Roles: Project Administration, Supervision, Writing – Review & Editing

Suwarna Dangore-Khasbage
Roles: Resources, Validation

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Datta Meghe Institute of Higher Education and Research collection.

Abstract

Oral submucous fibrosis (OSMF), a potentially malignant disorder, is developed by progressive fibrous tissue deposition in connective tissue along with atrophy of oral mucosa. Histological sections also show the mast cell infiltration in submucosa which may indicate their possible role in this entity. Abundant availability of biochemicals in mast cells like histamine and serine proteases like chymase may be released and play specific pathways in the disease pathophysiology. Possibly, if the histamine release has some part to play, diamine oxidase may also be found to have a relationship as it metabolizes histamine. The present study is proposed to identify the presence of chymase, histamine, and diamine oxidase in both, serum as well as tissue by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) respectively. This study may provide probable insight into the mast cell-related chemicals and their association with OSMF.

Keywords

Mast cells, Oral submucous fibrosis, Chymase, Histamine, Diamine oxidase

Corresponding author: Harshkant Gharote

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2024 Gharote H et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Gharote H, Bhowate R and Dangore-Khasbage S. Enzyme-linked immunosorbent assay and immunohistochemical analysis of mast cell related biochemicals in oral submucous fibrosis [version 3; peer review: 2 approved]. F1000Research 2024, 12:1288 (https://doi.org/10.12688/f1000research.141179.3) First published: 09 Oct 2023, 12:1288 (https://doi.org/10.12688/f1000research.141179.1) Latest published: 20 May 2024, 12:1288 (https://doi.org/10.12688/f1000research.141179.3)

Revised Amendments from Version 2

The valuable inputs from the reviewer pertaining to statistical analysis and method of analysis like use of software provided by the institute were updated in the new version. Citation of new information was added as suggested.

See the authors' detailed response to the review by Vini Mehta
See the authors' detailed response to the review by Xinjia Cai

Introduction

Oral submucous fibrosis (OSMF) is a potentially malignant disorder affecting the oral cavity and oropharynx causing extensive fibrous tissue deposition in submucosa. The presence of the mast cells in histological section of OSMF has been found to be related to various stages of OSMF. Although, studies have suggested that enzymes released by degranulation of mast cells in OSMF have some role in its initiation¹^–⁴ although the exact mechanism of action is not known. Therefore, it would be imperative to identify the presence of various biochemicals of mast cells in affected oral mucosa and perhaps the serum of the affected individuals. Hence, it can be stated that if there is histamine release during the initiation and progression of OSMF, possible increased levels of serum histamine levels can be observed in those patients. Additionally, in response to increased histamine levels, its metabolism will be initiated which is mediated through enzyme diamine oxidase (DAO). Therefore, variation in serum DAO levels may also be expected in those individuals.

Mast cells (MCs) are multifaceted immune cells subclassified based on their protease content as mast cell with tryptase only (MCT) and mast cell with tryptase and chymase (MCTC) and may have varied role in various pathogeneses including cancer. Mast cells exhibit increased accumulation within tumor microenvironments and attribute to prognosis, metastasis and reduced survival in several types of human cancer. mast cells can influence the tumor microenvironment and induce pro-tumor effects by conditioning the fate of tumor cells, initiation of angiogenesis and tissue remodeling though its bioactive molecules. including drug resistance.⁵^–⁷ Also, it has been observed that mast cells and fibroblasts show physical interactions and induce fibroblast contraction of collagen lattices.⁸ Further, OSMF shows high potential for malignant transformation and is correlated to a series of biochemical alterations seen during the progress of the disease. The incidence of mast cells in the affected mucosa may be implicated in the malignant transformation. Studies show that mast cell serine proteases like tryptase and chymase significantly impact angiogenesis thus affecting tumor development and progression.⁹ There is evidence that supports an association between mast cell chymase and tumor angiogenesis. While tryptase, a neutral serine protease, is the abundant mediator stored in the mast cell granules that mediate degranulation of mast cells in allergic diseases.¹⁰ Thus, among these, evaluation of serum chymase may provide insights into the function of mast cells in the initiation and progression of OSMF.

Protocol

The present study proposes that variations in the serum levels of diamine oxidase, histamine, and chymase may be noticed in OSMF. With this view, the present study will attempt to estimate the serum levels of chymase, histamine, and diamine oxidase in various stages of OSMF and compare them with the levels in healthy individuals and persons with areca habit without OSMF. Additionally, an immunohistochemical analysis will be performed for mast cell-related markers- chymase, diamine oxidase, and histamine to extrapolate their presence in serum.

The present research study has been approved by the Institutional Ethical Committee and will be conducted in the Department of Oral Medicine and Radiology, Sharad Pawar Dental College and Hospital, Wardha, India.

Aim: Assessment of variations in serum diamine oxidase, histamine, and chymase levels in the various stages of OSMF, areca chewers without OSMF, and healthy individuals by an enzyme-linked immunosorbent assay.

Objectives

1. To estimate serum histamine and diamine oxidase levels in the various stages of OSMF and between overall OSMF patients, areca chewers without OSMF, and healthy individuals.
2. To estimate serum chymase levels in the various stages of OSMF and between overall OSMF patients, areca chewers without OSMF, and healthy individuals.
3. To compare serum histamine and diamine oxidase levels in various stages of OSMF and between overall OSMF patients, areca chewers without OSMF, and healthy individuals.
4. To compare serum chymase levels in the various stages of OSMF, and between overall OSMF patients, areca chewers without OSMF, and healthy individuals.
5. To correlate serum diamine oxidase levels with serum histamine levels in the various stages of OSMF and between overall OSMF patients, areca chewers without OSMF, and healthy individuals.
6. To correlate serum histamine, chymase, and diamine oxidase levels in various stages of OSMF and between overall OSMF patients, areca chewers without OSMF, and healthy individuals.
7. To analyze the immunohistochemical expression of mast cell chymase, histamine, and diamine oxidase in OSMF, patients with areca habit without OSMF, and healthy individuals.
8. The serum values will be correlated to the immunohistochemistry expressions of the chymase, diamine oxidase and histamine to identify the possible relationship.

Method

Experimental design

The participants will be divided into three groups as OSMF group, individuals with areca habit without OSMF group, and Healthy individuals’ group. All individuals included will be above 18 years without any systemic or metabolic disorder. OSMF patients with a history of treatment will be excluded from the study. Written consent will be obtained after an explanation of the study procedure and protocol for the collection of serum samples and biopsy specimens.

Selection of OSMF patients

Individuals with OSMF will be selected following functional staging classification of proposed by More et al.¹¹ The classification of functional stages is given as follows:

Functional staging:

‐ M1: Interincisal mouth opening - up to or greater than 35 mm.
‐ M2: Interincisal mouth opening between 25 and 35 mm.
‐ M3: Interincisal mouth opening between 15 and 25 mm.
‐ M4: Interincisal mouth opening less than 15 mm.

Selection of individuals with areca habit without OSMF

Individuals with a history of areca consumption of more than one year duration, without any evidence of OSMF and other oral mucosal conditions like leukoplakia and lichen planus; and without history of any medical disorders

Selection of Healthy individuals

Individuals without OSMF and without habits and any systemic disorders/conditions.

The details of selected participants will be recorded in the case history proforma for recording clinical findings and investigations. The informed consent will be obtained before enrolling the participant for proposed investigations.

Exclusion criteria

1. The individuals with a history of the consumption of tobacco in any other form such as cigarette, bidi, and tobacco with lime.
2. The individuals with a history of any systemic disease.
3. The patients having history of receiving treatment for OSMF.
4. The patients having history for antihistaminic medications.

The proposed study, a case control study, will include OSMF patients, areca habitual without OSMF and healthy individuals. The population of Indian subcontinent is infinite, and the individuals affected by OSMF are variable from state to state, therefore no definite data is available. Yet, the latest published literature shows that the prevalence of OSMF in India is 7.21%,¹² the sample size can be calculated using the formula for known population proportion (p) with the help of SPSS software (IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY: IBM Corp). The sample size selection will be at 95 per cent confidence interval with margin of error (d) at ±10%. Thus, following formula can be used to calculate sample size:

n = \frac{z^{2} p (1 - p)}{d^{2}}

In the formula:

n = sample size

z = z score (1.96)

p = population proportion (0.0721)

d = margin of error (0.1)

n = \frac{{(1.96)}^{2} 0.0721 (1 - 0.0721)}{{0.1}^{2}}

n = 25.7

Thus, 26 individuals can be enrolled in each group for the study. Thus, a total sample size of 78 will be used for balanced study with each group consisting of 26 subjects.

Five histological sections each for overall OSMF patients, areca chewers without OSMF and healthy individuals will be subjected for immunohistochemical analysis. Thus, 15 histopathological slides will be subjected to immunohistochemical expression for each marker viz. histamine, diamine oxidase and chymase.

Materials

Commercially available ELISA kits will be procured for the estimation of serum histamine, chymase and diamine oxidase. The optical density will be measured by spectrophotometry at a wavelength of 450 nm ± 2 nm using an ELISA reader. The immunohistochemical analysis will be performed on formalin-fixed paraffin-embedded biopsy tissue sections of OSMF patients.

Procedure

Collection of serum samples: Blood sample will be drawn from antecubital vein using a 5 ml syringe and it will be taken into a vial containing a clot activator and serum gel separator. After that, it will be transferred into a centrifuge tube. The centrifugation of blood will be done for ten minutes, and serum will be obtained. Centrifugation is a procedure of using centrifugal force and the aim is to separate two unmixable liquids. There is sedimentation of heterogeneous mixtures in which more heavy constituents drift away from the axis of centrifuge and less heavy constituents drift towards the axis of centrifuge. The effective gravitational force on a test tube is increased which causes the precipitate to collect on the bottom of tube and the remaining supernatant liquid will be withdrawn with the help of a pipette.

Estimation of serum Diamine oxidase levels: This ELISA kit will use the Sandwich-ELISA principle. The ELISA plate provided is pre-coated with an antibody specific to Mouse DAO. Standards or samples will be added to the ELISA plate wells and combined with the specific antibody. Next, a biotinylated detection antibody specific for Mouse DAO and Avidin-Horseradish Peroxidase (HRP) conjugate will be added sequentially to each micro plate well and incubated. Free components are washed away. The substrate solution will be added to each well. Only those wells that contain Mouse DAO, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction will be terminated by the addition of stop solution and the color turns yellow. The optical density (OD) will be measured using spectrophotometry at a wavelength of 450 nm ± 2 nm. The OD value will be proportional to the concentration of Mouse DAO. The concentration of Mouse DAO in the samples will be calculated by comparing the OD of the samples to the standard curve.

Estimation of serum Histamine levels: After Histamine is quantitatively acylated, the subsequent competitive ELISA kit will be used the microtiter plate format with the antigen bound to the solid phase. The acylated standards, controls and samples and the solid phase bound analyte will compete for a fixed number of antiserum binding sites. After the system is in equilibrium, free antigen and free antigen-antiserum complexes will be removed by washing. The antibody bound to the solid phase will be detected by an anti-rabbit IgG peroxidase conjugate using 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate. The reaction will be monitored at 450 nm. Quantification of unknown samples will be achieved by comparing their absorbance with a reference curve prepared with known standard concentrations.

Estimation of serum chymase levels: This ELISA kit will use the Sandwich ELISA principle. The ELISA plate provided in this kit is precoated with an antibody specific to Human CMA1. Standards or samples will be added to the ELISA plate wells and combined with the specific antibody. Then, a biotinylated detection antibody specific for Human CMA1 and Avidin Horseradish Peroxidase (HRP) conjugate will be added successively to each micro plate well and incubated. Free components will be washed away. The substrate solution will be added to each well. Only those wells that contain Human CMA1, biotinylated detection antibody and Avidin HRP conjugate will appear blue in color. The enzyme substrate reaction will be terminated by the addition of stop solution and the color turns yellow. The optical density (OD) will be measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value will be proportional to the concentration of Human CMA1. The concentration of Human CMA1 in the samples will be calculated by comparing the OD of the samples to the standard curve.

Immunohistochemistry: The tissue sections will be deparaffinized in xylene solution and rehydrated through decreasing graded ethanol solution. Endogenous peroxidase activity will be inhibited by incubation for 10 minutes with 3% hydrogen peroxidase. The primary monoclonal antibodies used for mast cells will be anti-MC chymase, anti-MC diamine oxidase, and antihistamine antibodies. Staining will be performed at room temperature on an automatic staining workstation. Commercially available immunohistochemistry kits will be procured for these markers.

Statistical analysis

The statistical analysis will be carried out using SPSS version 27.0 (IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY: IBM Corp). The data obtained will be analysed using following tests:

1. Students t-test
2. Analysis of variance (ANOVA)
3. Pearson’s correlation

A p-value of 0.05 or lower with 95 percent confidence interval will be considered statistically significant for the analysis.

Expected outcomes

As numerous mast cells are found to be present histologically in OSMF, it is expected to see the possible increase in serum levels of diamine oxidase, histamine, and chymase. Further, the presence of these enzymes will be confirmed by immunohistochemical analysis of histological sections. positive results in both the methods will give an insight into the role of these enzymes in progression and the possible role in the malignant transformation of OSMF.

If the outcomes reveal statistically nonsignificant variation in serum levels of these biomolecules, role of mast cells in systemically influencing the initiation and progression of OSMF will be ruled out. Nonetheless, increased serum levels might be indicative of the active secretory action of mast cells and may provide an understanding of their role in progression of OSMF in oropharynx and esophagus. Tissue expression of histamine and chymase will establish a possible role of histamine receptors and chymase in induction of fibrosis and futuristic approach to malignant transformation of the OSMF.

Discussion

The prevalence of OSMF in India is 7.21% and shows higher rates of malignant transformation.¹² The presence of mast cells in histological sections reveal their strong association with the disease so that some biochemicals and enzymes might play role in the progression and malignant transformation.³^,¹³ Thus, detection of the mast cell-related chemicals in tissue and serum will delineate further mechanisms in pathogenesis. With special emphasis on diamine oxidase and its relation to histamine, the possible allergic response of mast cells to etiological factors like betel nut will be discerned. Further, possible detection of chymase in serum will define its elaborate role in local as well as a systemic mechanism in angiogenesis in OSMF.

Infiltration of mast cells in OSMF and other premalignant disorders like lichen planus and leukoplakia has been studied and are implicated in initiation and progression of these entities.⁴^,¹⁴^,¹⁵ The studies are conducted on expression of MCT and MCTC in OSMF using immunohistochemistry and observed a significant increase in expression of MCTC in OSMF.¹⁶ Further, serum histamine and tryptase levels have been assessed in oral squamous cell carcinoma (OSCC) along with its correlation with various histological zones/stages. While histamine was positively correlated to the depth of the invasion, serum tryptase had no correlation with various grades of OSCC.¹⁷^,¹⁸ Moreover, as mast cells are associated with collage metabolic disorders, it is suggested that they play similar role in pathogenesis of OSMF and labelled it as a collagen metabolic disorder.¹⁹ Nonetheless, the literature gap shows need to understand the role of chymase and histamine in pathogenesis of OSMF and its malignant transformation. Further, mast cell related research on their degranulation and release of bioactive molecules may provide an insight to understand the pathophysiology of OSMF as a collagen metabolic disorder.

Ethical considerations

The present study is approved by the Institutional Ethics Committee of Datta Meghe Institute of Higher Education and Research, Sawangi Wardha, India with the ethical approval reference no. DMIMS (DU)/Ph.D. Regn. /2021/1226.

Study status

The present study is in the process of data collection wherein serum samples are being collected from OSMF group, Areca habitual and control groups as per the design.

Data availability

Not applicable as it is a proposed protocol.

References

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2. Pujari R, Vidya N: Mast cell density in oral submucous fibrosis: a possible role in pathogenesis. Int. J. Health Sci (Qassim). 2013 Jan; 7(1): 23–29. PubMed Abstract | Publisher Full Text
3. Kumar LB, Mathew P, Madhavan N, et al.: Evaluation of mast cells and burning sensation in various stages of Oral Submucous Fibrosis. J. Oral Biol. Craniofac. Res. 2020 Oct-Dec; 10(4): 430–434. PubMed Abstract | Publisher Full Text | Free Full Text
4. Ankle MR, Kale AD, Nayak R: Mast cells are increased in leukoplakia, oral submucous fibrosis, oral lichen planus and oral squamous cell carcinoma. J. Oral Maxillofac. Pathol. 2007; 11: 18–22. Publisher Full Text
5. Zhao XO, Sommerhoff CP, Paivandy A, et al.: Mast cell chymase regulates extracellular matrix remodeling-related events in primary human small airway epithelial cells.J. Allergy Clin. Immunol.2022 Dec;150(6): 1534–1544. Epub 2022 Jun 30. PubMed Abstract | Publisher Full Text
6. Aponte-López A, Fuentes-Pananá EM, Cortes-Muñoz D, et al.: Mast Cell, the Neglected Member of the Tumor Microenvironment: Role in Breast Cancer. J. Immunol. Res. 2018 Feb; 2018: 2584243. PubMed Abstract | Publisher Full Text | Free Full Text
7. Maciel TT, Moura IC, Hermine O: The role of mast cells in cancers. F1000Prime Rep. 2015 Jan 5; 7: 09. PubMed Abstract | Publisher Full Text | Free Full Text
8. Reddy MGS, Kakodkar P, Mehta V: Mast cell mediated myofibroblast differentiation responsible for progression of oral submucous fibrosis. Oral Oncol. Rep. 2024; 9: 100216. Publisher Full Text
9. De Souza Junior DA, Santana AC, da Silva EZM , et al.: The Role of Mast Cell Specific Chymases and Tryptases in Tumor Angiogenesis. Biomed. Res. Int. 2015; 2015: 1–13. Publisher Full Text
10. Mahmood M, Rashid BM, Amin K, et al.: Correlation between Serum Tryptase Level and Disease Severity in Asthmatic Patients in the Sulaimani Governorate. Int. J. Med. Res. Health Sci. 2016; 2016(5): 34–41.
11. More CB, Gupta S, Joshi J, et al.: Classification System for Oral Submucous Fibrosis. J. Indian Aca. Oral Med. Radiol. 2012; 24(1): 24–29. Publisher Full Text
12. Rao NR, Villa A, More CB, et al.: Oral submucous fibrosis: a contemporary narrative review with a proposed inter-professional approach for an early diagnosis and clinical management. J. Otolaryngol. Head Neck Surg. 2020 Jan 8; 49(1): 3. PubMed Abstract | Publisher Full Text | Free Full Text
13. Agostinelli E, Tempera G, Viceconte N, et al.: Potential anticancer application of polyamine oxidation products formed by amine oxidase: a new therapeutic approach. Amino Acids. 2010 Feb; 38(2): 353–368. PubMed Abstract | Publisher Full Text
14. Kinra M, Ramalingam K, Sarkar A, et al.: Comparison of mast cell count and mast cell densit y in normal mucosa, oral leukoplakia, oral lichen planus, oral submucous fibrosis and oral squamous cell carcinoma - a study of 50 cases. JPSI. 2012; 1: 4–11.
15. Sundararajan A, Muthusamy R, Gopal Siva K, et al.: Correlation of Mast Cell and Angiogenesis in Oral Lichen Planus, Dysplasia (Leukoplakia), and Oral Squamous Cell Carcinoma. Rambam Maimonides Med. J. 2021 Apr 29; 12(2): e0016. PubMed Abstract | Publisher Full Text | Free Full Text
16. Yadav A, Desai RS, Bhuta BA, et al.: Altered immunohistochemical expression of mast cell tryptase and chymase in the pathogenesis of oral submucous fibrosis and malignant transformation of the overlying epithelium. PLoS One. 2014 May 29; 9(5): e98719. PubMed Abstract | Publisher Full Text | Free Full Text
17. Hasan NR, Dehuri P, Jena A, et al.: A Correlation of Serum Histamine and Mast Cell Count with the Established Prognosticators in Oral Cancer. J. Microsc. Ultrastruct. 2023 Jan 5; 11(2): 97–102. PubMed Abstract | Publisher Full Text | Free Full Text
18. Jaafari-Ashkavandi Z, Khademi B, Akbari S, et al.: Serum level of mast cell tryptase in patients with oral squamous cell carcinoma: lack of correlation with clinicopathologic factors. Asian Pac. J. Cancer Prev. 2013; 14(5): 2955–2958. PubMed Abstract | Publisher Full Text
19. Rajalalitha P, Vali S: Molecular pathogenesis of oral submucous fibrosis--a collagen metabolic disorder. J. Oral Pathol. Med. 2005 Jul; 34(6): 321–328. PubMed Abstract | Publisher Full Text

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Version 3

VERSION 3 PUBLISHED 09 Oct 2023

Author details Author details

¹ Oral Medicine and Radiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Higher Education and Research, Sawangi (Meghe), Wardha, Maharashtra, 442004, India

Harshkant Gharote
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation

Rahul Bhowate
Roles: Project Administration, Supervision, Writing – Review & Editing

Suwarna Dangore-Khasbage
Roles: Resources, Validation

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Article Versions (3)

version 3

Revised

Published: 20 May 2024, 12:1288

https://doi.org/10.12688/f1000research.141179.3

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Published: 29 Apr 2024, 12:1288

https://doi.org/10.12688/f1000research.141179.2

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Published: 09 Oct 2023, 12:1288

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© 2024 Gharote H et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Gharote H, Bhowate R and Dangore-Khasbage S. Enzyme-linked immunosorbent assay and immunohistochemical analysis of mast cell related biochemicals in oral submucous fibrosis [version 3; peer review: 2 approved]. F1000Research 2024, 12:1288 (https://doi.org/10.12688/f1000research.141179.3)

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Version 3

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Reviewer Report 30 May 2024

Vini Mehta, Dr. D. Y. Patil Dental College and Hospital, Pimpri, India

Approved

https://doi.org/10.5256/f1000research.166384.r280429

The authors have satisfactorily done all the corrections.
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Reviewer Report 10 May 2024

Vini Mehta, Dr. D. Y. Patil Dental College and Hospital, Pimpri, India

Approved with Reservations

https://doi.org/10.5256/f1000research.165338.r270235

1. Please club the objectives.
2. Use software for sample size estimation.
3. Will CTRI registration be done for this study?
4. Write which software will be used for statistical analysis? which version? Complete the statement regarding confidence interval and p values.
5. Add the following reference:
- Mast cell mediated myofibroblast differentiation responsible for progression of oral submucous fibrosis

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

Yes
Are the datasets clearly presented in a useable and accessible format?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Public Health

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 21 Jun 2024

Harshkant Gharote, Oral Medicine and Radiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Higher Education and Reasech, Sawangi (Meghe), Wardha, 442004, India

21 Jun 2024

Author Response

Thank you for Valuable inputs.
The software used for sample size formula is provided by the research methodology cell of the institute and the details are added to the manuscript. ... Continue reading Thank you for Valuable inputs.
The software used for sample size formula is provided by the research methodology cell of the institute and the details are added to the manuscript. Same software will be used to analyze the data.
The statement for the confidence interval and p-value are added.
There will be no CTRI registration for the study
The reference suggested has been included and cited in the manuscript.
There will be no CTRI registration for the study protocol.
Thank you for Valuable inputs.
The software used for sample size formula is provided by the research methodology cell of the institute and the details are added to the manuscript. Same software will be used to analyze the data.
The statement for the confidence interval and p-value are added.
There will be no CTRI registration for the study
The reference suggested has been included and cited in the manuscript.
There will be no CTRI registration for the study protocol.
Competing Interests: Nil Close
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Respond or Comment

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Author Response 21 Jun 2024

Harshkant Gharote, Oral Medicine and Radiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Higher Education and Reasech, Sawangi (Meghe), Wardha, 442004, India

21 Jun 2024

Author Response

Thank you for Valuable inputs.
The software used for sample size formula is provided by the research methodology cell of the institute and the details are added to the manuscript. ... Continue reading Thank you for Valuable inputs.
The software used for sample size formula is provided by the research methodology cell of the institute and the details are added to the manuscript. Same software will be used to analyze the data.
The statement for the confidence interval and p-value are added.
There will be no CTRI registration for the study
The reference suggested has been included and cited in the manuscript.
There will be no CTRI registration for the study protocol.
Thank you for Valuable inputs.
The software used for sample size formula is provided by the research methodology cell of the institute and the details are added to the manuscript. Same software will be used to analyze the data.
The statement for the confidence interval and p-value are added.
There will be no CTRI registration for the study
The reference suggested has been included and cited in the manuscript.
There will be no CTRI registration for the study protocol.
Competing Interests: Nil Close
Report a concern

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Reviewer Report 07 May 2024

Xinjia Cai, Peking University School and Hospital of Stomatology, Beijing, China

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https://doi.org/10.5256/f1000research.165338.r270102

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Reviewer Report 01 Apr 2024

Xinjia Cai, Peking University School and Hospital of Stomatology, Beijing, China

Approved with Reservations

https://doi.org/10.5256/f1000research.154596.r254085

This manuscript provides a study protocol for enzyme-linked immunosorbent assay and immunohistochemical analysis of mast cell-related biochemicals in OSMF, and although some plans for the experiments are provided, the details of the experimental methodology and subsequent analyses need to be further optimized, and the Discussion section of the article should have included a discussion of studies on mast cells and OSMF or oral potentially malignant disorders, which would have enabled the reader to understand the potential significance of the study.
However, studies related to mast cells in OSMF deserve to be investigated. Mast cells are myeloid-derived immune cells located primarily in tissues in contact with the external environment that can be stimulated by a variety of different triggers to act as sentinel immune cells, recruiting other immune cells into the affected microenvironment and altering their function by releasing chemokines, cytokines and other factors. The study of mast cells might help to elucidate the changes in the immune microenvironment in OSMF development and malignant transformation and provide an experimental basis for the development of immunotherapies.
Introduction:
Mast cells could be classified into two groups according to whether they contain tryptase without chymase (MC_T) or both tryptase and chymase (MC_TC), and according to the studies that have been reported, there were different roles for the two mast cell subtypes in cancer progression, and it is possible that both tryptase and chymase might play a role in the developments and malignant transformation of OSMF.
Objectives:
Since OSMF is a disease confined to the oral mucosa, it is suggested to correlate the serum levels and immunohistochemical expression levels of the mast cell secretions to clarify whether the changes in immunohistochemical expression levels would be more pronounced than the changes in serum levels.
As mast cells can be divided into two subtypes, MC_T and MC_TC, the analysis of changes in mast cell subtypes and their spatial localization using immunofluorescence could be one of the further progress in this study.
Method:
How was the P (population ratio) of 0.0721 in the sample size calculation determined and is it based on any references?
Please describe the tools or methods used to identify molecular expression after immunohistochemical staining of tissue sections.
Expected outcomes:
The effect of mast cells on the malignant transformation cannot be identified in the current study protocol. The prognostic information of the patient needs to be clarified (e.g. through regular clinical examination or follow-up, etc.), and correlation analyses between molecular expression and prognostic information need to be included in the study objectives, which may help to understand the immune mechanisms and microenvironmental alterations of malignant transformation in OSMF.
Discussion: The current Discussion section does not provide enough information. Studies have reported the role of mast cells in OSMF as well as in other oral potentially malignant disorders and oral cancer, and although this study was only a study protocol, the authors should have discussed the results of the previous studies, clarifying the differences between this study and the previous studies, the existence of any additions to the previous results, and the implications of the study in terms of inspiration for future research. In the current manuscript, understanding the potential mechanisms of mast cell-related biochemicals in OSMF pathogenesis or carcinogenesis is difficult, and deepening the Discussion section may help to remedy this.

Is the rationale for, and objectives of, the study clearly described?

Partly
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

Yes
Are the datasets clearly presented in a useable and accessible format?

Not applicable

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Malignant transformation of oral potentially malignant disorders.

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Author Response 29 Apr 2024

Harshkant Gharote, Oral Medicine and Radiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Higher Education and Reasech, Sawangi (Meghe), Wardha, 442004, India

29 Apr 2024

Author Response

Suitable changes are made with addition of relevant information in the introduction section as suggested.
Sample size calculation is elaborated with citation.
Expected outcomes have been explained in details in ... Continue reading Suitable changes are made with addition of relevant information in the introduction section as suggested.
Sample size calculation is elaborated with citation.
Expected outcomes have been explained in details in context to the results of the assays.
Discussion is supported with other studies with citations and the relevant information.
Suitable changes are made with addition of relevant information in the introduction section as suggested.
Sample size calculation is elaborated with citation.
Expected outcomes have been explained in details in context to the results of the assays.
Discussion is supported with other studies with citations and the relevant information.
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Author Response 29 Apr 2024

Harshkant Gharote, Oral Medicine and Radiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Higher Education and Reasech, Sawangi (Meghe), Wardha, 442004, India

29 Apr 2024

Author Response

Suitable changes are made with addition of relevant information in the introduction section as suggested.
Sample size calculation is elaborated with citation.
Expected outcomes have been explained in details in ... Continue reading Suitable changes are made with addition of relevant information in the introduction section as suggested.
Sample size calculation is elaborated with citation.
Expected outcomes have been explained in details in context to the results of the assays.
Discussion is supported with other studies with citations and the relevant information.
Suitable changes are made with addition of relevant information in the introduction section as suggested.
Sample size calculation is elaborated with citation.
Expected outcomes have been explained in details in context to the results of the assays.
Discussion is supported with other studies with citations and the relevant information.
Competing Interests: None Close
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Version 3

VERSION 3 PUBLISHED 09 Oct 2023

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Version 1 09 Oct 23	read

Xinjia Cai, Peking University School and Hospital of Stomatology, Beijing, China
Vini Mehta, Dr. D. Y. Patil Dental College and Hospital, Pimpri, India

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30 May 2024 | for Version 3

Vini Mehta, Dr. D. Y. Patil Dental College and Hospital, Pimpri, India

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Approved

The authors have satisfactorily done all the corrections.
The manuscript can be indexed in the current form.

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No competing interests were disclosed.

Reviewer Expertise

Public Health, Oral Cancer,

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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10 May 2024 | for Version 2

Vini Mehta, Dr. D. Y. Patil Dental College and Hospital, Pimpri, India

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Approved With Reservations

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

Yes
Are the datasets clearly presented in a useable and accessible format?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Public Health

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Author Response

21 Jun 2024

Harshkant Gharote, Oral Medicine and Radiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Higher Education and Reasech, Sawangi (Meghe), Wardha, 442004, India

Thank you for Valuable inputs.
The software used for sample size formula is provided by the research methodology cell of the institute and the details are added to the manuscript. Same software will be used to analyze the data.
The statement for the confidence interval and p-value are added.
There will be no CTRI registration for the study
The reference suggested has been included and cited in the manuscript.
There will be no CTRI registration for the study protocol.

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07 May 2024 | for Version 2

Xinjia Cai, Peking University School and Hospital of Stomatology, Beijing, China

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Approved

There are no further comments.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Malignant transformation of oral potentially malignant disorders.

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01 Apr 2024 | for Version 1

Xinjia Cai, Peking University School and Hospital of Stomatology, Beijing, China

25 Views Cite this report Responses(1)

Approved With Reservations

Is the rationale for, and objectives of, the study clearly described?

Partly
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

Yes
Are the datasets clearly presented in a useable and accessible format?

Not applicable

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Malignant transformation of oral potentially malignant disorders.

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Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Enzyme-linked immunosorbent assay and immunohistochemical analysis of mast cell related biochemicals in oral submucous fibrosis

Abstract

Keywords

Revised Amendments from Version 2

Introduction

Protocol

Objectives

Method

Experimental design

Selection of OSMF patients

Selection of individuals with areca habit without OSMF

Selection of Healthy individuals

Exclusion criteria

Materials

Procedure

Statistical analysis

Expected outcomes

Discussion

Ethical considerations

Study status

Data availability

References

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