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ORIGINAL ARTICLE
Year : 2021  |  Volume : 12  |  Issue : 3  |  Page : 136-140

An exfoliative cytology study to assess oral mucosa of oral submucous fibrosis patients in Chennai, Tamil Nadu: A preliminary study


Department of Oral Pathology, SRM Dental College and Hospital, Chennai, Tamil Nadu, India

Date of Submission31-Dec-2020
Date of Decision18-Jun-2021
Date of Acceptance18-Jun-2021
Date of Web Publication17-Sep-2021

Correspondence Address:
Dr. N Vaishnavi Dhanvantri
Department of Oral Pathology, SRM Dental College and Hospital, Chennai, Tamil Nadu
India
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DOI: 10.4103/srmjrds.srmjrds_139_20

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  Abstract 

Background: Oral submucous fibrosis (OSMF) is a chronic, insidious disease that affects the oral mucosa. It is a progressive high risk potentially malignant disorder that leads to oral mucosa scarring. Aim: In this study, we aimed to study and compare the differences in the buccal cytological smear and the nuclear and cytoplasmic changes seen in the exfoliated cells of patients with OSMF and healthy controls, during preliminary screening. Materials and Methods: Patients with clinical OSMF (reduced mouth opening, blanched buccal mucosa, burning sensation in the buccal mucosa, palpable vertical bands) were included in the study. The cytological smears were stained with Papanicolaou stain and studied under light microscopy. The nuclear and cytoplasmic changes were recorded. The cytologic features that were assessed included the presence of keratinized squames, intermediate cells, inflammatory cells, cytoplasmic creasing, peri nuclear halo, and abnormal chromatin. In addition, the presence and absence of candidal organisms in the smear were assessed. Results: The cytological features which were seen to predominate in the OSMF patients were cells of intermediate and keratinized buccal epithelial cells, cytoplasmic creasing, and presence of inflammatory cells. Conclusion: We conclude that the results of our study indicate that there are cytological changes associated with OSMF that can be assessed with exfoliative cytology. The cellular pattern alterations recorded in our study adds on to the current knowledge pool and literature on OSMF. Further studies are necessary to validate the significance of such cellular alterations.

Keywords: Cytology, oral submucous fibrosis, oral potentially malignant disorder


How to cite this article:
Dhanvantri N V, Karthik R, Rajkumar K. An exfoliative cytology study to assess oral mucosa of oral submucous fibrosis patients in Chennai, Tamil Nadu: A preliminary study. SRM J Res Dent Sci 2021;12:136-40

How to cite this URL:
Dhanvantri N V, Karthik R, Rajkumar K. An exfoliative cytology study to assess oral mucosa of oral submucous fibrosis patients in Chennai, Tamil Nadu: A preliminary study. SRM J Res Dent Sci [serial online] 2021 [cited 2021 Dec 9];12:136-40. Available from: https://www.srmjrds.in/text.asp?2021/12/3/136/326205




  Introduction Top


Dentists encounter a wide spectrum of oral mucosal lesions in their ever clinical practice. Considering the wide range of diseases found, an adequate diagnosis is essential as these lesions may vary in nature from simple to life-threatening ones. Chairside simple noninvasive tools like exfoliative cytology should be adopted in clinical practice to aid in early diagnosis of various lesions including premalignant disorders.

Oral submucous fibrosis (OSMF) is a chronic, insidious disease that affects the oral mucosa. It initially results in loss of fibroelasticity of the lamina propria and as the disease advances, results in fibrosis of the lamina propria and the submucosa of the oral cavity along with epithelial atrophy. It is a potentially malignant disorder that leads to stiffness of mucosa and restricted mouth opening.[1],[2] Areca nut is the major etiological factor. OSMF is now considered to be a collagen metabolic disorder.[3] Gutkha chewing habit among young Indians is known to be a major factor for OSMF cases amidst the various predisposing factors.[4],[5] An important feature of OSMF is epithelial atrophy. Oral mucosa scarring/fibrosis leads to limited mouth opening in OSMF patients that may predispose the patients to candidal growth.[5] It is essential to identify the association of candida in OSMF patients for a thorough understanding of the disease.

Exfoliative cytology aids in chairside diagnosis and identification of epithelial changes in various oral mucosal lesions and disorders. These changes are represented by variations in the nucleus, nuclear area and the nuclear-cytoplasmic ratio. The detection of qualitative and quantitative cellular changes by exfoliativecytology may assist in the diagnosis of a number of oral lesions. The cytoplasmic diameter, nuclear diameter, and nuclear to cytoplasmic ratio are key parameters in cytology and aid in detecting oral lesions including potential and frank malignancy. Alterations in the mean cytoplasmic, nuclear diameter of cells, and nuclear changes in the smear might be early indicators of dysplasia.

However, a combination of several structural changes including nuclear changes is essential for the diagnosis of premalignancy and malignancy.

Even after cessation of habits such as gutka chewing, OSMF does not seem to regress, nor does it regress spontaneously.[6] The submucous fibrosis persists and may increase in severity with time involving other oral mucosal regions. Success of the treatment options for this potentially malignant disorder is variable. The current therapies include Glucocorticoids (Local and systemic applications), placental extracts, and hyaluronidase used as intralesional injections. They however do not provide a complete cure. Early diagnosis and treatment of OSMF is essential. If OSMF patients show characteristic epithelial cellular changes in their buccal exfoliated cells, it would be a simple and noninvasive method, to aid in the early diagnosis of OSMF. This study is an attempt to use exfoliative cytology procedure for this potentially malignant disorder for early detection reducing their progression to malignancy.

Aim

To study and compare the differences in the buccal cytological smear and the nuclear and cytoplasmic changes seen in the exfoliated cells of patients with OSMF and healthy controls. In addition, we also aimed to assess and compare the presence or absence of candidal organisms in the smears.


  Materials and Methods Top


The study was conducted on patients attending the Outpatient department of SRM Dental College, Chennai, Tamil Nadu. Institutional ethical clearance was obtained. Patients were first asked about their demographic details and their medical history. All patients were screened for the presence of any oral lesions such as aphthous ulcers and other tobacco-associated lesions, and excluded if found any. The patients with other dental issues without any oral lesions were considered as normal controls (n = 50). Patients with clinical OSMF (reduced mouth opening, burning sensation in the buccal mucosa, blanched buccal mucosa, palpable vertical bands) were included in the study (n = 50). Informed consent was obtained from all the patients included in the study. Only the patients who had not undergone any treatment for OSMF were included in the study. Patients with any other clinically visible oral lesion, patients with any systemic illness, and those under long-term medications were excluded from the study. The OSMF patients were then asked to undergo a routine blood investigation as part of the treatment plan.

Oral smears are made using a tongue blade/wooden stick by scraping the buccal mucosa with a gentle pressure for 2–3 times and then smeared onto the slide. The slide is then immediately fixed using an alcohol spray. After the oral smears were taken, the patients received treatments based on their individual clinical staging as per the current protocol, including iron, multivitamins including lycopene, local submucosal injections (steroids, hyaluronidase) and surgical excision of the fibrous bands (in 4 cases).

The slides were stained by rapid Papanicolaou (PAP) method using the rapid PAP kit. The slides were then studied under light microscopy and the nuclear and cytoplasmic changes were recorded. The cytologic features that were assessed included the presence of keratinized squames, intermediate cells, inflammatory cells, cytoplasmic creasing, peri-nuclear halo, and abnormal chromatin. 100 cells were analyzed in each slide. The cytological changes were assessed qualitatively. In addition, the smears were also assessed for the presence of candidal hyphae. Each slide was assessed by two observers (pathologists) separately. No variations were seen in the slide report between the two. Statistical analysis was performed using Chi-square test, and a P < 0.05 was considered statistically significant.


  Results Top


The sample size was equally distributed between the OSMF patients and healthy controls with n = 50 each. The patient group comprised of 39 males and 11 females with a mean age of 31 years. The healthy control group included 25 males and 25 females with a mean age of 34 years.

Group-wise distribution of the cytological features is highlighted in [Table 1]. The cytological features which were seen to predominate in the OSMF patients were cells of keratinized buccal epithelial cells [Figure 1], and intermediate cells [Figure 2], cytoplasmic creasing [Figure 3], and presence of inflammatory cells [Figure 4]. Increased nucleus size was also observed in few cells. It was found that cytoplasmic creasing and inflammatory cells showed equal distribution. Peri-nuclear halo and abnormal chromatin condensation were seen in only 4 of the 50 cases (8%) [Figure 5]. There was no candidal organism detected in any of the OSMF patients or normal patients. The cytological changes seen in the buccal exfoliated squames were insignificant with a P = 0.12.
Figure 1: Numerous keratinized squamous cells stained orange to pink in the buccal smear of oral submucous fibrosis patient, PAP (×400)

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Figure 2: Numerous keratinized squames and intermediate cells (black arrows) in the buccal smear of oral submucous fibrosis patient, PAP (×400)

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Figure 3: Perinuclear halo and cytoplasmic creasing (black arrows) in the buccal smear of oral submucous fibrosis patient, PAP (×400)

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Figure 4: Inflammatory cells, in the buccal smear of oral submucous fibrosis patient, PAP (×400)

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Figure 5: Abnormal chromatin pattern - in the buccal smear of oral submucous fibrosis patient, PAP (×400)

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Table 1: Group wise distribution of the cytologic features

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  Discussion Top


Oral submucousfibrosis, a potentially malignant disorder of oral mucosa requires early diagnosis to start prompt treatment. Gutkha and other areca nut products usage and the frequency, duration, and the type of chewing habit determined the severity and progression of the lesion. Chewing for a long duration and swallowing the products posed a higher risk of severe lesion and cause early symptoms clinically.[6] In our study, 94% of the OSMF patients from Chennai, Tamilnadu used gutkha and betel quid that placed them at a higher risk of severe lesion and progression to malignancy.

Cellular morphology reflects basic defects at the molecular level. The assessment of the exfoliated cells of oral mucosa will assist in monitoring the status of the mucosa. A combination of several structural changes including nuclear changes is essential for the diagnosis of premalignancy and malignancy. To detect alterations in the nuclear and cellular dimensions, cytomorphometric analysis using exfoliative cytology smears can be performed on a regular basis. The exfoliated buccal cells of normal mucosa usually reveal different cells from the epithelium and color ranging from orange to pink depending upon the degree of maturation. If basal cells are found in the smear, their cytoplasm stains dark to deep blue in color with PAP stain.

In this study, the cytological features which were seen to predominate in the OSMF patients were keratinized cells (anucleated and nucleated cells staining orange to pink) and intermediate buccal epithelial cells that stained green to blue in color with PAP stain, and cytoplasmic creasing. The presence of increased number of intermediate cells could indicate early atrophic changes. Since very few cases showed the presence of inflammatory cells, abnormal chromatin pattern, and perinuclear halo, these cytological changes could be attributed to the general condition of the patient and may not be specific to OSMF.

The parameters-cell diameter and nuclear diameter have been used in morphometric analysis of cells in numeral studies.[7] A decrease in cytoplasmic diameter and an increase in nuclear diameter in OSMF patients were observed in a recent study by Jaitley et al. Such changes were concluded to be early indicators malignant potential of the lesions.[7] Changes, if any, in nuclear and cell size were not very prominent in our study. This emphasizes the fact that changes in nuclear-cytoplasmic ratio and pleomorphisms occur only in cases that have malignant transformation.

In this study, candidal organisms were not detected in any of the OSMF patients. Yeast cells like Candida albicans must adhere and anchor to some surface in the oral cavity to proliferate, or else they can be easily washed off by the flow of saliva. Thus these organisms have developed varied mechanisms to adhere and colonize and their virulence is affected by their ability to do so. The constant and prolonged chewing habits generally seen in OSMF patients lead to increased salivary rate and flow. This increased salivary flow rate might make it difficult for the candidal organisms to colonize and grow in the oral cavity of OSMF patients. Furthermore, betel quids have hydrophilic solvents that have inhibitory effects on candidal growth and colonization. Acidic pH is known to favor candidal adherence to epithelial cells. Gutkha has slaked lime that raises the pH and leads to alkalinity in the oral cavity, thus preventing candidal colonization.[4],[6] All of the above-mentioned factors are the probable reasons for the absence of candida in OSMF patients in our study. On the contrary, several studies show that fibrosis of minor salivary glands, atrophy of acinar cells with disease progression, and fibrosis of ducts of major salivary glands, could lead to decreased salivary flow rate. This variation between the study results could be due to the type of habit and duration of the habit and the severity of the disease.[8],[9]


  Conclusion Top


We conclude that the results of our study indicate that there are cytological changes associated with OSMF that can be assessed with exfoliative cytology. The cytological changes seen in the buccal exfoliated squames were in majority, nonspecific and could be applied to a number of other oral lesions. The cellular pattern alterations recorded in our study adds on to the current knowledge pool and literature on OSMF. It is necessary to validate the significance of such cellular alterations in relation to malignant transformation potential. To improve the diagnostic value of oral exfoliative cytology, quantitative techniques can be applied to buccal smears. Thus, further studies using quantitative techniques with a larger sample size and clinical staging of OSMF, are essential to identify the specific pathognomic changes in the buccal exfoliated squames in OSMF patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Chaudhuri G, Das R, Paul M, Kundu S. Oral submucous fibrosis associated with malignancy. SRM J Res Dent Sci 2014;5:55-8.  Back to cited text no. 1
  [Full text]  
2.
Warnakulasuriya S, Kujan O, Aguirre-Urizar JM, Bagan JV, González-Moles MÁ, Kerr AR, et al. Oral potentially malignant disorders: A consensus report from an international seminar on nomenclature and classification, convened by the WHO Collaborating Centre for Oral Cancer. Oral Dis 2020 Oct 31. doi: 10.1111/odi.13704. Epub ahead of print. PMID: 33128420.  Back to cited text no. 2
    
3.
Rajesh SB, Reddy SB, Ramamurthy TK, Srinivas K, Patil S. Cytomorphometric analysis of squames obtained from normal oral mucosa and lesion of Oral submucosa and lesions of oral submucous fibrosis. J Indian Aca Oral Med Radiol 2012;24:200-5.  Back to cited text no. 3
    
4.
Ramlal G, Reddy G, Venkateshwarulu N, Shalini K, Rajshekar Patil. Significance of oral brush biopsy of oral mucosal lesions in Gutka chewers: A prospective study. J Indian Aca Oral Med Radiol 2011;23:539 42.  Back to cited text no. 4
    
5.
Anila K, Hallikeri K, Shubhada C, Naikmasur V, Kulkarni R. Comparative study of candida in oral submucous fibrosis and healthy individuals. Rev Odonto Ciênc 2011;26:71-6.  Back to cited text no. 5
    
6.
Supadminidevi G, Jayanthi K, Bina S, Kamala R. Evaluation between betel quid and pan masala/Gutka Habitual chewers of OSMF. World J Dent 2012;3:37-40.  Back to cited text no. 6
    
7.
Jaitley S, Agarwal P, Upadhyay R. Role of oral exfoliative cytology in predicting premalignant potential of oral submucous fibrosis: A short study. J Cancer Res Ther 2015;11:471-4.  Back to cited text no. 7
    
8.
Abdul Khader NF, Dyasanoor S. Assessment of salivary flow rate and pH among areca nut chewers and oral submucous fibrosis subjects: A comparative study. J Cancer Prev 2015;20:208-15.  Back to cited text no. 8
    
9.
Prashant T, Rashmi W, Sunita K, Ajay C, Sanchit J, Chalapathi KV, et al. Comparative evaluation of alteration in salivary pH among gutkha chewers with and without oral submucous fibrosis and healthy subjects: A prospective case. Control Study 2020;3:8-16.  Back to cited text no. 9
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1]



 

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