|Year : 2015 | Volume
| Issue : 2 | Page : 86-93
Keratinocytes in oral submucous fibrosis: A cytomorphometric analysis using computer aided image analyser
Pratibha Kemtur, Swetha Acharya, Kaveri Hallikeri
Department of Oral Pathology and Microbiology, Rajiv Gandhi University of Health Sciences, SDM College of Dental Sciences and Hospital, Dharwad, Karnataka, India
|Date of Web Publication||14-Jul-2015|
Department of Oral Pathology and Microbiology, SDM College of Dental Sciences and Hospital, Dharwad - 580 009, Karnataka
Source of Support: None, Conflict of Interest: None
Aim: To examine and compare cytomorphometric changes in the keratinocytes obtained from buccal mucosa of oral submucous fibrosis (OSF) cases in different clinical stages with healthy subjects and oral squamous cell carcinoma (OSCC) cases.
Materials and Methods: Buccal mucosal smears of OSF cases [n = 30], OSCC cases [n = 30] and age-sex matched healthy controls [n = 30] were stained with Papanicolou stain and observed under light microscope (40x objective magnification) and 100 cells in each case were analysed cytomorphometrically using an image analyser. The average nuclear area (NA), cell area (CA), ratio of nuclear area/cell area, nuclear diameter (ND), cell diameter (CD) and ratio of nuclear diameter/cell diameter was obtained for each case. One-way ANOVA and Tukey-HSD procedure (P < 0.05) were used to analyze all the parametric variables.
Results: The comparison of mean values of the NA, CA, NA:CA, ND, CD, ND:CD shows statistically significant difference among the healthy, OSF and OSCC groups [P < 0.05]. OSF group showed significant increase in nuclear dimensions and N/C ratio and significant decrease in cell dimensions compared to healthy group (P < 0.05). Among the parameters analysed ND:CD and NA:CA showed a highly statistically significant difference between the OSF and healthy group (P < 0.001).
Conclusions: There are significant alterations in the cell and nuclear dimensions of the buccal mucosal cells in OSF patients. These alterations could be attributed to usage of various tobacco and areca nut preparations and due to the mucosal changes (atrophic epithelium, high epithelial turnover) in the OSF. Atrophic epithelium, in OSF is envisaged to predispose malignant transformation with persistent exposure oral carcinogens. As the mean values of CA, NA:CA in stage IV OSF are close to OSCC cases, it strengthens the view that advancement of fibrosis increases the risk of development of epithelial dysplasia.
Keywords: Cytomorphometry, keratinocytes, oral submucous fibrosis
|How to cite this article:|
Kemtur P, Acharya S, Hallikeri K. Keratinocytes in oral submucous fibrosis: A cytomorphometric analysis using computer aided image analyser. Saudi J Oral Sci 2015;2:86-93
|How to cite this URL:|
Kemtur P, Acharya S, Hallikeri K. Keratinocytes in oral submucous fibrosis: A cytomorphometric analysis using computer aided image analyser. Saudi J Oral Sci [serial online] 2015 [cited 2019 Dec 15];2:86-93. Available from: http://www.saudijos.org/text.asp?2015/2/2/86/160772
| Introduction|| |
Oral submucous fibrosis [OSF] is a chronic condition characterised by mucosal rigidity of varying intensity due to fibro-elastic transformation of the juxta-epithelial layer. This leads to restricted oral opening.  The pathogenesis of the OSF is believed to be multifactorial. Factors include areca nut chewing, ingestion of chillies, genetic and immunologic processes, nutritional deficiencies and other factors.  Data from recent epidemiological studies provide overwhelming evidence that areca nut is the main etiological factor for OSF.  Recent epidemiological data indicates that the number of cases of OSF has risen rapidly in India from an estimated 250,000 cases in 1980 to 2 million cases in 1993.  The reasons for the rapid increase of the disease are reported to be due to an upsurge in the popularity of commercially prepared areca nut preparations (pan masala, guthka) in India  and an increased addiction to this habit by young people due to easy access, effective price changes, and marketing strategies, which claim these to be safer products. ,
OSF is a well recognised potentially malignant condition. Malignant transformation rate was found to be in the range of 7-13%.  Many oral carcinomas arise within regions that previously had premalignancy.  Because of the premalignant nature of OSF, early detection and treatment is of paramount importance. Although surgical biopsy remains the most definitive method of diagnosis of premalignant and malignant lesions, exfoliative cytology has proved to be a reliable primary diagnostic test due to its simplicity and non invasiveness. 
Miller et al., were the first to study the cytology of the normal oral epithelium. The superficial epithelial cells contain nuclei, and thus, alterations in these cells can serve as reliable indicators of dysplastic or neoplastic changes.  Cowpe et al., pointed out that the application of quantitative techniques to cytology could markedly improve its diagnostic sensitivity.  Cowpe and colleagues first applied morphometry to oral smears. Cowpe et al., in a quantitative exfoliative cytology of normal oral squames: An age, site and sex related survey displayed a significant variation in nuclear area (NA) and cell area (CA) between different sites. Nuclear size varied significantly with advancing age; however, this was not the case for CA. There was no significant variation in either criterion between males and females.  Whereas Nayar et al.,  and Donald et al.,  noted age related variations in nuclear diameter (ND), cell diameter (CD) and N:C ratio and variations in the pattern of exfoliated normal buccal mucosal cells in female subjects and concluded it be probably as result of hormonal influence. Reddy et al., in a study on normal buccal mucosa under various age groups concluded that increase in ND and decrease CD are due to ageing process and decreased cell metabolic activity. 
Cowpe et al., used a planimetry method to measure NA and CA in a variety of normal and abnormal smears. The planimetry method was then replaced by an image analysis system. Their findings were that malignancies and some premalignancies displayed a reduction in cell size and an increase in nuclear size. 
In 1980s, cytological studies were restricted largely to the subjective or qualitative interpretation. But now, with advance technology, more reliable quantitative techniques like cytomorphometry, histometry, densitometry and use of computer-assisted image analyser, will nullify the interobserver variation. Objective measurements of quantitative parameters could be more useful than subjective parameters. Since the quantitative parameters are objective and reproducible; they are important adjuncts in making a cytopathological diagnosis. ,
Morphometry can be applied selectively on samples, which are difficult to assess accurately, like variation in cell and nuclear dimensions.  There are several studies in the literature that have used CD and ND parameters in morphometric analysis of cells in absence of the defined normal baseline values. For oral smears it is suggested that quantitative techniques, based on evaluation of parameters, such as NA, CA and NA:CA may increase sensitivity of exfoliative cytology for early detection of malignant transformation.  The present analysis employs exfoliative cytology and image analysis for scrutinizing the keratinocytes of the buccal mucosa to estimate the NA, CA, ND, and CD and to compute the changes in the above variables among the groups.
| Materials and Methods|| |
The samples for the analysis were obtained from the buccal mucosa of OSF cases of different clinical stages, OSCC patients and from age and sex matched healthy subjects following an informed consent. The samples for the study and control groups were taken from patients who report to the Department of Oral Diagnosis and Radiology and Department of Oral and Maxillofacial Surgery, of the institution.
The study group included 30 cases of OSF and 30 healthy subjects, 30 OSCC cases as negative and positive controls respectively. The OSF cases were divided into four stages based on mouth opening parameters of normal patients as suggested by Ranganathan et al., 2001.  We included 10 cases each from stage II, stage III and stage IV OSF. No patients belonging to stage I was included. Stage I includes individuals having only symptoms, with no demonstrable restriction of mouth opening. Stage I patients rarely report at such an early phase. OSF patients who had taken any treatment prior or with systemic illness were excluded. Clinically and histologically confirmed cases OSCC of buccal mucosa served as positive controls. OSF and OSCC patients with a history of chronic exposure to tobacco and/areca preparations were included in the study.
Age and sex matched healthy subjects without any exposure to tobacco and areca nut preparations and who are free from oral and systemic diseases served as negative controls. As alterations in cytomorphometry in the normal buccal mucosal cells may be due to aging, hormonal changes, tobacco habits, betel quid chewing, nutritional deficiencies (anemia) and systemic illness (diabetes) has been mentioned in the literature. ,,,,,
Thorough oral examination was done after recording a comprehensive case history. Buccal mucosal smear samples were collected using a wooden spatula after rinsing the mouth. Samples were spread evenly on a clean labeled glass slide and fixed. After fixation with 95% alcohol for 15 minutes smears were stained with Papanicolou stain.
Smears were coded to ascertain blinded protocol. From each subject hundred separate non overlap maturing kertinocytes with definite borders were selected at random from the stained smears under a research light microscope, 40× objective magnifications. Images of the cells were captured and stored, to evade repetition. The stored images were then subjected to morphometric analysis using Leica Qwin Plus version 3 Software. Parameters like NA, CA, ND and CD were measured for each cell using feature measurement [Figure 1],[Figure 2] and [Figure 3]. The outline of the cell and nucleus were traced with the digitizing pen stylus separately on the screen and the software automatically calculated the area. The area measurements were expressed in square microns. ND and CD were also measured, in microns for the same cell. The ND and CD were calculated taking the mean of diameters in two perpendicular planes. Mean NA, CA, ND, CD and NA:CA, ND:CD was calculated for each case.
|Figure 1: Measurement of Nuclear diameter using the image analysis software program|
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|Figure 2: Measurement of cell diameter using the image analysis software program|
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|Figure 3: Ratio of nuclear area/cell area using the image analysis software program|
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Statistical analysis was done using one way ANOVA (Analysis of Variance) for comparing the parameters for the multiple groups namely Control, OSF and SCC and also for the clinical stages II, III and IV of OSF. Pair-wise comparison between the groups was done using multiple comparison tests by Tukey-HSD procedure. The P value <0.05 was considered to be significant.
| Results|| |
Ninety subjects with age ranging from 25-55 years were enrolled in this study. All subjects were males and most of the patients in OSF and OSCC groups were chronic betel quid or gutkha chewers. Statistical analysis was carried out to find the difference between measured parameters of different groups. The descriptive statistics is shown in [Table 1] and [Table 2]. The result of one way ANOVA showed that the difference in mean NA, ND, NA, CA, NA:CA, ND:CD across all the three groups was statistically significant (P < 0.05).
|Table 1: Comparison of study parameters between the study and control groups|
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|Table 2: Comparison of study parameters between the study and control groups|
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The nuclear dimensions and ratio N/C dimensions of keratinocytes in OSF smears was much greater than that of healthy, but was lesser than OSCC smears. The mean value of cellular dimensions was lesser in OSF than healthy but greater than that of OSCC [Figure 4],[Figure 5] and [Figure 6].
|Figure 6: Mean value of ratio of nuclear diameter/cell diameter among the study groups|
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[Table 3] shows parameters like NA, NA:CA, ND, CD, ND:CD showed significant difference between healthy vs. OSF, healthy vs. OSCC and OSF vs OSCC groups [P < 0.05]. CA showed significant difference between healthy vs. OSF and healthy vs. OSCC [P < 0.05]. There was no significant difference between OSF vs OSCC groups for CA [P = 0.45].
Among the parameters analysed by Tukey-HSD comparisons of the three groups for the difference in NA:CA indicated that the OSF (M = 0.028) had significantly higher NA:CA than healthy (M = 0.021), (P < 0.001), no significant difference with OSCC (M = 0.034), (P < 0.001). OSF (M = 10.04, 0.16) had significantly higher ND and ND:CD than healthy (9.01, 0.13), (P < 0.001), significantly lower ND, ND:CD than OSCC(M = 0.034, 10.99, 0.18), (P < 0.001).
Among the three clinical stages II, III and IV of OSF subjects there was no statistical significant difference in the mean values of various parameters as shown in [Table 4]. [Table 5] depicts multiple comparisons of parameters using Tukey HSD procedure. The parameters like NA, NA:CA, ND, CD and ND:CD showed no statistical significant difference between IIvs. III, III vs. IV and IV vs. II OSF groups.
|Table 4: Comparison of study parameters between the three different stages of OSF|
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| Discussion|| |
Papanicolaou staining technique differentiates epithelial cells according to the degree of keratinisation within the cells. Cells of the basal layer stain green, fully keratinised cells stain red, and intermediate cells show shades of orange with this technique. The present study analysed the maturing kertinocytes mostly superficial cells which were orange to pink.
Since the epithelium in the OSF patients was atrophic and parched, it was difficult to take the scraping samples without hurting patients. The smear samples had less number of well-defined, non overlapping maturing keratinocytes than healthy. The intermediate and parabasal cells which are are turquoise green to blue were more compared to cells superficial cells, because of high epithelial turnover rate in OSF. Smears obtained from OSCC cases were with abundant inflammatory cells and debris apart from having dysplastic kertinocytes.
Significant decrease in cell dimensions, increase in the nuclear dimensions and ratios were observed in smears obtained from OSF cases, as compared to healthy controls [P < 0.05]. These changes in the OSF cases may be attributed to local trauma and mechanical injury to the oral mucosa caused by areca nut chewing due to its abrasive nature. This could be more severe in users of pan masala and guthka due to their fine particulate nature, with the high probability of particle adhesion to the traumatized mucosa, leading to morphological changes and membrane damage.  Guthka and pan masala could also result in the formation of high levels of reactive oxygen species (ROS) close to buccal mucosa, overhelming the protective enzymes and thus causing direct damage to the tissue.  Carcinogens reduce the ability of the cytoplasm to mature, so that there is a greater immaturity of the cytoplasm of the cell with greatly increased activity of the nucleus leading to increase in the nuclear parameters and decrease in cellular parameters.  Increase in nuclear size could be produced as an inflammatory change due to chronic irritation of the oral mucosa caused by tobacco habits. 
High proliferative activity and basal cell hyperplasia in conjunction with rapid exfoliation of superficial cells and epithelial atrophy suggestive of very high epithelial turnover in OSF have been mentioned in the literature.  The above mentioned changes in cytomorphometry observed in OSF could also be attributed to this.
It has been hypothesized that OSF was an Asian version of sideropenic dysphagia wherein the chronic deficiency leads to mucosal susceptibility to irritants such as chilli and areca nut. Several observations did not establish an etiologic or contributory role for nutritional deficiencies in OSF. It is probable that the nutritional deficiency among OSF patients may be secondary.  Nutritional deficiencies may not play a primary role but it could synergize the symptoms by contributing to epithelial atrophy.  In the present study the mean cell dimensions show a significant decrease in OSF than healthy smears. This may be in part due to micronutrient deficiency having an effect on integrity of oral epithelium in OSF cases. 
Boddington et al., in a study on changes in buccal cells in anemias noted that CD of keratinocytes in iron deficiency anaemic patients are likely to smaller than normal healthy patients. But the mean ND was within normal limits.  Sumathi et al., in an analysis of exfoliative buccal cells in iron deficiency anemic patients revealed significant increase in the average ND and N/C ratio of the anemic group when compared to the control group. 
Significant hematological abnormalities have been reported in OSF subjects.  Although OSF and iron deficiency anaemia exist as separate conditions, the clinical and histological findings of OSF mimic those of iron deficiency. Thus, this unclear line of demarcation between both these conditions still persists. 
The mean value of NA, ND, NA:CA and ND:CD of keratinocytes in OSF smears was much greater than that of health, but was lesser than OSCC. The ratios of NA:CA and ND:CD was highest OSCC group. Malignant cells show a significant increase in nuclear size as compared to normal cells. This increase in nuclear dimension is related to an increase in the nuclear contents required for replication.  Compared to the healthy subjects and OSF patients, means CA, CD were lowest in OSCC cases. It is noted that in cells with increased activity, the ability of cytoplasm to mature diminishes. The amount of cytoplasm produced in relation to nucleus is less, resulting in decreased cell dimensions. 
A gradual increase in nuclear dimensions from healthy to OSF, from OSF to OSCC smears were noted. Steady decrease in cell dimensions was observed normal to OSF, from OSF to OSCC smears. This is in accordance with Gao et al., conclusion that the pathologic grade of OSF is between normal mucosa and mild epithelial dyplasia.  These alterations may be owing to the genotoxic effects most likely caused by tobacco and areca nut specific nitrosamines and ROS generated by areca nut, catechu polyphenols and slaked lime as proposed by Nair et al. Oxidative stress and subsequent ROS generation can induce cell proliferation, cell senescence or apoptosis, depending upon the level of ROS; during chronic exposure, these events can lead to preneoplastic lesions in the oral cavity and subsequently to malignancy. 
A brief review of literature on cytomorphometric analysis in potentially malignant and malignant lesions has been tabulated in [Table 6]. ,,,,,,,,
|Table 6: Brief review of literature on cytomorphometric analysis in potentially malignant and malignant lesions|
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In premalignant and malignant lesions the epithelium shows alterations at the molecular level and this is reflected in the cellular morphology  as dysplastic changes like increased in nuclear cytoplasmic ratio, cellular and nuclear pleomorphism, and nuclear hyperchromatism. Karyorrhexis, karyolysis, micronucleus formation, pyknosis, binucleation, anucleation and broken egg appearance are the other nuclear abnormalities noted in the oral smears.  Hence sampling of individual cells might improve the chances of detecting dysplastic changes.
It has been mentioned in literature that smoking tobacco and betel quid/gutkha chewing can produce cytomorphometric alterations. ,,,, Cytomorphometric changes have been noted in several potentially malignant disorders such as OSF, leukoplakia and OLP. ,,,,,, However the synergistic effect of habits and OSF on the morphology of buccal mucosal cells has not been thoroughly investigated. Till date, morphometric analysis of keratinocytes obtained from OSF cases with chronic exposure to various tobacco and areca nut preparations, measuring all the nuclear and cell parameters, using image analyzer have not been cited in literature.
| Conclusion|| |
The present study shows that, the nuclear dimension increased and the cell dimension decreased in OSF subjects and to a greater degree in OSCC patients compared to healthy individuals. These can be attributed to the dysplastic changes in the epithelium. The mean values of few parameters in clinically advanced OSF stages were very close to those of OSCC values, indicating a greater degree of morphometric changes in the advanced stages.
CA and NA: CA showed no significant difference between OSF and OSCC group. The advancement of fibrosis increases the risk of development of epithelial dysplasia in OSF. This strengthens the view that cytomorphometric analysis can be used as an effectual screening tool for early detection of malignant transformation in the advanced OSF cases and follow up of patients who are at a high risk for malignancy.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]