|ORIGINAL CONTRIBUTION: CYTOMORPHIC CLINICS IN ONCOLOGY
|Year : 2015 | Volume
| Issue : 4 | Page : 288-291
Cytomorphometric analysis of buccal mucosa cells in tobacco chewers
D Parmar, Nilima Sawke, Gopalkrishna Sawke
Department of Pathology, People's College of Medical Sciences and Research Centre, Bhopal, Madhya Pradesh, India
|Date of Web Publication||28-Jul-2015|
Dr. Nilima Sawke
Department of Pathology, People's College of Medical Sciences and Research Centre, Bhopal, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Objective: The objective of the present study was to assess the effect of tobacco chewing on buccal mucosa by using cytomorphometry. Study Design: The study group consisted of 100 subjects divided into 50 tobacco chewer subjects (Group C) and 50 control subjects (Group N) aged between 31 and 80 years. We analyzed and compared the nuclear and cell morphometric features, that is, mean nuclear area (MNA), mean nuclear perimeter (MNP), mean of maximum nuclear diameter (Max-ND), mean of minimum nuclear diameter (Min-ND), mean cell area (MCA), mean cell perimeter (MCP), mean of maximum cell diameter (Max-CD), mean of minimum cell diameter (Min-CD) and nuclear to cell parameter ratio. Buccal epithelial cells of these individuals were collected with a brush and fixed smears were stained with Papanicolaou stain and cytomorphometric analysis performed using Image J image analysis software. Results: The mean of above nuclear and cell morphometric parameters and their ratio were found to be statistically significant by Univariate analysis of variance (ANOVA) being P value < 0.001. Conclusions: There is progressive increase in nuclear parameters, decrease in cellular parameters and increase in ratio of nuclear to cellular parameters in smears from tobacco chewers, as compared with normal subjects. It is possible to conclude that these adaptive changes in the cellular and nuclear parameters tend to be a progression toward dysplastic or premalignant change.
Keywords: Cytomorphometry, oral mucosa, tobacco chewing
|How to cite this article:|
Parmar D, Sawke N, Sawke G. Cytomorphometric analysis of buccal mucosa cells in tobacco chewers. Astrocyte 2015;1:288-91
| Introduction|| |
The strong association between cancers of the oral cavity and pharynx with the use of tobacco is well established. Oral cancer is one of the six most common cancers in the world, and is one of ten major causes of death across the globe.  All of the major forms of tobacco use are known to cause oral cancer. The magnitude of the risks associated with greater amounts or longer duration of tobacco usage are consistent with findings of oral cancer across numerous cultures. 
Tobacco contains carcinogens that influence the DNA repair, cell cycle control and may produce chromosomal aberrations.  Tobacco is initially placed between the teeth and gently chewed. It is then held against the buccal mucosa over a long duration and continued to be lightly chewed and sucked. The constituents may either be swallowed or spat out when desired. 
Most oral cancer lesions are preceded by premalignant changes in oral mucosa, which could be of great help in early diagnosis of these lesions. Buccal smear cytology, which is mostly based on the presence of nuclear or cytoplasmic alterations, can easily be used to detect premalignant changes at an early stage using computerized quantitative morphometric techniques. This cytological method is a simple, noninvasive and painless technique that also involves microscopic analysis of cells collected from the surface of the buccal mucosa. 
With advancements in the field of quantitative oral exfoliative cytology, various parameters such as nuclear size, cell size, nuclear-to-cytoplasmic ratio, nuclear shape, nuclear discontinuity, optical density, and nuclear texture can be evaluated collectively in order to confirm the diagnosis accurately. 
This aim of this study is to quantitate and evaluate the cytomorphological changes in the buccal mucosa in association with tobacco chewing.
The purpose of this study was to analyze the cytomorphometric change of buccal mucosa cells of tobacco chewers using computerized image analysis based on quantitative parameters such as nuclear and cell area, perimeter, minimum diameter, maximum diameter and their ratios, as well as to evaluate potential dysplastic transformation.
| Materials and Methods|| |
A total of 50 tobacco chewers (Group C) and 50 normal subjects (Group N) were selected for the study. The chewers had been using chewing tobacco for at least 10 years. Patients with systemic disease such as anemia or diabetes, clinically apparent oral mucosa lesions and previous benign or malignant lesions were excluded from this study. Both control and subjects of chewer groups were nonalcoholics and nonsmokers.
Informed consent was obtained from all patients before taking the cytological smears. The smears were taken from clinically normal buccal mucosa. The subject was asked to rinse the mouth with drinking water. Taking all the aseptic precautions, a wooden spatula was then used to scrape the sample area (inner side of the cheek) three to four times with firm pressure. The scrapings were smeared on to the center of glass slide. The slides were immediately sprayed with commercially available spray fixative to ensure proper fixation. All cytological smears were stained by Papanicolaou staining technique.
PAP stained smears were examined under a light microscope. Only cells that were fully included in the field of vision and with clearly defined cellular and nuclear outlines were selected. In our study, we have taken well preserved, well visualized keratinized pink cells with unfolded borders. And excluded blue/green (immature/basal/intermediate) cells from morphmetric analysis. A 640 × 400 pixel digital image was taken by a camera on the microscope with 10× eyepiece and 40× objective. Using the image J 1.47 image analysis software, morphometric analysis of around 50 cells/case was done [Figure 1].
|Figure 1: Nuclear and Cell morphometric analysis of buccal squamous cells of control and chewer group using Image J v 1.47 image analysis software.|
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The following nuclear and cell morphometric features were analyzed, that is, mean nuclear area (MNA), mean nuclear perimeter (MNP), mean of maximum nuclear diameter (Max-ND) mean of minimum nuclear diameter (Min-ND), mean cell area (MCA), mean cell perimeter (MCP), mean of maximum cell diameter (Max-CD), and mean of minimum cell diameter (Min-CD). Based on the analyzed values the nuclear to cell parameter ratios were calculated for each case.
The mean values were obtained in square micrometers for area and in micrometers for perimeter and diameter.
The data obtained was statistically analyzed and compared for the two groups.
| Results|| |
This study was conducted on 100 individuals, which included 50 chewers (Group C) and 50 controls (Group N).
In the present study, most of the chewers were in the age group of 41-50 years (38%), followed by 51-60 years (24%), 31-40 years (18%), 61-70 years (16%), and 71-80 (4%) [Table 1].
|Table 1: Age Wise Distribution of Tobacco Chewers (Group C) and Control Subjects (Group N) |
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The contents of [Table 2] show results of nuclear and cellular parameters, that is, MNA, MNP, Max-ND, Min-ND, MCA, MCP, Max-CD, and Min-CD, respectively.
|Table 2: Nuclear and Cellular Morphometric Parameters in Chewers (Group C) and Controls (Group N) Using Image J Software |
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The MNA (μm 2 ) for Group C and Group N were 85.04 ± 8.84 and 67.71 ± 10.04. The MNP (μm) was found to be 33.52 ± 1.94 and 31.06 ± 2.39 for Group C and Group N, respectively. Max-ND and Min-ND (μm) for Group C were 12.041 ± 0.968 and 9.123 ± 0.334, whereas for Group N they were 10.96 ± 0.824 and 7.987 ± 0.76, respectively (P < 0.001).
The MCA (μm 2 ) for Group C and Group N were 2556.14 ± 404.57 and 2834.87 ± 254.67. The MCP (μm) was found to be 193.1 ± 15.96 and 209.58 ± 15.76 for Group C and Group N, respectively. Max-CD and Min-CD (μm) for Group C were 67.939 ± 6.427 and 48.222 ± 3.821, whereas for Group N they were 74.652 ± 4.126 and 51.104 ± 3.635, respectively.
The nuclear versus cell parameter ratios of both the groups were calculated and are shown in [Table 3]. In Group C the ratio of nuclear to cell area was 0.0341 ± 0.007 compared with 0.0251 ± 0.0035 in Group N. Similarly in Group C the N/C ratio of perimeter, maximum diameter, and minimum diameter were 0.1748 ± 0.185, 0.179 ± 0.0242, and 0.1827 ± 0.0161, respectively. The corresponding values in Group N were 0.1489 ± 0.0139, 0.1496 ± 0.0129 and 0.162 ± 0.013, respectively (P < 0.001).
|Table 3: The Nuclear to Cell (N/C) Parameter Ratio of Area, Perimeter, Max-D and Min-D, Chewers (Group C) and Controls (Group N) |
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The data obtained was statistically analyzed and compared for the two groups using one-way analysis of variance (ANOVA), post hoc and was found to be statistically significant.
| Discussion|| |
Different forms of tobacco usage are prevalent in India, and many of them are specific to certain areas. 
The morphology of the exfoliated cells depends on the nature of the changes taking place in the epithelial layer. Applying this possibility, exfoliative cytological techniques have been applied to examine the effect of tobacco on the oral mucosa. ,
During transformation of normal tissue to premalignancy or malignancy, cellular changes occur at the molecular level before they are seen under the microscope and much before clinical changes become evident. Identification of high-risk oral lesions and intervention at premalignant stages could reduce the mortality and morbidity. Such patients can be screened by clinical examination and cytomorphometry at an early-stage disease and, if diagnosed, are curable. 
Cowpe et al. demonstrated that exfoliative cytology is capable of detecting malignant changes through estimation of ratio of nuclear size to cytoplasmic size, using planimeter method in Papanicolaou-stained smears.  Since then a number of studies had been carried out using the quantitative cytomorphometric techniques.
Ramaesh et al. used cytomorphometric techniques to assess nuclear diameter and cellular diameter in normal oral mucosa, in dysplastic lesions, and in oral squamous cell carcinoma. They found that cellular diameter was highest in normal mucosa, lower in dysplastic lesions and lowest in oral squamous cell carcinoma. By contrast, nuclear diameter was lowest in normal mucosa, higher in dysplastic lesions, and highest in oral squamous cell carcinoma. They concluded that exfoliative cytology is of value for monitoring clinically suspect lesions and for early detection of malignancy. 
In 2005, Einstein and Sivapathasundharam reported cytomorphologic alterations in the form of reduction in cellular diameter and increase in nuclear diameter in buccal squames of tobacco users in the south Indian population. 
In our study, we found significant quantitative alterations in the form of increased nuclear parameters (MNA, MNP, Max-ND and Min-ND), decreased cellular parameters (MCA, MCP, Max-CD and Min-CD), and increase in ratio of nuclear to cellular parameters in tobacco chewer group compared with control group. Increase in the nuclear parameters could be due to increased DNA content of the nucleus. Increase in ratio of nuclear to cellular parameters is due to the changes in nuclear and cytoplasmic size. These findings are consistent with the results reported by Cowpe et al.
Franklin and Smith reported that the N:C ratio has the advantage of relating nuclear volume to cytoplasmic volume and possibly represents the significant changes that occur in the cell, more accurately at a morphological level.  In our study, the N:C ratio was found to be statistically significant in differentiating changes occur in study subjects (group C) as compared with control (group N).
The results of our study reveal that the buccal mucosa cells of chewers had higher nuclear size parameters, that is, MNA, MNP, Min-ND and Max-ND, lower cell size parameters, that is, MCA, MCP, Max-CD and Min-CD and higher nucleus to cell parameter ratio, that is, area, perimeter, maximum and minimum diameter as compared with controls and the difference was found to be statistically significant. This can be attributed to increase in nuclear size and decrease in cell size in chewers.
| Conclusions|| |
There is progressive increase in nuclear parameters, decrease in cellular parameters and increase in ratio of nuclear to cellular parameters in smears from tobacco chewers, as compared with normal subjects. This indicates that there could be cause-effect relationship between tobacco consumption and quantitative alterations and cellular adaptation. It is possible to conclude that these adaptive changes in the cellular and nuclear parameters tend to be a progression towards dysplastic or premalignant change.
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[Table 1], [Table 2], [Table 3]