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 Table of Contents  
ORIGINAL RESEARCH
Year : 2021  |  Volume : 13  |  Issue : 1  |  Page : 53-59

Cytomorphometric analysis of exfoliated buccal mucosal cells in smokers and patients with hypertension: A quantitative analysis


1 Department of Oral Pathology & Microbiology, Government Dental College and Hospital, Hyderabad, Telangana
2 Department of Dentistry, Government Medical College, Nalgonda, Telangana
3 Department of Oral Pathology, S.V.S. Institute of Dental Sciences, Mahbubnagar, Telangana
4 S.V.S. Institute of Dental Sciences, Mahbubnagar, Telangana

Date of Submission29-Jun-2020
Date of Decision29-Jun-2020
Date of Acceptance28-Nov-2020
Date of Web Publication28-Jan-2021

Correspondence Address:
Dr. Suvarna Manthapuri
Department of Dentistry, Government Medical College, Nalgonda.
Telangana
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jioh.jioh_228_20

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  Abstract 

Aim: To examine and estimate cytomorphometric changes by using parameters such as cell area (CA), nuclear area (NA), cell diameter (CD), nuclear diameter (ND), and nuclear-cytoplasmic ratio (N/C) in exfoliated buccal mucosal cells in smokers, patients with hypertension, and healthy individuals by using Papanicolaou stain. Materials and Methods: This quantitative analysis was performed on totally 120 individuals who were randomly selected and were divided into four groups. Group 1: 30 healthy individuals free of hypertension and without any smoking habit comprised the control group; Group 2: 30 individuals with smoking habit and without hypertension; Group 3: 30 individuals with smoking habit and with hypertension; and group 4: 30 individuals with hypertension and without smoking habit. Buccal smears were taken and stained by Papanicolaou stain. Image analysis of 50 cells was done at 40× magnification with a digital image capture analysis software system (ProgRes capture pro, version 2.8.8). The obtained parameters were statistically compared among the groups by one-way ANOVA test and Kruskal–Wallis test. Results: Statistically significant increase in NA, ND, and N/C ratio values were found in smokers and smokers with hypertension, and minimal change was observed in patients with hypertension alone when compared with normal mucosa. Conclusion: Smoking habit alone and patients with hypertension along with smoking habit show significant cytomorphometric changes. The combined use of exfoliative cytology and cytomorphometric analysis gives an added advantage in monitoring clinically suspect lesions and early detection of malignancy in high-risk individuals such as smokers.

Keywords: Cytomorphometry, Hypertension, Papanicolaou Stain, Smokers


How to cite this article:
Srilatha T, Manthapuri S, Shylaja S, Ramanand OV, Reddy ES, Vamshi VR. Cytomorphometric analysis of exfoliated buccal mucosal cells in smokers and patients with hypertension: A quantitative analysis. J Int Oral Health 2021;13:53-9

How to cite this URL:
Srilatha T, Manthapuri S, Shylaja S, Ramanand OV, Reddy ES, Vamshi VR. Cytomorphometric analysis of exfoliated buccal mucosal cells in smokers and patients with hypertension: A quantitative analysis. J Int Oral Health [serial online] 2021 [cited 2021 Mar 5];13:53-9. Available from: https://www.jioh.org/text.asp?2021/13/1/53/308355


  Introduction Top


The anatomical and physiological functions of cells are altered due to pathological and systemic conditions such as diabetes and hypertension and also because of the long-term usage of tobacco, which poses an important risk factor for several oral diseases, including potentially malignant disorders (PMDs) and oral cancers.[1],[2] There is also strong evidence of the association between smoking and vascular impairment.[3]

Smoking often results in cellular irritation and increased proliferative activity of cells, leading to cellular changes.[4],[5] Hypertension is a systemic disease in which multiple factors are involved. It is characterized by oxidation and vasoconstriction of capillaries, which alters the microcirculation leading to atrophy and cellular alterations in the oral mucosa.[6] The study of cellular alterations through exfoliative cytology of oral mucosal cells is used in the detection of various disease conditions such as potentially malignant disorders and cancers.[7]

Measurement of cell parameters such as CA, NA, CD, ND, and N/C ratio through exfoliative cytology by using computerized quantitative imaging techniques such as cytomorphometric analysis may help in the early diagnosis of pre-cancer lesions and susceptibility for cancers in smokers and it can be used as an additional aid in monitoring lifestyle-associated diseases such as diabetes and hypertension.[6],[8]The effect of tobacco on buccal mucosal changes is well established; many previous studies were conducted on patients with diabetes, but the effect of hypertension on exfoliative buccal cells is debatable and is not much investigated. Hypertension accompanied by a habit of tobacco smoking may cause a cumulative effect on buccal mucosal cell changes.

The purpose of this study is to compare the calculated cytomorphometric changes in exfoliated buccal mucosal cells of smokers, patients with hypertension, and smokers with hypertension, with those of healthy individuals to assess the altered nature of buccal cells.


  Materials and Methods Top


Setting and design

This quantitative analysis was conducted in a total of 120 individuals attending the Outpatient department of SVS Medical and Dental College, Mahabubnagar from 2017 to 2018 for a period of six months. A total of 120 individuals were divided into four groups, consisting of Group 1: 30 healthy individuals free of hypertension and without any smoking habit comprised the control group; Group 2: 30 individuals with smoking habit and without hypertension; Group 3: 30 individuals with smoking habit and with hypertension; and Group 4: 30 individuals with hypertension and without smoking habit.

An inclusion criterion includes patients with hypertension with recently monitored blood pressure (BP), individuals with a habit of smoking, and smokers with known hypertension. For the control group, normal healthy adults with no history of smoking, hypertension, or any other illness were included. All individuals with clinically normal-appearing mucosa were included. Uncooperative patients, alcoholics, patients with anemia, patients with any other systemic illness and infections, and patients on medication other than for hypertension were excluded from the study.

Cytosmear preparation

After obtaining the informed consent of the patients, subjects were asked to rinse their mouth gently with water. Scrapings were obtained from buccal mucosa by using a slightly moistened wooden spatula. The collected material was smeared onto the pre-cleaned microscopic slides. Just before drying, the smears were fixed in an alcohol solution, then were stained with PAP stain. ((Nice Chemicals (P) Ltd. Manufacturer) Harris hematoxylin, Orange G6, 10% aqueous Orange G6 – 50 ml, Alcohol – 950 ml, Phosphotungstic acid – 15 g, EA 36 0.04 M, Light Green SF – 100 ml,0.3 M Eosin – 20 ml Phosphotungstic acid – 2 g, Alcohol – 750 ml, Methanol – 250 ml, Glacial acetic acid – 20 ml) [Figure 1]. The clear background of the obtained slides enhances the sensitivity and quality of results.
Figure 1: Collection of exfoliative cell of buccal mucosa and cytosmear preparation

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Cytosmear evaluation

All the stained slides were observed under a light microscope and then studied in a Zig-Zag pattern. Fifty clearly defined cells were measured in each case. Photomicrographs of individual unfolded cells with a clear outline were obtained at 40 × magnification. The captured pictures were stored after calculating NA and CA, as well as nuclear and cell diameter. The maximum and minimum values were measured after proper calibration with image analysis software, and the average value was taken. All measurements were imported from the image analysis software (ProgRes capture pro, version 2.8.8, SVS Dental College, Mahabubnagr, and Telangana, India) to a Microsoft Excel sheet for statistical analysis [Figure 2].
Figure 2: Image software analysis

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Cytomorphometric parameters studied

CA and NA

The cellular and nuclear outlines were traced by using a digitalized cursor with an interactive measurement tool in the software, and the NA and CA were calculated [Figure 3] and [Figure 4].
Figure 3: Cytomorphometric evaluation of CA in buccal smears of control group

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Figure 4: Cytomorphometric evaluation of NA in buccal smears of smokers group

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CD and ND

The CD and ND were measured by using a digitalized cursor with an interactive measurement tool by tracing two perpendicular lines (maximum and minimum diameter) by the software, and the mean values were taken [Figure 5] and [Figure 6].
Figure 5: Cytomorphometric evaluation of CD in buccal smears of smokers with hypertension group

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Figure 6: Cytomorphometric evaluation of ND in buccal smears of patients with hypertension group

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Cytoplasmic area (CyA)

The CyA was measured from the difference between CA and NA (CyA = CA − NA). The NC ratio was calculated by using the formula: NC = NA/CyA.

Statistical analysis

The obtained data were analyzed by Graph Pad Prism software version 6.0 (Telanagana, India). Data were summarized as mean ± SD for continuous data. The comparison between four groups was done by the one-way ANOVA test for continuous normal data and the Kruskal–Wallis test for continuous non-normal data and these were followed by the post hoc multiple-comparisons test. All P-values less than 0.05 were considered statistically significant.


  Results Top


A statistically significant reduction in CA from group 1 (778.10) to group 3 (715.40) was observed. In group 4 (760.40), minimal changes were seen when compared with group 1. There was a significant increase in NA values from group 1 (132.20) to group 3 (169.50), with a minimal reduction in the NA of group 4 (122.80). For the parameter CD, a significant progressive reduction in CD measurements from group 1 (237.30) to group 3 (197.50) was noticed along with a minimal decrease in CD for group 4 (227.60). A noticeable progressive increase was seen in the obtained ND values from group 1 (37.28) to group 3 (69.39). The least measurements were observed in group 4 (28.60). The NC ratio values exhibited a similar fashion of increase from group 1 (0.20) to group 3 (0.31). The least ratio was seen in group 4 (0.19) [Table 1].
Table 1: Comparison of cell parameters between controls and smokers, smokers with hypertension, and patients with hypertension

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  Discussion Top


Exfoliative cytology is an attractive option, as it is a quick, simple, painless, and noninvasive laboratory technique that is useful to identify the early changes in oral mucosal cells of tobacco users.[9] Usually, cells are attached through cell junctions and they exfoliate after maturation. The characteristic cellular and nuclear morphological appearance of cells of the healthy epithelium differs from cells that are derived from inflamed or malignant lesions.[10],[11]

Various adverse cardiovascular events are associated with tobacco usage and act as synergistic agents with hypertension and this increases the risk of coronary heart diseases along with dyslipidemia.[12] Smoking often causes an acute rise in heart rate and BP, and it has also been found to be associated with malignant hypertension.[13] Nicotine is a known alkaloid of tobacco and tobacco products possess an antiadrenergic action, mediating the local and systemic liberation of catecholamines by vasopressin release.[14] The exact relationship between smoking and cardiovascular diseases remain unclear, but paradoxically several epidemiological studies have found high BP in smokers than nonsmokers.[15],[16]

The results of our study showed a decrease in CA, CD, and an increase in NA, ND, and NC ratio in smokers and smokers with hypertension when compared with controls. The hypertensive group showed minimal cellular and nuclear parameter values when compared with all other study groups. To the best of our knowledge, this is the second study carried out in patients with hypertension.

Constant contact of tobacco and tobacco products with oral mucosa induces a local rise in temperature and inflammation for longer periods, resulting in a delayed cell division, which, in turn, increases the NA and ND due to an increase in nuclear content required for replication. In such cells with increased activity, the ability of cells to form cytoplasm decreases.[17],[18] The increased nuclear volume and decreased cytoplasmic volume possibly represent the significant changes that occur in the cells, which are more accurately identified at a morphological level.[4]

Ogden et al. (1990) conducted a study to evaluate the cytomorphometric changes in smokers and reported an approximately 5% increase in mean NA in smokers when compared with the control group. They concluded that these cellular changes can be partly attributed to the fact that tobacco consumption, which releases various byproducts such as nitrosamine and nitrosonornicotine, can influence the cellular morphometry.[19] These results are in accordance with the current study, showing an increase in NA in smokers.

Hande et al. (2010) carried out a cytomorphometric study in tobacco chewers and observed a progressive decrease in CA, an increase in ND, and an increase in NC ratio in tobacco users; they concluded that these changes could be the earliest indicators of cellular alterations. They suggested that there could be a relationship between tobacco usage and quantitative alterations within the cells that are characteristic of actively replicating cells[20]; we also noted similar results.

Goregen et al. (2011) conducted a study to examine the effects of tobacco on normal buccal mucosa through the cytomorphological analysis and reported a statistically significant rise in nuclear parameters such as NA, ND, and NC ratio in smokers, when compared with nonsmokers. They concluded that these cellular alterations with tobacco smoking lead to dysplastic changes.[21] Hashemipour MA et al. (2013) carried out a study to evaluate cytomorphometric changes in exfoliative buccal mucosa among smokers, opium addicts, and nonsmokers. They observed a statistically significant increase in NA, ND, and NC ratio, and a decrease in CA, CD in smokers when compared with nonsmokers; they also suggested a possible relationship between the number of cigarettes per day and an increase in the rate of cellular proliferation of oral mucosal cells.[22] Similar studies were conducted by Buch et al.,[23] Babuta et al.,[4] and Khot et al.,[24] and their results are also in accordance with the results of the current study.

Another study was conducted by Shetty et al. to evaluate the cytomorphometric changes in the oral squames from the buccal mucosa of tobacco users; they found a significant decrease in mean CA, CD, and an increase in mean NA, ND, and NC ratio in smokers when compared with nonsmokers. They concluded that these altered cellular changes in exfoliated cells suggest the usefulness of cytomorphometric analysis to estimate the cellular alterations in tobacco users.[25] The results just cited are in accordance with the results of the current study, where a progressive decrease in CA (778.10–722.40) and CD (237.30–200.80) and a progressive increase in the NA (132.20–162.60), ND (37.28–64.39), and NC ratio (0.20–0.29) are noticed in tobacco smokers.

Oral cancer risk in tobacco smokers depends on the number of cigarettes consumed per day and the duration of exposure to smoking. Usually, size of the nucleus and cytoplasm decline with age but there is a noticeable increase in nuclear size in smokers, which is a cellular adaptation in response to smoking in oral epithelial cells.[26] Smoking often results in a decreased turnover rate of cells, which leads to cells remaining in the cell cycle for longer periods and resulting in a delayed cell division, which, in turn, increases the nuclear size. Increased NC ratio has an advantage over other cell parameters, where it represents the significant changes that occur in the cell, which are more accurately identified at morphological level.[27]

Sameera et al. carried out a cytomorphometric study on exfoliated buccal mucosal cells in controls, diabetics, and patients with hypertension; they observed that there was no significant change in CD, ND, CA, NA, and NC ratio in patients with hypertension when compared with the control group.[6] We also noted no significant changes in the cytomorphometric values. However, significant cytomorphometric changes were observed in smokers with hypertension, suggesting that hypertension along with smoking habit may have a synergistic effect on oral mucosa, causing significant cellular changes.

Exfoliative cytology has value in diagnosis, as it is painless, reliable and an easily carried out procedure with less cost and irritants to the patient than biopsy.[28] Considering the cumulative advantages of exfoliative cytology, it can be used as a diagnostic tool for screening programs of high-risk populations.[29],[30]

With the study results cited earlier, we can conclude that hypertension alone may not show any significant morphometric changes in the buccal mucosal cells whereas smokers along with hypertension and smoking habit alone cause significant alterations. Our study is a preliminary attempt to investigate the effect of hypertension on buccal mucosal cells; further studies with a larger sample size are encouraged to justify the study results.


  Conclusion Top


Smoking habit alone and patients with hypertension along with smoking habit show significant cytomorphometric changes. The combined use of exfoliative cytology and cytomorphometric analysis gives an added advantage in monitoring clinically suspect lesions and early detection of malignancy in high-risk individuals such as smokers. Patients with hypertension along with smoking habit show significant cytomorphometric changes. Further prospective studies have to be conducted with a large sample size, and comparisons to other conditions that can cause similar cytomorphometric changes are needed to determine the predictive value of this method.

Future scope/clinical significance

Early precancerous lesions and oral cancers are often asymptomatic and subtle. Therefore, it is important for the clinician to diagnose suspicious conditions, especially in associated risk factors such as tobacco users and lifestyle-associated diseases such as hypertension. There is an imperative need to develop early diagnostic tests to evaluate the cellular alterations caused by smoking. Exfoliative cytology with cytomorphometry might aid in this aim.

Acknowledgment

Not applicable.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Authors’ contributions

Not applicable.

Ethical policy and institutional review board statement

The ethical clearance (with reference No. SVSIDS/OP/2/2016) was obtained from the institutional ethical committee.

Patient declaration of consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/ have given his/ her/ their consent for his/ her/ their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Data availability statement

Data can be available on valid request by contacting the corresponding author by mail.

 
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