ORIGINAL ARTICLE

Cytomorphometrical Analysis of Exfoliated

Buccal Mucosal Cells: Effect of Smoking
Sumit Babuta1, Rohin Garg2, Khushboo Mogra3, Neha Dagal4
1
Assistant Professor, Department of Anatomy, SMS Medical College, Jaipur, Rajasthan, India, 2Assistant Professor, Department of Anatomy,
Teerthanker Mahaveer Medical College & Research Centre Moradabad, U.P., India, 3PG Resident, Department of Anatomy, SMS Medical
College, Jaipur, Rajasthan, India, 4Senior Demonstrator, Department of Anatomy, SMS Medical College, Jaipur, Rajasthan, India

Introduction: Exfoliative cytology is a non-aggressive, non-invasive procedure with higher patient compliance and is therefore, an
attractive technique for the early diagnosis of oral lesions. The purpose of this study is to evaluate and compare cytological changes using
morphometric analysis of the exfoliated buccal mucosal cells in smokers, with results obtained for non-smokers. Methods: Smears
were collected from the clinically normal buccal mucosa of 120 individuals. Age range of subjects taken was 40-60 years. Smears
were then stained with Papanicolaou stain. Results: Mean NA for smokers was significantly elevated compared with the mean NA
for non-smokers. Mean CA in smokers was decreased as compared to non-smokers but the difference was not significant. Also,
N/C ratio was significantly elevated in smokers group. With increasing heavy exposure in duration of years, Cytomorphometric
changes show significant altered values for all three measured parameters (NA, CA and N/C ratio). Conclusion: Increase in NA
and decreased CA as well as altered N/C ratio would appear to be due to smoking tobacco. Cytomorphometric analysis can be
used regularly to detect these cell alterations. This method can also aid in motivating individuals to withdraw from adverse effects
of tobacco smoking. Currently, use of exfoliative cytology has increased as an adjunct to screening of precancerous lesions and
malignancies of the oral cavity.

Keywords: Cytomorphology, Exfoliative cytology, Oral mucosa, Tobacco smoking

INTRODUCTION morbidity and mortality rates of oral cancer have risen

despite advances in therapeutic techniques, leading to
Geographic variations exist among different countries increased treatment costs and complications. Hence,
of the world for the incidence of cancer of the head the early diagnosis of oral cavity cancers is of immense
and neck, also among different regions within a value in successful treatment of patients.1 In some
country. It indicates that environmental factors apparently healthy smokers, changes are observed in
may play a vital role in the pathogenesis of these the frequency of epithelial cell proliferation, the size of
malignancies. Tobacco smoking and alcohol intake nucleus and the size of nucleus in relation to cytoplasm.
In addition, an increase number of keratinized cells are
have been a ributed to as major risk factors. Strong
also observed.6-8
association between cancers of the oral cavity and
pharynx with the use of tobacco in any form is well
Exfoliative cytology is the microscopic examination
established. Epidemiological studies show that the risk
and measurements of shed or desquamated cells from
of developing oral cancer is five to nine times greater
the surface epithelium usually the mucous membrane.
for smokers than for non-smokers.1,2
Those cells that have been collected by scraping the tissue
surface or collected from body fluids such as sputum,
However incidence of head and neck cancers in some saliva, etc. are also studied. Continuous exfoliation of
communities are decreasing, the incidence of oral epithelial cells is a part of physiological turnover. Deeper
cavity cancers has not fallen in recent years, one reason cells which are strongly adhered in normal conditions
of which is the increased use of cigare es and tobacco become loose in the cases of malignancies and tends to
in those communities.3 Despite the implementation exfoliate or shed along with superficial cells.9
of cancer prevention programs in some countries and
the fact that the oral cavity is an accessible area (so The role of exfoliative cytology in the detection of
the patients can easily examine their oral cavities), oral neoplasms has created various controversies.
the majority of oral cavity cancers are diagnosed at Few researchers say that oral exfoliative cytology is a
advanced stages, resulting in poor prognosis and simple, non invasive, less time consuming procedure
survival rate among patients. 4,5 In addition, the with sensitivity of 89% and specificity of 89.5% while

Corresponding Author:
Dr. Rohin Garg, Assistant Professor, Teerthankar Mahaveer Medical College & Research Centre Delhi Road, Moradabad, U.P. India: 244001.
E-mail: [email protected]

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 Babuta, et al.: Cytomorphometrical Analysis of Buccal Mucosal Cells

others strongly criticize that there is no role for exfoliative included in the study, since this is the age group in which
cytology in early cancer detection since it is not 100% cancer of the buccal mucosa is associated.15
sensitive. Even though exfoliative cytology is not 100%
accurate, it has its own potential value in cases where Patients for the study group were selected because they
biopsy is contraindicated like in systemically compromised fulfilled the following criteria:
patients, inaccessible areas, recurrent malignancies and in 1. Smoked at least 20 cigare es, 3 cigars or 3 pipes per day
mass screening. The smear obtained by exfoliative cytology for at least 5 years.
can be analyzed quantitatively and qualitatively. With 2. Did not suffer from systemic diseases such as anemia
advancements in the field of quantitative oral exfoliative or diabetes.
cytology, various parameters such as nuclear size, cell 3. Had not received radiotherapy and/or chemotherapy in
size, nuclear-to-cytoplasmic ratio, nuclear shape, nuclear the last 6 months.
discontinuity, optical density and nuclear texture can be 4. Did not consume alcoholic drinks or using any drugs
evaluated collectively in order to confirm the diagnosis. affecting the oral epithelium.
Of these parameters, the nuclear size, cytoplasmic size
and their ratio have been shown to be significant in the Neither the smokers nor non-smokers had any oral lesions,
evaluation of oral lesions.10-13 systemic disease or even any histopathological dysplasia
in microscopic evaluation. The smears were taken from
Exfoliative cytology is a non-aggressive, non-invasive clinically normal buccal mucosa. Non-smokers were defined
technique with higher patient compliance and is therefore, by no use of cigare e or any addictive material and smoke-
an attractive technique for the early diagnosis of oral producing substances during the preceding year. Sampling
malignancies, including epithelial atypia and squamous cell was carried out from 9 to 11 a.m to exclude possibility of
carcinoma. However it has limited usage so far due to poor diurnal variations. Informed consent was obtained from
sensitivity and specificity in diagnosing oral malignancies. all the patients in the study. All the patients filled out a
The purpose of this study is to evaluate and compare form and specified their age, the frequency and duration
cytological changes using morphometric analysis of the of their smoking, and diseases, if any or any other relevant
exfoliated buccal mucosal cells in smokers, with results medical history.
obtained for non-smokers. This technique might yield
important information about the influence of tobacco upon 90 patients of smokers group were categorised in three
nuclear area (NA) and cytoplasmic area (CA), particularly subgroups based upon duration of history of smoking habit.
since these la er two variables are known to alter within Each group consists of 30 patients.
dysplastic tissue for which smoking is a potential etiologic Group A: 5-10 years history of exposure to smoking but
factor. not less than 5 years.
Group B: 11-20 years.
METHODS Group C: More than 20 years of exposure history.

This is Hospital based case-control analytical type of Oral examinations were performed using a mouth mirror
observational study to observe cytomorphological and artificial light. Subjects were asked to rinse their mouth
changes in exfoliated buccal mucosal cells of smokers. with water and a pre moistened wooden spatula was then
Smears were collected from the clinically normal buccal scraped firmly across the mucosa and the cells transferred to
mucosa of 120 individuals. Age range of subjects taken a dry glass slide fixed in 95% ethanol, followed by washing
was 40-60 years. These patients were attending the in running tap water for a further hour. Smears were then
outpatient department of SMS medical college and stained with Papanicolaou stain.
associated group of hospitals, Jaipur, Rajasthan, India.
Name, age, occupation and relevant medical history Then stained slides were subjected to research microscopy.
(including whether they smoked) were recorded. In Fifty randomly selected cells were measured in a stepwise
addition hemoglobin and full blood counts were carried manner moving the slide from the right upper corner to left
out for each patient, to exclude anemia. and then down to avoid measuring the same cell twice. Only
clearly defined cells were measured, excluding the clumped
Ninety subjects were placed in the smoker group and or folded cells and unusually distorted nuclei and cells.
thirty others in the non-smokers group. Women were not
included in the study due to cellular changes that occurs Cytomorphometric analysis was done by using Image J v 1.45
during menstruation, after menopause and also due to the image analysissoftware. The nuclear (NA) and cytoplastmic
possibility of pregnancy and other hormonal changes.14 (CA) areas were obtained by drawing round the nuclear
Furthermore, only those greater than 40 years of age were and cell boundaries using the digital cursor (Figures 1-4).

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 Babuta, et al.: Cytomorphometrical Analysis of Buccal Mucosal Cells

Outcome variables are were examined using t-test for equality of means. Differences
(a) Mean Nuclear area/50 cells were considered statistically significant when P < 0.05.
(b) Mean cytoplasmic area/50 cells
(c) Mean nuclear and cytoplasmic ratio. RESULTS

Statistical Analysis The age range of subjects taken was 40-60 years with a
All data were tabulated and statistical tests were performed mean age for smokers was 46.4 ± 4.9 years; the mean of
using SPSS. Significant statistical differences between groups non-smokers was 48.1± 5.7 years.

Table 1 contains the mean values for nuclear area (NA) and
cytoplasmic area (CA) and N/C ratio in smoker and control
group. Using a two sample “t-test” for independent samples
the mean NA for smokers was significantly elevated
compared with the mean NA for non-smokers. Mean CA
in smokers was decreased as compared to non-smokers
but the difference was not significant. Also, N/C ratio was
significantly elevated in smokers group.

Table 2 depicts cytomorphometric analysis of smokers based

upon duration of exposure. Table shows that with increasing

Figure 1: Cellular and Nuclear morphometric analysis ofexfoliated squamous

epithelial cell in buccal smears of smokers using Image J v 1.45 image analysis
software

Figure 4: Procedure for taking buccal mucosa smear by scraping

Table 1: Cytomorphometric analysis of the buccal mucosa

of smokers and controls
Variable Smokers Non-smokers
Figure 2: Only clearly defined cells were measured, excluding the clumped or Mean nuclear area 72.15 66.43
folded cells and unusually distorted nuclei and cells
Mean cytoplasmic area 2366.21 2492.51
Mean nuclear/ 0.030 0.027
mean cytoplasmic ratio (N/C)

Table 2: Cytomorphometric analysis of smokers based

upon duration of exposure
Variable Duration of exposure to smoking
Group A Group B Group C
5-10 years 11-20 years >20 years
Mean nuclear area 69.47 72.43 74.56
Mean cytoplasmic area 2455.54 2389.46 2253.64
Meannuclear/ 0.028 0.030 0.033
Figure 3: Armamentarium for PAP staining mean cytoplasmic ratio (N/C)

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 Babuta, et al.: Cytomorphometrical Analysis of Buccal Mucosal Cells

heavy exposure in duration of years, Cytomorphometric Ogden et al. investigated the effect of smoking on the
changes shows significant altered values for all three oral mucosa in individuals over 40 years of age using
measured parameters (NA, CA and N/C ratio). cytomorphology. They reported a 5% average increase in
the NA values of smokers when compared to non-smokers.22
DISCUSSION Goregen M et al. found 16.5% increase in the NA.23 Similar
findings were also reported by Seifi S et al.24 Our findings
Few studies in the past appear to have investigated at the are consistent with these studies; however, we observed a
effects of smoking tobacco on the oral mucosa, with regard 8.6% increase in the NA value of smokers over non-smokers.
to the use of exfoliative cytology technique. Application Also decreased CA was found in our study. This increase
of quantitative techniques has largely improved the in NA and decrease in CA can be a ributed to a cellular
potential accuracy of exfoliative cytology. Exfoliative adaptation that depends on smoking. Decrease in the
cytology is considered a moderate, easy and non-invasive cellular diameter and increase in the nuclear size are two
technique compared to conventional anatomopathological significant morphological changes that occur in actively
examination. proliferating cells.25

Wrubel & Scopp studied the keratinization of the hard Various other researchers also studied different parameters
palate and buccal mucosa, following smoking cessation, by cytomorphometrical analysis. Ramaesh et al. investigated
and found no definite changes. The karyopyknotic index that the nuclear diameter of the oral mucosa cells in
(KI) for smokers was no different from that for non- cigare e smokers, chewed betel quid, or practiced both
smokers.16 In contrast, Brown & Young, who investigated these habits, was significantly greater than control group
100 cells from the hard palate and buccal mucosa, found individuals. They also found that the cytoplasmic diameter
that the Kl for smokers was increased, as compared to non- was significantly smaller than that of the control group
smokers.17 Baric et al. who studied the prevalence of oral individuals.26 Similarly, Einstein and Sivapathasundraham
leukoplakia, found an increasing number of such lesions also analyzed the effect of smoking and betel quid
in those individuals who smoked tobacco. They observed chewing on the oral mucosa and determined an increase
that cigare e and cigar smokers had higher percentage in the average value of ND, and a decrease in cytoplasmic
buccal mucosal lesions whereas palatal lesions were more diameter values of smokers and individuals with both
common in pipe smokers.18 Hirayama in an extensive these habits.
investigation of oropharyngeal cancers in Central and
South East Asia, found a definite relationship between In our study we found that with increasing heavy exposure
non chewing smokers of tobacco and cancer of the buccal in duration of years, Cytomorphometric changes shows
mucosa.19 In present study, majority of our patients smoked significant altered values for all three measured parameters
cigare es. Hence according to the observations of Baric et (NA, CA and N/C ratio). Hashemipour et al. found similar
al.18 & Hirayama19 if one were looking for changes in the oral results.27 Zimmermann and Zimmermann14 and Ogden
mucosal cells of smokers, one would expect to find them in et al.22 acknowledge the presence of cell alterations related
cells removed from the buccal mucosa. Since present study to the number of cigare es smoked per day and mentions
focuses on normal buccal mucosa, patients with lesions that the number of cigare es smoked must be considered
such as epithelial dysplasia, leukoplakia, erythroplakia, as a factor. In present study it was found that there is a
and squamous cell carcinoma were not included. significant relationship between the duration of smoking
and the NA, CA & N/C ratio. A decrease in cellular size and
The effect of smoking, as a risk factor for oral malignancies, an increase in nuclear size are two important morphologic
depends on the number of cigare es smoked per day and changes which are a ributed to precancerous and cancerous
the duration of exposure to smoking. Individuals who changes. During the transition from the normal tissue
have been smoking for 10 years or more, and/or over 2 to precancerous and cancerous lesions, some cellular
packs a day are defined as heavy smokers.20,21 In this study, changes take place at the molecular level, which can be
individuals comprising the study group smoked at least 2 determined.28 Franklin and Smite reported that increased
pack a day and had been smoking for at least 5 years. nucleus/cytoplasm ratio might be due to changes in the size
of the nucleus relative to the size of the cytoplasm and is
In present study, samples were taken from those patients, possibly a reflection of significant changes in the cell at the
who were all greater than 40 years of age. Cowpe JG et al. morphologic level.29
showed that there were not any significant variations in NA
and CA, after the age of 40 yr.12 In present study, increase in Cytological preparations are of established value in the
NA and decreased CA as well as altered N/C ratio would diagnosis of a variety of disorders – local and systemic,
appear to be due to smoking tobacco. neoplastic, infectious, endocrine, genetic, etc. In this study,

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 Babuta, et al.: Cytomorphometrical Analysis of Buccal Mucosal Cells

all the smears were obtained by liquid-based cytology, a Cancer J Clin, 2002; 52: 195-215.
new method of preparing oral and cervical samples for 3. Saedi B, Razmpa E, Sadeghi M, Mojtahed M, Mojtahed A. The
epidemiology of laryngeal cancer in a country on the esophageal
cytological examination. This technique results in slides
cancer belt. Indian J Otolaryngol Head Neck Surg. 2009; 61:1-5.
with high cellularity dispersed in a homogeneous thin 4. Gupta PC, Metha FS, Pindborg JJ. Oral cancer in rural India. Lancet.
layer. Blood, inflammatory cells and mucus are reduced 1987;1:1087.
and distributed randomly throughout the slide. The clear 5. Sampaio HC, Loyola AM, Gomez RS, Mesquita RA. AgNOR count
background obtained enhances sensitivity and quality of in exfoliative cytology of normal buccal mucosa effect of smoking.
Acta Cytol. 1999; 43: 117-120.
the results.28,30
6. Johnson N. Tobacco use and oral cancer: A global perspective.
J Dent Educ. 2001; 65:328-339.
Diagnostic aids in the evaluation of oral mucosal lesions 7. Noufal A, George A, Jose M, Abdul Khader M, Jayapalan CS.
can serve an important role by identifying lesions that Cytomorphometric analysis of oral buccal mucosal smears in
need to be biopsied in spite of a “benign” appearance. tobacco and areca nut chewers who abused with and without
Early oral cancers and precancerous lesions are often betel-leaf. Substance Abuse J. 2013; 4:14-18.
8. Van Oijen MG, Gilsing MM, Rijksen G, Hordijk GJ, Slootweg PJ.
subtle and asymptomatic. In addition, histopathological
Increased number of proliferating cells in oral epithelium from
changes may be present in areas in which there is no smokers and exsmokers. Oral Oncol. 1998; 34:297-303.
clinical evidence of an oral lesion on visual examination 9. Sivapathasundharam B, Kalasagar M. Yet another article on
alone. Therefore, it is important for the clinician to exfoliative cytology. JOMFP, 2004;8(2):54-57.
maintain a high index of suspicion, especially if risk 10. Ramaesh T, Mendis BR, Ratnatunga N. Diagnosis of oral
factors such as tobacco use or alcohol abuse is present.31,32 premalignant and malignant lesions using cytomorphometry.
Otonto stomatologie tropicale, 1999; 22(85):23-8.
Consequently, there is an imperative need to develop 11. Einstein TB, Sivapathasundharam B. Cytomorphometric analysis
early diagnostic tests to evaluate the cellular/genotoxic of the buccal mucosa of tobacco users. Indian J Dent Res. 2005;
damage caused by smoking tobacco. Exfoliative cytology 16(2):42-46.
may aid in this goal. 12. Cowpe JG, Longmore RB, Green MW. Quantitative exfoliative
cytology of normal oral squames: an age, site and sex related
CONCLUSION 13.
survey. J R Soc Med 1985; 78:995-1004.
Cowpe JG, Longmore RB, Green MW, Quantitative exfoliative
cytology of abnormal oral mucosal smears, J R Soc Med, 1988,
The basic pathogenesis of any cell alteration begins at 81(9):509-513.
molecular level and initiates a cascade of reactions that 14. Zimmermann ER, Zimmermann AL. Effects of race, age, smoking
affect entire cell system. That culminates in altered cell habits oral and systemic disease on oral exfoliative cytology. J Dent
Res.1965; 44:627-631.
morphology. Cytomorphometric analysis can be used
15. Conley J, Saooyama JA. Squamous cell cancer of the buccal mucosa.
regularly to detect these cell alterations. Further, as the Arch Otolaryngol 1973; 73:333-8.
acceptance in reliability of measurable value increases, this 16. Wrubel GJ, Scopp IW. A study of the exfoliative cytology of the
method can also aid in motivating individuals to withdraw hard palate and buccal mucosa following cessation of smoking in
from adverse effects of tobacco smoking. Early diagnosis previous smokers. Dent Res 1960;40:341-5.
of oral lesions is an important aspect of health care. It has 17. Brown AM, Young GA. The effects of age and smoking on the
maturation of the oral mucosa. Acta Cytol 1970; 14:566-9.
been shown that smoking may cause various changes in 18. Baric JM, Alman JE, Feldman RS, Chauncey HH. Influence of
the cells of the oral mucosa, which can be determined by cigare e, pipe and cigar smoking, removable partial dentures and
exfoliative cytology. Diagnosis of the underlying pathology age as oral leukoplakia. Oral Surg 1982; 54:424-9.
is an important step in management of any disease. In early 19. Hirayama T. An epidemiological study of oral and pharyngeal
stages, malignancies of oral cavity sometimes demonstrate cancer in central and South East Asia. Bull WHO 1966; 34: 41-69.
20. Ayanian JZ, Cleary PD. Perceived risks of heart disease and cancer
slow growth and may not be noticed by the patient. In this
among cigare e smokers. JAMA 1999; 281:1019-21.
context, exfoliative cytology can be applied because it is 21. Saye e MA, Martin CS, Wer JM, Shiffman S, Perro MA. A multi-
simple, fast, inexpensive, and non-invasive and carries li le dimensional analysis of cue-elicited craving in heavy smokers and
risk. Currently, use of exfoliative cytology has increased tobacco chippers. Addiction 2001; 96:1419-32.
as an adjunct to screening of precancerous lesions and 22. Ogden GR, Cowpe JG, Green MW. Quantitative exfoliative
cytology of normal buccal mucosa: Effect of smoking. J Oral Pathol
malignancies of the oral cavity.
Copenhagen. 1990;19:53-55.
23. Goregen M, Akgul HM, Gundoğdu C; The cytomorphological
REFERENCES analysis of buccal mucosa cells in smokers; Turk J Med Sci. 2011;
41 (2):205-210.
1. Orellana-Bustos AI, Espinoza-Santander IL, Franco-Martínez 24. Seifi S, Feizi F, Mehdizadeh M, Khafri S, Ahmadi B; Evaluation of
ME, Lobos-James-Freyre N, Ortega-Pinto AV. Evaluation of Cytological Alterations of Oral Mucosa in Smokers and Waterpipe
keratinisation and AgNORs count in exfoliative cytology of normal Users; CELL JOURNAL (Yakhteh), 2014, 15 (4), p.302-09.
oral mucosa from smokers and non-smokers. Med Oral. 2004; 25. Frost JK. Pathologic processes affecting cells from inflammation to
9:197-203. cancer. In: bibbo Med, comprehensive cytopathology, 2nd edition,
2. Neville BW, Day TA. Oral cancer and precancerous lesions. CA Philadelphia.1997; 68-78.

| Jan - Jun 2014 | Vol 1 | Issue 1 | Acta Medica International 26

 Babuta, et al.: Cytomorphometrical Analysis of Buccal Mucosal Cells

26. Ramaesh T, Mendis BR, Ratnatunga N, Tha il RO. The effect of tobacco 30. Payne N, Chilco J, McGoogan E. Liquid-based cytology for
smoking and of betel chewing with tobacco on the buccal mucosa: A cervical screening. Cytopathology 2000; 11(6):469-70.
cytomorphometric analysis. J Oral Pathol Med 1999; 28:385-8. 31. Silverman SJR. Demographics and occurrence of oral and
27. Hashemipour MA, Aghababaie M, Mirshekari TR, Shekaari pharyngeal cancers. The outcomes, the trends, the challenge. J Am
MA, Arashlow MT, Arashlow FT, Gandjalikhan Nassab SAH; Dent Assoc 2001; 132:7S-11S.
Exfoliative Cytology of Oral Mucosa among Smokers, Opium 32. Ehrig T, Abdulkadir SA, Din is SM, Milbrandt J, Watson MA.
Addicts and Non-smokers: A Cytomorphometric Study;Archives Quantitative amplification of genomic DNA from histological
of Iranian Medicine; 2013; 16(12); p725-30. tissue sections after staining with nucleardyes and laser capture
28. Mehrotra R, Gupta A, Singh M, Ibrahim R. Application of cytology microdissection. J Mol Diagn,2001;3: 22-5.
and molecular biology in diagnosing of premalignant or malignant
oral lesions. Mol Cancer 2006; 23:5-11 How to cite this article: Babuta S, Garg R, Mogra K, Dagal N.Cytomorphometrical
analysis of exfoliated buccal mucosal cells: Effect of smoking. Acta Medica
29. Franklin CD, Smith CJ. Stereological analysis of histological
International 2014;1(1):26-31.
parameters in experimental premalignant hamster cheek pouch
Source of Support: Nil, Conflict of Interest: None declared.
epithelium. J Pathol. 1980; 130:201-215.

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