Table of Contents  
Year : 2019  |  Volume : 11  |  Issue : 2  |  Page : 84-88

Cytomorphometric Evaluation of Oral Exfoliated Cells − Its Correlation With Age of an Individual

Department of Oral and Maxillofacial Pathology and Oral Microbiology, Thai Moogambigai Dental College and Hospital, Chennai, Tamilnadu, India

Date of Submission16-Aug-2019
Date of Decision26-Sep-2019
Date of Acceptance24-Oct-2019
Date of Web Publication29-Jan-2020

Correspondence Address:
Dr. T. Radhika
Department of Oral Pathology and Microbiology, Thai Moogambigai Dental College and Hospital, Chennai, Tamilnadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jofs.jofs_101_19

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Introduction: Age is a vital tool in assessing the mental and physical growth of an individual. Exfoliative cytology, a non-invasive procedure, also has its potential implication in age assessment of an individual. This study aims to correlate and compare the average cell size from oral buccal smears of individuals of varying age groups using cytomorphometric analysis. Materials and Methods: In this study, 100 buccal smears were collected from normal individuals, dividing them into five age groups (Group 1: 10–20, Group 2: 21–30, Group 3: 31–40, Group 4: 41–50, Group 5: 50 and above). The collected smear samples were stained using papanicolau stain. The average sizes of the cells were measured using Dewinter’s image analysis software. Results: Cytomorphometry revealed a decrease in the average cell size as age increases. Analysis using Bonferonni and Post Hoc tests showed statistically significant decrease in the average cell size (P < 0.005). Conclusion: Cytomorphometric evaluation of exfoliated oral cells proves reliable tool for correlation of age. Cytomorphometric analysis of exfoliated cells of buccal mucosa serves a potential alternative non-invasive procedure in evaluation and correlation of age of an individual compared to the other screening modalities, which are usually either invasive or expensive.

Keywords: age estimation, cytomorphometric analysis, exfoliative cytology, image analysis, papanicolau stain

How to cite this article:
Radhika T, Hussain S, Adithyan S, Jeddy N, Lakshmi S. Cytomorphometric Evaluation of Oral Exfoliated Cells − Its Correlation With Age of an Individual. J Orofac Sci 2019;11:84-8

How to cite this URL:
Radhika T, Hussain S, Adithyan S, Jeddy N, Lakshmi S. Cytomorphometric Evaluation of Oral Exfoliated Cells − Its Correlation With Age of an Individual. J Orofac Sci [serial online] 2019 [cited 2023 Jun 9];11:84-8. Available from:

  Introduction Top

Age is a vital tool in assessing the mental and physical growth of an individual. Age of an individual is not only a source of information, it is also useful in documentation and authentication of the individual. Age and gender are two important parameters in the field of forensic sciences for the identification of a deceased individual where there is no other possible way of identification. Be it in case of deaths occurring from natural calamities, accidents, homicides, determination of age and gender of the deceased individual forms the preliminary step toward the progress of the investigation. Age estimation methods used in forensic investigations include extent of tooth wear, amount of secondary dentin formation, tooth-to-pulp ratio, DNA analysis, and by using the morphology of skeletal system. The pre-existing modalities of age estimation are either expensive or complex, or too simple and less accurate. Age estimation using amount of tooth wear, amount of secondary dentin formation, tooth-to-pulp ratio, etc., are less accurate whereas radiocarbon dating of tooth is not cost effective. Radiological examination of dental and skeletal development also proves less useful in adults than younger individuals.[1]

Exfoliative cytology is a non-invasive, cost-effective, and safe procedure. Though it has primarily been adopted for research and diagnosis pertaining to pathological conditions, cytological examination of normal cells also provides key information about an individual’s physical status. Exfoliative cytology also provides data on other conditions like diabetes mellitus, presence of any malignancies, effects of smoking and alcoholism, iron content in beta thalassemia etc.[2] With the introduction of cytomorphometric image analysis softwares, calculating the size of exfoliated cells has become much easier. Other cellular parameters such as nuclear size and perimeter and cellular perimeter have also been analyzed using cytomorphometry.[3] However, cell size has proved to be a valuable parameter in assessing the age of an individual. The results can be obtained on the very same day within few hours. The armamentarium required is also less and cost effective.

The human body produces cells which undergo changes from layer to layer as it moves from the basal level to top, until it finally exfoliates. However, the effect of senescence does not cause any discrepancies in cellular morphology, which makes it ideal for age estimation.[4] The turnover of cells in individual decreases with age in terms of number as well as size, which acts as an indication for age estimation. Exfoliative cytology is thus a simple, precise, and effective alternative to age estimation tools which involves the collection of buccal smears from the oral cavity of the subject under study, followed by scrutiny under a microscope to evaluate the size of these exfoliated cells and estimate the corresponding age of that individual.

Hence, the objective of this study is to estimate and compare the average cell size from oral buccal smears of individuals of varying age groups using cytomorphometric analysis.

  Materials and Methods Top

The study subjects were randomly selected from patients reporting to the outpatient (OP) department of our institution. The study sample included 100 samples out of which 50 were males and 50 were females. The samples were divided into five age groups (Group 1: 10–20 years, Group 2: 21–30 years, Group 3: 31–40 years, Group 4: 41–50 years, Group 5: 51–60 years with each group consisting of 20 individuals (10 males and females in each group). The maximum age in group 5 was 57 years. Subjects with any systemic illness and deleterious habits such as tobacco and alcohol consumption were excluded from the study. Informed consent was obtained from study participants. Ethical approval for this study (Dr.MGRDU/TMDCH/2019-20/020719001) was provided by the institutional ethical committee on 02 July 2019.

Oral smear samples were collected from the buccal mucosa of individuals from each age group. The smear samples were collected using moistened wooden spatula in a gentle scraping motion from normal healthy-looking buccal mucosa and were immediately smeared onto a clear glass slide. It was then fixed with 95% ethyl alcohol for 15 minutes before they were subjected to Papanicolau staining technique.

The stained smear samples were observed under a microscope for image analysis. Images were taken at 40x magnification, focusing them on a stage micrometer scale. An average of 20 clearly defined cells was examined and was measured in both horizontal and vertical axis in micrometer. Folded and clumped cells were not included in the measurement. The cells were marked manually using Dewinter’s image analysis software and images were captured and projected onto the computer for image analysis using Lawrence and Mayo LM-52-3001 microscope. The average cell sizes (Cs) were determined and statistically analyzed using one-way ANOVA and Bonferroni comparison tests.

  Results Top

The mean cell size in group 1 was 0.011 ± 0.002 mm/sq [Figure 1]a. In Group 2, the mean cell size was 0.009 ± 0.003 mm/sq [Figure 1]b. In Group 3, the mean cell size was 0.006 ± 0.002 mm/sq [Figure 1]c. In Group 4, the mean cell size was 0.005 ± 0.001 mm/sq [Figure 1]d. In Group 5, the mean cell size was 0.003 ± 0.001 mm/sq [Figure 1]e. [Table 1] shows the mean value and standard deviation of average cell size in every age group. Statistical analysis using one-way ANOVA, Bonferroni, and Post Hoc tests [Table 2] revealed statistically significant difference in the mean value of each group when individually compared with the other groups (P < 0.005), and confirmed that the cell size decreases with increase in age [Figure 2].
Figure 1 Decrease in average cell size among different age groups with increase in age.

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Table 1 Mean value of average cell size in every age group

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Table 2 Statistically significant difference in the mean value between age groups, where each group (primary group) is individually compared with the other age groups (other groups) using Bonferroni and Post Hoc Tests for multiple comparisons

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Figure 2 Correlation between average cell size and age groups.

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  Discussion Top

Age estimation is of paramount importance in the field of forensic medicine and plays a significant role in medicolegal issues.[1] Of all the available investigative modalities, exfoliative cytology stands out owing to its simplicity and ease in application. Exfoliative cytology is an effective, non-invasive procedure that has been standardized in screening and diagnosis of oral pathologies. Cytomorphometric analysis refers to image analysis of these exfoliated cells which gives us an insight upon the morphological changes taking place inside the cell, be it nuclear or cytoplasmic changes in cytological smears.

The histological section of an oral epithelium comprises of the following cells: stratum basale, stratum spinosum, stratum granulosum, and stratum corneum. The act of collecting smear samples through gentle scrapings may dislodge all the layers of the epithelium, including the stratum basale. The basal cells are small cells with a large nucleus that has a diameter which is one third or one fourth the size of the cell. It has a basophilic cytoplasm. The stratum spinosum or the prickle cell layer is larger than the basal layer but its nucleus is smaller than basal cells. The cell outline is irregular and more flatter.

The staining of these cells varies from pink to light orange. The cells of stratum corneum stain orange as they are orangeophilic. As age increases, the epithelial cells get compressed, flattened and pyknosis occurs, which involves the condensation of nuclear chromatin to a dense structure less mass. Upon further maturation, the nucleus completely disappears.[5]

A recent study by Shetty et al.[1] revealed similar results in which the average cell size seemed to decrease with increase in age groups. However, in our study, we have tried to standardize the ratio of male to female subjects among the 100 sample size, with more variables under consideration. Latti BR et al.[6] have effectively used cytomorphometry to measure and determine increase in cell size in patients with diabetes mellitus. Cytomorphometric analysis has also been used in conditions such as diabetes mellitus, tobacco and alcohol usage, iron content in beta thalassemia, malignant and premalignant conditions, etc.[7],[8] Bloching et al.[9] used cytomorphometry in exfoliative buccal cells to determine cellular changes in precancerous conditions using micronuclei (MN) assay. Nallamala S et al.[10], compared the accuracy of age estimation using exfoliative cytology and radiovisiography and showed exfoliative cytology to be more accurate and reliable tool when it comes to age estimation. A cytomorphometric study by Reddy et al. yielded results similar to our study. The study involved estimation of age by measuring changes in nuclear diameter and cell diameter in three age groups as opposed to our study where we have calculated the average cell size of 100 individuals divided into five age groups.[11] Technical innovations such as computer-aided image analysis is simple and effective than the conventional plainmetric methods.[12][5]The results of this study shows a significant age-related variation in the cell size which is independent of gender and can be ascribed to cellular senescence.[3] The cells in the oral epithelium only divide until a period of time, after which it ceases. The ability to turnover new cells decreases with age, resulting in the accumulation of senescent cells. The decrease of turnover rate and the cellular activity due to decrease in the cellular organelles could be the reason for decrease in cell size.

There is a significant decrease in the size of the oral exfoliated cells as age increases, when subjected to cytomorphometric analysis. Hence, cytomorphometric analysis of oral cells from buccal smears presents a negative correlation with age of an individual. Further studies on a larger sample size are recommended to yield a significant correlation and also to yield a regression formula for age estimation.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Shetty DC, Wadhwan V, Khanna KS, Jain A, Gupta A. Exfoliative cytology: a possible tool in age estimation in forensic odontology. J Forensic Dent Sci 2015;7:63-6.  Back to cited text no. 1
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Leekha S, Nayar AK, Bakshi P, Sharma A, Parhar S, Soni S. Estimation of iron overloads using oral exfoliative cytology in beta-thalassemia major patients. Cyto Journal 2016;13:6.  Back to cited text no. 2
Chaudhary R, Sahni P, Shylaja M.D., Patel A. Age estimation by exfoliative cytology: New era of noninvasive forensic science. Int J Forensic Odontol 2018;3:40-3.  Back to cited text no. 3
Priyadarshini C, Puranik MP, Uma SR. Dental age estimation methods: a review. Int J Adv Health Sci 2015;1:19-25.  Back to cited text no. 4
Anuradha A, Sivapathasundharam B. Image analysis of normal exfoliated gingival cells. Indian J Dent Res 2007;18:63-6.  Back to cited text no. 5
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Latti BR, Birajdar SB, Latti RG. Periodic Acid Schiff-Diastase as a key in Exfoliative cytology in diabetics: a pilot study. J Oral Maxillofac Pathol 2015;19:188-91.  Back to cited text no. 6
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Ogden GR, Cowpe JG, Green MW. Quantitative exfoliative cytology of normal buccal mucosa: Effect of smoking. J Oral Pathol Copenhagen 1990;19:53-5.  Back to cited text no. 7
Babuta S, Garg R, Mogra K, Dagal N. Cytomorphometrical analysis of exfoliated buccal mucosal cells: effect of smoking. Acta Med Int 2014;1:22-7.  Back to cited text no. 8
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Bloching M, Hofmann A, Lautenschläger C, Berghaus A, Grummt T. Exfoliative cytology of normal buccal mucosa to predict the relative risk of cancer in the upper aerodigestive tract using the MN-assay. Oral Oncol 2000;36:550-5.  Back to cited text no. 9
Nallamala S, Guttikonda VR, Manchikatla PK, Taneeru S. Age estimation using exfoliative cytology and radiovisiography: a comparative study. J Forensic Dent Sci 2017;9:144-8.  Back to cited text no. 10
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Reddy SV, Kumar SG, Vezhavendhan N. Cytomorphometric analysis of normal exfoliative cells from buccal mucosa in different age groups. IJCDS 2011;2:53-6.  Back to cited text no. 11
Hande KH, Chaudhary MS. Cytomorphormetric analysis mucosa of tobacco chewers. Rom J Morphol Embryol 2010;51:527–32  Back to cited text no. 12


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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