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Original Article
Exfoliative Cytology for Age Estimation: A Correlative Study in
Different Age Groups
Vadivel Ilayaraja, Thambu Keerthi Priyadharshini, Nalliappan Ganapathy, Andamuthu Yamunadevi,
Janardhanam Dineshshankar, Thangadurai Maheswaran
Department of Oral Background: Oral exfoliative cytology is a simple and relatively pain‑free procedure
Pathology and Microbiology, which can be carried out repeatedly with minimum discomfort to the patients. Oral
Vivekanandha Dental College exfoliative cytology has a sensitivity of 89.5%; its accuracy could be enhanced by
for Women, Tiruchengode, cytomorphometric analysis of the cells. Aim and Objective: The present study
Tamil Nadu, India Abstract
was undertaken to evaluate the cell diameter (CD), nuclear diameter (ND), and
nuclear‑cytoplasmic ratio (N:C) and their variation with age using buccal smears.
Materials and Methods: Buccal smears were collected from 100 apparently
normal healthy individuals. The patients were divided into five groups 10–
20, 21–30, 31–40, 41–50, and more than 50 years. The smears were fixed and
stained. The CD and ND were measured using MS paint tool. The CD, ND,
and nuclear‑cytoplasmic (N:C) ratio were calculated for each case. Results: The
cytomorphometric analysis of the exfoliated normal buccal mucosal cells revealed
that there is a decrease in CD and ND with increasing age. No variation is found
in N:C ratio. Conclusion: Age‑related changes are observed in buccal smears,
which could be helpful in age estimation.
Keywords: Cell diameter, cytomorphometric analysis, exfoliated buccal cells,
nuclear‑cytoplasmic ratio and nuclear diameter
Introduction Nuclear‑cytoplasmic ratio was considered to be more
[3]
n forensic medicine, estimating the chronological age of significant among all the parameters.
Ia person involved in judicial or legal proceedings is very The oral cavity can be an ideal site to observe the
[1]
important. Chronological age is determined by the date manifestations of aging. However, few of the associated
of birth and the period of time or number of years elapsed signs and symptoms provide themselves as a factor for
[2]
after that to any point of time. There is no medical test quantifying age. Epithelial cells undergo continuous
to find the accurate chronological age of a human being. renewal as a part of normal physiological turnover,
Anyway, this type of expert report is needed in everyday but as age progresses, the renewal capacity of tissues
practice in Justice Courts and other Public Institutions by is declined showing age‑related variation irrespective
[1] [5]
forensic physicians and Legal Medicine Institutes. of gender. Thus, cytomorphometric analysis of these
Exfoliative cytology is a noninvasive, simple, and less exfoliated cells can aid in age estimation of an individual
time‑consuming procedure which allows pain‑free by visualizing the cellular morphology.
collection of intact cells from various layers within
the epithelium for microscopic examination. It has Address for correspondence: Dr. Thambu Keerthi Priyadharshini,
[3,4] Department of Oral Pathology and Microbiology,
a sensitivity of 89% and specificity of 89.5%. In Vivekanandha Dental College for Women, Elaiyampalayam,
exfoliative cytology, various parameters such as nuclear Tiruchengode, Tamil Nadu, India.
size, cell size, cell and nuclear pleomorphism, nuclear E‑mail: drtkeerthi@gmail.com
membrane discontinuity, degenerative changes of This is an open access journal, and articles are distributed under the terms of the
nucleus, and nuclear‑cytoplasmic ratio were analyzed. Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows
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How to cite this article: Ilayaraja V, Priyadharshini TK, Ganapathy N,
DOI: 10.4103/jiadsr.jiadsr_29_17 Yamunadevi A, Dineshshankar J, Maheswaran T. Exfoliative cytology
for age estimation: A correlative study in different age groups. J Indian
Acad Dent Spec Res 2018;5:25-8.
© 2018 Journal of Indian Academy of Dental Specialist Researchers | Published by Wolters Kluwer ‑ Medknow 25
Ilayaraja, et al.: Age estimation using exfoliative cytology
Since there are conflicting data regarding the age changes diameter [Figure 2]. N:C ratio was then calculated.
in human buccal cell dimensions, the present study Mean of CD, ND, and N:C was calculated separately
was proposed to assess the age changes of exfoliated for all five groups. The obtained values were statistically
buccal cells which may be helpful in forensics for age analyzed using one‑way analysis of variance, to find
estimation. the difference in CD, ND, and N:C. Tukey’s HSD
Aim and objective (honest significance difference) post hoc test was done
To estimate the cell diameter and nuclear diameter and to identify the significance between various age groups.
nuclear‑cytoplasmic ratio of buccal cells of all the study Ethical clearance was obtained from the Institutional
participants. Ethics Committee.
To compare the cell diameter and Nuclear diameter and Results
nuclear‑cytoplasmic ratio between various age group. Buccal smears were collected from 100 subjects with
Materials and Methods 20 subjects in each age group and CD, ND, and N:C
A total of 100 apparently healthy subjects with 20 in ratio were measured. There was a statistically significant
each age group were selected randomly from Outpatient difference in ND and CD in different age groups. The
Department of Vivekanandha Dental hospital. The average CD of each group is shown in Table 1. Mean
subjects were divided into five age groups: CD was found to be 50.43 µm. CD appears to show a
• Group I – 10–20 years, gradual decrease with increase in age. Table 2 shows ND
• Group II – 21–30 years, measurements. Average ND is found to be 7.18 µm. ND
• Group III – 31–40 years, displays a variation with age groups. N:C for all five
• Group IV – 41–50 years groups was calculated manually and shown in Table 3.
• Group V – >50 years. Mean N:C is found to be 0.146. The N:C ratio is found
Samples were collected from the patients who visited to fluctuate in different age groups with no specific
our outpatient department. Clinically normal individuals pattern and is not statistically significant.(P = 0.490).
without past history of systemic disease or therapeutic
medications were included in the study. Patients with a
history of systemic illness, oral‑related habits such as
smoking, tobacco usage, and alcohol consumption were
excluded from the study.
Scrapings were made from the buccal mucosa with
moistened wooden spatula, then smeared on to a clear
glass slide and immediately fixed with alcohol (Biofix
spray fixative). Papanicolaou stain (Bio Lab Diagnostics)
was used for staining the slides. The cells were examined
using ×40 objective (Leica DMD108 microscope) and
projected onto the monitor through camera attached to the
microscope. A screenshot of each slide was captured and
transferred to the computer for image analysis [Figure 1].
For each subject, 25 cells were chosen. Unfolded cells Figure 1: Cytological smear taken from buccal mucosa
with clear outline were selected excluding the clumped or
folded cells. Cell diameter (CD), Nuclear diameter (ND), Table 1: Comparison of cell diameter within age groups
and nuclear‑cytoplasmic ratio (N:C) of 25 cells for each using one‑way analysis of variance and Tukey ‑ honest
subject were recorded, and the average was found for all significance difference procedure
cells belonging to each age group. Sampling was done Groups Age Mean (µm) SD Significance
in a stepwise manner, moving the slide from left upper I 10‑20 51.97 8.89 1 versus 2, 3, 4, 5*
corner to right and then down to avoid measuring the II 21‑30 54.77 8.95 2 versus 3, 4, 5*
same cells again. Photographs were exported to MS paint III 31‑40 53.82 10.54 3 versus 4, 5*
and viewed under gridlines command. A line is drawn IV 41‑50 50.21 11.52 4 versus 5*
along the maximum diameter of the cell and nucleus. V >50 41.40 10.40
Total 50.43 10.06 P<0.001 (HS)
Number of grids from one end to the other end of the *Statistically significant. SD: Standard deviation, HS: Highly
line is counted separately for each cell and nucleus significant
26 Journal of Indian Academy of Dental Specialist Researchers ¦ Volume 5 ¦ Issue 1 ¦ January‑June 2018
Ilayaraja, et al.: Age estimation using exfoliative cytology
Table 2: Comparision of nuclear diameter within age
groups using one‑way analysis of variance and Tukey ‑
honest significance difference procedure
Groups Age Mean (µm) SD Significance
I 10‑20 7.59 1.56 1 versus 2, 3, 4*, 5*
II 21‑30 8.08 1.65 2 versus 3, 4*, 5*
III 31‑40 8.01 1.99 3 versus 4*, 5*
IV 41‑50 6.40 1.04 4 versus 5
V >50 5.80 1.40
Total 7.18 1.52 P<0.001 (HS)
*Statistically significant. SD: Standard deviation, HS: Highly
Figure 2: Screenshot demonstrates the measurement of cell diameter and significant
nuclear diameter in MS paint tool with Grindline selection
Discussion Table 3 : Nuclear‑cytoplasmic ratio comparison within
Exfoliative cytology has been used as a standard screening age groups using one‑way analysis of variance
aid for oral malignancy and premalignancy. The normal Groups Age n Mean (µm) SD P
I 10‑20 25 0.149 0.029 0.490
exfoliative cytology of the oral epithelium had been II 21‑30 25 0.150 0.033
[5] III 31‑40 25 0.155 0.053
thoroughly studied by Miller and Montgomery. Earlier
cytomorphometric analysis was done using planimetric IV 41‑50 25 0.135 0.043
methods, but with time, planimetric methods have been V >50 25 0.145 0.037
replaced by computer‑assisted image analysis techniques, Total 125 0.146 0.039
[6] SD: Standard deviation
which are faster, more accurate, and reproducible.
Hence, cytomorphometric analysis or image analysis of
exfoliated cells has been suggested as a key approach to A study was done by Shetty et al., using buccal smear
[5] exhibited a significant difference of CD similar to our
define and identify the cellular and nuclear changes.
[5]
A number of studies have been carried out to evaluate study. The reason attributed for this variation is cellular
the influence of systemic and local factors on normal senescence. A basal cell can only divide for a set of
[7] [8] number, then the renewal capacity of tissues declines
cells. Factors such as smoking, tobacco chewing, with age, resulting in the accumulation of senescent cells.
[9] [10]
anemia, and diabetes mellitus have shown These cells which stay for a longer duration in oral cavity
cytomorphometric changes in exfoliated cells of oral succumb to the effect of various local environmental
cavity. It is also used in early detection of premalignant [5]
[11] factors.
and malignant conditions of oral mucosa. Fluctuations
in hormonal levels also had an influence over the buccal When considering the ND, our study shows a constant
[12] decrease in ND except for the first group and second
cell morphology. Other factors such as radiotherapy,
chemotherapy, and medications are the possible factors group. Lee et al. reported no significant variation in
[14]
that could contribute to the morphometric changes in ND with age. Cowpe in his pilot study performed
[13] with clinically normal buccal squames displayed no
the cells. The cytomorphometric analysis result of the
[15]
present study revealed that there was a decrease in CD statistically significant variation in ND. Nayar et al.
and ND across different age groups. showed significant variation with an increase in ND with
[16]
The results show that average cell size varied between age. Patel et al. presented significant variation in NA
[6]
different age groups. When considering the cell diameter, with age.
in our study, there was a constant decrease in CD with When we consider the N:C ratio, our study shows no
age progression. Lee et al. could detect no significant significant difference in the N:C ratio across different age
variation in cell size in relation to age, between 6 and groups and remains constant. The study done by Scott
[14] et al. reveals that there was a reduction in N:C ratio with
80 years. Cowpe stated that there is a significant
[17]
variation in CD in his study from the smears taken from advancing age from the cells of lingual mucosa. Reddy
[15] et al. showed increase in N:C with age as there was a
75 patients obtained from various sites. Nayar et al.
found significant variation with a decrease in CD with significant elevation in mean nuclear area and significant
[16] [3]
age similar to our study. In the study, among the 80 reduction in mean cytoplasmic area. In our study as
subjects of Karnataka population, Patel et al. indicated there was a proportionate decrease in the nuclear and cell
significant variation in cytoplasmic area with different diameter, the N:C ratio remains constant. Limitations of
[6]
age groups. this study include small sample size with only 20 subjects
Journal of Indian Academy of Dental Specialist Researchers ¦ Volume 5 ¦ Issue 1 ¦ January‑June 2018 27
Ilayaraja, et al.: Age estimation using exfoliative cytology
in each age group. In this study, measurements were not 4. Anuradha A, Sivapathasundharam B. Image analysis of normal
done in the age group of 1–10 years and subjects were exfoliated gingival cells. Indian J Dent Res 2007;18:63‑6.
not divided into different age groups beyond 50 years. 5. Shetty DC, Wadhwan V, Khanna KS, Jain A, Gupta A.
Exfoliative cytology: A possible tool in age estimation in forensic
Although there are numerous methods available for age odontology. J Forensic Dent Sci 2015;7:63‑6.
estimation such as visual examination, physical and 6. Patel PV, Kumar S, Kumar V, Vidya G. Quantitative
chemical methods, and histological and radiographic cytomorphometric analysis of exfoliated normal gingival cells.
methods, this semi‑invasive cytomorphometric method J Cytol 2011;28:66‑72.
can also be added to them. Thus, a combination of 7. Babuta S, Garg R, Mogra K, Dagai N. Cytomorphometrical
methods can reduce the biological variations and analysis of exfoliated buccal mucosal cells: Effect of smoking.
Acta Med Int 2014;1:22‑7.
uncertainty associated with age estimation. 8. Buch AC, Patel SS, Chandanwale SS, Kumar H, Patel KM,
Bamanikar SA. Study of oral exfoliative cytology in tobacco
Conclusion chewers of Western India. Int J Pharm Bio sci 2014;1:138‑43.
The present study shows that there is a correlation 9. Sumanthi J, Reddy GS, Anuradha CH, Sekhar PC, Prasad LK,
between CD, ND, and the age of the individual. As the Reddy BV, et al. A study on cytomorphometric analysis of
exfoliative buccal cells in iron deficiency anemic patients.
age progression, there is a constant decrease in CD and Contemp Clin Dent 2012;3:S156‑9.
ND. The cell size is influenced or altered in many other 10. Rivera C, Núñez‑de‑Mendoza C. Exfoliative cytology of
systemic conditions; hence, all those parameters have oral epithelial cells from patients with type 2 diabetes:
to be considered. One limitation of this study is that it Cytomorphometric analysis. Int J Clin Exp Med 2013;6:667‑76.
cannot be used in deceased individuals. This study has 11. Verma R, Singh A, Badni M, Chandra A, Gupta S, Verma R,
to be carried out in a large number of samples to get a et al. Evaluation of exfoliative cytology in the diagnosis of
oral premalignant and malignant lesions: A cytomorphometric
better objective baseline for estimating the age. analysis. Dent Res J (Isfahan) 2015;12:83‑8.
Financial support and sponsorship 12. Donald PM, George R, Sriram G, Kavitha B,
Nil. Sivapathasundharam B. Hormonal changes in exfoliated normal
buccal mucosal cells. J Cytol 2013;30:252‑6.
Conflicts of interest 13. Goregen M, Akgul HM, Gundogdu C. The cytomorphological
There are no conflicts of interest. analysis of buccal mucosa cells in smokers. Turk J Med Sci
2011;41:205‑10.
14. Lee LH, Pappelis AJ, Pappelis GA, Kaplan HM. Cellular and
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28 Journal of Indian Academy of Dental Specialist Researchers ¦ Volume 5 ¦ Issue 1 ¦ January‑June 2018
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