Quantitative cytomorphometric analysis of exfoliated normal gingival cells.

BACKGROUND: The use of oral exfoliative cytology as a diagnostic aid accentuates the need for establishing an accurate baseline, thereby enabling the comparison of abnormal oral tissue with established baseline. AIMS AND
OBJECTIVE: To detect any changes in the nuclear area (NA), cytoplasmic area (CA), and nuclear:cytoplasmic ratio (N:C ratio) values for clinically normal gingival smears in relation to age and sex of apparently healthy subjects.
MATERIALS AND METHODS: Gingival smears were collected from 80 (40 male, 40 female) apparently healthy subjects belonging to the age group of 0-20, 21-40, 41-60, and more than 60 years. Smear slides were fixed by using spray fixative. The smears were stained using Papanicolaou procedure. The cytoplasmic and NAs were measured using image analysis software. Statistical analysis of the data was done using one-way ANOVA with Tukey-HSD procedure and Student's t test.
RESULTS: The result showed that there was a significant difference (P<0.001) in NA, CA, and N:C in males of different age groups. There was a significant difference (P<0.001) in NA, CA, and N:C in females of different age groups. The difference in N:C between males and females was significant (P<0.001) in all the groups. The difference in NA, CA, and N:C with age irrespective of gender was significant (P<0.05). There was a significant difference (P<0.05) between males and females with respect to NA, CA, and N:C irrespective of age.
CONCLUSION: Age-and sex-related alterations were observed in gingival smears, which could be a baseline for these variables to compare identical measurements, made on pathologic smears of oral premalignant and malignant lesions.

Introduction

Oral cancer is one of the common cancers and constitutes a major health problem in developing countries, representing the leading cause of death. Although representing 2–4% of the malignancies in the western part of the world, the oral cancer accounts for almost 40% of all cancers in the Indian subcontinent.[1] The early diagnosis and the consequent treatment of precancerous lesions could prevent a large number of deaths due to this disease. Currently, a wide range of early diagnostic aids, such as toluidine blue, chemiluminescence, and tissue autofluorescence are being used for early detection and prompt treatment of the lesion.[2] In spite of all these procedures, clinical examination and histopathological studies of biopsy material are the classical diagnostic methods used for precancerous and cancerous oral lesions.[34]

In the last few years the interest for oral exfoliative cytology as a diagnostic and prognostic methodology and also for monitoring patients in oral precancer and cancer has re-emerged. However, in general, cytology has relied primarily on the personal judgement of the cytologist and not on the measurement of cell parameters.[5] Controversy has surrounded the use of cytology in the diagnosis of oral cancer. One of the main reasons for this being the occurrence of false-negative results.[45] A number of authors have suggested reasons for this, including inadequate sampling, technical error, misinterpretation of the findings, and bias.[6-8]

To minimise the false-negative results, many authors, such as Cowpe et al,[910] Goldsby et al,[11] and Ogd et al.[12] have suggested the utilisation of quantitative techniques, based on the evaluation of parameters, such as nuclear area (NA), cytoplasmic area (CA), and nucleus-to-cytoplasmic area ratio (NA/CA). This may increase the sensitivity of exfoliative cytology for early diagnosis of oral cancers, since these techniques are precise, objective, and reproducible and non-invasive.[9-12] Although, a number of quantitative cytomorphologic and cytomorphometric studies have been carried out on precancerous and cancerous lesions presenting in the oral cavity but few studies have been performed on clinically normal oral mucosa matched for age and sex. Moreover, authors, such as Morrison et al[13] and Cowpe et al,[10] have also clearly stressed the need of a baseline cytomorphologic data of normal oral squamous cells prior to the examination of pathological squamous cells, for comparison and evaluation of malignant epithelial cells. The aim of this study was to detect any changes in NA, CA, and nuclear:cytoplasmic ratio (N:C ratio) values, for clinically normal gingival smears in relation to age and sex. Thus, an attempt is made to establish a baseline for these variables with which, to compare identical measurements, made on pathological smears.

Materials and Methods

A total of 80 apparently healthy subjects were selected randomly from the OPD of the college. A prior written consent was taken based on “Declaration of Helsinki” (1964) from all included subjects.[14] The study was approved by ethical committee at the University.

Subjects were divided accordingly [Table 1]:

Table 1

Division of subjects based on their age and gender (n=80)

40 Male: further to age group of (10 each) 0–20, 21–40, 41–60, more than 60 years

40 Female: further to age group of (10 each) 0–20, 21–40, 41–60, more than 60 years

Subjects were included who were clinically healthy and presented normal gingival tissue with gingival index[15] (Loe and Silness) score less than 1.

Subjects were excluded who presented with history of alcohol intake, tobacco, or its products abuse in any form (present or past), any clinical pathological findings on the gingiva (acute or chronic in nature), history of anemia, diabetes mellitus, hepatitis, tuberculosis, acquired immune deficiency syndrome and leukemia or any other systemic or hormonal condition, which predisposes the gingival manifestation, and who were receiving or had received prior to study entry, systemic corticosteroids, immunosuppressive agents, radiation therapy, and/or chemotherapy.

Screening/baseline

At baseline for each subject name, age, gender, occupation, and relevant medical history were recorded. A thorough clinical examination was performed to rule out any pathologic changes in oral cavity, especially on gingival tissue. A thorough scaling (tooth cleaning) was performed to eliminate any inflammation from the gingival tissue. Subjects were recalled after 2-week intervals and gingival index[15] (Loe and Sillness) was recorded to include the subjects with a score of less than 1. Following confirmation of score less than 1, each subject was asked to rinse with normal saline (0.91% isotonic solution) to maintain the normal tonicity of superficial gingival epithelial cells. A sterilised disposable interproximal brush (STIM® Interprox Brushes, DENT–AIDS, New Delhi, India) was used to scrap the attached gingiva in upper anterior region [Figure 1]. Scrapings were smeared onto the slide. A minimum of 4 smear samples were taken from each subject that would be sufficient to give 200 cells per subject (50 cells per smears). In order to obtain cells from all the layers of the epithelium, a moderate pressure was applied while taking the smear. The smears were immediately fixed using spray fixative (RAPID PAP® Spray fixative, Biolab Diagnostics (I) Pvt Ltd, India) to avoid air drying. Papanicolaou technique (RAPID PAP® stain, Biolab Diagnostics (I) Pvt. Ltd., India) was used to stain the smear. The stained smears were examined under a microscope equipped with a 40× objective (Olympus WPlanFL 160/0, Tokyo, Japan) and a 2.25× video projection lens (Nikon CCTV/Microscope Adapter, Yokohama, Japan). The acquired images were transmitted to a video camera (CCD 72; Dage MTI, Michigan City, IN, USA) for display on a video monitor (Sony HR Trinitron, Tokyo, Japan). A screen shot of each slide was captured saved and transferred to the computer for image analysis.

Figure 1

Figure demonstrating collection of smear from the anterior attached gingiva using an interproximal brush

Image analysis

Two hundred cells per subject, which were unfolded with clear outline were selected for the study. Cells were analysed for CA, NA, and nucleocytoplasmic (N/C) ratio using a software (SCION image for window v.4.0.3.2, Scion Corporation, Frederick, MD, USA). The sampling was done in a stepwise manner, moving the slide from left upper corner to right and then down in order to avoid measuring the same cells again. For measurement, the software was calibrated and scale setting was changed from square pixels to square micrometers (μm2). The instructions were followed, as given in the manual of the software for measuring the cell sizes. The nucleus and the cell outline were traced using digital cursor on the screen and the software automatically calculated the cell and NA [Figure 2]. The N:C ratio was calculated manually. All the recorded data underwent statistical analysis.

Figure 2

Screen-shot demonstrating the measurement of nuclear area using a computer image analysis software

Statistical analysis

The various parameters were used to analyse the NA, CA, N:C ratio of each cells in relation to age and sex of apparently healthy subjects. One-way ANOVA test was used for analysis of CA, NA, and N:C ratio in relation of age and sex. Tukey–HSD procedure was used to identify the significance between various age groups and sex. Student's t test was used to evaluate the difference in CA, NA, and N:C ratio in male and female in various age groups. The level of significance was set at P≤0.05. Data was reported as mean and standard deviations.

Results

The nuclei of epithelial cells of healthy subjects was normal with no signs of morphological alterations.The statistical result showed that the difference in NA, CA, and N:C ratio in males of different age groups was statistically significant (P<0.001) [Table 2]. There was a significant difference (P<0.001) in NA, CA, and N:C ratio in females in different age groups [Table 3]. The difference in NA between males and females was significant (P<0.001) in 0–20, 21–40, and 41–60 age groups, whereas difference in CA was significant (P<0.001) in 0–20, 21–40, and >60 age groups and the difference in N:C was significant (P<0.01) in all the age groups [Table 4]. The difference in NA, CA, and N:C ratio with age irrespective of gender was significant (P<0.05) [Table 5]. There was a significant difference (P<0.05) between males and females in the terms of NA, CA, and N:C ratio irrespective of age [Table 6].

Table 2

Comparison within age groups in males using one-way ANOVA and Tukey-HSD procedure

Table 3

Comparison within age groups in females using one-way ANOVA and Tukey–HSD procedure

Table 4

Comparison between males and females in different age groups by Student's independent t-test

Table 5

Age variation of nuclear area, cell area, and nuclear-cytoplasmic ratio irrespective of gender using one-way ANOVA and Tukey–HSD procedure

Table 6

Variation of nuclear area, cell area and nuclear-cytoplasmic with respect to gender using one-way ANOVA and Tukey-HSD procedure

Discussion

The present study represents a quantitative technique applied to the smears collected from clinically normal gingival mucosa, matched for age and sex in an attempt to define a baseline for comparison with pathological smears. We hoped that if this was achieved, the diagnostic potential of exfoliative cytology in the diagnosis of early gingival malignancy would be greatly enhanced.

In recent years, there has been increasing interest in the role of exfoliative cytological technique as a screening aid for the oral malignancy and pre-malignancies. A growing body of literature has also accumulated regarding the role of exfoliative cytology as a standard technique in screening of oral pathologies. Moreover, cytomorphometric analysis or image analysis of exfoliated cells has also been suggested as a key approach to define and identify the cellular and nuclear changes in such cytological smears.[1011]

Previously, cytomorphometric analysis was done by using planimetric methods but with time, planimetric methods have been replaced by computer-assisted image analysis techniques, which are faster, more accurate, and more reproducible. In this regard authors, such as Ogden et al[12] suggested that this computer-assisted analysis of images formed by a microscope may increase the sensitivity of exfoliative cytology for early diagnosis of oral cancers, since these techniques are precise, objective, and reproducible. Similarly, Cowpe et al[910] also demonstrated that exfoliative cytology is capable of detecting malignant changes, through estimation of NA:CA ratio using the planimetric method in Papanicolaou-stained smears. This study concluded that 50 cells were sufficient to provide indication of malignant changes.[10] Since then, a number of studies have been carried out using this technique to evaluate the influence of systemic and local factors on normal cells. Cowpe et al[9] found that tissues undergoing malignant transformation typically show a reduction in CA before the reduction in NA. They also suggested that samples of healthy mucosa from the same patient provide the best control.[910]

The cytomorphometric result of the present study revealed an age-related significant variation in NA, CA, and N:C ratio, irrespective of gender. Age-related variation, irrespective of gender can be ascribed to cellular senescence.[1617] A basal cell can only divide for a set of number; then the renewal capacity of tissues declines with age, resulting in the accumulation of senescent cells. The cells which stay for a longer duration in the oral cavity succumb to the effect of various local environmental factors.[17]

Our result suggested that there was a significant difference in NA, CA, and N:C ratio in different age groups of female. This may be attributed to the variations in the hormonal levels in female throughout the life time. Both estrogen and progesterone promote protein anabolism and growth of the organ system, including oral cavity. Estrogen influences the cytodifferentiation of stratified squamous epithelium[1819] as well as the synthesis and maintenance of fibrous collagen.[19]

Our result showed a statistically significant comparative variation in CA and NA, between males and females in 0–20 age groups. These changes could be ascribed to the hormonal changes occurring at puberty. Data suggest that at puberty there is a significant increase in sex hormone in both male and female life. Changes in hormone levels have been related to an increased prevalence of gingivitis followed by remission.[1820] Increase in gingival inflammation is one of the factors that can increase NA and lead to a poorly preserved cytoplasm. However, these characteristics are typically found only in young cells and are not representative of cellular atypia.

In the second age group (21–40 years), there was a significant comparative variation in NA and N:C ratio between males and females. This may be attributed to the hormonal imbalances occurring during 21–40 years of age.[18] Mostly women in this age group are on oral contraceptives and often experience hormonal changes associated with pregnancy.[21-23] In the present study, the detailed history of oral contraceptives intake and pregnancy was not established. Therefore, these factors might have shown age-related variations in females in the age group of 21–40 years.

In the age group of 41–60 years there was statistically significant comparative variation in NA and N:C ratio between males and females. The observed variations could be ascribed to hormonal changes occurring during or after menopause in women's life.[25] The time frame between regular menstrual cycles and the cessation of menstrual periods, called perimenopausal transition, is 2–7 years. During this period, the concentration of circulating estrogen decreases, while follicle-stimulating hormone and luteinizing hormone concentrations increase.[2425] Consequently, the effects of estrogen on gingival tissue are reduced, therefore compromising the anti-inflammatory effect of this hormone on the gingival tissue and ultimately resulting in cytological variation in the gingival tissues.

There was a significant variation of NA and CA with respect to gender and irrespective of age. The result showed that NA and CA were significantly greater in females than in males of all age groups except in >60 age group. However, the difference in N:C ratio between males and females was not statistically significant. The variation of NA and CA with respect to gender can be ascribed to sexual dimorphism observed in human[26] and hormonal difference in individuals. Hormonal influence happens to be the differentiating factor for the cytomorphometric trends of males and females. Testosterone has been frequently associated with metabolism and maintenance of bone and connective tissue matrix unlike the female sex hormones, which have their effect on the epithelium.[18]

On comparing our results with the results of previous studies, our study results showed a similar result as shown by Anuradha and Sivapathasundharam[27] where similar age-related and sex-related alterations are observed in gingival smears. Our results are also similar to the findings of Ramaesh et al,[28] where they found that smears from normal buccal mucosa of healthy subjects display a wide variation in the size of the cells and nuclei.

Moreover, our result are also in agreement with results of Scott et al.[29] They showed a reduction in N:C ratio with advancing age at a similar rate in both males and females. Similarly Cowpe et al also showed a significant variation in nuclear diameter (ND) with age but there was no variation in cell diameter (CD), whereas the ND and CD varied significantly in various regions in the oral cavity.[10] In the current study, we found statistically significant maximum CA and NA values for age group of 21–40 years followed by 41–60, 0–21, and >60 years age group.

Our results also confirm the findings of Zitwack et al,[30] where animals treated with estrogen showed larger and more active cells in the vagina with similar changes in gingiva and buccal mucosa. Similarly, our results also confirm the findings of Nayar and Sivapathasundharam[31] where the ND was reported to increase with age and CD to decrease with age. The cell diameter in the age group 40–59 years, particularly was less in females than in males. Our result also showed similar trend but in the age group of 41–60 and >60 years, the NA was lesser than that in the 21–40 years age group. However, our result is not in agreement with the results of Lee et al[32] where no significant variations in ND and CD with age has been reported.

Another important method of quantifying cytological smears is to apply the technique of DNA cytophotometry. This technique has been used extensively in the past to characterize the proliferative activity of cell populations. However, with the occurrence of aberrant diploid DNA distributions in malignancy, the value of DNA measurement as a single diagnostic criterion of malignancy has reduced to some extent. Therefore, authors, such as Cowpe and Longmore[33] have expressed a need for the measurement of more variables than just DNA content, in order to increase the accuracy of cytology in the diagnosis of oral malignancy.[34] Goldsby et al[11] in their study on normal human exfoliative oral cells concluded that the measurement of nuclear and cell size and N:C ratio are the 3 most important factors to be considered while producing a baseline for normal oral squamous cells. Whereas Cowpe and Longmore[33] concluded that NA provides an accurate baseline against which future measurements on abnormal tissue can be compared. Therefore, only these factors are considered in the present study.

Several authors, such as Miller et al,[34] Montgomery,[35] and Cowpe et al,[10] have evaluated different sites in clinically healthy oral cavity that included buccal mucosa, floor of the mouth, hard and soft palate, lower labial mucosa, and dorsum of tongue. However, data on gingival tissue is scarce. Therefore, gingival tissue is considered in the present study to provide some initial experimental baseline data.

Data suggest that inflammation is one of the factors that can increase NA and lead to a poorly preserved cytoplasm, but these characteristics are typically found only in young cells and are not representative of cellular atypia.[36] Therefore, in the present study we performed a thorough scaling (tooth cleaning) to control the inflammatory factors. Moreover, we took the smears from attached gingiva of upper anterior teeth region to reduce the effect of localized inflammation on our results. We also detected a range of cellular age in the smears, and cytomorphometric measurements were generalized rather than restricted to a certain generation of cells, suggesting that the changes we observed are not just related to inflammation.

Our study is a preliminary attempt to define a baseline for pathological smears obtained from gingival tissue. The data presented in our study can be of more diagnostic value, if future pathological samples are obtained and processed in a similar manner to the present study. As each study differs in the number of cells being counted per slide, type of fixatives used, time lapse of staining following smear preparation and time lapse of collection of specimen, and cytomorphometric analysis. Therefore, there is a need to establish a guideline for cytological procedure and to standardize the cytological and image analysis procedures.

Conclusion

The present study shows age- and sex-related variations of NA, CA, and N:C in normal exfoliated gingival cells, which could be considered as a preliminary attempt to provide a baseline of quantitative cytomorphometric data, with which pathological smears can be compared. Thereby enhancing the diagnostic and screening potential of the oral exfoliative cytological technique. Moreover, the data presented in the present study can be utilised for the identification of premalignant/malignant gingival lesion in a more objective manner.