International Journal of Oral Health Sciences

: 2015  |  Volume : 5  |  Issue : 1  |  Page : 21--29

Evaluation of role of cytodiagnostic techniques in detection of oral premalignant and malignant lesions: Study of 50 cases

Anup Pravin Bhandari, Rasika Uday Gadkari 
 Department of Pathology, SRTR Government Medical College, Ambajogai, Maharashtra, India

Correspondence Address:
Anup Pravin Bhandari
D-602, Pearl Tower, Gadital, Hadapsar, Pune-411 028, Maharashtra


Aims and Objectives: In this study, we studied cytomorphological features and determined the sensitivity and specificity of cytology specimens obtained with simple instruments like toothbrush (modified brush biopsy), wooden spatula and analyzed without computer-assisted analysis. Settings and Design: Fifty patients attending outpatient clinics who exhibited oral lesions suspicious of premalignancy or malignancy were enrolled. All patients underwent scrapping by wooden ice cream stick and an oral brush biopsy using a baby toothbrush. The specimens were analyzed manually in a double-blinded fashion. Histopathological confirmation by a scalpel biopsy was obtained whenever possible. Chi square test and Fischer exact 't' test was used for statistical analysis. Results: In our study, brush cytology was better (P < 0.05) than scrape cytology in the criteria of cellularity, abnormal cell group adequacy and detection of higher grade of dysplasia. Also, we found that increased N: C ratio, nuclear pleomorphism, nucleoli and presence of micronuclei were the parameters statistically significant in oral carcinoma as compared to high grade dysplasia. Sensitivity of detecting malignancy by cytodiagnosis was 76.47% and false negative rate of 23.53% was present in our study. Conclusions: Modified brush biopsy is relatively easy, non invasive, OPD procedure, particularly an effective tool for screening small innocuous lesions for dysplasia/ malignancy as each of their evaluation by cumbersome and painful scalpel biopsy procedure is often impractical. Early diagnosis of dysplasia/ malignancy helps patient to aim for curative treatment. Though cytomorphological difference between high grade dysplasia and carcinoma is not very distinct, certain features like high grade nuclear pleomorphism, presence of high frequency of micronuclei, tumor diathesis and bizarre shaped cells like tadpole, spindle shape etc. are the features which are more significantly associated with malignancy.

How to cite this article:
Bhandari AP, Gadkari RU. Evaluation of role of cytodiagnostic techniques in detection of oral premalignant and malignant lesions: Study of 50 cases.Int J Oral Health Sci 2015;5:21-29

How to cite this URL:
Bhandari AP, Gadkari RU. Evaluation of role of cytodiagnostic techniques in detection of oral premalignant and malignant lesions: Study of 50 cases. Int J Oral Health Sci [serial online] 2015 [cited 2020 Sep 22 ];5:21-29
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Oral cancer is increasing worldwide, and it is sixth most common cancer in the world. In developing countries such as India, Sri Lanka, and Pakistan, it is the most common cancer in men accounting for 30% of all new cancer cases. In addition to increasing incidence, it also accounts for 50–70% of total cancer mortality which is very high when compared with other cancer-related mortality.[1] This lack of improvement in the prognosis of oral cancer despite advanced surgery and management techniques is primarily thought to be due to diagnostic delay as indicated by 5 years survival rate. The prognosis for patients with oral cancer that is treated early (stage I at diagnosis) is much better, with 5-year survival rates as high as 80%. In addition, the rate of recurrence after surgery is very high (3–7%) and is often the cause of death. All this warrants better and early diagnostic approach to oral cancer.[1]

Although, histological examination of tissue remains the gold standard for diagnosis of malignant oral lesions, a biopsy is an invasive technique with surgical implications, technique limitations, and psychological implications for most patients.[5] It also presents limitations when the lesions are large, where it is important to select the most appropriate site of biopsy. These issues underline the importance of discovering and developing new diagnostic methods, improving the existing ones and discovering new techniques. Among these newer techniques, exfoliative cytology has gained importance as a rapid and simple, noninvasive method.[2] Cytology has now been widely accepted as a tool in the early diagnosis of cancer and has taken its rightful place beside the other recognized branches of hospital diagnostic services.

When encountering an oral lesion without an obvious etiology, options for the clinician include an observation period of some defined time, toluidine blue staining to assist in the selection of the most suspicious site for biopsy, various fluorescent or chemiluminescent aids for visualization of the lesion, oral brush biopsy to evaluate the lesion or scalpel biopsy. There is sufficient evidence that visual inspection alone is not adequate to differentiate precancers and early oral cancers from benign lesions, regardless of the expertise of the clinician. Thus, oral lesions without an underlying etiology should not be observed or "watched" and instead, require evaluation to rule out dysplasia and carcinoma. Unlike cervical cytology, which has been shown to be an accurate method of detecting dysplasia, the examination of oral cytology specimens obtained by brushing of the oral mucosa with a spatula or cytobrush results in an unacceptably high number of errors.[3],[4],[5] A variety of ancillary techniques such as toluidine blue, p53 analysis, AgNOR, flow cytometry, and ploidy analysis have been investigated in addition to oral brush biopsy and have yielded promising results.[2]

The oral brush biopsy obtains a complete transepithelial biopsy specimen with cellular representation from each of the 3 layers (the superfical, intermediate, and parabasal/basal) of the lesion, thus providing a completely disaggregated biopsy sample. The method of obtaining cells from at least 2 layers of the epithelial tissue collected by any nonlacerational device such as a brush has been patented [6],[7] and developed as the oral brush biopsy with computer-assisted analysis (Oral-CDx, Suffern, NY). The test is indicated for oral lesions that are not clinically suspicious, despite the fact that such lesions may harbor dysplasia. Highly suspicious oral lesions should still be subjected to an immediate scalpel biopsy to diagnose dysplasia and carcinoma.

The oral brush biopsy with computer-assisted analysis has been found to be a simple, relatively inexpensive, highly sensitive, and specific test. The specimen is obtained by a specially designed brush and contrasts with an inadequate exfoliative cytology sample that consists only of cells from the superficial layer of the epithelium.

The use of a brush biopsy without computer-assisted analysis may have applications in resource challenged areas and could be a risk-free method of evaluating oral lesions. In this study, we determined the sensitivity and specificity of cytology specimens obtained with simple instruments like a toothbrush (modified brush biopsy), wooden spatula, and analyzed without computer-assisted analysis while confirming by histopathology study of a scalpel biopsy wherever possible in the evaluation of oral premalignant and malignant lesions.

 Subjects And Methods

This was hospital-based diagnostic test evaluation study (cross-sectional analytical study) carried out in Department of Pathology, Shree Ramanand Teerth Medical College and Hospital, Ambajogai, Maharashtra for 2½ years. All patients having oral lesions with an abnormal epithelial surface were included in the study (e.g., premalignant lesions such as leukoplakia, erythroplakia, submucous fibrosis, chronic hypertrophic candidiasis, lichen planus, and suspected malignant lesions) while submucosal lesions were excluded. Total 50 patients were studied in this study.


Clinical data collection

Detailed demographic information of each patient was obtained, including the patient's age, sex, dental hygiene, personal habits, and present complaints.

Emphasis was given to collecting information about consumption of tobacco and alcohol. The detailed clinical examination was performed on each patient to assess the site, size, and the clinical characteristics of the lesion as well as the extent of local infilitration (if any), cervical lymph node metastases (if any) and effectiveness of oral hygiene [Figure 1]a and [Figure 1]b. The study did not aim at monitoring the patients in the long-term.{Figure 1}

Detailed clinical evaluation as per proforma and written informed consent of each patient was taken, and the procedure was performed under aseptic precautions.


After rinsing the oral cavity thoroughly with water, the lesion was visualized under adequate illumination.


Appropriate areas of lesions were scraped first with a wooden spatula (ice cream stick) then with cytobrush [Figure 1]c. A commercially available hard nylon baby toothbrush was used to obtain a complete transepithelial biopsy with minimal discomfort. Using moderate pressure, the spatula/brush was repeatedly applied in one direction over the entire lesion many times until pinpoint bleeding was obtained, signaling entry into lamina propria and thus obtaining epithelial cells through the full thickness of the epithelium. All aseptic precautions were taken during procedure and instruments were discarded after the procedure. The material from the instrument was spread on the middle third of 2 clean, dried glass slides. The smears were fixed immediately with 100% ethanol and stained with Papanicolaou's method.

All specimens were examined manually independently by two different cytopathologists in a double-blind fashion. If there were any discrepancies, a third opinion was obtained to reach a final diagnosis. As the cytological slides were evaluated by cytopathologists and the patients were examined by the ENT or dental specialists, there was no chance of bias.

As the cellularity from this type of brush biopsy sample is scant, only slides with at least 30 well-preserved cells (i.e., not obscured by blood or exudate or necrosis) from deep epithelial layers (intermediate or parabasal-basal) were considered adequate. The minimum number of cells confirmed to the adequacy criteria previously followed by Navone et al.[8] Inadequate specimens were excluded from the data.

The following outcome measures were analyzed in the smears:

Cellularity Abnormal cell group adequacy (at least 30 well-preserved cells were considered adequate) Hyperchromatism, chromatin pattern, and distribution [Figure 2]d Nucleo: Cytoplasmic ratio (graded as N = normal N: C ratio, I [nuclear size approximately 1/3–1/2 to that of cell size], II [nuclear size approximately 1/2–2/3 to that of cell size]) [Figure 2]a and [Figure 2]b Variation in nuclear size and shape (nuclear pleomorphism) (graded as N = no pleomorphism, Grade I [mild pleomorphism], Grade II [high-grade pleomorphism]) The presence of prominent nucleoli [Figure 2]c The presence of micronuclei (MN) (According to the criteria put forward by Countryman and Heddle [9] size of MN should be at 1/3 of the size of the nucleus. We used same criteria regarding the assessment of the size of MN. Structures ranging in <1/3 of the size of the nucleus were considered as MN) [Figure 3] Nuclear membrane irregularity [Figure 2]d Mitosis Bi/multinucleation [Figure 4]a Keratinization [Figure 4]c The presence of inflammation Hemorrhage Necrosis Abnormal cell shapes, e.g. tadpole forms, and as well as a discrepancy in nucleo-cytoplasmic maturation, other specific findings [Figure 4]d.{Figure 2}{Figure 3}{Figure 4}

For frequency estimation of MN computerized image analysis is essential (as at least 10,000 cells should be screened for estimation of average MN/Cell [10]). In our study, due to unavailability of image analyzer, for manual screening we simplified grading for the presence of MN as positive (>10/100 cells)/negative (10< 100/cells) based on calculating average no. MN/100 cells. Cells showing clear-cut evidence of these changes, especially if present in the background of tumor diathesis, as evidenced by necrosis and dysplastic changes were categorized as malignant. Those exhibiting borderline changes were considered dysplastic while those with no specific changes were labeled negatively for dysplasia/malignancy (NDM).

For purposes of analysis, lesions were divided into low-grade dysplasia, high-grade dysplasia, and carcinoma. Lesions with inflammatory changes were included in low-grade dysplasia category. The lesions with no dysplastic changes were considered NDM. A biopsy was done, and the histopathological correlation obtained wherever possible.

Statistical methods

The statistical software namely GraphPad Prism 6 and GraphPad Instat (Graph Pad Software Inc., USA)were used for the analysis of the data and Microsoft Word and Excel have been used to generate graphs, tables, etc.

Chi-square test was used for comparison of brush cytology and scrape cytology while Fischer exact t-test is used for comparison of cytomorphological features of low-grade dysplasia, high-grade dysplasia, and carcinoma. P < 0.05 was considered as statistically significant.


In the present study, out of 50 cases, oral carcinoma was presented in 16 cases with the proportion of 32% while dysplasia was reported in 60% of cases. Oral malignancy was more common in males than in females in the ratio of 4.3:1 and mean age of the patient of oral malignancy in our study was 58.64 (±10.13) years while that for low and high-grade dysplasia were 39.61 (±12.80) and 49.13 (±13.68) years, respectively [Table 1].{Table 1}

The most common site involved in the present study for dysplastic/malignant lesions was tongue (28%) followed by buccal mucosa (20%) while least common site were soft and hard palate (4% each). In our study, the most common clinical presentation was an ulcer (50%) followed by growth (44%), reduced mouth opening (32%), white patch (18%), and pain (18%). Reduced mouth opening and ulcer (both 50%) were most common complaints in NDM cases while the white patch was most commonly associated with low-grade dysplasia (37%). Growth was a chief complaint in cases of high grade (64%) and carcinoma (69%). The pain was more associated with benign nature of disease with the maximum percentage in NDM (25%) while least in malignancy (6%).

In our study, 87.5% of oral carcinoma patients were addicted to tobacco chewing/smoking/alcohol with an average duration of addiction being 20.85 years while out of 30 patients with dysplastic features, 22 patients (73.34%) were having an addiction with average duration 14.36 years. The average duration of addiction in patients with NDM was 11.5 years.

In our study, when we compared wooden spatula with a brush, it was observed that brush cytology was significantly better (<0.05) than scrape cytology in the criteria of cellularity, abnormal cell group adequacy and detection of a higher grade of dysplasia on cytology [Table 2].{Table 2}

In the study between the comparison of carcinoma and high-grade dysplasia, we compared 12 parameters as enlisted in the table. Out of these, we found that increased N:C ratio, nuclear pleomorphism, nucleoli and presence of MN were the parameters statistically significant in oral carcinoma (P < 0.05).

A similar comparison between high-grade dysplasia and low-grade dysplasia showed that hyperchromasia increased N:C ratio, nuclear pleomorphism, the presence of mitosis, nucleoli, nuclear membrane irregularity, bi/multinucleation, keratinization, and necrosis were statistically significant finding in our study [Table 3].{Table 3}

[Table 4] shows out of 17 cases where subsequent scalpel biopsy was done, 4 cases turned out to be malignant which were earlier reported as high-grade dysplasia while out of 16 cytodiagnosis of carcinoma 13 cases were confirmed on histopathology.{Table 4}


The incidence of oral cancers and oral premalignant lesions is very high in India as compared with western population primarily because customs of tobacco chewing habits, betel nut consumption. Dysplasia and early carcinomas are asymptomatic and commonly misinterpreted as benign lesions or innocuous oral problems. The inconspicuous nature of these lesions or misleading perception of practitioners may primarily be responsible for the advanced stages of these tumors at the time of discovery from which complete recovery is not possible.[11]

Though scalpel biopsy followed by histopathology is considered as gold standard in diagnosing these lesions, it may not be feasible to do scalpel biopsy in all suspected cases (the patient maybe medically compromised or may refuse to undergo scalpel biopsy). In such cases, cytology may offer an attractive alternative. In our study, we aimed to study newer cytodiagnostic methods and its utility for early detection of oral carcinoma.

This study revealed a male:female ratio of 4.3:1 with the largest number of oral cancers developing in the fifth and sixth decades of life (mean age =58.64 years). This is consistent with an earlier report from this group that confirned that oral cancer in Northern India was a disease of middle-aged men.[12]

In this study, the buccal mucosa and tongue were the most frequently involved sites (20% and 28% respectively), while the palate was the least commonly involved site (4%). In contrast, the lateral tongue and floor of mouth are the more commonly involved sites in the west, the anterior two-thirds of the tongue is commonly involved in India; while the posterior lateral border and ventral surfaces are frequently involved in the United States (according to study by Nair et al.).[13] These regional differences may be attributed to the extensive use of chewing tobacco in the Indian subcontinent compared to smoking in the west with the incidence being highest at mucosal sites with prolonged contact with carcinogens.[14]

Association of oral cancer with consumption of alcohol is also lower in the Indian population as compared to tobacco consumption. Among various substances of abuse, tobacco consumption was the most common with 38 patients (76%) having used it in one form or another. Six patients (12%) gave a history of exposure to more than one carcinogen while 2 patients (4%) consumed heavy amounts of alcohol. Duration of addiction as measured in years was directly proportional to the grade of cytological atypia [Table 1]. In the study performed by Mehrotra et al.[12] tobacco consumption was the most common with 54% patients having used it in one form or another, 35.4% gave a history of exposure to more than one carcinogen while 5% patients consumed heavy amounts of alcohol.

Exfoliative cytology

The current general perception is that the sensitivity of exfoliative cytology is not sufficient to warrant its widespread use as a screening modality to triage visible lesions because of high false negative rates, which can exceed 30%.[15] Second, no established diagnostic criteria like in cervical cytology is present to grade dysplasia and its further management. In our study, we have tried to address both these problems.

It is well established that only 20% of cervical cytology cells collected on a variety of collection devices from the uterine cervix can be mechanically transferred to the flat surface of a glass slide, and the small portion of cells that do get onto slide are not randomly selected from the sampling device.[16] Another postulate suggested is that a potential source for false negative smears in oral cytology smears may be the low number of the cells that are transferred onto the glass slide. Presently, oral cells can be obtained by different physical systems of scraping the surface of the mucosa, by rinsing the oral cavity or even by taking a sample of saliva from the patients. Unlike the cervical Papanicolaou smear, exfoliative cytology in the oral cavity has proven to be a little value because of the poor results are due, in part to the fact that cytology instruments do not obtain a sample from the deepest layers of the oral lesion and also anatomy of mouth making it difficult to access posterior structures. Newer methods like brush biopsy may offer an alternative to obtain deeper and high number of cell yield.

The commercially available computer-assisted oral brush biopsy system, Oral-CDx, is a diagnostic procedure with high sensitivity and low false negative value for detection of dysplasia and innocuous-appearing oral cancers at an early, curable stage. Sciubba demonstrated a high sensitivity of 96% with a false negative rate of up to 4% by using this technique.[17] In all studies where brush biopsies and scalpel biopsies were performed concomitantly on the same lesion and by the same examiner, the results of the US pivotal clinical trial were replicated. The improved accuracy of this technique over cytology is attributed to the complete transepithelial cellular sample and the analysis of specimens with computer assistance.

The oral brush biopsy has broad potential to fill the "Diagnostic Gap" that currently challenges the early detection of oral cancer and can be an effective and noninvasive means of detecting dysplasia as well as early carcinoma in those patients who are either asymptomatic or in those with minor symptoms who do not warrant an immediate biopsy.[18] In their study reported that the mean delay time in diagnosing a carcinoma in different cases was 117.25 days because harmful lesions that were benign-appearing were not evaluated promptly.[19] suggested that the main reason for the use of oral brush biopsy is not to find a substitute for scalpel biopsy, but rather to take advantage of a first-level test that is able to identify dysplastic cells or molecular alterations which would be an indication for histological control, even in clinically apparently benign oral lesions. The oral brush biopsy has developed a bridge between the visual examination and referral for histological evaluation in the clinical assessment of a patient. It helps to determine if an oral lesion with innocuous clinical features requires surgical biopsy during the time of oral examination. The importance of the brush biopsy for evaluating harmless looking lesions has been emphasized in a multicenter study where nearly 5% of clinically benign-appearing mucosal lesions were sampled and later confirmed by scalpel biopsy to represent dysplastic epithelial changes or invasive cancer.[17]

Unlike the Oral-CDx brush, which was specifically designed to penetrate down to the basement membrane, we tried manual brush biopsy by an inexpensive baby toothbrush and compare its efficacy with conventional scrape cytology. In 50 patients we studied, it was observed that brush cytology was significantly better (P <0.05) than scrape cytology in the criteria of cellularity, abnormal cell group adequacy, and change in cytodiagnosis to a higher grade. Though hemorrhage observed was less in brush cytology (in 4 patients out of 50) it was not statistically significant. This showed that brush biopsy helps in collecting deeper cells where early dysplasia can be observed. Being de novo technique there are no published data to compare our results. Furthermore, the study showed 8% inadequate sample rate, a number that is greater than that reported for the commercial Oral-CDx brush biopsy. It is attributed to the fact that the number of cells examined in this study (minimum 30 well-preserved cells) was significantly less than hundreds of thousands of cells on a typical Oral-CDx slide that is subsequently examined by the computer.

Our other objective was to study various cytomorphological features of oral premalignant and malignant conditions and formulate a system for diagnosis based on 12 cytomorphological parameters [Table 3]. Out of 50 cases, 4 cases were reported NDM in which no abnormality was detected in any parameter. In the category of low-grade dysplasia (all inflammatory cases were included as there is always mild dysplasia associated with inflammation), 18 cases were reported where most of the inflammatory smears (7 cases) showed abundant inflammatory cells, necrotic debris, mild dysplastic changes in the form of nucleomegaly, hyperchromasia, and occasional presence of prominent nucleoli. Smears from leukoplakic lesions (7 cases) showed predominantly superficial cells with pronounced orangeophilia and cells with varying atypia ranging from low grade to high grade [Figure 5]a. When we compared low and high-grade dysplastic lesions dysplastic lesions, it was observed that presence of nuclear pleomorphism, mitosis, nuclear membrane irregularity, bi/multinucleation, necrosis, abnormal keratinization were the features more significantly associated with high-grade dysplasia [Figure 5]b and [Figure 5]c. Comparison of high-grade dysplasia and carcinoma revealed that nucleomegaly with nuclear size more than ½ of cell size (Grade II), high-grade nuclear pleomorphism, the presence of prominent nucleoli as well as increased frequency of MN in cells were the findings significantly associated with carcinoma [Figure 5]d.{Figure 5}

Certain cytomorphological features were characteristically seen in carcinoma patients like abnormal cell shapes, e.g., tadpole cells, spindle-shaped cells, and anisodiametric nuclei. Furthermore, cytoplasm had lost its translucent quality and was deeply keratinized [Figure 4].

Our hospital being a rural hospital with no radiotherapy facility, all suspected cancer cases are referred to higher centers. Therefore in our study, we were not able to get follow-up biopsy of all suspected patients. The sensitivity of brush biopsy in our study was 76.47% and false negative rate of 23.53%. Specificity and statistical significance could not be determined (no true negatives) as we lost the patients in the follow-up once the diagnosis of NDM was give on cytology.

In the two studies where brush biopsies and scalpel biopsies were performed concomitantly on the same lesion and by the same examiner, the sensitivity of Oral-CDx was shown to be >92%. Our study result had less sensitivity compared to this study, but it can be attributed to a number of reasons such as the use of toothbrush instead of specially designed brush that penetrates to the basement membrane, absence of computer-based analysis, and cytodiagnosis-histodiagnosis delay time (concomitant biopsy was not done). A computer specially designed to detect optical and sub optical changes unique to the abnormal oral epithelium, identified suspicious cells for a pathologist to evaluate.[17] This highly specialized neural network based image processing system specifically can detect as few as one or two abnormal individual cells among several hundred thousand normal cells typically found on an Oral-CDx brush biopsy specimen.[20] The brush biopsy with computer-assisted analysis has been shown to have a high positive predictive value of 30–38% (exceeding the Pap smear and mammography) and leads to earlier cancer diagnosis. Therefore, because of its less invasive nature, health care providers are more likely to use the brush biopsy on lesions that they would have not have otherwise been subjected for immediate biopsy for detection of dysplasia and early carcinoma among lesions that would otherwise not have been promptly evaluated.

A major limitation of our study is the inability to deduce exact sensitivity and specificity because of the absence of concomitant scalpel biopsy results in all patients, especially of NDM and low-grade dysplasia cases. In addition number of cases is low due to a significant dropout rate of many patients.

The main advantage of this study is that the technique used is quite simple and inexpensive so it can be used in any setup and easy to replicate. It has a definite advantage over conventional scrape cytology, and there are no obvious complications or side effects of this procedure.

No controversies were raised during the conduct of this study.


This study suggests that early detection of oral carcinoma is possible even at the precancerous stages by using noninvasive, painless outpatient-based procedures like a toothbrush oral brush biopsy. This technique showed a reasonable specificity and sensitivity compared to conventional scrape cytology that makes it a potentially practical tool in resource challenged countries like India, which bear the major brunt of the disease burden. Although the accuracy of this modified brush biopsy is less than that reported for the commercial brush biopsy with computer-assisted analysis, it is better for evaluation of lesions that would not ordinarily be subjected to early scalpel biopsy.

Though the cytomorphological difference between high-grade dysplasia and carcinoma is not very distinct, certain features like presence of MN and its frequency is an important clue to warrant scalpel biopsy for confirmation of malignancy. Finally, a major advantage of this technique is to reduce diagnostic delay thus helping significant improvement in morbidity and mortality due to oral cancer.

Future research directions

Oral carcinoma being ranked one in terms of incidence among males in India, it is of paramount importance to develop newer simpler and efficient cytotechniques to aid the early diagnosis. More studies are necessary to establish definite criteria regarding grading of lesions and its further management like in cervical cytology.

A reasonable approach might well be to combine ancillary methods with newer cytotechniques (brush biopsy, liquid-based morphology), noninvasive microbiopsies, AgNOR (for diagnosis and prognosis), immunocytochemistry, molecular biology, and so forth.[21]

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Brocklehurst P, Kujan O, O'Malley LA, Ogden G, Shepherd S, Glenny AM. Screening programmes for the early detection and prevention of oral cancer. Cochrane Database Syst Rev 2013;11:CD004150.
2Mehrotra R, Gupta A, Singh M, Ibrahim R. Application of cytology and molecular biology in diagnosing premalignant or malignant oral lesions. Mol Cancer 2006;5:11.
3Ogden GR, Cowpe JG, Green M. Cytobrush and wooden spatula for oral exfoliative cytology. A comparison. Acta Cytol 1992;36:706-10.
4Fischer DJ, Epstein JB, Morton TH Jr, Schwartz SM. Reliability of histologic diagnosis of clinically normal intraoral tissue adjacent to clinically suspicious lesions in former upper aerodigestive tract cancer patients. Oral Oncol 2005;41:489-96.
5Warnakulasuriya S. Histological grading of oral epithelial dysplasia: Revisited. J Pathol 2001;194:294-7.
6Storm P. Minimally Invasive Apparatus for Testing Lesions of the Oral Cavity and Similar Epithelium. Available from: [Last accessed on 2010 Feb 20].
7Storm P. Method for Detection of Abnormal Keratinization in Epithelial Tissue. Available from: [Last accessed on 2010 Feb 20].
8Navone R, Burlo P, Pich A, Pentenero M, Broccoletti R, Marsico A, et al. The impact of liquid-based oral cytology on the diagnosis of oral squamous dysplasia and carcinoma. Cytopathology 2007;18:356-60.
9Countryman PI, Heddle JA. The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutat Res 1976;41:321-32.
10Jadhav K, Gupta N, Ahmed MB. Micronuclei: An essential biomarker in oral exfoliated cells for grading of oral squamous cell carcinoma. J Cytol 2011;28:7-12.
11Guggenheimer J, Verbin RS, Johnson JT, Horkowitz CA, Myers EN. Factors delaying the diagnosis of oral and oropharyngeal carcinomas. Cancer 1989;64:932-5.
12Mehrotra R, Singh M, Kumar D, Pandey AN, Gupta RK, Sinha US. Age specific incidence rate and pathological spectrum of oral cancer in Allahabad. Indian J Med Sci 2003;57:400-4.
13Nair U, Bartsch H, Nair J. Alert for an epidemic of oral cancer due to use of the betel quid substitutes gutkha and pan masala: A review of agents and causative mechanisms. Mutagenesis 2004;19:251-62.
14Babu S, Bhat RV, Kumar PU, Sesikaran B, Rao KV, Aruna P, et al. A comparative clinico-pathological study of oral submucous fibrosis in habitual chewers of pan masala and betelquid. J Toxicol Clin Toxicol 1996;34:317-22.
15Folsom TC, White CP, Bromer L, Canby HF, Garrington GE. Oral exfoliative study. Review of the literature and report of a three-year study. Oral Surg Oral Med Oral Pathol 1972;33:61-74.
16Wilbur DC, Parker EM, Foti JA. Location-guided screening of liquid-based cervical cytology specimens: A potential improvement in accuracy and productivity is demonstrated in a preclinical feasibility trial. Am J Clin Pathol 2002;118:399-407.
17Sciubba JJ. Improving detection of precancerous and cancerous oral lesions. Computer-assisted analysis of the oral brush biopsy. U.S. Collaborative OralCDx Study Group. J Am Dent Assoc 1999;130:1445-57.
18Ahmed HG, Idris AM, Ibrahim SO. Study of oral epithelial atypia among Sudanese tobacco users by exfoliative cytology. Anticancer Res. 2003;23:1943-9.
19Scheifele C, Schmidt-Westhausen AM, Dietrich T, Reichart PA. The sensitivity and specificity of the Oral CDx technique: Evaluation of 103 cases. Oral Oncol 2004;40:824-8.
20Fist S. The oral brush biopsy: Separating fact from fiction. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:654-5.
21Lingen MW, Kalmar JR, Karrison T, Speight PM. Critical evaluation of diagnostic aids for the detection of oral cancer. Oral Oncol 2008;44:10-22.