Journal of Indian Academy of Oral Medicine and Radiology

: 2022  |  Volume : 34  |  Issue : 3  |  Page : 254--258

Reliability of toluidine blue and colposcopy in determining biopsy site among patients with oral leukoplakia – A comparative study

Karunakar Naik1, D Sujatha2, H Kiran Raj3, Anuradha Pai2, Pooja Naik4, M Shruthi5,  
1 Private Practitioner, UMA Multispeciality Clinic, Ankola, Uttar Kannada, Karnataka, India
2 Department of Oral Medicine and Radiology, The Oxford Dental College, Bengaluru, Karnataka, India
3 Department of Paediatrics, Srinivas Institute of Medical Sciences and Research Centre, Mangaluru, Karnataka, India
4 Department of Oral Pathology and Microbiology, Srinivas Institute of Dental Sciences, Mangaluru, Karnataka, India
5 Department of Oral Medicine and Radiology, Srinivas Institute of Dental Sciences, Mangaluru, Karnataka, India

Correspondence Address:
Pooja Naik
Srinivas Institute of Dental Sciences, Mangaluru, Karnataka


Background: To date, no simple and reliable method can replace oral biopsy site detection using Toluidine blue for early identification and treatment of potentially malignant oral disorders (OPMDs). Aims and Objectives: The study was conducted to assess the feasibility of using the colposcope and compare the findings with the toluidine blue staining method for selecting the biopsy site. Materials and Methodology: Study group included 30 patients; five patients dropped out as regression was seen in the lesion. Twenty patients with homogenous leucoplakia and five patients with nonhomogenous Leukoplakia formed a study group. An area with the most extensive cell change in the colposcopic examination was selected by the first version, Image J 1. x Software. After toluidine, blue staining was done. Dark blue-stained sites were considered positive, and site selection for the biopsy was made. Results: Normal vascular pattern in six (24%) cases, 10 (40%) cases showing punctuate vascular pattern, four (16%) of cases showed the mosaic vascular pattern, five (20%) of cases were with the atypical vascular pattern were assessed in colposcopic examination with statistically significant P value (0.009). Fourteen cases out of 20 cases of homogenous and all five cases of nonhomogenous leukoplakia were correctly diagnosed for the biopsy site with both toluidine blue and colposcopic techniques. Conclusion: In this study, Toluidine blue and colposcopic technique showed 76% accuracy for detecting the biopsy site. So colposcope can be an alternative diagnostic aid to toluidine blue in detecting biopsy sites for PMD.

How to cite this article:
Naik K, Sujatha D, Raj H K, Pai A, Naik P, Shruthi M. Reliability of toluidine blue and colposcopy in determining biopsy site among patients with oral leukoplakia – A comparative study.J Indian Acad Oral Med Radiol 2022;34:254-258

How to cite this URL:
Naik K, Sujatha D, Raj H K, Pai A, Naik P, Shruthi M. Reliability of toluidine blue and colposcopy in determining biopsy site among patients with oral leukoplakia – A comparative study. J Indian Acad Oral Med Radiol [serial online] 2022 [cited 2022 Dec 1 ];34:254-258
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Oral leukoplakia (OL) is the most commonly found Oral Potentially Malignant Disorder (OPMDs),[1] with a prevalence rate of 0.2–5.2% in India.[2] The pooled estimate of the annual rate of OL malignant transformation ranges between 0.13 and 10%.[2] Early identification and treatment of OPMDs are essential to stop its progression into invasive carcinoma.[3]

The detection of biopsy sites during oral examination is usually determined using Toluidine blue (TB).[4] It is reported to have a sensitivity of 97.8% and overall specificity of 100% while detecting premalignant or malignant lesions in the oral cavity.[5] The risk associated with DNA staining and false-positive staining is as high as 30% due to the enhanced staining of ulcerations and filiform papillae of the tongue.[6]

The colposcopy technique (CT) has recently gained prominence for detecting the biopsy regions in the vagina, cervix, and lower endocervical canal.[7] The stereoscopic magnification in colposcopy enables the evaluation of changes in surface topography and vascular patterns of the lining mucosa, thus, aiding in selecting the right site of biopsy, rulingout the possibility of taking a biopsy from the areas supposed to reveal epithelial dysplasia.[7],[8],[9],[10] The colposcopy technique has been applied to detect appropriate biopsy sites in the oral region and has successfully detected the presence of pre-malignant lesions. The technique showed a sensitivity of 64–71%, while the overall specificity was 91–93%.[11],[12]

The present study compares the TB staining technique with the CT technique for detecting the oral biopsy sites considering the sensitivity and overall specificity.

 Materials and Methodology

Approval for the study was granted by The Oxford Dental College and St. John's Medical College & Hospital Institutional Ethics Committee (approval date: November 8, 2013, approval number: 315/2012-2013) and confirming the standards of the Declaration of Helsinki and its subsequent revisions. The study was conducted among the patients attending the outpatient Clinic of Dental College and Hospital who fulfilled the inclusion criteria and accepted to participate. Considering sensitivity at 0.7 and specificity of 0.95, expected precision of 0.1 confidence of interval of 95% then 80% power and 10% drop out rate a sample size of 19 was estimated to have exact specificity of colposcopy in deciding the biopsy site. A total of 30 study participants were selected based on the age and sex-matched demographic data, history, and clinical examination. After obtaining written informed consent, the details regarding habits and clinical manifestations were recorded on the prescribed proforma, along with the inspection and manual palpation of the lesion. After the intraoral clinical examination, the lesion was initially categorized into homogenous and nonhomogenous types based on criteria suggested by Brouns et al.[13] Patients were counseled to quit the habit using interventional strategies 5-A's approach and educational materials. Out of 30 patients, 5 patients dropped out from the study as there was a regression seen in the lesion in subsequent visits after a 1-month follow-up. Out of 25 clinically diagnosed oral leukoplakia patients, 20 patients with homogenous leukoplakia and five with nonhomogenous leukoplakia formed a study group [Figure 1]a. After confirming the clinical diagnosis, the study participants were subjected to colposcopic examination, Toluidine blue staining, and routine blood investigations before the punch biopsy was performed for histopathological examination.{Figure 1}

Colposcopic examination

Patients with persistent lesions underwent colposcopic examination. The oral mucosa, which appeared abnormal, was slightly elevated and greyish-white plaque than the normal mucosa when it was wiped with saline. Acetic acid 5% was applied to the lesion for about 60 seconds. Areas that turn white after applying acetic acid were subjected to evaluate Abnormal vascular patterns by applying a blue (or green) filter and higher-power magnification [Figure 2].{Figure 2}

Reid's Criteria explain the vascular pattern, inter-capillary distance, surface pattern, and clarity of differentiation of the mucosal lesions.[14] When it is challenging to explain the pattern of the vessels, the term atypical vessels is applied. If one of the patterns is seen, this is an indicator for a biopsy and histopathological investigation [Figure 1]b. Based on colposcopic criteria given by Reid and his team, the sites predicted the huge cell changes were selected for biopsy. The photograph of the identified site was the normal resolution and was analyzed by ImageJ 1. X software.

Toluidine blue staining

The patients were instructed to rinse the oral cavity with water for 20 s to remove debris before rinsing with 1% acetic acid for 20 s. Toluidine blue (1% W/W) was applied as an oral rinse for 20 s, and then 1% acetic acid was used for 20 s to eliminate mechanically retained stains. The staining was interpreted based on the color acquired by the lesion; a dark blue (royal or navy) staining was considered positive, and light blue or no color was interpreted as negative [Figure 1]c. After the site was identified, the photograph was taken in the normal resolution, and analyzed images taken were compared on the computer screen by using adobe photoshop CS6, version 13 for the selection of biopsy site. If the same square was selected by both toluidine blue staining and colposcopy, the same area was considered for biopsy. If different squares were selected, then biopsy was performed from representative sites using a 6 mm diameter punch under local anesthesia [Figure 3] to confirm the histopathological diagnosis. As proposed by Rajendran et al.,[15] the changes were graded into hyperkeratosis without dysplasia and hyperkeratosis with mild & moderate dysplasia [Figure 1]d.{Figure 3}

Inclusion criteria

All clinically diagnosed cases of oral leukoplakia. Modified WHO diagnostic criteria of oral Leukoplakia were used. Patients in the 3rd to 6th decade were included in the study.

Exclusion criteria

The study group presented other associated lesions with oral Leukoplakia, underlying systemic diseases, and frank malignancies, and the patients undergoing treatment for oral Leukoplakia were excluded from the study.

Statistical analysis

The toluidine blue staining, colposcopic findings, and histopathologic findings were correlated and subjected to statistical analysis. All the findings were entered in Microsoft Excel. The analysis of data was done using IBM SPSS Software Chicago version 21.0. A descriptive analysis was carried out, and a Chi-square test was used. A P value less than 0.005 was considered significant.


Out of 25 subjects, 20 (80%) were homogenous, and 5five (20%) were nonhomogenous types. The age and sex distribution of homogenous and nonhomogenous Leukoplakia and the tobacco forms were assessed. Statistical significance was not observed regarding the demographic details [Table 1].{Table 1}

In histological findings of the colposcopic and toluidine blue examination, the findings of homogenous leukoplakia and nonhomogenous leukoplakia were assessed. P value (0.009) was found to be statistically significant [Table 2].{Table 2}

The vascular patterns observed in homogenous and nonhomogenous leukoplakic lesions during colposcopic examination shown in [Table 3]. P value (0.050) were statistically significant. A correlation of histopathologic findings and different vascular patterns found in the Colposcopic examination was done. P value (0.0531) was found to be statistically significant [Table 3].{Table 3}

The percentage-wise distribution of accuracy of the Toluidine blue and Colposcopic technique detecting biopsy sites among Oral Leukoplakia patients was assessed. Out of 25 leukoplakia cases, 19 (76%) cases were accurately diagnosed for the biopsy site with the Toluidine blue and Colposcopic Technique [Table 4].{Table 4}


Colposcopy is a simple, painless, chairside diagnostic method that aids in assessing cervical intraepithelial neoplasia and preclinical invasive carcinoma.[16] Hopman, in his study, stated that the final histopathological diagnosis of cervical intraepithelial neoplasia was correctly predilected by the colposcopic impression in 78.3% of the cases.[17] Considering its scope, a colposcopy can be a useful technique for determining the appropriate biopsy sites in the oral region and can help detect pre-malignant lesions and oral squamous cell carcinoma.

The present study showed a male: female ratio of 3:1, whereas the study conducted by Mishra M et al.[18] showed an equal male to female ratio of 1:1. The gender-specific distribution in the majority of the research studies differs, ranging from strong male dominance in various parts of India to nearly 1:1 in the western countries.[18],[19]

In the present study, the most representative site selected for biopsy by both the diagnostic tools were the same, so there was no difference in the histopathological findings with Colposcopic findings and toluidine blue staining. Compared the study by Nayyar AS et al. concluded that assessment of biopsy site using Colposcopic technique was found to be more significant in leukoplakia. In contrast, the clinical criterion was more suitable for carcinoma buccal mucosa cases after comparison with histopathological diagnosis.[20]

In this study, we could observe two basic capillaries in the normal mucosa: network capillaries and hairpin capillaries. The vascular patterns associated with abnormal epithelium in mild and moderate dysplasia included punctuation, mosaicism, and atypical vessels with statistical significance [Figure 4]. We didn't come across any cases of severe dysplasia or carcinoma in situ, so comments on the vascular patterns in those dysplastic changes are limited.{Figure 4}

Compared to our study Shetty et al.,[21] concluded that 78% showed vascular changes on colposcopy. Direct microscopy-guided biopsy was highly representative of the histologic findings than those chosen with regular clinical examination.

In the present study accuracy of the Toluidine blue and Colposcopic technique in detecting biopsy sites among Oral Leukoplakia was found to be as follows, out of 25 leukoplakia cases, 19 (76%) cases were accurately diagnosed for the biopsy site with both the Toluidine blue and Colposcopic Technique. Out of 20 cases of homogenous leukoplakia, 14 cases were correctly diagnosed with 76%, and out of 5 cases of nonhomogenous leukoplakia, all the nonhomogenous Leukoplakia cases were correctly diagnosed with 100% accuracy.


With similar results in both the diagnostic aids used for detecting biopsy sites, a colposcope can be an alternative diagnostic tool to the toluidine blue in detecting biopsy sites for PMDs. The colposcopic examination is feasible; it's a noninvasive procedure and helps in clear visualization of vascular patterns, which detects signs of progression from normal mucosa to dysplastic changes and precision in clinical diagnosis.

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Conflicts of interest

There are no conflicts of interest.


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