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 Table of Contents  
Year : 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

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

Date of Submission11-Mar-2022
Date of Decision09-Aug-2022
Date of Acceptance10-Aug-2022
Date of Web Publication26-Sep-2022

Correspondence Address:
Pooja Naik
Srinivas Institute of Dental Sciences, Mangaluru, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jiaomr.jiaomr_83_22

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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.

Keywords: Biopsy, colposcope, oral potentially malignant disorders, toluidine blue

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;34:254-8

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 2023 Feb 7];34:254-8. Available from: http://www.jiaomr.in/text.asp?2022/34/3/254/356972

   Introduction Top

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 Top

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: Non homogenous oral leukoplakia of the right buccal mucosa a) Non homogenous leukoplakia b) After the acetic acid wash c) After the toluidine blue staining d) Histopathologic picture showing keratosis with moderate dysplasia

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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: Homogenous oral leukoplakia of the right buccal mucosa a) After acetic acid wash b) with blue colour filter

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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: Site of punch biopsy shown after the procedure

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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.

   Results Top

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: Distribution of study population according to age, gender, and habits

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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: Histopathologic findings of Colposcopic and Toluidine blue examination guided biopsy sites in Oral Leukoplakia patients

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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: Correlation of histopathologic findings & different vascular patterns of Colposcopic examination among Oral Leukoplakia patients

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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: Accuracy of the Toluidine blue and Colposcopic Technique in detecting biopsy site among Oral Leukoplakia patients

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   Discussion Top

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: Distribution of vascular patterns in Oral Leukoplakia lesions with Colposcopic examination

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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.

   Conclusion Top

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.

   References Top

Reibel J, Prognosis of oral pre-malignant lesions. Significance of clinical, histopathological, and molecular biological characteristics. Crit Rev Oral Biol Med 2003;141:47-62.  Back to cited text no. 1
Sridharan G. Epidemiology, control and prevention of tobacco-induced oral mucosal lesions in India. Indian J cancer 2014;51:80-5.  Back to cited text no. 2
[PUBMED]  [Full text]  
Awadallah M, Idle M, Patel K, Kademani. Management update of potentially premalignant oral epithelial lesions. Oral Surg Oral Med Oral Pathol Oral Radiol 2018;125:628-36.  Back to cited text no. 3
Epstein JB, Scully C, Spinelli J. Toluidine blue and Lugol's iodine application in assessing oral malignant disease and lesions at risk of malignancy. J Oral Pathol Med 1992;21:160–3.  Back to cited text no. 4
Singh D, Shukla R. Utility of toluidine blue test in accessing and detecting intra-oral malignancies. Indian J Otolaryngol Head Neck Surg 2014;67:47-50.  Back to cited text no. 5
Gynther GW, Rozell B, Heimdahl A. Direct oral microscopy and its value in diagnosing mucosal lesions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000;90:164-70.  Back to cited text no. 6
Edebiri AA. The relative significance of colposcopic descriptive appearances in the diagnosis of cervical intraepithelial neoplasia. Int J Gynaecol Obstet 1990;33:239.  Back to cited text no. 7
Schmitt A. Colposcopy detection of atypical and cancerous lesions of the cervix. Obstet Gynecol 1959;13:66571.  Back to cited text no. 8
Coppleson M. The value of colposcopy in the detection of preclinical carcinoma of the cervix (Three years' experience at King George V Memorial Hospital, Sydney). J Obstet Gynaecol Br Emp 1960;67:1123.  Back to cited text no. 9
Stafl A, Mattingly RF. Colposcopic diagnosis of cervical neoplasia. Obstet Gynecol 1973;41:16876.  Back to cited text no. 10
Ujwala N, Singh NA, Milind N, Prafulla P, Vidhya P, Bhushan B, et al. Colposcopy in pre-malignant lesions and oral squamous cell carcinoma: Linking threads of clinical, histopathological and colposcopic inference. J Can Res Ther 2016;12:295-301.  Back to cited text no. 11
[PUBMED]  [Full text]  
Kesarwani P, Choudhary A. Colposcopy in screening dysplastic lesions of oral cavity: A study. Glob J Res Anal 2017;5:675-8.  Back to cited text no. 12
Brouns ER, Baart JA, Bloemena E, Karagozoglu H, Van der Waal I. The relevance of uniform reporting in oral leukoplakia: Definition, certainty factor and staging based on experience with 275 patients. Med Oral Patol Oral Cir Bucal 2013;18:e19-26.  Back to cited text no. 13
Sellors JW, R S. Colposcopy and Treatment of Cervical Intraepithelial Neoplasia: A Beginner's Manual: International Agency for Research on Cancer. Diamond Pocket Books (P) Ltd; 2003.  Back to cited text no. 14
Rajendran R. Oral leukoplakia (leukokeratosis): Compilation of facts and figures. J Oral Maxillofac Pathol 2004;8:58-68.  Back to cited text no. 15
  [Full text]  
Pallagatti S, Sheikh S, Puri N, Gupta D, Singh B. Colposcopy: A new ray in diagnosing oral lesions. Indian J Dent Res 2011;22:810-5.  Back to cited text no. 16
  [Full text]  
Hopman EH, Helmerhorst TJ. Colposcopic findings and nomenclature. CME J Gynecol Oncol 2005;101:11-3.  Back to cited text no. 17
Mishra M, Mohanty J, Sengupta S, Tripathy S. Epidemiological and clinicopathological study of oral leukoplakia. Indian J Dermatol Venereol Leprol 2005;713:161.  Back to cited text no. 18
Bokor-Bratic M. Prevalence of oral leukoplakia. Med Pregl 2003;56:552-5.  Back to cited text no. 19
Nayyar AS, Khan M, Bafna UD, Siddique A, Gayitri HC. Colposcopy: Gynecological vision in viewing oral lesions. Indian J Pathol Microbiol 2014;57:223-30.  Back to cited text no. 20
[PUBMED]  [Full text]  
Shetty DC, Ahuja P, Taneja DK, Rathore AS, Chhina S, Ahuja US, et al. Relevance of tumor angiogenesis patterns as a diagnostic value and prognostic indicator in oral precancer and cancer. Vasc Health Risk Manag 2011;7:41-7.  Back to cited text no. 21


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2], [Table 3], [Table 4]


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