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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 32  |  Issue : 3  |  Page : 253-258

Efficacy of toluidine blue, lugol's iodine and acetic acid for detecting oral lesions of Leukoplakia and erosive lichen planus – A cross-sectional study


1 Department of Oral Medicine and Radiology, Indira Gandhi Institute of Dental Sciences, Sri Balaji Vidyapeeth, Puducherry, India
2 Department of Oral Medicine and Radiology, Government Dental College, Kottayam, Kerala, India
3 Department of Oral Medicine and Radiology, Rajah Muthiah Dental College, Annamalai University, Chidambaram, Tamil Nadu, India
4 Department of Oral and Maxillofacial Surgery and Diagnostic Sciences College of Dentistry, Jouf University, Sakakah, Kingdom of Saudi Arabia (KSA), Kingdom of Saudi Arabia
5 Kiruthic Dental Care, Tanjavur, Tamil Nadu, India

Date of Submission07-Feb-2020
Date of Decision07-Jun-2020
Date of Acceptance27-Jun-2020
Date of Web Publication29-Sep-2020

Correspondence Address:
Dr. Sivasankari Thirunavukarasu
Indira Gandhi Institute of Dental Sciences, Sri Balaji Vidyapeeth (Deemed-to-be-University), Puducherry
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaomr.jiaomr_22_20

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   Abstract 


Background and Objectives: Early detection and treatment of oral cancer will significantly improve the survival rate and prognosis of the patients. Adjunctive diagnostic aids such as vital staining have been developed to supplement clinical examination and improve the diagnosis. The current study was conducted to evaluate the diagnostic efficacy of acetic acid (2%) (AA), lugol's iodine (3% dilution) (LIS), and toluidine blue (1%) (TBS) in oral leukoplakia (OL) and erosive variant of oral lichen planus (OLP). Materials and Methods: A hospital-based, cross-sectional study was conducted with 30 randomly selected subjects having clinically proven cases of OL and erosive OLP. Every patient underwent AA, LIS, and TBS application to their oral lesions in a sequential manner. Subsequently, clinical and histopathologic diagnosis was compared with staining results of each. Cases which were diagnosed as epithelial hyperplasia were considered as a control group. Data are presented in numbers and percentages. Chi-square test was used to compare between TBS, LIS and AA. Diagnostic efficiency and reliability was calculated by sensitivity and specificity test in terms of its utility in predicting the dysplastic nature of the lesion. Results: Sensitivity of both TBS and LIS staining was calculated as 90.48% whereas the specificity of the former test was 22.22% and latter was 11.11%. AA test showed a sensitivity of 57.14% and specificity of 33.33%. Multiple comparisons of staining with three agents and the histopathologic variants did not show any statistically significant difference. Conclusion: TBS and LIS staining showed high sensitivity in diagnosing OL and erosive OLP compared to AA. The specificity of all three modalities used was low.

Keywords: Lugols iodine, oral leukoplakia, oral lichen planus, sensitivity and specificity, toluidine blue


How to cite this article:
Thirunavukarasu S, Mathew P, Austin RD, Srivastava KC, Ramasamy S, Usha V. Efficacy of toluidine blue, lugol's iodine and acetic acid for detecting oral lesions of Leukoplakia and erosive lichen planus – A cross-sectional study. J Indian Acad Oral Med Radiol 2020;32:253-8

How to cite this URL:
Thirunavukarasu S, Mathew P, Austin RD, Srivastava KC, Ramasamy S, Usha V. Efficacy of toluidine blue, lugol's iodine and acetic acid for detecting oral lesions of Leukoplakia and erosive lichen planus – A cross-sectional study. J Indian Acad Oral Med Radiol [serial online] 2020 [cited 2020 Nov 1];32:253-8. Available from: https://www.jiaomr.in/text.asp?2020/32/3/253/296580




   Introduction Top


Oral cancer (OC) is a significant threat to public health all over the world, especially in the South-East Asian countries. In India, OC is the most prevalent cancer in males and the third most prevalent in females, indicating a major health problem constituting up to 40% of all cancers.[1] Oral Squamous cell carcinoma (OSCC) is the most common cancer of the oral cavity and represents about 90% of all oral malignancies.[2] Most OSCC are usually preceded by certain warning lesions referred as potentially malignant disorders (PMD). Oral Leukoplakia (OL), Erosive Lichen Planus, and Oral Submucous Fibrosis (OSMF) are some of the common PMD seen in the oral cavity. Clinician's approach towards early detection and subsequent treatment of these precursor lesions can significantly improve the survival rate and eventually the prognosis of such patients.[3] Various researches have been done to elucidate with the role of reactive oxygen species (ROS) and endogenous antioxidants as early diagnostic marker for oral leukoplakia using serum,[4] tissue,[5] and salivary samples.[6] However, periodic clinical examination of the oral cavity remains the mainstay in early detection of PMD.[7] Additionally, adjunctive diagnostic aids such as vital staining have been developed to supplement clinical examination in order to improve the diagnosis of PMD and early malignant lesions.[1] Vital staining commonly employs dyes like toluidine blue (TBS), acetic acid (AA), and Lugol's iodine (LIS).[8] Similar in-vivo staining has been extensively used in gynecologic practice to supplement visual inspection for detection of malignant changes in cervix.[9] Several recent studies suggested that visual examination supplemented by vital staining closely match the Pap smear in its performance in detection of early cervical cancer.[10]

Clinical significance:Early detection of oral cancer in the precursor stages is possible and this will significantly improve the survival rate of the patient.

TBS is an acidophilic metachromatic dye which has the ability to bind to acidic components like nuclear genetic material of the tissue, there by adhering more to the dysplastic and anaplastic cells containing quantitatively more nucleic acids than normal tissues. Also, the intercellular canals are wider in malignant epithelium than the normal epithelium, thereby enhancing the penetration of dye.[11] LIS is predominantly a cytoplasmic stain. Lugol's solution is chemically iodine–potassium iodide (I2 KI). The principle behind this staining is based on iodine–starch reaction in which iodine reacts with glycogen in the cytoplasm and there by staining the area.[12] Tissue glycogen content gets depleted with the degree of keratinization, resulting in minimal staining reaction of those areas. Importantly, the enhanced glycolysis in cancer cells also diminishes glycogen levels, thereby reducing iodine–starch reaction. Thus normal mucosa stains brown or mahogany due to its high glycogen content, while dysplastic tissue does not stain, and appears pale compared to the surrounding tissue.[13] Application of AA causes reversible coagulation/precipitation of cellular proteins thereby causing swelling of the epithelium and dehydration of the cells. The normal squamous epithelium appears pink due to the reflection of light from the underlying stroma, which is rich in blood vessels. If the epithelium contains a lot of cellular proteins, acetic acid coagulates these proteins, which may obliterate the color of the stroma resulting in to aceto whitening of the area.[14],[15]

These vital staining techniques of diverse mechanisms have been individually evaluated in the detection of PMD in various sets of population with conflicting results.[8],[16] Thus the current study attempts to compare the efficacy in terms of sensitivity and specificity of TBS (1%), LIS (50% dilution) and AA (3%) for the detection of oral lesions of Leukoplakia and erosive lichen planus in the South-Indian population. The study also strives to evaluate their utility in identifying the dysplastic area in an innocuous lesion.


   Materials and Methods Top


Study design

A hospital-based, cross sectional study was conducted at the department of Oral Medicine, Diagnosis, and Radiology.

Sample characteristics

A random sample of 30 subjects with clinically suspicious PMD, specifically homogenous oral leukoplakia and erosive variant of oral lichen planus were included in the study. The diagnostic criteria for OL and OLP given by Vanderwaal et al were followed (Van der waal et al. 2009).[17] On questioning, all patients with clinically proven lesion of oral leukoplakia had a history of tobacco usage either in smoking or and smokeless form. Patients who had undergone treatment for PMD lesions in the previous 6 months, or those who have associated chronic co-morbid systemic disease or were allergic to dyes were excluded from the study. Due to the ethical concern, the control group was not included in the study. For the purpose of validation, the contralateral side of few cases was used as control. With regard to sample size, a Post-hoc analysis was carried out with the help of software G Power 3.1.9.2 at CI (α) 0.05 and effect size (Cohen's d) of 0.5, considering a two-tail test. The power (1-β) of the study with 30 samples was 0.86, indicative of the high power of the study.

Study protocol

Initially, patients were screened for oral leukoplakia and erosive oral lichen planus in the outpatient clinic of Oral medicine, Diagnosis, and Radiology. Complete history with emphasize on adverse habits and thorough clinical examination was carried out for the patients with positive signs of these lesions. All subjects were informed in an understandable language about the objectives and protocol of the study. Subjects who voluntarily agreed to participate in the study were asked to sign the informed consent. Every participant was later evaluated for the allergic response to vital stain dye by using skin scratch test. On satisfactory response, every subject was given a “chit” with a code depicting the type of stain to be subjected. Accordingly, each patient underwent AA solution and LIS application to their suspected lesions on the first day. On the next day subjects were subjected to TBS. To evaluate the staining pre and post photographs were taken. In order to eliminate the bias, different examiners were involved in the screening and recruitment of patients as well in the procedure of stain application. All the examiners involved in the study were standardized about the identification of lesion, application, and assessment of the lesion after vital staining. The cronbac's alpha statistics of 0.87 was achieved when test-retest reliability with a sample of 10 lesions was done, showing a high level of agreement. These sample results were excluded from the data analysis of the study. After completion of the staining protocol and histopathological confirmation, appropriate treatment was instituted for all the subjects, which included tobacco cessation counselling for patient with presence of this adverse habit.

Staining procedure

Acetic acid (AA) application

It was carried out by applying 2% freshly prepared AA on suspicious areas and read after 2 minutes. If the lesion changed to opaque white (aceto white) color it was considered as positive for dysplastic epithelium [Figure 1].
Figure 1: Clinical photograph showing non-homogenous, clinical photograph showing acetic acid uptake

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Lugol's iodine (LIS)

LIS was applied over suspected areas (prepared by dissolving 5 gm of iodine and 10 gm of potassium iodide in 100 ml of distilled water), waited for 2 minutes and examined thoroughly followed by a photograph of the area. Areas which stained brown or mahogany were interpreted as normal and areas that did not stain and appeared pale compared to the surrounding tissue were considered dysplastic [Figure 2].
Figure 2: Showing non-homogenous leukoplakia, clinical photograph showing uptake of stain after application of Lugol's Iodine

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Toulidine blue staining (TBS)

The suspected areas were first applied with 1% AA with cotton bud for 20 seconds followed by rinsing with water. 1% TBS solution was applied with a cotton bud for 10–20 seconds and was decolorized with 2% acetic acid rinse for 20–30 seconds, and a photograph was taken. Dark blue colored stain retention was considered as positive for lesions suspicious of malignancy, light blue retention was considered as positive for premalignant lesions and the lesions without any retention of stain were considered as negative [Figure 3].[17]
Figure 3: Showing homogenous leukoplakia, clinical photograph showing uptake of stain after application of toluidine blue

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All the lesions that stained positive were re-stained after 2 weeks to reduce the false positive rate. Lesions which showed negative staining, clinical judgment directed the biopsy and the specimens were sent for histopathology examination. Clinical and histopathologic diagnosis were compared with staining results.

Data analysis

The collected data was analyzed using the Statistical package for social sciences (SPSS) version 21.0 software. Chi-square test was used to compare between TBS, LIS, and AA staining. Diagnostic efficiency was calculated by sensitivity and specificity test formulae. Multiple comparisons of staining with three agents and the histopathologic variants were done using Kruskal-Wallis test at significance level P < 0.05.


   Results Top


In this study, 30 potentially malignant disorders consisting of 7 OL (23.3%), 16 erythro-leukoplakia (53.3%), and 7 erosive OLP (23.34%) were evaluated using vital staining [Table 1]. Histopathologically, 9 cases (30.0%), showed only epithelial hyperplasia. Mild dysplastic features were seen in 4 cases (13.3%), whereas 15 cases (50%) exhibited moderate epithelial dysplasia. Only 2 cases (6.7%) showed severe dysplastic features [Table 2].
Table 1: Showing frequency distribution for type of cases included in the study and majority of the cases belonged to non-homogenous leukoplakia (53.33%)

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Table 2: Showing frequency distribution of histopathology grading and 50% of cases included in the study falls under moderate dysplasia. 30% of the cases belonged to epithelial hyperplasia and they were used as positive control group

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TBS and LIS stained positive in all cases of mild and severe epithelial dysplastic cases but both failed to identify 2 moderate dysplastic cases. Interestingly, TBS staining yielded false positive results in 7 epithelial hyperplasia cases whereas LIS yielded 8 false positive results. AA applications showed positive results only in two mild dysplasia, nine moderate dysplasia and one severe dysplasia cases. However, false positive results were shown only in six cases on AA application. Based on these findings the sensitivity of both TBS and LIS staining was calculated as 90.48% whereas the specificity of the former test was 22.22% and latter was 11.11%. AA test showed a sensitivity of 57.14% and specificity of 33.33% [Table 3].
Table 3: Showing Sensitivity and Specificity calculated for each vital staining technique

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Comparing percentage of TBS with LIS staining and AA with LIS staining did not show a statistically significant difference (P > 0.05). However, comparison of percentage of staining of TBS with AA showed a statistically significant difference (P < 0.05) [Table 4], [Table 5], [Table 6]. Multiple comparisons of staining with three agents and the histopathologic variants were done using Kruskal-Wallis test which did not show any statistically significant difference (P > 0.05) [Table 7].
Table 4: Showing comparison (cross tabulation) between Toluidine blue and Lugol's iodine and the difference in the percentage of staining was not statistically significant. (p>0.05)

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Table 5: Showing comparison (cross tabulation) between Lugol's iodine and Acetic acid in the difference in th percentage of staining was not statistically significant. (p>0.05)

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Table 6: Showing comparison (cross tabulation) between Toluidine blue and Acetic acid and the difference in the percentage of staining was statistically significant. (p<0.05)

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Table 7: Showing multiple comparisons of staining with three agents and the histopathologic variants using Kruskal-Wallis test yielding no statistically significant difference (p value>0.05)

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   Discussion Top


Majority of cases of oral cancer are diagnosed at an advanced stage as cytology-based screening programmes are ineffective in developing countries. Visual examination after vital staining with TBS and AA are well established early diagnostic aids in oral PMD. However, cytoplasmic stains like LIS are not commonly used in oral PMD examination.[18],[19] So, the current study was undertaken to compare the diagnostic efficacy of these three available staining modalities in detection of oral PMDs' namely OL and erosive OLP.

In our study, out of 30 premalignant lesions, on histopathological grading, 9 (30.0%) were hyperplasia, 4 (13.3%) were mild dysplasia; 15 were moderate dysplasia and 2 were severe dysplasia [Table 2].

In our study, TBS and LIS showed high sensitivity of 90.48% which reflects their ability to detect true positive lesions. However, the specificity of TBS was 22.22 which suggests it's inefficiency in detecting true negative lesions and that of LIS was still lower (11.11%). Understandably, there was no significant difference in the staining percentage (positive and negative) between TBS and LIS staining. Epstein et al. in their study showed sensitivity and specificity of TBS were 92.5% and 63.2% and that of LIS were 87.5% and 84.2%, respectively. Even though sensitivity of the tests was in agreement with our study, specificity of TBS and LIS was very high compared to our results. This difference in specificity of TBS may be due to false positive staining of inflammatory lesions which is the commonest side effect associated with TBS.[20] More false positive findings associated with LIS may be due to the fact that glycogen level may be reduced even in epithelial hyperplasia cases which acted as control group. Throughout the oral mucosa the content of glycogen varies with the keratinization. There is a debatable correlation between the degree of inflammation and glycogen content as well.[21],[22]

Warnakulasuriya and Newell Johnson (1996) studied 39 PMD with epithelial dysplasia using 1% modified TBS stain and reported a sensitivity and specificity of 79.5% and 62%, respectively. The sensitivity was in agreement with our study however specificity was higher than our results. They suggested that TBS staining is an invaluable option in the surveillance of high-risk subjects and in addition it has remarkable sensitivity in the detection of early PMD.[23]

AA application in the current study exhibited a sensitivity of 57.14 and sensitivity of 33.33. The decrease in sensitivity reflects in inefficiency in diagnosing true positive cases. The reason for this may be the non-keratinized/less keratinized lesions in the cases enrolled to the study group as the acetowhite appearance which indicates positive for dysplasia on acetic acid application is predominantly related to the reversible coagulation of protein content in epithelium.[24],[25] However the specificity of AA application was higher than TBS and LIS. This may be again related to the decreased keratin content in the epithelial hyperplasia group which acted as control in this study. Interestingly, there was no significant difference in the staining percentage (positive and negative) between AA and LIS. Nevertheless, on comparing TBS with AA, a significant difference in the percentage of staining was observed. Bhalang et al. conducted a study to assess the diagnostic accuracy of 5% AA for PMD diagnosis. The sensitivity and specificity were 83.33% and 84.21% respectively. Both were not in agreement with our study which may be related to the diverse clinical presentations of the lesions which we included in the study group.

On performing multiple comparative analyses between different staining modalities with histopathological grades, no significant difference was noticed. Despite having a small sample size with respect to number of variables, the present study has been able to differentiate hyperplastic with dysplastic lesions. However further evaluation of dysplastic lesions was restricted.

Conventionally, sensitivity and specificity of a test is the parameter to be evaluated for choosing a vital staining procedure for accurate diagnosis. Specificity measures the proportion of negatives which are correctly identified. In our study specificity was reduced because of the retention of dye in some benign lesions. Hence, our results suggested that the staining techniques we adopted, especially TBS and LIS are effective in detecting positive cases. In a clinical setting the false positives are of course, of less concern than false negative results because any positive should lead to confirmation on biopsy.[26],[27] The limitation of the study was smaller sample size in various staining groups and even in the category of histopathological grades. Follow up evaluation was not done for the confirmed dysplastic lesion. Such an analysis would have reinforced the result of the current study.


   Conclusion Top


TBS and LIS showed high sensitivity in diagnosing PMD like OL and erosive OLP compared to AA. The specificity of all three modalities used was low. Since none of the vital stains have both the parameters of an acceptable level, it is indicated that a combination of staining may yield better results. Future studies with larger sample and multicentric nature are warranted to comment on specificity and sensitivity for each staining with more conviction.

Declaration of patient consent

This study involving human participants was reviewed and approved by the ethical board. Written informed consent for participation was taken for this study in accordance with the national legislation and the institutional requirements. The data collected from this research is solely for the research and educational purposes.

Ethical considerations

Ethical approval was taken from the institutional Committee of Bioethics. This study was conducted within the framework of declaration of Helsinki.

Financial support and sponsorship

Nil.

Conflicts of interest

The author(s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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