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Acetic acid versus toluidine blue as screening tools for oral potentially malignant disorders

 Department of Oral Medicine and Radiology, Coorg Institute of Dental Sciences, Virajpet, Karnataka, India

Date of Submission12-Jan-2021
Date of Decision19-Jan-2021
Date of Acceptance14-Mar-2021
Date of Web Publication29-Jun-2022

Correspondence Address:
Veena S Narayanan,
Department of Oral Medicine and Radiology, Coorg Institute of Dental Sciences, Virajpet, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijc.IJC_42_21


Background: Diagnostic adjuncts such as toluidine blue have been investigated as screening tools that improve visual examination of potentially malignant disorders (PMD) and oral cancer. Acetic acid has been reported to be of value in the early detection of cervical cancers. This study assessed the utility of 5% acetic acid as a diagnostic adjunct in oral PMD and compared the accuracy of acetic acid with toluidine blue in the detection of dysplastic PMD and high-risk lesions.
Materials and Methods: This cross-sectional study was conducted at a dental hospital in a rural setting. Thirty-one patients with oral PMD formed the study group. Five percent acetic acid was applied to the lesions, followed by toluidine blue application and biopsy. Sensitivity, specificity, and positive and negative predictive values were computed considering true positives as stain uptake in dysplastic and high-risk PMD.
Results: The sensitivity, specificity, and positive and negative predictive values of acetic acid for identifying dysplastic or malignant lesions were 100%, 13.3%, 51.2%, and 100%, respectively, and that for toluidine blue were 75%, 100%, 100%, and 78.9%, respectively. The corresponding values for identifying high-risk PMD (lesions with moderate and severe dysplasia) using acetic acid were 100%, 9.1%, 25.9%, and 100%, respectively, and for toluidine blue were 85.7%, 81.8%, 60%, and 94.7%, respectively.
Conclusion: The utility of acetic acid in detecting dysplasia and high-risk PMD is severely limited due to its poor specificity. Compared with acetic acid, toluidine blue is a superior screening tool.

Keywords: Acetic acid, toluidine blue, epithelial dysplasia, oral potentially malignant disorders
Key Message: Although acetic acid is very sensitive in identifying dysplastic and high-risk PMD, its utility is limited due to its poor specificity. Compared with acetic acid, toluidine blue has better accuracy and hence is a more suitable diagnostic adjunct.

How to cite this URL:
Winton Kalluvelil SR, Narayanan VS. Acetic acid versus toluidine blue as screening tools for oral potentially malignant disorders. Indian J Cancer [Epub ahead of print] [cited 2022 Dec 4]. Available from:

  Introduction Top

Visual screening and clinical examination may fail to identify PMD or discriminate between PMD and other red and white lesions. Minimally invasive visual diagnostic aids such as toluidine blue, Lugol's iodine, and chemiluminescence have been used to enhance detection of PMD with variable accuracy.[1],[2],[3],[4],[5]

Toluidine blue being an acidophilic metachromatic dye has an affinity for nucleic acids deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Dysplastic and malignant cells retain this dye as they contain more nucleic acids quantitatively. Previous studies have reported varying sensitivity and specificity values for toluidine blue in the detection of oral malignant lesions.[1],[2],[3]

Acetic acid has been found to be a useful and cost-effective agent for the visual screening of cervical cancer in low resource settings.[6],[7] A few recent studies have suggested the use of acetic acid in oral cancers and have indicated that when applied to suspected cancerous lesions of the oral mucosa, acetic acid produces the appearance of “acetowhite” that aids in the visual identification of oral cancers.[8],[9]

However, the role of acetic acid as a visual screening tool for oral PMD is unclear. We investigated the utility of 5% acetic acid as a diagnostic adjunct in identifying dysplastic and high-risk oral PMD and compared the accuracy of acetic acid with that of toluidine blue.

  Materials and Methods Top

This cross-sectional study was conducted during the years 2016–2018 at a dental hospital located in a rural area in South India. The study protocol was reviewed and approved by the institutional review board.

Thirty-one patients attending the Department of Oral Medicine and Radiology were recruited for the study. A written informed consent was obtained from the participants. Patients above the age of 18 years with white or red and white oral lesions, suspicious of PMD (as described by Warnakulasuriya et al., 2007)[10] were included in the study. Patients with systemic disorders contradicting biopsy were excluded from the study.

The principal investigator trained in the specialty of oral medicine obtained a detailed history of all the patients and conducted a clinical examination of the suspected lesions. The findings were recorded in a proforma. The clinical diagnosis was confirmed by an oral medicine specialist with more than 10 years' experience.

The patients were asked to rinse their mouths with clean water. The lesions were dried using gauze and then photographed. This was followed by the application of the agents – acetic acid and toluidine blue.

Application of acetic acid: Five percent acetic acid was applied to the lesion with a sterile disposable cotton applicator for 60 seconds. A photograph of the lesion was taken again following this. The lesion was observed for any color changes following the application of acetic acid.

Outcome measures: The results of the acetic acid application were considered positive if the lesion appears opaque white (acetowhite) [Figure 1], [Figure 2], [Figure 3] and a negative if the lesion does not show any color change.
Figure 1: (a) Leukoplakia of right buccal mucosa with mild dysplasia; (b)“Acetowhite” appearance after acetic acid application; (c) After toluidine blue application (the area with dark blue uptake was considered “positive uptake” and subjected to incisional biopsy)

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Figure 2: (a) Speckled leukoplakia with moderate dysplasia; (b) After acetic acid application; C- After toluidine blue application

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Figure 3: (a) Early invasive squamous cell carcinoma - tongue (clinically diagnosed as leukoplakia); (b) After acetic acid application; (c) After toluidine blue application (biopsy was performed from the anterior stain-positive areas)

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Application of TB: After the application of acetic acid, 1% toluidine blue was applied to the lesion with a sterile disposable cotton applicator for 60 seconds and decolorized with acetic acid. A photograph of the lesion was taken following this procedure.

Outcome measures: The lesion was considered toluidine blue positive if it retained blue color after the staining [Figure 1], [Figure 2], [Figure 3]. Lesions with no uptake or questionable uptake constituted the negative toluidine blue category of lesions.

A biopsy was obtained from the lesion and subjected to histopathological examination. The site of the biopsy was selected based on the clinical appearance and dye retention. The histopathological diagnosis was considered the “gold standard” for reference. Lesions were grouped in two different ways:

  1. Those with no dysplasia and those with dysplasia or carcinoma
  2. Low-risk (lesions with no/questionable/mild dysplasia) and high-risk PMD (lesions with moderate/severe dysplasia). High-risk lesions with positive uptake were considered true positives

The clinical findings and histopathological findings were correlated with the mucosal changes following acetic acid and toluidine blue application.

The data were collected, coded, and subjected to SPSS (IBM Version 23) for statistical analysis.

The descriptive statistics included mean, frequency, and percentage. The sensitivity, specificity, positive predictive value, and negative predictive were calculated using the standard formulae for these test characteristics.

  Results Top

Thirty-one patients, including 24 men and 7 women, with clinically identified PMD were selected for the study. The age range was 31 to 73 years with a mean age of 49.7 years. On histopathological examination, 14 lesions were dysplastic, squamous cell carcinoma was observed in two lesions, and no dysplasia was present in 15 lesions. The frequency distribution of the histopathological diagnosis is presented in [Table 1].
Table 1: Distribution of histopathological diagnosis

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Twenty-two lesions were of the “low-risk” category, nine belonged to the “high-risk” category of lesions or carcinoma.

The distribution of acetic acid and toluidine blue screening test results in the various category of lesions (dysplastic lesions/carcinoma and nondysplastic; low-risk and high-risk lesions) are presented in [Table 2] and [Table 3].
Table 2: Distribution of acetic acid and toluidine blue screening test results for lesions with and without dysplasia/carcinoma

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Table 3: Distribution of acetic acid and toluidine blue screening test results for low-risk and high-risk potentially malignant disorders

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A comparison of sensitivity, specificity, and positive and negative predictive values for acetic acid and toluidine blue in detecting dysplasia/carcinoma and high-risk PMD lesions is presented in [Table 4] and [Table 5].
Table 4: Test characteristics of acetic acid and toluidine blue in detecting dysplastic lesions and early invasive carcinoma

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Table 5: Test characteristics of acetic acid and toluidine blue in detecting high-risk lesions and early invasive carcinoma

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Toluidine blue was found to have superior accuracy at identifying dysplastic lesions and high-risk PMD compared with acetic acid.

  Discussion Top

The present study was conducted to investigate the role of 5% acetic acid as a diagnostic adjunct in oral PMD and compare the accuracy of acetic acid with toluidine blue as a screening tool in detecting PMD with dysplasia and histologically high-risk lesions.

Among the 31 clinically identified PMD, 2 (6.5%) were early invasive squamous cell carcinoma histologically. Waldron and Shafer reported a prevalence of 3.1% invasive squamous cell carcinoma among the 3,256 clinically diagnosed cases of leukoplakia.[11] Fifteen (48.4%) lesions in the present study showed no dysplasia and dysplastic lesions accounted for 14 (45.2%) of the lesions. Histopathology is considered the gold standard in the diagnosis of PMD. However, several studies have reported high interobserver and intraobserver variability in the diagnosis of various grades of dysplasia. This led the working group at WHO Collaborating Centre for Oral Cancer and Precancer (2008) to arrive at a consensus view that a binary system of classification of dysplasia could improve agreement among pathologists. No, questionable, or mild dysplasia was categorized as low risk, while moderate or severe was categorized as high risk.[12] This system of classification has been used in the present study.

The major drawback we observed with the use of acetic acid in screening PMD was its very poor specificity. This is in contrast with the findings of Bhalang et al. who in their study observed a sensitivity of 83.33% and a specificity of 84.21% with 5% acetic acid in the detection of oral squamous cell carcinoma. They also observed a statistically significant association between clinical examination using 5% acetic acid and expression of p53 protein.[8]

In addition to dysplasia, the cell membrane permeability could be enhanced in inflammation. Lichen planus, which is an inflammatory condition, could appear acetowhite even in the absence of dysplasia. Chewing habits are often associated with chronic trauma of the mucosa and inflammation. Acetic acid causes osmolar changes in the cells. In the presence of increased cell membrane permeability, this leads to outward movement of water leading to cytoplasmic dehydration and collapse of the cell membrane resulting in coagulation of cellular proteins. This results in an altered refractive index of epithelium, which gives it a white appearance. Also, acetic acid removes the mucus by coagulating it and thus allowing the visualization of dysplastic areas.[9],[13]

In the present study, large numbers of false-positive results could have been associated with tissue changes other than dysplasia such as inflammation and trauma, especially in patients with chewing habits. The role of these factors in causing “acetowhite” appearance needs to be investigated further.

In a study conducted by Farah and McCullough, a visualization tool using an acetic acid wash and chemiluminescence was unable to discriminate between keratotic, inflammatory, premalignant, and malignant lesions. In their study, all oral mucosal white lesions were reported as acetowhite, resulting in very poor specificity (0%), though sensitivity was 100%.[14] These findings are comparable with that of our study.

In a study conducted to evaluate the utility of 5% acetic acid in tobacco-associated lesions, Vinuth et al. reported sensitivity and specificity of 97% and 50%, respectively, in Group 1 lesions (patients with chronic tobacco use and with clinically apparent normal mucosa). For Group 2 lesions (patients suspected of having oral cancer), the sensitivity and specificity were 95% and 60%, respectively. The authors attributed false-positive results to lesions with inflammation.[9] Direct comparisons with this study is not possible as the authors considered hyperkeratosis and mild dysplasia to be histopathologically positive, whereas in the present study, hyperkeratosis with no dysplasia was considered as negative and all cases with no dysplasia and mild dysplasia were categorized as low-risk lesions.

Sankaranarayanan et al. indicated that a lower specificity could limit the usefulness of visual inspection with 4% acetic acid in cervical cancer screening. The presence of infection, inflammation, and metaplasia of cervical mucosa has been suggested to affect the results. They suggested that the specificity of visual examination with acetic acid could be improved with standardized training, uniform and reproducible criteria, and the development of quality control procedures.[7]

Although a large number of acetowhite tissues were histologically hyperkeratosis (leukoplakia), and lichen planus with no dysplasia, the possibility that these areas could have early changes in the form of gene mutations cannot be ruled out. Bhalang et al. found a statistically significant association between positive staining with acetic acid and expression of p53.[8]

Toluidine blue is an acidophilic metachromatic dye that has the ability to bind acidic tissue components. Therefore, it binds to the nuclear material of the tissues having high DNA and RNA content.

The sensitivity and specificity of toluidine blue varied in earlier studies from 38% to 98% (median 85%) and 9% to 93% (median 67%), respectively, whereas the positive predictive value ranged from 33% to 93% (median 85%) and the negative predictive value from 22% to 92% (median 83%).[15],[16]

In the present study, the sensitivity of toluidine blue in detecting dysplastic lesions improved from 75% to 85.7% when they were grouped into high-risk lesions. A similar observation was reported by Awan et al. In their study, the sensitivity for detecting dysplasia was 56.8%, whereas for high-risk lesions it increased to 85%.[5]

In a study by Epstein et al., toluidine blue provided 100% true positives in identifying severe dysplasia and oral squamous cell carcinoma. Toluidine blue appeared to be more sensitive in identifying high-risk lesions.[17]

The higher specificity in our study could be attributed to the fact that questionable stain uptake was considered as negative staining. Accepting light blue staining as negative improves the specificity significantly.[17] To decrease the number of false-positive results, Mashberg recommended that all irritating, inflammatory, and traumatic lesions should be allowed to heal, and if not responding, they should be re-evaluated and stained after 10 to 14 days. This protocol reduced false positives by 8.5%.[1]

Discrepancies in the results may be due to the nature of the sample analyzed, variation in inclusion criteria of lesions, staining method employed, variation in interpretation of the color of the stain, and lack of histological correlation.

In the present study, none of the participants reported any adverse effects with acetic acid or toluidine blue application.

One of the limitations of this study was the small sample size. A larger sample size would have allowed for greater power of the study. Interobserver variations among pathologists in the diagnosis of dysplasia were not evaluated. Uniform and reproducible criteria need to be developed for the evaluation of stainability with vital stains. The subjectivity in visual examination was not investigated as a single examiner performed all the observations.

  Conclusion Top

The utility of acetic acid in detecting dysplasia and high-risk PMD is severely limited due to its poor specificity. Acetic acid was unable to discriminate between hyperkeratotic lesions with no dysplasia and lesions with dysplasia. Toluidine blue, due to its better specificity, could be a useful adjunct for chairside diagnosis of more advanced PMD. Studies with a larger sample size and use of molecular markers in the evaluation of the ability of vital stains to detect early changes associated with carcinogenesis would further clarify the role of these agents as diagnostic adjuncts.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Mashberg A. Final evaluation of tolonium chloride rinse for screening of high-risk patients with asymptomatic squamous carcinoma. J Am Dent Assoc 1983;106:319-23.  Back to cited text no. 1
Epstein JB, Feldman R, Dolor RJ, Porter SR. The utility of tolonium chloride rinse in the diagnosis of recurrent or second primary cancers in patients with prior upper aerodigestive tract cancer. Head Neck 2003;25:911-21.  Back to cited text no. 2
Onofre MA, Sposto MR, Navarro CM. Reliability of toluidine blue application in the detection of oral epithelial dysplasia and in situ and invasive squamous cell carcinomas. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:535-40.  Back to cited text no. 3
Gandolfo S, Pentenero M, Broccoletti R, Pagano M, Carrozzo M, Scully C. Toluidine blue uptake in potentially malignant oral lesions in vivo: Clinical and histological assessment. Oral Oncol 2006;42:89-95.  Back to cited text no. 4
Awan KH, Morgan PR, Warnakulasuriya S. Assessing the accuracy of autofluorescence, chemiluminescence and toluidine blue as diagnostic tools for oral potentially malignant disorders--a clinicopathological evaluation. Clin Oral Investig 2015;19:2267-72.  Back to cited text no. 5
Singh V, Sehgal A, Parashari A, Sodhani P, Satyanarayana L. Early detection of cervical cancer through acetic acid application--an aided visual inspection. Singapore Med J 2001;42:351-4.  Back to cited text no. 6
Sankaranarayanan R, Wesley R, Thara S, Dhakad N, Chandralekha B, Sebastian P, et al. Test characteristics of visual inspection with 4% acetic acid (VIA) and Lugol's iodine (VILI) in cervical cancer screening in Kerala, India. Int J Cancer 2003;106:404-8.  Back to cited text no. 7
Bhalang K, Suesuwan A, Dhanuthai K, Sannikorn P, Luangjarmekorn L, Swasdison S. The application of acetic acid in the detection of oral squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:371-6.  Back to cited text no. 8
Vinuth DP, Agarwal P, Kale AD, Hallikeramath S, Shukla D. Acetic acid as an adjunct vital stain in diagnosis of tobacco-associated oral lesions: A pilot study. J Oral Maxillofac Pathol 2015;19:134-8.  Back to cited text no. 9
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Warnakulasuriya S, Johnson NW, van der Waal I. Nomenclature and classification of potentially malignant disorders of the oral mucosa. J Oral Pathol Med 2007;36:575-80.  Back to cited text no. 10
Waldron CA, Shafer WG. Leukoplakia revisited. A clinicopathologic study 3256 oral leukoplakias. Cancer 1975;36:1386-92.  Back to cited text no. 11
Warnakulasuriya S, Reibel J, Bouquot J, Dabelsteen E. Oral epithelial dysplasia classification systems: Predictive value, utility, weaknesses and scope for improvement. J Oral Pathol Med 2008;37:127-33.  Back to cited text no. 12
Sankaranarayanan R, Wesley R, Somanathan T, Dhakad N, Shyamalakumary B, Amma NS, et al. Visual inspection of the uterine cervix after the application of acetic acid in the detection of cervical carcinoma and its precursors. Cancer 1998;83:2150-6.  Back to cited text no. 13
Farah CS, McCullough MJ. A pilot case control study on the efficacy of acetic acid wash and chemiluminescent illumination (ViziLite) in the visualisation of oral mucosal white lesions. Oral Oncol 2007;43:820-4.  Back to cited text no. 14
Epstein JB, Güneri P. The adjunctive role of toluidine blue in detection of oral premalignant and malignant lesions. Curr Opin Otolaryngol Head Neck Surg 2009;17:79-87.  Back to cited text no. 15
Patton LL, Epstein JB, Kerr AR. Adjunctive techniques for oral cancer examination and lesion diagnosis: A systematic review of the literature. J Am Dent Assoc 2008;139:896-994.  Back to cited text no. 16
Epstein JB, Silverman S Jr, Epstein JD, Lonky SA, Bride MA. Analysis of oral lesion biopsies identified and evaluated by visual examination, chemiluminescence and toluidine blue. Oral Oncol 2008;44:538-44.  Back to cited text no. 17


  [Figure 1], [Figure 2], [Figure 3]

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


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