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Table of Contents   
ORIGINAL RESEARCH  
Year : 2021  |  Volume : 32  |  Issue : 3  |  Page : 310-315
Diagnostic efficiency of methylene blue and lugol's iodine double staining method in oral leukoplakia in detecting dysplasia


1 Department of Oral Medicine and Radiology, Institute of Dental Sciences, Sehora, Jammu, India
2 Department of Oral Medicine and Radiology, Swami Devi Dyal Hospital and Dental College, Barwala, Haryana, India
3 Department of Pedodontics and Preventive Dentistry, Institute of Dental Sciences, Sehora, Jammu, India

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Date of Submission31-Mar-2019
Date of Decision07-May-2019
Date of Acceptance11-Nov-2021
Date of Web Publication23-Feb-2022
 

   Abstract 


Objectives: Early detection of dysplasia in oral potentially malignant disorders (PMD) might facilitate screening for possible subsequent malignant transformation. Vital staining is a non-invasive clinical adjunct used for determining the biopsy site, which facilitates early detection of dysplastic changes in PMD. Some authors suggested that double staining method has superior results over staining with a single dye. Aim: The aim of the study was to evaluate the accuracy of in vivo staining with methylene blue (MB) and Lugol's iodine (LI) double staining method in comparison with MB staining alone. Materials and Methods: Fifty patients of oral leukoplakia were recruited for the study. After obtaining written informed consent from the patients, the lesions were stained consecutively with 5% MB and 3% LI. The pattern of dye retention of MB alone, followed by MB and LI was noted. Incisional biopsy from the lesion was taken based on the retention of MB and the absence of staining of LI or by clinical judgement in case both stains were not retained. The clinical uptake of the stains was correlated with the degree of dysplasia on histopathological examination. Results: Out of 50 subjects, MB was retained in 47 cases (94%), while 3 cases (6%) failed to retain the dye. However, out of 47 cases, 20 cases had dark blue stain and were considered as MB positive, while the rest 27 cases had pale blue stain and were considered to be negative for MB staining. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy (DA) of MB staining were 57.82%, 100%, 100%, 26.53% and 46%, respectively. After applying both stains, i.e., double staining method, the outline of the lesion was better defined. Out of 50 PMD cases, 3 patients retained only LI and showed no dysplasia. Out of 47 cases which showed dysplasia, 27 cases showed pale blue retention of MB (-) and also were negative for LI. The rest 20 cases were positive for MB but negative for LI. The sensitivity, specificity, PPV and NPV with double staining technique were 100%. Conclusion: The accuracy and reliability of double staining method was superior to MB staining. Clinical Relevance: Increased accuracy of double staining method aids in better detection of dysplasia and is of great help to the clinician in deciding the nature of PMD in question.

Keywords: Double staining method, Lugol's iodine, methylene blue, oral potentially malignant disorders

How to cite this article:
Sharma D, Konidena A, Sharma A. Diagnostic efficiency of methylene blue and lugol's iodine double staining method in oral leukoplakia in detecting dysplasia. Indian J Dent Res 2021;32:310-5

How to cite this URL:
Sharma D, Konidena A, Sharma A. Diagnostic efficiency of methylene blue and lugol's iodine double staining method in oral leukoplakia in detecting dysplasia. Indian J Dent Res [serial online] 2021 [cited 2022 Oct 7];32:310-5. Available from: https://www.ijdr.in/text.asp?2021/32/3/310/338123



   Introduction Top


Oral squamous cell carcinoma is one of the most common neoplasms and ranked eighth in cancer incidence worldwide. The age standardised incidence rate of oral cancer in India is 12.6 per 100,000 population.[1] The concept of a two-step process of cancer development in the oral mucosa, i.e., the initial presence of a precursor (premalignant or precancerous) lesion subsequently developing into cancer, is well established.[2] Clinical observations indicate that a proportion of carcinoma cases are preceded by or co-exist with other distinct oral epithelial lesions. Hence, early detection of dysplasia in oral potentially malignant disorders (PMD) might facilitate screening for possible subsequent malignant transformation.[2],[3]

Simple visual examination is well known to be limited by subjective interpretation as well as by the potential of occurrence of dysplasia and early squamous cell carcinoma within areas of normal looking mucosa.[4] Early stages of dysplasia are difficult to detect as the lesion may not be palpable and colour changes are not very different from the surrounding mucosa.[5] Biopsy with histopathological examination is considered to be the gold standard to complement the clinical diagnosis and establish a definitive diagnosis.[5] Though biopsy is considered a gold standard, accurate selection of site for biopsy is the most important criterion to arrive at the correct diagnosis. As a consequence, adjunctive techniques have been recommended to increase the ability to differentiate between benign changes of the mucosa from dysplastic/malignant changes as well as to identify areas of dysplasia that are not visible to the naked eye.[4]

Vital staining is a non-invasive technique used for detecting dysplastic changes or helping the clinician to choose the appropriate site for biopsy, which can aid in early detection of oral cancers.[3],[4] In vivo stains are prompt resources helpful in diagnosing molecular changes or some specific chemical reactions taking place within cells or tissues or cytological details that go unnoticed during the process of carcinogenesis.[6] The various dyes studied in this context are toluidine blue, methylene blue (MB) and Lugol's iodine (LI), the most extensively studied being toluidine blue.[5],[6],[7],[8]

Toluidine blue (TB) and MB are acidophilic dyes that bind to double-helical DNA with a high affinity with similar physicochemical properties. Since MB is less toxic, cheaper and easily available than TB, it had been used to detect gastric, prostate, bladder, oral cancers and oral precancerous lesions for the identification of metaplasia, carcinoma or dysplasia.[9] Riaz et al. had studied its validity in detection of oral cancer and precancerous lesion, with a reported sensitivity of 90%.[10] To further increase the sensitivity of in vivo staining, Zhu et al.[11] had performed double staining technique.

Double staining technique includes staining of the abnormal mucosa with TB/MB and the adjacent normal mucosa by LI. Zhu et al.[11] had reported that double staining with MB and LI improves the ability to see the true size, borders of the lesions with a co-relation between mucosal staining and histology in oesophageal carcinoma. The basis of the mucosal double staining technique is that the PMD lesion is stained blue by MB stain while LI reversibly stains glycogen, brown. The application of this double staining technique using MB and LI in detecting oral lesions has so far not been addressed. Hence, the present study was conducted to evaluate the accuracy of in vivo staining with MB and LI double staining method by correlating the clinical uptake with histopathological diagnosis and comparing the results with MB staining alone.


   Materials and Methods Top


Ethical clearance was obtained from the institutional ethical committee and written informed consent was taken from all the study participants (SDD/13/Eth/008). All procedures performed in the study were conducted in accordance with the ethics standards given in 1964 Declaration of Helsinki, as revised in 2013.

Study design: An in vivo cross-sectional study was conducted in 50 patients with oral leukoplakia. Patients over 18 years of age, clinical diagnosis of homogeneous or non-homogeneous leukoplakia according to the criteria given by Warnakulasuriya et al.[12] were included in the study. Patients with a clinical diagnosis of oral cancer, patients suffering from systemic diseases contraindicating biopsy, known hypersensitivity to the stains were excluded from the study.

Method of staining and data collection: All the study subjects were subjected to complete clinical examination [Figure 1] with special reference to the details of habit history, the lesion characteristics including the location, type and size. After routine haematological investigations, the participants were subjected to staining followed by incisional biopsy. All the patients were directed to rinse their mouth with 1% lactic acid and distilled water for 30 seconds to remove the food debris. Mucosa of the target area was then gently dried with gauze. The MB dye (1%) was then applied directly on the lesion with cotton bud for 30 seconds. Patients were asked to rinse with 1% lactic acid for 30 seconds again to wash out the excess dye [Figure 2]. Pattern of dye retention was assessed by intensity of stain retention. Subsequently, 3% LI was applied to the lesional and perilesional sites with a cotton bud for 10–20 seconds [Figure 3].
Figure 1: Clinical picture of non-homogenous leukoplakia at the commissural area

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Figure 2: Clinical picture after methylene blue staining; Red outlined area shows darkly stained MB area, while the black outlined region shows pale stain of MB

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Figure 3: Clinical picture after double staining with MB followed by LI. Light blue outline shows mixed colour (where MB was retained), green outline is the surrounding region with pale yellow colour, while the surrounding mucosa outlined by black shows mahogany brown of LI

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Interpretation of stains: Dark royal blue/deep blue stains of MB were considered as positive retention. Wide, shallow, faint, pale royal blue stains or no retention of the stain were considered as negative for MB retention.[10] Areas staining dark brown with LI (positive) were considered to be normal. Pale yellow or unstained areas (negative) were considered to be dysplastic.[13] The interpretation of stains was conducted initially by two examiners (DS, AK) in all the cases. In case of disagreement between the examiners, third examiner (AS) evaluated the lesion and decision was taken in favour of majority opinion. Out of 50 cases, there was disagreement in only 2 cases, which was resolved by the third examiner.

Incisional biopsy was done under local anaesthesia. Biopsy site was selected based on the retention of MB and the absence of staining of LI. In cases where MB dye retention was absent, clinical judgment directed the biopsy. Histopathological grading of the biopsy specimens was done on the basis of degree of dysplasia into those with no dysplasia, mild dysplasia, moderate dysplasia and severe dysplasia, according to criteria given by Warnakulasuriya et al.[14]

Statistical analysis: It was conducted using SPSS version 22.0 and Medcal Software. Proportions were compared using Chi-square test. The results of positive/negative uptake of MB and LI in each lesion were correlated with the histopathological diagnosis. Performance of the staining methods was expressed as sensitivity, specificity, positive and negative predictive values and diagnostic accuracy (DA).


   Results Top


The study sample consisted of 50 male oral leukoplakia subjects, out of which, 32 had homogeneous while 18 presented with non-homogeneous variants. The mean age of total patients was 47.50 years, with an age range between 22 and 70 years. The descriptive data of the study population including age and gender is given in [Table 1].
Table 1: Descriptive data of the study population

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All subjects had a smoking habit with a mean duration of 18.18 years, with a mean frequency of 7.46 bidis per day. In addition to smoking, 6 patients had a habit of smokeless tobacco with a mean duration of 11.75 years and 7 had a habit of alcohol abuse [Table 2]. Smokeless tobacco users consumed 4 packets per day on an average, while alcohol users consumed alcohol with a mean frequency of 2.56 times per week. There were only 2 patients with all the three habits. All the patients presented with lesions on buccal mucosa alone.
Table 2: Distribution of habits according to type of habits

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Out of 50 samples, 15 cases of mild dysplasia, 25 with moderate and 7 with severe dysplasia were found when analysed histopathologically. Three patients showed hyperkeratosis without dysplasia. In the present study, there were no adverse reactions or persistent staining observed with both the dyes. Out of 50 subjects, MB was retained in 47 cases (94%), while 3 cases (6%) failed to retain the dye. However, out of 47 cases, 20 cases had dark blue stain and were considered as MB positive, while the rest 27 cases had pale blue stain and were considered to be negative for MB staining.

Out of 20 cases, which were positive for MB, 2 cases had mild dysplasia, 11 had moderate dysplasia and 7 had severe dysplasia [Table 3](a). Out of 27 cases, which were negative for MB, 13 had mild dysplasia, 14 had moderate dysplasia but none had severe dysplasia. The sensitivity was found to be 57.82%, while the specificity was 100%. The positive predictive value (PPV) and negative predictive value (NPV) were 100% and 26.53%, respectively. The DA was found to be 46% [Table 3](b).
Table 3:

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Out of 32 homogeneous leukoplakia cases, there was no retention in 3 cases, 22 cases showed pale blue MB stain, while 7 cases depicted dark blue stain. Out of these homogeneous leukoplakia cases, 3 cases had hyperkeratosis, 12 had mild dysplasia and 17 cases showed moderate dysplasia. Out of 18 non-homogenous leukoplakia cases, 5 cases showed pale blue MB stain, while 13 cases showed dark blue stain, out of which, 3 had mild dysplasia, 8 had moderate dysplasia and 7 had severe dysplasia.

The difficulties in double staining technique were the exact identification of colour. After applying both stains, i.e., double staining method, in the case of lesions that retained MB, further application of LI resulted in a dark shade, though perilesional mucosa was pale yellow. This may be misleading as the mixed colour obtained might be difficult to differentiate from mahogany colour due to retention of LI. The area between the positive retention of both stains may indicate the extent of the lesion. However, the outline of the lesion was better defined. Perilesional dark brown staining was found in all the subjects with LI. In 3 cases, lesional sites were stained dark brown by LI which were also negative for dysplasia. Out of 47 cases which showed dysplasia, 27 cases showed pale blue retention of MB (-) and also were negative for LI. The rest 20 cases were positive for MB but negative for LI. The sensitivity, specificity, PPV and NPV with double staining technique were 100% [Table 4].
Table 4: Diagnostic accuracy of double staining method

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   Discussion Top


Vital staining has a long and well-established place in the identification of non-visible, non-palpable, abnormal premalignant areas of mucosa in the oral cavity, large bowel, oesophagus and cervix.[15] Greater value of cytological examination lies in the ability to disclose the presence of intraepithelial non-invasive carcinoma when the clinical appearance is relatively innocent and cancer is not suspected.[3]

MB is an acidophilic dye that binds to the double-helical DNA with a high affinity, as deduced from the absorption and fluorescence spectral data.[9] It has been established the fact that Methylene shows proportionate binding to DNA nucleotide and this accounts for increase in intensity of stain colour with increase in chromatin material in premalignant and malignant cells.[10] It had also been hypothesised that the intercellular cannaliculi are much larger in malignant cells, due to the haphazard arrangement of tumour cells, thus allowing more intensive penetration of the dye.[3] The mechanism of LI staining is that iodine reacts with glycogen in the cytoplasm. The reaction, known as the iodine-starch reaction, is visualised by a colour change. The loss of cellular differentiation and the enhanced glycolysis in cancer cells do not promote the iodine-starch reaction. When it is applied on the suspicious lesions, normal mucosa stains brown or mahogany due to its high glycogen content, while dysplastic and cancer lesions do not stain, and appear pale compared with the surrounding tissue.[16]

All our patients had known risk factors for PMD including smoking or smokeless tobacco and/or alcohol. Tobacco consumption is the main etiologic factor inducing carcinogenesis in oral mucosa. A survey by WHO Southeast Asia (WHOSEA) has indicated that almost 50% of oral cancer in men and 25% cases of women in India are believed to be tobacco borne.[17] In the present study, majority of the PMD has single habit (70%). The most common habit was smoking tobacco (100%) followed by alcohol consumption (14%) and smokeless tobacco (12%). All the subjects in our study had lesions on the buccal mucosa, which was the most common site of involvement according to Sharma and Mubeen (70%),[3] Riaz et al. (84%)[10] and Cancela-Rodriguez et al.[18] This could be partly attributed to the ease of detection on buccal mucosa even during self-examination, when compared with other mucosal sites.

In the present study, there were no adverse reactions or persistent staining observed with both the stains in any patient, thus advocating the safety of both stains. Some previous studies report no adverse reaction with the use of both stains, while Reyes et al.[19] reported parenchymal necrosis of the breast following the use of MB. However, they had attributed it to contaminated solution.

In the present study, MB dye was applied first in all the cases and resulted in a positive test in 20 out of 47 cases, while wide, shallow faint stain retention (considered to be negative for MB retention) was seen in 27 cases. It may thus be inferred that the intensity of MB stain did not correlate with the degree of dysplasia, since both pale and dark blue stained lesions exhibited mild and moderate dysplasia.

In the current study, 20 of 47 pathologically proved PMD were positive with deep and focal MB staining. The overall sensitivity was 57.82%. In our study, all the three benign lesions showed negative staining; thus, the specificity was 100%. Our results were contrary to the findings of Riaz et al.[10] who found 91.3% sensitivity and 75% specificity. The differences in study results could be due to the differences in the inclusion criteria of the study sample taken and interpretation of the stain. The morphological description and the clinical classification of PMD were not mentioned in their study to analyse further.

High false negative results found in the present study can be due to the inclusion of more cases of homogeneous leukoplakia as study population, wherein the surface layer of keratin contains pyknotic or no nuclei.[3] Moreover, it could also be due to the fact that despite wide, shallow, faint retention of the stain was present in certain lesions (pale blue retention), they were considered as being negative for MB staining.[10] If even pinpoint or faint retention of the dye had been considered to be positive and only no retention of stain had been considered to be negative, the sensitivity and specificity of MB stain would have been higher in our study as only 3 cases did not show retention of the dye. It can thus be deduced from the present study that even faint staining with MB should be taken critically and the lesions should be followed up not to miss any probability of the lesion being dysplastic. This is well supported by the findings of Epstein et al.[20] who reported a prognostic relevance for pale blue stain for TB cases through loss of heterozygosity. They found that even pale blue stained lesions correlated well with loss of heterozygosity, a better and early marker for carcinomatous transformation.

In the present study, there were no false positive cases. This could be attributed to the absence of inflammatory or ulcerative lesions, irregular papillary or surfaces of the lesions in the study population or absence of dye retention in the fissures of leukoplakia, contamination of saliva and plaque or retention of dye material in papilla of minor salivary gland ducts.[3],[10]

In the present study, after applying both stains, i.e., double staining method, the outline of the lesion was better defined. In 27 MB negative cases, the lesional area stained pale yellow with LI (LI negative), while in 3 cases there was a dark brown stain (LI positive) observed. Epstein et al.[21] conducted a comparative study, in which TB and LI were used both in combination and separately in 59 patients. The sensitivity and specificity of TB were 0.925 and 0.632, respectively, while the sensitivity and specificity of LI were 0.875 and 0.842, respectively. When combined, the two stains showed a sensitivity of 0.850 and a specificity of 0.895.

In the present study, the DA of double staining method had superior results when compared with using MB dye alone. This is in accordance with the studies conducted by Nagaraju et al.[6] and Zhu et al.[11] The sensitivity, specificity, PPV and NPV with double staining technique were 100%, since false negative cases with MB stain retained pale brown stain of LI and hence gave a positive result. Our results could not be compared with other studies as double staining of MB with LI in oral premalignant lesions was not addressed earlier.

The results of our study were comparable to study done by double staining utilising TB and LI by Nagaraju et al.[6] with respect to sensitivity, but differed in specificity. According to Nagaraju et al.,[6] the DA of validity of double staining with TB and LI in PMD was 93% with sensitivity and specificity of 100% and 60%, respectively. However, accurate comparison was not possible with this study because their results comprised of overall accuracy of LI with TB within PMD and malignant lesions.

The limitations of the present study were small sample size and lack of correlation with molecular markers of the lesions, which assess the risk better. Also, the use of MB followed by LI was followed in all cases and not vice versa, evaluation of OED by two pathologists was not done and comparison of DA of MB and LI was not conducted with the malignant lesions as well as controls.


   Conclusion Top


The present study showed an uptake of staining of MB and LI in PMD. The accuracy and reliability of double staining method were superior to MB staining. The clinical uptake of the MB dye did not correlate with mild and moderate degree of dysplasia.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Correspondence Address:
Dr. Aravinda Konidena
Department of Oral Medicine and Radiology, Swami Devi Dyal Hospital and Dental College, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_291_19

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