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Clinical diagnosis of squamous cell carcinomas of the
oral mucosa is not difficult when the lesion is obvi-
ously invasive or when the patient experiences pain,
functional limitation, or regional lymphadenopathology.
Conversely, it is more difficult to diagnose dysplasias
and carcinomas mainly in potentially malignant epithe-
lial lesions (PMELs).
With the aim of improving the efficiency of these diag-
noses, techniques are being developed to complement
clinical examination and to facilitate the identification of
initial carcinomas. Several clinical studies have evalu-
ated the efficiency of in vivo staining with toluidine blue
in the detection of dysplasias and malignant lesions.
Although there is a consensus that this staining often
assists in the identification of these malignant lesions,
results have been diverse.1-17 This diversity of results is
probably due to variations in methodology, the popula-
tion studied, and the lesions analyzed. Warnakulasuriya
and Johnson16 emphasized that the majority of studies
analyzed few PMELs and large groups with clinically
suspected malignancy. In addition, in some of these
studies, biopsies of lesions that did not retain toluidine
blue were not performed.2,4,5,9 These factors may inter-
fere in the evaluation of staining, thus limiting the value
of results obtained. Thus, our objective in this study was
to determine the reliability of in vivo staining with tolu-
idine blue in the detection of oral epithelial dysplasia, in
situ carcinoma, and squamous cell carcinoma associated
with PMELs and superficial oral ulcerations suspicious
of malignancy.
PATIENTS AND METHODSFor this study 50 patients with PMELs and superfi-
cial oral ulcerations suggestive of malignancy were
selected from those treated at the Oral Medicine
Service, Faculty of Dentistry, Araraquara, So Paulo,
Brazil, from August 1993 to May 1995 (n = 1957). Not
included in this study were patients who refused to be
submitted to biopsy (n = 21), those who abandoned
treatment, or those who had clinically obvious invasive
carcinomas or lesions without risk or suspicion of
Reliability of toluidine blue application in the detection of oral
epithelial dysplasia and in situ and invasive squamous cell
carcinomas
Mirian Aparecida Onofre, DDS, PhD,a Maria Regina Sposto, DDS, PhD,b and Cludia Maria
Navarro, DDS, PhD,c So Paulo, BrazilSCHOOL OF DENISTRY ARARAQUARAUNESP
Objective. The objective of this study was to evaluate the reliability of in vivo staining with toluidine blue in the detection oforal epithelial dysplasia, in situ carcinoma, and invasive squamous cell carcinomas in potentially malignant epithelial lesions(PMELs) and superficial oral ulcerations suggesting malignancy.Study design. Fifty patients with PMELs and superficial oral ulcerations suggestive of malignancy were selected from thosetreated at the Oral Medicine Service, Faculty of Dentistry, Araraquara, Brazil. All lesions were submitted to staining with anaqueous solution of 1% toluidine blue, followed by biopsy and histologic analysis. The sensitivity, specificity, and positive andnegative predictive values were calculated.Results. Histologic diagnosis revealed that 14% of the lesions analyzed were in situ carcinoma and invasive squamous cell carci-nomas, 12% were epithelial dysplasias, 13% were keratosis, 40% were lichen planus, and 8% were other benign lesions. The sensi-tivity of the staining was 77%, the specificity 67%, and the positive and negative predictive values 43.5% and 88.9%, respectively.Conclusions. Staining with toluidine blue was demonstrated to be highly reliable in the detection of in situ carcinoma andinvasive squamous cell carcinoma, because false-negative results for the lesions did not occur. Toluidine blue staining is anadjunct to clinical judgment and not a substitute for either judgment or biopsy.(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:535-40)
Supported by grants CNPq no. 523164/96-3 and FAPESP no.
96/5927-8.
Presented at the 5th Biennial Congress of the European Association
of Oral Medicine, Gteborg, Sweden, Aug 24-26, 2000.aAssistant Professor, Oral Medicine Service, Department of
Diagnosis and Surgery, School of Dentistry AraraquaraUNESP, So
Paulo, Brazil.bAssociate Professor, Oral Medicine Service, Department of
Diagnosis and Surgery, School of Dentistry AraraquaraUNESP, So
Paulo, Brazil.cAssistant Professor, Oral Medicine Service, Department of
Diagnosis and Surgery, School of Dentistry AraraquaraUNESP, So
Paulo, Brazil.
Received for publication Jul 19, 2000; returned for revision Aug 23,
2000; accepted for publication Oct 30, 2000.
Copyright 2001 by Mosby, Inc.
1079-2104/2001/$35.00 + 0 7/13/112949
doi:10.1067/moe.2001.112949
535
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malignancy. Twenty-eight of the patients were men,
and 22 were women, 45 were white, 2 were black, 2
were mixed race, and 1 was Asian, with a mean age of
55.2 13.4 years. A clinical history of the lesion was
taken from each patient, and all were submitted to a
systematic oral examination. The criteria for clinical
diagnosis were (1) homogenous leukoplakia: apredominantly white, uniform, and flat lesion, not able
to be scraped, with a smooth, wrinkled, or corrugated
surface that may exhibit shallow cracks18,19; (2) nonho-
mogenous leukoplakia: a predominantly white or red
and white lesion with an irregular, nodular, or exophy-
tic surface18,19; (3) erythroplakia: a velvety red lesion
with imprecise borders that could not be diagnosed as
any other lesion18,19; (4) reticular lichen planus: a
predominantly white lesion with intertwining lines or
striae that confer a lacy or annular appearance19; (5)
erosive/ulcerated lichen planus: a predominantly red,
irregular erosion or ulceration associated with a retic-
ular form, especially in the peripheral region of thelesion and with pseudomembranes covering the ulcer-
ated areas19; (6) superficial ulcerations suspicious of
malignancy: localized, superficial lesions without inva-
sion or loss of mobility of neighboring chronic tissues
that do not heal after local treatment.
After clinical diagnosis the lesions were submitted to
topical application of an aqueous solution of 1% tolui-
dine blue, according to the technique shown in Table I.
The stain was prepared following the recommenda-
tions of Mashberg.8,10 In the lesions associated with
mechanical trauma, these sites were then eliminated,
and the staining was repeated after 14 days, with the
object of reducing the number of false-positive
results.8,10 The clinical diagnosis and staining results
were determined by 2 examiners, previously calibrated,
who were specialists in oral medicine, following the
recommendations made by Mashberg.7,10
The biopsy sites were selected on the basis of the
clinical appearance of the lesion and the staining result.
Areas retaining stain were biopsied. In sites where no
retention of stain occurred, clinical judgment directed
the biopsy. Erythematous, verrucous, depressed, ele-
vated, indurated, and diffuse margin areas were prefer-
entially removed.20-23 Lesions larger than 1 cm were
submitted to biopsy in various sites for representativeinvestigation of the entire lesion.
Stain-retained areas were placed in separate containers,
and the pathologist was not informed of the staining
result. The specimens were fixed in a buffered solution of
10% formalin and submitted to routine procedures
and hematoxylin-eosin staining for analysis by light
microscopy.
The lesions were histologically classified as (1) squa-
mous cell carcinoma: severe dysplasias involving the
entire extent of the epithelium without invasion of the
connective tissue (in situ carcinoma) or with invasion of
connective tissue (frank invasive carcinoma) or limited to
the juxtaepithelial area (initial invasive carcinoma)19,24,25;
(2) epithelial dysplasia: the parameters recommended by
the WHO24 were used; dysplasias were classified as
mild, moderate, and severe; (3) keratosis: the presence ofvarious degrees of keratosis and/or acanthosis with no
epithelial dysplasia or atypical cells; (4) lichen planus:
liquefying degeneration of basal cells, band-shaped
lymphocyte infiltrate in the connective epithelial tissue,
with or without saw-toothed projections, hyperkeratosis,
the presence of Civatte bodies, and separation between
the epithelium and connective tissue26,27; (5) other
benign lesions: lesions without epithelial dysplasia or
atypical cells that are not included among the previously
described groups. The histologic diagnoses were deter-
mined by a previously calibrated pathologist who was
blinded to the toluidine blue results.
In the cases where the tissue analyzed presenteddifferent histologic appearances, the most severe histo-
logic diagnosis was considered.18
The results of the clinical and histologic diagnoses and
the staining results were compared. The sensitivity, speci-
ficity, and the positive and negative predictive results
were calculated according to the method proposed by
Rosenberg and Cretin.12
RESULTSOf the 50 patients studied, 34 were smokers
(23 smoked paper cigarettes, 7 hand-rolled cigarettes,
and 4 pipes), 13 regularly consumed alcohol, and 13 had
related occupational activities that frequently exposed
them to the sun. The clinical diagnosis of the oral lesions
analyzed and the results of staining are presented in
Table II. All superficial ulcerations suggestive of malig-
nancy retained stain. The nonhomogenous leukoplakia
and the erosive/ulcerated lichen planus retained stain
with a higher frequency than did the homogenous leuko-
plakia. There was no retention of stain in the reticular
lichen planus.
The histologic diagnosis and the results of the toluidine
blue staining are demonstrated in Tables III and IV. The
histologic analysis demonstrated that among the lesions
analyzed, 14% (n = 7) were squamous cell carcinomas(1 in situ carcinoma and 6 initial invasive carcinomas).
All retained stain, indicating a 100% sensitivity of
staining for the detection of in situ carcinoma and inva-
sive squamous cell carcinoma, because false-negatives
did not occur. Of the superficial ulcerations, 57.1% (n = 4)
were invasive squamous cell carcinomas, and 15% (n = 3)
of nonhomogenous leukoplakias were carcinomas (1 in
situ carcinoma and 2 invasive carcinomas). Epithelial
dysplasias were detected in 12% (n = 6) of the lesions
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analyzed (4 mild and 2 moderate), yet just 3 retained
stain, demonstrating an occurrence of false-negativeresults and a staining sensitivity of 50% for this group of
lesions. Epithelial dysplasia occurred in 25% (n = 3) of
the homogenous leukoplakias and in 15% (n = 3) of the
nonhomogenous leukoplakias. In 74% (n = 37) of all
lesions evaluated, the histologic analysis revealed an
absence of epithelial dysplasia or atypical cells, and 40%
(n = 20) were lichen planus, 26% (n = 13) keratosis, and
8% (n = 4) other benign lesions (3 granular tissues and 1
ulcerated papilloma). Histologic analysis showed that 9
cases clinically diagnosed as leukoplakia were in fact
plaquelike lichen planus. Among keratosis, lichen planus,and other benign lesions, 13 retained stain; thus 35% of
results were false-positives, indicating a staining speci-
ficity of 65% (Table IV). The highest number of false-
positive results occurred in the lesions clinically diag-
nosed as nonhomogenous leukoplakia (n = 6). According
to the histologic analysis, toluidine blue staining was
positive in 6 cases of lichen planus.
In the total sample analyzed, 26% (n = 13) of results
were false-positives and 6% (n = 3) were false-
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Onofre, Sposto, and Navarro 537Volume 91, Number 5
Table I. Method of application of the 1% toluidine blue staining technique
Oral examination and annotation of location, size, clinical characteristics, and photographing of the lesion
Cleaning of the lesion with a cotton tip soaked in 10% H 2O2 (for the elimination of saliva, food, or tissue remains)
Cleaning of lesion with water jet
Cleaning of lesion with 1% acetic acid
Cleaning of lesion with water jet
Application of 1% aqueous solution of toluidine blue with cotton tip for 30 seconds
Cleaning of lesion with water jet
Application of 1% acetic acid with cotton tip for 30 seconds (for elimination of excess of stain)
Oral examination and annotation of location and size of the retained stained areas
Photographing of lesion
Table II. Clinical diagnosis of oral lesions and results of staining
Toluidine blue
Clinical diagnosis n (%) +
Homogenous leukoplakia 12 (24) 2 10
Nonhomogenous leukoplakia 20 (40) 10 10
Reticular lichen planus 4 (8) 4
Erosive/ulcerated lichen planus 7 (14) 4 3Superficial ulcerations 7 (14) 7
Total 50 (100) 23 27
Table III. Clinical and histologic diagnosis of oral lesions and results of staining
Clinical diagnosis
Homogenous Nonhomogenous Reticular Erosive/ulcerated Superficial
leukoplakia leukoplakia lichen planus lichen planus ulcerations
(n = 12) (n = 20) (n = 4) (n = 7) (n = 7)
Histologic diagnosis + + + + +
Squamous cell carcinoma* (n = 7)
Carcinoma in situ 1 Invasive carcinoma 2 4
Epithelial dysplasia (n = 6)
Mild 2 1 1
Moderate 1 1
Benign keratosis (n = 13) 4 4 5
Lichen planus (n = 20) 5 2 2 4 4 3
Other benign lesions (n = 4) 1 3
Total (n = 50) 2 10 10 10 4 4 3 7
*Initial invasive carcinoma.3 granular tissues and 1 ulcerated papilloma.
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negatives. The sensitivity of staining was 72% and the
specificity 65%, the positive predictive value was
43.5%, and the negative predictive value was 88.9%.
DISCUSSIONThe sample investigated in this study was composed
of 86% PMELs and 14% superficial oral ulcerations
suggestive of malignancy, differentiating this work
from the majority of previous works, which include in
their samples few PMELs and a large number of clini-
cally suspected malignancies or typically benign
lesions with no risk of malignancy. Individuals without
lesions (control group) were not included in this study,
because performance of biopsy in normal mucosa not
containing stain retention would not be ethical.
SensitivityStudies have demonstrated that toluidine blue has a
high sensitivity in its detection of malignant oral
lesions; values vary from 84% to 100% (Table V). In
this study, staining was seen to be highly efficient in the
detection of in situ carcinoma and invasive carcinomas,having a sensitivity of 100%, because no false-negative
results occurred among the lesions histologically diag-
nosed as carcinomas. Recently, Warnakulasuriya and
Johnson16 confirmed that oral squamous cell carci-
nomas can be detected with a sensitivity of 100% by
using a commercial rinse containing toluidine blue as
the active ingredient. A study developed by Epstein et al17
evaluated the utility of toluidine blue application
in aiding the recognition and diagnosis of clinically
evident lesions in patients previously treated for oral
cancer and undergoing post monitoring. Toluidine blue
stain identified all in situ carcinomas and invasive
malignant lesions, whereas the clinical examination
identified 78% of the in situ carcinomas or invasive
malignant lesions.
Few studies demonstrated false-negative results incarcinomas, and when they occurred frequencies varied
between 0.9% and 5.5% of the total of the sample
analyzed.5,8,10,11,13 Martin et al28 analyzed the sensi-
tivity of toluidine blue in the detection of epithelial
dysplasia and carcinoma areas in situ and in invasions of
both the normal mucosa as well as the altered adjacent
mucosa of 14 specimens of invasive carcinoma. Forty-
two percent of the results obtained for in situ carcinomas
were false-negatives, and 58% were false-negatives for
epithelial dysplasia. These results are considerably
higher than those presented in the majority of studies.
This discrepancy in results is probably due to the sample
analyzed and to the fact that these authors consideredonly lesions with stippled or dark blue staining as posi-
tive, and those lesions that were weakly stained were
considered as negative. This interpretation differs from
that offered by Mashberg,7,10 who suggested that
doubtful light blue stains should be considered as posi-
tive, at least until biopsy proves the contrary.
Among the lesions diagnosed as epithelial dysplasia
in our study (n = 6), 3 (1 mild and 2 moderate) did not
retain stain and were therefore false-negative results.
The number of cases of epithelial dysplasia was small
in this study, and therefore statistical tests could not be
applied. According to Warnakulasuriya and Johnson,16
false-negative results may occur as a result of a lack of
objective criteria for the evaluation of stain uptake.
Moreover, the exact mechanisms by which the dye
differentially stains malignant or dysplastic tissues
remains unknown. Mashberg10 reported that lesions
with limited dysplasia or atypia did not intensely retain
the stain. Warnakulasuriya and Johnson16 obtained
false-negative results in 26% of the lesions with histo-
logic diagnoses of dysplasia and agreed with the find-
ings of Mashberg.10 Thus, in PMELs some areas of
mild or moderate epithelial dysplasias may not be
detected by staining alone. The use of toluidine blue,
however, should not be excluded, because all in situcarcinomas and invasive carcinomas retain toluidine
blue stain.
SpecificityPrevious studies demonstrated a great variation in the
specificity of staining by toluidine blue of between 44%
and 100% (Table V); this is due to the diversity of lesions
included in the different studies. The lowest specificity
found, 44%, was obtained by us in a previous study.15
538 Onofre, Sposto, and Navarro ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGYMay 2001
Table IV. Histologic diagnosis of oral lesions, resultsof staining, sensitivity, specificity, and positive and
negative predictive values
Toluidine blue
n (%) +
Histologic diagnosisSquamous cell carcinoma 7 (14) 7
Epithelial dysplasia 6 (12) 3 3
Lichen planus 20 (40) 6 14
Keratosis 13 (26) 4 9
Other benign lesions 4 (8) 3 1
Total 50 (100) 23 27
Sensitivity
Carcinoma 7/7 (100)
Carcinoma in situ 1/1 (100)
Superficial invasive
carcinoma 6/6 (100)
Epithelial dysplasia 3/6 (50)
Mild dysplasia 3/4 (75)
Moderate dysplasia 0/2 (0)
Specificity 24/37 (67)
Positive predict ive value 10/23 (43.5)
Negative predictive value 24/27 (88.9)
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This result occurred because we considered in somecases the staining results of the first visit and not the
result of the day of the biopsy 7 to 14 days after the first
consultation. This caused a large number of false-positive
results, generally caused by the retention of stain in
inflammation and trauma areas. To decrease the number
of false-positive results and consequently increase the
specificity of staining, Mashberg8,10 recommends that all
irritating and inflammatory factors should be eliminated
in the lesions that retain stain. These lesions should be
reevaluated and stained after 10 to 14 days, and if the
stain is again retained, they should be considered as
suspicious of carcinoma. For practical reasons some
studies often do not follow this recommendation, conse-
quently increasing the number of false-positives and
reducing the specificity of staining. In this study the
staining specificity was increased to 65% because we
followed the recommendations of Mashberg,8,10 proving
that the variety of results presented in the literature
depends on the methodology used and the sample
analyzed. Our results demonstrated that in the lesions
without epithelial dysplasia or atypical cells, the level of
false-positive results was 35% (n = 13); 6 cases occurred
among the nonhomogenous leukoplakias, 4 among the
erosive/ulcerated lichen planus, and 3 among the superfi-
cial ulcerations suspicious of malignancy. Thus, 100% ofthe false-positive results occurred in lesions with ulcera-
tion or erythema. This result was higher than that
presented by Silverman et al11; they observed that 70% of
lesions with false-positive results presented these charac-
teristics. The higher level of false-positive results may
increase the number of biopsies performed in PMELs
and superficial ulcerations suspicious of malignancy.
This may be beneficial because all PMELs and superfi-
cial ulcerations suspicious of malignancies that do not
respond to treatment should be submitted to microscopicanalysis.22 In addition, the diagnosis of PMELs, based
only on clinical appearance, may lead to a misdiagnosis
and therapeutic errors.19
In this study the high level of false-positive results
led to a reduced positive predictive value. Results
showed that the lesions with retained stain demonstrate
a 43.4% probability of having areas with epithelial
dysplasia, in situ carcinoma, or invasive carcinoma.
The negative predictive value, however, was high, indi-
cating that lesions that do not retain stain demonstrate
an 88.9% probability of not having areas of epithelial
dysplasia or atypical cells. If the cases of carcinoma
only are evaluated this probability is 100%, because
false-negative results did not occur in these lesions.
Another important result to be emphasized is the
high prevalence, 26%, of epithelial dysplasia and squa-
mous cell carcinoma found in this study. Among the
PMELs, the prevalence was 20.9%, occurring predom-
inantly among the leukoplakias, a result similar to that
observed by us in a previous study19 in which the
prevalence found was 17.8%, confirming the necessity
of performing microscopic analysis in all the PMELs.
We concluded that staining with toluidine blue is
highly reliable for the detection of in situ carcinoma
and invasive carcinoma. From our point of view, stainingwith toluidine blue is an adjunct to clinical judgment
and not a substitute for either judgment or biopsy.
Staining should be routinely used as a method to assist
in the choice of biopsy sites and in the follow-up of
PMELs. We believe that toluidine blue serves the
important purpose of accelerating biopsy, particularly
in persistent lesions, and allows the selection of areas
of the lesion more likely to be demonstrated as malig-
nancies or dysplasias. In addition, our clinical experi-
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Onofre, Sposto, and Navarro 539Volume 91, Number 5
Table V. Studies evaluating the application of toluidine blue in oral lesions
Location of No. of lesions Prevalence of Sensitivity Specificity
Study study analyzed carcinoma/dysplasia (%) (%) (%)
Niebel and Chomet1 VH/USA 11 45/55 100 100
Shedd et al2 OM/USA 42 69/3 100 75
Myers4 CH/USA 70 72 100 100
Vahidy et al5 CH/Pakistan 1190 40 86 76Reddy et al6 OM/India 490 92 99 88
Mashberg8 VH/USA 105 49/3 96 95
Mashberg10 VH/USA 179 45/5 90 91
Silverman et al11 OM/USA 132 43/32 98 90
Epstein et al13 CH/Canada 59 41/25 92 63
Onofre et al15 OM/Brazil 44 18/9 92 44
Warnakulasuriya and Johnson16 OM/Sri Lanka 86 21/45 86 62
Epstein et al17 CH/Canada 81 27/28 100 52
Onofre et al OM/Brazil 50 14/12 77 65
The prevalence of carcinoma/dysplasia, the sensitivity, and the specificity were recalculated according to the method proposed by Rosenberg and Cretin.12
VH, Veterans hospital; OM, oral medicine service; CH, cancer hospital.
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ence has shown that the examiner should be experi-
enced in the practice of oral medicine, particularly in
the diagnosis and treatment of precancer and oral squa-
mous cell carcinomas, and should be trained to inter-
pret exactly the results of toluidine blue staining.
We are indebted to the Mario A.S. Paino Laboratory of
Clinical Pathology.
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Reprint requests:
Dr Mirian A. OnofreDepartamento de Diagnstico e CirurgiaFaculdade de Odontologia de Araraquara - UNESPRua Humait, 1680Araraquara - So PauloBrazilmaonofre@foar.unesp.br
540 Onofre, Sposto, and Navarro ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGYMay 2001