oral medicine

Editor:
JAMES W. LITTLE, D.M.D., M.S.D.
School of Dentistry
University of Minnesota
515 S.E. Delaware St.
Minneapolis, Minn. 55455

Toluidine blue staining in the detection of

oral precancerous and malignant lesions
S. Silverman, Jr., M.A., D.D.S.,* C. Migliorati, D.D.S.,** and J. Barbosa, D.D.S.,***
San Francisco, Calif

UNIVERSITY OF CALIFORNIA AT SAN FRANCISCO

One hundred thirty-two consecutive patients suspected of having malignant or precancerous oral lesions
were studied by comparing toluidine blue dye uptake clinically with a simultaneous biopsy. The
histopathologic diagnosis confirmed 57 squamous carcinomas, 42 epithelial dysplasias, and 33 benign
mucosal changes. Overall accuracy of the toluidine blue uptake was 91%. In the dysplastic and
malignant lesions the false negatives were 2%, and there were 30% false positives in the benign
lesions. It was concluded that toluidine blue staining is a useful adjunct to careful examination,
clinical judgment, and biopsy. (ORAL SWIG. 57:379-382, 1984.)

C linical identification of epithelial dysplasia and
early squamous carcinoma is often difficult because
of the varied appearances which may confuse them
with a number of similar-appearing benign lesions.
In addition, biopsies are frequently delayed because
of this uncertainty and becauseof attempts at initial
empirical remedies. It has been shown that vital
staining techniques often help in accelerating biop-
sies, diagnoses, and treatments.le4 Therefore, tech-
niques adjunctive to clinical judgments and micro-
scopic diagnoses have contributed to oral cancer
control.
Toluidine blue is a metachromatic dye of the
thiazine group that has been used effectively as a
nuclear stain because of its binding with DNA. In
vivo it has been associatedwith the early recognition
of asymptomatic oral squamous carcinomas.5-9How-
ever, questions have arisen relative to its accuracy
and thus its clinical usefulness.‘o
*Professor of Oral Medicine.
**Postdoctoral Scholar, Oral Medicine.
***Visiting Professor of Oral Medicine.
The purpose of this study was to assess the
usefulness of toluidine blue stain in a series of
patients with oral lesions suspectedof being precan-
cerous or malignant by comparing clinical impres-
sions, microscopic diagnoses, and staining reac-
tions.
MATERIALS AND METHODS
The study group comprised 132 consecutive
patients seen in the oral medicine clinic who were
suspectedof having oral carcinomas or precancerous
(dysplastic) lesions. Each patient was given a com-
plete examination, and a differential diagnosis was
formed. A biopsy was planned, since neoplastic
changes were included in the differential diagnoses.
For staining, a 1% aqueous toluidine blue dye was
applied for approximately 30 seconds,followed by a
tap water rinse, and then lightly blotted with 1%
acetic acid. Both solutions were applied with cotton-
tipped applicators. Any dye uptake was recorded by
photographs. If there was dye uptake, the biopsy
specimen was taken from that area; otherwise, clini-
cal judgment guided the biopsy site. The biopsy
379
380 Silverman, Migliorati, and Barbosa Oral Surg.
April. 1984

Fig. 1. A, This 43-year-old man had an “irritation” of the mouth floor for several months. Note the mild
keratotic change, which was soft to palpation and nonulcerated (arrow). B, Application of 1% aqueous
toluidine blue dye. C, Note the residual area of tissue affinity for the dye after decolorization with 1% acetic
acid. D, A biopsy specimen from the dye-positive area revealed squamous carcinoma

Table 1. Toulidine blue efficacy comparing staining Table II. Clinical findings, microscopic diagnoses,
reaction and microscopic diagnosis and toluidine blue staining in thirty-three benign
lesions
Toluidine
Principal blue uptake
Benign 33 (25%) 10 (30%) 30% false positive clinical (‘alse
Dysplasia 42 (32%) 42 (100%) None appearance Microscopic diagnosis positives)
Carcinoma 57 (43%) 55 (96%) 4% false negative
Total 132 91% 7% Keratosis 20 Hyperkeratosis 2 (10%)
Keratosis 1 Lichen planus 0
Keratosis and 2 Hyperkeratosis I (50%)
erythema
specimenswere fixed in 10% neutral buffered forma- Ulceration and 7 Nonspecific ulceration 6 (90%)
lin and sent to the oral pathology laboratory for erythema and inflammation
routine processing. Ulceration and -3 Erosive lichen planus 1 (33%)
erythema
RESULTS 33 10 (30%)

Table I compares the microscopic diagnosis with

toluidine blue uptake in the 132 patients. Reactivity
was accurate in 97 of 99 dysplastic and malignant
lesions and 23 of 33 benign lesion, for an overall
efficacy of 91% (Figs. 1 and 2). The majority of
errors involved false positives in the group of benign
lesions. Table II compares the microscopic diagnoses
with clinical appearance and dye reactivity in the 33
benign lesions. Areas clinically appearing as ulcer-
ation and erythema constituted 70% of the lesions
that involved false-positive dye uptake (Fig. 3).
DISCUSSloON
The usefulnessand reliability of toluidine blue dye
binding in tissues that have undergone malignant or
dysplastic changes have been demonstrated. The
overall 91% accuracy of the toluidine blue technique
supports its clinical usage to further confirm clinical
impressions regarding tissues at risk of malignancy,
as well as to help the clinician choose biopsy sites.
Since most, if not all, dysplastic and malignant tissue
Volume57 Toluidine blue staining of oral lesions 381
Number4

Fig. 2. A, This M-year-old woman had a 2-year history of mildly uncomfortable erythroleukoplakia (red
and white lesions) of the lateroventral tongue. B, Note the positive areaof irregular dye retention after the
application of 1% toluidine blue and decolorization with 1% acetic acid. C, A biopsy specimen from the
dye-retention area showed superficially invasive squamous carcinoma.

Fig. 3. A, This patient experienced severe pain associated with postradiation necrosis of the tongue. He
had been treated 1 year previously for a squamous carcinoma at this site with external radiation and an
interstitial radium needle implant. B, Two well-circumscribed false-positive areas of toluidine blue uptake
remained after dye application and decolorization. The patient has remained tumor free throughout a 2-year
follow-up.

present in the mucosal surface will stain blue, the dye
uptake also assisted in evaluation by marking lesion
margins. In addition, toluidine blue binding in oral
epithelium did not interfere with histologic staining
or interpretation.
Alternatively, toluidine blue rinses have been rec-
ommended for screening high-risk patients who may
have asymptomatic malignant lesions of the oral
cavity. ‘I Our experience has demonstrated that clini-
cal judgment based on careful oral examination is
the most critical and important procedure, with
toluidine blue staining used as a support measure.
This study was not designed to determine the
accuracy of the toluidine blue technique as a general
382 Silverman, Migliorati, and Barbosa Oral Surg
April. 1983

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staining was seen as a blue reaction outlining cyto- Reprint requests to.

plasmic and nuclear borders but only in the outer- Dr. S. Silverman, Jr.
Department of Oral Medicine
most two to three cell surface layers. One study University of California San Francisco
suggestedin vitro toluidine blue staining as a power- San Francisco. CA 94143
ful method aiding studies of the glycocalyx of eryth-
rocytes and other cells.‘5
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