Staining Techniques:
Screening Tests for Oral Cancer
JamesT. Helsper, M.D.
Study of the patient's saliva after
irrigation of the mouth with Gey's
balanced salt solution may be a useful
screening test for squamous cell or
epidermoid carcinoma in the oral cavity.
Likewise, the topical application of
toluidine blue, an acidophilic meta
chromatic nuclear stain, helps differ
entiate areas of carcinoma in situ or
invasive carcinoma from normal
tissue. However, these two procedures
—¿themouthwash technique and the
toluidine blue test—while helpful, are
nota substitute for biopsy and a
negative smear does not preclude the
presence of cancer.
The Mouthwash Technique
A large majority of oral neoplasms
are squamous cell or epidenmoid
carcinomas. Since these tumors con
tinually exfoliate malignant cells, it
was felt that examination of saliva
specimens taken from the entire oral
cavity following irrigation with some
form of mouthwash might be an effective
screening test for oral cancer.
Dr. Helsper is Associate Clinical Professor of
Surgery, University of Southern California School
of Medicine, Pasadena Tumor Institute, Pasadena,
California.
Dr. Eugene N. Myers, Department of Otolaryn
gology, Hospital of the University of Pennsylvania.
Philadelphia, Pennsylvania, has kindly supplied
Figures 3-7 to accompany the section on the
toluidine blue test.
172
Numerous mouthwash solutions have
been tested: Normal saline solution,
which is not truly osmotic, was found
unsatisfactory for oral irrigation; cells
suspended in this preparation generally
absorbed the fluid and burst within a few
minutes, making cytologic evaluation
impossible.' Then, at the suggestion of
the late Dr. Charles M. Pomerat.
Research Director of the Pasadena
Foundation for Medical Research, Gey's
balanced salt solution—a simple
culture fluid containing various amounts
of salts normally found in most cells
was tested and found to be a successful
mouthwash inrigant. (Table I.)
Although a direct biopsy is the most
accurate means of diagnosing a
Table 1—Gey's Balanced Salt Solution
Gm per Liter
NaCI 8.000
KCI 0.375
CaCl2 0.275
MgCl2.6H20 0.210
Na2HPO4.2H2O 0.150
KH2PO4 0.025
Glucose 2.000
NaHCO3 1.250
Dist. H2Oq.s. ad 1000.
Add 2.5 ml of 0.5 percent phenol red to this one
liter volume. Adjust the pH with NaOH to an
orange-pink, and use only solutions of this color.
Note: Solution must be prepared by adding the salts
in the order shown. Also, it may not be boiled
since a calcium precipitate will appear.
 -

‘¿V

‘¿-a

@ i.-.
Figs I and 2. Characteristic malignant cells as shown by the mouthwash technique.

suspicious oral lesion, the mouthwash difficult to recognize. The leukocytes.

technique has proved valuable in two plasma cells and red cells seen so
specific situations: (I) in the diagnosis prominently in specimens from oral
of patients with extensive leukoplakia scrapings are less common in mouth
who have negative biopsies. and (2) wash smears.
in the follow-up of patients after Although epidermoid carcinomas as
surgical or radiation therapy for oral small as 3mm have been detected by
cancer. Experience at two dental schools the mouthwash technique, only about an
and a large county hospital suggests 85 percent yield has been obtained from

that this method may also be useful as
a screening technique in asymptomatic
patients ,2 However, if malignant cells
are seen in a specimen. further at
tempts to discover the area of malignant
change, such as repeated careful
examinations of the oral cavity and
multiple biopsies, must, of course, be
carried out.
The technique is simple: have the
patient vigorously irrigate his mouth for
60 seconds with one ounce of Gey's
Solution at room temperature. A speci
men with a cell population typical
of the entire oral cavity is obtained,
placed in a large-mouthed jar and
refrigerated to prevent growth of
organisms. Mix the specimen with
equal parts of 95 percent alcohol.
Spin it down in a centrifuge and smear
and stain the specimen in the usual
Papanicolaou method.
Malignant cells will usually appear
singly—not in clumps—and present
little problem in identification. (Figs. I
and 2.) Occasionally, however, more
anaplastic and small cell types may be
patients with known oral cancer; there
is no accounting for those instances in
which carcinomas were missed.
However, despite the fact that the study
of smears is somewhat time-consuming
and requires an experienced examiner.
this technique has proved useful in the
diagnosis of patients with diffuse oral
leukoplakia in whom previous biopsies
have been negative and in the follow-up
of patients previously treated with
surgery or radiation.
The Toluidine Blue Test
The topical application of toluidine
blue, yet another technique in the
diagnosis of oral cancer, will stain an
area of carcinoma in situ or invasive
carcinoma. Toluidine blue will not
stain normal mucosa. Earlier reports by
Shedd and Strong demonstrated that this
test can detect squamous cell and

173
 I

Fig. 3. Exophytic squamous cancer of the tongue. Fig. 5. Sharplydemarcated squamous cell carcinoma
in the soft palate stains vividly.

Fig. 4. Ulceratedsquamous cancer of the tongue Fig. 6. Deeply ulcerated malignant melanoma.
stains vividly while the surrounding mucosa Surrounding normal mucosa does not stain.
covered malignant tissue does not stain.

Fig. 7. A large, ulcerated squamous cancer of the

floor of the mouth with two small satellite areas
posterior to the main tumor.

174
epidermoid carcinoma (Figs. 3,4, and 5)
as well as the usual benign oral lesions,
such as leukoplakia, lymphoid hyper
plasia, lichen planus and traumatic
ulcerations.35 Myers has demon
strated that toluidine blue can also
identify melanoma, fibnosarcoma and
lymphosarcoma, in addition to epider
moid cancer.6 (Fig. 6.)
In large, advanced cancers of the oral
cavity, inspection and biopsy will
generally provide sufficient information
for diagnosis. However, toluidine blue
may be indicated to diagnose leuko
plakic lesions or dysplasia; differ
entiate a traumatic or inflammatory
ulcer from cancer; determine the
margins of resection prior to excision;
and demonstrate a small second primary
or satellite lesion adjacent to a larger
lesion. (Fig. 7.) However, since
toluidine blue does not stain normal
mucosa, it is not of value in the diag
nosis of tumors which spread without
involving the overlying mucous
membrane.
To perform this test, have the
patient first rinse his mouth with water
and then swallow several sips of water.
Aspirate excess saliva with suction
and apply one percent acetic acid, a
mucolytic agent, with a cotton ap
plicator. If there is a large deposit of
fibnin or debris in an ulcer, also remove
this by suction. Next, place a small
amount of one percent toluidine blue
on the entire lesion as well as on
some of the surrounding oral mucosa.
Instruct the patient to rinse his mouth
with water thus washing away the
excess toluidine blue. If the lesion is
stained, the test is positive; biopsy im
mediately. Of course, you may also
biopsy many lesions which do not
take up the stain in order to document
their precise nature. Keep those negative
lesions which are not biopsied under
close supervision.
The efficacy of this procedure has
been tested in several series: Myers
demonstrates no false positives in his
study of 70 patients,6 although false
positives have been reported by other
investigators.3@4'7 Even though such
results are controversial, toluidine is,
nevertheless, a valuable preliminary
screening technique.
References
I. Helsper, J. 1.; Sharp, G. S., and Bullock, W. K.:
The mouthwash technic. A method of screening
for intra-oral carcinoma. Amer. J. Surg. 106:
802-806, 1963.
2. Helsper, J. 1., Sharp, G. S., and Bullock, W. K.:
Experiences with the tissue culture solution mouth
wash method for the early detection of oral cancer.
Acta.Cytol. 10:179-181,1966.
3. Shedd, D. P.; Hukill, P. B., and Bahn, S.:
In vivo staining properties of oral cancer. Amer.
J. Sung. 110:631-634, 1965.
4. Shedd, D. P.. etal.: Further appraisal of in vivo
staining properties of oral cancer. Arch. Sung.
(Chicago)95: 16-22, 1967.
5. Strong, M. S.; Vaughan. C. W., and Incze,J. S.:
Toluidine blue in the management of carcinoma of
the oral cavity. Arch. Otolaryng. (Chicago) 87: 527-
531, 1968.
6. Myers. E. N.: The toluidine blue test in lesions
of the oral cavity. Ca 20: 134-139, 1970.
7. Rosen, I. B.; Cornish, M., and Edelson, J.:
Detection of early oral cancer by toluidine blue.
J. Can. Dent. Ass. 37: 347-349, 1971.
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