Download Original Article Effective staining method with iodine for leukoplakia

J Med Dent Sci 2009； 56： 123－130
Original Article
Effective staining method with iodine for leukoplakia and lesions surrounding
squamous cell carcinomas of the tongue assessed by colorimetric analysis
Keiko Maeda1, Masashi Yamashiro1, Yasuyuki Michi1, Tetsuo Suzuki1, Yoshio Ohyama1,
Norihiko Okada2, and Teruo Amagasa1,
1) Maxillofacial Surgery, Maxillofacial Reconstruction and Function, Division of Maxillofacial and Neck
Reconstruction, Graduate School, Tokyo Medical and Dental University, Japan
2) Diagnostic Oral Pathology, Oral Restitution, Oral Health Science, Graduate School, Tokyo Medical and
Dental University, Tokyo, Japan
To determine whether staining with iodine solution
provides an efficient criterion for determining
the area of resection for the lesions surrounding
squamous cell carcinoma (SCC) and leukoplakia
of the tongue, we determined the optimum density
of iodine solution and staining procedure and
analyzed the color of lightly stained lesions (LSLs)
in relation to the histopathologic findings. Sixtyfive patients with SCC or leukoplakia of the tongue
were divided into two groups: lesions stained
with 3% Lugol solution and restained with either
5% Lugol solution (n=38) or 10% iodine glycerin
(n=27). Among the lesions stained with 5% Lugol
solution, significant differences were found in all
color values. Color difference values (ΔE *ab) using
3% and 5% Lugol solutions were significantly
different between epithelial hyperplasia/mild
epithelial dysplasia and moderate to severe
dysplasia (P < 0.05). According to the evaluations
of five clinicians in 46 LSLs, a distinctive
boundary was most often obtained using 5% Lugol
solution. These results suggest that the most
effective method for obtaining a clear boundary
and distinguishing moderate to severe dysplasia
from mild or no epithelial dysplasia according to
Corresponding Author：Keiko Maeda
Maxillofacial Surgery, Maxillofacial Reconstruction and Function,
Division of Maxillofacial and Neck Reconstruction, Graduate School,
Tokyo Medical and Dental University, Tokyo, Japan
1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
TEL：+81 (3) 5803 5500 FAX：+81 (3) 5803 0298
E-mail：[email protected]
Received May 7；Accepted September 18, 2009
the measured color value was to stain with 3%
followed by 5% Lugol solution.
Key words: squamous cell carcinoma of the tongue;
leukoplakia of the tongue; vital iodine
staining; iodine lightly stained lesions; color
measurement
Introduction
Vital iodine staining, known as the Schiller iodine
test, was first performed to detect early cervical
cancer in the 1930s, and this technique can be used
immediately before resection as a useful and minimally
invasive test to determine the area of resection for
esophageal cancer 1,2. Many studies have also reported
the usefulness of iodine staining of oral lesions using
current detection techniques such as narrow band
imaging (NBI) and magnifying endoscopy, which are
routinely used by clinicians 3-6 . However, there is
still no consensus on the optimal density or staining
sequence of iodine solutions for oral lesions．In this
study, we performed vital iodine staining with 3%
Lugol solution followed by either 5% Lugol solution or
10% iodine glycerin and determined the density and
staining sequence of iodine solutions that provided the
clearest boundary for lightly stained lesions (LSLs) in
order to more clearly determine the area of resection
of squamous cell carcinoma (SCC) with the surrounding
lesions or leukoplakia of the tongue. The color and
shape of the LSLs were often irregular as viewed
subjectively by each operator; however, our method
could distinguish each histopathologic group of LSLs
using differences in the objective values of lightness
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K.Maeda et al.
J Med Dent Sci
Figure 1 : A : A case showing staining with 3% and 5% Lugol solutions (SCC, T2N0M0)
(a) Before staining. Lesions that were clinically deemed cancerous were marked with a dotted line using methylrosanilium
chloride solution (Pyoktanin Blue®), (b) stained with 3% Lugol solution, (c) stained with 5% Lugol solution. The histopathologic
diagnosis of the lightly stained lesions was mild epithelial dysplasia (lesions marked with*).
B: A case showing staining with 3% Lugol solution and 10% iodine glycerin (SCC, T2N0M0)
(a) Before staining, (b) stained with 3% Lugol solution, (c) stained with 10% iodine glycerin
The histopathologic diagnosis of the lightly stained lesions was moderate epithelial dysplasia (lesions marked with*).
(L *) and hue (a *, b *). Here, we evaluated the color of
LSLs by colorimetry and determined the relationships
between the color of LSLs and the histopathologic
diagnosis, density of iodine solution, and sequence
of staining. The values obtained were compared with
those derived by subjective clinical judgment.
Patients and methods
Patients
Sixty-five patients (31 men and 34 women) who had
conditions diagnosed as SCC (n=45) or leukoplakia of
the tongue (n=20) and who underwent biopsy under
local anesthesia or surgery under general anesthesia
at the Maxillofacial Surgery, Tokyo Medical and
Dental University Hospital Faculty of Dentistry (Tokyo,
Japan), from July 2007 to April 2009 were included
in this study. This study was approved by the Ethics
Review Board of the Faculty of Dentistry (No. 277).
Details of the study were explained to the patients,
and informed consent was obtained prior to the start
of the investigation. The mean age of the patients was
59.2 years (range: 25 - 83 years). The selection criteria
were as follows: patients with SCC whose lesions were
classified as T1 (n=23) or T2 (n=22); patients with
leukoplakia who underwent biopsy for definite diagnosis
or were selected for surgical treatment; and patients
whose lesions could be measured and illuminated by
an artificial sunlight lamp. The patients were divided
into two groups: one group underwent staining of the
lesions with 3% followed by 5% Lugol solution (n=38),
and the other group underwent staining using 3% Lugol
solution followed by 10% iodine glycerin (IG) (n=27).
Staining solutions and procedures
Solutions of 3% Lugol, 5% Lugol, and 10% IG were
used for staining according to the following procedure.
First, the untreated lesion was photographed, and a
colorimeter (CS-100, KONICA MINOLTA HOLDINGS,
INC., Tokyo, Japan) was calibrated in the oral cavity.
Then, 3% Lugol solution was applied, and the area
was rinsed with water and dried with gauze. Color
measurement was performed after 2 minutes, then
the LSLs were photographed and sodium thiosulfate
was applied, and restaining with 5% Lugol or 10%
IG was performed using the same steps (Fig. 1). The
Effective iodine staining method for oral lesions
125
Figure 2 : (a) Before staining (SCC, T2N0M0).
(b) Stained with 5% Lugol solution. The color measuring point is indicated by *.
(c) Before resection. The incision line and boundary line between SLs and LSLs are indicated as a solid line and a
dotted line using methylrosanilium chloride solution (Pyoktanin Blue®), respectively. The color measuring point is
indicated by *.
(d) Re-stained specimen with 5% Lugol solution. The boundary line after resection corresponded to the dotted line
marked before resection. A shallow incision (yellow line) was made with a scalpel into the boundary line between
LSLs and SLs, and the areas to be measured were marked with a pin (white point).
(e) Specimen fixed in 10% buffered formalin. Sections of the specimen were made through the shallow incision line
(arrow) and pin hole (white dotted line).
(f) Arrow indicates the shallow incision line in the specimen showing the macroscopical boundary line between SLs
and LSLs (H-E staining × 40). Left side of the incised line shows LSLs.
boundary line between stained lesions (SLs) and LSLs
after staining was marked with a dotted line using
methylrosanilium chloride solution (Pyoktanin Blue ®)
(Fig. 2abc)
Color measurement
Color was measured using CS-100 according to the
following method described by Yamashiro7: an artificial
sunlight lamp (SOLAX XC-100, SERIC LTD., Tokyo,
Japan) was used to illuminate the lesion at a 45 °
angle and a distance of 1 m, and color was measured
in the vertical direction. A close-up lens No. 122
(measurement diameter: 3.2 - 4.3 mm) was attached
to the colorimeter, and color was measured within the
wavelength range of 323 - 368 mm. Numerical values
were recorded using the data processor DP-101
(KONICA MINOLTA HOLDINGS, INC.). Areas for color
measurement in LSLs were determined on the basis of
the following criteria: 1) lesions deemed to surround
tumorous lesions or leukoplakia clinically; 2) the size of
the LSL was more than 5 mm (measurement diameters
of close-up lens: 3.2-4.3 mm); and 3) areas within
approximately 10 mm from the clearer boundary line
between the LSLs and SLs. Moreover, when the LSLs
were small, one area was selected as the measuring
points, whereas when the LSLs were large, several
lesions were selected. In each test, five measurements
were taken to eliminate marked variations, and an
average value was calculated. For the light source, D55
was used and data were calculated in the Y, x, y mode.
Using a dental mirror-type standard white board, an
arbitrary standard calibration must be carried out for
each measurement under the same conditions as those
used for the test areas.
The CIE L*a*b* color system was used to describe the
developed color, as recommended by the Commission
Internationale dl’Eclairage in 1976. In this system, L*
indicates lightness, and a* and b* indicate hue. The
following formulas are used for converting Y, x, y into
L*, a*, b * 8:
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K.Maeda et al.
L* = 116 (Y/Yn)1/3 - 16
a* = 500 [(X/Xn)1/3 - (Y/Yn)1/3 ]
b* = 200 [(Y/Yn)1/3 - (Z/Zn)1/3 ]
Color differences refer to the quantitative differences
in color perception and were calculated using the
following formula:
ΔE* ab = [(ΔL *)2 + (Δa* )2 + (Δb* )2 ]1/2
Tissue processing and evaluation
Resected specimens were re-stained, and it was
confirmed that the boundary line between SLs and
LSLs after resection corresponded to the dotted
line marked before resection using methylrosanilium
chloride solution (Pyoktanin Blue®) (Fig. 2d). A shallow
incision was made with a scalpel into the boundary line
between LSLs and SLs and the areas to be measured
were marked with a pin and then the specimens were
fixed in 10% buffered formalin. Tissue sections (4 μ m
thick) were prepared and stained with H-E by routine
methods. Sections of the specimens were made through
the shallow incision line and pin hole (Fig. 2e), and all of
the final histopathologic diagnoses were made on the
basis of these stained sections (Fig. 2f).
For histopathologic evaluation of LSLs, SCC and
epithelial dysplasia were classified as mild, moderate, or
severe according to World Health Organization (WHO)
standards 9, and if atypical findings were not observed,
those with hyperkeratosis and acanthosis of the simple
elongated epithelial layer were grouped into epithelial
hyperplasia (HYP). When there were measured areas
of LSLs that differed from the histopathologic diagnosis,
the more severe histopathologic diagnosis of the lesion
was taken as the final diagnosis.
Clinical evaluation and statistical analysis
Clinical staging for cancer of the tongue was
performed according to the International Union Against
Cancer 10, and leukoplakia was classified according
to WHO criteria 9. Clinical evaluations as to the clarity
of the boundary line between SLs and LSLs were
scored as follows: Category Ⅰ, unclear boundary line;
Category Ⅱ, partially clear boundary line; Category
Ⅲ, clear boundary line all around the lesion. When the
clinical evaluations did not coincide among three or
more of five clinicians, the clarity of the boundary line
was classified as “None”. Scores that coincided among
three or more of five clinicians’ evaluations were used
as the average score, and an evaluation-concordance
ratio (%) was defined as the average evaluation-
J Med Dent Sci
concordance over four of five clinicians’ evaluations in
this study.
Color difference (ΔE* ab) was assessed on the basis of
National Bureau of Standards (NBS) units, as reported
by Koksal 11 and Seghi 12. The criteria for categorization
were as follows: trace (0 - 0.5), slight (0.5 - 1.5),
noticeable (1.5 - 3.0), appreciable (3.0 - 6.0), strong
(6.0 - 12.0), and very strong (> 12.0). It was possible
for five oral surgeons having more than 10 years
of clinical experience to evaluate the clarity of the
boundary lines with digital photography in 46 cases (5%
Lugol solution: 26 cases; 10% IG: 20 cases). For the
statistical analysis, the cases were divided into three
groups according to the different densities of iodine
solution: 3% Lugol, 5% Lugol, and 10% IG. Data were
analyzed using SPSS 16.0 J statistical software (SPSS
Inc., Chicago, IL, USA), and values were expressed
as mean ± standard deviation. P -values of the color
values (L*, a*, b* ) and color differences (ΔE* ab) are from
Dunnett’s multiple comparison test. Color values were
compared between SLs and each histopathologic group
of LSLs, and color differences were compared between
moderate to severe epithelial dysplasia (MOD/SED) and
HYP or mild epithelial dysplasia (MLD). Fisher’s exact
test was used for analysis of clinical evaluation of the
clarity of the boundary line of the LSLs. The level of
significance was set at P < 0.05 (two-sided).
Results
Classification of LSLs
LSLs were observed in 57 of 65 patients (87.7%) using
3% Lugol solution alone. The histopathologic diagnoses
for the 57 measured areas with detected LSLs were
HYP in 7 patients (12.3%), MLD in 16 patients (28.1%),
and MOD/SED in 34 patients (59.6%). LSLs were not
observed in 8 patients; the histopathologic diagnoses
for these 8 patients were SCC without surrounding
lesions in 6 patients, leukoplakia and ulcer in 1 patient
each. When staining with 5% after 3% Lugol solution,
LSLs were observed in 35 of 38 patients (92.1%). The
histopathologic diagnoses for the 35 patients with
detected LSLs were HYP in 4 patients (11.4%), MLD
in 11 patients (31.4%), and MOD/SED in 20 patients
(57.1%). LSLs were not observed in 3 patients, and
the histopathologic diagnosis was SCC in all three.
When staining with 10% IG after 3% Lugol solution,
LSLs were observed in 26 of 27 patients (96.3%). The
histopathologic diagnoses for the 26 patients with
detected LSLs were HYP in 3 patients (11.5%), MLD
in 7 patients (26.9%), and MOD/SED in 16 patients
Effective iodine staining method for oral lesions
127
Table 1. Color values and differences after staining
(61.5%). LSLs were not observed in 1 patient and the
histopathologic diagnosis was SCC.
LSLs color values
The results of the color values and differences in the
colors of the LSLs are summarized in Table 1. P -values
of the color values (L*, a*, b* ) and color differences
(ΔE* ab) are from Dunnett’s multiple comparison test.
Color values were compared between SLs and each
histopathologic group of LSLs, and the color differences
(ΔE* ab) were compared between MOD/SED and HYP
or MLD. After staining with 3% Lugol solution, the L*
(lightness) values in each histopathologic group of
LSLs and the b* value in the MOD/SED group showed
significant differences compared with the SLs (P <
0.05). When staining using 5% after 3% Lugol solution,
all color values (L*, a*, b* ) showed a significant
difference between SLs and each histopathologic group
of LSLs, whereas when staining using 10% IG after
3% Lugol solution, there was no significant difference
except in the L* value between the MOD/SED group of
LSLs and SLs.
The mean values of color difference (ΔE* ab) between
SLs and HYP or MLD using 3% Lugol solution were
6.39 and 8.86, respectively, and that between SLs
and HYP using 5% after 3% Lugol solution was 11.36,
and these values were assessed as “strong” according
to the NBS units. Other color difference values were
>more than 12.0 and were assessed as “very strong”.
When using the 5% after 3% Lugol solutions, there were
significant differences between the MOD/SED and MLD
groups (P = 0.001 and P = 0.042, respectively) and the
HYP group (P = 0.002 and P = 0.036, respectively),
whereas with 10% IG after 3% Lugol solution, there
was no significant difference between the MOD/SED
and MLD groups (P = 0.818) or between the MOD/SED
and HYP groups (P = 0.328).
Clinical evaluation of LSLs
The clinical evaluations of the clarity of the boundary
lines between LSLs and SLs were scored as follows:
with 3% Lugol solution there were 5 cases (10.9%)
in category Ⅰ, 12 cases (26.1%) in category Ⅱ, 23
cases (50.0%) in category Ⅲ, and 6 cases (13.0%)
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K.Maeda et al.
on which the clinicians could not agree. With the 5%
Lugol solution, 2 cases (7.7 %) were in category Ⅰ, 3
cases (11.5%) in category Ⅱ, and 21 cases (80.8%) in
category Ⅲ. After staining with 10% IG, 1 case (5.0%)
was in category Ⅰ, 2 cases (10.0%) in category Ⅱ,
and 17 cases (85.0%) in category Ⅲ. When staining
with 3% Lugol solution followed by 5% Lugol solution
or 10% IG was compared with staining with 3% Lugol
solution, the clarity of the boundary line in category Ⅲ
was increased in 34.6% (9/26) and 30.0% (6/20) of the
cases, respectively, after the second staining (Table 2).
Six lesions stained with 3% Lugol solution fell into the
category of “None” for clarity.
The evaluation-concordance ratios (%) among 4 of
5 clinicians were 45.7% (21/46 cases) for 3% Lugol
solution, 73.1% (19/26 cases) for 5% Lugol solution,
Table 2. Clinical evaluation of LSLs a
and 70.0% (14/20 cases) for 10% IG. P-values were
determined using two-tailed Fisher’s exact test, and
significant differences were found between 3% Lugol
solution and 5% Lugol solution/ 10% IG groups (P <
0.001) .
Discussion
Most studies of vital iodine staining using endoscopy
in the upper gastrointestinal tract used 1-3% iodine
solutions, whereas 2-3% Lugol solutions or 10% IG
has been used for oral lesions. The densities and
J Med Dent Sci
compositions of iodine solutions vary among authors
(Table 3) 3-6,13-20. The technical term of unstained lesions
(USLs) was used in previous reports, but the regions
called USLs were lightly stained by iodine in our
colorimetric study. Therefore, we termed unstained
lesions as lightly stained lesions. To the best of our
knowledge, although malignant lesions have been
detected and LSLs have been histopathologically
classified, there have been few studies objectively
quantifying the color of LSLs and comparing different
densities of iodine solutions and staining sequences to
optimize the clarity of staining of LSLs 21,22.
In this study, significant differences were found in the
L* values between MOD/SED and SLs with each density
of iodine solution. When stained with 3% Lugol solutions
or 5% after 3% Lugol solutions, there were significant
differences between the color difference (ΔE* ab )
values of MLD and MOD/SED, but none was found in
staining with 10% IG after 3% Lugol solutions. When
staining with 5% Lugol solution, significant differences
were found in all color values and differences. These
results suggest that it might be possible to distinguish
each histopathologic group of LSLs on the basis of
the values of lightness (L* ) and hue (a*, b* ) by staining
with 3% Lugol solution followed by 5% Lugol solution.
In contrast, the use of 10% IG would not produce
a distinguishable color difference between each
histopathologic group of LSLs and SLs because of the
very strong black-brown staining.
The number of cases with a clear boundary line
increased by 30% after the second staining. Such
an increase in clarity would improve the evaluationconcordance ratios and provide a more accurate area
of minimum resection. One of the probable reasons for
the increased clarity might be that the shapes of the
LSLs were roughly visible after the first staining, and
staining for the second time enhanced the sharpness of
the boundary line.
The evaluation-concordance ratios (%) of the
boundary lines using 3% Lugol solution, 5% Lugol
solution, and 10% IG were 45.7%, 73.1%, and 70.0%,
respectively. Staining with 5% Lugol solution had the
highest concordance ratio, and it was clear that the
staining sequence with different densities of iodine
reduced the inconsistency of the evaluations by each
clinician regarding the determinations for resection.
Kurita et al. have reported favorable staining with 10%
16
IG . However, in our experiments, LSLs are stained
strongly dark over time with 10% IG, making it difficult
to differentiate LSLs from SLs, and it is often difficult
to draw a resection line without bleaching. Moreover,
129
Effective iodine staining method for oral lesions
Table 3. Reports of incidence of LSLs* by vital staining
when iodine staining is performed in patients, high-dose
iodine and glycerin remain in the mucosa and even
after gargling with sodium thiosulfate and water, many
patients complain of irritation and pain, except when the
patients are under general anesthesia. Iodine staining
should be avoided in conscious patients with wounds,
such as ulcers, or after radiotherapy. When iodine
staining is performed macroscopically in the oral cavity
without an endoscope, and if the LSLs borders are not
clearly seen using 3% Lugol solution, it is necessary to
sequentially increase the iodine concentration.
The rates of malignant transformation of oral
leukoplakia were reported to be 7.9% (n = 444) in our
institution over the last 30 years 23, and Petti reported
annual malignant transformation rates from 0.69% to
2.03% (95% CI) 24. In order to reduce recurrence and
malignant transformation when resection is selected
as the treatment for oral leukoplakia, it is important
to resect the LSLs with epithelial dysplasia with a
sufficient safety margin.
The oral cavity has various functions such as
mastication, speech, and swallowing and is also
esthetically important. Therefore, it is desirable to
minimize resection in order to obtain a favorable
postoperative quality of life. However, there is no
appropriate standard for the distance between the
surgical margin and the border of the LSLs. The
results of this study indicate that the evaluation of
the boundary line between SLs and LSLs will differ by
each clinician if it is indistinct after iodine staining, and
that the evaluation-concordance ratio will increase if
the boundary line is distinct. From the above results, it
may be concluded that the most distinct boundary line
could be observed by restaining with 5% Lugol solution
after staining with 3% Lugol solution. Consequently, it is
expected that this method will minimize the deviation in
excision range among clinicians. Moreover, this method
may enable clinicians to distinguish the MOD/SED group
from the MLD or HYP group in LSLs.
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