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1207
Laser Physics, Vol. 14, No. 9, 2004, pp. 1207–1213.
Original Text Copyright © 2004 by Astro, Ltd.
English Translation Copyright © 2004 by
MAIK “Nauka /Interperiodica”
(Russia).
INTRODUCTION
The existing extensive experience in diagnosing and
treating oncological diseases makes it possible to
assess the importance and complexity of the timely
diagnostics of cancer and precursors of cancer. The
existing means for intraoperative diagnostics in gyne-
cology (visual examination, including endoscopic
study, palpation, and urgent histological study) are
insufficient for surgeons, since urgent histological or
cythological studies, as well as visual and palpatory
examinations, often yield erroneous results, leading to
inadequate decisions at important diagnostic stages.
Thus, the importance of early and precise diagnostics in
gynecology has stimulated a search for fundamentally
new diagnostic procedures. In the development of new
diagnostic tools, attention is mostly given to optical
methods [1].
Fluorescence diagnostics (FD) involves a spectral
analysis of living tissues. Optical diagnostics is based
on a selective excitation of fluorescence in tissues using
a laser or an alternative light source with a certain
wavelength. One can use both endogenous and exoge-
nous photosensitizers. The selectivity of their distribu-
tion in the body is related to biochemical and physio-
logical differences between tissues and the properties
of the intercellular medium. Spectroscopic analysis of
fluorescence signals makes it possible to distinguish
between healthy and pathological tissues. Fast data pro-
cessing enables one to make corrections to the treat-
ment [2].
One of the topical problems in FD is the search for
optimal photosensitizers.
δ
-5-Aminolevulinic acid
(5-ALA) and the Alasens preparation based on 5-ALA
cannot be classified as photosensitizers. However, these
substances induce the intracellular synthesis of proto-
porphyrin IX (PP IX), which exhibits high-intensity
fluorescence and photodynamic activity. The high
accumulation of PP IX in rapidly proliferating cells and
its rapid utilization in normal cells serve as the biolog-
ical basis for FD. The aforesaid processes account for a
high fluorescence contrast of the pathological tissue vs.
the normal tissue. Rapid production of PP IX and its
rapid utilization in normal cells yielding photoinactive
heme (this accounts for the absence of phototoxicity)
have led to extensive clinical study of FD using 5-ALA
[3–7].
It is known that blue and green light used for fluo-
rescence excitation and fluorescence imaging are
readily absorbed by blood hemoglobin. This leads to a
decrease (down to 0.5 mm) in the measurement depth
in the tissues under study [8] and causes errors in esti-
mates of the real concentration of PP IX owing to the
variation in the hemoglobin content in tissues [9]. The
aforesaid facts necessitate spectral analysis of the fluo-
rescence. We measure fluorescence spectra using red
laser excitation with a wavelength of 633 nm, so the
depth of optical biopsy of the tissues is about 1–3 mm.
To increase the efficiency of early and differential
diagnostics of gynecological diseases, we develop an
FD method that can be used in gynecological opera-
BIOPHOTONICS
Laser Fluorescence Spectroscopy with 5-Aminolevulinic Acid
in Operative Gynecology
L. A. Belyaeva
1, 2
, L. V. Adamyan
1
, A. A. Stepanyan
1
, K. G. Linkov
2
, and V. B. Loshchenov
2
1
Department of Reproductive Medicine and Surgery, Faculty of Postgraduate Education,
Moscow State University of Medicine and Dentistry, Moscow, Russia
2
Natural Sciences Center, Prokhorov General Physics Institute, Russian Academy of Sciences,
ul. Vavilova 38, Moscow, 119991 Russia
e-mail: l_b@bk.ru
Received September 10, 2003
Abstract
—A method for the fluorescence intraoperative diagnostics of gynecological diseases using a laser–
fiber spectrum analyzer and Alasens-induced protoporphyrin IX (PP IX) is developed, and the efficiency of the
method is estimated. In the spectroscopic study, the fluorescence of PP IX is excited using laser radiation with
a wavelength of 633 nm and is measured during laparoscopy, laparotomy, hysteroscopy, physition examination,
and
ex vivo
in 75 patients with genital endometriosis and various pathologies of the ovary, vulva, and cervix and
body of the uterus. The spectroscopic results are compared to histological diagnosis. The data obtained show
laser fluorescence spectroscopy using Alasens to be very effect in differential diagnostics of ovary diseases, cer-
vical and vulvar cancer, and metastases. Additional study is needed to estimate the applicability of the method
under consideration for intraoperative diagnostics of active peritoneal endometriosis. A target biopsy of visually
intact fragments can be realized with the aid of fluorescence diagnostics based on laser fluorescence spectros-
copy of the Alasens-induced PP IX in organs and tissues. High accumulation of PP IX in normal and patholog-
ical endometrium impedes application of the fluorescence method for purposes of differential diagnostics but
opens up prospects for photodynamic ablation of endometrium.
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LASER PHYSICS
Vol. 14
No. 9
2004
BELYAEVA
et al
.
tions and that is based on laser spectroscopic measure-
ments of the concentration of Alasens-induced PP IX in
tissues. For this purpose, we carry out a spectroscopic
study, estimate the fluorescence spectral properties of
tissues in the organs of female genitals, and compare
the results obtained with histological data. We study
normal tissues and tissues from patients with
endometriosis ovarial, endometrial, cervical, and vul-
var benign and malignant tumors. We develop new
methods to measure fluorescence in order to optimize
the FD procedures.
MATERIALS AND METHODS
We perform fluorescence intraoperative measure-
ments in 75 gynecological patients in the course of lap-
arotomy, laparoscopy, hysteroscopy, and physition
examination. The age of the patients ranges from 20 to
77 years. Below, we present detailed information on the
patients and demonstrate examples and the results
obtained.
All patients undergo conventional a scheduled exam-
ination prior to operation. None of the patients with por-
phyria. During operations, FD supplements conven-
tional diagnostic methods. We analyze spectroscopic
characteristics of tissues in real time. The mean time of
optical express diagnostics of tissues is 2–3 min.
We employ 5-ALA; the commercial name of the
preparation produced by NIOPIK (Moscow, Russia) is
Alasens.
Equipment
For the intraoperative FD of gynecological diseases,
we employ conventional surgical instruments and
equipment and an LESA-01-Biospec electronic laser
spectral setup. The main components of this setup are
shown in Fig.1 and comprise (i) a helium–neon laser
with a wavelength of 632.8 nm and an output power of
1–10 mW, (ii) a fiber-optic multichannel catheter as a
system consisting of two parts (the laser system is used
to deliver the laser radiation to the object under study,
and the detecting and measuring system is used to
detect fluorescence and scattered laser light and to
transmit it to the detector), (iii) a set of color filters with
a transmission band of 630–750 nm, (iv) a multichannel
spectrum analyzer allowing for fast spectral measure-
ments, and (v) a PC. The data obtained are processed
using original computer codes.
Method for Intraoperative FD
Patients were informed about the procedure. After
obtaining the patient’s consent, we orally introduce
about 40 ml of Alasens at a doze of 25 mg/kg. The diag-
nostic procedures are started from three to eight hours
after the preparation is introduced. During gynecologi-
cal operations, we perform the fluorescence spectral
analysis as follows. A diagnostic fiber (with a diameter
of 1.8 mm) delivering radiation from a helium–neon
laser is brought in light contact with the tissue under
study. In the case of laparotomy and external examina-
tion, we do this directly by hand. In the case of laparos-
copy, we employ a additional troacar and an aspirating
needle. In the case of hysteroscopy, we introduce the
fiber via the operation channel. For spectral measure-
Fig. 1.
LESA-01-Biospek spectroscopic system.
LASER PHYSICS
Vol. 14
No. 9
2004
LASER FLUORESCENCE SPECTROSCOPY 1209
ments, we need to switch off the source of light for a
short period of time. For comparative analysis, we per-
form a preoperative measurement of the accumulation
of the Alasens-induced PP IX in the skin of the internal
surface of the forearm and in the mucous of the
patient’s lip and measure the autofluorescence of the
healthy skin. We also perform a spectral analysis of the
removed tissues immediately after operation.
Algorithm to Process FD Results
FD results are given in fluorescence spectra, in
which we plot the wavelength in nanometers and the
intensity of fluorescence and scattered laser radiation in
arbitrary units on the
x
and
y
axes, respectively. The
sharp peak at 633 nm corresponds to the scattered laser
light, whereas the fluorescence is seen as a broad band.
We analyze the spectral shapes and signal amplitudes.
To construct histograms, we calculate the fluorescence
coefficient:
(1)
We use the fluorescence coefficient as a diagnostic
criterion for the PP IX relative concentration in tissues.
The values of
k
f
are normalized by the value of the coef-
ficient corresponding to normal skin of the arm.
To estimate the difference between the fluorescence
intensities of normal and pathological tissues, we
employ the diagnostic contrast coefficient:
(2)
k
f
Area_under_Fluorescense_curve
Area_under_Laser_scattered_peak
.=
k
dc
k
f
pathol()
k
f
norma()
.=
We analyze the laser fluorescence spectral data for
2000 points of the tissues under study (20–40 points per
patient).
RESULTS AND DISCUSSION
To estimate the efficiency of laser fluorescence
spectroscopy, we employ such parameters as sensitivity
and specificity. The sensitivity shows if FD can be used
for diagnosing a certain pathology. The specificity
characterizes the ability of the method to rule out the
possibility of the pathology.
Normal Tissues
The processing of spectral data yields the minimum
accumulation of PP IX in normal peritoneum; serous
tissue of the uterus, uterine tube, and myomas; and in
normal ovary tissues (
k
f
= 2.1–3.8). Based on these
data, we can develop a method for panoramic intraperi-
toneal fluorescence visualization that enables one to
compare normal tissue with pathological tissue exhibit-
ing anomalously high-intensity fluorescence. We find a
high accumulation of the Alasens-induced PP IX in the
fimbrii. The spectral analysis shows that the PP IX
accumulation in normal endometrium (
k
f
= 11.6) is
higher than that in myometrium (
k
f
= 3.6) by a factor
of 3.5.
Ovarian Diseases
Benign ovarian pathologies (
n
= 22) (follicular cyst,
corpus luteum cyst, endometrioid cyst, fibrothecoma,
[0] h-n
[2] lips-p
[3] big t-r ovarii
[9] periton-n
3600
3400
3200
3000
2800
2600
2400
2200
2000
1800
1600
1400
1200
1000
800
600
400
200
600 650 700 750
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
0
2 3 9
Fig. 2.
Patient M. aged 40 years. Diagnosis: corpus luteum cyst with hemorrhage.
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LASER PHYSICS
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2004
BELYAEVA
et al
.
benign ovarian cystadenomas) are characterized by a
low intensity of the PP IX fluorescence (
k
f
= 3). In the
case of endometrioid cysts and cysts with hemorrhage,
we occasionally observe high values of
k
f
related to the
contribution of autofluorescence (this is clearly seen
from the spectral shapes in Fig. 2).
For the borderline ovarian tumors (
n
= 3), the fluo-
rescence coefficient is
k
f
= 29.5 and the diagnostic con-
trast coefficient with respect to the normal ovarian tis-
sue is
k
dc
= 9 (Fig. 3).
It is demonstrated that a high fluorescence intensity
of the Alasens-induced PP IX is typical of ovarian can-
2000
620
1800
1600
1400
1200
1000
800
600
400
200
640 660 680 700 720 740 760 780
[0] h-n
[5] c-r-ovar
[7] ovar
800
2200
2400
2500
2600
3000
[10] tub-ser
[11] tub-met-ser
[16] n-serosa-uter
[17] serosa-met
[24] periton-n
[25] perit-met
[27] met-salnk
0
5
10
15
20
25
30
25
0 5 7 10 11 16 17 24 25 27
Fig. 4.
Patient D. aged 56 years. Diagnosis:
T
3
c
N
0
M
1
ovarian cancer.
2000
620
1800
1600
1400
1200
1000
800
600
400
200
640 660 680 700 720 740 760 780
[0] h-n
[3] ovar-n-left
[10] kistoma-right
0
2
4
6
8
10
12
14
16
18
20
22
24
26
0 3 10
Fig. 3.
Patient M. aged 45 years. Diagnosis: ovarian serous cystadenoma of borderline malignancy.
LASER PHYSICS
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No. 9
2004
LASER FLUORESCENCE SPECTROSCOPY 1211
cer (
n
= 9):
k
f
= 45.6 and
k
dc
= 17.2 (Fig. 4). We also
demonstrate statistically valid differences between the
results obtained for ovarian metastases and the normal
ovarian tissues. We can also reliably distinguish
between the metastases of lymph nodes, peritoneum,
and greater omentum, on the one hand, and the normal
unchanged tissues, on the other hand. The method
under consideration demonstrated 100% sensitivity
and specificity in the differential diagnostics of cysto-
mas with cancer and ovarian metastases with border-
line cystadenomas versus normal ovary and benign
cysts.
[0] h-n
[13] endocerv-suspicion
[16] ectocerv-suspicion
2800
2600
2400
2200
2000
1800
1600
1400
1200
1000
800
600
400
200
600 650 700 750
0 13 17
5
10
15
20
25
30
35
0
[17] endocer
[18] ectocer
16
18
Fig. 6.
Patient K. aged 55 years. Diagnosis:
T
1
A
1
N
0
M
1
cervical cancer.
[0] h-n
[6] endom-n
[3] endom-left-and
3600
3400
3200
3000
2800
2600
2400
2200
2000
1800
1600
1400
1200
1000
800
600
400
200
600 650 700 750
0 6 7
5
10
15
20
25
30
35
0
Fig. 5.
Patient Yag. aged 44 years. Diagnosis:
T
1
N
0
M
0
adenocarcinoma. For this patient, the scheduled histological examination
revealed microfocuses of high-grade differentiated adenocarcinoma without invasions in myometrium (predominantly, in the left
uterine corner).
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2004
BELYAEVA
et al
.
Endometrium Pathologies
For endometrial cancer (
n
= 11), the mean fluores-
cence coefficient is
k
f
= 37.3. For the local form of
byendometrial cancer, the diagnostic contrast coeffi-
cient is
k
dc
= 5.9 (Fig. 3).
For the atypical hyperplasia of endometrium (
n
= 2),
the mean fluorescence coefficient is
k
f
= 31.4. We can-
not determine the diagnostic contrast coefficient owing
to the diffuse form of the pathology.
In the case of endometrium polyp (
n
= 9), we also
observe a relatively high intensity of the Alasens-
induced fluorescence (
k
f
= 26.4). However, the diagnos-
tic contrast coefficient (polyp vs. normal endometrium)
is
k
dc
= 2.7.
Uterine sarcoma (
n
= 1) is characterized by a rela-
tively low fluorescence intensity of the Alasens-
induced PP IX.
In patients with first-stage uterine cancer, cancer
in
situ
, and third-stage dysplasia (
n
= 13), we find that the
fluorescence intensity of pathological tissues is greater
than that of normal tissues by a factor of 5.5 (on the
average) (Fig. 6).
Figure 7 shows the dependence of the diagnostic
contrast coefficient (with respect to intact tissue) of the
Alasens-induced PP IX on the stage of the pathological
process. Note that the values above the level of 5.3 are
related to T
1A, B
invasive cervical cancer. The sensitivity
and specificity of the method in the differential diag-
nostics of cervical cancer versus normal epithelium are
estimated as 93 and 99%, respectively.
For cancer of the vulva (n = 4), the mean diagnostic
contrast coefficient (with respect to normal tissue) is
k
dc
= 5.23 (Fig. 8).
The spectroscopic data allow us to differentiate
between cancer of the vulva and normal tissue with a
sensitivity and a specificity of 100%.
For endometriosis of the peritoneum, uterine tubes,
and ovary (n = 11), the diagnostic contrast coefficients
(with respect to intact tissue) are k
dc
= 5.2, 3.8, and 4.0,
respectively. The diagnostic contrast coefficient can
2000
620
1800
1600
1400
1200
1000
800
600
400
200
640 660 680 700 720 740 760 780
[8] vulva-t-r-left
[12] vulva-n-right
[18] met-i-u-left
0
2
4
6
8
10
12
14
16
18
20
22
24
26
8 12 18
0
600
28
21 26
[21] l-u-n-right
[26] h-n
Fig. 8. Patient T. aged 66 years. Diagnosis: T
3
N
1
M
0
cancer of vulva.
9
8
7
6
5
4
3
2
k
dc
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Number of patient
Fig. 7. Plot of k
dc
vs. the stage of cervical pathology: n = 1,
CIN I; n = 2, CIN III; n = 3–8, cancer in situ; and n = 9–14,
T
1A, B
cervical cancer.
LASER PHYSICS Vol. 14 No. 9 2004
LASER FLUORESCENCE SPECTROSCOPY 1213
also take a value of 1.6, which corresponds to the
absence of reliable difference. A possible reason for
this is the proliferative activity of endometriosis.
Leiomyoma on the side of the serous tissue of the
uterus is characterized by a relatively low k
f
. In the case
of submucosa localization of nodes and, probably, in
the case of an actively growing tumor, the fluorescence
coefficient can be increased by a factor of 5.4.
Alasens is tolerated well by the majority of patients.
In a few cases, it causes nausea. At a dose of 30 mg/kg,
one patient exhibited hyperemia of the face and chill,
which appeared four hours after ingestion and lasted
about one day with the introduction of antihistamines.
To optimize the FD methods in the diagnostics of
pathologies, we develop a new procedure for the fluo-
rescence measurement of tissue microsamples (biop-
sies, samples of diagnostic curettage, puncture mate-
rial). It is difficult to measure the fluorescence spectra
of the aforesaid samples owing to the smallness of the
volume under study. A direct concentration measure-
ment at an object plate yields a relatively high error
owing to the losses of laser radiation (part of the laser
beam passes by the sample under study, and part is
reflected from the glass surface). For the spectroscopic
measurements of small volumes of biological tissues,
we employ specially designed object plates with an
absorbent coating. The sample under study is placed in
a glass container with a volume of 1 mm
3
and is covered
with a cover glass. This makes it possible to substan-
tially improve the results obtained.
The results of test measurements show that the
above procedure yields an error of 10–20% with respect
to the results from fluorescence spectrometry of the tis-
sues, whereas in the absence of the special substrate,
the relative error of measurements is 100–150%.
To measure lateral surfaces (e.g., the mucous tissue
of the cervical channel), we employ specially designed
heads mounted on the optical fiber. These heads make
it possible to take measurements at an angle of 70° rel-
ative to the axis.
CONCLUSIONS
In the course of surgical operations, optical express
biopsy makes it possible to differentiate between, on
the one hand, benign cysts and cystomas and, on the
other hand, ovarian cancer and borderline ovarian
tumors and to diagnose micrometastases in the ovary,
peritoneum, and omentum. In the cases of endometrial,
cervical, vulva cancer, the diagnostic contrast coeffi-
cients are determined. It is expedient to develop fluo-
rescence diagnostics for screening programs aimed at
early diagnosis of premalignancies of the cervix and
vulva. Apparently, the differences in PP IX accumula-
tion in endometrioid focuses of peritoneum, uterine
tubes, and ovary are related to the proliferative activity
of endometriosis. A combination of spectrometry and
fluorescence imaging will make it possible to diagnose
pathologies that are invisible to the eye, to estimate the
proliferation of a pathological process, and to monitor
tissues after treatment. The results on a relatively high
accumulation of the Alasens-induced PP IX in
endometrium and a relatively low concentration of
PP IX in myometrium are evidence of the promising
character of the photodynamic ablation of endometrium.
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. method that can be used in gynecological opera-
BIOPHOTONICS
Laser Fluorescence Spectroscopy with 5-Aminolevulinic Acid
in Operative Gynecology
L. A and precursors of cancer. The
existing means for intraoperative diagnostics in gyne-
cology (visual examination, including endoscopic
study, palpation, and
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