Laser Fluorescence Spectroscopy with 5-Aminolevulinic Acid in Operative Gynecology pdf

Optical diagnostics is based on a selective excitation of fluorescence in tissues using a laser or an alternative light source with a certain wavelength.. We measure fluorescence spectra

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 fluofluo-rescence 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 Belyaeva1, 2, L V Adamyan1, A A Stepanyan1, K G Linkov2, and V B Loshchenov2

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

1208 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 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 kf 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)

Area_under_Laser_scattered_ peak

-

=

kdc kf(pathol)

kf(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 (kf = 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 (kf = 11.6) is higher than that in myometrium (kf = 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

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Fig 2. Patient M aged 40 years Diagnosis: corpus luteum cyst with hemorrhage.

1210 BELYAEVA et al.

benign ovarian cystadenomas) are characterized by a

low intensity of the PP IX fluorescence (kf = 3) In the

case of endometrioid cysts and cysts with hemorrhage,

we occasionally observe high values of kf 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 kf = 29.5 and the diagnostic con-trast coefficient with respect to the normal ovarian tis-sue is kdc = 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

[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: T3c N0M1 ovarian cancer.

2000

620

1800

1600

1400

1200

1000

800

600

400

200

[0] h-n [3] ovar-n-left [10] kistoma-right

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Fig 3. Patient M aged 45 years Diagnosis: ovarian serous cystadenoma of borderline malignancy.

LASER FLUORESCENCE SPECTROSCOPY 1211

cer (n = 9): kf = 45.6 and kdc = 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

5 10 15 20 25 30 35

0

[17] endocer [18] ectocer

Fig 6. Patient K aged 55 years Diagnosis: T1A1N0M1 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

5 10 15 20 25 30 35

0

Fig 5. Patient Yag aged 44 years Diagnosis: T1N0M0 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).

1212 BELYAEVA et al.

Endometrium Pathologies

For endometrial cancer (n = 11), the mean

fluores-cence coefficient is kf = 37.3 For the local form of

byendometrial cancer, the diagnostic contrast

coeffi-cient is kdc = 5.9 (Fig 3)

For the atypical hyperplasia of endometrium (n = 2),

the mean fluorescence coefficient is kf = 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 (kf = 26.4) However, the diagnos-tic contrast coefficient (polyp vs normal endometrium)

is kdc = 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

kdc = 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 kdc = 5.2, 3.8, and 4.0, respectively The diagnostic contrast coefficient can

2000

620

1800

1600

1400

1200

1000

800

600

400

200

[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

0

600

28

[21] l-u-n-right [26] h-n

Fig 8 Patient T aged 66 years Diagnosis: T3N1M0 cancer of vulva.

9

8

7

6

5

4

3

2

kdc

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Number of patient

Fig 7 Plot of kdc 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 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 kf 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 mm3 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

REFERENCES

1 N Rammanujam, Encyclopedia of Analytical Chemistry

(Wiley, Chichester, 2000)

2 R Baumgartner, Proc SPIE 3563, 90 (1999).

3 C J Kelty, N J Brown, M R A Reed, and R Ackroyd,

Photochem Photobiol Sci 1, 158 (2002).

4 M K Fehr, P Wyss, Y Tadir, et al., Photomedicine in

Gynecology and Reproduction (Basel, Karger, 2000).

5 E Malik, A Meyhofer-Malik, C Berg, et al., Hum.

Reprod 15, 584 (2000).

6 P Hillemanns, H Weingandt, H Stepp, et al., Am J.

Obstet Gynecol 184 (5), 1046 (2001).

7 L A Belyaeva, L V Adamyan, V B Laschenov, et al.,

Fluorescence diagnostics in oncological gynecology,

Proc of SPIE vol 5068 (2003), Saratov Fall Meeting, 2002; Optical Technologies in Biophysics and Medicine,

ed V V Tuchin (SPIE, Bellingham, WA, 2003), pp 55– 60

8 P Wyss, L O Svaasand, Y Tadir, et al., Hum Reprod.

10 (1), 221 (1995).

9 B Loschenov, V I Konov, and A M Prokhorov, Laser

Phys 10, 1188 (2000).