Photobleaching measurements of pigmented and vascular skin lesions: results of a clinical trial

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1 Photobleaching measurements of pigmented and vascular skin lesions: results of a clinical trial Alexey Lihachev a, Kristine Rozniece b, Janis Lesins a, Janis Spigulis a a Institute of Atomic Physics and Spectroscopy, University of Latvia, Raina blvd.19, Riga, Latvia, LV-1586 b Skin Diseases and Sexually Transmitted Disease Clinic, Briana str.2, Riga, Latvia, LV-11; ABSTRACT The autofluorescence photobleaching intensity dynamics of in vivo skin and skin pathologies under continuous 532 nm laser irradiation have been studied. Overall the 141 human skin malformations were investigated by laser induced skin autofluorescence photobleaching analysis. Details of equipment are described along with some measurement results illustrating potentiality of the technology. Keywords: in vivo skin, photobleaching, autofluorescence, pigmented skin 1. INTRODUCTION Timely detection and evaluation of skin pathologies is a topical problem of modern clinical diagnostics, because number of occurrence of such pathologies (incl., deadly melanomas) in Europe and throughout the world keeps on growing. The most precise method at present to identify the skin diseases is the biopsy a sample removal from the damaged skin area with consequent histological analysis. It is a painful, destructive, time-consuming and rather expensive method. Modern bio-photonics researches are developing new non-invasive optical biopsy methods, which are faster and more patientfriendly. Possible applications of laser excited skin autofluorescence photobleaching parameters in diagnostics cause special interest at present. Fluorescence intensity decrease during a lasting optical excitation is well known as photo-bleaching effect. Laser-excited tissue autofluorescence photo-bleaching (AFPB) have been studied extensively over recent decades. Most of the authors observed that the bleaching fluorescence intensity can be well described by double exponential equation in two phases. The intensity usually tends to a constant fluorescence background level after a while. Some researchers have dealt with AFPB studies by laser excitation at ultra-violet (337 nm), violet (45 nm), blue (442 nm), green (532 nm) and red (632 nm) wavelengths, both under continuous and pulsed excitations in a wide range of power densities (1 to 5 mw/cm 2 ) [1-3]. Mechanism of the skin AFPB effect has not been explained in details so far, but several hypotheses are examined experimentally. Summarizing the available literature data it was stated that the skin AF photobleaching effect application opportunities for skin diagnostics were not considered in the researches done during the time period from 1993 to 25. Our research as a continuation of previous studies [4-6] is aimed to develop the AFPB analysis in order to find a potential method for diagnostics of skin pathologies. Safety and well being of human subjects involved in a clinical trial have been provided according to permission of the local ethics committee. 2. MATERIALS AND EQUIPMENT This clinical trial comprised 141 pigmented and vascular lesions was performed in Skin Diseases and Sexually Transmitted Disease Clinic by laser induced autofluorescence phobleaching analasys. Overall 2 angiomas, 4 dermatofibromas, 3 basaliomas, 5 giant congenital nevi, 46 papillary nevi, 68 hiperpigmented nevi, 11 dysplastic nevi and 2 blue nevi were investigated in this study. Clinical trial was performed with Ethics Committee permission and written patient consent. The measurement setup comprises a cw laser (532 nm) with focusing output SMA-connector, Y-shaped optical fiber bundle for delivery of the laser radiation to the skin probe and the fluorescent light to spectrometer AvaSpec (via Clinical and Biomedical Spectroscopy and Imaging II, edited by Nirmala Ramanujam, Jürgen Popp, Proc. of SPIE-OSA Biomedical Optics, SPIE Vol. 887, 8872F 211 SPIE-OSA CCC code: /11/$18 doi: / SPIE-OSA/ Vol F-1

2 a laser-blocking filter) which was connected to PC. The emitting and detecting fibers were placed 3 mm from the skin. The typical excitation time was 1 minute, the spectrometer integration time.5 sec, laser power density on the skin ~65mW/cm 2, area of the excited skin surface ~.3cm 2 [4]. Sequences of autofluorescence spectra with.5 sec. intervals were taken from the pathologic skin areas at different locations of the body. Intensity-time dependent array is normalized by maximum AF intensity; the data is fitted to the exponential bleaching expression exp (1) with subsequent extraction of bleaching rate τ 1, parameter A and absolute AF changes. This results in curves of AFPB dynamics and corresponding bleaching rates. 3. RESULTS AND DISCUSSION AF photobleaching can be characterized for all examined pathologies. Fig.1. presents healthy skin, pigmented nevus and cherry angioma autofluorescence intensity dynamics. The figure shows distinguishable differences of photo-bleaching, where AF intensity of healthy skin bleaches exponentially while AF intensity of pigmented nevi varies around the initial value. Each of skin pathologies has a specific AFPB characteristic, especially differ the dynamic features of pigmented cellular nevus and cherry angioma. In case of pigmented cellular nevus the initial intensity of AF is remarkably low and it tends to hold constant whereas the intensity of cherry angioma (vascular formation) rapidly falls. Results include three skin photo-types of patients (confirmed by dermatologist), i.e., Fig.1 represents cherry angioma of skin phototype II and pigmented cellular nevus of III phototype. Fluorescence intetsity (norm.u.) Pigmented cellular nevus 1.5 Cherry angioma 1. Healthy skin Time (sec) Fig.1. Dynamics of the autofluorescences intensity for some skin pathologies and healthy skin, 532 nm laser excitation, registration at 6 nm, power density 65 mw/cm 2. SPIE-OSA/ Vol F-2

3 τ 1 average value, sec ±3. ±4. ±4. ±3. ±2. ±2. ±3. ±2. Giant congenital nevus Displastic nevi Fig.2 Photobleaching parameter τ1 averaged values for different skin pathologies, 532 nm excitation, power density ~ 65 mw/cm 2. Figure 2 show the averaged photobleaching parameter τ 1 values with statistical dispersion for eight skin pathologies groups under 532 nm laser excitation. The AF photobleaching parameter τ 1 values for all seven skin pathologies groups proved to be within the interval of 7 13 sec. 45 ± A average value, r.v Displastic nevi ±44 ±18 Giant congenital nevus ±4 ±2 ±58 ±18 ±7 Fig.3 Photobleaching parameter A averaged values for different skin pathologies, 532 nm excitation, power density ~ 65 mw/cm 2. Figure 3 presents the parameter A averaged values with statistical dispersion for all skin types is within the borders from 25 to 43. SPIE-OSA/ Vol F-3

4 5 ±1 Avarage intensity changes, % ±7 ±4 ±1 ±9 Giant conginetal nevus ±9 Displastic nevus ±11 ±6 Fig.4. AF average intensity changes for different skin pathologies, excitation 532 nm, power density ~ 65 mw/cm 2, registration wavelength 6 nm. Figure 4 presents the AF absolute changes with statistical dispersion for skin pathologies groups under 532 nm laser excitation. 4. CONCLUSSIONS Results of the present study show considerable sensitivity of skin pathologies of the AF photbleaching analysis method. This methodology has a great potential for fast and patient-friendly diagnostics in dermatology. Dynamics of AFPB intensity are mainly dependent on a variety of skin chromophore distribution of various pathologies. The more homogeneous is a certain pathology structure, the more regular is its bleaching. The results prove that increased melanin content in the skin hampers the photobleaching process under the green (532 nm) excitation. The smallest intensity bleaching has been observed in cases of highly pigmented blue nevus, though the most rapid bleaching refers to vascular formations as angioma. Clinical trial results prove the potential of skin autofluorescence photobleaching parameter τ 1 for application in the clinic diagnostics as the additional criterion for quantitative evaluation of pigmented pathologies. In perspective, this methodology could be used for evaluation of skin melanin concentration, which would be especially useful for early diagnosing of melanomas. Since the AFPB mechanism is still under discussion, additional studies by use of this methodology would promote a better understanding of the AFPB mechanisms that take place in the human skin. ACKNOWLEDGMENTS The financial support of European Social Fund (grant 29/211/1DP/ /9/APIA/VIAA/77) is highly appreciated. REFERENCES 1. H. Zeng, C. E. MacAulay, B. Palcic, and D. I. McLean. Laser induced changes in autofluorescence of in-vivo skin, Proc. SPIE, Vol. 1882, (1993). 2. A. Stratonnikov, V. S. Polikarpov, and V. B. Loschenov. Photobleaching of endogenous fluorochroms in tissues in vivo during laser irradiation, Proc. SPIE, Vol. 4241, (21). 3. E. V. Salomatina and A.B. Pravdin. Fluorescence dynamics of human epidermis (ex vivo) and skin (in vivo), Proc. SPIE, Vol. 568, (23). SPIE-OSA/ Vol F-4

5 4. A. Lihachev, J. Spigulis. Skin autofluorescence fading at 45/532 nm laser excitation, IEEE Xplore, 1.119/NO, (27). 5. A. Lihachev, J. Lesins, D. Jakovels, J. Spigulis. Low power cw-laser signatures on human skin. Quantum Electron, 4 (12), (21). 6. J. Spigulis, A. Lihachev, and R. Erts. Imaging of laser-excited tissue autofluorescence bleaching rates, Appl Optics, Vol. 48, Issue 1, D163-D168 (29). SPIE-OSA/ Vol F-5