Abstract
In this study we proved the efficiency of the fluorimetric detection of a minimum number of malignant cells ex vivo. The goal of this work was to investigate whether the combination of photodynamic diagnosis (PDD) with oral brush biopsy might become a suitable chair-side tool to detect early oral carcinoma. Small numbers (100–500) of established human tumour cells—small cell lung carcinoma (OAT 75), transitional cell carcinoma of the bladder (SW1710) and human embryonic kidney cells (HEK293)—were incubated with 2 mM 5-aminolaevulinic acid (5-ALA). In addition, 50 brush biopsies from volunteers were prepared. After 2 h and 3 h of incubation, all samples were investigated by spectrofluorometry. Measurements were performed in capillaries. For excitation (405 nm) and detection of fluorescence spectra, a fibre microprobe–spectrofluorometer system (fibre 400 µm) was used. A minimum of 100 malignant cells and 3 h of incubation with ALA were needed to detect a typical spectrum for protoporphyrin IX (PPIX). Some epithelial samples from brush biopsy showed strong (bacteria related) PPIX autofluorescence, which increased after the addition of 5-ALA. From the testing of various antibiotics and antiseptics it emerged that 0.4 mM chlorhexidine strongly reduced fluorescence in brush biopsies from healthy volunteers, whereas the fluorescence signal of established cancer cell lines decreased only a little. The experiments revealed that, by means of an optical microprobe, very few cancer cells (100) can be detected. The addition of chlorhexidine before the incubation of brush biopsies with 5-ALA increases the reliability of the test by largely reducing the autofluorescence signal due to the presence of bacteria. Chair-side diagnostics of epithelial carcinoma seem feasible.
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References
Nanda K, McCrory DC, Myers ER, Bastian LA, Hasselblad V, Hickey JD, Matchar DB (2000) Accuracy of the papanicolaou test in screening for and follow-up of cervical cytological abnormalities. Ann Intern Med 132:810–819
Svirsky JA, Burns JC, Carpenter WM et al (2002) Comparison of computer-assisted brush biopsy results with follow up scalpel biopsy and histology. Gen Dent 50:500
Rick GM, Slater L (2003) Oral brush biopsy: the problem of false positives. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 96:252. doi:10.1016/S1079-2104(03)00362-7
Moonen PMJ, Witjes JA (2006) Diagnosis of bladder cancer—fluorescence cystoscopy and urinarymarkers. Business briefing: European Kidney and Urological Disease 2006, pp 76–79
Andersson-Engels S, Berg R, Svanberg K, Svanberg S (1995) Multi-colour fluorescence imaging in connection with photodynamic therapy of δ-amino levulinic acid (ALA) sensitised skin malignancies. Bioimaging 3:134–143. doi:10.1002/1361-6374(199509)3:3<134::AID-BIO4>3.3.CO;2-T
Schleier P, Berndt A, Zinner K, Zenk W, Dietel W, Pfister W (2006) ALA-based fluorescent diagnosis of malignant oral lesions in the presence of bacterial porphyrin formation. Proc SPIE 6139:613908
Ohara H, Okamoto T (1977) A new in vitro cell line established from human oat cell carcinoma of the lung. Cancer Res 37:3088–3095
Kennedy JC, Marcus SL (1996) Photodynamic therapy (PDT) and photodiagnosis (PD) using endogenous photosensitization induced by 5-ALA: mechanisms and clinical results. J Clin Laser Med Surg 14:289–304
Bunke A, Zerbe O, Schmid H, Burmeister G, Merkle HP, Gander B (2000) Degradation mechanism and stability of 5-aminolevulinic acid. J Pharm Sci 89(10):1335--1341
Ebhara A, Liaw L-H, Krasieva TB, Wilder Smith P (2003) Detection and diagnosis of oral cancer by light-induced fluorescence. Lasers Surg Med 32:17–24
Mitchell DA, Mitchell L (1995) Oxford handbook of clinical dentistry, 2nd edn. Oxford University Press Inc., New York, pp. 210–211
Todar K (2002) Todar’s online textbook of bacteriology. University of Wisconsin
Lennon AM, Buchalla W, Brune L, Zimmermann O, Gross U, Attin T (2006) The ability of selected oral microorganisms to emit red fluorescence. Institute of medical microbiology. 40:2–5
http://www.ouhsc.edu/histology/Text%20Sections/Cytology.html. University of Oklahoma Health Sciences Center Interactive Histology At Department of Cell Biology histology loan collection, Dr. Allan F. Wiechmann. Copyright © 2008 The Board of Regents of the University of Oklahoma.
Cawson RA, Odell EW (2002) Cawson’s essential of oral pathology and oral medicine, 7th edn. London, Churchill Livingstone, pp 180–195
Wang X (2004) Pharmacokinetics and selectivity of ALA- induced porphyrin synthesis after topical application of hexyl-aminolevulinic-acid in cervical intraepithelial neoplasia. Dissertation. Department of Obstetrics and Gynecology, Grosshadern, Ludwig-Maximilians-University, Munich
Vervoorts A, Richter C, Bayer R (2005) New tool for optical biopsy based on endogenously produced protoporphyrin IX. Institute for Laser Medicine, Düsseldorf. www.ilm.uni-duesseldorf.de/Publications/pub_05/Ipa2005.pdf. Accessed September 2005
Roy K, Bottril I, Ingrams DR, et al (1995) Diagnostic fluorescence spectroscopy of oral mucosa. Proc SPIE 2395:135–142
Gibbons RJ, van Houte J (1971) Selective bacterial adherence to oral epithelial surfaces and its role as an ecological determinant. Infect Immun 3(4):567–573
Koenig K, Schneckenburger H (1994) Laser-induced autofluorescence for medical diagnosis. J Fluoresc 4:17–40. doi:10.1007/BF01876650
Nitzan Y, Kauffman M (1999) Endogenous porphyrin production in bacteria by δ-aminolaevulinic acid and subsequent bacterial photoeradication. Lasers Med Sci 14:269–277
de Veld DCG, Skurichina M, Witjes MJH, Duin RPW, Sterenborg HJCM, Star WM, Roodenburg JLN (2003) Autofluorescence characteristics of healthy oral mucosa at different anatomical sites. Lasers Surg Med 32:367–376
Nitzan Y, Salmon-Divon M, Shporen E, Malik Z (2004) ALA induced photodynamic effects on gram positive and negative bacteria. Photochem Photobiol Sci 3:430–435
Tonzetich J, Friedman SD (1965) The regulation of metabolism by the cellular elements in saliva. Ann N Y Acad Sci 131:815–829. doi:10.1111/j.1749-6632.1965.tb34847.x
Onizawa K, Saginoya H, Furuya Y, Yoshida H (1996) Fluorescence photography as a diagnostic method for oral cancer. Cancer Lett 108:61–66
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Hamad, L.O., Vervoorts, A., Hennig, T. et al. Ex vivo photodynamic diagnosis to detect malignant cells in oral brush biopsies. Lasers Med Sci 25, 293–301 (2010). https://doi.org/10.1007/s10103-009-0712-1
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DOI: https://doi.org/10.1007/s10103-009-0712-1