5-aminolevulinic acid photodynamic therapy for the treatment of high-grade gliomas.

INTRODUCTION: Photodynamic therapy (PDT) is a two-step treatment involving the administration of a photosensitive agent followed by its activation at a specific light wavelength for targeting of tumor cells. MATERIALS/METHODS: A comprehensive review of the literature was performed to analyze the indications for PDT, mechanisms of action, use of different photosensitizers, the immunomodulatory effects of PDT, and both preclinical and clinical studies for use in high-grade gliomas (HGGs). RESULTS: PDT has been approved by the United States Food and Drug Administration (FDA) for the treatment of premalignant and malignant diseases, such as actinic keratoses, Barrett's esophagus, esophageal cancers, and endobronchial non-small cell lung cancers, as well as for the treatment of choroidal neovascularization. In neuro-oncology, clinical trials are currently underway to demonstrate PDT efficacy against a number of malignancies that include HGGs and other brain tumors. Both photosensitizers and photosensitizing precursors have been used for PDT. 5-aminolevulinic acid (5-ALA), an intermediate in the heme synthesis pathway, is a photosensitizing precursor with FDA approval for PDT of actinic keratosis and as an intraoperative imaging agent for fluorescence-guided visualization of malignant tissue during glioma surgery. New trials are underway to utilize 5-ALA as a therapeutic agent for PDT of the intraoperative resection cavity and interstitial PDT for inoperable HGGs. CONCLUSION: PDT remains a promising therapeutic approach that requires further study in HGGs. Use of 5-ALA PDT permits selective tumor targeting due to the intracellular metabolism of 5-ALA. The immunomodulatory effects of PDT further strengthen its use for treatment of HGGs and requires a better understanding. The combination of PDT with adjuvant therapies for HGGs will need to be studied in randomized, controlled studies.

Near-infrared autofluorescence imaging to detect parathyroid glands in thyroid surgery.

Objective To identify and save parathyroid glands during thyroidectomy by displaying their autofluorescence. Methods Autofluorescence imaging was carried out during thyroidectomy with and without central lymph node dissection. After visual recognition by the surgeon, the parathyroid glands and the surrounding tissue were exposed to near-infrared light with a wavelength of 690-770 nm using a modified Karl Storz near infrared/indocyanine green endoscopic system. Parathyroid tissue was expected to show near infrared autofluorescence at 820 nm, captured in the blue channel of the camera. Results We investigated 41 parathyroid glands from 20 patients; 37 glands were identified correctly based on near-infrared autofluorescence. Neither lymph nodes nor thyroid revealed substantial autofluorescence and nor did adipose tissue. Conclusions Parathyroid tissue is characterised by showing autofluorescence in the near-infrared spectrum. This effect can be used to identify and preserve parathyroid glands during thyroidectomy.

[Optical diagnostic methods for early tumour diagnosis in the upper aerodigestive tract: Quo vadis?]. / Optische Diagnoseverfahren zur Tumorfrühdiagnostik im oberen Luft-Speise-Weg: Quo vadis?

BACKGROUND: Optical diagnostic methods may simplify and improve the early diagnosis of tumours of the upper aerodigestive tract; however, these have not yet found their way into clinical routine. OBJECTIVE: This article aims to define the problems that have prevented routine use of optical diagnostic methods so far, as well as listing and also explaining potential trendsetting approaches to overcome these difficulties. MATERIALS AND METHODS: The study is based on a combined analysis of publically accessible databases (PubMed MEDLINE, Thompson Reuters Web of Science, SPIE. Digital Library; full time period available; search strings: "oral cavity", "pharynx", "larnyx", "optical diagnosis", "optical biopsy", "optical coherence tomography", "confocal endomicroscopy", "fluorescence endoscopy", "narrow band imaging", "non-linear imaging", "fluorescence lifetime imaging"), as well as personal experiences. RESULTS: Both conceptual and methodical problems were determined, and possible solutions based on current developments are discussed. CONCLUSION: Optical diagnostic methods have the potential to revolutionise early diagnosis of upper aerodigestive tract malignancies, providing the different hurdles listed in this review can be overcome.

3D optical coherence tomography of cervical intraepithelial neoplasia--early experience and some pitfalls.

OBJECTIVES: To compare two different systems for optical coherence tomography for the diagnosis of cervical dysplasia and to assess potential benefits of three-dimensional imaging. MATERIALS AND METHODS: OCT images were taken from unsuspicious and suspicious areas of fresh conisation specimens using two different imaging systems, one with the capability to produce three-dimensional images. All OCT images were separately evaluated by two blinded investigators based on a 6-grade classification (normal, inflammation, CIN 1, CIN 2, CIN 3, squamous carcinoma) and later compared to the corresponding histology. Sensitivity and specificity of OCT in detecting cervical dysplasia were determined. RESULTS: OCT images using both OCT systems were taken from 46 sites in ten conisation specimens and later compared to the corresponding histology. CIN lesions were diagnosed correctly by the two-dimensional OCT system with a sensitivity and specificity of 91% and 78% accordingly. Using the three-dimensional system sensitivity and specificity were 82% and 86% accordingly. CONCLUSIONS: Both OCT systems used were highly sensitive in identifying cervical intraepithelial neoplasia. Despite technical problems experienced in the present series, we believe that three-dimensional imaging has the potential to further improve the accuracy of optical coherence tomography.

Diagnostic efficacy of backscattering intensity measurements in optical coherence tomography of cervical intraepithelial dysplasia.

BACKGROUND AND OBJECTIVES: The aim of this study was to determine the diagnostic efficacy of backscattering intensity measurements in optical coherence tomography in identifying different grades of cervical intraepithelial dysplasia. STUDY DESIGN/MATERIALS AND METHODS: OCT images were taken from 153 unsuspicious and suspicious areas of 30 fresh conisation and hysterectomy specimens, evaluated by two blinded investigators using a six-grade classification (normal, inflammation, CIN1, CIN2, CIN3, squamous carcinoma) and later compared to the corresponding histology. Differences between judgments based on either the histology or the OCT images were investigated employing Correspondence Analysis (CA). Further, we explored the extent as to which backscattering intensity profiles of OCT images contained the essential information required for a reliable and valid diagnosis, using Linear Discriminant Analysis (LDA). RESULTS: The CA of histology- and OCT-based judgments suggests that the diagnostic process may be characterized in terms of two stochastically independent underlying ("latent") variables, the first of them reflecting the definiteness with which CIN classes are identified, the second reflecting a bias towards diagnosing inflammation on the side of the OCT-based judgments. This finding is supported by the results of LDAs, where histology and OCT categorizations differ in particular with respect to the positions of inflammation and CIN1. Possibly, a second canonical variable has to be assumed accounting for the evaluation of carcinoma. CONCLUSIONS: The systematic differences between histology-based and OCT-based diagnoses suggest that the use of available information is influenced by perceptual and/or cognitive biases. Apart from this it seems that the profiles appear to provide a remarkably large amount of information determining the main course of the diagnostic process.

Optical coherence tomography for bladder cancer -- ready as a surrogate for optical biopsy? Results of a prospective mono-centre study.

INTRODUCTION: New modalities like Optical Coherence Tomography (OCT) allow non-invasive examination of the internal structure of biological tissue in vivo. The potential benefits and limitations of this new technology for the detection and evaluation of bladder cancer were examined in this study. MATERIALS AND METHODS: Between January 2007 and January 2008, 52 patients who underwent transurethral bladder biopsy or TUR-BT for surveillance or due to initial suspicion of urothelial carcinoma of the bladder were enrolled in this study. In total, 166 lesions were suspicious for malignancy according to standard white light cystoscopy. All suspicious lesions were scanned and interpreted during perioperative cystoscopy using OCT. Cold cup biopsies and/or TUR-B was performed for all these lesions. For this study we used an OCT-device (Niris, Imalux, Cleveland, US), that utilizes near-infrared light guided through a flexible fibre-based applicator, which is placed into the bladder via the working channel of the cystoscope. The technology provides high spatial resolution on the order of about 10-20 microm, and a visualization of tissue to a depth of about 2 mm across a lateral span of about 2 mm in width. The device used received market clearance from the FDA and CE approval in Germany. The diagnostic and surgical procedure was videotaped and analyzed afterwards for definitive matching of scanned and biopsied lesion. The primary aim of this study was to determine the level of correlation between OCT interpretation and final histological result. RESULTS: Of 166 scanned OCT images, 102 lesions (61.4%) matched to the same site where the biopsy/TUR-BT was taken according to videoanalysis. Only these video-verified lesions were used for further analysis. Of all analyzed lesions 88 were benign (inflammation, edema, hyperplasia etc.) and 14 were malignant (CIS, Ta, T1, T2) as shown by final histo?pathology. - All 14 malignant lesions were detected correctly by OCT. Furthermore all invasive tumors were staged correctly by OCT regarding tumor growth beyond the lamina propria. There were no false negative lesions detected by OCT. Sensitivity of OCT for detecting the presence of a malignant lesion was 100% and sensitivity for detection of tumor growth beyond the lamina propria was 100% as well. Specificity of OCT for presence of malignancy was 65%, due to the fact that a number of lesions were interpreted as false positive by OCT. CONCLUSION: As a minimally invasive technique, OCT proved to have extremely high sensitivity for detection of malignant lesions as well as estimation of whether a tumor has invaded beyond the lamina propria. However, specificity of OCT within the bladder was impaired (65%), possibly due to a learning curve and/or the relatively low spatial resolution and visualization depth of the OCT technology. Further studies and technical development are needed to establish an adequate surrogate for optical biopsy.

Endoscopic measurements of free-flap perfusion in the head and neck region using red-excited Indocyanine Green: preliminary results.

BACKGROUND: Free-tissue transfer has become a standard procedure for reconstructive surgery in the head and neck area. Flap failures are relatively rare (or=64% for all other examinations. CONCLUSIONS: It was possible to prove the feasibility of endoscopic ICG fluorescence angiography in patients undergoing free-flap transfer to the UADT. The method provides instant information about the perfusion state of the tissue and is easily performed without greater patient discomfort or risk of side effects. Due to the endoscopic approach, the method seems highly promising for this indication and merits further evaluation.

In vitro and in vivo evaluation of photodynamic techniques for the experimental treatment of human hepatoblastoma and neuroblastoma: preliminary results.

PURPOSE: The purpose of this study was to test the susceptibility of human hepatoblastoma and neuroblastoma cells to photodynamic diagnostics (PDD) and photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) as a photosensitizer. METHODS: Cell cultures of human hepatoblastoma (HuH6) and neuroblastoma (MHH-NB-11) were incubated with 5-ALA at increasing concentrations to measure the cellular kinetics of photosensitization. After optimizing incubation parameters, the cell cultures were then irradiated with increasing light doses and cell viability was measured by CTB assay. Human fibroblastic cells served as controls. So far, only the hepatoblastoma cell line has been tested in vivo. After injection of HUH6 cells in immunoincompetent rats, the efficacy of PDT was assessed. Photosensitization was achieved by intraperitoneal injection of 5-ALA. The pharmacokinetics of different tissues was studied. In a second study, a PDT of implanted hepatoblastoma, liver and peritoneum was performed. The irradiated areas were excised 48 h after treatment and studied by microscopy. RESULTS: Cell culture experiments demonstrated a selective fluorescence for both tumor lines compared to controls. The photosensitized tumor cells demonstrated marked reductions in cell viability at significantly lower irradiation doses than the fibroblasts under PDT. The specificity of fluorescence was confirmed in vivo for hepatoblastoma, and all the sensitized and irradiated tumors showed marked phototoxic necrosis. CONCLUSION: Human hepatoblastoma and neuroblastoma demonstrate marked and specific fluorescence after the application of 5-ALA, making PDD possible. Cell death occurred in both cell lines after PDT in vitro. Additionally, hepatoblastoma was susceptible to PDT in an animal model. Further studies will be necessary to determine the role of PDT and PDD in a clinical setting.

[Fluorescence diagnosis and photodynamic therapy in urology]. / Fluoreszenzdiagnostik und photodynamische therapie in der urologie.

Urology is a preferential domain of endoscopy and as such an important research field for photodynamic procedures. An important milestone in the long-lasting and successful history of "photodynamics" in urology is the European approval of hexaminolevulinate (HAL, Hexvix) for fluorescence cystoscopy. All clinical studies carried out so far have demonstrated a significant increase in sensitivity of fluorescence versus standard cystoscopy for the detection of bladder cancer, especially concerning carcinoma in situ. The majority of the randomised, two-armed studies additionally show significantly reduced rates of residual tumour and recurrences. Tumor-selective fluorescence can also be observed in the kidney and prostate. Intraoperative fluorescence detection might thus simplify the achievement of high rates of R0 resections. Apart from the diagnostic potential of "photodynamics", also some possible therapeutic indications will be mentioned, including photodynamic therapy of bladder cancer and prostate cancer. Whereas initial clinical experience has been obtained for photodynamic therapy of bladder cancer, clinical studies for other indications are currently being designed. By providing an overview over methods and procedures as well as hitherto the available clinical results, we hope to provide reader with a basis for obtaining his/her own judgement.

[Fluorescence cystoscopy at bladder cancer: present trials]. / Die Fluoreszenzzystoskopie beim Harnblasenkarzinom : Aktueller Stellenwert.

Bladder cancer is a frequent disease and represents the second most common genitourinary neoplasm. Although many aspects of the management of not-muscle-infiltrating bladder cancer are now well established, significant challenges remain, which influence patient outcome. Early detection and treatment of recurrent disease is required to optimize bladder preservation, reduce patient morbidity and increase quality of life and survival. Fluorescence cystoscopy, often referred to as "photodynamic diagnosis" (PDD) with intravesical application of photosensitizing agents has been developed in order to enhance the early detection of bladder cancer. Since March 2005 the hexyl-ALA ester (Hexvix) has been approved for the diagnosis of bladder cancer in 27 EU/EEA countries through the European Mutual Recognition Procedure. There is growing evidence that PDD enhances the detection of bladder cancer, particularly of high-grade flat lesions. Furthermore, transurethral resection of bladder tumor under fluorescence guidance has been shown to reduce the risk of recurrent tumors. Nevertheless, a resulting relatively decreased number of recurrences have still to be verified in prospective randomized trials.

[Fluorescence cytology. Improvement of urinary cytology]. / Die Fluoreszenzzytologie. Eine Weiterentwicklung der Urinzytologie.

Urothelial cancer of the bladder is a frequent disease, and urinary cytology often is used as a routine diagnostic tool. But this technique has an impaired sensitivity in low-grade tumours, and as a subjective method it is highly dependent on the experience of the cytologist. Here we present the technique of fluorescence cytology as an improvement of conventional cytology. This method is potentially able to compensate for the disadvantages of urinary cytology as it is an automated process that uses the principles of 5-Ala-induced photodynamic diagnosis (PDD).

Low frequency of colorectal dysplasia in patients with long-standing inflammatory bowel disease colitis: detection by fluorescence endoscopy.

BACKGROUND AND STUDY AIM: Patients with long-standing inflammatory bowel disease (IBD) have an increased risk of developing colonic dysplasias. Dysplastic changes in flat mucosa are likely to be missed by conventional colonoscopy. Endoscopic fluorescence imaging, using 5-aminolevulinic acid (5-ALA) as photosensitizer, has evolved as a new technique to differentiate between normal colonic mucosa and dysplasia. We combined this technique with random biopsies to prospectively evaluate the occurrence of dysplasias in patients with long-standing IBD. PATIENTS AND METHODS: 52 colonoscopies were performed in 42 consecutive patients (n = 28 with ulcerative colitis, n = 11 with Crohn's colitis, n = 3 with indeterminate colitis; mean age 43 years, range 21 - 78) with long-standing IBD colitis (median disease duration 14 years, range 3 - 40). All patients were in clinical remission. Patients were examined using both conventional white light and by fluorescence colonoscopy using oral 5-ALA. Four biopsies were taken every 10 cm from mucosa of normal appearance. In addition, macroscopically suspicious and fluorescence-positive areas were biopsied. RESULTS: A total of 688 biopsies of red-fluorescent (n = 20) and nonfluorescent (n = 662) areas of mucosa were taken. Dysplasia was detected histopathologically in only two of the biopsies. These biopsies were taken from two polypoid lesions which were fluorescence-negative. CONCLUSIONS: The rate of colonic dysplasia in patients with long-standing IBD colitis may be lower than previously reported.

Oedema formation in experimental photo-irradiation therapy of brain tumours using 5-ALA.

BACKGROUND: Five-aminolevulinic acid (5-ALA) induces the specific accumulation of photosensitising porphyrins in malignant gliomas and has been explored for photo-irradiation therapy of these tumours. However, information is unavailable on whether and to what extent this treatment modality may induce the formation of brain oedema, and how potential oedema might be treated. METHODS: Rats were implanted with C6 gliomas. Eight days later magnetic resonance images (MRI) were obtained. On day 9 rats received 100 mg 5-ALA/kg b.w. and were craniotomized for photo-irradiation of tumours 6 hours later (100 J/cm2, 635 nm argon-dye laser). Part of the animals was treated with daily dexamethasone injections (0.3 mg/kg), beginning 6 hours before phototherapy. 72 hours later, brains were removed and dissected according to tumour dimensions on pre-therapy MRI into "tumour", "brain around tumour" (BAT), residual cortex and basal ganglia, for measurements of water contents. Measurements were also performed in untreated animals with tumours, with or without steroid treatment and in control animals. An additional group of animals lacking tumours, with or without steroid treatment, underwent 5-ALA-phototherapy to determine effects on normal brain. RESULTS: C6 gliomas induced brain oedema, which responded to steroid treatment. 5-ALA-phototherapy resulted in additional oedema, which responded partly to steroids. 5-ALA-phototherapy of normal brain increased water content moderately in irradiated cortex. This oedema was also partly counteracted by steroids. CONCLUSIONS: Photo-irradiation therapy with 5-ALA induces oedema which is partly counteracted by steroid therapy. The possibility of steroid resistant oedema formation should be considered when planning human trials with this treatment modality.

5-Aminolevulinic acid-induced fluorescence in bronchial tumours: dependency on the patterns of tumour invasion.

5-ALA-induced protoporphyrin IX (PPIX) fluorescence kinetics was quantified by fluorescence microscopy in three-dimensional organ co-cultures of human bronchial epithelium, which were infiltrated by four different lung tumour cell lines (EPLC-M31, LCLC-103H, NCI-H125 and NCI-H841). Corresponding fluorescence measurements were performed in monolayer cultures of these tumour cell lines and BEAS-2B cells as a model for normal bronchial epithelium by flow cytometry. Significant differences of fluorescence intensities (FI) between the tumours were detected in organ co-cultures as well as in single cell measurements. Relative FI values in organ co-cultures (FI(EPLC-32M1)>FI(LCLC-H103)>FI(NCI-H125)>FI(NCI-H841)) did not correspond to the measurements in single cells (FI(LCLC-H103)>FI(NCI-H125)>FI(NCI-H841)>FI(EPLC-32M1)). Histology of organ co-cultures revealed different patterns of invasion and tumour cell densities depending on the tumour type. After correction of FI in the co-cultures to tumour cell density the correlation coefficient for fluorescence values between both models increased considerably. Thus, additionally to distinctive features of 5-ALA metabolism, patterns of tumour invasion may be a factor determining 5-ALA-induced fluorescence. Considering these results, a pronounced heterogeneity of 5-ALA-induced fluorescence might be expected in different bronchial tumours in vivo. This could interfere with the diagnostic reliability of 5-ALA-induced fluorescence for early tumour detection.

Fluorescence diagnosis: a novel method to guide radical inguinal lymph node dissection in penile cancer.

In penile cancer there is still a diagnostic dilemma between over treatment of lymph node-negative patients and the missing of occult metastases by watchful waiting. In the current study the value of fluorescence diagnosis during radical inguinal lymph node dissection was evaluated. Five patients with penile cancer were elected to undergo groin dissection. All patients received 5-aminolevulinic acid (5-ALA) orally before the operation for fluorescence diagnosis. Intraoperatively, fluorescence detection of the lymph nodes was performed by visual detection and spectroscopy. Two of the five patients had positive inguinal lymph nodes. Fluorescence in tumor-bearing tissue was detectable in the exposed lymph nodes. Protoporphyrin IX (PPIX) is accumulated in tumor-positive lymph nodes, making fluorescence diagnosis in penile cancer possible. More studies with higher patient numbers are necessary to evaluate optimal dosage and excitation conditions to detect tumor-bearing nodes in vivo.

Fluorescence-guided resections of malignant gliomas--an overview.

Radical resections of contrast-enhancing tumour in patients with malignant gliomas may be pertinent for survival but are often difficult to achieve due to uncertainties in distinguishing tumour margins intra-operatively. In this respect a number of novel methods are being examined which aim at enhancing resections. Among these methods, resections that exploit the accumulation of fluorescent porphyrins within malignant glioma tissue in response to exogenous administration of a metabolic percursor, 5-aminolevulinic acid, may offer particular advantages. This article summarises the clinical background and current status of 5-ALA drug development for fluorescence-guided resections of malignant gliomas and analyses the available literature with regard to possible mechanisms that govern the highly specific accumulation of fluorescent porphyrins in malignant glioma tissue in response to 5-ALA administration.

Whole bladder photodynamic therapy with 5-aminolevulinic acid using a white light source.

OBJECTIVES: To determine whether whole bladder photodynamic therapy after intravesical administration of 5-aminolevulinic acid using a white light source would destroy urothelial carcinoma. We sought to define the optimal target group of patients for this therapy. The side effects of treatment were also assessed. METHODS: We performed whole bladder photodynamic therapy with 100 J/cm(2) white light 2 to 4.5 hours after intravesical administration of 17% 5-aminolevulinic acid in 12 patients with recurring, multifocal, Stage pTa, grade I to III, urothelial tumors of the bladder and carcinoma in situ. RESULTS: Immediately after whole bladder irradiation, histologic examination of biopsies taken from flat suspicious lesions showed no viable cells; remnants of malignant cells were found in papillary tumors. Of the 12 patients, 11 returned for follow-up examination. At a median follow-up of 18 months (range 3 to 25), 3 of the 7 patients with carcinoma in situ and 2 of the 4 patients with papillary tumors were free of disease. In all patients, urinary frequency and urgency subsided within 3 weeks. No decreased bladder capacity or systemic side effects were observed. CONCLUSIONS: Our preliminary data show that whole bladder photodynamic therapy with intravesically applied 5-aminolevulinic acid using a white light source is effective in destroying flat malignant lesions of the bladder such as carcinoma in situ. The procedure is easy to perform and is not associated with any major side effects. The findings warrant long-term and multicenter studies.

New concept for the development of a confocal endomicroscope.

It is one of modern medicine's top priorities to reduce the stress for the patient during diagnosis and therapy by designing minimally invasive applications of medical techniques. Microsystems technology has always held the key to minimally invasive solutions in surgery and micro-endoscopy. The new measuring system KOMED is currently developed for the application of in-vivo cellular microscopy. This approach is based on the concept of a miniaturised fibre-optical confocal microscope. The application range of this microsensor system includes the minimally invasive optical biopsy for purposes such as the early detection of tumour cells. In this paper the concept of the KOMED system is described, as well as technical challenges to be solved in the project.

Localization and staging of cervical intraepithelial neoplasia using double ratio fluorescence imaging.

A phase zero evaluation of a new fluorescence imaging technique for diagnosing cervical intraepithelial neoplasia (CIN) was performed. The fluorescence imaging prototype performed quantitative imaging of Protoporphyrin induced by a topically applied aminolevulinic acid using double ratio (DR) fluorescence imaging technique developed by our group. A total of 38 patients were in the protocol, with 16 colposcopically selected for biopsy. Fluorescence images of these 16 patients were taken, 19 sites were biopsied, and the disease was staged histopathologically. DR fluorescence imaging of the cervix using our general purpose prototype appeared to be cumbersome but feasible. In four cases strongly localized fluorescent hotspots were observed at the location where the disease was colposcopically visible. In the other cases the fluorescence showed a more diffuse multifocal image. The value of the DR determined at the site of biopsy correlated in a statistically significant way with the histopathologically determined stage of the disease [Spearman rank correlation, r=0.881, p<0.001 (confidence interval 0.7044-0.9552)]. This suggests that noninvasive staging of CIN using this technique is feasible. We believe that the results of this study justify the development of a dedicated device that combines regular white light colposcopy with DR fluorescence imaging.

Quantification of 5-aminolevulinic acid induced fluorescence improves the specificity of bladder cancer detection.

PURPOSE: 5-Aminolevulinic acid induced fluorescence endoscopy has outstanding sensitivity for detecting early stage bladder cancer. Nevertheless, a third of the lesions that show specific fluorescence are histologically benign. We decreased the false-positive rate of 5-aminolevulinic acid induced fluorescence endoscopy by incorporating protoporphyrin IX fluorescence quantification into the standard cystoscopy procedure. MATERIALS AND METHODS: In 25 cases (53 biopsies) of a history of or suspicion for bladder cancer 5-aminolevulinic acid induced fluorescence endoscopy and fluorescence image quantification were performed. For fluorescence image quantification images obtained with a target integrating color charge-coupled device camera were digitized and stored in a personal computer. Red-to-blue ratios were calculated from fluorescence positive lesions and results were correlated with hematoxylin and eosin histology. RESULTS: Malignant fluorescence positive lesions showed significantly stronger fluorescence intensity than fluorescing lesions with benign histology. A threshold was established that decreased the false-positive rate by 30% without affecting sensitivity. CONCLUSIONS: Fluorescence image quantification is a new endoscopic method for objectively selecting multicolor fluorescence bladder lesion images for biopsy. It has the potential of eliminating human error by different surgeons with variable experience in fluorescence endoscopy.