ObjectivesFluorescence spectroscopy of human urine is a method with the potential to gain importance as a diagnostic tool in the medical field, e.g., for measuring Coproporphyrin III (CPIII) as an indicator of cancer and acute types of porphyria. Food can change human urine's color, which could influence the urine fluorescence spectrum and the detection of CPIII in urine. To determine if there is a noticeable influence on the urine fluorescence spectrum or on the detection of CPIII in urine, 16 vitamin supplements, and three food items were tested. Such investigation may also prevent false interpretation of measured data.MethodsUrine samples were collected before and after (overnight, ca. 8 h) intake of each test substance. Samples were investigated by fluorescence spectrum analysis. At excitation wavelengths from 300 to 500 nm and emission wavelengths from 400 to 700 nm excitation-emission-matrices were measured. Data obtained from urine before intake were compared to the data from overnight urine. Furthermore, the investigation of any interference with the CPIII concentration was performed at an excitation wavelength of 407 +/- 3 nm and emission wavelengths of 490-800 nm.ResultsOnly vitamin B2, but none of the other tested substances, showed noticeable influence on the urine fluorescence spectrum. None of the tested substances showed noticeable interference with the recovery rate of CPIII.ConclusionsThe correct interpretation of measured data by fluorescence spectroscopy is possible with the exception if vitamin B2 supplementation was performed; thus, the consumption of vitamin B2 supplements before fluorescence testing of the patient's urine should be avoided and/or must be requested. CPIII concentrations could reliably be measured in all cases.

5-aminolevulinic acid (5-ALA) mediated interstitial photodynamic therapy (iPDT) is undergoing clinical trials for the treatment of malignant gliomas. 5-ALA iPDT is based on the creation of reactive oxygen species (ROS) via excitation of 5-ALA mediated protoporphyrin IX (PpIX) in the tumor cells and causing a phototoxic reaction. After iPDT local chemo-radiation is performed as adjuvant therapy. 16 newly diagnosed glioblastomas and 44 malignant glioma recurrences treated with 5-ALA iPDT in Munich were retrospectively analyzed for treatment outcome, spectral online monitoring and changes in the MRI. iPDT for newly diagnosed glioblastomas showed a median overall survival (OS) of 28 months, 16.4 months progression free survival (PFS), respectively. 43.8% patients with newly diagnosed glioblastoma experienced a long term PFS > 24 months. In addition, the methylation of the MGMT promotor showed to be a prognostic factor for prolonged survival (p=0.04). In case of recurrent malignant gliomas PFS after iPDT was 7.1 months with 25% >24 months survival after iPDT (17.9% PFS > 24 months). Analysis of spectral online monitoring showed that a measured decrease of the laser light transmission between the cylindrical diffuser fibers, used for the irradiation, can be associated with silent hemorrhages visible in terms of T1-hyperintensity in the MRI after iPDT. Overall 5-ALA iPDT is a promising tool for the treatment of glioblastomas and other malignant gliomas with prolonged survival and minimally invasive surgery.

ObjectivesA fast, simple, versatile, and reliable method to record light emission intensity profiles of cylindrical light diffusers (CDFs) in air and transparent liquids has been developed.MethodsA fluorescent color glass filter (RG695) converts red light emitted by a cylindrical diffuser fiber into near-infrared light in an emission angle-independent manner. The red light was provided from a diode laser system at 635 nm. Near-infrared fluorescence from the RG695 was imaged with a camera. Images from this camera were processed to obtain emission intensity profiles. Cylindrical diffuser fiber profiles of four different manufacturers were compared.ResultsThe proposed method provides angle-independent intensity profiles of cylindrical diffuser fibers with a single camera shot. It could be demonstrated that dependent on the underlying principle of how the diffuser fiber tips emit light, the emission profile can change significantly in media with different refractive indices.ConclusionsBy converting the light emitted by a diffuser fiber tip into fluorescence light one can eliminate the dependence of the recorded profile on the emission angle from the diffusor. This approach allows for easily taking into account refraction-index (mis)matching by placing the equipment into a suitable liquid. The proposed measurement principle bears potential for quality assurance measurements of CDFs used for interstitial laser thermotherapy or photodynamic therapy.

Different clinical aspects of photodynamic therapy (PDT), like treatment planning, treatment and dosimetry protocols, spectral on-line-monitoring (SOM) as well as follow-up evaluation of clinical outcome, are of interest regarding further iPDT developments. For three specific applications, interstitial PDT (iPDT) for prostate cancer in urology, intraluminal PDT for cholangiocarcinoma in gastroenterology and stereotactic iPDT for glioblastoma in neurosurgery, the clinical scenario is described. If all information arising around a PDT intervention is combined, as shown on the example iPDT for glioblastoma, further insight about the involved processes and mechanisms can be derived and utilized to develop the therapeutic approach further as a whole.

Stereotactic interstitial photodynamic therapy using 5 aminolevulinic acid is a more upcoming approach for the treatment of malignant gliomas, whose treatment is remaining a major challenge in brain tumor therapy. The therapeutic outcome of 16 patients who underwent 5-ALA iPDT for newly diagnosed glioblastomas, are presented. In addition to the basic survival analysis, MRI data was analyzed concerning image changes after iPDT and the possibility to use these changes as prognostic factor for therapy response. Overall the iPDT showed a progression-free survival (PFS) of 16.4 months and an overall survival (OS) of 28.0 months. A PFS longer than 2-years was seen for 43.8% of iPDT patients. In contrast to this complete tumor resection with consecutive chemoradiation shows 8.9% 2-year PFS. Standard MRI-related prognostic factors of the tumor resection like necrosis-tumor ratio, tumor volume and post-treatment contrast enhancement are not useful for iPDT prognosis. This shows that the MRI interpretation has to be different compared to conventional tumor therapy. The survival results showed that iPDT is a potential treatment option especially for tumors, where standard therapy is not possible. Further studies are needed.

BACKGROUND AND OBJECTIVES: Fluorescence-guided resection (FGR) of malignant gliomas with five-aminolevulinic acid (5-ALA) is an established method using surgical microscopes equipped with filter systems for observing fluorescence. Over the past decade, new technologies have been introduced for the same purpose, with available publications evaluating their clinical efficacy based on varying criteria. This study aims to review technologies and concepts of validation in the context of 5-ALA-mediated FGR. METHODS: A systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement was performed to identify devices capable of detecting 5-ALA-induced fluorescence. Articles found eligible for this review were analyzed, focusing on the methods of validation used for novel devices. A qualitative analysis is presented. RESULTS: Using predefined eligibility criteria, 22 studies were analyzed. Publications on the following visualization devices were reviewed: FL400 (Leica Microsystems), Aeos (Aesculap), BLUE400 and BLUE400 AR Filter System (Carl Zeiss Meditec AG), Endoscope with D-Light C (Karl Storz), Fiberscope N-4L (Machida), ORBEYE 4K 3D Digital Video Microscope (Olympus), and several customized surgical loupe systems. In many cases, validation seemed unstandardized, with inherent biases and limited reproducibility. CONCLUSION: This review illustrates the significance of device validation within the framework of FGR. It emphasizes the criticality of validating devices in accordance with established standard, i.e. the BLUE400 filter system, which was employed in the approval studies of 5-ALA. Furthermore, standardized concepts of validation are required to assess whether new devices are, in fact, a reliable or superior alternative in the field of FGR. Published guidelines should be considered when performing future studies.

Applications of photodynamic therapy and diagnosis are widely spread over many medical disciplines [1]. Optical dosimetry calculations that include the estimation of thermal effects [2-3] are inevitable to guarantee a sufficient disease control while at the same time preserving tissue at risk [4]. Optical monitoring in an interstitial setting has turned out to be very elucidating, both regarding the initial state of the tissue to be treated, and regarding therapy-induced changes of the optical properties of the tissue [5-7], beyond the assessment of photobleaching. This information can be utilized immediately in the surgery room, to individualize, monitor and optimize the therapy session, as well as to warrant and optimize patient safety. Furthermore, by combining all available information around a PDT intervention, further insight about the involved processes and mechanisms can be gained [5-6], which can be utilized to develop the therapeutic approach further as a whole. As an example, the quite promising clinical outcome of iPDT treatments on malignant brain tumors will be summarized [7-8] and evaluation concepts based on spectral online monitoring and medical imaging [5-7], before and after the intervention, will be illustrated. iPDT-induced effects, including conversion of hemoglobin, will be discussed in relation to tumor coverage by the illumination volume and the location of recurrences.

Brain tumor treatment via interstitial photodynamic therapy (iPDT) needs precise treatment light delivery, which is essential for the conduction of the therapy [1]. The light delivery and the resulting light dosimetry are highly dependent on the optical tissue properties of the tumor tissue and the surrounding brain tissue. Employing intraoperative spectral online monitoring (SOM), it looks possible to assess the treatment light transmittance between the used light applicators and monitor potential changes during therapy [2]. Changes have been observed during clinical iPDT-illumination and can be interpreted as changes in the optical tissue properties [2, 3]. In vitro experiments mimicking the clinical iPDT-illumination situation using liquid optical tissue phantoms, including blood, showed SOM intensity changes in transmittance. Due to simultaneous remission spectroscopy, this can be related to the deoxygenation of hemoglobin and its oxidation to methemoglobin (MetHb) [4]. The analysis of data from clinical iPDT-procedures confirmed this interpretation. Based on intraoperative SOM data, changes in the optical absorption coefficient have been calculated and correlated with newly diagnosed early visible intrinsic T1-hyperintensity in the treatment volume [3]. The intrinsic T1 hyperintensity is clinically an indicator of the formation of MetHb after silent hemorrhages, which may occur during iPDT. As the T1 hyperintensity was early visible in the MRI, the corresponding early appearance of MetHb was in context with the iPDT and consistent with the in vitro experiments. Further in vitro experiments showed that changes in optical tissue properties and hemoglobin oxidation is not only possible due to ROS production during iPDT but also due to a slight temperature increase during iPDT by 4°C [5]. These results give more insight into mechanisms occurring during iPDT irradiation, but the impact on treatment outcome has still to be assessed.

An innovative treatment modality for brain tumors is interstitial photodynamic therapy [1,2]. The analysis of patients undergoing iPDT contain the assessment of the outcome, and qualitative changes in the MRI due to iPDT have already been reported [1,3].

Background Chlorin e6 trisodium salt (Ce6) is a newly developed hydrophilic photosensitizer designed to mediate anticancer photodynamic therapy (PDT). The response of different cancer types and strategies to boost anticancer efficiency of Ce6-PDT are poorly studied. Objectives This study aimed to investigate the response of different cancer types to Ce6-PDT, identify the unresponsive ones, and develop a nanosystem for response enhancement. Methods Sk-Br-3, MCF-7, U87, and HF-5 cells were tested in 2D cell cultures. Ce6 uptake, PDT-mediated phototoxicity, ROS production, caspase 3/7 levels, and cell death mode were examined. Furthermore, U87 spheroids were treated with Ce6-PDT. Mesoporous silica nanoparticles (MSN) were synthesized and loaded with Ce6. Cellular uptake and phototoxicity of MSN-Ce6 were compared to free Ce6 in vitro and in vivo. Results Ce6 was detectable in the cell cytoplasm within 15 min. U87 cells showed the highest Ce6 cellular uptake. Upon Ce6-PDT, U87 cells were the most responsive ones with an 11-fold increase in ROS production. Here, 5 µM Ce6 and 4 J/cm 2 were enough to reach IC50. Ce6-PDT induced both necrotic and caspase-dependent apoptotic cell death and 75% reduction of spheroids volume. Also, MCF-7 and HF-5 cells responded well to Ce6-PDT treatment. Sk-Br-3 breast cancer cells, on the other hand, were the least responsive ones with 80% viability after treatment (5 µM Ce6, 8 J/cm 2 ). However, MSN-Ce6 conjugates increased Sk-Br-3 cellular uptake of Ce6 sevenfold decreasing the IC50 irradiation dose by an order of magnitude. In a very aggressive breast cancer rat model , MSN-Ce6-PDT treatment led to suppression of tumor volume by 50% and elevation of both Bax and caspase 3 by 90% compared to the control while the corresponding values for Ce6-PDT were 30% and 70%, respectively. Conclusion The newly developed hydrophilic chlorin and even more its MSN conjugate show high activities in anticancer PDT.

Background: Frequent blood donors are at high risk of developing iron deficiency. Currently, there is no potent screening during blood donation to detect iron deficient erythropoiesis (IDE) before anemia develops and deferral from donation is inevitable. Study Design and Methods: In addition to capillary and venous hemoglobin, the iron status of 99 frequent blood donors was assessed by various venous blood parameters and zinc protoporphyrin IX (ZnPP). ZnPP was determined by high-performance liquid chromatography (HPLC) and a new prototype fiber-optic device was employed for non-invasive measurements of ZnPP through the blood collection tubing (NI-tubing) and on lip tissue (NI-lip). We aimed to evaluate the feasibility and diagnostic value of the NI-tubing measurement for early detection of severe iron deficiency in blood donors. Results: NI-tubing and HPLC reference measurements of ZnPP showed narrow limits of agreement of 12.2 μmol ZnPP/mol heme and very high correlation (Spearman’s Rho = 0.938). Using a cutoff of 65 μmol ZnPP/mol heme, NI-tubing measurements (n = 93) identified 100% of donors with iron deficiency anemia (IDA) and an additional 38% of donors with IDE. Accordingly, NI-tubing measurements would allow detection and selective protection of particularly vulnerable donors. Conclusion: NI-tubing measurements are an accurate and simple method to implement ZnPP determination into the routine blood donation process. ZnPP was able to identify the majority of subjects with IDE and IDA and might therefore be a valuable tool to provide qualified information to donors about dietary measures and adjustments of the donation interval and thereby help to prevent IDA and hemoglobin deferral in the future.

Background: The treatment of glioblastomas, the most common primary malignant brain tumors, with a devastating survival perspective, remains a major challenge in medicine. Among the recently explored therapeutic approaches, 5-aminolevulinic acid (5-ALA)-mediated interstitial photodynamic therapy (iPDT) has shown promising results. Methods: A total of 16 patients suffering from de novo glioblastomas and undergoing iPDT as their primary treatment were retrospectively analyzed regarding survival and the characteristic tissue regions discernible in the MRI data before treatment and during follow-up. These regions were segmented at different stages and were analyzed, especially regarding their relation to survival. Results: In comparison to the reference cohorts treated with other therapies, the iPDT cohort showed a significantly prolonged progression-free survival (PFS) and overall survival (OS). A total of 10 of 16 patients experienced prolonged OS (≥ 24 months). The dominant prognosis-affecting factor was the MGMT promoter methylation status (methylated: median PFS of 35.7 months and median OS of 43.9 months) (unmethylated: median PFS of 8.3 months and median OS of 15.0 months) (combined: median PFS of 16.4 months and median OS of 28.0 months). Several parameters with a known prognostic relevance to survival after standard treatment were not found to be relevant to this iPDT cohort, such as the necrosis–tumor ratio, tumor volume, and posttreatment contrast enhancement. After iPDT, a characteristic structure (iPDT remnant) appeared in the MRI data in the former tumor area. Conclusions: In this study, iPDT showed its potential as a treatment option for glioblastomas, with a large fraction of patients having prolonged OS. Parameters of prognostic relevance could be derived from the patient characteristics and MRI data, but they may partially need to be interpreted differently compared to the standard of care.

Purpose Innovative, efficient treatments are desperately needed for people with glioblastoma (GBM). Methods Sixteen patients (median age 65.8 years) with newly diagnosed, small-sized, not safely resectable supratentorial GBM underwent interstitial photodynamic therapy (iPDT) as upfront eradicating local therapy followed by standard chemoradiation. 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX was used as the photosensitizer. The tumors were irradiated with light at 635 nm wavelength via stereotactically implanted cylindrical diffuser fibers. Outcome after iPDT was retrospectively compared with a positively-selected in-house patient cohort (n = 110) who underwent complete tumor resection followed by chemoradiation. Results Median progression-free survival (PFS) was 16.4 months, and median overall survival (OS) was 28.0 months. Seven patients (43.8%) experienced long-term PFS > 24 months. Median follow-up was 113.9 months for the survivors. Univariate regression revealed MGMT-promoter methylation but not age as a prognostic factor for both OS (p = 0.04 and p = 0.07) and PFS (p = 0.04 and p = 0.67). Permanent iPDT-associated morbidity was seen in one iPDT patient (6.3%). Patients treated with iPDT experienced superior PFS and OS compared to patients who underwent complete tumor removal (p < 0.01 and p = 0.01, respectively). The rate of long-term PFS was higher in iPDT-treated patients (43.8% vs. 8.9%, p < 0.01). Conclusion iPDT is a feasible treatment concept and might be associated with long-term PFS in a subgroup of GBM patients, potentially via induction of so far unknown immunological tumor-controlling processes.

BackgroundDiagnosis of diffuse parenchymal lung disease (DPLD) is based on clinical evaluation, radiological imaging and histology. However, additional techniques are warranted to improve diagnosis.Aims and objectiveProbe based confocal laser endomicroscopy (pCLE) allows real time in vivo visualisation of the alveolar compartment during bronchoscopy based on autofluorescence of elastic fibres. We used pCLE (Cellvizio®, Mauna Kea Technology. Inc, Paris, France) to characterise alveolar patterns in patients with different types of DPLD.MethodsIn this pilot study we included 42 therapy naive patients (13 female, age 72.6 +/- 2.3 years), who underwent bronchoscopy for workup of DPLD. pCLE images were obtained during rigid bronchoscopy in affected lung segments according to HR-CT scan, followed by cryobiopsies in the identical area. Diagnoses were made by a multidisciplinary panel. The description of pCLE patterns was based on the degree of distortion of the hexagonal alveolar pattern, the density of alveolar structures, the presence of consolidations or loaded alveolar macrophages (AM). The assessment was performed by 2 investigators blinded for the final diagnosis.ResultsThe normal lung showed a typical alveolar loop pattern. In amiodarone lung disease loaded AM were predominant. COP showed characteristic focal consolidations. IPF was characterized by significant distortion and destruction, NSIP showed significant increase in density, and chronic HP presented with consolidations, mild distortion and density.ConclusionpCLE shows potential as an adjunctive bronchoscopic imaging technique in the differential diagnosis of DPLD. Structured and quantitative analysis of the images is required.

In a former study, interstitial photodynamic therapy (iPDT) was performed on patients suffering from newly diagnosed glioblastoma (n = 11; 8/3 male/female; median age: 68, range: 40–76). The procedure includes the application of 5-ALA to selectively metabolize protoporphyrin IX (PpIX) in tumor cells and illumination utilizing interstitially positioned optical cylindrical diffuser fibers (CDF) (2–10 CDFs, 2–3 cm diffusor length, 200 mW/cm, 635 nm, 60 min irradiation). Intraoperative spectral online monitoring (SOM) was employed to monitor treatment light transmission and PpIX fluorescence during iPDT. MRI was used for treatment planning and outcome assessment. Case-dependent observations included intraoperative reduction of treatment light transmission and local intrinsic T1 hyperintensity in non-contrast-enhanced T1-weighted MRI acquired within one day after iPDT. Intrinsic T1 hyperintensity was observed and found to be associated with the treatment volume, which indicates the presence of methemoglobin, possibly induced by iPDT. Based on SOM data, the optical absorption coefficient and its change during iPDT were estimated for the target tissue volumes interjacent between evaluable CDF-pairs at the treatment wavelength of 635 nm. By spatial comparison and statistical analysis, it was found that observed increases of the absorption coefficient during iPDT were larger in or near regions of intrinsic T1 hyperintensity (p = 0.003). In cases where PpIX-fluorescence was undetectable before iPDT, the increase in optical absorption and intrinsic T1 hyperintensity tended to be less. The observations are consistent with in vitro experiments and indicate PDT-induced deoxygenation of hemoglobin and methemoglobin formation. Further investigations are needed to provide more data on the time course of the observed changes, thus paving the way for optimized iPDT irradiation protocols.

Glioblastoma multiforme is a malignant neoplasia with a median survival of less than two years and without satisfactory therapeutic options. The so-called glioblastoma stem cells escape the established radio- and chemotherapies and lead to tumor recurrence in most cases. The alkaloid Shikonin with its various anti stem cell properties and the interstitial photodynamic therapy with 5-aminolevulinic acid seem to be promising new options in the therapy of glioblastoma. In this study, in vitro investigations were performed to observe the influence of Shikonin on viability, proliferation, induction of apoptosis and the capability of forming tumor spheres in U-87 MG and the primary glioblastoma cell line GB14. The combined effect with the chemotherapeutic temozolomide and photodynamic treatment on the mRNA expression of glioma specific stem cell markers and further examined intracellular protoporphyrin IX accumulation under Shikonin treatment was analyzed. Shikonin effectively inhibited the capability of forming tumor spheres and enhanced temozolomide effectiveness in the reduction of proliferation and in the induction of apoptosis. Additionally, Shikonin increased the mRNA expression of the tumor suppressing Neurofibromatosis type 1 (NF1) gene and showed modulating effects on intracellular protoporphyrin IX.

Losses in treatment light transmission during interstitial PDT were found to correlate with T1 hyperintensity in post-therapeutic non-contrast-enhanced T1-weighted MRI. This finding might be related to iPDT-induced early formation of methemoglobin.

Increased formation of fluorescent zinc protoporphyrin IX (ZnPP) inside erythrocytes indicates functional iron deficiency. Two-wavelength excitation spectroscopy detects ZnPP even through intact skin. Its suitability was successfully tested in women, surgical patients, infants and blood donors.

The temperature dependency (25°C – 50°C) of optical tissue properties was investigated on artificial and animal tissue samples. An increase of optical properties with temperature was found for all tissue phantoms and most tissue samples.