Protoporphyrin IX Concentration Research Articles

Tumor Models for Training of Fluorescence-Guided Brain Tumor Resection

BACKGROUND AND OBJECTIVES: Neurosurgical resection of brain tumors is a technically demanding task with a steep initial learning curve. Experience has been shown to improve surgical performance and increase the rates of resection. High-grade gliomas (HGG) are responsible for 65% of all primary malignant central nervous system tumors and are the commonest surgically treated brain tumor worldwide. There is increasing evidence that maximal safe surgical resection of HGG impacts on overall survival. The delineation of tumor tissue (and hence the extent of resection) is improved by the use of intraoperative tumor fluorescence, which is achieved by preoperative oral administration of 5-aminolevulinic acid. This however relies on the surgeon developing a skillset of operating under different light conditions for which specific training and experience are required. METHODS: We propose a novel, affordable, and highly adaptable model for recreating these surgical conditions and thus simulate fluorescence-guided HGG resection outside the operating theater for trainee neuro-oncological surgeons. We present a model incorporating plant-based polysaccharides, doped with protoporphyrin IX (PpIX), into cadaveric brains of animals. RESULTS: We show that the concentrations of polymer and PpIX can be titrated (using previously published data) to produce mechanical and fluorescence properties that model tumor tissue. We validate the simulation in a neurosurgical skills laboratory. Furthermore, we used a mini spectrometer with handheld probe to collect fluorescence signals and validate the spectroscopic signal from the PpIX. CONCLUSION: It is hoped that this model system will be useful in neurosurgical teaching and training courses and to neurosurgeons who want to try out new techniques or equipment in anatomy laboratories.

Novel Iron-Chelating Prodrug Significantly Enhanced Fluorescence-Mediated Detection of Glioma Cells Experimentally In Vitro.

(1) Background: The protoporphyrin IX (PpIX)-mediated fluorescence-guided resection and interoperative photodynamic therapy (PDT) of remaining cells may be effective adjuvants to the resection of glioma. Both processes may be enhanced by increasing intracellular PpIX concentrations, which can be achieved through iron chelation. AP2-18 is a novel combinational drug, which ester-links a PpIX precursor (aminolaevulinic acid; ALA) to an iron-chelating agent (CP94). (2) Methods: Human glioma U-87 MG cells were cultured in 96-well plates for 24 h and incubated for 3 or 6 h with various test compound combinations: ALA (±) CP94, methyl aminolevulinate (MAL) (±) CP94 and AP2-18. PpIX fluorescence was measured at 0, 3 or 6 h with a Bio-tek Synergy HT plate reader, as well as immediately after irradiation with a 635 nm red light (Aktilite CL16 LED array), representing the PDT procedure. Cell viability post-irradiation was assessed using the neutral red assay. (3) Results: AP2-18 significantly increased PpIX fluorescence compared to all other test compounds. All treatment protocols effectively achieved PDT-induced cytotoxicity, with no significant difference between test compound combinations. (4) Conclusions: AP2-18 has potential to improve the efficacy of fluorescence-guided resection either with or without the subsequent intraoperative PDT of glioma. Future work should feature a more complex in vitro model of the glioma microenvironment.

Distribution and potential roles of microbial protoporphyrin IX in marine sediments

ABSTRACT Protoporphyrin IX (PPIX) serves as a pivotal precursor in the biosynthesis of porphyrins, playing a crucial role in both biological metabolism and biogeochemical cycling. This study investigates the spatial dynamics and co-occurrence patterns of PPIX within the microbial community of marine sediment. The concentrations of PPIX ranged from 7.38 - 91.33 ng/g. Clear patterns in spatiotemporal dynamics in the PPIX were observed, with a more pronounced spatial rather than seasonal variation. Notably, the PPIX and its derivatives in brackish sediment samples exhibited distinct characteristics from those found in saltwater samples. The Spearman test found that PPIX has a positive relationship with chlorophyll-a and pheophytin-a but not with chlorophyll-b or pheophytin-b. Significant positive correlations were identified between PPIX and nitrogen nutrients. Network and variation partitioning analyses highlighted the potential interactions between PPIX and microbial lineages. These findings indicate that PPIX is necessary for microorganisms and widely distributed in sedimentary environments. Additionally, this study underscores the influence of organic matter on microbial community composition and its responsiveness to environmental variations. These insights contribute to a comprehensive understanding of the potential ecological roles of PPIX within natural ecosystems.

Smartphone-based dual radiometric fluorescence and white-light imager for quantification of protoporphyrin IX in skin.

The quantification of protoporphyrin IX (PpIX) in skin can be used to study photodynamic therapy (PDT) treatments, understand porphyrin mechanisms, and enhance preoperative mapping of non-melanoma skin cancers. We aim to develop a smartphone-based imager for performing simultaneous radiometric fluorescence (FL) and white light (WL) imaging to study the baseline levels, accumulation, and photobleaching of PpIX in skin. A smartphone-based dual FL and WL imager (sDUO) is introduced alongside new radiometric calibration methods for providing SI-units of measurements in both pre-clinical and clinical settings. These radiometric measurements and corresponding PpIX concentration estimations are applied to clinical measurements to understand mechanistic differences between PDT treatments, accumulation differences between normal tissue and actinic keratosis lesions, and the correlation of photosensitizer concentrations to treatment outcomes. The sDUO alongside the developed methods provided radiometric FL measurements () with a demonstrated sub nanomolar PpIX sensitivity in 1% intralipid phantoms. Patients undergoing PDT treatment of actinic keratosis (AK) lesions were imaged, capturing the increase and subsequent decrease in FL associated with the incubation and irradiation timepoints of lamp-based PDT. Furthermore, the clinical measurements showed mechanistic differences in new daylight-based treatment modalities alongside the selective accumulation of PpIX within AK lesions. The use of the radiometric calibration enabled the reporting of detected PpIX FL in units of with the use of liquid phantom measurements allowing for the estimation of in-vivo molar concentrations of skin PpIX. The phantom, pre-clinical, and clinical measurements demonstrated the capability of the sDUO to provide quantitative measurements of PpIX FL. The results demonstrate the use of the sDUO for the quantification of PpIX accumulation and photobleaching in a clinical setting, with implications for improving the diagnosis and treatment of various skin conditions.

5-aminolevulinic acid, fluorescein sodium, and indocyanine green for glioma margin detection: analysis of operating wide-field and confocal microscopy in glioma models of various grades.

Surgical resection remains the first-line treatment for gliomas. Several fluorescent dyes are currently in use to augment intraoperative tumor visualization, but information on their comparative effectiveness is lacking. We performed systematic assessment of fluorescein sodium (FNa), 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX), and indocyanine green (ICG) fluorescence in various glioma models using advanced fluorescence imaging techniques. Four glioma models were used: GL261 (high-grade model), GB3 (low-grade model), and an in utero electroporation model with and without red fluorescence protein (IUE +RFP and IUE -RFP, respectively) (intermediate-to-low-grade model). Animals underwent 5-ALA, FNa, and ICG injections and craniectomy. Brain tissue samples underwent fluorescent imaging using a wide-field operative microscope and a benchtop confocal microscope and were submitted for histologic analysis. Our systematic analysis showed that wide-field imaging of highly malignant gliomas is equally efficient with 5-ALA, FNa, and ICG, although FNa is associated with more false-positive staining of the normal brain. In low-grade gliomas, wide-field imaging cannot detect ICG staining, can detect FNa in only 50% of specimens, and is not sensitive enough for PpIX detection. With confocal imaging of low-intermediate grade glioma models, PpIX outperformed FNa. Overall, compared to wide-field imaging, confocal microscopy significantly improved diagnostic accuracy and was better at detecting low concentrations of PpIX and FNa, resulting in improved tumor delineation. Neither PpIX, FNa, nor ICG delineated all tumor boundaries in studied tumor models, which emphasizes the need for novel visualization technologies and molecular probes to guide glioma resection. Simultaneous administration of 5-ALA and FNa with use of cellular-resolution imaging modalities may provide additional information for margin detection and may facilitate maximal glioma resection.

Cell senescence-associated porphyrin metabolism affects the efficacy of aminolevulinic acid-photodynamic diagnosis in bladder cancer

Aminolevulinic acid-photodynamic diagnosis (ALA-PDD) is a promising alternative method to detect cancer cells because of its high specificity and low rate of side effects. Exogenous ALA is administered and accumulates as protoporphyrin IX (PpIX) in cancer cells, which then emit red fluorescence following light irradiation to enable surgeons to accurately identify and remove cancerous tissue. Recent reports suggested that PpIX failed to accumulate in some patients who underwent ALA-PDD. We hypothesized that cell senescence, which is a relatively inactive state, affects porphyrin accumulation in bladder cancer cells. In this study, we evaluated the relationship between cell senescence and porphyrin accumulation in affecting the efficacy of ALA-PDD. First, we utilized three bladder cancer cell lines to evaluate senescence-related indicators and establish a cell senescence model. Then, we identified the differences in porphyrin production and the proteins involved in porphyrin accumulation between old and young cells. We found that compared with young cells, old cells possessed higher concentration of PpIX and had lower ABCG2 expression. The increase in PpIX levels following ABCG2 inhibition is three times higher in old cells than in young cells, suggesting that cell senescence was closely related with porphyrin accumulation in cancer. In conclusion, we found that the efficacy of ALA-PDD and porphyrin accumulation was relatively high in senescent cancer cells and that inhibition of ABCG2 could improve the efficacy of ALA-PDD in young bladder cancer cells.

SURG-24. QUANTITATIVE ANALYSIS OF TUMOR CELL DENSITY AND PROTOPORPHYRIN IX FLOURESCENCE IN GLIOMA PATIENTS USING PAIRED STIMULATED RAMAN HISTOLOGY AND TWO-PHOTON EXCITATION FLUORESCENCE MICROSCOPY

Abstract Fluorescence guidance is widely utilized to improve the precision of cancer surgery. 5-aminolevulinic acid, the most widely used fluorophore in glioma surgery, is thought to cause selective accumulation of fluorescent protoporphyrin IX (PpIX) in tumor cells by exploiting pathologic alterations in heme biosynthetic pathways. PpIX-induced fluorescence is highly specific for densely tumor-infiltrated tissue but less effective for visualizing the tumor periphery. To improve clinical detection of PpIX, we developed a microscope to perform paired stimulated Raman histology and two-photon excitation fluorescence microscopy (TPEF) and validated it in 175 fresh, unprocessed core tumor specimens from 75 high-grade glioma patients and three central nervous system lymphoma patients across three institutions. Surprisingly, intracellular PpIX accumulation was observed primarily in cells with histiocytic, rather than neoplastic morphology, and the number of cells concentrating PpIX within the cytoplasm was associated with the abundance of CD163 positive cells (p< 0.02). There was no correlation between the degree of tumor cellularity and the concentration of PpIX across all imaged specimens (R=-0.21). Our findings encourage reconsideration of the existing theory of 5-ALA-induced tumor cell fluorescence in gliomas and demonstrate how 5-ALA and TPEF imaging can provide a window into the immune microenvironment of human gliomas.

Special issue on enhanced photodynamic therapy: Part II

Special issue on enhanced photodynamic therapy: Part II

Protoporphyrin IX delayed fluorescence imaging: a modality for hypoxia-based surgical guidance.

.SignificanceHypoxia imaging for surgical guidance has never been possible, yet it is well known that most tumors have microregional chronic and/or cycling hypoxia present as well as chaotic blood flow. The ability to image oxygen partial pressure () is therefore a unique control of tissue metabolism and can be used in a range of disease applications to understand the complex biochemistry of oxygen supply and consumption.AimDelayed fluorescence (DF) from the endogenous molecule protoporphyrin IX (PpIX) has been shown to be a truly unique reporter of the local oxygen partial pressure in tissue. PpIX is endogenously synthesized by mitochondria in most tissues, and the particular property of DF emission is directly related to low microenvironmental oxygen concentration. Here, it is shown that PpIX has a unique emission in hypoxic tumor tissue regions, which is measured as a DF signal in the red to near-infrared spectrum.ApproachA time-gated imaging system was used for PpIX DF for wide field direct mapping of changes. Acquiring both prompt and DF in a rapid sequential cycle allowed for imaging oxygenation in a way that was insensitive to the PpIX concentration. By choosing adequate parameters, the video rate acquisition of images could be achieved, providing real-time tissue metabolic information.ResultsIn this report, we show the first demonstration of imaging hypoxia signals from PpIX in a pancreatic cancer model, exhibiting contrast relative to surrounding normal oxygenated tissues. Additionally, tissue palpation amplifies the signal and provides intuitive temporal contrast based upon neoangiogenic blood flow differences.ConclusionsPpIX DF provides a mechanism for tumor contrast that could easily be translated to human use as an intrinsic contrast mechanism for oncologic surgical guidance.

Double dose of 5-aminolevulinic acid and its effect on protoporphyrin IX accumulation in low-grade glioma.

Administration of 5-aminolevulinic acid (5-ALA) does not regularly elicit fluorescence in low-grade glioma (LGG) at currently established doses and timing of administration. One explanation may be differences in blood-brain barrier (BBB) integrity compared to high-grade glioma. The authors hypothesized that for a BBB semipermeable to 5-ALA there might be a relationship between plasma 5-ALA concentration and its movement into the brain. A higher dose would elicit more 5-ALA conversion into protoporphyrin IX (PPIX). The authors present a case series of patients harboring LGG who received higher doses of 5-ALA. Patients undergoing surgery for indeterminate glioma later diagnosed as LGG were included in this study. 5-ALA was administered at a standard dose of 20 mg/kg body weight (bw) 4 hours prior to induction of anesthesia. A subgroup of patients received a higher dose of 40 mg/kg bw. Fluorescence was evaluated visually and PPIX concentration (cPPIX) was determined ex vivo by hyperspectral measurements in freshly extracted tissue. All adverse events were recorded. A total of 23 patients harboring diffuse low-grade astrocytomas (n = 19) and oligodendrogliomas (n = 4) were analyzed. Thirteen patients received 20 mg/kg bw, and 10 patients received 40 mg/kg bw of 5-ALA. In the 20 mg/kg group, 30.8% (4 of 13) of tumors harbored areas of visible fluorescence, compared to 60% of cases (n = 6 of 10) with 40 mg/kg bw. The threshold to visibility was 1 μg/ml in both groups. Measured over all biopsies, the mean cPPIX was significantly higher in the double-dose group (1.8 vs 0.45 μg/ml; p < 0.001). In non-visibly fluorescent tissue the mean cPPIX was 0.146 μg/ml in the 20 mg/kg and 0.347 μg/ml in the 40 mg/kg group, indicating an increase of 138% (p < 0.001). These observations demonstrate different regions with different levels of PPIX accumulation in LGG. With higher 5-ALA doses cPPIX increases, leading to more regions surpassing the visibility threshold of 1 μg/ml. These observations can be explained by the fact that the BBB in LGG is semipermeable to 5-ALA. Higher 5-ALA doses result in more PPIX conversion, an observation with implications for future dosing in LGG.

P07.07.A Fluorescence lifetime imaging for improved visualization of 5-ALA fluorescence during biopsies of suspected brain tumors

Abstract Background Stereotactic brain tumor biopsies represent a standard neurosurgical technique enabling a precise histopathological tumor diagnosis which is of utmost importance for optimal further treatment allocation. However, non-diagnostic biopsies are not uncommon in routine clinical practice. To overcome this shortcoming, serial biopsies or intraoperative histopathology are typically performed prolonging the operating times and potentially increasing the risk of complications. Recently, the value of 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) fluorescence for visualization of diagnostic tumor tissue was demonstrated in navigation-guided brain tumor biopsies. However, a not negligible proportion of brain tumors lack visible fluorescence. In some of these tumors, a subvisual concentration of PpIX could be detected by spectroscopic analysis. Thus, detection of such subvisual PpIX accumulation in tumor samples in biopsies by the new “Fluorescence Lifetime Imaging” (FLIM) technique might lead to further improvement of this approach. Material and Methods Patients that underwent navigation-guided biopsy of a suspected brain tumor were eligible for inclusion in this study. After administration of 5-ALA, the tissue samples were checked for visible fluorescence during the navigation-guided stereotactic biopsy with a sterile microscope. In each sample the observed fluorescence signal was documented. Additionally, the tissue samples were investigated ex-vivo by FLIM for detection of PpIX accumulation. In each case, a histopathological diagnosis was established according to the WHO classification of CNS tumors. Results A total number of 20 tissue samples could be retrieved in 16 patients with suspected brain tumors. The biopsy with assistance of 5-ALA was feasible in all patients and no relevant side effects occurred. Visible PpIX fluorescence could be observed in 75% of cases. In contrast, 25% of samples did not show visible fluorescence. In one specimen, no fluorescence signal could be evaluated as the sample was covered by too much blood. However, significant accumulation of PpIX could be detected by FLIM in all samples in which evaluation was possible (n=19). A final histopathological diagnosis was feasible in all patients. Conclusion Fluorescence Lifetime Imaging is a promising new technique for improved detection of brain tumor tissue during navigation-guided biopsies. Thus, this new technique might lead in future to reduced operating times and complication rate by minimizing the cases that need serial biopsies or intraoperative histopathology.

Endothelin-1 depletion of cartilage oligomeric matrix protein modulates pulmonary artery superoxide and iron metabolism-associated mitochondrial heme biosynthesis.

This study examines if heme biosynthesis-associated iron metabolism is regulated in pulmonary arteries by endothelin-1 (ET1) potentially through modulating cartilage oligomeric matrix protein (COMP) availability. Our studies in organoid-cultured endothelium-rubbed bovine pulmonary arteries (BPAs) observed COMP depletion by siRNA or hypoxia increases NOX2 and superoxide and depletes mitochondrial SOD2. ET1 also increases superoxide in a manner that potentially impairs mitochondrial heme biosynthesis. In this study, organoid culture of BPA with ET1 (10 nM) increases superoxide in the mitochondrial matrix and extramitochondrial regions associated with COMP depletion, and COMP (0.5 μM) inhibited these superoxide increases. As mitochondrial matrix superoxide could impair heme biosynthesis from protoporphyrin IX (PpIX) by decreasing Fe2+ availability and/or ferrochelatase (FECH), we studied ET1, COMP, and COMP siRNA effects on the expression of FECH, transferrin receptor-1 (TfR1, an indicator of iron availability) and soluble guanylate cyclase (sGC, a key heme-dependent protein), and on measurements of PpIX (HPLC) and heme content. ET1 decreased FECH, heme, and sGC, and increased TfR1 and iron. COMP reversed these effects of ET1, and COMP decreased PpIX and increased heme in the absence of ET1. COMP siRNA increased PpIX detection and TfR1 expression and decreased the expression of FECH and sGC. Nitric oxide (spermine NONOate) relaxation of BPA was inhibited by ET1, and this was attenuated by COMP during exposure to ET1. Thus, COMP depletion by ET1 or siRNA modulates pulmonary artery iron metabolism, which results in loss of heme biosynthesis and heme-dependent cGMP mechanisms.

Three-dimensional quantification of protoporphyrin IX in photodynamic therapy using SFDI/DFT: A pilot experimental validation

Photodynamic therapy (PDT) dosimetry, including light dose, photosensitizer dose and tissue oxygen, has been a research focus in PDT. In this work, we present a three-dimensional (3D) quantification of protoporphyrin IX (PpIX) using combined spatial frequency domain imaging (SFDI) and diffuse fluorescence tomography (DFT). The SFDI maps both the distributions of tissue absorption and scattering properties at three wavelengths and accordingly provides the optical background for DFT and extracts the tissue oxygenation for assessing the therapeutic outcomes, while DFT dynamically monitors the 3D distribution of PpIX dose from measured fluorescence signals for the procedure optimization. A pilot in vivo application in tumor nude models showed that the proposed SFDI/DFT is able to dynamically trace changes in the PpIX concentration and tissue oxygen during the treatment, rendering it a potentially powerful tool for PDT to improve clinical efficacy.

Association of ABCG2 polymorphisms with susceptibility to anti-tuberculosis drug-induced hepatotoxicity in the Chinese population

The accumulation of endogenous hepatotoxin protoporphyrin IX (PPIX) in the liver was proposed to be a novel mechanism of anti-tuberculosis drug-induced hepatotoxicity (ATDH). ATP-binding cassette transporter G2 (ABCG2) plays an important role in modulating PPIX concentrations. This study aimed to explore the role of ABCG2 genetic polymorphisms in the risk of ATDH in Chinese patients. A 1:4 matched case–control study was performed among 202 ATDH cases and 808 controls. Conditional logistic regression model was used to estimate the association between genotypes and the risk of ATDH by odds ratios (ORs) with 95% confidence intervals (CIs). Male patients with CC genotype of rs2622605 had an increased risk of ATDH (adjusted OR = 1.615, 95% CI: 1.119–2.332, p = 0.011). The peak value of alkaline phosphatase (ALP) was significantly higher in male patients with CC genotype of rs2622605 than in those with TT + TC genotype during antituberculosis treatment (102.0 U/L vs. 98.0 U/L, p = 0.029). This is the first attempt to evaluate the association between ABCG2 genetic variants and the risk of ATDH. Based on the 1:4 matched case–control study, the polymorphism at rs2622605 in the ABCG2 gene may be associated with the susceptibility to ATDH in Chinese male patients.

Identification of excimer delayed fluorescence by Protoporphyrin IX: A novel access to local chromophore concentration?

Protoporphyrin IX (PpIX) is a molecule produced in the mitochondria following the administration of its approved precursor, aminolevulinic acid (ALA). Strong light absorber at different wavelengths in the visible range, PpIX is extensively used as a photosensitizer (PS) for Photodynamic Therapy (PDT). PpIX is also an ideal molecular probe for the quantification of the tissue oxygen partial pressure (pO2), as its delayed fluorescence (DF) is quenched by oxygen, creating a direct relationship between the DF lifetime and the pO2. A limitation of both techniques is the ignorance of the PpIX concentration in tissues when the pO2 is measured or during PDT. In this study, the prompt (PF) and delayed fluorescence of PpIX dissolved in DiMethylFormamide (DMF) were acquired, in absence of oxygen, at different PpIX concentrations. Measurements of the PpIX emission for different excitation energies and temperatures, as well as spectral considerations led to the conclusion that E-type (thermal) DF was the dominant DF mechanism at low PpIX excited states concentrations (density of absorbed energy Hε[PpIX]<1 μJ. cm-3, H:excitation radiant exposure per pulse, ε: molar extinction coefficient at excitation wavelength) while P-type (Triplet Triplet Annihilation) DF took place at higher excited states concentrations (Hε[PpIX]>10 μJ. cm-3). The gradual development of a strong, red-shifted structureless DF peak at 670nm, invisible in the PF and absorption spectra, strongly points towards the first observation of PpIX excimer DF (EDF). It appears that, similarly to other aromatic molecules, PpIX excimers can be formed either by the encounter of two molecules in the first excited triplet state T1, or by the reaction of an excited singlet S1 with a triplet T1. Excimer DF could be beneficially used to determine the local concentration of PpIX, as the initial DF intensity ratio I0670/I0630 is linearly correlated with the local PpIX concentration, and thus rises up to the challenge of PpIX based pO2 measurement and PDT. This work could also pave the way for a fine comprehension of the production, diffusion and catabolization of PpIX in biological tissues.

Photodynamic Activity of Protoporphyrin IX-Immobilized Cellulose Monolith for Nerve Tissue Regeneration.

The development of nerve conduits with a three-dimensional porous structure has attracted great attention as they closely mimic the major features of the natural extracellular matrix of the nerve tissue. As low levels of reactive oxygen species (ROS) function as signaling molecules to promote cell proliferation and growth, this study aimed to fabricate protoporphyrin IX (PpIX)-immobilized cellulose (CEPP) monoliths as a means to both guide and stimulate nerve regeneration. CEPP monoliths can be fabricated via a simple thermally induced phase separation method and surface modification. The improved nerve tissue regeneration of CEPP monoliths was achieved by the activation of mitogen-activated protein kinases, such as extracellular signal-regulated kinases (ERKs). The resulting CEPP monoliths exhibited interconnected microporous structures and uniform morphology. The results of in vitro bioactivity assays demonstrated that the CEPP monoliths with under 0.54 ± 0.07 μmol/g PpIX exhibited enhanced photodynamic activity on Schwann cells via the generation of low levels of ROS. This photodynamic activation of the CEPP monoliths is a cell-safe process to stimulate cell proliferation without cytotoxic side effects. In addition, the protein expression of phospho-ERK increased considerably after the laser irradiation on the CEPP monoliths with low content of PpIX. Therefore, the CEPP monoliths have a potential application in nerve tissue regeneration as new nerve conduits.

Fluorescence real-time kinetics of protoporphyrin IX after 5-ALA administration in low-grade glioma.

5-Aminolevulinic acid (5-ALA) induces fluorescence in high-grade glioma (HGG), which is used for resection. However, the value of 5-ALA-induced fluorescence in low-grade glioma (LGG) is unclear. Time dependency and time kinetics have not yet been investigated. The purpose of this study was to investigate real-time kinetics of protoporphyrin IX (PpIX) in LGG based on hyperspectral fluorescence-based measurements and identify factors that predict fluorescence. Patients with grade II gliomas and imaging from which HGGs could not be completely ruled out received 5-ALA at 20 mg/kg body weight 4 hours prior to surgery. Fluorescence intensity (FI) and PpIX concentration (CPpIX) were measured in tumor tissue utilizing a hyperspectral camera. Apparent diffusion coefficient (ADC)-based tumor cell density, Ki-67/MIB-1 index, chromosomal 1p/19q codeletion, and 18F-fluoroethyl-l-tyrosine (18F-FET) PET values and their role for predicting fluorescence were evaluated. Eighty-one biopsies from 25 patients were included. Tissues with fluorescence demonstrated FI and CPpIX maxima between 7 and 8 hours after administration. When visible fluorescence was observed, peaks of FI and CPpIX were observed within this 7- to 8-hour time frame, regardless of any MRI gadolinium contrast enhancement. Gadolinium enhancement (p = 0.008), Ki-67/MIB-1 index (p < 0.001), 18F-FET PET uptake ratio (p = 0.004), and ADC-based tumor cellularity (p = 0.017) significantly differed between fluorescing and nonfluorescing tissue, but not 1p/19q codeletions. Logistic regression demonstrated that 18F-FET PET uptake and Ki-67/MIB-1 index were independently related to fluorescence. This study reports a fluorescence-based assessment of CPpIX in human LGG tissues related to 18F-FET PET uptake and Ki-67/MIB-1. As in HGGs, fluorescence in LGGs peaked between 7 and 8 hours after 5-ALA application, which has consequences for the timing of administration.

Detection improvement of gliomas in hyperspectral imaging of protoporphyrin IX fluorescence – in vitro comparison of visual identification and machine thresholds

Background5-aminolevulinic acid (5-ALA) – precursor of protoporphyrin IX (PpIX) – is utilized in fluorescence guided surgery (FGS) of high-grade gliomas. PpIX is used to identify traces of glioma during resection. Visual inspection of the fluorescence seems inaccurate in comparison to optic techniques such as hyperspectral imaging (HSI). AimTo characterize the limits of PpIX fluorescence detection of (i) visual evaluation and (ii) HSI analysis and to (iii) develop a classification system for visible and non-visible PpIX fluorescence. MethodsSamples with increasing concentrations (C) of PpIX and non-fluorescent controls were evaluated using a surgical microscope under blue light illumination. Similar samples were imaged with a HSI system tuned to PpIX fluorescence peak wavelength (635 nm) and control (RGB) channels. Samples’ intensities were defined, leading to 96 analysed pixels after batching. ResultsThree expert neurosurgeons assessed the PpIX samples (n = 16) and controls (n = 8) with unanimous decisions (ICC = 0.704), resulting in 63% recognition rate, 48% sensitivity, 92% specificity, 92% positive predictive value (PPV) and 47% negative predictive value (NPV). HSI image analysis, comparing mean relative values, resulted in 96%, 100%, 86%, 94%, 100%, respectively. Minimum PpIX concentration detection for experts was 0.6–1.8 μmol/l and HSI's 0.03–0.15 μmol/l. ConclusionsPpIX concentrations of low-grade gliomas, and those reported on glioblastoma infiltration zones, are below experts’ detection threshold. HSI analysis exceeds the performance of expert's visual inspection nearly by 20-fold. Hybrid FGS-HSI systems should be investigated in parallel to long-term outcomes. Described methods are applicable as a standard for calibration, testing and development of subvisual FGS techniques.

5-Aminolevulinic Acid-Induced Porphyrin Contents in Various Brain Tumors: Implications Regarding Imaging Device Design and Their Validation.

Fluorescence-guided resections using 5-aminolevulinic acid (5-ALA)-induced tumor porphyrins have been established as an adjunct for malignant glioma surgery based on a phase III study using specifically adapted microscopes for visualizing fluorescing protoporphyrin IX (PPIX). New hardware technologies are being introduced, which claim the same performance as the original technology for visualizing fluorescence. This assumes that qualitative fluorescence detection is equivalent to the established standard, an assumption that needs to be critically assessed. To determine PPIX concentrations (cPPIX) in tissue that can be detected visually using the established BLUE400 filter system (Carl Zeiss Meditec, Oberkochen, Germany) as a basis for defining the performance of this system. Utilizing a hyperspectral imaging system, tumor samples from patients harboring different tumor tissues, with or without visible fluorescence, were analyzed. Absolute values of cPPIX were calculated after calibrating the system with fluorescence phantoms with known cPPIX. A total of 524 tumor samples from 162 patients were analyzed. Visual fluorescence under the BLUE400 filter was documented by experienced neurosurgeons. A 0.9μg/ml threshold of cPPIX was defined as the minimal concentration required to detect and discriminate visual fluorescence. This is the first report providing data on the threshold of cPPIX, which is visually detected using the current generation of microscopes, thus defining the specificity and sensitivity of this technology as initially tested in a randomized trial. Novel technologies should show similar characteristics in order to be used safely and effectively. If more sensitive, such technologies require further assessments of tumor selectivity.

Ultraviolet A phototest positivity is associated with higher free erythrocyte protoporphyrin IX concentration and lower transferrin saturation values in erythropoietic protoporphyria.

BackgroundErythropoietic protoporphyria (EPP) is a rare disorder of heme biosynthesis hallmarked by early‐onset photosensitivity and mainly due to defective ferrochelatase activity leading to increased erythrocyte protoporphyrin IX (PPIX) levels. Evidence regarding the relationship between erythrocyte PPIX concentration and photosensitivity is limited.MethodsTo investigate the relationship between free erythrocyte PPIX (FEP) concentration; routine laboratory tests, particularly iron metabolism biomarkers; and ultraviolet (UV) A/visible light phototesting findings, 20 genetically confirmed EPP and one XLPP treatment‐naive patients were included in our study. They underwent UVA and visible light phototesting. On the same day, blood samples were collected for measurement of FEP, serum iron, transferrin, transferrin saturation, and ferritin, 25‐hydroxyvitamin D, and liver enzyme levels.ResultsMedian FEP concentration at the time of phototesting was 57.50 (IQR: 34.58‐102.70) μg/g of Hb. UVA and visible light phototesting were positive in 9 (42.9%) and 8 (38.1%) patients, respectively. Median FEP concentration was significantly higher in UVA phototest–positive patients than in those negative (64.37 [IQR: 57.45‐121.82] vs 45.35 [IQR: 24.53‐74.61] μg/g of Hb, respectively; P = .04486). Similarly, UVA photosensitive individuals had significantly lower median serum iron levels (61.5 [IQR: 33.5‐84] μg/dL vs 109 [IQR: 63.25‐154] μg/dL, respectively; P = .01862) and transferrin saturation values (15.005 [IQR: 7.0775‐18.41] % vs 29.645 [IQR: 17.8225‐34.3575] %; P = .0109) than those negative.ConclusionsOur study demonstrates that UVA phototest positivity is associated with higher FEP concentration and lower transferrin saturation and serum iron concentration in EPP.