Confocal Laser Endomicroscopy Images Research Articles

P13.05.A Image annotation guideline for invivo confocal laser endomicroscopy, interrater reliability and how to learn from medical consensus for machine learning algorithms

Abstract Background Intraoperative confocal laser endomicroscopy (CLE) is an in vivo imaging technique increasingly studied in neurosurgery and neuropathology. It can be affected by artifacts introduced by the CLE device or related to the intraoperative setting. We developed and evaluated an image annotation guideline (AGL) to detect and eliminate images bearing no valuable information as a result of such artifacts. Images ware classified into good and bad quality, based on defined technical criteria, which are also considered relevant by clinical experts. Material and Methods Datasets were created from intraoperative CLE in vivo specimens of patients resected for brain tumors. The process from data collection to development of the ML algorithm followed 7 steps: data quality specification, image and metadata collection, AGL development, annotation, data allocation for clinical validation, clinical validation, and, optionally, algorithm development. Final diagnoses were obtained by pathological analysis. Artifacts were grouped into three categories: diminished signal-to-noise-ratio (dSNR), optical distortions (movement/perturbations), and contrast/brightness artifacts. Images were annotated by 4 medical data annotators (T4). For clinical validation, 500 images were excluded from the training data and additionally annotated by 3 board certified neuropathologists (NPs 1-3) with experience in CLE imaging, to determine the medical consensus on good and bad images. All raters (NPs) were compared against each other and against T4; T4 was also compared against the medical consensus. Cohen’s Kappa and overall percentage agreement (OPA) were used to evaluate inter-rater reliability. Positive percent agreement (PPA) and negative percentage agreement (NPA) were also used to evaluate agreement between medical consensus and T4. Results 21,616 CLE images and corresponding clinical metadata were collected from 94 patients and annotated. For each case between 27 and 815 CLE images were acquired over the course of the surgery (mean=175 images per case, SD=170.6). 11% and 13% of images were labeled as dSNR and distortion, respectively, and 34% as class contrast. 42% of the images represented the good quality images. Interrater agreement between the 3 NPs ranged between 0.30 and 0.59. Agreement between T4 and the medical consensus was substantial (Cohen’s Kappa >=0.61). OPA between T4 and the medical consensus was 80.60%, PPA 72.34% and NPA 87.92%. Conclusion Annotations according to a well-structured and expertly curated AGL show higher values for Cohen’s Kappa and Overall Percent Agreement (OPA) with the medical consensus, than that of individual experts among one another. Such an AGL can be considered appropriate and produces on par results with annotations by a group of experts in the field and can be further employed for training machine learning (ML) algorithms.

P05.01.B Distribution of the staining agent sodium-fluorescein in cerebral neoplasia - a comparison between intraoperative confocal laser endomicroscopy and in vitro fluorescence microscopy

Abstract Background While conventional frozen sections and other ex vivo microscopy always come along with artefacts, in vivo confocal laser endomicroscopy (CLE) offers less invasive, less manipulated imaging of living tissues. Understanding the distribution of the staining agent sodium-fluorescein (NaFl) plays a key role in establishing in vivo CLE as a new opportunity for real-time transmitted, in vivo imaging. By comparing the distribution of NaFl in in vivo and ex vivo CLE to conventional fluorescence microscopy of NaFl incubated tumor cell cultures we gain a better understanding of the staining mechanism. Material and Methods Sodium-fluorescein is the water-soluble sodium salt of fluorescein and is used as a fluorescent tracer in neurosurgery. The staining agent was applied intravenously at the beginning of the surgical procedure. In vivo CLE of the lesion was performed 30 to 50 minutes later and compared to ex vivo CLE imaging and conventional fluorescence microscopy. In addition, different tumor cell lines derived from malignant gliomas and carcinomas, respectively, were incubated with NaFl in vitro and the uptake of the fluorescent dye was monitored over time. Results From initial results, the intraoperative images showed specific fluorescein distribution depending on the architecture of the tumor entity. In most cases, glial tumors demonstrated higher accumulation of the staining agent in the extracellular tumor matrix, whereas the cells of carcinoma metastases appeared to take up NaFl intracellularly. These results were corroborated by NaFl uptake in cell culture experiments. Compared to ex vivo CLE, in vivo imaging offered a faster assessment of the tissue, brighter images and higher staining levels. Due to movement artefacts and the narrow intraoperative imaging time frame, in vivo CLE images were sometimes impaired by lower image quality. Conclusion The specific distribution of the fluorescent agent NaFl allowed for a discrimination between the different neoplastic entities. Images from in vivo and ex vivo confocal laser endomicroscopy showed NaFl uptake in concordance with the results of NaFl incubated cell cultures. Intraoperative, in vivo confocal laser endomicroscopy shows promising first results in the understanding of brain tumor histomorphology in situ. Being faster and less manipulated by artefacts than ex vivo investigations, it opens up wider opportunities for research and intraoperative diagnostics.

Bronchoscopic Journey of in vivo Real-Time Microscopic Imaging in ILD: A Case Series

Background: Patients with interstitial lung diseases (ILDs) frequently present with nondiagnostic high-resolution CT (HRCT) scan and bronchoalveolar lavage (BAL) results, resulting in the need for invasive surgical or cryo-lung biopsy that is associated with significant morbidity. Confocal laser endomicroscopy (CLE) and optical coherence tomography (OCT) are high-resolution laser and light-based techniques that provide real-time imaging of the alveolar compartment during bronchoscopy with a different depth and field of view. Objectives: The aim of the study was to correlate OCT and CLE imaging to HRCT imaging in ILD. Methods: This is a retrospective case series of 20 ILD patients who underwent alveolar CLE and OCT imaging during a standard bronchoscopy with BAL, followed by a lung biopsy when indicated. CLE and OCT imaging were compared to four main HRCT patterns and histology. The final diagnosis was based on the multidisciplinary discussion diagnosis. Results: Bronchoscopic CLE and OCT imaging were feasible and safe and provided additional high-detailed anatomical information compared to the HRCT. Bronchoscopic real-time CLE was capable of identification of “alveolar cells” (ground glass opacities) and lung fibrosis (increased alveolar elastin fibers). Bronchoscopic real-time OCT allowed for visualization of “patchy fibrotic disease”, “honeycombing” (microcysts), and mucosal granulomas in the airways. Conclusions: Bronchoscopic CLE and OCT of the alveolar compartment is feasible and safe and enables minimally invasive, high-resolution detection of specific ILD features with the potential to improve ILD diagnostics and monitoring and decrease the need for surgical or cryo-lung biopsies.

Characterization of ex vivo and in vivo intraoperative neurosurgical confocal laser endomicroscopy imaging.

BackgroundThe new US Food and Drug Administration-cleared fluorescein sodium (FNa)-based confocal laser endomicroscopy (CLE) imaging system allows for intraoperative on-the-fly cellular level imaging. Two feasibility studies have been completed with intraoperative use of this CLE system in ex vivo and in vivo modalities. This study quantitatively compares the image quality and diagnostic performance of ex vivo and in vivo CLE imaging.MethodsImages acquired from two prospective CLE clinical studies, one ex vivo and one in vivo, were analyzed quantitatively. Two image quality parameters – brightness and contrast – were measured using Fiji software and compared between ex vivo and in vivo images for imaging timing from FNa dose and in glioma, meningioma, and intracranial metastatic tumor cases. The diagnostic performance of the two studies was compared.ResultsOverall, the in vivo images have higher brightness and contrast than the ex vivo images (p < 0.001). A weak negative correlation exists between image quality and timing of imaging after FNa dose for the ex vivo images, but not the in vivo images. In vivo images have higher image quality than ex vivo images (p < 0.001) in glioma, meningioma, and intracranial metastatic tumor cases. In vivo imaging yielded higher sensitivity and negative predictive value than ex vivo imaging.ConclusionsIn our setting, in vivo CLE optical biopsy outperforms ex vivo CLE by producing higher quality images and less image deterioration, leading to better diagnostic performance. These results support the in vivo modality as the modality of choice for intraoperative CLE imaging.

Deep learning for size and microscope feature extraction and classification in Oral Cancer: enhanced convolution neural network

Background and Aim: Deep learning technology has not been implemented successfully in oral cancer images classification due to the overfitting problem. Due to the network arrangement and lack of proper data set for training, the network might not produce the required feature map with dimension reduction which result in overfitting problems. This research aims to reduce the overfitting by producing the required feature map with dimension reduction through using Convolutional Neural Network. Methodology: The proposed system uses the Enhanced Convolutional Neural Network and the autoencoder technique to increase the efficiency of feature extraction process and compresses the information. In this technique, unpooling and deconvolution is done to generate the input data to minimize the difference between input and output data. Furthermore, it extracts characteristic features from the input data set which regenerates the input data from those features by learning a network to reduce the overfitting problem. Results: Different value of accuracy and processing time is achieved using different sample group of Confocal Laser Endomicroscopy (CLE) images. Based on result, it shows that the proposed solution is better than the current system. Also, the proposed system has improved the classification accuracy by 5 ~ 5.5% in average and reduced the processing time by 20 ~ 30 milliseconds in average. Conclusion: The proposed system is focused on accurately classifying the oral cancer cells of different anatomical locations from the CLE images. Finally, this study enhances the accuracy and processing time using autoencoder method and solve the problem of overfitting.

Real-time correction of transverse dislocation and distortion in probe-based confocal laser endomicroscopy imaging

High speed scanning system based on resonant scanner can improve the imaging speed of probe-based confocal laser endomicroscopy (pCLE), which makes pCLE be a very effective tool to observe cell tissue morphology in vivo. However, since the speed of resonant scanner changes nonlinearly, transverse distortion will occur when the digitizer collects the image data at a constant rate. Moreover, accurate turning time is hard to be obtained due to the limitation of scanner hardware, although synchronous square wave signals are used for reference. Therefore, it is difficult to accurately segment the bidirectional scanned data into two adjacent lines of the image, which results in image dislocation. In this work, the offset value of each line is obtained by estimating the overall dislocation and local dislocation of the image respectively. Then, the start time of the sampling and sampling interval are adjusted based on the scanner speed to correct the image in real-time by FPGA. As a result, the dislocation and distortion of the image are corrected at video rates.

Introduction of In Vivo Confocal Laser Endomicroscopy and Real-Time Telepathology for Remote Intraoperative Neurosurgery-Pathology Consultation.

Precise communication between neurosurgeons and pathologists is crucial for optimizing patient care, especially for intraoperative diagnoses. Confocal laser endomicroscopy (CLE) combined with a telepathology software platform (TSP) provides a novel venue for neurosurgeons and pathologists to review CLE images and converse intraoperatively in real-time. To describe the feasibility of integrating CLE and a TSP in the surgical workflow for real-time review of in vivo digital fluorescence tissue imaging in 3 patients with intracranial tumors. Although the neurosurgeon used the CLE probe to generate fluorescence images of histoarchitecture within the operative field that were displayed on monitors in the operating room, the pathologist simultaneously remotely viewed the CLE images. The neurosurgeon and pathologist discussed in real-time the histological structures of intraoperative imaging locations. The neurosurgeon placed the CLE probe at various locations on and around the tumor, in the surgical resection bed, and on surrounding brain tissue with communication through the TSP. The neurosurgeon oriented the pathologist to the location of the CLE, and the pathologist and neurosurgeon discussed the CLE images in real-time. The TSP and CLE were integrated successfully and rapidly in the operating room in all 3 cases. No patient had perioperative complications. Two novel digital neurosurgical cellular imaging technologies were combined with intraoperative neurosurgeon-pathologist communication to guide the identification of abnormal histoarchitectural tissue features in real-time. CLE with the TSP may allow rapid decision-making during tumor resection that may hold significant advantages over the frozen section process and surgical workflow in general.

Real-time intraoperative surgical telepathology using confocal laser endomicroscopy.

Communication between neurosurgeons and pathologists is mandatory for intraoperative decision-making and optimization of resection, especially for invasive masses. Handheld confocal laser endomicroscopy (CLE) technology provides in vivo intraoperative visualization of tissue histoarchitecture at cellular resolution. The authors evaluated the feasibility of using an innovative surgical telepathology software platform (TSP) to establish real-time, on-the-fly remote communication between the neurosurgeon using CLE and the pathologist. CLE and a TSP were integrated into the surgical workflow for 11 patients with brain masses (6 patients with gliomas, 3 with other primary tumors, 1 with metastasis, and 1 with reactive brain tissue). Neurosurgeons used CLE to generate video-flow images of the operative field that were displayed on monitors in the operating room. The pathologist simultaneously viewed video-flow CLE imaging using a digital tablet and communicated with the surgeon while physically located outside the operating room (1 pathologist was in another state, 4 were at home, and 6 were elsewhere in the hospital). Interpretations of the still CLE images and video-flow CLE imaging were compared with the findings on the corresponding frozen and permanent H&E histology sections. Overall, 24 optical biopsies were acquired with mean ± SD 2 ± 1 optical biopsies per case. The mean duration of CLE system use was 1 ± 0.3 minutes/case and 0.25 ± 0.23 seconds/optical biopsy. The first image with identifiable histopathological features was acquired within 6 ± 0.1 seconds. Frozen sections were processed within 23 ± 2.8 minutes, which was significantly longer than CLE usage (p < 0.001). Video-flow CLE was used to correctly interpret tissue histoarchitecture in 96% of optical biopsies, which was substantially higher than the accuracy of using still CLE images (63%) (p = 0.005). When CLE is employed in tandem with a TSP, neurosurgeons and pathologists can view and interpret CLE images remotely and in real time without the need to biopsy tissue. A TSP allowed neurosurgeons to receive real-time feedback on the optically interrogated tissue microstructure, thereby improving cross-functional communication and intraoperative decision-making and resulting in significant workflow advantages over the use of frozen section analysis.

Confocal Laser Endomicroscopy Can Improve the Diagnosis Rate and Range Assessment of Patients With Conflicting Chronic Atrophic Gastritis Results of White Light Endoscopic and Pathological Diagnosis.

Background and AimsChronic atrophic gastritis (CAG) is closely related to the development of gastric cancer. However, the diagnostic accuracy of white light endoscopy (WLE) biopsy for CAG is poor. The diagnostic role and efficacy of confocal laser endomicroscopy (CLE) in CAG missed under WLE biopsy remain unclear.MethodsThis study is a single-center prospective study that included 21 patients from 1,349 patients who underwent WLE and biopsy and whose WLE results confirmed CAG, but pathological results did not. Then, all these patients received CLE examination and underwent targeted biopsies and five-point standard biopsies. The sensitivity, specificity, and accuracy of CLE diagnosis and targeted biopsy were analyzed.ResultsThe pathological results of five-point standard biopsies in 21 patients confirmed CAG, and 17 patients (81.0%) were confirmed to have intestinal metaplasia (IM). According to the image diagnosis of CLE, there were 19 cases (90.5%) of CAG and 14 cases (66.7%) of IM among these 21 patients. According to the targeted biopsy of CLE, 17 cases (81.0%) of CAG and 14 cases (66.7%) of IM were diagnosed. There was no significant difference between CLE image diagnosis and five-point standard biopsies in terms of atrophy severity score (p = 0.927), IM severity score (p = 0.250), atrophy scope score (p = 0.781), and IM scope score (p = 0.195). For CAG, the sensitivity and accuracy of CLE image diagnosis were higher than those of CLE targeted biopsies (90.5% vs. 81.0%, p = 0.331), but for IM, the diagnosis was the same.ConclusionsCLE can improve the diagnosis rate of CAG and can increase the comprehensive assessment of the scope and severity of CAG.

Hybrid confocal Raman endomicroscopy for morpho-chemical tissue characterization

Confocal laser endomicroscopy (CLE) offers imaging of tissue microarchitecture and has emerged as a promising tool for in vivo clinical diagnosis of cancer across many organs. CLE, however, can show high inter-observer dependency and does not provide information about tissue molecular composition. In contrast, Raman spectroscopy is a label-free optical technique that provides detailed biomolecular compositional information but offers limited or no morphological information. Here we present a novel hybrid fiber-optic confocal Raman endomicroscopy system for morpho-chemical tissue imaging and analysis. The developed confocal endomicroscopy system is based on a novel detection scheme for rejecting Raman silica fiber interference permitting simultaneous CLE imaging and Raman spectral acquisition of tissues through a coherent fiber bundle. We show that this technique enables real-time microscopic visualization of tissue architecture as well as simultaneous pointwise label-free biomolecular characterization and fingerprinting of tissue paving the way for multimodal diagnostics at endoscopy.

Image-guided in-Vivo Needle-Based Confocal Laser Endomicroscopy in the Prostate: Safety and Feasibility Study in 2 Patients.

Purpose: To assess the safety and technical feasibility of in-vivo needle-based forward-looking confocal laser endomicroscopy in prostate tissue. Methods: For this feasibility study, 2 patients with a suspicion of prostate cancer underwent transperineal needle-based confocal laser endomicroscopy during ultrasound-guided transperineal template mapping biopsies. After intravenous administration of fluorescein, needle-based confocal laser endomicroscopy imaging was performed with a forward-looking probe (outer diameter 0.9 mm) in 2 trajectories during a manual push-forward and pullback motion. A biopsy was taken in a coregistered parallel adjacent trajectory to the confocal laser endomicroscopy trajectory for histopathologic comparison. Peri- and postprocedural adverse events, confocal laser endomicroscopy device malfunction and procedural failures were recorded. Needle-based confocal laser endomicroscopy image quality assessment, image interpretation, and histology were performed by an experienced confocal laser endomicroscopy rater and uro-pathologist, blinded to any additional information. Results: In both patients, no peri- and post-procedural adverse events were reported following needle-based confocal laser endomicroscopy. No confocal laser endomicroscopy device malfunction nor procedural failures were reported. Within 1.5 min after intravenous administration of fluorescein, needle-based confocal laser endomicroscopy image quality was sufficient for interpretation for at least 14 min, yielding more than 5000 confocal laser endomicroscopy frames per patient. The pullback confocal laser endomicroscopy recordings and most of the push-forward recordings almost only visualized erythrocytes, being classified as non-representative. During the push-forward recordings, prostate tissue was occasionally visualized in single frames, insufficient for histopathologic comparison. Prostate carcinoma was identified by biopsy in one patient (Gleason score 4 + 3 = 7, >50%), while the biopsy from the other patient showed no malignancy. Conclusion: Needle-based confocal laser endomicroscopy imaging of in-vivo prostate tissue with a forward-looking confocal laser endomicroscopy probe is safe without device malfunctions or procedural failures. Needle-based confocal laser endomicroscopy is technically feasible, but the acquired confocal laser endomicroscopy datasets are non-representative. The confocal laser endomicroscopy images’ non-representative nature is possibly caused by bleeding artifacts, movement artifacts and a lack of contact time with the tissue of interest. A different confocal laser endomicroscopy probe or procedure might yield representative images of prostatic tissue.

Confocal laser endomicroscopy in gastro-intestinal endoscopy: technical aspects and clinical applications.

Confocal laser endomicroscopy (CLE) is an advanced endoscopic imaging technology that provides a magnified, cellular level view of gastrointestinal epithelia. In conjunction with topical or intravenous fluorescent dyes, CLE allows for an "optical biopsy" for real-time diagnosis. Two different CLE system have been used in clinical endoscopy, probe-based CLE (pCLE) and endoscope-based CLE (eCLE). Using pCLE, the device can be delivered: (I) into the luminal gastrointestinal tract through the working channel of standard endoscopes; (II) into extraluminal cystic and solid parenchymal lesions through an endoscopic ultrasound (EUS) needle; or (III) into the biliary system through an endoscopic retrograde cholangiopancreatography (ERCP) accessory channel. With eCLE, the probe is directly integrated into the tip of a conventional endoscope, however, these endoscopes are no longer commercially available. CLE has moderate to high diagnostic accuracy for neoplastic and inflammatory conditions through the gastrointestinal tract including: oesophageal, gastric and colonic neoplasia, pancreatic cysts and solid lesions, malignant pancreatobiliary strictures and inflammatory bowel disease. Some studies have demonstrated the diagnostic benefit of CLE imaging when combined with either conventional white light endoscopy or advanced imaging technologies. Therefore, optical biopsies using CLE can resolve diagnostic dilemmas in some cases where conventional imaging fails to achieve conclusive results. CLE could also reduce the requirement for extensive tissue sampling during surveillance procedures. In the future, CLE in combination with molecular probes, could allow for the molecular characterization of diseases and assess response to targeted therapy. However, the narrow field of view, high capital costs and specialized operator training requirements remain the main limitations. Future multi-center, randomized trials with a focus on conventional diagnostic applications, cost-effectiveness and standardized training will be required for definitive evidence. The objective of this review is to evaluate the technical aspects and current applications of CLE in patients with gastrointestinal and pancreatobiliary diseases and discuss future directions for this technique.

Evaluation of the diagnostic value of confocal laser endomicroscopy images in brain tumors

Evaluation of the diagnostic value of confocal laser endomicroscopy images in brain tumors

Intensity-based nonrigid endomicroscopic image mosaicking incorporating texture relevance for compensation of tissue deformation

Image mosaicking has emerged as a universal technique to broaden the field-of-view of the probe-based confocal laser endomicroscopy (pCLE) imaging system. However, due to the influence of probe-tissue contact forces and optical components on imaging quality, existing mosaicking methods remain insufficient to deal with practical challenges. In this paper, we present the texture encoded sum of conditional variance (TESCV) as a novel similarity metric, and effectively incorporate it into a sequential mosaicking scheme to simultaneously correct rigid probe shift and nonrigid tissue deformation. TESCV combines both intensity dependency and texture relevance to quantify the differences between pCLE image frames, where a discriminative binary descriptor named fully cross-detected local derivative pattern (FCLDP) is designed to extract more detailed structural textures. Furthermore, we also analytically derive the closed-form gradient of TESCV with respect to the transformation variables. Experiments on the circular dataset highlighted the advantage of the TESCV metric in improving mosaicking performance compared with the other four recently published metrics. The comparison with the other four state-of-the-art mosaicking methods on the spiral and manual datasets indicated that the proposed TESCV-based method not only worked stably at different contact forces, but was also suitable for both low- and high-resolution imaging systems. With more accurate and delicate mosaics, the proposed method holds promises to meet clinical demands for intraoperative optical biopsy.

Intranasal inoculation with Bordetella pertussis confers protection without inducing classical whooping cough in baboons.

The resurgence of whooping cough in many countries highlights the crucial need for a better understanding of the pathogenesis of respiratory infection by Bordetella pertussis. Exposure of baboons to B. pertussis by the intranasal and intra-tracheal routes is a recently described preclinical model that reproduces both B. pertussis infection of humans and whooping cough disease. Here, we tested both intranasal and intranasal+intra-tracheal exposure routes and assessed their impact on disease development and immunity. Young baboons were intranasally exposed to the B1917 clinical isolate, representative of circulating strains in Europe, or its green-fluorescent protein expressing derivative. Animals were followed for pertussis symptoms and bacterial colonization and by in vivo probe-based confocal laser endomicroscopy (pCLE) imaging. Sero-conversion and protection against subsequent infection were then evaluated. Seroconversion and bacterial colonization of both the nasopharynx and trachea was observed in baboons exposed to B. pertussis by the intranasal route only, and also in those animals challenged by both the intranasal and intra-tracheal routes together. However, baboons exposed solely by the intranasal route developed only mild clinical symptoms, with no paroxysmal cough. These animals were protected against re-infection by B. pertussis. Intranasal exposure of baboons to B. pertussis does not induce disease but elicits immune mechanisms that protect them from subsequent exposure to the bacteria. These findings suggest that the intranasal route of inoculation in this non-human primate model could be used in the pre-clinical evaluation of nasal candidate vaccines against pertussis.

Redosing of Fluorescein Sodium Improves Image Interpretation During Intraoperative Ex Vivo Confocal Laser Endomicroscopy of Brain Tumors

BackgroundFluorescein sodium (FNa) is a fluorescence agent used with a wide-field operating microscope for intraoperative guidance and with confocal laser endomicroscopy (CLE) to evaluate brain tissue. Susceptibility of FNa to degradation over time may affect CLE image quality during prolonged surgeries. This study describes improved characteristics of CLE images after intraoperative redosing with FNa.MethodsA retrospective analysis was performed using CLE images obtained ex vivo from samples obtained during tumor resections with FNa-based fluorescence guidance with a wide-field operating microscope. The comparison groups included CLE images acquired after FNa redosing (redose imaging group), images from the same patients acquired after the initial FNa dose (initial-dose imaging group), and images from patients in whom redosing was not used (single-dose imaging group). A detailed assessment of image quality and interpretation regarding different FNa dosage and timing of imaging after FNa administration was conducted for all comparison groups.ResultsThe brightest and most contrasting images were observed in the redose group compared to the initial-dose and single-dose groups (P<0.001). The decay of FNa signal negatively correlated with brightness (rho = -0.52, P<0.001) and contrast (rho = -0.57, P<0.001). Different doses of FNa did not significantly affect the brightness (P=0.15) or contrast (P=0.09) in CLE images. As the mean timing of imaging increased, the percentage of accurately diagnosed images decreased (P=0.03).ConclusionsThe decay of the FNa signal is directly associated with image brightness and contrast. The qualitative interpretation scores of images were highest for the FNa redose imaging group. Redosing with FNa to improve the utility of CLE imaging should be considered a safe and beneficial strategy during prolonged surgeries.

High-Dose Fluorescein Reveals Unusual Confocal Endomicroscope Imaging of Low-Grade Glioma.

Background: Fluorescence-guided brain tumor surgery using fluorescein sodium (FNa) for contrast is effective in high-grade gliomas. However, the effectiveness of this technique for visualizing noncontrast-enhancing and low-grade gliomas is unknown. This report is the first documented case of the concurrent use of wide-field fluorescence-guided surgery and confocal laser endomicroscopy (CLE) with high-dose FNa (40 mg/kg) for intraoperative visualization of tumor tissue cellularity in a nonenhancing glioma.Case Description: A patient underwent fluorescence-guided surgery for a left frontal lobe mass without contrast enhancement on magnetic resonance imaging. The patient received 40 mg/kg FNa intravenously at the induction of anesthesia. Surgery was performed under visualization with a Yellow 560 filter and white-light wide-field imaging. Intraoperative CLE produced high-quality images of the lesion 1.5 h after FNa injection. Frozen-section analysis demonstrated findings comparable to those of intraoperative CLE visualization and consistent with World Health Organization (WHO) glioma grades II–III. The patient recovered without complications. Analysis of the permanent histologic sections identified the tumor as an anaplastic oligodendroglioma, IDH-mutant, 1p/19q co-deleted, consistent with WHO grade III because of discrete foci of hypercellularity and increased mitotic figures, but large regions of the lesion were low grade.Conclusions: The use of high-dose FNa in this patient with a nonenhancing borderline low-grade/high-grade glioma produced actionable wide-field fluorescence imaging using the operating microscope and improved CLE visualization of tumor cellularity. Higher doses of FNa for intraoperative CLE imaging and possible simultaneous wide-field fluorescence surgical guidance in nonenhancing gliomas merit further investigation.

Cross-sectional imaging of intestinal barrier dysfunction by confocal laser endomicroscopy can identify patients with food allergy in vivo with high sensitivity

Food allergy (FA) affects approximately 3 to 4% of the adult population in westernized countries. Suspected FA is even more prevalent and requires extensive diagnostic work-up. Within this study, we evaluated whether assessment of the integrity of the epithelial barrier by confocal laser endomicroscopy (CLE) during colonoscopy can be used as a screening tool to identify patients with FA. 60 patients with suspected FA were prospectively included. Serology with total and food-specific IgE, anti-tissue transglutaminase, skin prick testing, food intolerance tests, food intake registration and assessment of clinical complaints were performed. During colonocopy, standardized CLE was performed in the terminal ileum and at two colorectal sites. Analysis of CLE images included functional (i.e. presence of epithelial barrier dysfunction) and quantitative parameters of intestinal architecture. 27 of 60 patients (45%) were diagnosed with FA. Barrier dysfunction was analyzed on 65.837 ileal and on 93.251 colonic images. 96% of patients with FA exhibited functional and structural barrier defects while barrier dysfunction was found in only 33% of patients without FA (p < 0.0001). Visualizing barrier dysfunction with CLE for in vivo diagnosis of FA had a sensitivity and specificity of 96% and 67%, respectively, with a positive and negative prediction of 70% and 96%, respectively. Parameters intrinsic to the crypt architecture including crypt diameter, intercrypt distance, crypt lumen diameter and colonic vasculature were not different between patients with and without FA. CLE-based imaging of the intestinal barrier during colonoscopy might help in stratifying patients with suspected FA for further diagnostic work-up.

Fluorescein-Guided Panendoscopy for Head and Neck Cancer Using Handheld Probe-Based Confocal Laser Endomicroscopy: A Pilot Study.

BackgroundWhite-light endoscopy and microscopy combined with histological analysis is currently the mainstay for intraprocedural tissue diagnosis during panendoscopy for head and neck cancer. However, taking biopsies leads to selection bias, ex vivo histopathology is time-consuming, and the advantages of in-vivo intraoperative decision making cannot be used. Confocal laser endomicroscopy (CLE) has the potential for a rapid and histological assessment in the head and neck operating room.MethodsBetween July 2019 and January 2020, 13 patients (69% male, median age: 61 years) with newly diagnosed head and neck cancer (T3/T4: 46%) underwent fluorescein-guided panendoscopy. CLE was performed from both the tumor and margins followed by biopsies from the CLE spots. The biopsies were processed for histopathology. The CLE images were ex vivo classified blinded with a CLE cancer score (DOC score). The classification was compared to the histopathological results.ResultsMedian additional time for CLE during surgery was 9 min. A total of 2,565 CLE images were taken (median CLE images: 178 per patient; 68 per biopsy; evaluable 87.5%). The concordance between histopathology and CLE images varied between the patients from 82.5 to 98.6%. The sensitivity, specificity, and accuracy to detect cancer using the classified CLE images was 87.5, 80.0, and 84.6%, respectively. The positive and negative predictive values were 87.0 and 80.0%, respectively.ConclusionCLE with a rigid handheld probe is easy and intuitive to handle during panendoscopy. As next step, the high accuracy of ex vivo CLE image classification for tumor tissue suggests the validation of CLE in vivo. This will evolve CLE as a complementary tool for in vivo intraoperative diagnosis during panendoscopy.

P397 Response to biologics in IBD patients assessed by Computerized image analysis of Probe Based Confocal Laser Endomicroscopy with molecular labeling

Abstract Background The increase in therapeutic choices in inflammatory bowel diseases (IBD) imposed the identification of personalized therapeutic strategy. Confocal laser endomicroscopy (CLE) is a new endoscopic tool developed to obtain virtual in vivo histology. This study aimed to identify CLE in vivo and ex vivo features predictive of response for patients starting biologics. Methods We performed a prospective observational study at a single tertiary referral centre. 29 IBD patients (14 ulcerative colitis-UC and 15 Crohn’s Disease-CD) underwent colonoscopy with CLE before and 12-14 weeks after starting anti -TNF or 16 weeks after anti α4β7 integrin. CLE parameters analyzed were: crypt distribution along the mucosal surface, crypt area (CA), eccentricity, diameter, inter-cryptic distance (ICD), vessel tortuosity (VT), fluorescein leakage through the colonic mucosa (FLCM). Ex-vivo binding activity of fluorescein labelled biologics on corresponding biopsies was also assessed. Mosaicism reconstruction of CLE images were analyzed using a dedicated software algorithm (CellvizioViewer, Mauna Kea Technologies, ParisFrance). A dedicated Graphical User Interface was designed to enable a semiautomated analysis. Results After treatment, VT resulted significantly changed in overall IBD population (p&amp;lt;0.05), FLCM were reduced in UC patients (p&amp;lt;0.05), whilst CA, eccentricity and ICD decreased in CD patients (p&amp;lt; 0.05). The univariate regression analysis identified FLCM as the most accurate parameter for predicting responsiveness (AUROC) 83%, accuracy 83%, positive predictive value-PPV 94% and negative predictive value-NPV 57%). FLCM and ICD were the best discriminants in responders Vs non responders in UC patients (AUROC 85%, accuracy 85%, PPV 100% and NPV 71%); whilst VT, CA and ICD in CD patients (AUROC 95%, 86% and 83%; accuracy 90%, 90% and 88%; PPV 100%, 100% and 86%; and NPV 75%, 75% and 100%, respectively). Ex vivo confocal imaging revealed that UC patients, especially those responders, had higher basal fluorescent intensity signals with a significant reduction after biological treatment (p&amp;lt; 0.05), whereas in CD patients no significant change was found. An increased mucosal binding to the fluorescent labelled biological agent was associated to a higher likelihood of response to the treatment; this effect was higher in UC patients (AUROC 81%, accuracy 77%, PPV 100%, NPV 63%) compared to CD patients (AUROC 64%, accuracy 79%, PPV 80%, NPV 50%). Conclusion FLCM and ICD were the best discriminants of response in UC, while VT, CA and ICD in CD. A higher mucosal binding to a biological agent before treatment was observed in responders UC patients but not in CD patients.