Diagnostic Accuracy of a Confocal Laser Endomicroscope for In Vivo Differentiation Between Normal Injured And Tumor Tissue During Fluorescein-Guided Glioma Resection: Laboratory Investigation.

OBJECTIVE: Glioma resection with fluorescein sodium (FNa) guidance has a potential drawback of nonspecific leakage of FNa from nontumor areas with a compromised blood-brain barrier. We investigated the diagnostic accuracy of in vivo confocal laser endomicroscopy (CLE) after FNa administration to differentiate normal brain, injured normal brain, and tumor tissue in an animal glioma model. METHODS: GL261-Luc2 gliomas in C57BL/6 mice were used as a brain tumor model. CLE images of normal, injured normal, and tumor brain tissues were collected after intravenous FNa administration. Correlative sections stained with hematoxylin and eosin were taken at the same sites. A set of 40 CLE images was given to 1 neuropathologist and 3 neurosurgeons to assess diagnostic accuracy and rate image quality (1-10 scale). Additionally, we developed a deep convolution neural network (DCNN) model for automatic image classification. RESULTS: The mean observer accuracy for correct diagnosis of glioma compared with either injured or uninjured brain using CLE images was 85%, and the DCNN model accuracy was 80%. For differentiation of tumor from nontumor tissue, the experts' mean accuracy, specificity, and sensitivity were 90%, 86%, and 96%, respectively, with high interobserver agreement overall (Cohen κ = 0.74). The percentage of correctly identified images was significantly higher for images with a quality rating >5 (104/116, 90%) than for images with a quality rating ≤5 (32/44, 73%) (P = 0.007). CONCLUSIONS: With sufficient FNa present in tissues, CLE was an effective tool for intraoperative differentiation among normal, injured normal, and tumor brain tissue. Clinical studies are warranted to confirm these findings.

Immediate ex-vivo diagnosis of pituitary adenomas using confocal reflectance microscopy: a proof-of-principle study.

OBJECTIVE The objective of this study was to evaluate the feasibility of using confocal reflectance microscopy (CRM) ex vivo to differentiate adenoma from normal pituitary gland in surgical biopsy specimens. CRM allows for rapid, label-free evaluation of biopsy specimens with cellular resolution while avoiding some limitations of frozen section analysis. METHODS Biopsy specimens from 11 patients with suspected pituitary adenomas were transported directly to the pathology department. Samples were immediately positioned and visualized with CRM using a confocal microscope located in the same area of the pathology department where frozen sections are prepared. An H & E-stained slide was subsequently prepared from imaged tissue. A neuropathologist compared the histopathological characteristics of the H & E-stained slide and the matched CRM images. A second neuropathologist reviewed images in a blinded fashion and assigned diagnoses of adenoma or normal gland. RESULTS For all specimens, CRM contrasted cellularity, tissue architecture, nuclear pleomorphism, vascularity, and stroma. Pituitary adenomas demonstrated sheets and large lobules of cells, similar to the matched H & E-stained slides. CRM images of normal tissue showed scattered small lobules of pituitary epithelial cells, consistent with matched H & E-stained images of normal gland. Blinded review by a neuropathologist confirmed the diagnosis in 15 (94%) of 16 images of adenoma versus normal gland. CONCLUSIONS CRM is a simple, reliable approach for rapidly evaluating pituitary adenoma specimens ex vivo. This technique can be used to accurately differentiate between pituitary adenoma and normal gland while preserving biopsy tissue for future permanent analysis, immunohistochemical studies, and molecular studies.

Immediate Label-Free Ex Vivo Evaluation of Human Brain Tumor Biopsies With Confocal Reflectance Microscopy.

Confocal microscopy utilizing fluorescent dyes is widely gaining use in the clinical setting as a diagnostic tool. Reflectance confocal microscopy is a method of visualizing tissue specimens without fluorescent dyes while relying on the natural refractile properties of cellular and subcellular structures. We prospectively evaluated 76 CNS lesions with confocal reflectance microscopy (CRM) to determine cellularity, architecture, and morphological characteristics. A neuropathologist found that all cases showed similar histopathological features when compared to matched hematoxylin and eosin-stained sections. RNA isolated from 7 tissues following CRM imaging retained high RNA integrity, suggesting that CRM does not alter tissue properties for molecular studies. A neuropathologist and surgical pathologist masked to the imaging results independently evaluated a subset of CRM images. In these evaluations, 100% of images reviewed by the neuropathologist and 95.7% of images reviewed by the surgical pathologist were correctly diagnosed as lesional or nonlesional. Furthermore, 97.9% and 91.5% of cases were correctly diagnosed as tumor or not tumor by the neuropathologist and surgical pathologist, respectively, while 95.8% and 85.1% were identified with the correct diagnosis. Our data indicate that CRM is a useful tool for rapidly screening patient biopsies for diagnostic adequacy, molecular studies, and biobanking.