ABSTRACT
Extracellular vesicles (EVs) are highly sought after as a source of biomarkers for disease detection and monitoring. Tumor EV isolation, processing, and evaluation from biofluids is convoluted by EV heterogeneity and biological contaminants and is limited by technical processing efficacy. This study rigorously compares common bulk EV isolation workflows (size exclusion chromatography, SEC; membrane affinity, MA) alongside downstream RNA extraction protocols to investigate molecular analyte recovery. EV integrity and recovery is evaluated using a variety of technologies to quantify total intact EVs, total and surface proteins, and RNA purity and recovery. A comprehensive evaluation of each analyte is performed, with a specific emphasis on maintaining user (n = 2), biological (n = 3), and technical replicates (n≥3) under in vitro conditions. Subsequent study of tumor EV spike-in into healthy donor plasma samples is performed to further validate biofluid-derived EV purity and isolation for clinical application. Results show that EV surface integrity is considerably preserved in eluates from SEC-derived EVs, but RNA recovery and purity, as well as bulk protein isolation, is significantly improved in MA-isolated EVs. This study concludes that EV isolation and RNA extraction pipelines govern recovered analyte integrity, necessitating careful selection of processing modality to enhance recovery of the analyte of interest.
Subject(s)
Extracellular Vesicles , Glioblastoma , Humans , Glioblastoma/genetics , Glioblastoma/metabolism , Extracellular Vesicles/chemistry , Extracellular Vesicles/metabolism , Chromatography, Gel , RNA/analysis , RNA/metabolism , Membrane Proteins/analysis , Membrane Proteins/metabolismABSTRACT
Pulmonary neuroendocrine neoplasms comprise ~20% of all lung tumors. Typical carcinoid, atypical carcinoid, small cell carcinoma, and large cell neuroendocrine carcinoma represent the 4 major distinct subtypes recognized on resections. This review provides a brief overview of the cytomorphologic features and the 2021 World Health Organization classification of these tumor types on small biopsy and cytology specimens. Also discussed are the role of immunohistochemistry in the diagnosis and molecular signatures of pulmonary neuroendocrine tumors.
