Tools for novel liquid biopsy approaches in Lung Cancer Diogo Fortunatoa, Laura Bianciardib, Gaia Papinic, Mattia Criscuolic, Davide Zoccod and Natasa Zarovnib Exosomics/University of Siena; bExosomics; cExosomics Siena, University of Siena; dExosomics SienaaSide scatter module for enhanced DcR3 Proteins site detection of Extracellular Vesicles by flow cytometry. Tina Van Den Broecka, Ludovic Monheimb, Ihor Berezhnyya, Oleg Guryeva, Tatyana Chernenkoa, Marybeth Sharkeya and Geoffrey OsborneaaIntroduction: Mounting clinical evidence suggests that liquid biopsy may perhaps revolutionize the way cancer sufferers are at present managed. Within this context, our study aims to assess and reinforce exceptional and complementary benefits of EV/exosome-based approaches, via identification and quantitative detection of non-small cell lung cancer (NSCLC) EV biomarkers. Existing technologies and methods for exosome isolation from complicated B7-H3/CD276 Proteins supplier biological samples (i.e. plasma), have shown to become unreliable. There is a have to substantially strengthen them to allow multiparameter EV evaluation. Thus, additionally to EV-biomarker discovery, we are testing plasma processing and preanalytical tools, devices and optimized immunoaffinity protocols that tackle basic obstacles, such as complicated matrix effects. Our goal is usually to deliver an EV immunocapture approach with adequate sensitivity, specificity and robustness for clinical grade diagnostic applications. Solutions: Size-based vs. immunocapture procedures for exosome isolation. Enzymatic and immunological assays for plasma pre-clearing; Flow cytometry, ELISA, nanoparticle tracking analysis, Western Blot, SPR and ddPCR for antibody and exosome characterization. Results: Exosomes derived from NSCLC cell lines show distinct membrane markers recognized by a panel of proprietary Abs, screened by flow cytometry, SPR, IP, ELISA and PCR. We developed and tested a screening platform based on endogenously labelled EVs to determine NSCLC EV antigens. Selected antibodies is going to be made use of to develop an immune-isolation protocol, coupled to state-of-the-art analytics to get a fast and sensitive readout, hence enabling a comparative evaluation of a repertoire of plasma pre-analytical protocols. Summary/conclusion: Different plasma pre-analytical protocols are ranked and orthogonally combined to optimally counteract matrix effects, increment EVBD Biosciences; bBD Life Sciences, Erembodegem, BelgiumIntroduction: EVs are nanosized (20 5000 nm) membrane vesicles released from cells which will transport cargo such as miRNA and proteins among cells as a potent way of intercellular communication. Presently, flow cytometry could be the only higher throughput approach capable of single particle cell surface phenotyping and sorting using the possibility of concentration determination. Unfortunately, the drawback of standard flow cytometry is lack of sensitivity to detect smallest particles, especially for those using a size significantly less than or equal for the dimensions on the excitation laser wavelength. Methods: BD has created an accessory side scatter (SSC) module for enhanced scatter detection of small particles by flow cytometry: the SP SSC module. The SP SSC module should be utilized in combination having a laser power of at the least 100 mW. Little particle detection enhancement is achieved by considerably growing the signal-to-noise ratio of the SSC. Outcomes: The SP SSC module might be installed on most commercially accessible BD flow cytometers, which have sufficient laser energy, as a.