Abstract 4880: Parsortix™ system: Analytic performance evaluation using ovarian cancer cell lines
Authors: Hustler Arianna , Wishart Gabrielle , Templeman Amy , Sales Erica Bravo , Torrecillas Sara Arocas , Mumford Kyra , O’Shannessy Daniel J. 
Published: August 2019

Abstract
Background: Ovarian cancer has the worst prognosis among gynecologic malignancies, in part due to presentation with later stage disease in a majority of women. Much effort has been expended towards early detection, but it remains a significant issue and challenge. One approach which may have potential in improving the detection of ovarian cancer, particularly in women who present with an adnexal or pelvic mass, is the isolation and interrogation of circulating tumor cells (CTCs) from peripheral blood. The microfluidic Parsortix™ system isolates rare cells from biological fluids, particularly blood, on the basis of cell size and deformability.
Objectives: The primary aim of the present study was to evaluate the performance of a modified Parsortix™ system for the isolation of ovarian cancer cell lines spiked into blood drawn from healthy volunteers (HVs). Parameters such as sensitivity, determined by dilution linearity, reproducibility and stability (time after blood draw) were assessed. In addition, the ability to molecularly interrogate Parsortix™ harvests by RT-qPCR for the CTC-specific genes Epithelial Cell Adhesion Molecule (EpCAM) and CytoKeratin (CK) was assessed.
Methods: Two ovarian cancer cell lines, CaOV3 and SKOV3, were used as model CTCs. Live cells were spiked into HV blood samples collected into K2EDTA vacutainers and stored for up to 120h. The spiked blood samples were then processed using modified Parsortix™ instruments (optimized to speed-up blood processing) and separation cassettes with a 6.5µm critical gap size. Harvests were evaluated for reproducibility, linearity, and stability by counting harvested cells and/or by using an optimized RT-qPCR assay. For experiments where cell counting and percentage recovery were assessed, cancer cells (CaOV3 or SKOV3) labelled with CellTracker™ Green were utilized and counted via fluorescence microscopy. When RT-qPCR was used to assess the recovered cells, unlabeled Parsortix™-isolated cells were lysed in a LiDS buffer, mRNA isolated using Dynabeads™, and cDNA synthesis performed followed by RT-qPCR for selected gene products.
Results: The sensitivity of the Parsortix™ system for the isolation/harvest of CaOV3 and SKOV3 cells was approximately 1 cell/mL blood. There was no appreciable effect of storage of the spiked K2EDTA blood samples on recovery of the pre-labelled CaOV3 cells for up to 120h hours after collection/spiking, with either room temperature or refrigerated (4oC) blood storage. The isolation/harvest of model CTCs was reproducible with a CV of ≤14%. RT-qPCR, while gene dependent, was shown to be linear with model CTC input and showed a sensitivity of approximately 1 cell for EpCAM.
Conclusions: These data demonstrate both the robustness and sensitivity of the Parsortix™ system for the isolation and interrogation of CTCs, and further studies, specifically in the area of ovarian cancer, are ongoing.
Citation Format: Arianna Hustler, Gabrielle Wishart, Amy Templeman, Erica Bravo Sales, Sara Arocas Torrecillas, Kyra Mumford, Daniel J. O’Shannessy. Parsortix™ system: Analytic performance evaluation using ovarian cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4880.