Liquid Biopsy Workflow for CTC Enrichment

Two EDTA-coated vacutainer tubes (Becton Dickinson) of 7.5 ml of peripheral blood and one additional 10 mL CellSave Preservative tube (Menarini-Silicon Biosystems) were collected per patient at different time points: 20 samples before the start of systemic therapy (visit 1, V1), 20 samples after 1 cycle of chemotherapy (visit 2, V2), and 13 samples after clinical progression (visit 3, V3) determined by CT-Scan. Blood samples were processed within two hours after withdrawal. All samples were anonymised and encoded before the analysis.
In total, 53 samples were included in this study. Blood samples were analysed in parallel with the label-independent antibody cocktail RosetteSep™ CTC Enrichment Cocktail Containing Anti-CD56 (STEMCELL Technologies) (V1, n = 20; V2, n = 20 and V3, n = 13) and with the EpCAM-based CellSearch® System (Menarini Silicon Biosystems) as previously described [22 (link)] (V1, n = 19; V2, n = 20 and V3, n = 13).
In addition, the PBMCs of the patients were isolated from one 7.5 mL EDTA tube of peripheral blood by density gradient centrifugation protocol (Lymphoprep™, STEMCELL Technologies) in SepMate™ tubes (STEMCELL Technologies) according to manufacturer’s instructions.
Enriched cells isolated with RosetteSep™ and PBMCs were placed in RNAlater™ Solution (Invitrogen, ThermoFisher Scientific) and kept at -80ºC until further analyses.