CTC Immunostaining, enumeration, and PD-L1 expression analysis. (A) Gallery of cells collected through Vortex technology from patient samples. Healthy WBCs and lung cancer cell lines are used as staining controls. Cells were immunostained for PD-L1-AF647, CK-FITC, CD45-PE and DAPI, then classified according to quantitative criteria previously published23. (B) Following this method, 10 healthy donors blood samples and 31 samples from 22 patients were processed and CTCs enumerated. A healthy threshold can be defined from the healthy donors as 1.32 CTCs/mL (average + 2 SD). Using such a threshold, 14 of 27 patient samples were positive for CTCs (52%). (C) CTCs are collected with high purity, with 0–9.67 CTCs/mL among 1–93 WBCs/mL. (D) CTCs captured from different patients at different points in the course of their therapy display varying levels of PD-L1 expression. Each bar represents a distinct patient and a distinct time point.