Celltracks analyzer 2

A solution of H1975 cells stained with Cell Tracker™ Green (Thermo fisher scientific) was loaded on the single-cell picking system and single cells were collected and added to individual wells of a 96-well microplate. The existence of a single cell in each well was confirmed by fluorescent microscopy. To quantify tumor cells identified using the single-cell picking system, approximately 1500 or a small number of (2–25) A549 cells were spiked into 7.5 mL of whole blood from a healthy donor (HD), which was collected in a CellSave Preservative Tube (Janssen Diagnostics). A549 cells spiked in HD blood were processed using the CellSearch® CTC Kit (Janssen Diagnostics), and A549 cell counts were determined using the CellTracks Analyzer II® (Janssen Diagnostics). Enriched cells were loaded onto the single-cell picking system and re-counted. CTC counts obtained by CellSearch® and the single-cell picking system were compared. When a small number of cells, i.e., A549 cells, were spiked, single cells were recovered and the recovery rate was calculated.

The enrichment and enumeration of tumor cells from whole blood were performed using the FDA-approved CellSearch® System (Janssen Diagnostics, Raritan, NJ, USA). First, 7.5 mL of the whole blood sample was processed using the CellSearch® CTC Kit (Janssen Diagnostics). In this assay, EpCAM-based immunomagnetically enriched cells were fluorescently counterlabeled with DAPI to stain nuclei, phycoerythrin (PE)-conjugated antibodies directed against cytokeratins 8/18/19, and allophycocyanin (APC)-conjugated antibodies directed against CD45 to stain the remaining WBCs. After enrichment, isolated fluorescently labeled cells were resuspended in a MagNest^Ⓡ Cartridge Holder (Janssen Diagnostics) and analyzed (i.e., identified and enumerated) using the CellTracks Analyzer II^Ⓡ (Janssen Diagnostics) according to the manufacturer’s instructions.

Collection of blood and immunofluorescent (IF) staining of CTC was performed using the CellSearch® system (Janssen Diagnostics, LLC), as previously reported [4 (link), 26 (link)]. Candidate cells (CTC +/− vimentin, or CTC +/− Ki67 – all cytokeratin positive) were identified using the CellTracks Analyzer II (Janssen Diagnostics, LLC) according to methods and IF expression criteria previously reported [27 (link)]. The number of CTCs is presented per 7.5 ml of blood. Unless otherwise stated in the text, patients in this study were defined as Ki67- or vimentin-CTC positive if they had ≥1 vimentin-positive or Ki67-positive CTCs. For Additional file 1: Figure S3, two separate patient cohorts were assessed in exploratory analyses: for (a) and (c), patients with ≥5 total CTCs and ≥1 vimentin-positive or Ki67-positive CTC were compared with those patients with ≥5 total CTCs and no vimentin-positive or Ki67-positive CTC; for (b) and (d), the 10 CTC-positive patients with the highest proportions of vimentin/Ki67-positive CTCs were compared with the 10 CTC-positive patients with the lowest proportions of vimentin/Ki67-positive CTCs.