Fig. 2: DTC-Flow assay workflow and gating strategy for analysis of bone marrow aspirates.

a Schematic depicting the DTC-Flow assay “stain/lyse/no wash” sample processing protocol. b Overall DTC-Flow gating strategy. The third dot plot shows the establishment of DTC gating cutoffs for HER2 and EpCAM. The polygonal gate shown in blue reflects the DTC gating cutoffs, defined by background staining levels in healthy donor control bone marrow aspirates (BMAs) (blue shaded area, “non-DTCs”), that were used to classify each bone marrow cell (BMC) as a DTC or not a DTC. The quadrant gates shown in black, defined using control BMAs in which HER2 and EpCAM antibodies were omitted from the staining protocol, were used to define cells as positive or negative for EpCAM and HER2 staining. c Dot plots showing background levels of HER2 and EpCAM expression (blue shaded area) in CD45– BMCs from representative control donors. Arrows indicate the “EpCAM-low” normal BMC population present in most control BMAs. d Dot plots showing HER2 and EpCAM expression levels in representative human breast cancer cell lines, relative to the gating cutoff established for CD45– cells from control BMAs. Percentages of cells from each cell line with HER2 and/or EpCAM levels above the control bone marrow gating cutoff are shown.