Enumeration of CTCs (by IE/FC and CellSearch) and DTCs was performed by
investigator JHS who was blinded to the study endpoints.
Blood and bone marrow samples were subjected to the IE/FC assay to
enumerate CTCs and DTCs, respectively (18 (link)–20 (link),22 (link)). Briefly, two distinct monoclonal antibodies
against EPCAM, one conjugated to immunomagnetic beads (MJ37) and the other
conjugated to phycoerythrin (EBA-1) were added to whole blood or bone marrow.
The sample was then placed in a magnet to capture cells labeled with the
magnetic bead-antibody conjugated. The supernatant containing cells that were
unbound (including red blood cells) was aspirated. Magnetic separation was
repeated twice to further enrich for EPCAM-expressing cells. A nucleic acid dye
(Thioflavin-T, BD Biosciences) and a monoclonal antibody to the
leukocyte-specific marker CD45 (2D1) conjugated to
peridinin-chlorophyll-protein-Cy5.5 were added to the sample. The enriched
sample was transferred to a BD TruCount™ (BD Biosciences) tube, and flow
cytometric analysis was performed using the BD FACSCalibur™ (BD
Biosciences). CTCs and DTCs were defined as nucleated cells that are
EPCAM-positive and CD45-negative.
investigator JHS who was blinded to the study endpoints.
Blood and bone marrow samples were subjected to the IE/FC assay to
enumerate CTCs and DTCs, respectively (18 (link)–20 (link),22 (link)). Briefly, two distinct monoclonal antibodies
against EPCAM, one conjugated to immunomagnetic beads (MJ37) and the other
conjugated to phycoerythrin (EBA-1) were added to whole blood or bone marrow.
The sample was then placed in a magnet to capture cells labeled with the
magnetic bead-antibody conjugated. The supernatant containing cells that were
unbound (including red blood cells) was aspirated. Magnetic separation was
repeated twice to further enrich for EPCAM-expressing cells. A nucleic acid dye
(Thioflavin-T, BD Biosciences) and a monoclonal antibody to the
leukocyte-specific marker CD45 (2D1) conjugated to
peridinin-chlorophyll-protein-Cy5.5 were added to the sample. The enriched
sample was transferred to a BD TruCount™ (BD Biosciences) tube, and flow
cytometric analysis was performed using the BD FACSCalibur™ (BD
Biosciences). CTCs and DTCs were defined as nucleated cells that are
EPCAM-positive and CD45-negative.
