1832 Materials
- Anti-EpCAM ferrofluid which contains a suspension of 0.022%
magnetic particles conjugated to a mouse monoclonal anti-
body specific for the cell surface marker EpCAM present on
epithelial cells in a buffer containing 0.03% bovine serum albu-
min (BSA) and 0.05% ProClin® 300 preservative. - Staining reagent consists of a small amount (~0.0006%) of
mouse monoclonal antibodies specific to cytokeratins conju-
gated to phycoerythrin (PE); 0.0012% mouse anti-CD45
monoclonal antibody conjugated to allophycocyanin (APC) in
buffer containing 0.5% BSA and 0.1% sodium azide. - Nucleic acid dye: Contains 0.005% 4′,6-diamidino-2-
phenylindole, dihydrochloride (DAPI), and 0.05% ProClin®
300. - Capture enhancement reagent: Contains 0.02% proprietary
reagent for controlled ferrofluid aggregation, 0.5% BSA, and
0.1% sodium azide in buffer. - Permeabilization reagent: Contains 0.011% proprietary per-
meabilization reagent and 0.1% sodium azide in buffer. - Cell fixative: Contains 25% proprietary fixative ingredients,
0.1% BSA, and 0.1% sodium azide in the buffer. - Dilution buffer: Contains buffer with 0.1% sodium azide.
- Conical centrifuge tubes (15 mL) and conical tube caps.
- Circulating tumor cell control kit.
- Cartridges and cartridge plugs.
- Horizontal swing out style rotor centrifuge capable of 800 × g.
- Test tube racks.
- Calibrated micropipettes and tips.
- Vortex mixer.
3 Methods
- Approximately ≥7.5 mL blood is required for isolating >5
CTCs in cancer patients. - The enumeration of circulating tumor cells (CTCs) of epithe-
lial origin is performed with help of specific markers such as
CD45−, EpCAM+, and cytokeratins 8, 18+, and/or 19+ in
the fresh whole blood from patients diagnosed with carcinoma
(see Note 1). - CTCs enumerated using an automated CellSearch™ assay fol-
lowed by CTC separation using flow cytometry and fluorescence-
3.1 Enumeration
of CTCs
Circulating Tumour Cells