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Table 2 CTC Assays and Technologies

From: Considerations in the development of circulating tumor cell technology for clinical use

Assay/Technology Name Manufacturer/Developer Assay Outcome Target Cancer(s) Technology/Process
AdnaTest AdnaGen, Langenhagen, Germany Enrichment/Characterization Breast, Prostate, Colon Immunomagnetic-based EpCAM enrichment using labelled beads incubated with the whole blood sample. Unlabeled cells are removed; labelled cells are then lyzed. RNA is isolated, followed by multiplex RT-PCR (GA733-2, HER2, MUC1) to detect specific tumor biomarkers.
Anti-EpCAM/Anti-CK Antibody CTC Enrichment Glenn Deng, Stanford University, Stanford, CA Enrichment/Enumeration Metastatic Breast Cancer CTC enrichment assay using the combination of anti-CK and anti-EpCAM antibodies. Image analysis performed using the Ariol® system. CTC identification with brightfield and fluorescence labelled anti-CK, anti-CD45, and 4′,6-diamidino-2-phenylindole (DAPI) images.
ApoStream TM ApoCell Enrichment Prostate, Lung Isolation of CTCs in a whole blood sample using dielectrophoretic field flow fractionation (DFFF), which separates cells based on differing dielectric properties. See also DFFF technology entry below.
autoMACS/MACS (Magnetic Activated Cell Sorting System) Mitenyi Biotec, Bergisch Gladbach, Germany Enrichment ---------- Utilizes an immunomagnetic column to capture cells with various antigens (EpCAM, pan-CK, HER2/neu, or CD45). Manual or semi-automated system. These viable cells are available for subsequent analysis following enrichment.
Bioflux Fluxion Biosciences, South San Francisco, CA Enumeration ---------- Well Plate Microfluidic™ technology to obtain physiologically-relevant data from cell-based assays. Data acquisition obtained in brightfield, phase, fluorescence, and confocal high-resolution microscopy.
CEER (Collaborative Enzyme Enhanced Reactive) Immunoassay Prometheus Laboratories Inc. Characterization ---------- Multiplexed protein microarray platform that measures the expression and activation of specific cancer pathways with high levels of sensitivity and specificity.
  Bayer Schering Pharma AG, Germany    
CELLection™ Epithelial Enrich Traci Libby, Invitrogen, Carlsbad, California Enrichment ---------- Positive isolation. Obtain up to 5 log enrichment of viable epithelial tumor cells that are suitable for immunocytochemical staining or any other downstream application.
CellSearch™ Veridex Enrichment/Enumeration Metastatic Breast, Colon, Prostate, Lung, Melanoma, Urothelial Cancer Automated immunomagnetic enrichment and staining system for quantification of CTCs in whole blood samples. CTCs are enriched using ferrofluids coupled to anti-EpCAM antibodies and identified by cytokeratin staining using fluorescent anti-CK antibodies, as well as counterstaining with anti-CD45 antibodies. Currently, the only diagnostic test cleared by the FDA.
ClearCell System (CTChip and Clearcell Unit) Clearbridge Biomedic, Singapore Enrichment ---------- Detects and isolates intact, viable CTCs from small quantities of whole, unprocessed blood. Isolated CTCs can then be stained directly on the CTChip® for identification, or retrieved for further molecular analysis.
CTC Chip Dan Haber and Mehmet Toner, Dana Farber and MGH, Boston, MA Enrichment/Enumeration Breast, Colon, Lung, Prostate, Pancreas Enrichment using microfluidic technology—whole blood is pumped across a silicon-etched chip that contains 78,000 microposts fitted with anti-EpCAM antibodies. EpCAM-positive cells attach to the microposts and are then detected by a camera. Includes a chamber to enclose the fluid and chip and a pneumatic pump.
  Developers:    
  On-Q-ity, Waltham, MA    
  ICx Biosystems, San Diego, CA    
  Johnson & Johnson    
CTC Membrane Microfilter Richard Cote, Ram Datar, University of Miami, FL Enumeration Prostate Stepwise photolithography process that produces controlled-size pores designed to exploit cell size differences between tumor and normal blood cells. Combined with quantum dot-based immunofluorescence detection for CTC characterization.
Cytoscale CTC Assay Hsian-Rong Tseng, University of California, Los Angeles Enumeration Prostate, Breast, Colon and Kidney Antibody cell-surface marker capture enhanced by nanostructures; immunohistochemistry staining for cell identification.
DEPArray Nicolo Manares, Silicon Biosystems, SpA, Bologna, Italy Enumeration ---------- Cell microarray for individual cell manipulation and detection. The base is a microelectronic active silicon substrate embedding control circuitry for addressing each individual dielectrophoretic (DEP) cage (cage size can be set to accommodate a single cell). The system allows detection and sorting of rare cells and sorting by morphological parameters such as shape, nucleus-to-cytoplasm ratio, fluorophores co-localization (by image-based selection).
Dielectrophoretic Field Flow Fractionation (DFFF) Peter Gascoyne, MD Anderson Cancer Center, Houston, TX Enumeration ---------- Cell-separation technique that exploits the differences in density and dielectric properties of cells to aid isolation of CTCs from clinical blood specimens. See also ApoStream™ assay above.
Dylight Technology Medical University Graz, Austria Enumeration Breast Immunofluorescence method for identifying CTCs that utilizes staining for multiple markers, including CD44, ALDH1, and CK using DyLight dyes; and subsequent analysis by novel DyLight technology.
Dynabeads® CD45 Traci Libby, Invitrogen, Carlsbad, California Enrichment ---------- Dynabeads® are coated with anti-CD45 monoclonal antibody for efficient depletion of human leucocytes in whole blood samples to enrich epithelial tumor cells.
Dynabeads® Epithelial Enriched Traci Libby, Invitrogen, Carlsbad, California Enrichment ---------- Dynabeads® are coated with the monoclonal antibody BerEP4 against the human epithelial antigen, EpCAM. Enriched tumor cells are lysed for mRNA isolation and RT-PCR amplification.
Epic HD-CTC Assay Epic Sciences, Inc. Enumeration/Characterization Prostate, Breast, Pancreas CTC detected in peripheral blood through red blood cells lysis and fluorescentlylabeled antibodies. See also FAST Cytometer entry.
EPISPOT (EPithelial ImmunoSPOT) Catherine Alix-Panabieres and Klaus Pantel, Laboratoire de Virologie, Hôpital Lapeyronie, CHU Montpellier, France & UKE, Hamburg, Germany Characterization Breast, Prostate, Colon After depletion of CD45 positive cells, remaining cells in a whole blood sample are cultured for 24 hours on a membrane coated with antibodies that detect proteins shed from viable CTCs by secondary antibodies labelled with fluorochromes.
FAST Cytometer (Fiberoptic Array Scanning Technology) Peter Kuhn, Scripps Institute, La Jolla, CA Enumeration/Characterization Metastatic Breast Cancer Fluorescence cytometry combined with an automated digital microscopy imaging system. Immunofluorescently labelled CTCs are detected on a glass slide using laser-printing optics, which can scan 300,000 cells per second. See also Epic HD-CTC Assay entry.
  Robert Bruce, Scripps Palo Alto Research Center, Palo Alto, CA    
Flow Cytometry Jeannie Gaylor, Becton-Dickinson, San Jose CA Enrichment/Enumeration ---------- Multiple reagents and systems adaptable to analysis or sorting of CTCs.
HB-CTC (Herringbone-Chip) Mehmet Toner and Daniel Haber, Massachusetts General Hospital (MGH) and Harvard Medical School Enrichment/Enumeration ---------- A high-throughput microfluidic mixing device which provides an enhanced platform (over the CTC-chip) for CTC isolation where microvortices are utilized to significantly increase the number of interactions between target CTCs and the antibody-coated chip surface.
ISET (Isolation by Size of Epithelial Tumor cells) Metagenex, Paris, France Enrichment ---------- CTCs are separated from other cells in whole blood by size via vacuum filtration. This technique is gentle and produces viable cells that can be further analyzed following enrichment.
IsoFlux™ Rare Cell Access System Fluxion Biosciences, South San Francisco, CA Enrichment ---------- Proprietary microfluidic technology to isolate rare cells with high efficiency. The system incorporates CellSpot™ Technology to produce a highly concentrated sample that is optimized for downstream molecular analyses.
Laser Scanning Cytometry Maintrac, Bayreuth Germany Enumeration/Characterization Breast, Colon, Prostate, Sarcoma Custom laboratory analysis service performed on slides using a variety of fluorochrome-labelled antibodies or other techniques (e.g., estrogen receptor, HER2, prostate-specific antigen, FISH, terminal dUTP nick end labelling). Traceable single cell detection within 1 million cells.
Laser Scanning Cytometry (LSC) ApoCell Enumeration ---------- Proprietary microscope-based immunofluorescent image analysis.
Lymphoprep™ (Ficoll-Isopaque) Axis-Shield PoC, Oslo, Norway Enrichment ---------- Separates mononuclear cells from other cells in whole blood based on cell density.
MagSweeper Stephanie Jeffrey and Ronald W. Davis, Stanford University, Stanford, CA Enrichment/Enumeration Metastatic Breast Automated immunomagnetic enrichment―gently enriches target cells and eliminates cells that are not bound to magnetic particles. Isolated cells can be extracted individually based on their physical characteristics to deplete any cells nonspecifically bound to beads. Processes 9 mL of blood per hour and captures >50% of circulating epithelial cells as measured in spiking experiments.
Multiphoton Intravital Flow Cytometry Philip Low, Purdue University, West Lafayette, IN Enumeration Prostate Noninvasively counts rare CTCs in vivo as they flow through the peripheral vasculature. The method involves intravenous injection of a tumor-specific fluorescent ligand followed by multiphoton fluorescence imaging of superficial blood vessels to quantitate the flowing CTCs.
Nanodetector Gilupi, Potsdam, Germany Enrichment/Enumeration Breast, lung, prostate The nanodetector (functionalized structured medical wire, FSMW) is inserted into the patient’s arm vein via a standard 20-gauge needle. The nanodetector consists of a medical stainless steel wire, coated with a gold layer and a hydrogel functionalized with an anti-EpCAM antibody. During the 30 min application in the vein, up to 1,500 mL of blood including the respective CTC pass the nanodetector and enable a high number of CTC to be bound by the anti-EpCAM antibody.
Negative Enrichment OMS Jeffrey Chalmers, Cleveland Clinic, Cleveland, OH Enrichment/Enumeration Head & Neck, Breast Red cell lysis, immunomagnetic labelling and subsequent depletion of CD45+ cells (leukocytes). Remaining cells may be further characterized (epithelial cells, cells undergoing EMT).
  PerCelleon, LLC    
  Stem Cell Technologies    
Nucleopore Assay Whatman International Ltd., UK Enrichment ---------- CTCs are separated from other cells in whole blood by size via vacuum filtration.
OncoCEE-BR™ Biocept, Inc. and Clarient, Inc. Enumeration/Characterization Breast OncoCEE™ captures CTCs via a microfluidic system that uses multiple antibodies for capture followed by detection using CEE-Enhanced staining and then detects their HER2/neu status via FISH.
OncoQuick Greiner Bio-One, Germany, North Carolina Enrichment Breast, Colon, Others Centrifugal separation using optimized liquid media based on tumor cell buoyant density only. Achieves enrichment of up to 6 logs from approximately 104 total mononuclear cells. Validated with spiking studies.
Optofluidic Intracavity Spectroscopy (OFIS) David Kisker eOptra, Longmont, CO Enumeration ---------- OFIS has been used to investigate the properties of several tumor cell lines and compared the results to cells from peripheral blood. The results suggest that a unique optical signature may be a characteristic of many tumor cells. This signature may offer a complementary tool to molecular methods for detection and enumeration of CTCs. In addition, by using dielectrophoresis to trap and steer cells, it is possible that induced changes in the OFIS spectrum may detect other characteristics of tumor cells, as well as transport and sort them according to those characteristic properties.
Photoacoustic Detection John Viator, University of Missouri, Columbia, MO Enumeration Melanoma, Breast Cancer Flowmetry system in which blood samples are irradiated with laser light, and photoacoustic waves from cancer cells are detected and counted (uses melanin in melanoma, gold-tagging of other cancer cells).
RARE (RosetteSep-Applied Imaging Rare Event) StemCell Technologies, Vancouver, BC Enrichment ---------- Negative selection technique where tetrameric antibody complexes crosslink CD45-expressing leukocytes to red blood cells in whole blood. These complexes pellet to the bottom of the tube when centrifuged due to increased density, enriching CD45-negative cells (CTCs).
RoboSep/EasySep™ Stem Cell Technologies, Vancouver Enrichment Myeloma, Epithelial Tumors, CD45 depletion Immunomagnetic nanoparticle-cell complexes are captured in tubes and unlabeled cells are poured off. Adaptable to custom CTC antibody surface antigens. Manual or semi-automated systems.
ScreenCell® Cyto, ScreenCell® CC, ScreenCell® MB ScreenCell Company Biopark 12 rue J-A de Baïf 75013, Paris Enrichment ---------- The ScreenCell® Cyto device isolates rare tumor cells, with a high recovery rate. The ScreenCell® CC device allows isolation of either fixed or live cells. Fixed cells are well preserved morphologically. Immunocytochemistry and FISH assays can be performed directly on the filter. Isolated live cells are able to grow in culture. High-quality genetic materials (DNA, RNA) can be obtained directly from tumor cells isolated on the ScreenCell® MB device filter. The ScreenCell® devices may be able to simplify and improve noninvasive access to tumor cells due to their reduced size, versatility, and capacity to isolate CTCs within minutes.
Single Cell Gene Expression with BioMark™ Real-Time PCR System Fluidigm Corporation, South San Francisco, CA Characterization ---------- Allows high-throughput cell-line studies to determine individual cell behavior and is suited to determine single-gene cell expression levels in CTCs. Results are presented as a heat map, with individual assays on the X-axis and individual cell samples on Y-axis. The intersection of each assay and sample is an individual real-time qPCR reaction.
Supervised* Automated Microscopy Iqbal Habib, Ariol, Genetix, Boston MA, San Jose CA Enumeration/Characterization Breast, Others? Commercial component to automatically track, review, and enumerate immunocytochemically stained candidate CTCs. Nuclear, shape factor morphology image analysis system with computer display.
TelomeScan (OBP-401) Oncolys BioPharma, Tokyo Enumeration/Characterization Gastric, Breast Uses a virus vector for CTC detection. The virus is incubated with whole blood sample for 24 hrs and replicates with cancer cells, incorporating the GFP marker into them. CTCs are then detectable by fluorescence system analysis of cell preparation on slide. Potential for in vivo transfection and detection of GFP + CTCs in capillary bed of patient.
Vita-Assay [Functional Collagen Adhesion Matrix (CAM)] Wen-Tien Chen, Vitatex, Stony Brook, NY Enumeration Prostate CAM ingestion. Enables molecular characterization of captured cells.
  1. (Commercial and Being Developed for Commercial Application).