Table 1 Current technologies for CTC detection

CellSearch^® system

Immunomagnetic beads: EpCAM-Ab-coupled ferrofluid

Immunocytochemistry: Positive for CK8, 18, 19 Negative for CD45 Nucleus positive for DAPI

Semi-automated system with FDA approval for metastatic breast, colon and prostate cancer. CTC can be enumerated and visualized [2]

CTC-chip

Microposts: EpCAM-Ab- coupled microposts

Immunocytochemistry: Positive for CK8, 18, 19 Negative for CD45 Nucleus positive for DAPI

High detection rate (approximately 100%) even in M₀-patients warrants further investigations on assay specificity; the Herringbone second generation of this microchip is more specific. Needs to be validated in clinical trials [67–70]

CTC-chip Ephesia

Column of nanobeads: EpCAM-Ab-coupled ferrofluids

Immunocytochemistry: Positive for CKs Negative for CD45 Nucleus positive for DAPI

Lack of validation studies in clinical settings [71]

MagSweeper

Immunomagnetic beads: EpCAM-Ab-coupled ferrofluids

Microscope visualisation: Morphology

Isolation of CepC with a high degree of purity. Analysis of large blood volume [72]

Laser scanning cytometry Maintrac^®

RBC lysis

Immunocytochemistry: Positive for EpCAM Negative for CD45

High incidence of positive events up to 3 logs higher CTC counts than those obtained with other techniques warrants further investigations of assay specificity [73]

Ikoniscope^® imaging system

Ficoll-Isopaque or filtration with track-etched membranes

Immunocytochemistry: Positive for EpCAM, CK7/8 PSA (prostate only) FISH: chromosomes 7 and 8 Nucleus positive for DAPI

Two epithelial specific Abs and FISH to detect chromosomal abnormalities in CTCs [74]

Ariol^® system

RBC lysis, then immunomagnetic beads: CK-Ab- + EpCAM-Ab- coupled ferrofluids

Immunocytochemistry: Positive for CK8, 18, 19 Negative for CD45 Nucleus positive for DAPI

Detection of EpCAM⁺ and EpCAM- CTCs [75]

AdnaTest

Immunomagnetic beads: MUC1-, EpCAM-Ab-coupled microbeads

Molecular biology: RT-PCR Positive for at least one of the following markers: MUC1, HER2, EpCAM

AdnaTest also does not quantify the tumour cell load, false positive results due to unspecific amplification, no further analysis possible [76]

Functional assays

EPISPOT assay

Rosette plus Ficoll: Depletion of CD45⁺ cells

Secretion of proteins: CK19, MUC1, Cath-D (breast); CK19 (colon); PSA (prostate); TG (thyroid)

Detection of viable epithelial secreting-cells; unbiased enrichment independent of CTC/DTC phenotype [41, 77]

Vita-Assay™ or Collagen Adhesion Matrix (CAM) technology

Invasion capacity: Ingestion of fluorescent CAM fragments (CAM+)

Immunocytochemistry: Positive for EpCAM, ESA, pan-CK 4, 5, 6, 8, 10, 13 and 18 Negative for CD45

Detection of CTCs with the invasive phenotype in blood [78]

Others

ISET

Cell size

Immunocytochemistry: Positive for CK Nucleus: Mayer's hematoxylin

Sensitivity threshold of one carcinoma cell per milliliter of blood; HER2 amplification determined by real-time PCR on DNA extracted from CK immunostained cells (CTCs) collected by laser microdissection from selected ISET-positive filters; the possibility of false-positive diagnosis stresses the need for using ancillary methods to improve this approach [79–81]

FAST (fiber-optic array scanning technology)

No pre-enrichment

Immunofluorescence: Positive for CK Nucleus positive for DAPI Morphology

Rare cells detected by laser scanning to almost 1,000 times faster than digital microscopy [82, 83]

DEP-FFF (dielectrophoretic field-flow fractionation)

Phenotype - membrane capacitance

Immunocytochemistry: Wright stain

No need for labeling or modification of CTCs; PBMC/CTC ratio is enriched more than 2000-fold; CTCs isolated by DEP are viable and suitable for a wide spectrum of analyses [84]

Versatile label free biochip

Cell size deformability

Immunofluorescence: Positive for CK Negative for CD45 Nucleus positive for DAPI Morphology

Label free selection and CTCs are viable after blood processing [85]