Table 1 Summary of the CTC isolation and detection technologies

 CellSearch® system

consists of ferrofluids coated with epithelial cell specific EpCAM antibodies; fluorescent labeling; immunohistochemical techniques

omitting CTCs expressing low levels of EpCAM; low frequency and large blood volumes demand; cells captured appear to be more apoptotic

FDA approved for advanced prostate, breast and colorectal cancers

≥85

[5, 7]

 Weissenstein et al

a combination of anti-EpCAM and anti-cytokeratin magnetic cell separation

cells captured appear to be apoptotic

CTC levels were measured in MBC patients to assess prognostic value

78-90

[16]

 MagSweeper

anti-EpCAM antibody-targeting immunomagnetic beads; characterize cells for multiple marker

omitting CTCs expressing low levels of EpCAM; cells captured appear to be apoptotic

capture live CTCs from BC patients for single cell analyses

55-69

[53]

 IsoFlux™ Rare Cell Access System

a combination of flow control and immunomagnetic capture; Multiple kits for lab usage are available for cell enrichment and downstream analysis

Maximum daily analysis is 12 samples biomarker heterogeneity of CTCs

patients presenting ≥4 CTCs per blood draw were analyzed with prostate and colorectal cancers;

KRAS mutations rate in CRC were described

≥74

[90]

 AdnaTest®

incubate blood samples with an antibody mixture (e.g. anti-EpCAM and anti-MUC1); a magnetic particle concentrator extracts the labeled cell detect CTCs via RT-PCR assay for tumor-associated transcripts

biomarker heterogeneity of CTCs; magnetic beads may attach to the tube wall

a combined analysis of CellSearch® and AdnaTest® leads to an improved detection of CTCs in mCRC patient;

prognosis prediction and efficacy evaluation in breast, prostate cancer

88

[43, 89, 109]

 CTC-μChip

incubation with anti-EpCAM targeting immunomagnetic nanobeads; characterize gene expression using RT-ddPCR

omitting CTCs expressing low levels of EpCAM

CTC enumeration and genetic analysis in blood of patients with prostate cancer

> 90

[91, 110]

 DynaBeads®

bind to desired target and beads responding to magnetic field

Only 3 types of DynaBeads are available for human tumor cell isolation

enumerate CD4+ T lymphocytes in HIV-1-infected individuals;

not test in cancer patients

44 ± 23

[94]

 MACS

CD45 leukocyte depletion method; utilize cytokeratin immunocytochemistry to analyze enriched cells

CTC expressing CD45 maybe remove from the sample; erythrocyte lysis cause damage to CTCs

CTC enumeration used in breast, lung, liver esophageal cancer patients;

morphologically intact tumor cells were not detected in the clinical application

70-88

[111, 112]

 EasySep

anti-CD45 for removal of blood leukocytes

CTC expressing CD45 maybe remove from the sample

CTCs were detected in all of the BC patients (23/23)

24 ± 19

[94, 113]

 GILUPI CellCollector

ex vivo Functionalized Structured Medical Wire is antibody coated and applied into peripheral arm vein

Isolation in vivo and overcomes sample blood volume limitations

Only used for extraction of CTCs directly from patient’s bloodstream

in vivo isolation of CTCs in patients with different stages of prostate cancer;

distinguish between CTCs isolated from benign and malignant nodules

41

[106, 114, 115]

 3DPIC

incubation with anti-EpCAM targeting immunomagnetic nanobeads

Utilize ATP luminescence assay for the detection of cancer cells in blood

extracellular ATP derived from non-CTCs may interfere with the measurement

the ATP luminescence assay can detect as low as 10 cells in blood;

not test in cancer patients

80

[92]

 microfluidic ratchet mechanism

distinguish CTCs based on cell deformability; deform cells in continuous flow without accumulating cells in the separation microstructure; the separated cells are available for downstream characterization

cellular damage; throughput limitation

detect CTC in a considerable proportion with clinically localized PC patients

> 90

[67, 116]

 ISET®

Utilizes a filter-based, size exclusion approach to isolate epithelial cells; high throughput

morphology and size heterogeneity; damage or fragment CTCs on the result of multi-step cell processes

ISET® has a relatively good detection rate for CTCs in BC and NSCLC patients;

fail to provide more information on pathological staging and molecular classification

75

[63, 64]

 Metacell Filtration®

Size based separation technique driven by capillary-action; allow cytomorphological and immunocytochemical analysis of CTCs

Filters have a larger pore size (8 μm)

CTCs were detected in 66.7% evaluable PaC patients and the captured cancer cells displayed plasticity

66.7

[60, 117]

 ScreenCell

size-based microfiltration; high CTC capture efficiency with processing 3 ml of blood per sample

unable to capture CTCs smaller than WBCs; erythrocyte lysis may cause damage to CTCs

the presence of CTCs does not influence prognosis in operated patients with NSCLC

89

[9, 118]

 Parsotrix™

size and compressibility-based platform for CTCs isolation; ability to capture CTC clusters; harvests CTCs with both epithelial and mesenchymal features

CTC heterogeneity regarding size

Parsortix-enriched and stained cells were successfully transferred with preservation of cell morphology;

not tested in clinical application

> 90

[65, 119]

 Dielectrophoresis (DEP)

isolation based on polarizability and size; discriminate between cells of similar size having different morphological origins

requires specific parameters such as cell type and electric field frequency; the extent to which DEP will be applicable of CTC isolation in different types of cancer is unclear

concentrate MCF7 cancer cells from leukocytes;

not test in cancer patients

> 90

[51, 120]

 OncoQuick

polypropylene tube is inserted above the separation medium which allows for elimination of unwanted blood cells;

High throughput, inexpensive

loss of sample while depleting mononuclear cells; detection depends upon only cytokeratine-20 biomarker

detect epithelial cells by RT-PCR targeting CEA, CK20, and TEM-8 in colorectal carcinoma patients;

CTCs in breast cancer are correlated to bone marrow micrometastases

87

[55, 121]

 Ficoll

density gradient centrifugation

numerous cytospins had to be evaluated because of the low sensitivity; numerous “contaminating” MNCs in the enriched cell fraction lead to false-positive results

detection of CTCs is of prognostic relevance in BCBM patients

84

[55, 122]

 AccuCyte®

density-based cell separation; allows virtually complete harvesting of the red blood cells without cell lysis or wash steps

cellular damage; viable cells recovery rate

the median CTC count was 5 circulating prostate cancer cells/7.5 mL (range, 0-20)

90-91

[69, 123]

 RosetteSep

unneeded cells are cross-linked with RBCs by specific antibodies to form a dense immune rose structure; unlabeled and highly purified target cells are left at the interface between plasma and density gradient centrifuge during density gradient centrifugation

cause inherent cell loss and morphologic changes during the spinning and wash steps

CTCs were detected in 54% (15/28) of MBC patients, 64% (16/25) of advanced stage HNC patients

36 ± 18

[9, 70, 95]

 SPPCN

based on the surface charge of cancer cells serum protein-coated electrically charged nanoparticles can trap different cancer cells

repeated magnetic separation and washing cause cells loss

2-8 CTCs has been isolated from 1 mL of blood;

only 0-1 CTC was detected from 10 healthy donors’ blood samples

50-89

[75]

 DEP-FFF Device

DEP crossover frequencies of CTCs that are distinct from those of peripheral blood cell subpopulations and would permit them to be isolated from blood.

throughput limitation;

Cannot be routinely applied in the biomedical and basic science labs

offer higher discrimination and throughput than earlier DEP trapping methods;

not test in cancer patients

92

[7]

 ApoStream®

using dielectrophoretic technology in a microfluidic flow chamber; overcomes throughput limitations; high precision and linearity of recovery of viable; cancer cells

may cause cellular damage

be used to detect FRα(+) CTCs and may have clinical utility for assessing FRα levels in cancer patients;

detect EMT-CTCs among patients after neoadjuvant chemotherapy

75.4 ± 3.1;

71.2 ± 1.6

[72, 124, 125]

 ELISPOT

enriches cells via a depletion of the CD45+ hematopoietic cells and detects proteins shed/ secreted/ released from single epithelial cancer cells; a multi-parameter analysis revealing a CTC/DTC protein fingerprint

requires efficient antigen binding and specific epitope presentation; high antigen levels demand; transition into in vitro cultures decrease cell viability and reduce detection rates

measure the release of cytokeratin-19 (CK19) and mucin-1 (MUC1) in BC;

measure the release of PSA in prostate cancer;

–

[78]

 CAM assay

based on CTC invasiveness compared to other cells; effective enrichment and identification based on CTC invasiveness; downstream analysis is possible.

isolation step requires more than 12 hours; biomarker dependent

capture invasive CTCs in mCRPC, mNSCLC and mPDAC

54 ± 9

[79]

 Nanoroughened Surfaces

utilize the differential adhesion preference of cancer cells to nanorough surfaces

adhesion strength of cancer cells might be affected by nanotopographic sensing; may cause cellular damage

efficiently capture different kinds of cancer cells (MCF-7, MDA-MB-231, Hela, PC3, SUM-149);

not test in cancer patients

> 80

[79]

 TelomeScan

Detects elevated telomerase activity via a telomerase-specific replication selective adenovirus

May also detect hematopoietic stem cells for false-positive results

The sensitivity of CTC detection was 69.1% in NSCLC patients;

Patients with positive EMT-CTCs at baseline had poor response to chemotherapy and decreased PFS

97

[81, 82, 126]

 eLoaD microfluidic platform

Anti-EpCAM was immobilized on gold electrodes; quantifies CTCs by using label-free electrochemical impedance;

CTCs expressing low levels of EpCAM are unlikely to be captured

perform five different assays in parallel with linear dynamic range between 16,400 and (2.6 ± 0.0003) × 10⁶ cancer cells/mL of blood;

not test in cancer patients

87

[98]

 NanoVelcro

utilize an anti-EpCAM-coated SiNS to achieve significantly enhanced capture of CTCs Thermoresponsive NanoVelcro chips have demonstrated the capture and release of CTCs at 37 and 4 °C

Only EpCAM-positive CTCs are detected

clinical applications of each generation for various types of solid cancers (prostate cancer, pancreatic cancer, lung cancer, and melanoma)

> 85

[99]

 iMECH

deformation-based separation of CTCs from whole blood; enable label-free biomechanical profiling of individual cells for distinction; provide a low-cost yet high-throughput for single-cell level metastatic detection

detect non-metastatic cells for false-positive results; may cause cellular damage

MDA-MB-231 and MDA-MB-468 cells exhibit a loss of resistance;

not test in cancer patients

95 identified as metastatic

[100]

 Size-Selective Microcavity Array

separate cancer cells from the blood in accordance with differences in the size and deformability; approximately 98% of viable recovered cells; fast samples processing speed (200-1000 μL/min)

clogging of cavities; size-heterogeneity

detect approximately 97% of NCI-H358 cells in 1 mL whole blood spiked with 10-100 lung cancer cells;

not test in cancer patients

> 80

[127]

 PDMS microfiltration chip

PDMS microfiltration membrane; size-based separation of CTCs from whole blood

size-heterogeneity; balance the recovery rate and purity

achieved great recovery from lung cancer cells spiked blood samples;

a high processing throughput of 10 mL/h;

not test in cancer patients

> 90

[53]