A summary of the various isolation techniques being utilized
| Technique | Description | Reference |
| Physical properties based assays | ||
| Dean Flow Fractionation (DFF) | The device maintains to process 3 mL of whole blood in an hour using centrifugal forces with >90% CTC recovery. The continuous collection of sorted CTCs and short residence time in the device significantly shortens the CTCs exposure time to constant shear in the channel, thus minimizing any undesirable shear induced changes to the CTCs' phenotype. | 25 |
| Cell density-based enrichment | Density gradient separation of CTCs from other cells in the blood may be performed using commercially available density gradient liquid separation kits. This process generates a layered separation of cell types based on their density. Limitations include a possible loss of CTCs due to an unwanted migration into the plasma fraction, as well as the formation of nonspecific aggregates containing CTCs at the bottom of the gradient. | 26–28 |
| Size-based cell enrichment by filtration | Direct enrichment of epithelial cells by filtration is based on the observation that the vast majority of peripheral blood cells are among the smallest cells in the human body. They can be eliminated by blood filtration using polycarbonate membrane calibrated pore filters. This method is quite simple, involving one single step. | 29–34 |
| Selective size amplification (SSA) | It offers advantages not only in resolving the trade-off between recovery rate and purity—optimizing both—but also in reducing the mechanical stress exerted upon the CTCs during filter transit. The major reasons for this enhanced performance include distinctive size discrimination between WBCs and CTCs as well as the benefits of the solid microbeads mitigating cell deformation within the MOA filter gap. | 35 |
| 3D microfiltration | This device consists of two parylene membrane layers with pores and a gap precisely defined by photolithography. The positions of the pores are shifted between the top and bottom membranes. The bottom membrane supports captured cells and minimizes stress, which is concentrated on the cell membrane and sustains cell viability during filtration under very low pressure. | 36 |
| ISET (isolation by size of epithelial tumor cells) | Size-based enrichments of CTCs have been described by membrane filter devices such as ISET. | 29,30,37 |
| NanoVelcro CTC Chip | By switching device temperature in a physiologically endurable range (i.e. 4-37°C), thermoresponsive conformational changes of nanosubstrate-grafted polymer brushes alter the accessibility of capture agent to specifically capture (37°C) and release (4°C) CTCs to give viable CTCs in desired purity. | 38 |
| Telomescan | A novel cancer detection platform that measures telomerase activity from viable CTCs captured on a parylene-C slot microfilter. Using a constant low pressure delivery system, the new microfilter platform is capable of cell capture from 1 mL of whole blood in less than 5 min, achieving 90% capture efficiency. Addition of an adenovirus-containing GFP to peripheral blood assay, incubation with cancer cells allows precise enumeration and visualization of CTCs. | 39,40 |
| Affinity based assays | ||
| CellSearch | The only FDA-approved technology for CTC detection is based on immunomagnetic enrichment. It employs an immunomagnetic enrichment step to isolate cells that express the epithelial cells' adhesion molecule (EpCAM). Additionally, to be identified as a CTC, the cell must contain a nucleus, express cytoplasmic cytokeratin, and have a diameter larger than 5 μm. This technology has demonstrated the prognostic utility of enumerating and monitoring CTC counts in patients with metastatic breast, prostate, and colorectal cancers. Semiautomated analyzer enriches CTCs with ferrofluid nanoparticles coated with anti-EpCAM antibodies, then CD45-, CK8+, CK18+ and CK19+ cells are counted by a four-color semiautomated fluorescence microscope. | 41–47 |
| CTC-chip | Capture of CTCs by EpCAM-coated microposts under strict manipulation of velocity and shear force. It enables a high yield of capture (median, 50 CTCs per milliliter) and purity (ranging from 10% to 50%), most likely caused by the gentle one-step microfluidic processing. Captured cells remain viable after capture, although the absence of cell fixation currently limits the time allowed between blood collection and microfluidic analysis to a few hours. Captured CTCs are visualized by staining with antibodies against cytokeratin or tissue-specific markers. For CTC enumeration, the entire device is imaged at multiple planes using a semiautomated imaging system while on-chip lysis allows for DNA and RNA extraction and molecular analyses. Nuclear fluorescence and CK stain for positive selection and CD45 stain for negative selection; CTCs captured are directly recognized by cameras, based on morphology, viability and expression of tumor markers. It has a total of 98% cell viability and high detection rate, making further analysis possible. | 48–51 |
| Herringbone-chip | Its chambers were made of transparent materials, allowing imaging of the captured CTCs, using traditional histopathological stains, transmitted light microscopy and immunofluorescence-conjugated antibodies. The Herringbone-chip has been tested in metastatic prostate and lung cancer patients, verifying results with those obtained with the CTC-chip method of analysis: Herringbone-chip shows higher flow rates and higher CTC capture efficiency and purity. | 52,53 |
| AdnaTest | Immunomagnetic separation with EpCAMs and MUC1 coupled antibodies; further analysis by isolation, direct lysis, mRNA extraction and application of multiplexed RT-PCR for HER2, EpCAM and MUC-1. Possibility to characterize CTCs for stem cells and epithelial mesenchymal transition. It lacks flexibility and automation. Cannot enumerate cells due to lysis. False-positive results due to the expression of the same antigens on nontumor cells; false-negative results due to loss of antigens on CTCs. | 54–58 |
| EPISPOT (Epithelial ImmunoSPOT) | Detects only viable cells after the depletion of CD45- positive cells, and was introduced for CTC analyses. Avoiding direct contact with the target cells, this technique assesses the presence of CTCs based on secreted or released proteins during 48h of short-term culture. | 59,60 |
| Collagen Adhesion Matrix (CAM) assay | It has been reported in breast, prostate and ovarian cancer: CAM ingestion and epithelial immunostaining identifies CTCs based on their invasive properties in vitro. | 61 |
| MAINTRAC | A specialized laser scanning cytometer provides another EpCAM-based approach. | 62 |
| Biocept | Utilizes proprietary antibody based enrichment technique to detect rare CTCs found in a patient’s blood sample (1 in 1 million). | 63 |
| Photoacoustic flowmetry | Making use of the broadband absorption spectrum of melanin, it has been tested to detect melanoma cells and has been combined with nanoparticles targeting cell surface antigens to broaden its applicability in CTC detection. | 64,65 |
| MagSweeper | A magnetic stir bar coated with an antibody to EpCAM. The device can process 9 mL of blood per hour and purified cells of interest can be individually selected for subsequent molecular analysis, since the MagSweeper technology preserves cell function and does not perturb gene expression. | 66–70 |
| DEPArray (Silicon Biosystems) | An automated system with fluorescence imaging that captures cells in a chip based upon electric movement. DEPArray achieved 100% purity, eliminating all white blood cells (WBC), in the isolation of a mixed population of tumor cell lines downstream of CellSearch enrichment. This enabled molecular profiling of pure tumor cells from whole blood spiked tumor cell lines. | 71 |
| CTC-iChip | Whole blood is now processed through a microscale system at speeds of 8 mL/hour while preserving the high sensitivity afforded by microfluidic isolation techniques. Furthermore, rapid and gentle isolation of CTCs, as well as their collection in suspension, increases the integrity of these cells and their RNA quality. Moreover, the system can be run in either a positive selection or a negative depletion mode. The robustness of this platform was demonstrated by staining CTCs per clinical pathology protocols, which yielded high-quality diagnostic images. The negCTC-iChip allowed for isolation of CTCs from a nonepithelial cancer (melanoma) and from cancer that has undergone EMT and lost virtually all detectable EpCAM expression (TNBC). Limitations: low CTC purity to facilitate routine molecular analyses of CTCs and total blood volume needed to enable early cancer detection. | 72 |
| Negative depletion CTC enrichment strategy | Relies on the removal of normal cells using immunomagnetic separation in the blood of cancer patients. This method is based on the combination of magnetic and fluid forces in an axial, laminar flow in long cylinders placed in quadrapole magnets. | 73 |
| Millennium Sciences IsoFlux | The blood is centrifuged. Immunomagnetic particles are added to the PMBC layer that target the cells of interest. It is then transferred into a microfluidic cartridge. A permanent magnet is placed on the roof of the channel to attract the labeled target cells. | 74 |
| Cynvenio Liquid Biopsy platform | This platform uses high throughput sheath flow microfluidics for the positive selection of CTC populations. Furthermore the platform quantitatively isolates cells useful for molecular methods such as detection of mutations in 50 oncogenes. | 75 |
| Photoacoustic flowmetry | Making use of the broadband absorption spectrum of melanin, it has been tested to detect melanoma cells and has been combined with nanoparticles targeting cell surface antigens to broaden its applicability in CTC detection. | 64,65 |
| Cytometric assays | ||
| FACS (Fluorescence-activated cell sorting) | It enables simultaneous analysis of multiparameters, such as size, viability, DNA content and expression of different markers for CTCs detection. It has high specificity, but low sensitivity. | 76–79 |
| Slide-based automated scanning microscopes (Ikoniscope and Ariol) | Maximizes scanner utilization with brightfield-multi-channel fluorescent and FISH capture capabilities. Introduced for detecting CTCs; still need to be validated. | 80,81 |
| Fiber-optic array-scanning technology (FAST) | It involves deposition of nucleated cells on the surface of a large glass slide, with scanning of cells positive for epithelial or tumor-specific antigens. Ultra-high-speed automated digital microscopy using fiber-optic array scanning technology has been developed to detect CTCs mounted directly on a slide that are labeled by antibodies with fluorescent conjugates. | 82,83 |
| Multiphoton intravital flow cytometry | It detects CTCs tagged in vivo using injected fluorescent ligands as they flow through the vasculature. | 48 |
| Functional based assays | ||
| Folate-conjugated nanotubes and magnetic uPA-conjugated nanoparticles + photoacoustic flow cytometry assay | This assay has been validated in a mouse model. Most cancer cells express folate receptors and high levels of the urokinase plasminogen activator (uPA) receptors. Thus, CTCs can be dually targeted in vivo (in the bloodstream) with folate-conjugated nanotubes and magnetic uPA-conjugated nanoparticles and subsequently detected with two-color photoacoustic flow cytometry. Future studies on humans will inform whether this new platform can diagnose tumor cell dissemination. | 84 |
| Molecular detection | ||
| RT-PCR | It allows the analysis of expression of candidate genes specific to epithelial tumor cells by mRNA evaluation, often combined with other enrichment techniques. It has high sensitivity. Disadvantages include RNA degradation, false-positive results due to nonspecific amplification, contaminations and pseudogenes; false negative results due to low expression levels. | 85–91 |
| Enzyme-linked immunosorbent spot technology | Immunological assay based on the ELISA (identification and count of cells able to secrete proteins like MUC1 and CK19 in short-term culture), after immunomagnetic depletion of CD45+ cells. Disadvantages include: CTC isolation not possible, further analysis not available, need of active protein secretion and technically challenging. | 55–57 |
| QuantiGene ViewRNA CTC Platform | CTC is isolated by size; sample is prepared (fixed, baked, permeabilized and protease digested) to enable RNA accessibility. Target RNA Probe Sets are hybridized followed by a sequential hybridization of signal amplification and detection components. Once processed, filters are transferred to a microscope slide for image processing and analysis. | 92 |
| CK19 mRNA Assay | Assays targeting specific mRNAs are the most widely used alternative to immunological assays to identify CTCs. In breast cancer, the CK19 mRNA has been most frequently used in clinical studies. Many transcripts (e.g. encoding CK18, CK19, CK20, Mucin-1, prostate-specific antigen and carcinoembryonic antigen), however, are also expressed at low levels in normal blood and BM cells 93, so quantitative RT-PCR assays with validated cutoff values are required to overcome this problem. | 93 |