Table 4.

Evolving methods for CTC detection and characterization.

Evolving Methods for CTC Detection and Characterization
		Name	Commercially Available Formats or Providers	Mode of Enrichment	Mode of Detection	Antibodies	Material	Advantages	Disadvantages	References
Enrichment techniques	Morphology-based approaches	Deepcell	Deepcell platform (Deepcell, Inc., Menlo Park, CA, USA)	Isolation of viable cells based on morphological distinction	Images are analyzed using deep learning AI	-	Blood and other body fluids	Permits cluster analysis and further molecular characterization of CTCs	Not clinically validated	[115]
		Nanotube-CTC-chip	-	CTCs adhere to a carbon nanotube surface via filaments extending from the main body of the cell	CTCs are immunostained on-chip and analyzed using automated fluorescence microscopy	DAPI, CKs 8/18, Her2, EGFR, and anti-CD45	Blood	Antigen- and size-independent capture	RBC lysis is necessary	[23]
		TetherChip	-	CTCs are captured based on the affinity of their microtentacles for a polyelectrolyte multilayer	Immunofluorescence staining with Hoechst, WGA, and GFP followed by fluorescence microscopy analysis	-	Blood	Preserves microtentacle structure after fixation and isolation from blood; enables testing of functional phenotypes in CTCs	Only tested on cell lines at the time of writing	[24]
	Immunology-based approaches	GILUPI CellCollector	GILUPI CellCollector (GILUPI GmbH, Potsdam, Germany)	CTCs captured by antibodies immobilized on a hydrogel	Immunofluorescence staining and molecular analysis (e.g., PCR, sequencing, gene expression analysis)	EpCAM	Blood	Enriches CTCs directly from bloodstream rather than volume-limited blood samples; enrichment time is 30 min	Used only for enrichment of CTCs directly from patient’s bloodstream	[116,117]
		3D conductive scaffold microchip	-	CTCs are captured on a 3D conductive scaffold made from porous polydimethylsiloxane with immobilized gold nanotubes (Au-NT) coated with an anti-EpCAM antibody	Immunocytochemistry using FITC-CK, PE-CD45, and DAPI	EpCAM	Blood	Captured cells can be reversibly released with high viability; high sensitivity	CTC clusters released less efficiently than single CTCs because of re-capture by the 3D scaffold	[118]
		3D nanoforest array	-	Cellular filopodia of CTCs interact with lateral branches of Zn(OH)F nanowires conjugated to an anti-EpCAM antibody	Immunofluorescence staining and fluorescence microscopy analysis	EpCAM, CD45, CK	Blood	Large binding surfaces provide many binding sites for CTC capture	Only tested on cell lines at the time of writing	[119]
		3D-printed functionalized device	-	3D-printed channel whose inner surface was functionalized with anti-EpCAM	Confocal laser scanning microscopy	EpCAM	Blood	Microfluidic device with a large binding surface area	Only tested on cell lines at the time of writing	[120]
Detection techniques		Epic Sciences	Epic Sciences (Epic Sciences, Inc., San Diego, CA, USA)	-	Pyxis™—whole slide fluorescent scanner	Cytokeratin, CD45, DAPI, and specific antibodies	Blood	Enrichment-free; cancer profiling combining CTC technology with circulating tumor DNA (ctDNA) and immune cell analysis	Samples must be sent to the company for analysis, only for prostate and breast cancer	[121,122,124]
		AI nanoarray	-	Detects both cancer cells and VOCs from cancer cells and their microenvironment	Gas chromatography linked with mass spectrometry	-	Blood	High sensitivity and specificity for early detection	Only tested on cell lines and a mouse model at the time of writing	[125,126]
Approaches combining CTC enrichment and detection		3D-printed microfluidic device	-	WBCs are captured in the device’s immunocapture channels; RBCs, platelets, and all nucleated cells migrate to a membrane micropore filter	CTCs are immunostained on-chip and analyzed using fluorescence microscopy	CD45	Blood	Label-free negative depletion of CTCs; isolation of very small CTCs	Only tested on cell lines at the time of writing	[127]
		CTCelect	CTCelect system (Fraunhofer Institute for Microengineering and Microsystems, IMM, Mainz, Germany)	Combines immunomagnetic enrichment with microfluidic sorting of fluorescence-activated cells	Fluorescence microscopy	EpCAM	Blood	Fully automated; permits further molecular characterization of CTCs	Captures only single cells, not clusters	[128,129]
		VyCAP	VyCAP technology (VyCap B.V., Enschede, The Netherlands)	Size-based filtration through a microsieve filter chip	Fluorescence microscopy with automated imaging system	CK, CD16, and CD45. Other cancer-specific labels can also be used (e.g., MUC-1, PDL-1)	Blood	Fully automated; filtration under low pressure, which minimizes damage to captured cells	Not clinically validated	[130,131]
		MyCTC chip	-	CTCs are captured on microfluidic chip with a polydimethylsiloxane upper layer and a rigid cyclic olefin copolymer underlayer	Cultivation of captured CTCs	-	Blood or other body fluids	Label- and antigen-free; captures clusters with high efficiency	Not clinically validated	[132]