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recovered from the medium.

Although cell filtration and centrifugation force have been investigated on the basis of these properties
in past decades, it has been demonstrated that variations in CTC size have identified, and CTCs after
[29]
undergoing EMT could be as deformable as leukocytes . Therefore, new approaches have been developed
to improve specificity of CTC enrichment.

Detection techniques
After CTC enrichment, CTCs are detected by many different assays. Recent CTC identification assays
combine enrichment and detection processes (e.g., CellSearch System, ISET, AdnaTest, CTC-chip, and
EPISPOT). Other detection technologies include immunocytological techniques, molecular techniques, and
functional assays.

Immunocytological techniques
Immunocytological techniques detect CTCs using antibodies against various antigens. These provide
characteristics with high accuracy and subpopulation quantification with high specificity for simultaneous
analysis with multiple parameters. However, the drawback of these techniques is lower sensitivity compared
with molecular techniques.

Fluoroscence-assited cell sorting (FACS) is widely used to separate a specific cell population by using
antibodies. Since FACS can analyze many parameters simultaneously, it is a versatile method with a wide
range of applications. FACS sorts each cell individually, meaning that throughput of FACS is limited.
[30]
Moreover, sorting conditions may be harmful to certain types of cells .
Fiber-optic array scanning technology (FAST; SRI International, Menlo Park, CA) can more efficiently
analyze large numbers of immunofluorescent-labeled cells in peripheral blood. FAST applies laser-based
techniques to scan broad fields of view, and can detect and characterize CTCs extremely quickly and
accurately. As FAST can analyze larger volumes of peripheral blood, it does not require an additional
[31]
enrichment step and reduces the risk of cell loss .
Fluorescence in situ hybridization (FISH) can precisely detect specific DNA sequences within chromosomes
by using fluorescent probes. However, FISH requires high proficiency, and sometimes cannot provide clear
results. To overcome these problems, a novel technology named Ikoniscope® (Ikonisis, New Haven, CT)
[32]
was developed for rare cell detection . This system can detect one CTC per milliliter of peripheral blood.
However, cells no longer have viability after FISH; therefore this technology has limited application for
analyzing CTC.

Molecular techniques
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) can analyze the expression
of specific markers in CTCs. Specificity of qRT-PCR has been reported to be superior to that of
[33]
immunohistochemistry . Nowadays, a multiplex RT-PCR approach combined with liquid bead array
detection has been developed to perform simultaneous amplification and detection of multiple biomarkers.
However, there are several limitations, such as the contamination of non-malignant cells, the high rate of
[34]
false positives, and amplification of cell-free nucleic acids . In addition, once RNA has been collected from
cells, the cells cannot undergo advanced analysis.

Functional assay
[35]
Epithelial immunospot (EPISPOT) detects specific tumor marker proteins secreted by CTCs . Only viable
CTCs are detected by EPISPOT because non-viable CTCs are not enough to detect secretion of proteins.
[36]
EPISPOT is much more sensitive than ELISA when detecting secretion of CK19 from CTCs . However,