the ClearCell FX technology uses Dean Flow and
hydrodynamic focusing within a curved channel
for differential collection of CTCs and WBC
streams. 25 However, this method includes lysing
the RBCs prior to isolation of CTCs.
Such technologies or approaches have the
advantages of being less complicated, sometimes
rapid, and requiring minimal equipment.
However, some of these approaches may be prone
to clogging and the release of the CTCs into
suspension for further analysis is challenging.
a substantial number of leucocytes, and CTCs
need to be identified specifically at the single-cell
level by a robust and reproducible method that
can distinguish them from normal blood cells.
Immunological techniques
CTCs can be detected by using a combination
of membrane and/or intra-cytoplasmic anti-
epithelial, anti-mesenchymal, anti-tissue-specific
marker or anti-tumour-associated antibodies. 11
However, many CTC assays use the same
identification step as the CellSearch ® system: cells
are fluorescently stained for cytokeratins (CK), the
common leucocyte antigen CD45 and a nuclear
dye (DAPI). Through multicolour image analysis
with a fluorescence microscope, CTCs are defined
as CK + /CD45 − /DAPI + cells. Although some antigens
are applicable to various different cancer types
(for example, CK for breast, colon and prostate
cancer and other epithelial tumours), tissue-
specific antigens are also suitable (PSA for
prostate cancer).
Ultra-high speed automated digital microscopy
using fiber optic array scanning technology
(FAST) has been developed to detect CTCs that
are labelled by antibodies with fluorescent
conjugates. 31,32 Other slide-based automated
scanning microscopes have been introduced
for detecting CTCs, including the Ikoniscope ®
imaging system and the Ariol ® system, 33,34 and
show promising results that still need to be
validated in large clinical studies. Some
researchers also use flow cytometry technology,
which offers high throughput detection and
labeling of several targets at the same time (9 or
12 targets). 35 Moreover some cytometers allow cell
sorting for post-processing analysis. 36 Indeed,
immunological detection offers the advantage of
allowing isolation of stained CTCs for subsequent
molecular characterisation. While manual
isolation by micromanipulation of CTCs is
possible, 37 it is rather arduous and time-
consuming; an alternative automated single cell
selection device has therefore been developed.
The DEPArray™ technology, based on
a dielectrophoresis strategy by trapping single
cells in DEP cages, 38 is designed for single-cell
recovery of CTCs. Multiple clinical studies have
used DEPArray™ to detect and recover single
CTCs for subsequent genetic analyses following
enrichment. 39–41 The use of this technology for
CTC recovery will likely be limited to samples
with a relatively high number of CTCs due to
a cell-loss of approximately 40% during sample
loading. 42
Dielectrophoresis selection
An innovative approach for cell separation,
dielectrophoresis (DEP), exploits the distinct
electrical fingerprints of different cells, which
depend on their composition (for example, cell
membrane, nucleus, organelles), morphology
(for example, size, shape), and phenotype.
ApoStream ® , a commercial system for CTC
enrichment, applies the first strategy through
the use of a microfluidic device using DEP
field-flow assist cell separation technique. 26
Cluster selection
Few methods have been developed for specialised
detection of CTC clusters. In most cases, CTC
clusters were observed when detecting individual
CTCs. Multiple studies using microfiltration for
CTC isolation have reported capturing CTC
clusters (microemboli). 19,27–29 These reports
indicate that CTC clusters stay intact when
isolated with microfiltration methods, whereas
other enrichment strategies might either fail to
capture clusters or break them apart. Sarioglu
et al reported the development of a novel
three-dimensional microfiltration system (the
Cluster-Chip) specifically designed to capture CTC
clusters. 30 The simple, but sophisticated, design
of the Cluster-Chip captures these circulating
tumour microemboli using multiple rows of
shifted triangular pillars.
Strategies for CTC detection
After enrichment, the CTC fraction still contains
Table 1
Strategies for circulating tumour cell
enrichment and detection
Enrichment
Biological properties
– Positive selection
Cell Search
IsoFlux
Magswipper AdnaTest
Ephesia CTC-Chip MACS
– Negative selection
RosetteSep MACS
EasySep
CTC-iChip
Detection
• Immunological
• Molecular
• Functional
– In vivo
Tumour xenograph
– In vitro
EPISPOT
Invasion assay
Nucleic acid-based strategies
Nucleic acid-based CTC detection methods are the
most widely used alternatives to immunological
assays. These technologies identify specific
tumour DNA or mRNA to confirm the presence
of CTCs indirectly. 43 Detection involves designing
specific primers supposedly associated with
CTC-specific genes. These genes either code for
tissue-, organ-, or tumour-specific proteins, or,
more specifically, contain known mutations,
translocations, or methylation patterns found in
cancer cell