HHE Pathology and diagnostics supplement 2018 | Page 10

other solid tumours) and other leukocyte
antigens, to achieve a CTC-enriched sample.
The RosetteSep TM CTC Enrichment Cocktail
(STEMCELL Technologie) offers a unique method
for further depletion of unwanted cells by
integrating immunoaffinity-based enrichment
with density centrifugation. RosetteSep™ targets
unwanted cells and forms a pellet with red blood
cells (RBCs) through tetrameric antibody
complexes that target an extensive mixture of
specialised antigens. An immunomagnetic version
is also available – the EasySep™ system – which
contains magnetic nanoparticles and tetrameric
antibody complexes targeting CD45, for example.
Some technologies are flexible and can function
either as positive or as negative enrichment
systems by applying different antibodies
(for example, replacing anti-EpCAM, specific
for epithelial cells with anti-CD45, specific for
leucocytes). For example, the CTC-iChip could
use an immunomagnetic selection with
functionalised beads against EpCAM or CD45
and CD66b. 17
Positive selection
Mostly used, immunomagnetic systems target
an antigen with an antibody, which is coupled
to a magnetic bead, and the antigen–antibody
complex is isolated via exposure to a magnetic
field. Positive selection is usually carried out with
antibodies against the epithelial cell adhesion
molecule (EpCAM). Among the current EpCAM-
based technologies, the FDA-approved
CellSearch™ system has gained considerable
attention over the past ten years and is frequently
compared with all new CTC detection methods as
the gold standard. However, capturing CTCs
lacking EpCAM has involved the use of antibody
cocktails against various other epithelial cell
surface antigens (for example, EGFR, MUC1), or
against tissue-specific antigens (for example, PSA,
HER2) and against mesenchymal or stem cell
antigens (Snail, ALDH1). 11 Many commercial
platforms for CTC detection using positive
magnetic enrichment are available, including:
• Based on EpCAM: MagSweeper 12 or MACS
technologies from Miltenyi 13
• Using different cocktails of antibodies:
IsoFlux TM , 14 AdnaTest from Adnagen. 15
At present, there is a focus on the development
of microfluidics devices (‘chips’) such as Ephesia
Chip 16 or CTC-iChip. 17
Interestingly, a unique in vivo device, GILUPI
CellCollector ® , was developed to capture CTCs
directly in the vein of patients, with a structured
medical Seldinger guidewire functionalised with
an antibody targeting EpCAM to trap CTCs. This
wire allows screening of a large volume of blood
during the 30-min collection period.
Positive selection of CTCs requires an
assumption about the unknown nature of CTCs in
an individual blood sample. This bias is avoided
by negative selection in which the blood sample
is depleted of unwanted cells.
Negative selection
Negative enrichment uses an indirect method to
isolate CTCs: they target and remove background
cells, such as leucocytes, using antibodies against
CD45 (which is not expressed on carcinomas or
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HHE 2018 | hospitalhealthcare.com
Physical properties
Numerous marker-independent techniques have
been developed for CTC isolation and detection.
Label-free enrichment processes based on physical
properties, such as density, size, deformability,
and electric charge, avoid molecular bias induced
by variability of cell biomarker expression
associated with tumour heterogeneity.
Density selection
Density gradient centrifugation was one of the
first methods recorded for CTC isolation. 18
Although not originally developed for CTC
isolation, Ficoll-Paque ® , a density gradient
medium for the separation and isolation of
mononuclear cells, has been used in research for
some time. However, after such a non-specific
pre-analytical process, CTCs are still present in
a large number of leucocytes; indeed, only
erythrocytes and polynuclear cells are depleted
using Ficoll-Paque centrifugation. To improve this
enrichment, a subsequent positive or negative
step is usually required and performed. Designed
for CTC isolation, OncoQuick (Greiner Bio-One)
employs a liquid separation medium that has
been optimised for the specific enrichment of
CTCs only, based on their buoyant density under
appropriate conditions and no additional step
is required because even the leucocytes are
eliminated from the cell monolayer between
the plasma and the Ficoll.
Size selection
Microfiltration technologies, based on the
precedent that CTCs generally exhibit a
larger morphology than leukocytes, such
as ScreenCell ® , 19 ISET ® , 20 CellSieve™, 21,22 or
Parsortix™ 23 involve flowing blood through pores
or microfluidic steps of calibrated size to trap
larger cells (the CTCs) while smaller cells pass
through.
Some other size-based microfluidic devices use
inertial focusing to separate CTCs from blood.
Vortex technology relies on inertial microfluidics
and laminar microscale vortices to position cells
along channel walls upstream of micro-vortices
designed to stably trap CTCs. 24 In the same way,