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Methodology in Detection of Circulating Her2-neu, or carcinoembryonic antigen (CEA) expressed
Tumor Cells on a particular type of cancer cells. Immunomagnetic
isolation technique utilizes monoclonal antibodies that
In general, methodology in the detection of CTCs are labeled magnetic microbeads and separates CTCs
consists of two steps, that is, enrichment and detection from the leukocytes background by magnetic force. To
process. The enrichment process is required because separate leukocytes, the negative selection is performed
of the rarity of CTCs in peripheral circulation using an anti-CD45 antibody recognizing surface marker
6
7
(one CTC per 1 × 10 to 1 × 10 mononuclear cells). of leukocytes. Based on this technique, the Magnetic
To enrich CTCs from blood mononuclear cells, density Activated Cell Sorting System (MACS Miltenyi Biotec,
TM
gradient centrifugation (Ficoll-Hypaque or OncoQuick Bergisch Gladbach, Nordrhein-Westfalen, Germany) is
separation), immunomagnetic or size fi ltration procedures a useful technology for detecting and analyzing CTCs
are used. [6,7] After enrichment, the identifi cation of CTCs because it avoids cell lysis and enables cell count by
is then performed. For identifi cation techniques, nucleic immunocytochemistry and immunofl uorescence assay.
[14]
acid methods and cytometric methods are usually used. CellSearch System (Veridex, Warren, NJ) approved
TM
Recently, the development of molecular techniques by the US Food and Drug Administration (FDA) is
can make molecular and genetic analysis of CTCs a semi-automated analyzer enriching the CTCs with
after enrichment and identifi cation of CTCs, leading to ferrofl uid nanoparticles coated with anti-EpCAM
developing CTC characterization.
antibodies. This system is proved to be useful for
Enrichment Techniques detecting and analyzing CTCs with patients with breast,
[15]
colorectal and prostate cancer in the clinic. However,
Cell morphologic-based enrichment Alunni-Fabbroni and Sandri argued that this technology
Isolation of CTCs using the size of epithelial tumors has two possible limitations, that is, there is no “universal
is based on the size of tumor cells without functional marker” available for each type of tumor, while
modifi cation and complex enrichment procedures. It epithelial marker (EpCAM) could be down-regulated
is usual to utilize 5-8 μm probe fi lters to enable and to in epithelial tumor cells after tumor cells undergo
[16]
separate small leukocytes from the large epithelial cell epithelial-mesenchymal transition (EMT). Thus, this
and the isolation sensitivity threshold is approximately method could only detect selected CTCs. [17,18]
one tumor cell per milliliter. [8,9] These techniques have Enrichment Techniques
a valuable advantage in isolation of CTCs without
damaging cells and enable further immunocytochemical Nucleic acid-based analysis
or immunofl uorescence evaluation of CTCs. Although it Reverse transcriptase polymerase chain reaction (RT-PCR)
is easily handled and cheap, it is considered to be not based techniques can increase the specifi city of the
highly sensitive and poorly specifi c.
molecular methods to discriminate between the higher
Furthermore, density gradient separation using levels of molecular changes in cancer patients and the low
Ficoll-Hypaque is an alternative technique to separate background level in normal cells. Expressions of epithelial
CTCs and mononuclear cells from other blood cells and or tumor-specifi c markers are detected using RT-PCR
granulocytes. However, Ficoll-Hypaque can be toxic to to evaluate and identify CTCs. Nowadays, multiplex
CTCs. CTCs can also be easily to lose due to the migration RT-PCR approach has been established to screen at the
of cells to the plasma layer. OnkoQuick was developed same time more than one single biomarker. Furthermore,
to avoid the cross-contamination of different layers, quantitative RT-PCR improves the specifi city of detection
resulting in higher recovery rate of CTCs. [10,11] Recently, for CTCs by defi ning a cut-off value for biomarker
RosetteSep (Stem Cell Technologies, Vancouver, expression. However, there are some limitations of this
TM
British Columbia, Canada) developed a novel method method: (1) contamination of non-malignant epithelial
based negative selection to improve the specifi city of cells such as skin cells; (2) false positive due to unspecifi c
standard gradient separation. [12,13] markers; and (3) amplifi cation of cell-free nucleic acids.
Therefore, it is essential to select the appropriate marker
Immunomagnetic circulating tumor cell that is expressed specifi cally by tumor cells to boost the
enrichment
specifi city and reliability of CTC detection.
The immunomagnetic CTC enrichment technique is a Cytometric-based analysis
magnetic bead-based separation method. To date, there
have been two methods to identify CTCs expressing Cytometric-based technique can isolate and count CTCs
targeting-specifi c biomarkers. One is using the epithelial using monoclonal antibodies against various antigens.
cell-specifi c marker, e.g. epithelial cell adhesion To detect CTCs, CK and EpCAM are most commonly
molecule (EpCAM) or cytokeratin (CK) expressed on the used. It enables to keep CTCs intact during analysis
surface of tumor cells from epithelial origin. Another is because cell lysis is not necessary. Furthermore, this
using the tumor-specifi c markers, such as α-fetoprotein, technique provides information of high statistical

Journal of Cancer Metastasis and Treatment ¦ Volume 1 ¦ Issue 3 ¦ October 15, 2015 ¦ 131