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Detection Technologies on Circulating Tumor Cell
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Detection Technologies on Circulating Tumor Cell . Classification, advantages and limitations of circulating tumor cell detection technologies!
Circulating tumor cells (CTCs) are tumor cells that shed naturally from the original or metastatic tumor tissue and circulate in the blood, and can lead to new metastasis.
CTC detection is a key tool to fight cancer and can be used for early diagnosis, treatment monitoring, recurrence monitoring and medication guidance.
Researchers published an article entitled “Current detection technologies for circulating tumor cells” in the authoritative magazine “Chemistyr Society Reviews” (IF=42.846), classifying the currently reported CTC detection technologies and summarizing the advantages and limitations.
The currently developed CTC detection technology is generally divided into four steps:
The capture step is the specific interaction between CTCs and materials (such as physical interactions and antibody/antigen interactions), such as magnetic beads, microfluidic chips;
the enrichment step refers to the separation of CTCs from blood;
after enrichment, CTCs It can be detected by fluorescence (such as fluorescence microscope, fluorescence spectrophotometer and flow cytometer) [1, 2], surface enhanced Raman scattering (SERS)  or electrical impedance ;
CTCs can be further released after release Phenotype identification and molecular analysis (such as RNA analysis and cell metabolism analysis).
Current CTC detection technology classification technology tree
The important technical indicators of these CTCs detection technologies include recovery, purity and limit of detection (LOD), as shown in Table 1.
The recovery rate is defined as the percentage of enriched CTCs in the total number of CTCs in the blood, also known as capture efficiency or enrichment efficiency.
Purity refers to the percentage of the number of enriched CTCs to the total number of cells in the enriched sample. LOD is the lowest concentration of CTCs that can be detected in the blood.
01 Non-enriched CTC technology
The detection of CTCs without an enrichment step is also called direct detection, including scanning confocal microscopy and surface enhanced Raman scattering (SERS) technology.
Confocal Microscopy Technology
Confocal microscopy technology is a fast and automated high-throughput screening method, developed based on microfluidics and multi-wavelength scanning confocal detection technology . Cells in the blood are first simultaneously labeled with multiple antibodies, which bind to different fluorescent agents. The blood is pumped out through the microfluidic channel and scanned with a confocal microscope. According to the fluorescence signal and labeling method, the number of CTCs can be automatically counted and reported.
(1) Due to the limited flow rate in the microfluidic channel, the detection time for each sample is too long (analyzing 7.5 mL of blood takes about 3.5 hours);
(2) The false negative rate reaches more than 40%;
(3) Because Several fluorescent dyes combined with antibodies are needed to label cells in the blood, resulting in high sample detection costs;
(4) CTCs cannot be separated for downstream phenotype identification and molecular analysis.
SERS direct detection
SERS is another method for rapid analysis of CTCs in peripheral blood. It is based on highly specific and sensitive SERS active nanoparticles with targeted ligands to identify CTCs in healthy blood cells.
First, the peripheral blood sample is added to the peripheral blood lymphocyte separation medium, and then centrifuged at room temperature.
The low-density cell layer containing white blood cells (WBCs) and CTCs is transferred to a new tube, incubated with SERS active nanoparticles, and washed with phosphate buffered saline (PBS), and finally the SERS spectrum of the sample is analyzed by the Raman system [ 6, 7].
The characteristics are:
(1) The CTC detection process is not easy to automate;
(2) Ligands are required to capture CTCs in the blood, and the cost of detection reagents is high;
(3) CTCs cannot be separated for downstream phenotyping and molecular analysis.
02 Need enriched CTC technology
The low concentration of CTCs in the blood can be enriched in two different ways.
One is negative enrichment, capturing non-target cells and eluting target cells; the other method is positive enrichment, capturing CTCs and eluting healthy blood cells.
Lara et al. developed a negative enrichment technology for the first time to obtain rare cells in the blood. The enrichment process includes red blood cell lysis, CD45 antibody screening for white blood cells, flow through the system for immunomagnetic separation, and finally automatic cell counting, filtration and visual counting or Cell spiral analysis carries out the cell analysis process .
In order to improve the efficiency of CTC negative enrichment, Hyun  and others developed a geometrically activated surface interaction (GASI) microfluidic chip, which uses herringbone to effectively capture a large number of blood cells (3a).
The channel is a herringbone structure, which can produce lateral flow and promote effective contact between the antigen on the cell and the antibody on the channel surface (3b and c).
This is the first attempt to use a negative enrichment microfluidic chip to enrich CTCs from metastatic cancer patients.
The GASI chip successfully enriched CTCs, and the same method may help collect other types of circulating rare cells, whose phenotype is not yet clear.
In 1 ml of blood, the number of separated CTCs ranged from 1 to 51.
The features of negative-enriched CTC detection technology include:
(1) CTCs-independent biomarker expression (such as EpCAM);
(2) Complete CTCs can be collected, and downstream research such as cell and molecular analysis can be performed;
(3) Easy to automate .
Positive enrichment captures CTCs and eluates healthy blood cells, which is divided into in vivo enrichment and in vitro enrichment.
In vivo enrichment
In vivo, the positive enrichment of CTCs captures and enriches a large number of rare CTCs. Galanzha et al.  proposed a method of magnetically capturing CTCs in the bloodstream of mice, and then performing rapid photoacoustic detection.
This method integrates multiple targeting, magnetic enrichment, signal amplification and multi-color recognition in vivo, enabling CTCs to be concentrated from a large amount of blood in the blood vessels of tumor-bearing mice, which may show potential for early diagnosis of cancer and prevention of human metastasis .
The characteristics of in vivo enrichment in CTC detection are:
(1) the enrichment time is very long;
(2) the technology is immature, and there is no data report on the purity of CTC;
(3) the cost is very high: CTC capture requires antibodies.
In vitro enrichment
1. No need for ligand capture.
In vitro CTC detection without specific ligand binding is called “physical capture”, which mainly relies on the physical properties of CTCs (cell density, cell size, and nanorod surface) to capture.
(1) The expression of biomarkers that do not depend on CTCs (such as EpCAM);
(2) The enriched CTCs are complete;
(3) The enrichment time is short and can be used for downstream analysis;
(4) Low cost. The disadvantage is that these technologies are difficult to commercialize, and the purity of the CTCs obtained by enrichment is low.
2. Capture with ligand.
2.1 Single method of enrichment.
It means that only one of the following enrichment technologies with ligand capture is used for the detection of CTCs: density gradient sedimentation, size exclusion filtration, barcode particles, self-propelled micromachines, magnetic beads and microfluidic chips.
(1) Density gradient precipitation:
The ligand is bound to the surface of the microspheres with high density to capture CTCs, and through selective precipitation, the microsphere-CTC complex is effectively separated from healthy blood cells.
1) The use of ligands increases the cost of testing;
2) Microbeads may affect light scattering and quenching, thereby interfering with fluorescence detection;
3) Beads may be internalized into CTCs, thereby affecting the activity of CTCs;
4) Free microbeads mixed with microbead-CTC complex will interfere with the accurate detection and analysis of CTCs.
(2) Magnetic beads:
Ligands are bound to the surface of magnetic beads to capture CTCs, and the magnetic bead-CTC complex is separated from healthy blood cells by an external magnetic field. This is a relatively mature technology.
- 1) High testing cost;
- 2) The internalization of microbeads may affect the feasibility of CTC;
- 3) Rely on the expression of CTC markers, such as EpCAM;
- 4) The entire process (CTC capture, enrichment and detection) is not easy to automate.
- 3) Microfluidic chip:
Microfluidics can achieve precise control on extremely small sizes. The amount of blood sample taken from the patient is small, and the ligand is connected to the microfluidic chip to capture CTCs in the flowing blood, thereby separating CTCs from healthy blood cells.
There are many different types of microfluidic chips based on ligand capture.
The main difference lies in the design of the microchip, including the channel structure and substrate coating. Nagrath et al. developed a unique microfluidic chip for CTC enrichment.
The chip is composed of a series of microcolumns functionalized with anti-EpCAM antibodies . The two basic parameters that determine the efficiency of cell capture on the CTC chip are:
- 1) Flow rate: because it affects the duration of cell-microcolumn contact;
- 2) Shear force:It must be low enough to ensure maximum cell-microcolumn attachment. Under precisely controlled laminar flow conditions, the microfluidic chip successfully identified CTCs in peripheral blood of cancer patients in 115 out of 116 (99%) samples, with a purity ranging from 5-1281 CTCs per milliliter. About 50.
The characteristics of microfluidic chip enrichment are:
- I) Limited sample size;
- II) Long time for CTC enrichment;
- 3) The shear force must be low enough to ensure maximum cell substrate attachment.
2.2 Bimodal enrichment
In order to improve the efficiency of CTC enrichment in blood samples, two of the above six enrichment techniques can be combined.
Combinatorial enrichment technology is called dual-modal enrichment, such as density gradient sedimentation plus size exclusion filtration.
However, the technology is not yet mature and has not been extensively studied.
CTCs detection is a key tool to fight cancer, and it plays a key role in early cancer diagnosis, treatment monitoring, recurrence monitoring, and medication guidance. “Rareness” is the biggest difficulty in CTCs detection.
How to accurately capture a few CTCs from hundreds of millions of blood cells? Enrichment efficiency is extremely important.
At present, there are many detection technologies for CTCs on the market, and the negative enrichment technology is recognized as the most efficient and most commonly used method for enrichment.
Independent of the size, physical and chemical properties and surface markers of CTCs, all types of CTCs can be wiped out.
Detection Technologies on Circulating Tumor Cell
(source:internet, reference only)