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Thanks to various advantages, such as retention of cell morphology, antigen independence, and high
sensitivity and specificity, membranous filter devices, for example, isolation by size of epithelial tumor cells
(ISET) (Rarecells Diagnostics, Paris, France), have caught more attention in CTC researches. In a comparative
study, Bai et al. estimated the clinical effect of CTCs by using CellSearch system and ISET devices among
[47]
patients with renal cell carcinoma (RCC), discovering that ISET was more appropriate for RCC patients.
CTCBIOPSY (Wuhan YZY Medical Science and Technology Co., Ltd., China) is a commercial one-step
ISET device which could complete automatic detection and identification within 10 min . The Parsortix
[48]
technology (ANGLE plc) incorporated a microscope slide sized cassette for CTC separation based on cell
size and compressibility . Owing to the excellent capture performance and the advantage of easy retrieval
[49]
of viable CTCs for downstream analysis, the FDA clearance process of this device for diagnose is underway.
The ClearCell FX system (Clearbridge BioMedics, Singapore), one of the first automated cell separation
and retrieval systems, is a new label-free and size-based technology with extremely high recovery rates
by dean flow fractionation . These devices, together with other similar size exclusion platforms, such as
[50]
ScreenCell (ScreenCell, France) based on microporous membrane filter , CellSieveTM (Creatv Microtech),
[51]
and MetaCell (Ostrava, Czech Republic), constitute the next generation label-free CTCs enrichment
technologies, demonstrating CTC isolation and detection with high efficiency, purity and viability.

Moreover, size difference can be combined with other physical features to improve capture yield. For example,
a recent wedge-shaped microfluidic device [Figure 2G] based on the difference in size, as well as rigidity and
nuclear/cytoplasmic ratio between CTCs and normal blood cells, was fabricated to enhance CTC isolation,
exhibiting excellent capture performance with ≥ 85% capture efficiency . Similarly, benefiting from those
[52]
multiple biophysical properties, Liu et al. [Figure 2H] developed a pyramid-shaped microchamber to
[53]
achieve a more than 85% capture efficiency and a 93% recovery yield. In addition, vortex technology has
been exploited and validated for isolating CTCs based on differences in size, shape and deformability by
inertial microfluidics and laminar micro-vortices. VTX-1 liquid biopsy system [Figure 2I] was developed for
fully automated isolation and enumeration of CTCs with either high recovery mode or high purity mode in
the vortex microfluidic chip .
[54]

Density-based CTC isolation
The density of nucleated CTCs lies between plasma and red blood cells, and within the scope of white blood
cells. Quantitative buffy coat analysis by centrifuging for separation was established by Stephen C. Wardlaw
in 1983 . AccuCyte separation based on this principle is the first step of the commercial RareCyte Platform
[55]
(RareCyte, Inc. Seattle) , coupled with fluorescence analyzing (CyteFinder system) and picking (CytePiker)
[56]
to count and retrieve cells for downstream single-cell characterization, overcoming the limitation of capture
methods which are dependent on sizes that might miss the small sized-CTCs and immunomarkers that might
not be expressed on some subpopulations. Some commercial density gradient solutions, such as Ficoll-Paque
(GE Healthcare) and Percoll (GE Healthcare), provide simple-to-use and inexpensive methods for separating
CTCs in the mononucleocyte layer from granulocytes and erythrocytes. OncoQuick (Greiner Bio-One/
Hexal Gentech, Germany) consists of a sterile tube with a porous barrier inserted above separation medium,
allowing the simple, rapid and highly efficient enrichment of CTCs through density-based centrifugation
and size-based separation.

Dielectrophoresis based CTC isolation
The overlap of size or density between CTCs and normal cells may affect the efficiency of these size-/density-
based approaches. Electrical properties of CTCs have been applied to discriminate them from other normal
cells using dielectrophoresis (DEP). Based on conventional DEP devices, microchips are used to manipulate
electric fields to achieve higher capture efficiency and recovery rate. Nguyen et al. fabricated a microchip
[57]
to guide target lung CTCs to sensing electrodes by DEP and hydrodynamic forces, achieving a LOD of
3 cells and an efficiency over 90% at 50 kHz electric field intensity within 10 minutes. The commercial