**3. The main elements in negative selection plus microfluidic CTCs isolation**

#### **3.1. Immunomagnetic beads‐based methods**

**Enrichment strategy**

156 Tumor Metastasis

Label‐free (gradient)

[93].

**System Detection markers Pros Cons**

Size High recovery rate,

CK‐19, BST1, PTPRC High sensitivity;

**Table 1.** Overview of analytical methodologies for the detection and molecular characterization of CTCs.

Another report addressed the differences between positive and label‐free method was reported by Qin et al. [50]. They designed a micropore filtration platform (using resettable cell traps) to perform CTC isolation by the characteristic of CTCs (size and deformability) from patients with castrate resistant prostate cancer. Compared with CellSearch™, the method could capture CTCs 10 times more than CellSearch™ can achieve. The method was also proven to be able to

Interestingly, some investigators compared the isolation efficiency of ISET and CellSearch™ systems [76, 93] and one team concluded that a combination of ISET plus CellSearch™ would have better performance in CTC detection in NCSCL patients than ISET or CellSearch™ alone

One question which is often and needed to be asked is that how to choose a best platform for upcoming studies and trials. Before answering the question, the readers/investigators should fully understand the differences, pros and cons among these methods. Then you should choose

DEP force Size, surface electricity, viability

ODEP force Size, surface electricity, viability

Ficoll + RT‐PCR CK‐19, HER2, h‐MAM,

B726P

OncoQuick CCNE2, DKFZp762E1312, EMP2

EpCAM

perform subsequent molecular analyses.

MAL2, PPIC and SLC6A8, hMAM, and

CEA, maspin, GABA A,

Tracheal carina‐ inspired bifurcated (TRAB) microfilter system

Ficoll + RT ‐qPCR

perform FISH, DNA/RNA

Low throughput; time‐

Relatively low throughput

Require clinical trial

not really capture CTCs

No morphology confirmation; not really capture CTCs

No morphology confirmation; not really capture CTCs

No morphology confirmation; not really capture CTCs

validation

High sensitivity No morphology confirmation;

consuming

Rapid processing, can isolate single cell very

Rapid processing, can isolate single cell very precisely; can differ viable

acceptable purity; viable

from dead cells

analysis

precisely

isolation

quantification

High sensitivity; quantification

High sensitivity; quantification

The method is in fact derived from conventional cytological diagnostics for bone marrow and hematologic malignancies. However, when investigators attempted to apply this method to CTC filed, they faced a big problem—the CTCs were so rare to identify in thousands of blood smear slides. Therefore, an alternative method was to exam samples after series of centrifu‐ gation, density separation (i.e., in buffy coat or peripheral blood mononuclear cells, PBMC layer), and red blood cells removal. The vast majority of the following detection techniques of CTCs in these prepared samples has long been based on sensitive immunocytochemical (ICC) analysis using antibodies against different epithelial antigens [29, 31, 239–242]. Whether positive selection or negative selection procedures using immunomagnetic beads before ICC analysis are both helpful and critical for efficient CTC identification [29, 31]. Zigeuner et al. [31] found that immunomagnetic cell enrichment significantly improves the sensitivity of detection of CTCs cells added to mononuclear cells compared to immunocytochemistry method.

Although the exact procedures of immunomagnetic beads separation protocol was variable with the beads and antibiotics but they have general principles and we would take the procedures of Dynabeads as an example (modified from Naume et al.'s work in 1997 [29]). The main procedures are preparation of beads, incubation with samples and beads, using a magnetic field or column (depends on chosen systems) for target cells isolation by washing out other cells which did not conjugated with beads. If the target cells are those we do not want to analyze, the procedure is defined to be a negative selection. Conversely, if the cells are the targets in the study, it is a positive selection.

#### *3.1.1. Preparation of the magnetic beads*

Rat antimouse (RAM) IgGl‐coated M280 Dynabeads coupled to BerEP4 mAb (Product No. 112.07), M450 Dynabeads coated with an anti‐CD45 mAb that recognizes all isoforms of CD45 (Product No. 111.19), and Neodynium Magnetic Particle Concentrators were sup plied by Dynal (Oslo, Norway). Coating of the M280 Dynabeads with antiepithelial mAb was per‐ formed according to the manufacturer's instructions. Briefly, the RAM M280 Dynabeads were incubated with either BerEP4, 9189, or MOC31 mAbs at a concentration of 1/u.g/107 beads for 30 min at 4°C under gentle rotation, followed by three magnet washes in PBS/0.1% HSA and then stored at 4°C. Before use, the Dynabeads were washed once with separation medium.
