**3.3. Perspective for future of CTC technology: combinations of several methods**

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.

separation medium to a concentration of 2 ×107 MNC/ml (0.5 ml volume) and incubated with RAM IgGl M280 Dynabeads coated with either BerEP4 317G5, or MOC31 mAb. The bead

bead/cell suspension was incubated under gentle rotation for 30 min at 4°C. The sample was then diluted to 3 ml and placed against a magnet for 7 min to recover the rosetted cells, followed by two additional washes as follows. The supernatant was removed, and the rosetted cells were resuspended in 3 ml separation medium, followed by treatment with the magnet (7 min). To facilitate ICC TC detection, the positive LMS product was finally resuspended to contain 5–7 × 106 beads/ml, and 0.5 ml aliquots were centrifuged onto each cytospin slide for further

separation medium to a concentration of 2 × 107 MNC/ml (0.5 ml volume) and incubated with anti‐CD45‐conjugated M450 Dynabeads at a bead/cell ratio of 2.5:1, 5:1, or 10:1. The bead/cell suspension was incubated under gentle rotation for 45 min. The solution was then diluted to about 30 ml, and the magnet was applied for 5 min, with initial rotation of the tubes onto the magnet to reduce trapping of tumor cells. The supernatant was collected and centrifuged at 450*g* for 10 min, counted, and resuspended in 10% FBS in PBS to 1 × 106 cells/ml. Then, cytospins containing 5 × 105 cells were prepared. All the slides were air‐dried overnight and stained by

In novel era of huge advances of microfluidic devices as mentioned in the section of "Label‐ free isolation strategy" [45, 46, 56, 60, 173–186]. In fact, the vast majority of microfluidic devices were designed based on EpCAM‐ or CK‐identifying mechanism, which is positive selection method. The CTC‐Chip [25, 243], and the herringbone chip [244, 245] have been proven effective to isolate CTCs with both high CTC purity (50–62%) [25, 245] and high recovery rate (90–95%) [244, 245]. There are several microfluidic devices designed to use positive selection strategy for proof‐of‐concept purpose [34, 57, 59, 124, 184, 210, 246–269] and for specific cancer in clinical trials, (e.g., breast [270, 271], pancreas [272, 273], ovarian [274], prostate [275], esophageal cancer [270], gastric [271], colorectal cancer [276], cancer of unknown primary [277]) and for mutational analysis [278]. Moreover, combined preparation using positive, negative, or label‐free selection methods with microfluidic devices for better performance is

peripheral blood mononuclear cells (PBMNC) were resuspended in cold

peripheral blood mononuclear cells (PBMNC) were resuspended in cold

b/ml, as described in individual experiments. The

*3.1.2. Positive immunomagnetic separation technique*

*3.1.3. Negative immunomagnetic separation technique*

**3.2. Microfluidic‐based methods for the high purity CTC isolation**

concentration varied from 2.5 to 40 × 106

immunocytochemical analysis.

A total of 1 × 107

158 Tumor Metastasis

A total of 1 × 107

immunocytochemistry.

also feasible and have been reported [93].

Our perspectives for future CTC isolation is mainly combined methodologies instead of conventional ones based on a single isolation strategy.

Yamamoto et al. [49] displayed a combination of size‐based filtration plus a magnetic column method for CTC isolation. The combined use of the column and filter decreased the required time for the spiked cancer cell capture, and the recovery rate of the spiked cancer cells from blood was significantly higher using the combination process (80.7%) than that using the filter alone (64.7%). Moreover, the recovered CTCs are more abundant by the combination process. Another combination was ISET and CellSearch™ systems [76, 93] and the combination had better performance in CTC detection in non‐small cell lung cancer (NCSCL) patients than ISET or CellSearch™ alone [93]. Furthermore, density separation plus flow cytometry or cell sorting systems have been postulated to be potential tools of CTC isolation and identification consid‐ ering their high sensitivity and purity since 1998 [27]. This method could be seen as a combi‐ nation of negative selection strategy and a positive confirmation with surface markers, such as EpCAM or cytokeratins. Later in 2011, leukapheresis plus fluorescence‐activated cell sorting (FACS) elutriation were also reported to be effective for large volume blood process for CTC isolation with molecular analysis [211]. These studies illustrated the possibility and better efficacy the combination can achieve, therefore, in our opinion, to find a suitable combination of CTC isolation protocols considering the balance of efficiency, time, sample and costs is very important in the future CTC field.

Intriguingly, several liquid biopsies, as aforementioned, could be combined to be tested in a single sample and at the same time. To realize the goals and minimize the blood sample required, Chudziak et al. [248] reported a novel device, Parsortix system, could negatively select CTCs and perform cfDNA analysis simultaneously. The system recovered more CTCs than CellSearch™ system in the comparison.
