*3.1.2. Positive immunomagnetic separation technique*

A total of 1 × 107 peripheral blood mononuclear cells (PBMNC) were resuspended in cold 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 concentration varied from 2.5 to 40 × 106 b/ml, as described in individual experiments. The 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 immunocytochemical analysis.

### *3.1.3. Negative immunomagnetic separation technique*

A total of 1 × 107 peripheral blood mononuclear cells (PBMNC) were resuspended in cold 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 immunocytochemistry.

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

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 also feasible and have been reported [93].

However, there are several drawbacks or limitations of microfluidic devices reported [267]. First of all, reports in literature, however, have revealed that EpCAM or CKs are not expressed in all cancer cells (e.g., sarcoma, melanoma, or CTCs bearing EMT), and therefore some kinds of CTCs cannot be targeted via the positive selection‐based microfluidic device [279]. Secondly, several microfluidic chips could identify with microtubes or micropoles with or without EpCAM conjugation. It seems to be difficult to release captured CTCs from the chips. The efficiency of identification will not be equal to recovered cells for further molecular or genetic analysis. Thirdly, almost more than 80% of microfluidic devices are still in proof‐of‐concepts phase and comes from a single team or laboratory. It might be because that the advances of new innovation always come up faster than validation reports. However, we do need well‐ designed and well‐conducted prospective clinical studies to critically elucidate the clinical impacts of the microfluidic devices. The investigators could consider to learn from the developing history of CellSearch™ system.
