Is the CTC enrichment kit severely inadequate in detecting mesenchymal phenotype cells?

EpCAM-only enrichment does indeed miss a portion of mesenchymal CTCs; this is a common shortcoming of immunomagnetic bead methods. It is advisable not to abandon the existing system yet; instead, two-stage enrichment can be performed: first use EpCAM magnetic beads to capture typical epithelial CTCs, then subject the remaining supernatant to secondary enrichment using mesenchymal markers such as vimentin, N-cadherin, or FAP. This is relatively low-cost and allows retention of both cell populations. If switching kits directly, prioritize domestic or imported products with dual-marker or multi-marker combinations, and ask clearly about the antibody clone numbers and whether they have been validated with EMT cell lines. Using TGF-β-induced mesenchymal cells or cells with known mesenchymal phenotypes spiked into healthy blood to assess recovery rate is more direct.

Sample stability at room temperature for 24 hours depends not only on the enrichment reagent, but more importantly on the blood collection tube preservation solution. It is recommended to use CTC-specific preservation tubes containing mild fixatives and anticoagulant components, which can maintain morphology and nucleic acids; however, if downstream single-cell sequencing is to be performed, it is necessary to confirm whether the fixative is reversible. After receipt, perform low-speed centrifugation as soon as possible to remove red blood cells and avoid hemolysis. Another approach for detecting mesenchymal CTCs that are missed is negative enrichment—using CD45 magnetic beads to remove leukocytes, followed by density gradient centrifugation to recover residual cells. This does not rely on EpCAM, but purity will be lower, requiring subsequent identification or single-cell picking. For off-the-shelf channels, it is advisable to inquire with multiple distributors; those that can provide same-lot testing reports and logistics temperature records should be prioritized.

For downstream single-cell sequencing, the enriched cells should not be fixed too rigidly, and preservation solutions that affect RNA quality should not be used. It is recommended to choose kits with releasable magnetic beads or gentle elution to preserve cell viability. The 24-hour room-temperature stability should be tested with your own time gradient: after blood collection, perform enrichment at 0, 6, 12, and 24 hours, and compare cell yield, viability staining, and RNA RIN values. If imported products cannot arrive within three weeks, you can ask regional distributors about domestic products in stock, such as companies making CTC-related products like Huadesen, Geno, and Jingzhun Medical, and ask whether they have small-size trial samples or same-city inventory. First use a small amount of blood for feasibility validation, then schedule the formal experiment.

The antibody-based method is indeed prone to missing EMT cells; it is recommended to add physical screening or a negative enrichment combination.

We have compared them, and a certain imported epithelial marker kit has a significantly higher missed detection rate for mesenchymal cells than negative enrichment.