**3.2 Detection of CAR-positive cells**

This test needs to be repeated as it involves CAR-T product quality evaluation and dynamics assay at different time points. After successful preparation of CAR-T, quality evaluation should be carried out before the infusion. The proportion and count of CAR-positive cells are the most important parts of them [1–4, 21–27, 34, 35]. Values detected by MFC and qPCR are well consistent [22]. Compared with the qPCR method, MFC may have a lower sensitivity [34]. However, MFC has unique advantages. Through multicolor and gating technology, MFC can clearly mark the proliferate, activated, or suppressive subgroups of CAR-T cells [36, 37, 69–75]. In rare cases, MFC can identify CAR-positive transduced leukemic cells [76, 77]. In addition, quality testing can also be carried out to detect other cells in the CAR-T product. The high speed, simplicity, and low cost of MFC facilitate its application in CAR-T studies because these assays need to be repeated.
