**3.1 Evaluation of lymphocyte activity and function**

The detection of lymphocyte activity and function by MFC mainly includes release of cytotoxic proteins (granzyme, perforin), degranulation (CD107a), expression of surface activation markers (PD1, CD25, CD38, HLA-DR, CD69, etc.) [15, 27, 36, 37], killing ability assays through apoptotic cells detection, expression of death signal molecules (Fas-L or TRAIL) [36, 37], new kinds of nuclear dye to stain dead and live cells in a high throughput format [36, 37, 68], and production of various inflammatory cytokines [4, 25–30, 36, 37].

These tests mainly involve the selection of the cell source for CAR-T and the quality evaluation of the product after successful preparation. Each study may apply a different method, and some studies do not choose MFC method. The advantage of MFC is that it can evaluate cell subsets and effector targets simultaneously [4, 25–30, 36, 37].

The choice of autologous or allogeneic cells for CAR-T has its own advantages and disadvantages. Compared to allogeneic CAR-T cells, autologous CAR-T cells are safer. However, CAR-T cell preparation may be compromised when patients have the following conditions: elderly patients, high tumor load, multiple treatments, low number and poor viability of lymphocytes, etc., in which case other immunotherapeutic products may be chosen [4, 41].

After successful CAR-T preparation, not only the proportion of CAR-positive cells affects the efficacy, but also the killing activity of lymphocytes [34–37].
