*Recent Developments in Application of Multiparametric Flow Cytometry in CAR-T… DOI: http://dx.doi.org/10.5772/intechopen.108836*

to detect tumor cells and/or target antigen-positive cells and those to evaluate the immune system. The previous category includes tumor cell immunophenotyping to select promising targets [17, 18], minimal residual/measurable diseases (MRD) detection [3, 19, 20], and recovery kinetics of target antigen-positive cells [3, 21, 22]. The latter category includes lymphocyte activity and function evaluation, CAR-positive cell assay [21–25], immune cell subsets detection [21–31], lymphocyte killing function assay [27], cytokines detection [26–29], tumor microenvironment (TME) evaluation, and immunosuppressive signals detection [23, 26].

If according to time points, the application progress of MFC in CAR-T can be divided into four stages: CAR-T research and development, patient enrollment of clinical research, quality evaluation after product preparation, and posttreatment evaluation.

These tests involve almost all aspects of MFC, including routine clinical testing items and special research items, and are carried out repeatedly at different time points, even with some overlaps. See **Table 1**.

However, even in routine items, the special nature of CAR-T brings technical challenges. The analysis of the immunophenotype of tumor cells needs a very accurate gate setting because the coexistence of target antigen negative subclone may become the source of recurrence [9–12]. The evaluation of CAR-T products before infusion and MRD detection after CAR-T immunotherapy need to be careful of the target antigen-negative malignant and benign cells [3, 19, 20]. The presence of CAR-T cells may affect the MRD detection of T lymphocytic malignancies. In the identification of CAR-T products and early immunological evaluation after treatment, it is necessary to evaluate the composition and activating status of CAR-positive and CAR-negative cells simultaneously [32–35].
