**Abstract**

In recent years, chimeric antigen receptor (CAR) modified T-cell (CAR-T) immunotherapy has achieved great success in cancer treatment, especially in some hematologic malignancies. Multiparametric flow cytometry (MFC) is a key immunologic tool and plays an important role in every step of CAR-T design, development, and clinical trials. This chapter discusses the application and new developments of MFC in CAR-T, including the selection of CAR-T targets, the enrollment of patients, the detection of minimal/measurable residual disease (MRD), the quality evaluation of CAR-T product, the detection of immune cell subsets and cytokines, and the study of immune checkpoint and immune suppressive microenvironment.

**Keywords:** chimeric antigen receptor, immunotherapy, multiparametric flow cytometry, hematological malignancies, immune

### **1. Introduction**

Chimeric antigen receptor (CAR) modified T-cell (CAR-T) has been a remarkable achievement in the field of cancer therapy in recent years [1–7], especially for the treatment of refractory and relapsed B-cell acute lymphoblastic leukemia (ALL) [1–3]. CD19-CAR-T alone or bridging allogeneic hematopoietic stem cell transplantation (allo-HSCT) can greatly improve the complete remission (CR) rate and overall survival (OS) rate. CAR-T therapy for other malignancies is being explored as well [4–7]. However, the main problem with CAR-T therapy is its high relapse rate, which involves a variety of mechanisms. Current researches focus on improving CAR-T structure, selecting new targets, and eliminating the inhibitory immune microenvironment [8–14].

Multiparametric flow cytometry (MFC) is an immunological technique developed in the 1970s and has become an indispensable methodology for clinical diagnosis and basic research [15, 16]. CAR-T is one kind of immunotherapy, and MFC plays a pivotal role in the entire process of CAR-T [3, 17–29]. These applications involve multiple aspects of MFC, and basically can be divided into two categories, that is, protocols



*Note. CAR: chimeric antigen receptor, CSF: cerebrospinal fluid, MRD: minimal residual/measurable diseases, and TME: tumor microenvironment.*
