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Advancing Cell Therapy

Flow cytometry has proven to be an invaluable tool for developing new cell therapies

Research into cell therapies, particularly chimeric antigen receptor (CAR) T cells, is experiencing exponential growth. Scientists in industry and academia will have conducted more than 1,200 cell therapy clinical trials in 2021—the majority of these being Phase 2 studies.1,2 As more and more products enter the clinical development pipeline, robust techniques are needed to characterize these novel products. One approach that has found broad application in both research and manufacturing of cell therapies is flow cytometry (FCM).3 

Uses of flow cytometry in cell therapy

FCM is unique because it allows the user to simultaneously analyze multiple parameters in individual cells, providing a comprehensive breakdown of heterogeneous cell populations.2 As a result, FCM can be used throughout the cell therapy development process, including for the following applications:2 

•    Identifying and quantifying antigen-bearing cells in donor samples 

•    Assessing cell viability and functionality 

•    Evaluating CAR expression on therapeutic cells 

•    Estimating effector cell activation and cytotoxic killing 

•    Quantifying tumor expression and immunosuppressive molecules 

•    Monitoring cell persistence and therapeutic effect in patients

Determining cell counts and viability are essential techniques in cell culture. Consequently, they are widely used throughout the cell therapy development process, from the initial manipulation to the final release for infusion. 

These techniques help standardize the input of cells for manufacturing and, further, help to select cells and determine the dosing of donor lymphocyte infusions. FCM can rapidly determine cell counts and viability and is sensitive even when assessing samples taken from lymphopenic patients, where detecting lymphocyte populations can be challenging.4

FCM complements qPCR analysis during post-treatment patient monitoring

Monitoring patients after the infusion is also essential to track the proliferation and persistence of the CAR T cells, which are crucial to the antitumor effect. Monitoring proliferation and persistence uses both FCM and qPCR on collected blood samples. The benefit of FCM over qPCR is that FCM looks at individual cells in samples. In contrast, qPCR is dependent on nucleic acids extracted from blood samples; therefore, qPCR results cannot distinguish between populations subsets. FCM is advantageous as the proportion of CD4+ and CD8+ T lymphocytes among viable cells can be determined using this technique.5 

Another advantage of FCM is that it can detect potentially life-threatening CAR-positive, transduced leukemic cells. Because they also express CAR and cannot be distinguished with qPCR,2 these cells can go undetected by the therapeutic CAR T cells, making them resistant to treatment. Finally, FCM functions at the protein level, providing additional information on CAR T cell functionality.6 

Given the novelty of available cell therapies, long-term monitoring of patients after treatment is also critical. For example, CAR T cell therapy has successfully led to the remission of certain B-cell leukemias and lymphomas that are particularly difficult to treat. However, it is not uncommon for these patients to experience adverse side effects that can become life-threatening. A solution is to use FCM to monitor patients after treatment to detect immediate and long-term complications.6

Overcoming FCM hurdles for cell therapy use 

Although FCM is crucial for the characterization of cell therapy products, it still has several limitations. These hurdles include the limited availability of positive controls and detection reagents, and the need for new gating and analysis strategies compared to routine cell phenotyping.2

The need for new gating and analysis strategies to monitor residual disease in patients who have received CAR T cell therapy also requires consideration. One such example is the cell surface marker CD19—a target for immunotherapy and a gating marker used in FCM analysis to enrich for neoplastic B cell populations when detecting residual disease. However, CAR T cells target both normal and abnormal cells expressing CD19, making gating with this marker challenging.7 Therefore, to enable the monitoring of patients, FCM assays need flexibility and redundancy to monitor cell therapies.

Another limitation is that the persistence of infused CAR T cells in a patient’s circulation decreases over time. While the CAR T cells can still be detected using qPCR, they eventually fall below the limit of detection of FCM. That said, in preclinical trials, researchers have found ways to improve the detection of CAR T cells via FCM by tagging CAR T cells with enhanced green fluorescent protein (EGFP).8 The expression of EGFP in the CAR T cells is detected when samples are analyzed by FCM, keeping the cells above the limit of detection.

Despite these limitations, strides have been made to adapt FCM to the needs of cell therapy. For example, high-speed sampling has been introduced to enable high throughput processing using 96- or 384-well plate formats, a process already in place for the phenotypic characterization of expanded mesenchymal stem cells.9 Another advancement is using preformulated dry antibody panels to help standardize FCM assays and reduce the impact of inter-operator and inter-assay variations.5 Such standardization makes FCM the preferred assay for cell manufacturers.4 Given its broad use, FCM has proven to be an invaluable tool for developing new cell therapies.


1.     Alliance for Regenerative Medicine. Regenerative Medicine in 2021: A year of Firsts & Records. Accessed August 18, 2021.

2.     Maryamchik E, Gallagher KME, Preffer FI, Kadauke S, Maus MV. New directions in chimeric antigen receptor T cell [CAR-T] therapy and related flow cytometry. Cytometry B Clin Cytom. 2020;98(4):299-327.

3.     Litwin V, Hanafi L, Mathieu M, Pouliot P, Boulais P. Strategies for successful monitoring of CAR T-cells by flow cytometry. Cytotherapy. 2020;22(5):S130.

4.     Mfarrej B, Gaude J, Couquiaud J, Calmels B, Chabannon C, Lemarie C. Validation of a flow cytometry-based method to quantify viable lymphocyte subtypes in fresh and cryopreserved hematopoietic cellular products. Cytotherapy. 2021;23(1):77-87.

5.     Demaret J, Varlet P, Trauet J, et al. Monitoring CAR T-cells using flow cytometry. Cytometry B Clin Cytom. 2021;100(2):218-224.

6. Blache U, Weiss R, Boldt A, et al. Advanced flow cytometry assays for immune monitoring of CAR-T cell applications. Front Immunol. 2021;12:1285.

7.     Cherian S, Stetler-Stevenson M. Flow cytometric monitoring for residual disease in B lymphoblastic leukemia post T cell engaging targeted therapies. Curr Protoc Cytom. 2018;86(1):e44.

8.     Batista L, Brand H, Fantacini D, Covas D, Souza L. Evaluation of anti-CD19 CAR-T Cell persistence and efficacy using a developed multiparametric flow cytometry and qPCR tracking platform for preclinical and clinical studies. Cytotherapy. 2021;23(4):13-14.

9.     Reis M, McDonald D, Nicholson L, et al. Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry. Sci Rep. 2018;8(1):1-12.