From Ex Vivo to In Vivo: Enabling Efficient and Accurate Whole-Blood Monitoring of Anti-CD19 CAR-T Cells

Introduction

Chimeric antigen receptor T cell (CAR-T) therapy has become a transformative approach in the treatment of multiple malignancies and autoimmune diseases^【1】. As the field evolves from conventional ex vivo engineered cell therapy toward emerging in vivo CAR-T strategies, accurate quantification of CAR-expressing T cells in peripheral blood becomes a critical analytical requirement for evaluating pharmacokinetics, efficacy, and safety. Across these therapeutic modalities, flow cytometry-based detection of CAR-positive T cells in blood is widely used to assess in vivo CAR transduction and pharmacokinetics.

Analytical approaches for CAR-T monitoring

CAR-T cell monitoring is commonly performed using flow cytometry (FCM) and PCR-based methods (qPCR or ddPCR)^【2,3】. PCR-based assays quantify CAR transgene copy number, while flow cytometry enables direct detection of CAR-expressing cells at the protein level.

High-affinity anti-idiotype antibodies have enabled sensitive detection of CAR expression by flow cytometry, with reported detection limits as low as ~0.05% and good concordance with ddPCR measurements^【4】.

However, FCM performance can be impacted by sample matrix and staining format. While peripheral blood mononuclear cell (PBMC)-based workflows are widely adopted, whole blood offers greater physiological relevance but introduces additional analytical complexity.

Whole-blood analytical challenges

Compared with PBMC preparations, whole blood presents a more physiologically representative but analytically complex matrix, characterized by:

- High background from erythrocytes and plasma components

- Reduced detectability of CAR-positive populations, especially for low CAR-expressing whole blood samples

- Increased gating complexity due to heterogeneous cell populations

- Potential variability introduced by multi-step sample processing including red blood cell (RBC) lysis

These factors affect assay robustness and inter-study comparability.

Evaluation of CAR detection reagents in whole blood

To assess analytical robustness in whole blood, we evaluated three CAR-binding detection reagents:

- PE-labeled CD19 protein (Cat. No. CD9-HP2H3, ACROBiosystems)

- PE anti-FMC63 mouse monoclonal antibody (Cat. No. FM3-HPY53, ACROBiosystems)

- PE anti-FMC63 rabbit monoclonal antibody (Cat. No. FM3-PFM721, ACROBiosystems)

Each reagent was tested under two conditions:

- Native whole-blood samples

- 20% CAR-T cell spike-in whole-blood model

This design enabled assessment assay specificity and accuracy across different product formats targeting the same CAR epitope.

Theoretical consistency

- All three detection reagents showed accurate CAR-positive cell frequency measurements consistent with theoretical spike-in proportions (20%)

- Quantitative performance remained stable in a complex whole blood matrix

High signal-to-noise ratio

- Clear separation between CAR-positive and CAR-negative populations, ensuring precise gating of CAR+ cells

- No background binding detected in whole blood negative control samples

Straightforward workflow

- Direct detection of whole blood samples without RBC lysis

- Avoid the loss of PBMC especially T cell subsets

- Minimize impact on cell fitness during PBMC isolation

Validation Data

Accurate Detection of Anti-CD19 CAR-T Cells in Whole Blood Using PE-Labeled Human CD19 (20-291) Protein, His Tag

After Fc receptor blockade, whole blood only control (Figure A) and whole blood spiked with CAR-T cells (Figure B) were first stained with PE-Labeled Human CD19 (20-291) Protein, His Tag (Cat. No. CD9-HP2H3), and then stained with 7-AAD. After 100-fold dilution, acquire data using a CytoFLEX S and then analyze the data with FlowJo. CAR-T cells were identified as PE-positive events within the live CD45+CD3+ (or CD45+) lymphocyte population. The proportion of CD45+CAR+ cells is around 20%, which is largely consistent with the theoretical spike-in value.

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Accurate Detection of Anti-CD19 CAR-T Cells in Whole Blood Using PE-Labeled Monoclonal Anti-FMC63 Antibody, Mouse IgG1 (Y45) (Site-specific conjugation) (Preservative free)

After Fc receptor blockade, whole blood only control (Figure A) and whole blood spiked with CAR-T cells (Figure B) were first stained with PE-Labeled Monoclonal Anti-FMC63 Antibody, Mouse IgG1 (Y45) (Site-specific conjugation) (Preservative free) (Cat. No. FM3-HPY53) , and then stained with 7-AAD. After 100-fold dilution, acquire data using a CytoFLEX S and then analyze the data with FlowJo. CAR-T cells were identified as PE-positive events within the live CD45+CD3+ (or CD45+) lymphocyte population. The proportion of CD45+CAR+ cells is around 20%, which is largely consistent with the theoretical spike-in value.

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Accurate Detection of Anti-CD19 CAR-T Cells in Whole Blood Using PE-Labeled Monoclonal Anti-FMC63 Antibody, Rabbit IgG (1G10)

After Fc receptor blockade, whole blood only control (Figure A) and whole blood spiked with CAR-T cells (Figure B) were first stained with PE-Labeled Monoclonal Anti-FMC63 Antibody, Rabbit IgG (1G10) (Cat. No. FM3-PFM721), and then stained with 7-AAD. After 100-fold dilution, acquire data using a CytoFLEX S and then analyze the data with FlowJo. CAR-T cells were identified as PE-positive events within the live CD45+CD3+ (or CD45+) lymphocyte population. The proportion of CD45+CAR+ cells is around 20%, which is largely consistent with the theoretical spike-in value.

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These results confirm that ACROBiosystems CD19 CAR detection reagents (PE-labeled CD19 protein (Cat. No. CD9-HP2H3), PE anti-FMC63 mouse monoclonal antibody (Cat. No. FM3-HPY53), PE anti-FMC63 rabbit monoclonal antibody (Cat. No. FM3-PFM721) can effectively and accurately detect CD19-CAR+ T cells from whole blood samples without RBC lysis. Single step and time-saving staining workflow enhances assay accuracy and consistency by eliminating PBMC isolation step and its impact on T cell fitness. Additionally, we also detected the CAR expression using the corresponding product in samples treated with RBC lysis. The results were consistent with those obtained without lysis, demonstrating that our products are well suited for various application scenarios.

Application relevance

Whole-blood CAR-T detection supports multiple applications, including:

- Assessment of CAR expression post in vivo CAR delivery

- Pharmacokinetic and pharmacodynamic evaluation of ex vivo and in vivo CAR-T therapies

Conclusion

Our validation data demonstrated that the anti-FMC63 antibody and CD19 protein enable highly accurate detection of CAR-positive T cells in whole blood samples. This approach is applicable for in vivo quantification of CAR positivity in blood samples for both ex vivo and in vivo CAR-T products, satisfying the requirements for CAR-T cell positivity assessment and in vivo pharmacokinetic monitoring. In addition, consistent performance across different reagents offers robust data support for flexible reagent selection.

Reference

1. Capell CR, et al. Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol. 2023;20:359--371. 16

2. Wang W, Xu D. Do You Know How Many CAR-T Cells You Have? Advantages and Disadvantages of Flow Cytometry vs qPCR. CTS. 2026. 31

3. Masilamani M, et al. Bioanalytical Methods for Characterization of CAR-T Cellular Kinetics: Comparison of PCR Assays and Matrices. Clin Pharmacol Ther. 2023;114(3):664--672. 30

4. Cheng J, et al. Monitoring anti-CD19 chimeric antigen receptor T cell population by flow cytometry and its consistency with digital droplet polymerase chain reaction. Cytometry A. 2023;103(1):16--26. 41