Magnetic enrichment–coupled flow cytometry for highly sensitive detection of persistent CAR-T cells in spiked and clinical peripheral blood samples
4 affiliations
- 1Research and Development Department, Abu Dhabi Stem Cells Center
- 2Yas Clinic Khalifa City (YCKC) Hospital
- 3Director Office, Emirates Drug Establishmnet (EDE), Masdar city
- 4Department of Pediatrics, Yong Loo Lin school of Medicine, National University of Singapore
Abstract
Background: Long-term monitoring of chimeric antigen receptor T-cell (CAR-T) persistence is essential for assessing therapeutic efficacy, particularly when circulating CAR-T cell levels decline following infusion. Although quantitative real-time PCR (qPCR) provides highly sensitive detection of CAR transgenes, it lacks single-cell resolution, phenotypic characterization, and the ability to assess cell viability.
Methods: In this study, we adapted and validated an automated enrichment-mode on MACSQuant flow cytometry that integrates automated immunomagnetic enrichment with multiparametric flow cytometric analysis to enhance the sensitivity of detecting and quantifying rare CAR-T cell populations. The assay’s limit of detection and potential clinical utility were evaluated using CAR-T cell spike-in experiments in peripheral blood mononuclear cell (PBMC) backgrounds and clinical blood samples obtained from patients who received CD19-targeted CAR-T cell therapy. Results were compared with a complementary qPCR assay for vector copy number (VCN) detection.
Results: The enrichment-based workflow enabled same-day detection of viable CAR-T cells by selectively enriching CAR-T cells while excluding non-target peripheral blood leukocytes, thereby substantially improving detection sensitivity at very low cell frequencies in post-infusion patient samples. Concordant CAR-T cell detection was observed between the enrichment-coupled flow cytometry workflow and qPCR. While qPCR served as an orthogonal approach for sensitive transgene detection, the enrichment-based flow cytometry workflow provided additional single-cell information, including viable CAR-T cell frequency and phenotypic characterization.
Conclusion: By combining enhanced sensitivity with single-cell and functional resolution, this enrichment-coupled flow cytometry workflow complements qPCR-based genomic detection and may serve as a rapid, accurate, and clinically valuable tool for the longitudinal monitoring of CAR-T cell persistence in patients receiving CAR-T cell therapy.
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BibTeX
@article{hadi2026magnetic,
title = {{Magnetic enrichment–coupled flow cytometry for highly sensitive detection of persistent CAR-T cells in spiked and clinical peripheral blood samples}},
author = {Loubna Abdel Hadi and Gokul Thangamani and Samira Sheikh and Syed Mazher Hussain and Alvin Jon Ingel and Shahid Imtiaz Ahmed and Antonio Alfonso Bencomo Hernandez and Fatima Mohammed Al Kaabi and Yendry Ventura Carmenate and Rupert Handgretinger},
journal = {Frontiers in Immunology},
year = {2026},
month = oct,
doi = {10.3389/fimmu.2026.1872290},
url = {https://www.frontiersin.org/articles/10.3389/fimmu.2026.1872290/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Magnetic enrichment–coupled flow cytometry for highly sensitive detection of persistent CAR-T cells in spiked and clinical peripheral blood samples
AU - Loubna Abdel Hadi
AU - Gokul Thangamani
AU - Samira Sheikh
AU - Syed Mazher Hussain
AU - Alvin Jon Ingel
AU - Shahid Imtiaz Ahmed
AU - Antonio Alfonso Bencomo Hernandez
AU - Fatima Mohammed Al Kaabi
AU - Yendry Ventura Carmenate
AU - Rupert Handgretinger
PY - 2026
DA - 2026/10/21
JO - Frontiers in Immunology
DO - 10.3389/fimmu.2026.1872290
UR - https://www.frontiersin.org/articles/10.3389/fimmu.2026.1872290/full
AB - Background: Long-term monitoring of chimeric antigen receptor T-cell (CAR-T) persistence is essential for assessing therapeutic efficacy, particularly when circulating CAR-T cell levels decline following infusion. Although quantitative real-time PCR (qPCR) provides highly sensitive detection of CAR transgenes, it lacks single-cell resolution, phenotypic characterization, and the ability to assess cell viability. Methods: In this study, we adapted and validated an automated enrichment-mode on MACSQuant flow cytometry that integrates automated immunomagnetic enrichment with multiparametric flow cytometric analysis to enhance the sensitivity of detecting and quantifying rare CAR-T cell populations. The assay’s limit of detection and potential clinical utility were evaluated using CAR-T cell spike-in experiments in peripheral blood mononuclear cell (PBMC) backgrounds and clinical blood samples obtained from patients who received CD19-targeted CAR-T cell therapy. Results were compared with a complementary qPCR assay for vector copy number (VCN) detection. Results: The enrichment-based workflow enabled same-day detection of viable CAR-T cells by selectively enriching CAR-T cells while excluding non-target peripheral blood leukocytes, thereby substantially improving detection sensitivity at very low cell frequencies in post-infusion patient samples. Concordant CAR-T cell detection was observed between the enrichment-coupled flow cytometry workflow and qPCR. While qPCR served as an orthogonal approach for sensitive transgene detection, the enrichment-based flow cytometry workflow provided additional single-cell information, including viable CAR-T cell frequency and phenotypic characterization. Conclusion: By combining enhanced sensitivity with single-cell and functional resolution, this enrichment-coupled flow cytometry workflow complements qPCR-based genomic detection and may serve as a rapid, accurate, and clinically valuable tool for the longitudinal monitoring of CAR-T cell persistence in patients receiving CAR-T cell therapy.
SN - 1664-3224
ER -