Work

Magnetic enrichment–coupled flow cytometry for highly sensitive detection of persistent CAR-T cells in spiked and clinical peripheral blood samples

Year: 2026

Type: article

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.

Source: Frontiers in Immunology

DOI: 10.3389/fimmu.2026.1872290