119 Auto-imaging cytometry on the slide (ICOS) for quality control of CAR-T products

<h3>Background</h3> For CAR-T cell manufacturing in both commercial and academic settings, transduction efficiency is crucial for CAR-T products effectiveness. Current methods like flow cytometry and PCR are time consuming and/or prone to variability due to manual intervention. We developed auto-imaging Cytometry On the Slide (ICOS), an efficient workflow for rapid CAR-T cells affixation on glass slides, followed by automated multiplex IHC (mIHC) staining, whole-slide imaging, and high-throughput immune phenotyping analysis, optimizing quality control and reducing time and manual effort. <h3>Methods</h3> As a proof of concept, GMP-grade dual-targeting aCD22-aCD19 CAR-T products (NCT05429905) consented for research were spiked into the buffy coat of healthy individual at specified ratios (0%, 1%, 2%, 5%, 20%, 50%, 100%). Approximately 300,000 cells per mixed sample were spread into a monolayer on positively charged slides and stored at -20°C until fixation. Slides underwent fluorescent mIHC staining for CD19 CAR/CD4/CD8/DAPI using a Leica BondMax (Leica Biosystems). Fluorescent mIHC images were acquired with an Axioscan 7 Slide Scanner (Zeiss) and analyzed with HALO software (Indica Labs). Aliquots from each sample were evaluated using a 6-plex flow cytometry panel for comparison. <h3>Results</h3> The ICOSs were successfully stained and imaged, preserving cell morphology and clearly identifying immune markers (CD19 CAR⁺CD4⁺, CD19 CAR⁺CD8⁺) (figure 1). The proportion of CD19 CAR⁺ cells detected using ICOSs and flow cytometry (table 1) demonstrated a very strong linear correlation, with a Pearson’s squared correlation coefficient of 0.9933, indicating that ICOS yields results comparable to that of flow cytometry. <h3>Conclusions</h3> ICOS involves a consistent and efficient workflow that preserves cell morphology and effectively detects CAR-T cells, which is crucial for downstream qualitative and quantitative analysis to evaluate transduction efficiency. We believe the ICOS might serve as a novel quality check tool to monitor the manufacturing of CAR-T cell products in the future, with the added dimension of cell morphology. The small sample volume and high precision will allow this technique to be applied to cerebrospinal fluid and bone marrow.