Abstract 2188: Enabling sensitivity, repeatability and reproducibility required for immuno-oncology research on the CyTOF XT Pro mass cytometer

Understanding the functional phenotype of immune cells from patients with cancer is critical for identifying mechanism of action, determining disease prognoses and monitoring the clinical efficacy of immunotherapies. The ability to reproducibly identify functional markers with low expression and use functional markers to identify rare cell populations can be challenging with fluorescence flow cytometry due to spectral spillover and autofluorescence. Mass cytometry overcomes many challenges of fluorescence-based cytometry, as low signal spillover and the absence of autofluorescence negate the need for spectral compensation and unmixing. To minimize technical variation, metal-tagged antibody cocktails and stained samples can be frozen for later use and acquisition. Antibody panels that detect both surface and intracellular markers can be rapidly designed, conveniently stained and acquired in a single tube to provide streamlined workflows with high consistency. In this study, human whole blood and PBMC samples were stained with antibody panels containing up to 50 surface and cytoplasmic targets. Stained samples were frozen and acquired on a later date using CyTOF™ XT PRO and CyTOF XT systems to assess repeatability and reproducibility, and to ensure data quality was not compromised when samples were acquired at 4x speed with the CyTOF XT PRO system. Population frequencies and resolution indices for markers were assessed by manual gating. There was no significant difference between population frequencies analyzed between the two CyTOF systems. Both the CyTOF XT and CyTOF XT PRO system had high sensitivity and dynamic range for both abundant and low-abundance markers. The cell population frequency and staining intensity results generated from the CyTOF XT and CyTOF XT PRO system were highly repeatable and reproducible across multiple instruments. Overall, these studies find that the CyTOF XT PRO system generates comparable signal resolution to the CyTOF XT system with up to 4x increased event-rate acquisition. Automated acquisition by the CyTOF XT PRO system enables researchers to accurately and reproducibly streamline human immunophenotyping and functional profiling, leading to accelerated discoveries. Citation Format: Stephen Li, Rita Straus, Michael Sullivan, Andrea McEwan, Ling Wang, Christina Loh, Alexander Loboda. Enabling sensitivity, repeatability and reproducibility required for immuno-oncology research on the CyTOF XT Pro mass cytometer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2188.