Abstract 1885: Semi and fully automated immunostaining sample preparation platforms improve live leukocyte recovery, reproducibility, and cytometry data quality

Abstract

Limited innovation in automated cell and organelle sample preparation methodology limits the effectiveness of modern analytical methods, such as single-cell ‘omics, flow and mass cytometry. These techniques traditionally rely on manual centrifugation-based protocols for cell washing and suspension preparation, hampering researchers’ access to the reproducibility and scalability benefits of automation. We have developed a suite of cell suspension preparation systems that enable semi and full automation of cell washing protocols. These Laminar Wash࣪ technologies robustly, gently, and efficiently remove debris, dead cells, and unbound reagent using laminar flow and liquid handling robotics, rather than turbulent and harsh pelleting-plus-pipetting methods. Adaptation of standard protocols to Laminar Wash automation typically improves repetitive immunostaining processes and workflows, in terms of reduced hands-on time and inter- and intra-operator variability. We demonstrate the superior live cell retention and reproducibility of Laminar Wash over centrifugation in processing murine and humanized mouse peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) for flow cytometry. Furthermore, we show how Laminar Wash improves flow cytometry data quality, in terms of debris removal and separation of immune cell subsets for both PBMCs and TILs. Overall, these results show how Laminar Wash methodology assists in standardizing sample preparation for cytometric analysis, an important and unmet need in cancer immunotherapy discovery and manufacturing workflows. Citation Format: Melvin Lye, Christoph Eberle, Ann Wang, Geoffrey K. Feld, Namyong Kim. Semi and fully automated immunostaining sample preparation platforms improve live leukocyte recovery, reproducibility, and cytometry data quality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1885.

A novel mass cytometry protocol optimized for immunophenotyping of low-frequency antigen-specific T cells

“…Similar to the results observed using debris removal solution ( Figure 4 ), laminar flow washes of samples after thawing and resting led to efficient sample debarcoding ( Figure 5A ) and correct representation of each barcodes in respective stimulation conditions. This is most probably due to the already described beneficial effect of laminar flow washes on the elimination of cell debris ( Lye et al., 2022 ). In addition, we observed approximately 2%–8% CD4 + or CD8 + CD25 + CD69 + T cells after stimulation with peptivator or allergen ( Figure 5B ).…”

“…Removal of dead cells and debris enhanced debarcoding efficiency in this dual-barcoding approach. Additional cell sparing was achieved by introducing a laminar flow-based cell washing system in our protocol, which avoids cell loss associated with multiple centrifugation steps ( Lye et al., 2022 ). Our improved method was connected to reduced cell loss, as well as efficient debarcoding and immunophenotyping of low-frequency antigen-specific T cells by means of mass cytometry.…”