Abstract 2068: Multi-pass flow cytometry for high-marker panels with minimal spillover spread
Authors: Brown Trevor , Forward Sarah , Assita Emane Rose , Abbott Geoffrey , Fahlberg Marissa , Kwok Sheldon J. 
Published: March 2024

Abstract
Multiparametric flow cytometry on immune cells is essential in cancer research, disease diagnosis, and treatment monitoring. Collecting data on &gt;10 markers at a time is ideal due to deeper cell characterization and assay sensitivity, however, high-marker panels are subject to significant spillover spread from spectrally overlapping fluorophores that compromise data quality, standardization, and interpretation. Clinical flow cytometry still relies on the use of multi-tube low-marker (&lt;10) panels that minimize spillover spread and ease analysis and interpretation of results. To improve and expand the clinical utility of flow cytometry, there is a need for higher marker measurements without use of highly overlapping fluorophores, which would also serve to streamline the use of samples and reagents.
Here, we present the development of a novel barcoding method using micron-sized laser particles (LPs) that confer an optical signature to individual cells at high-throughput, enabling tracking of cells through multiple passes in a flow cytometer. This process is possible using a custom cytometer equipped to detect LPs and capture samples. Sequential staining of samples with releasing or photobleaching antibodies/fluors between cycles enables merging of multiple smaller panels that each have minimal spillover, resulting in a high-marker dataset with drastic reduction in spillover compared to conventional single-acquisition flow cytometry.
We demonstrate the utility of this technology through three different assays. Human PBMCs were barcoded with LPs and stained with 1) a panel of releasable antibodies (Miltenyi Biotec, 10 markers, 2 passes), 2) a laboratory-designed panel that utilized photobleaching (32 markers, 3 passes), and 3) a panel of dried and validated DuraClone antibodies that utilized photobleaching (Beckman Coulter, 18 markers, 2 passes). For each assay, cells were stained, acquired, photobleached/released, captured, re-stained, re-acquired and steps were repeated either once (for 2 passes) or twice (for 3 passes). In all cases, we found drastically reduced spillover, better data quality, and identified the same cell subtypes detected by conventional flow cytometry. Reproducibility was high across multiple (3+) replicates with CVs &lt;30% among all populations with &gt;1% frequency of total. New cell populations were identified that would not be possible with separate acquisitions of each pass.
Recent advances in flow cytometry now allow more markers to be analyzed together, but at the cost of increasing spectral overlap and spillover spread. Multi-pass flow cytometry confines spillover to individual panels, reducing overall interference and simplifying analysis. This enhances data quality and usability while streamlining resources, facilitating the clinical uptake of complex flow cytometry in cancer research, diagnosis, and treatment.
Citation Format: Trevor Brown, Sarah Forward, Emane Rose Assita, Geoffrey Abbott, Marissa Fahlberg, Sheldon J. Kwok. Multi-pass flow cytometry for high-marker panels with minimal spillover spread [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2068.