CYTO 2026 Scientific Tutorial: Panel Gains Without the Pains: Smarter Re-Optimization for High-Parameter Flow

Panel Gains Without the Pains: Smarter Re-Optimization for High-Parameter Flow

Presenters:
Diana L. Bonilla Escobar, PhD, Scientific Director, Cytek Biosciences
Kamila Czechowska, PhD, Chief Diagnostic Product Development Officer, Metafora Biosystems
Megan McCausland, BSc, Scientific Advisor, Flow Cytometry, IQVIA Laboratories
Veronica Nash, PhD, Director of Flow Cytometry (Cellular Biomarkers), GSK

Abstract:
Assay optimization is essential for achieving reproducible and accurate results in multicolor flow cytometry. Recent advancements in cytometer hardware and fluorochrome chemistries, coupled with the ability to use an expanded number of fluorophores simultaneously, have enabled highly multiparametric assays. These capabilities create opportunities to re-optimize existing panels by adding new markers to gain deeper insights, without compromising resolution or data quality. However, expanding an optimized and/or validated panel is often challenging. Issues such as reagent availability, clone specificity, titer adjustments, and performance variability must be addressed, alongside technical problems like steric hindrance, reagent interactions, and increased spillover spreading. These challenges underscore the need for clear guidance when modifying complex panels. This tutorial will outline a structured approach for re-optimizing previously optimized and/or validated assays, including strategies for selecting optimal fluorochrome replacements or additions, evaluating reagent compatibility, and maintaining optimal resolution across all parameters in an expanded panel. We will walk through real case examples showing how updated panels were revalidated to ensure comparable marker performance, consistent population resolution, and robust identification of all relevant cell subsets after adding new markers. In these examples, careful panel redesign and iterative testing preserved data integrity even as dimensionality increased. Based on these findings, we will propose a practical workflow and a set of best-practice guidelines for panel expansion and re-optimization. This “panel expansion” workflow will serve as a template that participants can apply in their own labs – an increasingly important skill as new fluorochromes and next-generation instruments continue to push the boundaries of high-dimensional flow cytometry.

Keywords: High Parameter, Data Analysis, Optimization

CMLE Credit: 1.5