Recommendations for Accurate Target Expression Evaluation by Quantitative Flow Cytometry

ABSTRACT Among various cellular characteristics, flow cytometry can evaluate antigen expression through qualitative or quantitative approaches. For relative quantification, fluorescence intensity (FI) values are converted into quantitative measurements using appropriate reference materials. To quantitatively estimate antigen density or define ligand‐binding sites per cell, antibody binding capacity (ABC) values serve as the preferred metric. Standardizing assays through the conversion of arbitrary FI units into quantitative data is essential for consistency. However, reported ABC values for well‐characterized antigens vary across the literature. This study addresses the challenges in achieving robust and reproducible quantitative flow cytometry data, offering methodological recommendations for accurately assessing target expression. Our research includes a comprehensive investigation of multiple factors, such as conventional and full‐spectrum instruments, antibodies, reagents, matrices, cell density/confluency, cellular autofluorescence, and quantitative kits, to identify the primary sources of variation in ABC calculations. By implementing a systematic and integrated approach, we aim to ensure the generation of reliable and reproducible ABC values. Longitudinal studies provide strong evidence of assay robustness, while the established protocol further supports biomarker evaluation across different matrices and various stages of drug development.