Single-EV Analyses Require Rigorous Antibody Qualification: PD-L1 Profiling in Cell Models and Patient Plasma

Imaging flow cytometry (IFCM) has emerged as a powerful method for high-throughput phenotyping of small extracellular vesicles (sEVs) at single-vesicle resolution. However, the reliability and sensitivity of IFCM critically depend on the performance of the antibodies used. In this study, we systematically compared several commercially available anti-PD-L1 antibodies for their ability to detect PD-L1-positive sEVs. Despite being derived from the same clone, antibodies from different manufacturers showed striking differences in labelling efficiency. These findings demonstrate that antibody validation for cellular targets does not ensure suitability for single-EV analysis and underscore the need for rigorous, application-specific qualification. To address this, we developed a robust IFCM workflow in full alignment with MIFlowCyt-EV reporting standards. Using a qualified anti-PD-L1 antibody, we achieved reproducible detection of PD-L1⁺ sEVs directly in unprocessed plasma samples from tumour patients. To assess the quantitative potential of single-EV analysis, we compared this approach with bead-based capture and performed dilution experiments. While bead-based methods yielded a relatively narrow range of mean fluorescence intensities across samples, IFCM revealed distinct PD-L1⁺ sEV levels, supporting its semi-quantitative capability. Finally, we applied this workflow to plasma samples from patients with head and neck, lung, and breast cancer, as well as healthy donors. In subsets of all three cancer cohorts, elevated PD-L1⁺ sEV levels were observed, suggesting potential clinical relevance. While in-depth clinical correlations lie beyond the scope of this study, our findings establish a validated, standardized protocol for IFCM-based single-EV profiling and highlight the central role of antibody quality in ensuring analytical accuracy.