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

Area of Science:

• Extracellular vesicle (EV) research
• Immunophenotyping
• Biomarker discovery

Background:

• Imaging flow cytometry (IFCM) offers high-throughput, single-vesicle analysis of extracellular vesicles (sEVs).
• Antibody performance is critical for IFCM reliability and sensitivity in sEV detection.
• Existing antibody validation for cellular targets may not translate to sEV analysis.

Purpose of the Study:

• To systematically compare commercial anti-PD-L1 antibodies for sEV detection using IFCM.
• To develop and validate a standardized IFCM workflow for sEV profiling.
• To assess the potential of IFCM for semi-quantitative analysis of PD-L1 positive sEVs in patient plasma.

Main Methods:

• Systematic comparison of anti-PD-L1 antibodies for labeling PD-L1 positive sEVs.
• Development of a robust IFCM workflow adhering to MIFlowCyt-EV standards.
• Application of the validated workflow to analyze PD-L1 positive sEVs in unprocessed plasma from cancer patients and healthy donors.
• Comparison of IFCM with bead-based methods and dilution experiments for quantitative assessment.

Main Results:

• Significant differences in labeling efficiency were observed among anti-PD-L1 antibodies from different manufacturers.
• A validated IFCM workflow enabled reproducible detection of PD-L1 positive sEVs in patient plasma.
• IFCM demonstrated semi-quantitative capability by revealing distinct PD-L1 positive sEV levels, unlike bead-based methods.
• Elevated PD-L1 positive sEV levels were detected in plasma subsets from head and neck, lung, and breast cancer patients.

Conclusions:

• Antibody suitability for sEV analysis requires rigorous, application-specific validation beyond standard cellular assays.
• A standardized IFCM workflow provides a reliable method for single-EV profiling.
• Elevated PD-L1 positive sEVs in cancer patient plasma suggest potential clinical relevance.
• The quality of antibodies is paramount for accurate analytical results in single-EV studies.