Resolving the dilution paradox to improve the interpretation of extracellular vesicle biomarker studies.

Background: Concentrations of extracellular vesicles (EVs) and other particles are measured in plasma for biomarker exploration. A commonly used method, flow cytometry, requires plasma dilution to ensure single-particle detection. Since plasma EVs are outnumbered by variable concentrations of lipoproteins, dilution differs between the plasma samples. Dilution can result in misidentification of fluorescent background signals as labeled EVs. This phenomenon, called the dilution paradox, leads to overestimation of plasma EV concentrations, and likely impacts conclusions from earlier performed biomarker studies. Objectives: This study reevaluated earlier conclusions from our clinical biomarker studies Antiplatelet Therapy Effect on Extracellular Vesicles (AFFECT EV) and Circulating Nanotraces to Identify the Cause of Stroke (CINTICS), by taking the dilution paradox into account. Methods: We developed a model that quantifies the fluorescent background and estimates whether a flow cytometry measurement is dominated by fluorescent background, that is, if the measurement is unreliable. This model was applied to the original datasets of the AFFECT EV and CINTICS studies to identify and exclude unreliable measurements. We investigated whether exclusion of unreliable data affects the original conclusions. Results: Our model estimated that 47% (1156/2457) of the evaluated measurements are unreliable, and conclusions from both biomarker studies required adjustment. Conclusion: Our model improves reliability and reproducibility of EV concentration measurements using flow cytometry. We recommend to reanalyze earlier EV flow cytometry studies using our model and to use a fixed dilution factor in future EV flow cytometry studies to enable reliable EV concentration measurements.