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

Joyce Rops, Naomi C Buntsma, Aleksandra Gąsecka, Rienk Nieuwland, Aleksandra Rosiek, Yvo B W E M Roos, A Yaël Nossent, Nyika D Kruyt, Edwin van der Pol, Ceren Eyileten

Journal: Research and practice in thrombosis and haemostasis 2026;10(4):106636

PMID: 42395915

View on PubMed

Abstract

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.

Address: Laboratory of Experimental Clinical Chemistry, Laboratory Specialized Diagnostics & Research, Department of Laboratory Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.; Biomedical Engineering & Physics, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.; Amsterdam Cardiovascular Sciences, Atherosclerosis and Ischemic Syndromes, Amsterdam, Netherlands.; Department of Neurology, Leiden University Medical Center, Leiden, Netherlands.; Laboratory of Experimental Clinical Chemistry, Laboratory Specialized Diagnostics & Research, Department of Laboratory Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.; Biomedical Engineering & Physics, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.; Amsterdam Cardiovascular Sciences, Atherosclerosis and Ischemic Syndromes, Amsterdam, Netherlands.; Department of Neurology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.; Amsterdam Neuroscience, Neurovascular Disorders, Amsterdam, Netherlands.; Laboratory of Experimental Clinical Chemistry, Laboratory Specialized Diagnostics & Research, Department of Laboratory Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.; Department of Cardiology, Medical University of Warsaw, Warsaw, Poland.; Laboratory of Experimental Clinical Chemistry, Laboratory Specialized Diagnostics & Research, Department of Laboratory Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.; Amsterdam Cardiovascular Sciences, Microcirculation, Amsterdam, Netherlands.; Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, Netherlands.; Department of Child and Adolescent Psychiatry, Institute of Psychiatry and Neurology, Warsaw, Poland.; Amsterdam Cardiovascular Sciences, Atherosclerosis and Ischemic Syndromes, Amsterdam, Netherlands.; Department of Neurology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.; Amsterdam Neuroscience, Neurovascular Disorders, Amsterdam, Netherlands.; Department of Nutrition, Exercise and Sports, University of Copenhagen, Copenhagen, Denmark.; Department of Neurology, Leiden University Medical Center, Leiden, Netherlands.; Laboratory of Experimental Clinical Chemistry, Laboratory Specialized Diagnostics & Research, Department of Laboratory Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.; Biomedical Engineering & Physics, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.; Amsterdam Cardiovascular Sciences, Atherosclerosis and Ischemic Syndromes, Amsterdam, Netherlands.; Department of Experimental and Clinical Pharmacology, Centre for Preclinical Research and Technology (CePT), Medical University of Warsaw, Warsaw, Poland.; Genomics Core Facility, Center of New Technologies (CeNT), University of Warsaw, Warsaw, Poland.

DOI: 10.1016/j.rpth.2026.106636

MeSH Terms: Biomarkers, Extracellular Vesicles, Flow Cytometry, Humans, Reproducibility of Results, Stroke, Indicator Dilution Techniques