Calibration of Flow Cytometers Enables Reproducible Measurements of Extracellular Vesicle Concentrations and Reference Range Establishment
The concentration of cells is a key component of modern blood tests. Given the biomarker potential of extracellular vesicles (EVs) in blood, we aimed to establish reference ranges for blood cell-derived EVs using flow cytometry. To address the orders-of-magnitude variability in reported EV concentrations between different flow cytometers (FCMs), we first validated a calibration methodology to enable reproducible EV concentration measurements. The methodology was evaluated in an interlaboratory comparison study and shows that calibration reduces the median absolute deviation of EV concentrations measured on 25 different FCMs from 67 % to 25 %-31 %. The calibration methodology was then used to determine reference ranges of erythrocyte-, leukocyte-, and platelet-derived EVs in human blood plasma in a cohort of healthy individuals (n = 224). This study demonstrates that calibration enables comparable concentration measurements of blood cell-derived EVs, thereby bringing EVs one step closer to clinical applications.
Authors
- Ton G. van Leeuwen (ORCID: https://orcid.org/0000-0002-5642-1133)
- Annette Meyer
- Zoltán Varga (ORCID: https://orcid.org/0000-0002-5741-2669)
- Sumeet Poudel (ORCID: https://orcid.org/0009-0006-7617-9838)
- Rienk Nieuwland (ORCID: https://orcid.org/0000-0002-5671-3400)
- Desmond Pink (ORCID: https://orcid.org/0000-0002-7543-3199)
- Tina Van Den Broeck
- Erika G. Marques de Menezes (ORCID: https://orcid.org/0000-0002-3273-6755)
- Pia Siljander (ORCID: https://orcid.org/0000-0003-2326-5821)
- John P. Nolan (ORCID: https://orcid.org/0000-0002-9669-382X)
- Françoise Dignat‐George (ORCID: https://orcid.org/0000-0001-7006-4462)
- Inge Nelissen (ORCID: https://orcid.org/0000-0002-1248-3463)
- Britta Bettin (ORCID: https://orcid.org/0000-0003-3353-2129)
- Bernd Giebel (ORCID: https://orcid.org/0000-0003-2446-948X)
- Randy P. Carney (ORCID: https://orcid.org/0000-0001-8193-1664)
- Vera A. Tang (ORCID: https://orcid.org/0000-0003-3930-9139)
- Pascale Berckmans (ORCID: https://orcid.org/0009-0008-5858-0892)
- Lili Wang (ORCID: https://orcid.org/0000-0003-2456-898X)
- Joanne Lannigan (ORCID: https://orcid.org/0000-0002-3981-8681)
- Joshua A Welsh (ORCID: https://orcid.org/0000-0002-1097-9756)
- Romaric Lacroix (ORCID: https://orcid.org/0000-0002-5756-470X)
- Bo Li (ORCID: https://orcid.org/0000-0002-6129-321X)
- Tobias Tertel (ORCID: https://orcid.org/0000-0001-8659-8610)
- Philip J. Norris (ORCID: https://orcid.org/0000-0003-0526-2088)
- Dorothée Faille (ORCID: https://orcid.org/0000-0003-4414-739X)
- Sean Cook
- Katariina Maaninka (ORCID: https://orcid.org/0000-0001-6341-3871)
- Edwin van der Pol (ORCID: https://orcid.org/0000-0002-9497-8426)
- Christian Gollwitzer (ORCID: https://orcid.org/0000-0002-6437-8825)
- Jennifer Jones (ORCID: https://orcid.org/0000-0002-9488-7719)
- Kim Falkena
- Edit I. Buzás (ORCID: https://orcid.org/0000-0002-3744-206X)
- Fabrice Lucien (ORCID: https://orcid.org/0000-0001-6149-345X)
- Rachel R. Mizenko (ORCID: https://orcid.org/0000-0002-9991-8799)
- Nadine Ajzenberg (ORCID: https://orcid.org/0000-0001-8960-1766)
- Yohan Kim (ORCID: https://orcid.org/0000-0002-4009-1694)
- Jovan P. Antović (ORCID: https://orcid.org/0000-0002-9860-1108)
- Stéphane Robert
Institutions
- Semmelweis University (HU)
- Physikalisch-Technische Bundesanstalt (DE)
- National Institutes of Health (US)
- Amsterdam University College (NL)
- National Institute of Standards and Technology (US)
- Mayo Clinic (US)
- Karolinska University Hospital (SE)
- University of Helsinki (FI)
- Inserm (FR)
- University of California, San Francisco (US)
- Université Paris Cité (FR)
- Aix-Marseille Université (FR)
- Finnish Red Cross (FI)
- Pacific Research Institute (US)
- Karolinska Institutet (SE)
- Budapest University of Technology and Economics (HU)
- Sorbonne Paris Cité (FR)
- Assistance Publique – Hôpitaux de Paris (FR)
- Nanfang Hospital (CN)
- Amsterdam Neuroscience (NL)
- European Media Laboratory (Germany) (DE)
- BD Biosciences (United States) (US)
- Vitalant (US)
- Laboratoire de Recherche Vasculaire Translationnelle (FR)
- Flometrics (United States) (US)
- National Cancer Institute (US)
- Hôpital Bichat-Claude-Bernard (FR)
- Cellectar Biosciences (United States) (US)
- Amsterdam University Medical Centers (NL)
- Alphabio (FR)
- Hôpital de la Timone (FR)
- Scintillon Institute (US)
- Amsterdam University of Applied Sciences (NL)
- Alberta Oil Sands Technology and Research Authority (CA)
- Southern Medical University (CN)
- University of Duisburg-Essen (DE)
- Flemish Institute for Technological Research (BE)
- HUN-REN Research Centre for Natural Sciences (HU)
- University of California, Davis (US)
- University of Amsterdam (NL)
- Amsterdam UMC Location Vrije Universiteit Amsterdam (NL)
Publication Details
- Journal
- Journal of Extracellular Vesicles
- Published
- 2025-12-01
- DOI
- https://doi.org/10.1002/jev2.70189
- Citations
- 1
- Primary Topic
- Extracellular vesicles in disease
- Type
- article
- Field-Weighted Citation Impact
- 0.81
Funders
- European Commission
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek
- European Metrology Programme for Innovation and Research