Plasma extracellular vesicle test sample to standardize flow cytometry measurements

Background: Extracellular vesicles (EVs) in body fluids are explored as disease biomarkers, but EV concentrations measured by flow cytometers (FCMs) are incomparable. Objectives: To improve data comparability, new reference materials with physical properties resembling EVs and reference procedures are being developed. The validation of new reference materials and procedures requires biological test samples. We developed a human plasma EV test sample (PEVTES) that i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry-compatible, and iv) is stable. Methods: The PEVTES was prepared from human plasma of 3 fasting donors. EVs were immunofluorescently stained with antibodies against platelet-specific (CD61) and erythrocyte-specific (CD235a) antigens or lactadherin. To reduce the concentration of soluble proteins, lipoproteins, and unbound reagents, stained EVs were isolated from plasma by size-exclusion chromatography. After isolation, the PEVTES was filtered to remove remnant platelets. PEVTESs were diluted in cryopreservation agents, dimethyl sulfoxide, glycerol, or trehalose and stored at -80 degrees C for 12 months. After thawing, stained EV concentrations were measured with a calibrated FCM (Apogee A60-Micro). Results: We demonstrate that the developed PEVTES resembles subcellular particles in human plasma when measured using FCM and that the concentrations of prestained platelet-derived, erythrocyte-derived, and lactadherin+ EVs in the PEVTES are stable during storage at -80 degrees C for 12 months when stored in trehalose. Conclusion: The PEVTES i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry-compatible, and iv) is stable. Therefore, the developed PEVTES is an ideal candidate to validate newly developed reference materials and procedures.