Abstract Number: OC 39.3
Meeting: ISTH 2021 Congress
Theme: Diagnostics and OMICs » Biomarkers of Thrombosis and Hemostasis
Background: Neutrophil-derived extracellular vesicles (NDEV) are a subset of circulating extracellular vesicles (EV). Although elevation of their plasmatic levels has been reported in a variety of inflammatory and cardiovascular disorders such as septic shock, hypercholesterolemia, stroke, or atherosclerosis, they currently remain one of the least studied EV populations.
Aims: The objective of the present study is to improve NDEV detection in plasma samples, using a new strategy combining size exclusion chromatography (SEC) and antibodies combination to enhance the flow cytometry (FCM) fluorescent signal. This new strategy was tested to improve the selective sorting of NDEV from plasma.
Methods: A large panel of monoclonal antibodies (54 specificities) was tested on purified NDEV generated from human circulating neutrophils by reference to monocyte and lymphocyte EV. Size exclusion chromatography (SEC), antibody combination and fluo-sensitive FCM were used to increase separation index (SI) in FCM between background noise and specific labeling. The new combined strategy was used for Jet-in-Air NDEV selective sorting from plasma of sepsis patients.
Results: The antibody screening identified CD15, CD66b and CD66c as the best specificities for the detection of NDEV while preserving a good specificity. We showed that combining SEC and CD15/CD66b/CD66c antibody combination allowed not only to reduce the background noise by removing the unbound antibody but also to enhance the fluorescence signal intensity. The use of instrument with fluo-sensitive detectors allowed significant increase in NDEV count in plasma from sepsis patients compared to standard flow cytometer. Finally, combining all these technological advances, NDEV from sepsis plasma were efficiently isolated by cell sorting.
Conclusions: Combining pre-analytical optimization, broad antibody screening, fluo-sensitivity cytometry led to improve the detection of NDEV by enhancing the FCM fluorescent signal and make possible selective EV sorting at a level required for relevant biomarker detection in clinical practice.
To cite this abstract in AMA style:
Bonifay A, Robert S, Champagne B, Petit P-, Eugène A, Chareyre C, Vallier L, Lacroix R, Dignat-George F. New strategy to improve the detection of large neutrophil-derived extracellular vesicles in plasma by fluo-sensitive flow cytometry [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/new-strategy-to-improve-the-detection-of-large-neutrophil-derived-extracellular-vesicles-in-plasma-by-fluo-sensitive-flow-cytometry/. Accessed December 5, 2021.« Back to ISTH 2021 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/new-strategy-to-improve-the-detection-of-large-neutrophil-derived-extracellular-vesicles-in-plasma-by-fluo-sensitive-flow-cytometry/