Abstract Number: PB0448
Meeting: ISTH 2020 Congress
Background: Neutrophil extracellular Traps (NET) are involved in various clinical settings such as thrombosis. They have been recently used as thrombosis biomarkers.
Aims: We aimed to develop a new reliable and reproducible method to quantify NETs using flow cytometry (FCM), and compare it to a quantitative ELISA assay for MPO/DNA complexes.
Methods: We used whole blood from 32 healthy donors and MPA to activate NETosis. To validate our gating protocol, we used various markers of detection and quantification of NETs (Myeloperoxydase (MPO), Propidium iodide (PI) and citrullined histone H3 (H3c)) and gated on the neutrophil window (targeting NET bound to neutrophil) and on cell fragments window (circulating NET remnants).
We then applied this test to 21 patients with non–CML myeloproliferative neoplasms (MPN), who are known to have more NETs than healthy controls.
Results: The triparametric gate MPO/IP/H3c+ showed significantly more events on MPA- stimulated neutrophil versus unstimulated samples (median: 10% vs 0.48%, p< 0.05) and offers the best specificity of quantification. Using the Bland-Altman difference plot, we observed that this triparametric gate offered the best statistical consistency, when compared to ELISA. However we did not report a significant correlation between both techniques.
Lastly, using FCM, we detected more NETs in MPN patients samples compared with healthy controls, both in the triparametric gate on neutrophil NET window and NET remnants.
Conclusions: NETs detection by FCM, is a recent, specific and reliable quantification methods of NETosis that allows rapid quantification of NETosis from whole blood. In this study, we have for the first time quantified NETs remnants by FCM. FCM and ELISA lack of correlation may be linked to the heterogeneity and complexity of NETs structures, which can affect MPO/AND complexes dosing. Finally, cytometric NETs detection on whole blood is probably the most closely reflect of NETosis in vivo.
To cite this abstract in AMA style:Chaghouri P-, Labrouche-Colomer S, Lechevalier N, Vial J-, James C. Quantification of Neutrophil Extracellular Trap by Flow Cytometry: Technical Development [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/quantification-of-neutrophil-extracellular-trap-by-flow-cytometry-technical-development/. Accessed September 24, 2023.
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