Quantification des Neutrophil Extracellular Traps en cytométrie en flux : mise au point de la technique

Abstract : Neutrophil extracellular Trap (NET) are a network of extracellular decondensed chromatin fibers associated with granular proteins produced by neutrophils when activated. The main function of NETs is to eliminate microbes within the innate immunity framework. In case of deregulation, this phenomenon can take part in the pathogenesis of many diseases, including auto-immune diseases, thrombosis and cancers. The exploration of NETosis requires an objective, reliable and reproducible quantification method. In this work, we developed a new technique to quantify NETs using flow cytometry (FCM) on whole blood, which was compared to a quantitative plasma assay of MPO/ADN complexes by ELISA. We applied this test to a sample of patients with non-CML myeloproliferative neoplasms (MPNs). We set up a technical protocol with neutrophils stimulation on whole blood using PMA and validated the cytometry gating protocol by testing various markers of detection and quantification of NETs: myeloperoxydase (MPO), propidium iodide (PI) and the anti-histone H3 citrullined antibody (H3c), gated on the neutrophil window population (aiming for neutrophil bound NETs) and on the neutrophil fragments window (aiming for circulating NETs remnants). The triparametric gate MPO/IP/H3c+ applied to both windows (neutrophil and remnants) offers the best specificity for these criteria. However, we didn’t manage to show in our study a significant correlation between the results obtained with FCM and the ones obtained in ELISA, whatever the choice of gate in FCM (Pearson correlation coefficient r non-significant with p>0.05). We then compared the two measurements methods using the Bland-Altman difference plot to study associated agreement between them, which highlighted an absolute systematic error (d) between the average differences of the two techniques, with a minimal value in the triparametric gate MPO/IP/H3c+ (- 1.6 in neutrophil NET window and -3.1 in the NET remnants window). Besides this systematic error d, we find out the existence of a second proportional error, affecting extreme values. Nevertheless, the triparametric gate MPO/IP/H3c+ on CMF offered the best statistical consistency with 95% of the values of differences between the 2 techniques within acceptable range of 95 % CI of limits of agreement LoA (defined by d+/- 1.96xSD). Lastly, this technique successfully showed a significant difference in NETs detection between MPN and healthy control samples using the triparametric gate MPO/IP/H3c+ on the neutrophil NET window (2.614%NET +/- 0.3346 vs 0.54969%NET +/- 0.05158 ) and NET remnants window ( 4.5310% NET +/- 2.0291 vs 1.9153%NET +/- 0.9833 ) at p<0.05.
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Master Thesis
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Pierre-Elian Chaghouri. Quantification des Neutrophil Extracellular Traps en cytométrie en flux : mise au point de la technique. Médecine humaine et pathologie. 2018. ⟨dumas-02088328⟩

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