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    Poster Networking

    Hemostatic Systems in Cancer, Inflammation and Immunity

    Poster Networking Session

    PB0728 - Models for in vitro investigation of Neutrophil activation

    Monday, June 26, 2023
    18:30 – 19:30 ET
    Room: Exhibition

      Co-Author(s)

    • Iman AL DYBIAT, PhD (she/her/hers)

      Team leader, Research & Dedevelopment
      Diagnostica Stago
      Gennevilliers, Ile-de-France, France

      • Co-Author(s)

    • CF

      Corentin FRANCO

      Project manager, Research & Dedevelopment
      Biocytex
      Marseille, Provence-Alpes-Cote d'Azur, France

    • AM

      Antonio MORENO

      Internship
      Biocytex
      Marseille, Provence-Alpes-Cote d'Azur, France

    • SM

      Shahsoltan MIRSHAHI

      Head of Cancer and Hemopathy Studies
      Diagnostica Stago
      Paris, Ile-de-France, France

    • FM

      Floriane MAS

      Project Manager, Research & Dedevelopment
      Biocytex, Marseille, France
      Marseille, Provence-Alpes-Cote d'Azur, France

    • TB

      Tarik BOURICHE

      Head of Hemostasis and Hematology Department, Research & Research & Dedevelopment
      Biocytex
      Marseille, Provence-Alpes-Cote d'Azur, France

      • Presenting Author(s)

    • Joffrey Feriel, PharmD

      Scientific Manager
      Diagnostica Stago
      ASNIERES-SUR-SEINE, Ile-de-France, France

      • Co-Author(s)

    • GC

      Geneviève CONTANT

      Prospective Research DRP Director
      Diagnostica Stago
      Gennevilliers, Ile-de-France, France

    Background: As a response to inflammatory stimuli, polymorphonuclear cells (PMNs, neutrophils) are able to release their content, called neutrophils extracellular traps (NETs). A role for NETs is emerging in the pathogenesis of many diseases, they are involved in host defense and are associated with thrombotic, infectious, or autoimmune diseases. They may play a major role in immunothrombosis.
    To release NETs, PMNs undergo dramatic morphological changes, leading to cell death by the loss of intracellular membranes before the plasma membrane integrity is compromised. This NETosis process lasts minutes to hours after stimulation, involving successive chromatin decondensation, granules disintegration and internal components ejection to the extracellular space.

    Aims: To investigate in vitro neutrophil activation models.

    Methods: EDTA Whole Blood was activated by Phorbol 12-myristate 13-acetate (PMA) and N-Formyl-Met-Leu-Phe (fMLP) molecules in different time and concentration conditions. PBS 1% or DMSO was added to the blood as a control. PMNs activation was determined by: i) their cellular volume and fluorescent signal increase using blood count analyzers (Sysmex and Diagnostica Stago), ii) confocal microscopy, using Anti-lamin B and DAPI for nuclear labeling, iii) time-lapse microcinematography to film first observations of neutrophil activation after incubation with fMLP.

    Results: Neutrophils stimulated with 200ng/mL PMA showed a significant increase of fluorescent signal from 15 to 30 minutes incubation time (+5%) and of cellular volume up to 60% to non-activated cells. After stimulation with 12.5 to 25ng/mL PMA, PMNs activation was evidenced up to 2h à 37°C (fluorescence signal +6%).
    In the 1mM fMLP model, the fluorescent signal increased up to 30 mins of incubation (+2 to +4.7 %). Decondensed nuclear materials of PMNs were recognized after labeling with DAPI and Anti-lamin B. Cinematography showed a direct effect of fMLP on neutrophil nuclear content release

    Conclusion(s): Both PMA and fMLP could be potential models for in vitro investigation of neutrophil activation.