Development of a polychromatic flow cytometry protocol to examine neutrophil activation during anaphylaxis. (#R21)

 

Abbie Creamer 1 2 3 Shelley F Stone 1 2 3 Simon GA Brown 1 2 3 4
  1. Centre for Clinical Research in Emergency Medicine, PERTH, WA, Australia
  2. University of Western Australia, PERTH, WA, Australia
  3. Harry Perkins Institute of Medical Research, PERTH, WA, Australia
  4. Emergency Medicine, Royal Perth Hospital, PERTH, WA, Australia

Background. Anaphylaxis is a severe allergic reaction that affects multiple organ systems, and is characterised by hypotension, bronchospasm, and upper airway obstruction. The mechanisms that rapidly amplify a localised allergic response into a systemic life-threatening reaction are poorly understood, specifically the role of peripheral blood leukocytes (PBLs). The abundance and importance of neutrophils in the innate immune response suggest they may play a role in amplifying the immune reaction during anaphylaxis. Flow cytometry presents an exciting opportunity to investigate the activation of neutrophils during acute human anaphylaxis. Aim. To develop a polychromatic flow cytometry protocol for analysing neutrophil activation in lysed human whole blood, to be implemented on patients with anaphylaxis. Methods. We selected a panel of markers to identify neutrophils and investigate their degree of activation. Optimisation of antibody concentration was achieved by individual titration, before testing the entire panel to confirm there was no significant spillover. A variety of different stimulation conditions were tested with the goal of achieving adequate activation, minimal cell death and ease of use in a clinical setting. Results. Optimal stimulation was observed upon addition of 0.5μg/mL lipopolysaccharide (LPS) with Brefeldin A (BFA) added to blood within 15 mins of collection, for 4 hours before red cell lysis and antibody staining. This protocol has been easily implemented in a clinical setting and has produced consistent quality flow cytometry data across several patients. Future Applications. We will recruit a minimum of 10 anaphylaxis patients which will be compared to patients with other inflammatory conditions.

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