Research paper Oxidative burst assessment and neutrophil–platelet complexes in unlysed whole blood Ariadna Avendaño a , Irene Sales-Pardo a , Lorena Marin a , Pedro Marin b , Jordi Petriz a, ⁎ a Institut de Recerca de l'Hospital Universitari Vall d'Hebron, Barcelona, Spain b Cryopreservation Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain article info abstract Article history: Received 5 May 2008 Received in revised form 22 August 2008 Accepted 2 September 2008 Available online 25 September 2008 Background: Methods currently employed for measuring reactive oxygen species production can lead to both cellular depletion and in artifactual activation. The objective of this study was to design a methodology allowing the measurement of oxidative burst (OB) with minimal sample manipulation. Methods: To that purpose a flow cytometry technique developed in our laboratory, based on nucleic acid staining to discriminate erythrocytes and debris, was employed. DRAQ5 dye and PECy5-CD45 monoclonal antibody (MoAb) were simultaneously used in FL3 to identify the leukocyte population and the PE-CD14 MoAb emission was detected in FL2 for monocytes. OB was measured by using the fluorogenic probe dihydrorhodamine 123, a marker of hydrogen peroxide production. Phorbol myristate acetate (PMA), Opsonized Zymosan (OZ), fMLP and calcium ionophore A23187 activators were also used in this study. For OB assays, dose–response curves were performed for each activator. In addition, the effect of activator concentration on annexin V binding, as a measure of phosphatidylserine translocation, was evaluated. Results: With this method no-lysis and no-wash steps are required, thus avoiding an unwanted damage to leukocytes. PMA and Zymosan produced an increase in annexin V binding, while fMLP and calcium ionophore did not. Conclusions: This study reports a feasible and reproducible new flow cytometry assay for assessing OB of neutrophils and monocytes with minimal sample manipulation. In addition, under PMA and OZ conditions, the number of neutrophils showing annexin V binding was strikingly increased. This effect is not related with a phagocytic overstimulation, but with an increased neutrophil-platelet complexes formation. © 2008 Elsevier B.V. All rights reserved. Keywords: Oxidative burst Neutrophil–platelet complexes Flow cytometry Whole blood DRAQ5 1. Introduction Phagocytes play a major role in host defense. The response of phagocytes to pathogens include: phagocytosis, proteolytic destruction via the action of the granules content, and the damage induced by the hydroxyl radical and the superoxide and hydrogen peroxide generated by NADPH oxidase (Chanock et al., 1994). After neutrophil activation in response to infection, or other stimuli, there is an increase in oxygen consumption which produces these different oxygen species, a phenomenon known as oxidative burst (Mollinedo et al., 1999). Patients with defective intracellular mechanisms for killing microorganisms have an increased susceptibility to infections. The most common of such disorders is chronic granulomatous disease (CGD), in which the NADPH-oxidase function is impaired due to mutations in the genes that encode their components (Smith and Curnutte, 1991; Lekstrom-Himes and Gallin, 2000). Because of the relevance of an early detection of phagocyte disorders, many clinical studies are investigating the oxidative burst potentials (Kraft and Kress, 2005; Amer and Fibach, 2005; Osar et al., 2004; Arellano-Rodrigo et al., 2006; Wehlin et al., 2005; Panasiuk et al., 2005; Matheson et al., 2003; Noguera et al., 2001; Siddiqi et al., 2001). Journal of Immunological Methods 339 (2008) 124–131 ⁎ Corresponding author. Laboratori 123, Institut de Recerca Hospital Vall d'Hebron, P° Vall d'Hebron 119-129, 08035 Barcelona. Tel./fax: +34 932 746 873. E-mail address: jpetriz@ir.vhebron.net (J. Petriz). 0022-1759/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.09.003 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim