Déficit d'activité microbicide des phagocytes mesuré par cytométrie en flux

Microbicidal activity by phagocytes is critical in protecting against bacterial or fungal infection. It is mainly due to the production or Free radical species induced by the phagocytosis. It is possible to measure directly the capacity to produce free radicles by flow cytometry (FCM) using substrates such as Dihydro-rhodamine 123 (DHR123) that become fluorescent under oxido-reduction in the presence of free radicals. This production can be triggered by bacteria or chemicals but the assay needs standardization to reach an ISO15189 accreditation level for clinical diagnosis. In this paper, we shortly describe the principle of this assay adapted from a commercial kit with some improvements. The test is directly performed on whole blood with a minimal preparation and only 20 minutes of incubation. It is calcium-dependent. As a result, CD16+ granulocytes give a straightforward response under induction by Phorbol Myristate Acetate. The response to E. coli is more heterogeneous and sometimes even stronger. Peripheral blood monocytes only give a low response. Interestingly, the response is significantly reduced in patients who had recent sever sepsis demonstrating immuno-paralysis as previously shown with other parameters. Part of this functional defect is due to a reduced opsonic activity in plasma proteins. The test appears to be robust and quite easy to be implemented in laboratory provided a minimal experience in Flow cytometry and functional tests. It is a good tool to detect primary as well as secondary deficiency in phagocytosis.