Microbicidal activity measured by flow cytometry: Optimization and standardization for detection of primary and...

Microbicidal activity is related to the production of reactive oxygen species (ROS) that can be measured by flow-cytometry using rhodamine 123 (R123). Few assays have been proposed to measure ROS production, usually on heparinized samples but none of them is standardized. Here we propose to improve the test by selecting polymorphonuclears (PMN) and monocytes, labelled and activated in one step to keep the test short, and to standardize the process even between different systems (i.e. Navios™ and FACSCanto™) using fluorescence intensity target setting ("FITS"). We applied this test on 15 patients without inflammation, 19 patients from an intensive care unit (ICU) and 11 healthy volunteers.

Provided calcium restitution, we show that the test can be performed on EDTA that is a better sample preservative. The results were highly correlated between instruments (r²=0.898). PMN CD16 (and not CD14) expression was altered under stimulation with E. coli (MdFI=239.3±93.5) or PMA (139.7±76.8) as compared to resting sample (307.6±145.1). RH123 was strongly and homogeneously induced by PMA (14.2±6.6) and more heterogeneously by E. coli (MdFI 21.9±23.4) as compared to unstimulated PNN (0.9±1.3, p<0.0001). The test is useful not only for genetic disorders but also for secondary deficiencies as observed in ICU (E. coli RH123 MFI=10.5±11.1 patients vs 30.1±26.5 in healthy donors). In ICU, CD16 expression was already altered on unstimulated samples (MdFI=197.4±131.2 vs 418, 2±81.3 in healthy donors; p≤0.0001). Bacterial stimulation was dependent of the complement that partly explains deficiency to bacterial stimulus in ICU patients.