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

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. RESULTS: 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.

New tools in cytometry.

Cytometry aims to analyze cells, of any type, using dedicated instruments. The quantitative aspect makes flow cytometry (FCM) a good complementary tool for morphology. Most of the identification tools are based on immunostaining of cell structure details and more and more tools are available in terms of specificities and labels. FCM is under exponential development thanks to technical, immunological and data analysis progresses. Actual generations are now routinely using 6 to 10 simultaneous immuno-labeling on 20 to 100,000 cells, at high speed and short sample preparation and can easily detect rare events at frequency below 10-4 cells. Data interpretation is complex and requires expertise. Mathematical tools are available to support analysis and classification of cells based. Cells from tissues can also be analyzed by FCM after mechanical and or enzymatic separation, but in situ cells can also be analyzed with the help of cytometry. Very new instruments bring spectral analysis, image in flow and mass spectrometry. Medical applications are very broad, notably in hemopathies, immunology, solid tumors, but also microbiology, toxicology, drug discovery, food and environmental industry. But, the limit of FCM is its dependence on operator from sample preparation, instrument settings up to data analysis and a strong effort is now under progress for standardization and constitution of international data bank for references and education.