PLoS One. 2015 May 15;10(5):e0127209. doi: 10.1371/journal.pone.0127209.
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Alfredo Corell's curator insight,
September 29, 2013 11:51 AM
Cytometry Part B: Clinical CytometrySpecial Issue: Validation of Cell-Based Fluorescence Assays: Practice Guidelines from the International Council for Standardization of Haematology and the International Clinical Cytometry SocietySeptember/October 2013
Volume 84, Issue 5 Pages 279–357 Issue edited by: Marie C. Béné, Gerald E. Marti |
Alfredo Corell's curator insight,
January 29, 2014 1:28 PM
This issue of Cytometry contains three reviews that further celebrate the longstanding, intense, and happy marriage between Cytometry and Immunology, as highlighted recently during the XV International Congress of Immunology, held in Milan, Italy, August 22–27, 2013. Scanning the scientific contributions of the Conference reveals that cytometric technology was used in the vast majority of abstracts and presentations. In other words, very few scientists did not switch on one or more lasers! It is obvious, that nowadays very few studies in the field of Immunology are possible without techniques based upon single cell analysis. |
Microparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results.
MATERIALS AND METHODS:We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM).
RESULTS:Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins).
CONCLUSION:We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.