title: Avoiding false positive antigen detection by flow cytometry on blood cell derived microparticles: the importance of an appropriate negative control.
abstract: BACKGROUND
  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.
  
  
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Clinical Cell Therapy, Université Libre de Bruxelles (ULB), Jules Bordet Institute, Brussels, Belgium.
authors.forename: Emerence
authors.initials: E
authors.lastname: Crompot
authors.affiliation: Laboratory of Clinical Cell Therapy, Université Libre de Bruxelles (ULB), Jules Bordet Institute, Brussels, Belgium.
authors.forename: Michael
authors.initials: M
authors.lastname: Van Damme
authors.affiliation: Hematology Department, Jules Bordet Institute, Brussels, Belgium.
authors.forename: Hugues
authors.initials: H
authors.lastname: Duvillier
authors.affiliation: Laboratory of Clinical Cell Therapy, Université Libre de Bruxelles (ULB), Jules Bordet Institute, Brussels, Belgium.
authors.forename: Karlien
authors.initials: K
authors.lastname: Pieters
authors.affiliation: Laboratory of Molecular Parasitology, IBMM, Université Libre de Bruxelles (ULB), Gosselies, Belgium.
authors.forename: Marjorie
authors.initials: M
authors.lastname: Vermeesch
authors.affiliation: Laboratory of Molecular Parasitology, IBMM, Université Libre de Bruxelles (ULB), Gosselies, Belgium.
authors.forename: David
authors.initials: D
authors.lastname: Perez-Morga
authors.affiliation: Hematology Department, Jules Bordet Institute, Brussels, Belgium.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Meuleman
authors.affiliation: Department of Hemato-Oncology, Grand Hôpital de Charleroi, Gilly, Belgium.
authors.forename: Philippe
authors.initials: P
authors.lastname: Mineur
authors.affiliation: Hematology Department, Jules Bordet Institute, Brussels, Belgium.
authors.forename: Dominique
authors.initials: D
authors.lastname: Bron
authors.affiliation: Laboratory of Clinical Cell Therapy, Université Libre de Bruxelles (ULB), Jules Bordet Institute, Brussels, Belgium.
authors.forename: Laurence
authors.initials: L
authors.lastname: Lagneaux
authors.affiliation: Laboratory of Clinical Cell Therapy, Université Libre de Bruxelles (ULB), Jules Bordet Institute, Brussels, Belgium.
authors.forename: Basile
authors.initials: B
authors.lastname: Stamatopoulos
mesh_terms: D017304:Annexin A5 / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology*; D001792:Blood Platelets / Q000276:immunology; D055252:Cell-Derived Microparticles / Q000276:immunology*; D005189:False Positive Reactions; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007159:Immunologic Tests / Q000379:methods; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology; D008214:Lymphocytes / Q000276:immunology; D008863:Microspheres; D009000:Monocytes / Q000276:immunology
chemical_list: D017304:Annexin A5; D000911:Antibodies, Monoclonal; D015703:Antigens, CD