title: Immuno-analysis of microparticles: probing at the limits of detection.
abstract: Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic in vitro system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ~60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30-40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data.
authors.affiliation: Vascular Immunology Unit, Sydney Medical School, The University of Sydney, NSW, Australia.|Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Sharissa L
authors.initials: SL
authors.lastname: Latham
authors.affiliation: Vascular Immunology Unit, Sydney Medical School, The University of Sydney, NSW, Australia.
authors.forename: Natalia
authors.initials: N
authors.lastname: Tiberti
authors.affiliation: Australian Centre for Microscopy and Microanalysis, The University of Sydney, NSW, Australia.
authors.forename: Naveena
authors.initials: N
authors.lastname: Gokoolparsadh
authors.affiliation: Beckman Coulter Australia, 23-27 Chaplin Drive, Lane Cove West, NSW 2066, Australia.
authors.forename: Karen
authors.initials: K
authors.lastname: Holdaway
authors.affiliation: Inserm, U1016, Institut Cochin, Paris, France.|Cnrs, UMR8104, Paris, France.|Univ Paris Descartes, Paris, France.
authors.forename: Pierre Olivier
authors.initials: PO
authors.lastname: Couraud
authors.affiliation: Vascular Immunology Unit, Sydney Medical School, The University of Sydney, NSW, Australia.
authors.forename: Georges E R
authors.initials: GE
authors.lastname: Grau
authors.affiliation: Vascular Immunology Unit, Sydney Medical School, The University of Sydney, NSW, Australia.|Faculty of Science, School of Life Sciences, University of Technology Sydney, NSW, Australia.
authors.forename: Valery
authors.initials: V
authors.lastname: Combes
mesh_terms: D017304:Annexin A5 / Q000276:immunology / Q000378:metabolism; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D000954:Antigens, Surface / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D055252:Cell-Derived Microparticles / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000071063:Endoglin; D005434:Flow Cytometry; D006046:Gold / Q000737:chemistry; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000276:immunology / Q000378:metabolism; D057230:Limit of Detection; D016253:Microscopy, Immunoelectron; D008894:Milk Proteins / Q000276:immunology / Q000378:metabolism; D010718:Phosphatidylserines / Q000276:immunology / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism
chemical_list: D017304:Annexin A5; D015703:Antigens, CD; D000954:Antigens, Surface; C064206:ENG protein, human; D000071063:Endoglin; C098942:MFGE8 protein, human; D008894:Milk Proteins; D010718:Phosphatidylserines; D011956:Receptors, Cell Surface; D018799:Intercellular Adhesion Molecule-1; D006046:Gold