title: Imaging flow cytometry elucidates limitations of microparticle analysis by conventional flow cytometry.
abstract: Microparticles (MPs) are submicron vesicles released from cell membranes in response to activation, cell injury, or apoptosis. The clinical importance of MPs has become increasingly recognized, although no standardized method exists for their measurement. Flow cytometry (FCM) is the most commonly used technique, however, because of the small size of MPs, and the limitations of current FCM instrumentation, accurate identification is compromised by this methodology. We decided to investigate whether the use of FCM combined with imaging, such as is possible with the ImagestreamX imaging FC (ISX), would be a more sensitive approach to characterizing MPs. Combining FCM with imaging eliminates some of the limitations demonstrated by conventional FCM, whereas also providing morphological confirmation and the ability to distinguish true single events from aggregates and cell debris. The detection limit of standard nonspecialized FCM is suboptimal when compared to ISX. Evaluating MPs below 0.200 µm and sizing remain a challenge as some MPs remain below the detection limit of ISX. Standardized calibrators, that more closely reflect the physical characteristics of MPs, need further development.
authors.affiliation: Department of Medicine, University of Virginia, Charlottesville, Virginia.
authors.forename: Uta
authors.initials: U
authors.lastname: Erdbrügger
authors.forename: Christine K
authors.initials: CK
authors.lastname: Rudy
authors.forename: Mark E
authors.initials: ME
authors.lastname: Etter
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Dryden
authors.forename: Mark
authors.initials: M
authors.lastname: Yeager
authors.forename: Alexander L
authors.initials: AL
authors.lastname: Klibanov
authors.forename: Joanne
authors.initials: J
authors.lastname: Lannigan
mesh_terms: D000465:Algorithms; D017304:Annexin A5 / Q000737:chemistry; D001792:Blood Platelets / Q000378:metabolism; D002138:Calibration; D002462:Cell Membrane / Q000502:physiology; D055252:Cell-Derived Microparticles; D003952:Diagnostic Imaging / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D008081:Liposomes; D008863:Microspheres; D057146:Multivesicular Bodies / Q000502:physiology; D010316:Particle Size; D013194:Staining and Labeling / Q000379:methods
chemical_list: D017304:Annexin A5; D008081:Liposomes
keywords: detection limits; imaging flow cytometry; microparticle detection; microvesicles