title: Red cell microparticle enumeration: validation of a flow cytometric approach.
abstract: OBJECTIVE
  There is growing interest in the clinical application of red blood cell (RBC) microparticle (MP) enumeration as they have been postulated to be effectors of coagulation and inflammation following transfusion and in sickle cell disease. No uniform approach in MP enumeration exists and a key limitation is the lack of an internal validation process. We present and validate a flow cytometric approach where an internal standard is utilized.
  
  
  METHODS
  Glycophorin A(+) Annexin V(+) events were enumerated using MPs isolated from RBC units or plasma samples obtained from volunteers. A mixture of absolute counting (7·6 μm) and calibration beads (0·5, 0·9 and 3 μm) at a fixed ratio was added to each sample.
  
  
  RESULTS
  RBC MPs were initially selected based upon a fluorescence threshold, and the 0·5- and 0·9-μm beads defined the upper and lower light scatter distribution of MPs. The ratio of 7·6:3-μm bead events was used as an internal standard to validate the precision of MP enumeration across samples (coefficient of variation = 2·5-7·2%) and remained constant in both platelet-rich plasma (PRP) and platelet-free plasma (PFP). RBC MP counts increased in both PRP and PFP obtained from whole blood stimulated with ionophore and increasing calcium concentrations, with PRP showing higher MP counts than PFP at every concentration studied.
  
  
  CONCLUSIONS
  This method is a useful strategy to detect RBC MP counts across bio-samples provided that the flow cytometer can reliably discriminate the size of the calibration beads.
authors.affiliation: Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Xiong
authors.forename: T B
authors.initials: TB
authors.lastname: Oriss
authors.forename: J P
authors.initials: JP
authors.lastname: Cavaretta
authors.forename: M R
authors.initials: MR
authors.lastname: Rosengart
authors.forename: J S
authors.initials: JS
authors.lastname: Lee
mesh_terms: D017304:Annexin A5 / Q000097:blood; D002138:Calibration; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D004906:Erythrocyte Count; D004912:Erythrocytes / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006021:Glycophorins / Q000378:metabolism; D006801:Humans; D008863:Microspheres; D010316:Particle Size; D053657:Platelet-Rich Plasma / Q000378:metabolism; D012015:Reference Standards
chemical_list: D017304:Annexin A5; D006021:Glycophorins