title: Detection of microparticles from human red blood cells by multiparametric flow cytometry.
abstract: BACKGROUND
  During storage, red blood cells (RBC) undergo chemical and biochemical changes referred to as "storage lesions". These events determine the loss of RBC integrity, resulting in lysis and release of microparticles. There is growing evidence of the clinical importance of microparticles and their role in blood transfusion-related side effects and pathogen transmission. Flow cytometry is currently one of the most common techniques used to quantify and characterise microparticles. Here we propose multiparametric staining to monitor and quantify the dynamic release of microparticles by stored human RBC.
  
  
  METHODS
  RBC units (n=10) were stored under blood bank conditions for up to 42 days. Samples were tested at different time points to detect microparticles and determine the haemolysis rate (HR%). Microparticles were identified by flow cytometry combining carboxyfluorescein diacetate succinimidyl ester (CFSE) dye, annexin V and anti-glycophorin A antibody.
  
  
  RESULTS
  We demonstrated that CFSE can be successfully used to label closed vesicles with an intact membrane. The combination of CFSE and glycophorin A antibody was effective for monitoring and quantifying the dynamic release of microparticles from RBC during storage. Double staining with CFSE/glycophorin A was a more precise approach, increasing vesicle detection up to 4.7-fold vs the use of glycophorin A/annexin V alone. Moreover, at all the time points tested, we found a robust correlation (R=0.625; p=0.0001) between HR% and number of microparticles detected.
  
  
  CONCLUSIONS
  Multiparametric staining, based on a combination of CFSE, glycophorin A antibody and annexin V, was able to detect, characterise and monitor the release of microparticles from RBC units during storage, providing a sensitive approach to labelling and identifying microparticles for transfusion medicine and, more broadly, for cell-based therapies.
authors.affiliation: Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Grisendi
authors.affiliation: R&D Filter Department, Fresenius HemoCare Italy, Cavezzo, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Finetti
authors.affiliation: BD Biosciences Italy S.p.A, Buccinasco, Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: Manganaro
authors.affiliation: Division of Transfusion Medicine, University Hospital of Modena, Modena, Italy.
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Cordova
authors.affiliation: Division of Transfusion Medicine, University Hospital of Modena, Modena, Italy.
authors.forename: Giuliano
authors.initials: G
authors.lastname: Montagnani
authors.affiliation: Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Carlotta
authors.initials: C
authors.lastname: Spano
authors.affiliation: Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Malvina
authors.initials: M
authors.lastname: Prapa
authors.affiliation: Department of Surgery, Oncology and Gastroenterology University of Padova, Istituto Oncologico Veneto IRCCS, Padua, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Guarneri
authors.affiliation: The Research Institute, Nationwide Children's Hospital, Columbus, Ohio, United States of America.
authors.forename: Satoru
authors.initials: S
authors.lastname: Otsuru
authors.affiliation: The Research Institute, Nationwide Children's Hospital, Columbus, Ohio, United States of America.|Division of Hematology/Oncology/BMT, Nationwide Children's Hospital, Columbus, Ohio, United States of America.
authors.forename: Edwin M
authors.initials: EM
authors.lastname: Horwitz
authors.affiliation: R&D Filter Department, Fresenius HemoCare Italy, Cavezzo, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Mari
authors.affiliation: Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Dominici
mesh_terms: D001793:Blood Preservation; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D013997:Time Factors