title: Improved circulating microparticle analysis in acid-citrate dextrose (ACD) anticoagulant tube.
abstract: BACKGROUND
  Recently extracellular vesicles (exosomes, microparticles also referred to as microvesicles and apoptotic bodies) have attracted substantial interest as potential biomarkers and therapeutic vehicles. However, analysis of microparticles in biological fluids is confounded by many factors such as the activation of cells in the blood collection tube that leads to in vitro vesiculation. In this study we aimed at identifying an anticoagulant that prevents in vitro vesiculation in blood plasma samples.
  
  
  METHODS
  We compared the levels of platelet microparticles and non-platelet-derived microparticles in platelet-free plasma samples of healthy donors. Platelet-free plasma samples were isolated using different anticoagulant tubes, and were analyzed by flow cytometry and Zymuphen assay. The extent of in vitro vesiculation was compared in citrate and acid-citrate-dextrose (ACD) tubes.
  
  
  RESULTS
  Agitation and storage of blood samples at 37 °C for 1 hour induced a strong release of both platelet microparticles and non-platelet-derived microparticles. Strikingly, in vitro vesiculation related to blood sample handling and storage was prevented in samples in ACD tubes. Importantly, microparticle levels elevated in vivo remained detectable in ACD tubes.
  
  
  CONCLUSIONS
  We propose the general use of the ACD tube instead of other conventional anticoagulant tubes for the assessment of plasma microparticles since it gives a more realistic picture of the in vivo levels of circulating microparticles and does not interfere with downstream protein or RNA analyses.
authors.affiliation: Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary. Electronic address: gyorgyben@gmail.com.
authors.forename: Bence
authors.initials: B
authors.lastname: György
authors.affiliation: Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary.
authors.forename: Krisztina
authors.initials: K
authors.lastname: Pálóczi
authors.affiliation: Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Kovács
authors.affiliation: Semmelweis University, Department of Laboratory Medicine, Budapest, Hungary.
authors.forename: Eszter
authors.initials: E
authors.lastname: Barabás
authors.affiliation: Semmelweis University, Department of Laboratory Medicine, Budapest, Hungary.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Bekő
authors.affiliation: Semmelweis University, Department of Laboratory Medicine, Budapest, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Várnai
authors.affiliation: Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary.
authors.forename: Éva
authors.initials: É
authors.lastname: Pállinger
authors.affiliation: Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Szabó-Taylor
authors.affiliation: Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary.
authors.forename: Tamás G
authors.initials: TG
authors.lastname: Szabó
authors.affiliation: Military Hospital, National Health Institute, Department of Obstetrics and Gynecology, Budapest, Hungary.
authors.forename: Attila A
authors.initials: AA
authors.lastname: Kiss
authors.affiliation: Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Falus
authors.affiliation: Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary. Electronic address: edit.buzas@gmail.com.
authors.forename: Edit I
authors.initials: EI
authors.lastname: Buzás
mesh_terms: D000293:Adolescent; D000328:Adult; D000925:Anticoagulants / Q000378:metabolism*; D001792:Blood Platelets / Q000166:cytology* / Q000187:drug effects; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D019343:Citric Acid / Q000378:metabolism*; D055354:Exosomes / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005947:Glucose / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D055815:Young Adult
chemical_list: D000925:Anticoagulants; C002113:acid citrate dextrose; D019343:Citric Acid; D005947:Glucose
keywords: ACD; AX; CPDA; CTAD; EDTA; EV; Extracellular vesicle; FITC; Flow cytometry; ISTH; International Society on Thrombosis and Haemostasis; MP; Microparticles; Microvesicles; PCR; PFP; PMP; PS; PerCP; RMS; RT; acid ‐ citrate ‐ dextrose; annexin V; citrate - theophylline - adenosine - dipyridamole; citrate- phosphate -dextrose - adenin; ethylenediaminetetraacetate; extracellular vesicles; fluorescein isothiocyanate; microparticles; peridinin chlorophyll protein; phosphatidylserine; platelet free plasm; platelet microparticle; polymerase chain reaction; room temperature; root mean square