title: Impact of sample processing on the measurement of circulating microparticles: storage and centrifugation parameters.
abstract: BACKGROUND
  Microparticles (MPs) have been shown to be markers of cellular activation and interactions. Pre-analytical conditions such as the centrifugation protocol and sample storage conditions represent an important source of variability in determining MPs values. The objectives of this study were to evaluate the influence of sample storage conditions and centrifugation speed and temperature on the determination of MPs in plasma.
  
  
  METHODS
  Citrate-anticoagulated blood samples obtained from 21 healthy subjects were centrifuged under four different protocols involving different speeds (2500 g or 1500 g) and temperatures (4 °C or 20 °C) to isolate platelet-poor plasma (PPP). The number of MPs in fresh and frozen-thawed PPP were analyzed by flow cytometry, and MPs-mediated procoagulant activity was determined by a thrombin generation test and phospholipid-dependent procoagulant tests.
  
  
  RESULTS
  The number of MPs and their procoagulant activity were affected by freeze-thaw cycling and centrifugation speed but not by centrifugation temperature. Sample freezing increased MPs number (six-fold) and thrombin generation (four-fold), and decreased clotting time (two-fold). Low centrifugation speed caused an increase in MPs number and a parallel increase in MP-mediated procoagulant activity.
  
  
  CONCLUSIONS
  Sample storage conditions and centrifugation speed are important processing conditions affecting MPs number and activity. Before any study, the protocol for MPs isolation should be optimized to ensure a reliable characterization of MPs, which could provide important information for diagnostic purposes and for understanding the pathogenesis of diseases.
authors.forename: Virtudes
authors.initials: V
authors.lastname: Vila-Liante
authors.forename: Verónica
authors.initials: V
authors.lastname: Sánchez-López
authors.forename: Vicenta
authors.initials: V
authors.lastname: Martínez-Sales
authors.forename: Luis A
authors.initials: LA
authors.lastname: Ramón-Nuñez
authors.forename: Elena
authors.initials: E
authors.lastname: Arellano-Orden
authors.forename: Alejandra
authors.initials: A
authors.lastname: Cano-Ruiz
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Rodríguez-Martorell
authors.forename: Lin
authors.initials: L
authors.lastname: Gao
authors.forename: Remedios
authors.initials: R
authors.lastname: Otero-Candelera
mesh_terms: D001793:Blood Preservation; D055252:Cell-Derived Microparticles / Q000737:chemistry*; D002498:Centrifugation; D005260:Female; D005434:Flow Cytometry; D064368:Healthy Volunteers; D006801:Humans; D008297:Male; D013696:Temperature