title: Comparison of established and novel methods for the detection and enumeration of microparticles in canine stored erythrocyte concentrates for transfusion.
abstract: BACKGROUND
  Microparticles (MP) are submicron, phosphatidylserine (PS)-bearing lipid vesicles with physiologic and pathologic roles in coagulation and inflammation. Microparticles accumulate in packed RBC (pRBC) stored for transfusion, potentially increasing recipient morbidity. Historically, canine MP have been detected with the PS label annexin V in supernatant samples. Other detection methods are available but have not been evaluated in dogs.
  
  
  OBJECTIVE
  The purpose of the study was to detect and enumerate MP in canine pRBC using annexin V, lactadherin, and bio-maleimide to compare label performance and assess microparticle accumulation under standard storage conditions.
  
  
  METHODS
  Microparticles (0.5-1.0 μm) in canine dog erythrocyte antigen 1.1 positive, nonleukoreduced pRBC were labeled with FITC-annexin V, FITC-lactadherin, and the fluorescent dye bio-maleimide, and were counted using flow cytometry at 3 time points (days 7, 21, and 35) of storage. Unprocessed pRBC, rather than supernatant, were used.
  
  
  RESULTS
  Annexin V and bio-maleimide labeling produced comparable microparticle counts (P = .16), while lactadherin labeling resulted in higher microparticle counts than annexin V (P = .002) and bio-maleimide (P = .006), particularly on day 7. Bio-maleimide- and annexin V-based microparticle counts increased significantly from day 7 to 35 (P = .04), and increases from day 21 to 35 approached statistical significance (P = .05).
  
  
  CONCLUSIONS
  Bio-maleimide- and annexin V-mediated microparticle counts were comparable in unprocessed canine pRBC using flow cytometry. Whether the increased microparticle counts with lactadherin were due to increased sensitivity for small, PS-bearing MP or due to labeling of membrane fragments and debris requires further investigation.
authors.affiliation: Veterinary Clinical Sciences, Washington State University College of Veterinary Medicine, Pullman, WA, USA.
authors.forename: Meredeth C
authors.initials: MC
authors.lastname: McEntire
authors.affiliation: Veterinary Clinical Sciences, Washington State University College of Veterinary Medicine, Pullman, WA, USA.
authors.forename: K Jane
authors.initials: KJ
authors.lastname: Wardrop
authors.affiliation: Veterinary Microbiology and Pathology, Washington State University College of Veterinary Medicine, Pullman, WA, USA.
authors.forename: William C
authors.initials: WC
authors.lastname: Davis
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000009:adverse effects*; D001777:Blood Coagulation; D001792:Blood Platelets / Q000166:cytology; D001793:Blood Preservation / Q000379:methods / Q000662:veterinary*; D001896:Boron Compounds / Q000009:adverse effects*; D055252:Cell-Derived Microparticles / Q000187:drug effects*; D004285:Dogs / Q000097:blood*; D017707:Erythrocyte Transfusion / Q000662:veterinary; D004912:Erythrocytes / Q000166:cytology; D005434:Flow Cytometry / Q000662:veterinary; D008301:Maleimides / Q000009:adverse effects*; D010718:Phosphatidylserines / Q000097:blood
chemical_list: D017304:Annexin A5; D001896:Boron Compounds; D008301:Maleimides; D010718:Phosphatidylserines; C530427:bodipy FL N-(2-aminoethyl)maleimide
keywords: Annexin; erythrocyte concentrate; lactadherin; storage lesion; transfusion