title: Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
abstract: BACKGROUND
  Analyses of circulating cell membrane-derived microvesicles (MV) have come under scrutiny as potential diagnostic and prognostic biomarkers of disease. However, methods to isolate, label and quantify MV have been neither systematized nor validated.
  
  
  OBJECTIVE
  To determine how pre-analytical, analytical and post-analytical factors affect plasma MV counts, markers for cell of origin and expression of procoagulant surface phosphatidylserine.
  
  
  RESULTS
  Peripheral venous blood samples were collected from healthy volunteers and patients with cardiovascular disease and/or diabetes. Effects of blood sample collection, anticoagulant and sample processing to platelet free plasma (PFP), and MV isolation, staining and storage (freeze-thaw) and cytometer design were evaluated with replicate samples from these populations. The key finding is that use of citrate or EDTA anticoagulants decreases or eliminates microvesicles from plasma by inducing adhesion of the microvesicles to platelets or other formed elements. Protease inhibitor anticoagulants, including heparin, preserve MV counts. A centrifugation protocol was developed in which recovery of isolated MV was high with resolution down to the equivalent light scatter of 0.2 μm latex beads. Each procedure was systematically evaluated for its impact on the MV counts and characteristics.
  
  
  CONCLUSIONS
  This study provides a systematic methodology for MV isolation, identification and quantification, essential for development of MV as diagnostic and prognostic biomarkers of disease.
authors.affiliation: Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, United States. jaya.m@mayo.edu
authors.forename: Muthuvel
authors.initials: M
authors.lastname: Jayachandran
authors.forename: Virginia M
authors.initials: VM
authors.lastname: Miller
authors.forename: John A
authors.initials: JA
authors.lastname: Heit
authors.forename: Whyte G
authors.initials: WG
authors.lastname: Owen
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000925:Anticoagulants / Q000494:pharmacology; D015415:Biomarkers / Q000097:blood / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D001800:Blood Specimen Collection / Q000379:methods; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D055252:Cell-Derived Microparticles / Q000187:drug effects / Q000378:metabolism*; D002951:Citrates / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005260:Female; D005615:Freezing; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008297:Male; D008875:Middle Aged; D010718:Phosphatidylserines / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D055815:Young Adult
chemical_list: D000925:Anticoagulants; D015415:Biomarkers; D002951:Citrates; D010718:Phosphatidylserines; D011480:Protease Inhibitors; D006493:Heparin; D004492:Edetic Acid
references.citation: Keio J Med. 2004 Dec;53(4):210-30
references.pmid: 15647627
references.citation: Semin Thromb Hemost. 2010 Nov;36(8):807-18
references.pmid: 21049381
references.citation: Platelets. 2009 May;20(3):225-6; author reply 227-8
references.pmid: 19437341
references.citation: Trends Cell Biol. 2009 Feb;19(2):43-51
references.pmid: 19144520
references.citation: Blood Rev. 2007 May;21(3):157-71
references.pmid: 17118501
references.citation: Thromb Haemost. 2001 Apr;85(4):639-46
references.pmid: 11341498
references.citation: J Thromb Haemost. 2009 Jan;7(1):190-7
references.pmid: 18983485
references.citation: J Thromb Haemost. 2004 Oct;2(10):1842-51
references.pmid: 15456497
references.citation: Thromb Res. 2006;117(6):705-11
references.pmid: 15990159
references.citation: Nanomedicine. 2011 Dec;7(6):780-8
references.pmid: 21601655
references.citation: Semin Thromb Hemost. 2007 Nov;33(8):771-9
references.pmid: 18175282
references.citation: Anticancer Res. 2008 Mar-Apr;28(2A):1107-12
references.pmid: 18507061
references.citation: J Lab Clin Med. 2000 Apr;135(4):339-46
references.pmid: 10779050
references.citation: Clin Cancer Res. 2009 Nov 15;15(22):6830-40
references.pmid: 19861441
references.citation: Thromb Res. 2010 Apr;125(4):332-9
references.pmid: 20117824
references.citation: Blood. 1997 Feb 15;89(4):1121-32
references.pmid: 9028933
references.citation: Platelets. 2008 Aug;19(5):365-72
references.pmid: 18791943
references.citation: Vasc Health Risk Manag. 2010 Dec 06;6:1125-33
references.pmid: 21191433
references.citation: Br J Haematol. 2007 Apr;137(1):36-48
references.pmid: 17359370
references.citation: Vasc Health Risk Manag. 2008;4(4):769-74
references.pmid: 19065994
references.citation: J Thromb Haemost. 2010 Feb;8(2):315-23
references.pmid: 19840362
references.citation: Thromb Haemost. 2008 Oct;100(4):663-9
references.pmid: 18841290
references.citation: Thromb Res. 2011 Jan;127(1):39-46
references.pmid: 21106230
references.citation: Thromb Haemost. 2011 Mar;105(3):396-408
references.pmid: 21174005
references.citation: Blood. 2003 Oct 1;102(7):2678-83
references.pmid: 12805058
references.citation: Arterioscler Thromb Vasc Biol. 2006 Dec;26(12):2594-604
references.pmid: 16990554
references.citation: J Thromb Haemost. 2011 Jun;9(6):1216-24
references.pmid: 21481178
references.citation: Blood Coagul Fibrinolysis. 2002 Jul;13(5):393-7
references.pmid: 12138366
references.citation: Climacteric. 2009 Apr;12(2):177-84
references.pmid: 19051075
references.citation: Water Res. 2008 Aug;42(14):3757-66
references.pmid: 18662824
references.citation: Am J Physiol Heart Circ Physiol. 2008 Sep;295(3):H931-H938
references.pmid: 18621859
references.citation: Cardiovasc Res. 2003 Aug 1;59(2):277-87
references.pmid: 12909311
references.citation: Circulation. 2000 Feb 29;101(8):841-3
references.pmid: 10694520
references.citation: Thromb Res. 2011 Apr;127(4):370-7
references.pmid: 21257195
references.citation: Crit Rev Oncol Hematol. 2007 May;62(2):126-36
references.pmid: 17293122