title: Techniques to improve detection and analysis of extracellular vesicles using flow cytometry.
abstract: Extracellular vesicles (EVs) range in size from 50 nm to 1 µm. Flow cytometry (FCM) is the most commonly used method for analyzing EVs; however, accurate characterization of EVs remains challenging due to their small size and lack of discrete positive populations. Here we report the use of optimization techniques that are especially well-suited for analyzing EVs from a high volume of clinical samples. Utilizing a two pronged approach that included 1) pre-filtration of antibodies to remove aggregates, followed by 2) detergent lysis of a replicate sample to account for remaining false positive events, we were able to effectively limit false positive non-EV events. In addition, we show that lysed samples are a useful alternative to isotypes for setting gates to exclude background fluorescence. To reduce background, we developed an approach using filters to "wash" samples post-staining thus providing a faster alternative to ultracentrifugation and sucrose gradient fractionation. In conclusion, use of these optimized techniques enhances the accuracy and efficiency of EV detection using FCM.
authors.affiliation: Blood Systems Research Institute, San Francisco, California.
authors.forename: Heather C
authors.initials: HC
authors.lastname: Inglis
authors.affiliation: Blood Systems Research Institute, San Francisco, California.|Department of Laboratory Medicine, University of California, San Francisco, Califoria.
authors.forename: Ali
authors.initials: A
authors.lastname: Danesh
authors.affiliation: Blood Systems Research Institute, San Francisco, California.
authors.forename: Avani
authors.initials: A
authors.lastname: Shah
authors.affiliation: Division of Critical Care, Department of Pediatrics, University of Montreal, Quebec, Canada.
authors.forename: Jacques
authors.initials: J
authors.lastname: Lacroix
authors.affiliation: Division of Critical Care, Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Philip C
authors.initials: PC
authors.lastname: Spinella
authors.affiliation: Blood Systems Research Institute, San Francisco, California.|Department of Laboratory Medicine, University of California, San Francisco, Califoria.|Department of Medicine, University of California, San Francisco, California.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Norris
mesh_terms: D000906:Antibodies / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology; D000067128:Extracellular Vesicles; D005374:Filtration / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000906:Antibodies; D015703:Antigens, CD
keywords: antibody aggregates; extracellular vesicles; filtration; flow cytometry; microparticles
references.citation: Stem Cells Dev. 2013 Mar 1;22(5):758-71
references.pmid: 23034046
references.citation: Arch Pathol Lab Med. 2010 May;134(5):771-5
references.pmid: 20441510
references.citation: Thromb Res. 2008;122(5):604-9
references.pmid: 18262600
references.citation: Circ Res. 2012 Jan 20;110(2):356-69
references.pmid: 22267840
references.citation: J Cell Biol. 2013 Feb 18;200(4):373-83
references.pmid: 23420871
references.citation: Thromb Res. 2010 Mar;125(3):e110-6
references.pmid: 19939440
references.citation: Thromb Haemost. 2010 May;103(5):1044-52
references.pmid: 20390225
references.citation: Neth J Med. 2009 Jul-Aug;67(7):266-73
references.pmid: 19687520
references.citation: Nat Rev Drug Discov. 2013 May;12(5):347-57
references.pmid: 23584393
references.citation: Cancer Res. 2007 Apr 1;67(7):2912-5
references.pmid: 17409393
references.citation: Nanomedicine. 2012 Jul;8(5):712-20
references.pmid: 22024193
references.citation: Semin Thromb Hemost. 2010 Nov;36(8):807-18
references.pmid: 21049381
references.citation: Clin Exp Metastasis. 1999 Mar;17(2):131-40
references.pmid: 10411105
references.citation: PLoS One. 2012;7(10):e47365
references.pmid: 23077602
references.citation: J Thromb Haemost. 2012 May;10(5):919-30
references.pmid: 22394434
references.citation: J Immunol Methods. 2012 Jan 31;375(1-2):207-14
references.pmid: 22075275
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: Cytometry A. 2010 Jun;77(6):502-14
references.pmid: 20235276
references.citation: Thromb Haemost. 2012 Aug;108(2):373-83
references.pmid: 22740162
references.citation: Trends Cell Biol. 2009 Feb;19(2):43-51
references.pmid: 19144520
references.citation: Clin Cancer Res. 2005 Feb 1;11(3):1010-20
references.pmid: 15709166
references.citation: Membranes (Basel). 2011 Dec 27;2(1):1-15
references.pmid: 24957960
references.citation: J Proteome Res. 2009 Jul;8(7):3346-54
references.pmid: 19413345
references.citation: J Exp Med. 2002 May 20;195(10):1303-16
references.pmid: 12021310
references.citation: Semin Thromb Hemost. 2010 Nov;36(8):888-906
references.pmid: 21049390
references.citation: Vasc Health Risk Manag. 2008;4(4):769-74
references.pmid: 19065994
references.citation: Blood. 2012 Mar 1;119(9):2174-5; author reply 2175-6
references.pmid: 22383792
references.citation: PLoS One. 2012;7(11):e49726
references.pmid: 23185418
references.citation: Cancer Immunol Immunother. 2010 Jun;59(6):841-50
references.pmid: 20043223
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: Nephrol Dial Transplant. 2011 May;26(5):1474-83
references.pmid: 21324974
references.citation: Vox Sang. 2009 Apr;96(3):206-12
references.pmid: 19175566
references.citation: Cancer Res. 2011 Aug 1;71(15):5346-56
references.pmid: 21670082
references.citation: Methods Mol Biol. 2008;484:79-93
references.pmid: 18592174
references.citation: Mol Ther. 2010 Sep;18(9):1606-14
references.pmid: 20571541
references.citation: Subcell Biochem. 2007;43:21-34
references.pmid: 17953389
references.citation: J Thromb Haemost. 2010 Dec;8(12):2596-607
references.pmid: 20880256
references.citation: Biol Chem. 2013 Oct;394(10):1253-62
references.pmid: 23770532
references.citation: Cytometry A. 2011 Dec;79(12):990-9
references.pmid: 21990118
references.citation: Thromb Haemost. 2007 Jan;97(1):67-80
references.pmid: 17200773
references.citation: PLoS One. 2012;7(3):e33115
references.pmid: 22431999
references.citation: Am J Pathol. 2013 May;182(5):1489-93
references.pmid: 23499459
references.citation: Anticancer Res. 1998 Sep-Oct;18(5A):3433-7
references.pmid: 9858920
references.citation: BMC Cell Biol. 2013 Apr 22;14:23
references.pmid: 23607880
references.citation: Blood. 2014 Jan 30;123(5):687-96
references.pmid: 24335232
references.citation: Front Physiol. 2012 Sep 07;3:354
references.pmid: 22973237
references.citation: Pharmacol Rev. 2012 Jul;64(3):676-705
references.pmid: 22722893
references.citation: Kidney Int. 2010 Oct;78(8):810-6
references.pmid: 20686450
references.citation: Int J Immunopathol Pharmacol. 2012 Jan-Mar;25(1):75-85
references.pmid: 22507320
references.citation: Nat Rev Immunol. 2009 Aug;9(8):581-93
references.pmid: 19498381
references.citation: Transfusion. 2013 Jan;53(1):156-66
references.pmid: 22624624
references.citation: Thromb Res. 2008;122(5):691-700
references.pmid: 18304614
references.citation: Cell Death Dis. 2011 Sep 29;2:e211
references.pmid: 21956548
references.citation: Arterioscler Thromb Vasc Biol. 2011 Jan;31(1):27-33
references.pmid: 21160065
references.citation: Cancer Res. 2004 Oct 1;64(19):7045-9
references.pmid: 15466198
references.citation: Cancer Res. 2011 Jun 1;71(11):3792-801
references.pmid: 21478294
references.citation: J Extracell Vesicles. 2013 May 27;2:
references.pmid: 24009894
references.citation: Blood. 2011 Jan 27;117(4):e39-48
references.pmid: 21041717
references.citation: Eur Heart J. 2011 Aug;32(16):2034-41
references.pmid: 21186238
references.citation: J Thromb Haemost. 2013 Apr 2;:
references.pmid: 23551930
references.citation: Thromb Res. 2011 Apr;127(4):370-7
references.pmid: 21257195
references.citation: Cell Mol Life Sci. 2011 Aug;68(16):2667-88
references.pmid: 21560073
references.citation: J Proteomics. 2010 Sep 10;73(10):1907-20
references.pmid: 20601276
references.citation: Transfusion. 2011 Apr;51(4):894-900
references.pmid: 21496052
references.citation: Transfus Med Rev. 2011 Jul;25(3):197-205
references.pmid: 21550205
references.citation: Nat Protoc. 2012 Jun 14;7(7):1311-26
references.pmid: 22722367
references.citation: Gastroenterology. 2005 Jun;128(7):1796-804
references.pmid: 15940614
references.citation: Clin Cancer Res. 2003 Nov 1;9(14):5113-9
references.pmid: 14613988