Immuno-analysis of microparticles: Probing at the limits of detection

Latham, SL; Tiberti, N; Gokoolparsadh, N; Holdaway, K; Couraud, PO; Grau, GER; Combes, V

Immuno-analysis of microparticles: Probing at the limits of detection

Latham, SL Tiberti, N

Gokoolparsadh, N Holdaway, K Couraud, PO Grau, GER Combes, V

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Publication Type:: Journal Article
Citation:: Scientific Reports, 2015, 5
Issue Date:: 2015-11-10

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Field	Value	Language
dc.contributor.author	Latham, SL	en_US
dc.contributor.author	Tiberti, N https://orcid.org/0000-0002-2981-5233	en_US
dc.contributor.author	Gokoolparsadh, N	en_US
dc.contributor.author	Holdaway, K	en_US
dc.contributor.author	Couraud, PO	en_US
dc.contributor.author	Grau, GER	en_US
dc.contributor.author	Combes, V https://orcid.org/0000-0003-2178-3596	en_US
dc.date.available	2015-10-07	en_US
dc.date.issued	2015-11-10	en_US
dc.identifier.citation	Scientific Reports, 2015, 5	en_US
dc.identifier.uri	http://hdl.handle.net/10453/41497
dc.description.abstract	Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic in vitro system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ∼60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30-40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data.	en_US
dc.relation.ispartof	Scientific Reports	en_US
dc.relation.isbasedon	10.1038/srep16314	en_US
dc.subject.mesh	Cell Line	en_US
dc.subject.mesh	Humans	en_US
dc.subject.mesh	Gold	en_US
dc.subject.mesh	Phosphatidylserines	en_US
dc.subject.mesh	Annexin A5	en_US
dc.subject.mesh	Milk Proteins	en_US
dc.subject.mesh	Intercellular Adhesion Molecule-1	en_US
dc.subject.mesh	Receptors, Cell Surface	en_US
dc.subject.mesh	Antigens, CD	en_US
dc.subject.mesh	Antigens, Surface	en_US
dc.subject.mesh	Microscopy, Immunoelectron	en_US
dc.subject.mesh	Flow Cytometry	en_US
dc.subject.mesh	Cell-Derived Microparticles	en_US
dc.subject.mesh	Limit of Detection	en_US
dc.subject.mesh	Endoglin	en_US
dc.title	Immuno-analysis of microparticles: Probing at the limits of detection	en_US
dc.type	Journal Article
utslib.citation.volume	5	en_US
utslib.for	0605 Microbiology	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Life Sciences
utslib.copyright.status	open_access
pubs.publication-status	Published	en_US
pubs.volume	5	en_US

Abstract:

Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic in vitro system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ∼60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30-40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data.

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http://hdl.handle.net/10453/41497