Immuno-analysis of microparticles: probing at the limits of detection
Sharissa L Latham1,2, Natalia Tiberti1, Naveena Gokoolparsadh3
1Vascular Immunology Unit, Sydney Medical School, The University of Sydney, NSW, Australia.
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.
Insights
High-sensitivity flow cytometry (FCM) has limitations in detecting small microparticles (MPs) with low antigen levels, even with improved resolution. Probe selection and data interpretation are crucial for accurate MP analysis.
Area of Science:
- Biomedical Engineering
- Cell Biology
- Analytical Chemistry
Background:
- Flow cytometry (FCM) accuracy for microparticle (MP) analysis is debated due to pre-analytical variables and instrument limitations.
- Immuno-electron microscopy (Immuno-EM) offers a comparative standard for MP characterization.
Purpose of the Study:
- To assess the strengths and limitations of probe selection and high-sensitivity FCM for MP analysis.
- To compare FCM findings with Immuno-EM using a simplified in vitro MP generation system.
Main Methods:
- Utilized a simplified in vitro system for generating microparticles (MPs).
- Performed comparative analysis between high-sensitivity flow cytometry (FCM) and immuno-electron microscopy (Immuno-EM).
- Examined phosphatidylserine ligands (annexin V, lactadherin), CD105, and CD54 expression on MPs.
Main Results:
- Phosphatidylserine ligands (annexin V, lactadherin) provided the most specific MP labeling, detecting ~60% of MPs.
- CD105 and CD54 expression accounted for only 30-40% of MPs, with CD54 enhanced after TNF activation.
- Greatest discrepancies between FCM and Immuno-EM were observed for surface antigen-only labeled populations.
Conclusions:
- High-sensitivity FCM struggles to detect small MPs expressing low antigen levels, despite resolution improvements.
- Careful selection of endothelial MP probes and interpretation of data are essential for accurate analysis.
- Annexin V binding is influenced by TNF activation, highlighting context-specific probe interactions.
