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Isotype controls in phenotyping and quantification of microparticles: a major source of error and how to evade it
Arne Trummer1, Christiane De Rop, Andreas Tiede
1Department of Hematology, Hemostasis and Oncology, Hannover Medical School, Hannover, Germany. trummer.arne@mh-hannover.de
Background:
The characterisation and quantification of cell-derived microparticles (MPs) using flow cytometry are often complicated by a low staining intensity and a non-discrete signal pattern of many cell surface antigens. Fluorescence-labelled isotype controls (ICs) are commonly used to set limits for the discrimination of antigen positive vs. negative events.
Objectives:
The influence of different ICs on the characterisation and quantification of MPs was studied. Antigen negative MPs stained with an antibody of interest were evaluated as an alternative control.
Methods:
MPs were prepared from platelets, endothelial cell lines and leucemic cell lines and stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labelled antibodies or isotype controls. Results are given as the mean fluorescence intensity (MFI) or percentage of "false-positive" events above a fluorescence intensity > 1.
Results:
Using identical instrument settings, seven different ICs (FITC-conjugates N = 3, PE-conjugates N = 4) resulted in a wide range of MFI and percentage of positive events with a mean coefficient of variation (CV) of 0.77. Instead, NMPs showed less variability with a mean CV of 0.50 and allowed a reliable and reproducible quantification of MPs when set as controls with < 2% false-positive events above an FI > 1. As a result, the expression of certain antigens (e.g. CD62P) was lower compared to previous reports in the literature.
Conclusions:
Diversity in the staining intensity of isotype controls is a potential source of error in the characterisation and quantification of MPs by flow cytometry. The use of antigen negative MPs to adjust instrument settings is suggested.
Insights
Isotype controls introduce variability in microparticle (MP) quantification via flow cytometry. Using antigen-negative MPs as controls offers a more reliable method for accurate MP characterization and analysis.
Area of Science:
- Biotechnology
- Immunology
- Cell Biology
Background:
- Flow cytometry analysis of cell-derived microparticles (MPs) faces challenges due to low antigen staining intensity.
- Fluorescence-labeled isotype controls (ICs) are standard for distinguishing antigen-positive from negative events.
Purpose of the Study:
- To investigate the impact of various isotype controls on MP characterization and quantification.
- To assess antigen-negative MPs as a potential alternative control for flow cytometry.
Main Methods:
- Microparticles were generated from platelets and cell lines.
- Samples were stained with FITC or PE-labeled antibodies and isotype controls.
- Results were quantified using mean fluorescence intensity (MFI) and percentage of false-positive events.
Main Results:
- Seven different isotype controls exhibited significant variability (CV of 0.77) in MFI and positive event percentages.
- Antigen-negative MPs demonstrated lower variability (CV of 0.50), enabling reproducible quantification (<2% false positives).
- This led to lower reported expression levels for certain antigens like CD62P.
Conclusions:
- Variations in isotype control staining intensity can be a significant error source in MP flow cytometry.
- Employing antigen-negative MPs for instrument setting adjustment is recommended for improved accuracy.
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