Isotype controls in phenotyping and quantification of microparticles: a major source of error and how to evade it.

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.