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Updated: Apr 18, 2026
Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery
Published on: October 4, 2018
Detection and quantification of microparticles from different cellular lineages using flow cytometry. Evaluation of
Matthieu Rousseau1, Clemence Belleannee2, Anne-Claire Duchez1
1Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Faculté de Médecine de l'Université Laval, Québec, QC, Canada.
Abstract:
Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.
Insights
Secreted phospholipases A2 (sPLA2) can interfere with microparticle quantification in biological fluids. This study highlights the impact of sPLA2 enzymes on detecting microparticles, crucial for biomarker development.
Area of Science:
- Cell Biology
- Biochemistry
- Immunology
Background:
- Microparticles (MPs) are extracellular vesicles released from cell membranes, acting as biomarkers in various physiological and pathological conditions.
- Quantifying MPs in biological fluids is challenging due to their small size and interfering soluble factors.
- Secreted phospholipases A2 (sPLA2) are enzymes found in biological fluids that hydrolyze membrane phospholipids, including phosphatidylserine, a common MP marker.
Purpose of the Study:
- To investigate the impact of specific sPLA2 enzymes (groups IIA, V, and X) on the quantification of microparticles.
- To determine how sPLA2 enzymes affect microparticle detection using flow cytometry, considering different species, sources, and detection methods.
- To provide analytical considerations for accurate microparticle measurement in samples containing sPLA2.
Main Methods:
- Utilized flow cytometry with enhanced resolution for microparticle detection.
- Generated microparticles from various human and mouse cell sources (platelets, red blood cells, endothelial cells, thymocytes, male reproductive tract cells).
- Assessed the effect of sPLA2 enzymes on microparticle quantification based on phosphatidylserine exposure and protein antigen detection.
Main Results:
- Certain sPLA2 enzymes, when enriched in biological fluids, significantly impair microparticle quantification.
- The extent of impairment varies depending on the species, the source of microparticles, and the detection method used (e.g., phosphatidylserine vs. protein markers).
- sPLA2 enzymes can interfere with both surface phosphatidylserine-based and protein antigen-based microparticle detection.
Conclusions:
- The presence of sPLA2 enzymes in biological samples necessitates careful consideration during microparticle quantification.
- Analytical methods for microparticle analysis should account for potential interference from sPLA2 to ensure accurate results.
- Understanding sPLA2's impact is crucial for reliable use of microparticles as biomarkers in clinical and research settings.
