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Updated: Jun 5, 2026
Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry
Published on: March 17, 2015
Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay
Lisa Ayers1, Malcolm Kohler, Paul Harrison
1Department of Clinical Immunology, Churchill Hospital, Oxford, UK. lisa.ayers@nhs.net
Introduction:
Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification.
Materials And Methods:
Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated.
Results:
Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV+MPs (P=0.002) and platelet-derived MPs (PMPs) (P=0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV+MPs (P=0.0004) and PMPs (P=0.0004). A single freeze thaw cycle of samples led to an increase in AnnV+MPs (P=0.0020) and PMPs (P=0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels.
Conclusions:
This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease.
Insights
Standardizing the measurement of circulating cell-derived microparticles (MPs) is crucial for understanding their role in disease. This study identified key pre-analytical variables that significantly impact MP quantification, highlighting the need for consistent protocols.
Area of Science:
- Biomedical research
- Flow cytometry
- Clinical diagnostics
Background:
- Circulating cell-derived microparticles (MPs) are implicated in various diseases.
- Elevated MP levels are observed in numerous pathological conditions.
- Accurate measurement of MPs is essential but hindered by a lack of standardization.
Purpose of the Study:
- Establish a reliable flow cytometric assay for measuring distinct MP subtypes.
- Identify critical variables affecting MP quantification.
- Improve standardization in MP measurement for disease research.
Main Methods:
- Phenotypic identification of circulating MPs (platelet, endothelial, leukocyte, annexin-V positive).
- Investigated effects of time from venepuncture to centrifugation.
- Assessed impact of washing steps, centrifugation number, freezing, and thawing on MP levels.
Main Results:
- Increased time to centrifugation elevated MP levels.
- Washing and double centrifugation decreased annexin-V positive (AnnV+) and platelet-derived MPs (PMPs).
- Freeze-thaw cycles increased AnnV+ and PMPs, while long-term storage decreased MP levels.
Conclusions:
- Minor protocol variations significantly impact MP quantification.
- This study is among the first to standardize circulating MP measurement.
- Standardization is vital for MP technology development and comparative research in disease states.
