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J Extracell Vesicles. 2014 Feb 4;3. doi: 10.3402/jev.v3.20795. eCollection 2014.

A flow cytometric method for characterization of circulating cell-derived microparticles in plasma.

Author information

1
Danish PhD School of Molecular Metabolism, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark.
2
Department of Endocrinology M, Odense University Hospital, Odense, Denmark.
3
Department of Biomedicine, Aarhus University, Aarhus, Denmark ; Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.
4
Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark.

Abstract

BACKGROUND AND AIM:

Previous studies on circulating microparticles (MPs) indicate that the majority of MPs are of a size below the detection limit of most standard flow cytometers. The objective of the present study was to establish a method to analyze MP subpopulations above the threshold of detection of a new generation BD FACSAria™ III digital flow cytometer.

METHODS:

We analyzed MP subpopulations in plasma from 24 healthy individuals (9 males and 15 females). MPs were identified according to their size (<1.0-µm), by Lactadherin-FITC labelling, and by exposure of cell-specific markers. The sensitivity of the flow cytometer was tested against that of a previous-generation instrument FC500. Reproducibility of the FACSAria and our set-up was investigated, and the percentage of phosphatidylserine (PS) exposing MPs binding Lactadherin was determined.

RESULTS:

By using a flow cytometric approach we identified and quantitated MPs derived from platelets, monocytes, erythrocytes and endothelial cells. In addition, levels of tissue factor-positive MPs were determined. The FACSAria demonstrated improved sensitivity and increased MP detection range compared to the FC500 instrument. The reproducibility of PS+PMP and PS+MP measurements was 11.7 and 23.2%, respectively. When expressed as a percentage of total MPs, the PS-positive MP population represented 15.1±5.5%, and PS-positive MPs were significantly increased in men.

CONCLUSION:

We have established a method to measure MPs above the detection limit of a new generation flow cytometer and derived from a number of cell-types in a healthy population of men and women.

KEYWORDS:

coincidence occurrence; endothelial cell-derived; erythrocyte-derived; extracellular vesicles; flow cytometry; lactadherin; monocyte-derived; platelet-derived; tissue-factor

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