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Thromb Res. 2014 Feb;133(2):285-92. doi: 10.1016/j.thromres.2013.11.010. Epub 2013 Nov 25.

Improved circulating microparticle analysis in acid-citrate dextrose (ACD) anticoagulant tube.

Author information

1
Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary. Electronic address: gyorgyben@gmail.com.
2
Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary.
3
Semmelweis University, Department of Laboratory Medicine, Budapest, Hungary.
4
Military Hospital, National Health Institute, Department of Obstetrics and Gynecology, Budapest, Hungary.
5
Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary. Electronic address: edit.buzas@gmail.com.

Abstract

INTRODUCTION:

Recently extracellular vesicles (exosomes, microparticles also referred to as microvesicles and apoptotic bodies) have attracted substantial interest as potential biomarkers and therapeutic vehicles. However, analysis of microparticles in biological fluids is confounded by many factors such as the activation of cells in the blood collection tube that leads to in vitro vesiculation. In this study we aimed at identifying an anticoagulant that prevents in vitro vesiculation in blood plasma samples.

MATERIALS AND METHODS:

We compared the levels of platelet microparticles and non-platelet-derived microparticles in platelet-free plasma samples of healthy donors. Platelet-free plasma samples were isolated using different anticoagulant tubes, and were analyzed by flow cytometry and Zymuphen assay. The extent of in vitro vesiculation was compared in citrate and acid-citrate-dextrose (ACD) tubes.

RESULTS:

Agitation and storage of blood samples at 37 °C for 1 hour induced a strong release of both platelet microparticles and non-platelet-derived microparticles. Strikingly, in vitro vesiculation related to blood sample handling and storage was prevented in samples in ACD tubes. Importantly, microparticle levels elevated in vivo remained detectable in ACD tubes.

CONCLUSIONS:

We propose the general use of the ACD tube instead of other conventional anticoagulant tubes for the assessment of plasma microparticles since it gives a more realistic picture of the in vivo levels of circulating microparticles and does not interfere with downstream protein or RNA analyses.

KEYWORDS:

ACD; AX; CPDA; CTAD; EDTA; EV; Extracellular vesicle; FITC; Flow cytometry; ISTH; International Society on Thrombosis and Haemostasis; MP; Microparticles; Microvesicles; PCR; PFP; PMP; PS; PerCP; RMS; RT; acid ‐ citrate ‐ dextrose; annexin V; citrate - theophylline - adenosine - dipyridamole; citrate- phosphate -dextrose - adenin; ethylenediaminetetraacetate; extracellular vesicles; fluorescein isothiocyanate; microparticles; peridinin chlorophyll protein; phosphatidylserine; platelet free plasm; platelet microparticle; polymerase chain reaction; room temperature; root mean square

PMID:
24360116
DOI:
10.1016/j.thromres.2013.11.010
[Indexed for MEDLINE]

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