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

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.© 2013 Elsevier Ltd. All rights reserved. IntroductionExtracellular vesicles (EVs) are membrane surrounded structures of various sizes (30-5000 nm) that have received significant attention recently [1]. EVs may be classified on the basis of their biogenesis, diameter and membrane markers. The two best characterized types of EVs include exosomes of endosomal origin and plasma membrane-derived microparticles (MPs) (recently often referred to also as microvesicles or ectosomes in the literature) [1]. EVs are present in all biological fluids including blood plasma, synovial fluid, cerebrospinal fluid, urine, tears and breast milk [1]. MPs are in between 100 and 1000 nm in diameter, and they are also detectable by flow cytometry [2,3]. Thus, MP profiles are easily analyzed in the routine clinical laboratory practice, and represent novel biomarkers of various diseases. In circulation, most MPs are derived from platelets, red blood cells, endothelial cells and leukocytes. Because of their abundance, platelet-derived MPs (PMPs) received the highest attention during the past few years. Elevated PMP counts are characteristic for nearly all autoimmune disorders [4,5], and also for several cardiovascular and metabolic diseases [1]. EVs not only contain proteins but also RNA molecules [1]. Extracellular RNA (exRNA) in blood plasma is encapsulated in EVs, or bound to either proteins or HDL molecules [6]. exRNAs, particularly miRNAs, are specific and sensitive biomarkers...

Measurement of microvesicle levels in human blood using flow cytometry

“…Storage of red cell concentrates lead to an exponential increase in red cell microvesicles, with a 20-fold increase from 6 to 50 days of storage (17). Vigorous agitation for 1 2 2 h lead to 200 2 900% increases in platelet and/or annexin V1 microvesicles (11,14). Transportation of sample tubes horizontally lead to higher platelet microvesicle counts than transportation of sample tubes in a vertical position.…”

“…This has been accomplished using electron microscopy by observing the lipid membrane structure of the microvesicles or using lipid binding dyes such as annexin V, lactadherin, and PKH67. An alternate approach is to use a less specific method to count microvesicles and then to treat the sample with a detergent that disrupts membranes followed by repeat measurement to show what fraction of the count was due to lipid vesicles (11,35). One difficulty with use of detergents is they generate a large number of small membrane fragments.…”

Microparticles in cancer: A review of recent developments and the potential for clinical application

“…Furthermore, an ongoing Phase II trial which assesses the efficacy of dendritic cell derived exosomes as autologous therapeutic vaccines in advanced non-small cell lung carcinoma (NSCLC) patients to stimulate their natural defences to prevent tumor progression [71]. The preclinical [72] and clinical data [72] …”

Therapeutic and diagnostic applications of extracellular vesicles

“…Besides the purification protocol, also other parameters, among which the sample collection procedure (e.g. the type of anticoagulant during blood collection [250]), processing time and sample store conditions [65,86] can influence the outcome of biomarker identification studies [251]. In response to this unmet need, an ISEV position paper was issued describing guidelines on how to handle different biological fluid samples and emphasizing the importance of a comprehensive experimental description to enhance reproducibility [252].…”