Abstract
Microparticles (MP) are small (100-1000 nm) membrane vesicles shed by cells as a response to activation, stress or apoptosis. Platelet-derived MP (PMP) has been shown to reflect the pathophysiological processes of a range of cardiovascular diseases and there is a potential clinical value in using PMPs as biomarkers, as well as a need to better understand the biology of these vesicles. The current method for isolating MP depends on differential centrifugation steps, which require relatively large sample volumes and have been shown to compromise the integrity and composition of the MP population. We present a novel method for rapid, non-contact capture of PMP in minute sample volumes based on a microscale acoustic standing wave technology. Capture of PMPs from plasma is shown by scanning electron microscopy and flow cytometry. Furthermore, the system is characterized with regards to plasma sample concentration and flow rate. Finally, the technique is compared to a standard differential centrifugation protocol using samples from both healthy controls and ST-elevation myocardial infarction (STEMI) patient samples. The acoustic system is shown to offer a quick and automated setup for extracting microparticles from small sample volumes with higher recovery than a standard differential centrifugation protocol.
Highlights
As a proof-of-principle of the technique and to visualize the trapping of platelet-derived MPs in the cluster of seed particles, cell-free plasma was stained with CD42a-PE and injected into the system under a fluorescence microscope
To demonstrate that the captured material was Platelet-derived microparticles (PMP), the experiment was repeated with unstained plasma, the cluster was released in phosphate buffered saline (PBS), stained and analyzed by flow cytometry
Analysis of the cluster after trapping with a scanning electron microscope (SEM) clearly showed the 12 μm seed particles surrounded by an aggregate of microparticle-sized objects, with a size range of 300 nm–1 μm (Fig. 2C)
Summary
Microparticles (MPs, known as microvesicles) are a class of extracellular vesicles released from various cell types upon activation, stress or apoptosis.[1,2,3] These 100–1000 nm membrane vesicles are present in the circulation of healthy individuals but their levels are altered as a consequence of various pathophysiological processes.[4,5,6,7] The biological role of MPs is not fully understood, but recent evidence suggests that they are key mediators of cell–cell communication, transferring mRNA, microRNA and proteins between cells,[8,9,10] regulating processes such as antigen presentation,[11] inflammation,[12] and hemostasis.[13]One concern regarding the analysis of MPs in general is the lack of a consensus protocol for sample preparation.[28,29] As demonstrated by Lacroix et al, pre-analytical parameters such as centrifugation speed, agitation of tubes or a delay in sample preparation can have a considerable effect on the outcome of the analysis.[29]. We demonstrate that by using large seed particles that can be retained against flow, acoustic trapping is capable of capturing microparticles on the seed particle cluster from plasma.
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