Standardized Isolation and Identification of Microparticles

Abstract and Introduction

Abstract

Microparticles (MPs) are small plasma membrane-derived vesicles that can expose molecules originating from their parental cells. As vectors of biological information they are likely to play an active role in both homeostasis and pathogenesis, making them promising biomarkers and nanomedicine tools. Therefore, there is an urgent need for standardization of MP isolation and analysis protocols to propel our understanding of MP biology to the next level. Based on current methodology and recent insights, this review proposes an optimized protocol for the isolation and biochemical characterization of MPs.

Introduction

Microparticles (MPs) constitute a promising alternative microparticulated drug delivery system, with significant advantages over liposomes and nanoparticles.^[1,2] Since MPs are an integral part of current blood transfusion products, their biocompatibility poses a relatively small challenge compared with other delivery vehicles. In particular, erythrocyte transfusion units provide a reliable natural source of large quantities of erythrocyte-derived MPs, often termed red cell MPs (RMPs). Their negative surface charge and rapid clearance by the reticulo–endothelial system may be used for efficient targeting of resident and circulating macrophages as well as the vascular endothelium.^[3] This makes them promising agents for use in nanomedicine for the treatment of vascular diseases, as immunosuppressors in inflammatory and autoimmune diseases,^[4,5] and as delivery platforms for imaging agents.^[6]

MPs were first described by Wolf in 1967, when he observed a halo of debris surrounding activated platelets that he termed 'platelet dust'.^[7] Since then, the techniques that are available for MP detection have greatly improved and diversified, and now include flow cytometry, dynamic light scattering, nanoparticle tracking analysis, fluorescence correlation spectroscopy, immunoblotting, mass spectrometry, transmission electron microscopy and atomic force microscopy.^[8,9]

Unfortunately, MP nomenclature has also diversified with time, replacing 'dust' for an array of terms including nanoparticle, microvesicle, exosome-like vesicle, ectosome, oncosome, texosome, prostasome, epididymosome and dexosome, depending on the sample source or isolation protocol used. To add to the confusion, MPs are often described as exosomes, smaller (40–100 nm) particles of endocytic origin and apoptotic blebs (50–5000 nm) released by dying cells.^[8,10,11]

Most commonly, MPs are defined as plasma membrane-derived vesicles with a diameter of 100–1000 nm that expose molecules specific to the parental cell, with the majority residing in the 100–200 nm range.^[8,10,12] Depending on their origin MPs may contain an array of signaling molecules, including receptors, cytokines, mRNA, miRNA and bioactive lipids. This molecular composition renders MPs as vectors of biological information. As such, they play an active role in homeostasis and pathogenesis, the latter including atherosclerosis, various malignancies, autoimmune disorders and infection.^[10,13] Therefore, MPs may be harnessed as diagnostic and/or prognostic biomarkers for disease.^[14–17]

The majority of the blood-borne MPs are generated by erythrocytes and platelets.^[12,18,19] RMPs are generated as part of the physiological erythrocyte aging process,^[20–22] and platelet-derived MPs (PMPs) are involved in coagulation after vascular injury.^[12] During storage in the blood bank, erythrocytes and platelets continuously shed MPs, which might be responsible for some of the side effects observed after transfusion.^[23,24]

As a consequence, proper identification and quantification of MPs is of great value, but the methods that are currently applied to isolate, characterize and quantify MPs are far from standardized, and contain several technical hurdles.^[8,25] The call for a consensus on both MP nomenclature, and methods for isolation and identification has, therefore, increased over the last ten years, and was one of the main topics during the first annual meeting of the International Society for Extracellular Vesicles.^[26] A widely supported consensus on these aspects will prove vital for future MP research, as the current lack of methodological clarity obfuscates the identification and molecular elucidation of their generation and biological function.

This review briefly discusses the possibilities for RMPs and PMPs in future science and medicine, while focusing on the technical limitations and challenges in isolation and flow cytometry analysis of MPs from plasma and transfusion products.