ABSTRACT

Under resting conditions a dynamic asymmetric steady state between the outer and inner membrane lipid leaflets is maintained via enzyme activity. The exact mechanism(s) for MV generation is not fully known, but membrane enzymes such as scramblase, gelsolin (present only in platelets), aminophospholipidtranslocase, calpain, and floppase are thought to be involved and result in the expression of negatively charged phospholipids on the cell surface, coupled with membrane blebbing.1-4 In a clinical setting, EVs may have significant potential for earlier and more sensitive diagnostics and therapeutic monitoring.5,6 EVs could potentially serve as unique biomarkers that describe the current metabolic state of their host cell at the time of their formation. Although platelet MVs are among the most widely studied, they can also originate from many different cell types, including leukocytes, endothelial cells, monocytes, tumor cells, and erythrocytes. There now exists a better understanding of the metabolic function of EVs and their possible diagnostic utility.1,6-8 Although EVs can be found circulating in low levels in healthy individuals, several investigators have documented elevated levels of EVs in diseases with vascular involvement and hypercoagulable states such as disseminated intravascular coagulation (DIC), acute coronary syndrome, systemic inflammatory disease, and kidney disease.6-14 EVs have also been associated with inflammation, cellular activation and dysfunction, angiogenesis, and transport of cellular receptors.15-18 The major impediment to advancements in the clinical diagnostic exploitation of EVs has been the inability to simultaneously and accurately determine their, size, number, phenotype, and other characteristics such as charge. Although flow cytometry is currently considered the technique of choice for EV characterization, conventional flow cytometers cannot routinely examine EVs below 0.5 µm, including the exosome and small MV populations. This is particularly relevant since many groups have reported large numbers of EVs in the 0.05-0.3 µm range using alternative methodologies (e.g., electron microscopy). However, recent advances in flow cytometry with the advent of modern digital instruments now facilitate analysis of EVs in the 0.2-0.5 µm range. This coupled with other types of instrumentation suggests that there is significant biological information and EVs below the traditional cutoff of cytometers. Two instruments capable of analyzing smaller EVs will be discussed herein.