Extracellular vesicles, specifically microparticles (MPs), are rapidly gaining attention for their capacity to act as biomarkers for diagnosis, prognosis, or responsiveness to therapy in lung disease, in keeping with the concept of precision medicine. However, MP analysis by high-sensitivity flow cytometry (FCM) is complicated by a lack of accurate means for MP enumeration. To address this gap, we report here an enhanced FCM MP gating and enumeration technique based on the use of novel engineered lipid bilayer microspheres (LBMs). By comparison of LBM-based MP enumeration with conventional bead- or fluorescent-based FCM enumeration techniques and a gravimetric consumption gold standard, we found LBMs to be superior to commercial bead preparations, showing the smallest fixed bias and limits of agreement in Bland Altman analyses. LBMs had simultaneous capacity to aid FCM enumeration of MPs in plasma, BAL, and cell culture supernatants. LBM enumeration detected differences in MP counts in mice exposed to intraperitoneal lipopolysaccharide or saline. LBMs provided for 1) higher sensitivity for gating MPs populations, 2) reduced background within MP gates, 3) more appropriate size, and 4) an inexpensive alternative amenable to different fluorescent tags. LBM-based MP enumeration was useful for a series of different FCM systems assessed, whereas LBM gating benefited high- but not low-sensitivity FCM systems compared with fluorescence gating. By offering exclusive advantages over current means of gating and enumerating MPs, LBMs are uniquely suited to realizing the potential of MPs as biomarkers in biological lung fluids and facilitating precision medicine in lung disease.
Keywords: biomarker; enumeration; extracellular vesicles; lipid bilayer microspheres; microparticles.
Copyright © 2016 the American Physiological Society.