Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging.
Here researchers from Semmelweis University, Hungary studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, they found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. They identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. These data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.
LDL mimics EVs during TRPS and FCM
(A) Commercially available human LDL analyzed by TRPS. Note that particles with different sizes were detected within the MV size range (100–800 nm) (measured on NP150 and NP300 nanopore membranes, black and gray bars, respectively). (B) FCM analysis of LDL particles conjugated to latex beads. Commercial LDL stained for apoB and apoCII, but not for the EV markers CD9 and CD63 (black filled histograms: antibody control, empty histograms: LDL). (C) LDL detection by FCM without bead conjugation at different dilutions. Note that the measured event number increases with the dilution, suggesting a swarm effect. (D) The signal obtained from commercial LDL is also partially sensitive to 0.1% Tx-100 at a physiological concentration (2 mg/mL) and in the 10 × diluted sample (0.2 mg/mL) as well (*P < 0.05, **P < 0.01, Mann-Whitney test).