Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection

Abstract

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 we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We 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. Our 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.

Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins

“…2d, e). It is, however, important to note that when the degree of contamination is evaluated by measuring lipoproteins such as Apo-A, Apo-B, and Apo-E, proteins such as Apo-E have been reported to be present on exosomes, specifically those released from pigment cells [18], and EVs isolated from plasma can be covered with LDL [13], thus making it difficult to discriminate between lipoprotein particle contaminants and EV-associated lipoproteins.…”

Exosomes and Cardiovascular Protection

“…However, as mentioned, none of these methods are perfect and lead to co-isolation of sEVs containing some degree of microvesicles [5, 6]. Density gradient purification may be used to increase the purification of lighter vesicles from more dense proteins, but is time-consuming and low throughput.…”

Microfluidics‐based on‐a‐chip systems for isolating and analysing extracellular vesicles

“…However, this method can co-isolate other components, including viruses, protein aggregates, very large proteins (such as von Willebrand factor and chylomicrons) or low-density lipoproteins [70–73]. Other disadvantages include the requirement for specialised equipment, the time required and the loss of small EVs [69].…”