Isolation of Extracellular Vesicles from human plasma samples: The importance of controls

There are a number of methods for the isolation of extracellular vesicles (EV) which include the traditional ultracentrifugation to column-based kits available from different companies. Isolation of EVs from complex fluids, such as blood, has several challenges as the detection of low abundance molecules can easily be masked by more abundant proteins, when performing mass spectrometry. For this reason, several commercially available kits contain Thromboplastin D (TP-D) to promote clotting, thus removing clotting factors and abundant proteins resulting in increased detection of proteins. Our study demonstrates that plasma pretreated with Rabbit brain derived TP-D (the most common additive), generated a dynamic range of proteins compared to plasma alone, however, most of these proteins were contaminants introduced from the TP-D (99.1% purity). As an alternative, we tested recombinant TP and demonstrated that although it did not introduce any significant contaminants, we did not see any difference in the detection of proteins. Thus TP-D is not required, and any protein additives must be carefully screened.