Table 4.

 Challenges of Extracellular Vesicle (EV) Isolation and Analysis and Proposed Recommendations

Method	Challenges	Recommendations
Isolation of EV cargo	Poor consistency among studies and highly variable isolation protocols Lack of predetermined handling and storage conditions Purification of EV preparations Consideration of the confounding patient-related and environmental variables	Use of optimized and standardized protocols Standard storage protocols Inclusion of strategies to remove potential contaminants (use of RNase, DNase, proteinase treatment) Careful selection of patient population and controls to minimize the influence of external variables (eg, age-dependent clonal heterogeneity)
Mutation detection (PCR)	Low input analyte Limited reproducibility Choice of blood component Low sensitivity and specificity	Use of ultrasensitive modalities with a lower mutant allele frequency (MAF) detection Large-scale validation studies (intra- and inter-institutional collaboration) More consistent and frequent use of plasma (vs serum) Methods to remove heterogeneous background and reduce the signal-to-noise ratio
Sequencing	Size selection bias (eg underrepresentation of medium size RNA) GC content bias Adapter dimers in ligation-based library preparation kits Lack of reproducible and standard bioinformatics pipelines Variability secondary to use of different sequencing platforms Limited reproducibility and clinical translation of findings (novel biomarkers)	Serial extractions of different-sized populations from the same patient sample. Careful selection of purification kit based on the population of interest. Comparison of different extraction protocols Modification of the kits to reduce ligation bias Use of approved and standard databases for mapping to reduce variability, reliable statistical tools, consistent normalization methods, inclusion of reference genes Use of identical sequencing technologies for accurate inter-study comparisons Validation using reliable techniques (PCR, Western blot, etc.)
Proteomics	Variability in isolated EV populations Limited proteome sequence coverage Paucity of information on glioma-specific protein mutations and protein-protein interactions Delineation of glioma-specific EV proteins from non-tumor markers	Minimize confounding variables (patient-related) and tailor the isolation protocol Use of high-throughput and accurate MS-based methods for a limited quantity of isolated proteins Proteogenomics; integrated approach incorporating targeted proteomics and RNA-seq data Development of sensitive and robust targeted proteomics in combination with downstream validation studies for functional characterization