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A Method to Analyze Urinary Extracellular Vesicles

Extracellular vesicles (EVs) play an important role in cell-to-cell communication carrying molecular messages which reflect physiological and pathological conditions of the parent cells. EVs have been identified in all body fluids; and among them, urine stands out as a sample that is easy and inexpensive to obtain and can be collected over time to monitor changes. Various protocols have been established to study urinary extracellular vesicles (UEVs) and they have shown great potential as a biomarker source for clinical applications, not only for urological, but also non-urological diseases. Due to the high variability and low reproducibility of pre-analytical and analytical methods for UEVs, establishing a standardized protocol remains a challenge in the field of diagnosis. Here, we review UEV studies and present the techniques that are most commonly used, those that have been applied as new developments, and those that have the most potential for future applications. The workflow procedures from the sampling step to the qualitative and quantitative analysis steps are summarized along with advantages and disadvantages of the methodologies, in order to give consideration for choosing the most promisingly suitable method to analyze human UEVs.


Introduction
considered including accuracy, precision, specificity, limits of detection and quantification. Comprehensive standardized guidelines for isolation of EVs 23 and over all analytical processes 24,25 have been provided that suggest all experimental details as well as critical evaluations in the EV analysis procedures. Some researchers have discussed the development of a variety of UEV isolation and interrogation approaches, and highlighted potentials and limitations. 26 Others have addressed the whole range of methods that are used for human UEV isolation and characterization. 27 In this review, we consolidate and select common protocols and up-to-date technologies that have been using specifically in UEV analysis. We present the overall workflow procedures from the sampling step to the output of qualitative and quantitative analysis results, in order to give some direction to researchers for choosing a method that is appropriate for meeting analysis needs.

Techniques of pretreatment
Since the biggest advantage to using urine sample is that it can be simply collected from an examinee into a sterilized container without any external instrumental needs, the ideal analysis methodology would isolate EVs directly from urine without any extensive pretreatment steps. Comparing to other liquid biopsy samples (blood, plasma, breast milk), urine has a narrower spectrum of interference from matrix components and requires less pretreatment before subjecting it to further analytical process. [28][29][30] Most pretreatment studies have used a short low-speed centrifugation at 2,000xg to remove large-sized components (cells, apoptotic bodies, and cell debris). 31-33 Some researchers have reported a unique pretreatment approach for increasing the secretion of UEVs and enrichment of prostatic markers using a digital rectal examination (DRE) to stimulate the prostate before urine collection. 34, 35 For preventing the degradation of UEVs, it is recommended that the urine be pelletized and stored at below -80°C to get 100% recovery, maintain uniform and unchanging morphology of the UEVs. 26 The functional properties ammonio)-1-propane sulfonate (CHAPS), 41 sodium chloride (NaCl), 42 and zinc sulfate (ZnSO4); 33 these treatments to inhibit protein-UEV entrapment are optional. It was found that the DTT treatment led to a relative increase in the number of UEVs, but it did not increase the yield of RNA. 43 Thus, the decision of whether or not to use protease inhibitors depends on the type of follow-up analyzes.

Techniques of isolation
Before carrying out any experiment, consideration must be given to what target information is expected to be obtained from UEVs in the downstream analysis, e.g. the morphology, variety of components, and proteomic and genomic information. The frozen urine pellets are used after thawing then in a 37 °C water bath for 10 min, 44  were reported to improve isolation yield and purity. 40,47,48,59 The precipitation techniques are based on the fact that UEVs change their solubility and/or aggregate with substances e.g. polymers, protamine, and sodium acetate.
ExoQuick is one widely used commercial reagents for exosome isolation. 60 The urine sample is incubated with precipitate-forming solution, then the mixture is centrifuged at low speed (1,500-10,000xg), and finally pellets of exosomes are collected. ExoQuick offers a tool for quick and effective isolation with high quantity and quality of exosomes, and miRNA and mRNA molecules, suggesting that the protocol is suitable for RNA profiling. [61][62][63] One disadvantage of ExoQuick is that some proteins can also be coprecipitated.
Immuno-based techniques offer highly specific isolation using surface affinity between UEVs, surface proteins, and antibodies conjugated with magnetic beads or other materials. 64,65 However, these highly specific materials are high-cost. Recently, Wu et al. 66 developed a novel method called EVTRAP based on functionalized magnetic beads that have a unique affinity toward lipid-coated EVs for capture and isolation of EVs from urine samples. They found a significant improvement over current standard techniques with highly efficient capture giving more than 95% recovery yield.
Microfluidics techniques are integrated platform that have been developed within the lab-on-a-chip concept promoting high robustness, sensitivity, and selectivity of UEV analysis. As noted earlier, the ideal analysis methodology is to detect EVs directly without any extensive pretreatment steps from urine, and numerous studies have tried to find out whether realizing this is possible. In 2017, we proposed a novel approach to

Techniques of downstream analysis
The  Table 2. Each of these techniques provides different types of measurement information e.g. morphology and biomolecular components that could reflect different disease states. Importantly, the possible errors from the pre-analysis and isolation processes have to be considered to ensure accuracy in the downstream analysis.

Analytical Sciences
Advance Due to its high-throughput with particle size distribution and concentration provided, currently NTA has become the most commonly used quantitative analysis for UEVs. 40,44,79,80 Some detergent such as Triton X is applied in NTA to gain more output for confirmation of EV-lipid moieties and specific particle fluorescent labeling. 81,82 However, similar to DLS, the impurities contained in isolated UEVs and dye aggregates might interfere and produce overestimation by NTA.
Tunable resistive pulse sensing (TRPS) and scanning ion occlusion sensing (SIOS) are non-optical, emerging, and potential techniques that has been used to determine individual EVs by measuring changes in electrical current when the particles pass through an adjustable nanopore. These methods provide high-throughput analysis of the vesicles, including size distribution, concentration, and surface charge (zeta-potential) of

Analytical Sciences
EVs without labelling. 83,84 This capability makes it a useful and applicable technique for UEV research. 76,85 Flow cytometry (FCM) is a technique that allows the analysis of physical characteristics such as the size of particles using multiple-angle scattering light and also chemical properties of UEV components such as proteins and nucleic acids using fluorescent dyes. Due to its advantage of high-throughput measurements, flow cytometry has been applied to the reproducible analysis of clinical samples. 59, 65,76,86 Notably, the overestimated scatter signals from high concentrations of EVs must be considered. The critical parameters necessary to increase UEV detection capacity by flow cytometry should be defined.
The pioneer publication on large-scale proteomics and phosphoproteomics of EVs in human urine appeared in 2009 and reported the use of liquid chromatography-mass spectrometry / mass spectrometry (LC-MS/MS). 87 Recently, improvements in mass spectrometry technology have enabled high-throughput and large-scale quantitation and validation of candidate protein biomarkers in UEVs. 66,[88][89][90][91] Western blot (WB) is another popular technique adopted to determine the protein composition that is suitable for confirming the correct isolation, despite being laborious and non-quantitative and sometimes yielding poor results, due to the quality of the antibodies used or to low exosome content in the sample. 40,45,76 Quantitative reverse transcriptase-PCR (qRT-PCR) is a conventional detection and quantification approach for UEV-derived nucleic acids including RNA, microRNA and DNA in terms of gene expression and mutational profiles. [92][93][94] In cases that the concentration of nucleic acid isolated is too low to be directly detected, a preamplification protocol needs to be performed. 95 The normalization of the input to the volume of sample input is also recommended. 96 Moreover, the evaluation of this approach relies on the probe or primers used which are limited. Microarray genomic analysis is another technology that offers a way to assess thousands of transcripts in UEVs using a gene expression array. This technique also relies on specific fluorophore probes and required a costly instrument to interpret the fluorescent signal from hybridization of miNAs and mRNAs with the probes. 67 Next generation sequencing (NGS) is the latest fast-growing technology that has enabled characterization of the whole spectrum of nucleic acids in a given sample, 97 and it was consistently applied to demonstrate the presence of the complex RNA molecules within the EV population released by various cells. A recent review of Turchinivich et al. 98 presented the state-of-the-art in the field of EVs-associated RNA transcriptomes using next-generation sequencing.

Conclusions and Perspectives
Analysis of urinary extracellular vesicles (UEVs) to understand human health was built goal would be development of a universal method for UEV analysis.