Problems with nanoparticle tracking analysis (NTA) of urine extracellular vesicles (uEVs)

Urinary extracellular vesicle (uEV) proteins may be used as specific markers of kidney damage in various pathophysiological conditions. The nanoparticle-tracking analysis (NTA) appears to be the most useful method for the analysis of uEVs due to its ability to analyze particles below 300 nm. The NTA method has been used to measure the size and concentration of uEVs and also allows for a deeper analysis of uEVs based on their protein composition using fluorescence measurements. However, despite much interest in the clinical application of uEVs, their analysis using the NTA method is poorly described and requires meticulous sample preparation, experimental adjustment of instrument settings, and above all, an understanding of the limitations of the method. We present the problems encountered during analysis with possible solutions: the choice of sample dilution, the method of the presentation and comparison of results, photobleaching, and the adjustment of instrument settings for a specific analysis.

Figure 1. Expressions of specific markers CD63 in protein-standardized samples detected with Western blot analysis; anti-CD 63 (HPA010088, Sigma-Aldrich, Saint Louis, MO, USA, 1:1000); secondary antibodies conjugated to horseradish peroxidase (554021, BD Pharmingen (BD Biosciences, San Jose, CA, USA) 1:10000).

Nanoparticle-Tracking Analysis of uEVs. A NanoSight NS300 instrument (Malvern Panalytical, Malvern, UK) was used to determine the concentrations and sizes of the uEVs in the samples. The total number of extracellular vesicles was measured during the continuous flow of samples delivered from a syringe pump.

Figure 2. Determination of the size and concentration of uEVs: dilution factor—1:100; laser—405 nm.

Figure 3. Effect of dilution on total number of particles per milliliter and size of uEVs in nanoparticle tracking analysis: sample dilutions—1:100, 1:500, and 1:1000; laser—488 nm.

Figure 5. Fluorescence-based nanoparticle-tracking analysis of CD 63 expression in uEVs: without 500 nm long-pass filter; with 500 nm long-pass filter; comparison of sizes and concentrations of uEVs without and with 500 nm long-pass filter; dilution factor—1:100; laser—488 nm; anti-CD 63 (HPA010088, Sigma-Aldrich); secondary antibodies conjugated to Alexa Fluor 488 fluorescent dye (ab150073-500, Abcam, Cambridge, MA, USA).

Figure 6. Fluorescence-based nanoparticle-tracking analysis of podocin expression in uEVs: without 500 nm long-pass filter; with 500 nm long-pass filter; comparison of sizes and concentrations of uEVs without and with 500 nm long-pass filter; dilution factor—1:100; laser—488 nm; anti-podocin (P0372, Sigma-Aldrich); secondary antibodies conjugated to Alexa Fluor 488 fluorescent dye (ab150073-500, Abcam, Cambridge, MA, USA).