Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery

Circulating extracellular vesicles as biomarkers are a relatively new field in biomarker research with immense potential. Our protocol aims to provide a hands-on approach to implement extracellular vesicle bound biomarker discovery and detection in an emergency department setting. Emerging evidence has shown that the molecular cargo found in extracellular vesicles and circulation contains proteins and RNA of interest in multiple disease contexts.

Several new extracellular vesicle, enclosed biomarkers have been linked to diagnosis and prognosis of various cardiovascular disease entities. Major challenges in the field of circulating extracellular vesicle biomarker discovery are preservation of extracellular vesicles and plasma acquisition dependent on EV release dynamics. Therefore, a stringent protocol embedded in clinical routine is necessary to avoid EV degradation and maintain reproducible results, particularly when applying a broadscale analysis methods such as proteomics.

Discovery of protein biomarkers enclosed within circulating EV is heavily dependent on the time of initial blood draw relative to symptom onset, and sample storage duration due to natural EV degradation. This protocol offers a hands-on protocol of plasma EV isolation and introduction into proteomics analysis to be integrated in the clinical workflow of an emergency department. Our forthcoming research will focus on integrating different patient collectives in our methodology among which are patients with decompensated heart failure, arrhythmias, or tachycardia cardiomyopathy, as well as longitudinal studies of patients undergoing bypass surgery.

Using this protocol, we aim to identify novel EV bound biomarkers contributing to diagnosis and prognostication of aforementioned pathologies. To begin centrifuge the human blood plasma, using an ultra centrifuge with a fixed angle rotor without breaking. After the debris and apoptotic body settle, transfer the supernatant to a new tube and ultra centrifuge at 100, 000 G for two hours at four degrees Celsius without breaking to isolate extracellular vesicles.

Remove the tube carefully after the ultracentrifugation. Using an electronic pipette, gently aspirate the supernatant, leaving approximately one milliliter of extracellular vesicle depleted plasma. Switch to a 1000 microliter pipette to carefully pipette the remaining plasma without disturbing the extracellular vesicle pellet.

Gradually tilt the tube during pipetting to ensure no plasma remains and resuspend the pellet in PBS. Next, dilute the extracellular vesicle stock solution with ice cold PBS at different ratios to achieve a final count range of 10 to 100 nanoparticles per frame during tracking. Switch on the NTA instrument and launch the applicable software on the connected computer.

Insert the chamber into the laser casket and click on start camera to activate it. Draw one milliliter of PBS into a syringe and connect it to the front tube. Flush the tubing system thoroughly, ensuring no air bubbles remain in the chamber.

Monitor the camera to detect any air bubbles or contaminating particles. Now draw up the prepared extracellular vesicle working dilution into a one milliliter syringe and inject it into the chamber. Go to the capture and process tabs to adjust the screen gain for matching the monitor's brightness.

Modify the camera level to enhance nanoparticle differentiation on the screen. Set an appropriate detection threshold in the process tab to differentiate particles from background noise. Focus the camera using the focus wheel on each side of the instrument until nanoparticles appear sharp on the screen.

After setting a constant temperature of 22 degrees Celsius for all measurements in the hardware settings, click on the run button to initiate measurement. Record at least three videos of 30 seconds each for every sample run. Click on change to input the samples dilution factor, and finally click export to save the results.

Using nanoparticle tracking analysis, extracellular vesicles were identified and visualized based on brownie and motion. The analysis revealed an average particle concentration of 1.2 times 10 to the power of 10 particles per milliliter in plasma from healthy individuals demonstrating the presence of extracellular vesicles. The average size of plasma extracellular vesicles was measured at 184.7 nanometers, consistent with typical extracellular vesicle dimensions.