FastEV & EV Array

FastEV evaluated with EV Array

EV contents of FastEV isolates were studied with Extracellular Vesicle Array (EV Array). EV Array targeted 14 EV-enriched marker proteins.

Methods: profiling EV-enriched markers with EV Array 

Starting material in FastEV was 160 µl plasma/condition and isolations were done on 96-well plates. FastEV isolates, ultracentrifugation (UC) and polyethylene glycol (PEG) controls were applied to EV array in equal amounts relative to starting plasma volume. Results show data from 62,5% of isolate corresponding to 100 µl of original plasma. However, amounts corresponding to 40 µl of plasma produced good signals as well. Commercial precipitation method, SmartSEC and plasma controls were loaded in different quantities, thus their signals are not directly comparable to FastEV samples

For combined analysis of two EV Array rounds (heatmap), log2-transformed data was normalised using limma function normalizeBetweenArrays and quantile method. Batch effect between the different rounds was removed using limma removeBatchEffect function.

Figure: Principle of EV Array. The EV Array is composed of different capture antibodies printed in the bottom of a 96-well target plate.​

Step 1: 50-100 µL plasma or other body fluids (urine, saliva, BALF, etc.) are applied incubated 2 hours to overnight.​

Step 2: The EVs are detected with a cocktail of biotinylated antibodies.​

Step 3: The presence and thereby phenotype of EVs is visualized after incubation with Cy3-labeled Streptavidin using a fluorescence scanner.

FastEV accesses EV subpopulations

FastEV shows a capacity to enrich subpopulations of EVs, for example blood cell -derived EV populations. FastEV isolates were profiled with EV Array and it showed that they contained EV-enriched markers in different proportions depending on the condition used.

Figure: Enrichment of EV subpopulations by FastEV. The signals of 14 EV markers varied between 14 FastEV conditions, controls and different plasma samples (round 1 and round 2 present data from two different plasma pools). The markers include general EV enriched proteins and some derived from different blood cells (red (CD235+) and white (CD45+) blood cells, endothelium (CD31+) and platelets (CD41+).

Bubble size indicates signal strength. Signals form UC and PEG controls are comparable to FastEV, whereas signals from other controls (Commercial precipitation method, SmartSEC and plasma) are not directly comparable as they were applied in different quantities to the EV Array.  Data is from 1-2 technical replicates per sample. PEG, polyethylene glycol, UC, ultracentrifugation.

FastEV enriches and depletes EVs from plasma

EV Array signals from FastEV were normalized between runs and compared to plasma. FastEV conditions showed unique combinations of enriched and depleted EV-markers. Overall, the EV profiles of most FastEV conditions appeared similar to EV control obtained by ultracentrifugation (UC), and different from polyethylene glycol control (PEG). PEG control appeared most similar to plasma.

Figure: EV enrichment by FastEV relative to plasma. ​Heatmap of fold changes (log2 scale) vs plasma for 14 EV-proteins in EV Array. Most conditions enriched particularly CD9 and CD41 and depleted Annexin V and CD31. CD81, CD82 and LAMP2 were among the most variable targets. Yellow color indicates level most similar to plasma, red indicates enrichment and blue depletion relative to plasma. PEG, polyethylene glycol, UC, ultracentrifugation.