Background
In an age of disease biomarker discovery there are many challenges for biobankers in the stardardisation of methods that provide suitable material for numerous downstream uses. For cell-free DNA plasma analysis the advent of specialised cell stabiliser tubes have offered convenience to delay sample processing through membrane stabilisation, particularly valuable to offer testing to rural communities. This study assessed the feasibility of using these tubes for EV isolation.
Methods
Six tubes (EDTA and two proprietary tubes R and S) were used to collect blood from three healthy donors, one processed immediately for plasma (Day 0: 1,500g and 10,000g), another stored for 3 days (Day 3). The 10,000g pellet was retained as a crude microEV pellet. NanoEV were isolated from 2mL plasma using size exclusion chromatography. EVs were assessed for protein using BCA and dot blotting, for particle number using nanoparticle tracking analysis (NTA). Hemolysis was also assessed using spectroscopy at 414nm.
Results
NanoEV particle counts did not differ between tube type or with increased processing time from immediate to 3 day delay. The protein quantities associated with these nanoEVs increased with time however there was no obvious difference between collection tube types. Hemolysis was increased at the 3 day timepoint as compared to the Day 0 samples. MicroEVs were increased in both particle number and protein quantity at Day 3 relative to Day 0. Cell surface markers identified an increase in CD61+ nano and microEVs at day 3, indicative of platelet activation in all tube types. CD235a+ and CD45+ nanoEVs were also more prevalent at Day 3.
Conclusions
Delay to processing in all blood collection tubes increased the production of micro particles from platelets, leukocytes and red blood cells. The nanoEVs did not increase in overall particle number but we hypothesise that lysed cell proteins become EV surface associated. The specialised tube additives did not limit particle production from nucleated or non-nucleated blood cells during delayed processing relative to standard, inexpensive EDTA vacutainers. Both specialised blood tubes can however be used instead of EDTA for plasma micro and nanoEV analysis if rapidly processed after collection.