ABSTRACT
In the last few years, several studies have emphasized the existence of injury-specific EV "barcodes" that could have significant importance for the precise diagnosis of different organ injuries in polytrauma patients. To expand the research potential of the NTF (network trauma research) biobank of polytraumatized patients, the NTF research group decided to further establish a biobank for EVs. However, until now, the protocols for the isolation, characterization, and storage of EVs for biobank purposes have not been conceptualized. Plasma and serum samples from healthy volunteers (n = 10) were used. Three EV isolation methods of high relevance for the work with patients' samples (ultracentrifugation, size exclusion chromatography, and immune magnetic bead-based isolation) were compared. EVs were quantified using nanoparticle tracking analysis, EV proteins, and miRNAs. The effects of different isolation solutions; the long storage of samples (up to 3 years); and the sensibility of EVs to serial freezing-thawing cycles and different storage conditions (RT, 4/-20/-80 °C, dry ice) were evaluated. The SEC isolation method was considered the most suitable for EV biobanking. We did not find any difference in the quantity of EVs between serum and plasma-EVs. The importance of particle-free PBS as an isolation solution was confirmed. Plasma that has been frozen for a long time can also be used as a source of EVs. Serial freezing-thawing cycles were found to affect the mean size of EVs but not their amount. The storage of EV samples for 5 days on dry ice significantly reduced the EV protein concentration.
Subject(s)
Biological Specimen Banks , Extracellular Vesicles , Multiple Trauma , Humans , Extracellular Vesicles/metabolism , Multiple Trauma/metabolism , Multiple Trauma/blood , Specimen Handling/methods , Chromatography, Gel/methods , Male , Ultracentrifugation/methods , MicroRNAs/blood , MicroRNAs/genetics , Adult , FemaleABSTRACT
Background: Polytrauma is one of the leading mortality factors in younger patients, and in particular, the presence of cardiac damage correlates with a poor prognosis. Currently, troponin T is the gold standard, although troponin is limited as a biomarker. Therefore, there is a need for new biomarkers of cardiac damage early after trauma. Methods: Polytraumatized patients (ISS ≥ 16) were divided into two groups: those with cardiac damage (troponin T > 50 pg/mL, n = 37) and those without cardiac damage (troponin T < 12 pg/mL, n = 32) on admission to the hospital. Patients' plasma was collected in the emergency room 24 h after trauma, and plasma from healthy volunteers (n = 10) was sampled. The plasma was analyzed for the expression of HFABP, GDF-15 and uPAR proteins, as well as miR-21, miR-29, miR-34, miR-122, miR-125b, miR-133, miR-194, miR-204, and miR-155. Results were correlated with patients' outcomes. Results: HFABP, uPAR, and GDF-15 were increased in polytraumatized patients with cardiac damage (p < 0.001) with a need for catecholamines. HFABP was increased in non-survivors. Analysis of systemic miRNA concentrations showed a significant increase in miR-133 (p < 0.01) and miR-21 (p < 0.05) in patients with cardiac damage. Conclusion: All tested plasma proteins, miR-133, and miR-21 were found to reflect the cardiac damage in polytrauma patients. GDF-15 and HFABP were shown to strongly correlate with patients' outcomes.