Impact of preanalytical conditions on plasma concentration and size distribution of extracellular vesicles using Nanoparticle Tracking Analysis.
Author(s): Jamaly S, Ramberg C, Olsen R, Latysheva N, Webster P, Sovershaev T, Brækkan SK, Hansen JB
Publication: Sci Rep, 2018, Vol. 8, Page 17216
PubMed ID: 30464183 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate the effects of anticoagulant type and of freezing platelet-poor plasma (PPP) before centrifugation to obtain platelet free plasma (PFP) on the concentration and size distribution of extracellular vessicles (EVs). The effects of fasting and consumption of a high fat meal prior to collection on the concentration of triglycerides in serum, plasma very low density lipoprotein (VLDL) particle count, and EV concentration were also investigated.
Conclusion of Paper
Total concentration and size distribution of EVs were not significantly affected by anticoagulant type or by freezing of PPP. The average size and size distribution of the EVs appeared smaller when analyzed using SEM rather than by nanoparticle tracking analysis. Centrifugation at 20,000 x g for 30 min did not alter the concentration of triglycerides found in diluted (1:5) or undiluted plasma. Consumption of a high fat meal resulted in a significant increase in serum triglycerides (peaked at 4 h and returned to baseline after 8 h) but did not affect the concentration of EVs or VLDL particles. The concentration of EVs in plasma was weakly to modestly correlated with the concentration of serum triglycerides and plasma VLDL particles after fasting, but strongly correlated in specimens collected 4 h after consumption of a high fat meal. The average diameter of VLDL particles also increased after ingestion of the high fat meal.
Studies
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Study Purpose
The purpose of this study was to investigate the effects of anticoagulant type and of freezing PPP before centrifugation to obtain PFP on the concentration and size distribution of EVs as determined by SEM and NTA. Blood was collected from ten healthy volunteers using a 21-gauge needle at 8:30 AM first into a discard tube and then into Vacutainer tubes containing sodium citrate, sodium heparin, K2EDTA, or buffered citrate-theophylline-adenosine-dipyridamole (CTAD) (draw order unspecified). Tubes were inverted and stored upright for <30 min before centrifugation. PPP was prepared by centrifugation at 3000 × g for 10 minutes and then centrifuged at 13,500 x g for 2 min to obtain PFP immediately or after 48 h at -70°C. PFP was then diluted in Hanks/Hepes buffer or Dullbecco’s phosphate buffered saline (DPBS). EVs were pelleted by centrifugation at 20,000 x g for 30 min at room temperature. The pellet was either resuspended in DPBS, snap-frozen, and stored at -70°C until further analysis or resuspended in Hanks/Hepes buffer, fixed in 4% formaldehyde, and analyzed by transmission electron microscopy. TEM was conducted on the resuspended EVs and on ultrathin sections of EVs adsorbed into an epoxy resin substrate containing colloidal gold. EV concentration and size distribution were determined using nanoparticle tracking analysis after thawing at room temperature. Immuno-electron microscopy was conducted on EVs fixed with 1% buffered glutaraldehyde and adsorbed onto formvar/carbon coated specimen grids before immunolabelling with an anti-annexin V antibody. EV morphology was investigated on EVs fixed with 1% buffered glutaraldehyde using SEM.
Summary of Findings:
Total concentration and size distribution were comparable when EVs were frozen as PPP or PFP, regardless of anticoagulant choice. The concentration EVs across all categories was lowest when blood was anticoagulated with sodium citrate and the concentration of large particles was 2-fold higher when anticoagulated with heparin rather than any other anticoagulant but the differences in EV concentration and size distribution among anticoagulants were not significant. The average size and size distribution of the EVs appeared smaller when analyzed using SEM rather than nanoparticle tracking analysis. TEM showed the majority of EVs were spherical and were not aggregated.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Morphology Electron microscopy Cell count/volume Electron microscopy Cell count/volume Light scattering Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Citrate
Citrate-theophylline-adenosine-dipyridamole
EDTA
Heparin
Storage Storage conditions As PPP
As PFP
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
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Study Purpose
The purpose of this study was to investigate the effects of fasting and consumption of a high fat meal on the concentration of triglycerides in serum and plasma, VLDL particle count in plasma, and EV concentration and also examined the correlations of EV concentration with serum triglycerides and VLDL particle count. Blood was collected using a 19-gauge needle from 40 heathy patients into EDTA, citrate, and serum tubes after an overnight fast and again at 0, 2, 4, 6, and 8 h after consumption of a high fat meal. Serum and plasma were prepared by centrifugation at 2000 x g for 15 min and frozen at -70 until analysis. Serum lipids were determined using an ABX Pentra 400 analyzer and lipoprotein particle size was determined by NMR of citrated plasma. EVs were pelleted by centrifugation at 20,000 x g for 30 min at room temperature. The concentration of EVs in plasma was determined by nanoparticle tracking analysis in citrated plasma.
Summary of Findings:
Centrifugation at 20,000 x g for 30 min did not alter the concentration of triglycerides found in diluted (1:5) or undiluted plasma. Consumption of a high fat meal resulted in a significant increase in serum triglycerides (peaked at 4 h and returned to baseline after 8 h) but did not affect the concentration of EVs or VLDL particles. The concentration of EVs in plasma after fasting was weakly to modestly correlated with the concentration of serum triglycerides (r=0.35 P=0.03) and plasma VLDL particles (r=0.43, P=0.005). However, 4 h after meal consumption the concentration EVs was strongly strongly correlated with serum triglyceride concentration (r=0.77, P<0.0001) and plasma VLDL particles (r=0.81, P<0.0001). The concentration of triglycerides and VLDL particles in plasma accounted for 13% and 19%; respectively, of the variation in EV concentration when fasting and 59% and 66%; respectively, of the variation in EV concentration 4 h after eating. The average diameter of VLDL particles also increased after ingestion of the high fat meal (42 nm to 55 nm, P<0.0001).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Light scattering Lipid Clinical chemistry/auto analyzer Lipid NMR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Time of biospecimen collection 0 h after consumption of high fat meal/Fasting
2 h after consumption of high fat meal
4 h after consumption of high fat meal
6 h after consumption of high fat meal
8 h after consumption of high fat meal
Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared