Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Handling and storage of human body fluids for analysis of extracellular vesicles.

Author(s): Yuana Y, Böing AN, Grootemaat AE, van der Pol E, Hau CM, Cizmar P, Buhr E, Sturk A, Nieuwland R

Publication: J Extracell Vesicles, 2015, Vol. 4, Page 29260

PubMed ID: 26563735 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of delayed centrifugation of blood, centrifugation duration and speed, and freeze-thaw cycling on the yield and composition of EVs from cell-free supernatants derived from platelets and erythrocytes concentrates, plasma, urine, and saliva. Erythrocytes and platelets were removed by double centrifugation at 1550 x g for 20 min of purchased, out-of-date erythrocyte and platelet concentrates to obtain cell-free supernatants. Blood was collected into citrate tubes from five healthy donors after overnight fast and was stored for 5 min or 1 h before isolation of plasma by double centrifugation at 1550 x g for 20 min. Urine and saliva from five healthy donors was stored on ice until removal of cells by centrifugation at 180 x g (urine) or 300 x g (saliva) followed by centrifugation at 1550 x g for 20 min. EVs were then obtained from cell-free supernatants by centrifugation at 18,890 x g or 100,000 x g for 30 min or 2 h, and from plasma by centrifugation at 18,890 x g for 2 h. EVs were resuspended by pipetting up and down before measurement. Cell-free supernatants, plasma, saliva, and urine were used fresh, frozen at -20 or-80˚C, or snap-frozen in liquid nitrogen (-196˚C) and stored at -80˚C for 6 months or 1 year before analysis.

   Summary of Findings:

   RPS and NTA detected 10^10-11 erythrocyte and platelet particles per mL, but flow cytometry only found 10^5-6 particles of each type per mL. The authors attribute this discrepancy to differences in the diameter of the smallest detectable particles and that RPS and NTA may detect particles that are not EVs. Centrifugation speed and duration had little effect on the EV count, diameter, and clumping in cell-free supernatants from erythrocyte concentrates but when cell-free supernatants derived from platelet concentrates were centrifuged for 2h, the number of EVs as determined by flow cytometry increased by 200% (18,800 x g) to 517% (100,000 xg) and the number of EVs as determined by RPS and NTA decreased by >35 and >70%, respectively. Importantly, the authors note this difference may be due to aggregation, as aggregates are harder to resuspend and consequently would not be seen in the small volumes used for RPS and NTA, yet aggregation allows for particles below the size cut-off for flow cytometry to be counted. When plasma was centrifuged, EV aggregates were observed regardless of whether the plasma was diluted prior to centrifugation.

   Storage of blood for 1 h versus 5 min prior to centrifugation had no effect on EV count (by any method) or mean EV diameter in plasma.

   A single freeze-thaw cycle of cell-free supernatants derived from erythrocyte or platelet had little effect on the EV counts by RPS and NTA and no effect on EV morphology. However, the erythrocyte EV count decreased by 10-fold when freeze-thaw cycled at -20˚C and analyzed by flow cytometry and the mean diameter of erythrocyte EVs decreased slightly with a freeze-thaw cycle, particularly when snap-frozen and analyzed by RPS.  Similarly, there was no effect of thawing or of freezing plasma at -20, -80, or -196˚C for 1 or 6 months on EV count by RPS and NTA or on CD61+ and CD235+ EVs by flow cytometry, but the number of lactadherin+ EVs detected by flow cytometry increased, regardless of frozen storage temperature and duration. Freeze-thaw cycling reduced the EV count by RPS in urine by 3-fold (p<0.05) and in saliva by 2-fold (p<0.05), regardless of temperature, reduced the lactadherin+ EV count in saliva by 2-fold (p<0.05) but not in urine, and increased the EV count by NTA in urine and saliva by 2-fold (p<0.05, both). Further, the particle diameter by RPS increased 17% after freeze-thawing, regardless of freezing temperature, but the particle diameter by NTA was unaffected.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood
   • Bodily Fluid - Saliva
   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Flow cytometry
   Cell count/volume	Fluorescent microscopy
   Cell count/volume	Electron microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Centrifuged Centrifugation delays investigated Multiple durations compared Multiple speeds compared Not centrifuged
   Storage	Short-term storage solution	Plasma diluted 8 fold in citrate PBS Plasma not diluted
   Storage	Storage duration	0 months 1 month 6 month
   Storage	Storage temperature	-20˚C -80˚C -196˚C
   Storage	Time at room temperature	5 min 1 h
   Storage	Freeze/thaw cycling	0 cycles 1 cycle
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh) Snap frozen
   Flow cytometry Specific	Technology platform	TEM Resistive pulse sensing Nanoparticle tracking analysis

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