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EDTA stabilizes the concentration of platelet-derived extracellular vesicles during blood collection and handling.

Author(s): Buntsma NC, Gąsecka A, Roos YBWEM, van Leeuwen TG, van der Pol E, Nieuwland R

Publication: Platelets, 2021, Vol. , Page 1-8

PubMed ID: 34697987 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare EV populations between untreated blood and blood after platelet activation, plasma subjected to a single freeze-thaw cycle and fresh plasma, plasma from blood that underwent PTS and blood that was transported manually, and plasma from blood that experienced a centrifugation delay of up to 24 h at 4°C or room temperature. Blood from 7 healthy patients was collected in trisodium citrate and K₂EDTA tubes after discarding the first 2 mL. To test the effect of platelet activation, specimens were incubated with TRAP-6 for 30 min and compared to untreated controls. To test the effect of pneumatic tube transport, case-matched blood of each tube type from 6 patients was transported by pneumatic tube or transported by hand in a vertical position. To determine the effect of delayed separation of plasma, case-matched tubes from six donors were stored for 0, 6, or 24 h at room temperature or 4°C before plasma separation. To test the effect of freeze-thaw cycling EV concentrations were measured in fresh plasma and again after snap-freezing in liquid nitrogen, storage at -80°C, and thawing at 37°C. Plasma was obtained from all specimens by dual-centrifugation at 2500 g for 15 min, and then snap-frozen in liquid nitrogen and stored at -80°C. Concentrations of EVs were measured directly in blood by flow cytometry.

   Summary of Findings:

   Unstimulated EDTA and citrate blood had comparable concentrations of EVs derived from platelets (CD61+), activated platelet (P-selectin+), erythrocyte (CD235+) and leukocyte (CD45+). Platelet stimulation with TRAP-6 increased the concentration of platelet- and activated platelet (P-selectin+)-derived EVs; the increase was greater in citrate blood than EDTA blood (4.2-fold versus 1.3-fold, P=0.016; and 9.6-fold and 1.9-fold, P=0.031). The concentration of platelet-derived EVs was higher in citrate plasma than EDTA plasma, but no obvious differences were observed for other EV sources. Plasma from citrate blood transported by PTS had 1.9-fold higher concentration of platelet-derived EVs than case-matched plasma that was manually transported as blood (P=0.031) but concentrations of other EV sources(?) were unaffected. In contrast, the concentration of platelet-derived EVs was higher in plasma from EDTA blood transported by PTS than in case-matched specimens transported manually (P=0.031). Citrate and EDTA plasma that underwent a freeze-thaw cycle had lower concentrations of platelet- (26%, P=0.002 and 23%, P=0.002, respectively), erythrocyte- (31%, P=0.010 and 8% P=0.031, respectively), and leukocyte- (36%, P=0.010 and 27%, P=0.004, respectively) derived EVs than case-matched fresh specimens, but concentrations of activated platelet-derived EVs were comparable. A 6 h room temperature delay to centrifugation led to a 1.5-fold increase in platelet-derived EV concentrations in citrate plasma (P=0.031) and a 1.3-fold increase in erythrocyte-derived EV concentrations in EDTA plasma (P=0.031), but concentrations of other EV sources were not affected by a 6 h blood centrifugation delay at room temperature. A progressive increase in platelet-derived EV concentration was found when blood (EDTA or citrate) was stored for 24 h at room temperature before centrifugation. A 6 h 4°C centrifugation delay of citrate blood led to increases in platelet- and leukocyte- derived EV concentrations (10.4-fold, P=0.031 and 1.6-fold P=0.031, respectively), but a 6 h 4°C centrifugation delay of EDTA blood led to an increase in platelet- and erythrocyte-derived EV concentrations (5.4-fold, P=0.094 and 2.3-fold, P=0.031, respectively). A 24 h centrifugation delay at 4°C, resulted in increases in platelet-, erythrocyte-, and leukocyte-derived EVs in both EDTA and citrate plasma.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	Potassium EDTA Sodium citrate
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Storage	Freeze/thaw cycling	0 cycles 1 cycle
   Storage	Storage duration	0 h 6 h 24 h
   Storage	Storage temperature	4°C Room temperature
   Storage	Within hospital transportation method	Hand-delivered Pneumatic tube system
   Biospecimen Aliquots and Components	Blood processing method	TRAP-6 treated Untreated

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