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Platelet removal from human blood plasma improves detection of extracellular vesicle-associated miRNA.

Author(s): Bracht JWP, Los M, van Eijndhoven MAJ, Bettin B, van der Pol E, Pegtel DM, Nieuwland R

Publication: J Extracell Vesicles, 2023, Vol. 12, Page e12302

PubMed ID: 36788785 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare platelet and erythrocyte concentrations, extracellular vesicle (EV) distributions, and EV- and platelet-associated miRNA expression before and after filtration of plasma and SEC-enriched EVs. Importantly, plasma was obtained by two different centrifugation protocols (ISTH and non-ISTH).  Blood was collected from three healthy donors into EDTA tubes.  Plasma was obtained using the ISTH protocol (2x centrifugation at 2500 g for 15 min with no brake) and the non-ISTH protocol (centrifugation at 900 g for 7 min with maximum brake followed by centrifugation 2500 g for 10 min with maximum brake). Importantly, in the ISTH protocol exactly 10 mm was left behind, but the non-ISTH protocol left approximately 10 mm behind. Plasma obtained using each protocol was then either filtered through a 0.8 µm filter directly or after EV enrichment by size-exclusion chromatography. The concentrations of platelets (CD61+, CD41+), activated platelets (CD61+ and CD62p+), erythrocyte ghosts (CD235a+), and EVs were quantified by flow cytometry. miRNA expression was evaluated using real-time PCR amplification of let7a-5p, miR-21-5p and miR-223-3p.

   Summary of Findings:

   Plasma obtained by the ISTH protocol contained 3×10⁶ platelets/ml and 8 × 10⁵ erythrocyte ghosts/ml and non-ISTH plasma contained similar concentrations of platelets and erythrocyte ghosts. Platelets (6 × 10⁶ /mL) and erythrocyte ghosts (3 × 10⁵/mL) were both present after EV enrichment by SEC, regardless of the centrifugation protocol used to obtain plasma. Filtration of plasma or SEC-enriched EVs removed >97% of platelets (P=0.03, both) and erythrocyte ghosts (P=0.05 and P=0.04, respectively) without decreasing the concentration of platelet-derived (CD61 +) or erythrocyte-derived (CD235a+) EVs.  Importantly, 1-2% of platelet were activated (CD61+ and CD62p+) both before and after filtration, indicating that filtration did not cause activation or selectively remove activated or non-activated platelets. The size distribution of EVs was also unaffected by filtration. Cq values of EV-associated (let7a-5p and miR-21-5p) and platelet-associated (miR-223-3p) miRNAs decreased by 1-2 cycles after filtration of plasma or SEC-enriched EVs, but the change in miR-21-5p was not significant in plasma. The concentration of platelets in plasma and SEC-enriched EVs was strongly correlated with the Cq for miR-223-3p (R² =0.76, P=0.023 and R² =0.73, P=0.030, respectively), miR-21-5p (R² =0.82, P=0.013 and R² =0.92, P=0.002, respectively) and let-7a-5p (R² =0.90, P=0.004 and R² =0.97, P<0.001, respectively), although miRNA levels and concentrations of erythrocyte ghosts or EVs were not correlated in plasma or SEC-enriched EVs. Finally, the concentrations of platelet-derived (CD61+), erythrocyte-derived (CD235a+) and leukocyte-derived (CD45+) EVs did not correlate with miRNA concentrations.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   Cell count/volume	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Multiple brake speeds compared Multiple durations compared Multiple speeds compared
   Biospecimen Aliquots and Components	Filtration	0.8 µM filter No filter
   Real-time qRT-PCR Specific	Targeted nucleic acid	let7a-5p miR-21-5p miR-223-3p
   Analyte Extraction and Purification	Analyte isolation method	SEC None
   Biospecimen Aliquots and Components	Blood and blood products	Extracellular vesicles Plasma

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