Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Removal of platelets from blood plasma to improve the quality of extracellular vesicle research.

Author(s): Bettin B, Gasecka A, Li B, Dhondt B, Hendrix A, Nieuwland R, van der Pol E

Publication: J Thromb Haemost, 2022, Vol. 20, Page 2679-2685

PubMed ID: 36043239 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared platelet count and platelet- and erythrocyte- derived EV counts collected as part of three studies; results obtained before and after plasma filtration were also compared. This study used blood collected from three different cohorts. In the Antiplatelet Therapy Effect on Extracellular Vesicles (AFFECT EV) study, blood specimens were collected in trisodium citrate S-Monovette tubes using 19-gauge needles on three different days from 21 healthy volunteers and 60 patients following an acute myocardial. In the Healthy Donor study, blood specimens were collected with a 21-gauge needle from 10 healthy donors after an overnight fast in sodium citrate Vacuette, acid citrate dextrose solution A Vacutainer (ACD-A), K2EDTA Vacutainer, and K2EDTA Vacuette with separator tubes. In the study that investigated EV reference ranges, blood specimens were collected from 224 healthy volunteers using a 21-guage needle into EDTA tubes. For all three studies, plasma was separated by dual centrifugation at 2500g for 15 min.The AFFECT EV and EV reference range studies used Rotina 380R centrifuges and the Healthy Donor study used a 5810 R centrifuge. Plasma from the AFFECT and Healthy Donor studies were stored at -80°C, but plasma from the EV reference range study was analyzed immediately (fresh). To test the effect of filtration, platelets were removed from aliquots of fresh plasma collected as part of the Healthy Donor study using a 0.8 µm Isopure filter. Platelets were enumerated using flow cytometry and antibodies against CD-61. EVs were counted by flow cytometry using antibodies against CD-61 and CD235a to identify platelet- and erythrocyte-derived EVs, respectively.

   Summary of Findings:

   Platelet count varied widely in plasma specimens collected from the three different studies 6.0 × 10⁴ to 1.0 × 10⁸/mL. In plasma specimens from the AFFECT study that underwent a single freeze-thaw cycle, platelet count was modestly correlated with the concentration of platelet-derived EVs (R²=0.56), but not erythrocyte-derived EVs.  When plasma was filtered after centrifugation, the platelet count was 146-fold lower than when it was not filtered (P=0.0013), although platelet-derived EV concentration was only 0.24% lower (P=0.982).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Cardiovascular Disease
   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Filtration	Not filtered Filtered

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