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Choice of blood collection methods influences extracellular vesicles counts and miRNA profiling.

Author(s): Tran V, de Oliveira-Jr GP, Chidester S, Lu S, Pleet ML, Ivanov AR, Tigges J, Yang M, Jacobson S, Gonçalves MCB, Schmaier AA, Jones J, Ghiran IC

Publication: J Extracell Biol, 2024, Vol. 3, Page e70008

PubMed ID: 39440167 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the abundance, size, antigenic profile, and miRNA expression among extracellular vesicles (EVs) isolated from EDTA, citrate, and heparin plasma and serum obtained from healthy men and women and examined whether differences were present based on sex, and individual centrifugation steps during EV separation from other particles, and inclusion of an additional freeze-thaw cycle after 3 months of frozen storage. Blood was collected from 20 healthy fasting volunteers (15 men and 5 women; 23-52 years of age) using a 21-gauge butterfly needle into K₂EDTA, Sodium Citrate (3.2%), PST Gel Lithium Heparin and Serum SST Vacutainer tubes. All tubes were inverted 6-8 times before separation of plasma and serum by centrifugation at 500 g for 10 min at room temperature; cellular material was then removed by centrifugation at 2,500 g for 5 min followed by 12,500 g for 15 min at room temperature. To test if further centrifugation could reduce lipoprotein contamination, an additional centrifugation at 260,000 g for 24 h was performed for some specimens. To investigate the effects of freeze-thaw cycling and frozen storage, specimens were analyzed before and after frozen storage at -20°C for 3 months. RNA was isolated using the miRNeasy Serum/Plasma Advanced Kit and reverse transcribed using the TaqMan microRNA reverse transcription Kit. Levels of 28 miRNAs were quantified in triplicate using real-time PCR using TaqMan-advanced miRNA assays and normalized to miR-4433b-3p. EVs were isolated from the plasma/serum of four volunteers by centrifugation at 2500 g for 10 min and then 12,000 g for 20 min followed by size exclusion chromatography with qEV original 35 nm EV isolation columns. EVs were stained with Acoerela 490 dye and characterized by flow cytometry using a CytoFLEX S. EVs from the plasma/serum of 6 volunteers were further characterized by MACSPlex Human Exosome Multiplex Assay Kit, which uses 39 fluorescent capture beads with antibodies against CD9, CD63, and CD81. EVs were visualized by dark field microscopy using an Olympus BX62 microscope.

   Summary of Findings:

   The presence of red blood cell ghosts, platelets and cell debris in plasma obtained by centrifugation at 500 g for 10 min followed by 2,500 g for 5 min were visible by dark field microscopy, but these were effectively removed by subsequent centrifugation at 12,500 g for 15 min. While light scattering particles remained after centrifugation at 12,500 g for 15 min, they were also present after an additional centrifugation step at 260,000 g for 24 h. The authors postulated that these light scattering particles are likely low-density lipoprotein complexes, as the Acoerela-labelled EVs were pelleted by the ultra high-speed centrifugation step. More EVs were isolated from serum (6.74 × 105 events/uL) than citrate (1.25 × 105 events/uL), heparin (7.88 × 104 events/uL), or EDTA (7.56 × 104 events/uL) plasma (P<0.01, all). The relative EV diameter range was higher for EVs from serum (80.8–210.2 nm) than those from citrate (155.4–171.9 nm), heparin (149.8–164.6 nm), or EDTA (161.1–181.3 nm) plasma, but only the difference between serum and EDTA EVs was significant (P<0.05). The mean fluorescent intensity of Acoerela labeling was also higher for EVs from serum than plasma. The effects of freeze-thaw cycling on EV surface marker detection were small. Overall, antigenic expression levels were very strongly correlated (r=0.9461-0.9962) in specimens before and after an additional freeze-thaw cycle, regardless of anticoagulant (serum and EDTA, citrate and heparin plasma) or patient sex; however, small changes in CD63/CD3 detection were observed regardless of patient gender, and changes in CD9/ROR1 detection were observed in specimens from females. Clustering based on antigenic profile did not separate specimens based on patient sex.

   EVs captured and characterized by MACSPlex analysis were very similar among plasma obtained with different anticoagulants, indicating similar surface antigens. Serum had more EVs captured with antibodies against CD2, CD41b, CD41, CD62p, or HLA-ABC and detected with antibodies against CD9 and/or CD63 than plasma, but the significance of the differences was dependent on anticoagulant type; the authors noted that the detection antibody signal was not evenly distributed, indicating at that not all  epitopes on EVs were detected.

   miRNA expression was highest in EVs from EDTA plasma and lowest in those from heparin plasma; principal component analysis based on miRNA expression clustered specimens by anticoagulant.  A few miRNAs that had higher expression in serum than EDTA plasma were identified (miR-511-5p, miR−381-3p, miR −1-5p, miR −511-3p, miR −4662a-5p, and miR−548j-5p).

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   Cell count/volume	Light scattering
   Protein	Flow cytometry
   RNA	Low density array

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient gender	Female Male
   Biospecimen Acquisition	Type of collection container/solution	K2EDTA Vacutainer SST Vacutainer tubes Sodium Citrate Vacutainer tubes PST Gel Lithium Heparin tube
   Storage	Freeze/thaw cycling	1 cycle 2 cycles
   Biospecimen Aliquots and Components	Centrifugation	Different number of centrifugation steps compared
   Storage	Storage duration	Unspecified An additional 3 months frozen storage
   Biospecimen Acquisition	Anticoagulant	Lithium heparin Sodium citrate None Potassium EDTA

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2. Study Purpose

   This study compared the antigenic composition of EVs isolated from serum, K₂EDTA plasma, and plasma from different Streck cell stabilization tubes (DNA BCT, RNA Complete, and Nucleic Acid).  Blood was collected from five volunteers using a 21-gauge butterfly needle into Streck DNA BCT, Streck RNA Complete, and Streck nucleic acid tubes as well K₂EDTA and SST Vacutainer tubes. Plasma was separated using manufacturer protocols (Streck RNA Complete BCT and DNA BCT tubes: 300 g for 20 min followed by 5000 g for 10 min; Streck RNA and nucleic acid tubes: 1800 g for 15 followed by 2800 g for 15 min). RNA was isolated using the miRNeasy Serum/Plasma Advanced Kit. EVs isolated from plasma/serum were characterized using 10 (for abundant) and 50 µL (for scarce) by the MACSPlex Human Exosome Multiplex Assay Kit, which uses 39 fluorescent capture beads with antibodies against CD9, CD63, and CD81.

   Summary of Findings:

   EVs isolated from plasma from Streck cell-stabilizing tubes had more platelet-derived EVs (CD62P and CD49a) than EDTA plasma, with the largest difference relative to EVs from EDTA plasma observed when Streck DNA BCT were used and the smallest difference when Streck Nucleic Acid tubes were used. Further analysis found no significant differences between EVs isolated from EDTA and Streck Nucleic Acid plasma tubes, but a trend toward higher variability in relative fluorescent intensity between the two volumes used as input (10 versus 50 µL) was observed, which the authors state may indicate dose-dependent interference of the proprietary fixative with antibody binding.

    

   Biospecimens

   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Streck/BCT
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Flow cytometry
   Cell count/volume	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	Streck RNA Complete tube Streck Nucleic Acid tube Streck DNA BCT K2EDTA Vacutainer SST Vacutainer tubes

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