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Irreversible alteration of extracellular vesicle and cell-free messenger RNA profiles in human plasma associated with blood processing and storage.

Author(s): Kim HJ, Rames MJ, Tassi Yunga S, Armstrong R, Morita M, Ngo ATP, McCarty OJT, Civitci F, Morgan TK, Ngo TTM

Publication: Sci Rep, 2022, Vol. 12, Page 2099

PubMed ID: 35136102 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The study compared EV profiles and mRNA expression among case-matched single-spun and double-spun plasma obtained using four different anticoagulants (ACD-A, K₂EDTA, heparin, and sodium citrate) before and after a single-freeze thaw event and in single-spun plasma after inclusion of a post-thaw centrifugation step. Blood was collected from 3 or more healthy volunteers using a 21-gauge butterfly needle into ACD-A, K₂EDTA, heparin, or sodium citrate tubes. All tubes were transported vertically and stored for less than 1 h before separation of plasma by centrifugation at 1000 g for 10 min with the highest acceleration and deceleration setting. Double-spun plasma was obtained by re-centrifugation at 15,000 g for 10 min. To test the effects of freezing, aliquots of single-spun and double-spun plasma were frozen at -80°C and thawed once before analysis. Some aliquots of single-spun plasma were subjected to a second centrifugation at 15,000 g for 10 min at room temperature after thawing. Platelet count was analyzed using a hemocytometer. EVs were characterized by flow cytometry using antibodies against CD-9, CD63 and CD41. Particle diameter was analyzed by light scatter on a flow cytometer. RNA was extracted from plasma using the Norgen Plasma/serum Circulating and Exosomal RNA Purification Kit and sixteen mRNAs were quantified using custom TaqMan assays.

   Summary of Findings:

   The hemocytometer showed that single-spun K₂EDTA plasma retained 313 ± 74 thousand platelets per μl, but 99.99% of these were removed after a second centrifugation. Similarly, the count of CD41+ platelets was 127 ± 32 thousand/μl after one spin, but the second spin removed 99.90% of the remaining platelets.  Double-spun K₂EDTA also had fewer 1000-3000 nm EVs than single-spun K₂EDTA plasma but the number of 500-1000 nm EVs was comparable between single-spun and double-spun K₂EDTA plasma. There were distinct subpopulations of EVs, with fewer 150-1000 nm EVs that were CD41+ than CD9+ or CD63+, in both single and double spun K₂EDTA plasma. After a single freeze-thaw event of single-spun K₂EDTA plasma, the number of CD9+ EVs that were 150-1000 nm increased 5.5-fold and those that were 1000-3000 nm increased 3-fold, but in double-spun K₂EDTA plasma no significant increase in CD9+EVs was found after a single freeze-thaw event. After a single freeze-thaw event of singe-spun K₂EDTA plasma, the number of CD63+ EVs and CD41+ EVs between 1000-3000 nm increased 2.4- and 16.3-fold, respectively, but a single freeze-thaw event did not affect the number of CD63+ EVs or CD41+ EVs between 150-1000 nm in single-spun K₂EDTA plasma or either population in double-spun K₂EDTA plasma.

   A second centrifugation and a single freeze-thaw event elicited generally similar effects among the anticoagulants tested but the magnitude of the effects depended on the anticoagulant used and EV type.  Regardless of anticoagulant, a freeze-thaw event had little to no discernable effect on EV counts in double-spun plasma. A single freeze-thaw event of single-spun plasma caused a >2-fold increase in 150–1000 nm CD9+ and CD41+ EVs, regardless of the anticoagulant used but did not change the number of 150–1000 nm CD63+ EVs; differences in 1000-3000 nm EVs in single-spun plasma were less pronounced. The smallest differences observed among 1000–3000 nm CD9+ and CD41+ EVs following a freeze-thaw cycle of single-spun plasma occurred in sodium citrate plasma, while the smallest differences in 1000–3000 nm CD63+ EVs was observed in ACD plasma.  

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • Frozen

   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 Acquisition	Anticoagulant	Acid-citrate-dextrose Heparin Potassium EDTA Sodium citrate
   Biospecimen Aliquots and Components	Centrifugation	Different number of centrifugation steps compared
   Storage	Freeze/thaw cycling	0 cycles 1 cycle
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)
   Storage	Storage conditions	Before second centrifugation After second centrifugation

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