Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Platelets confound the measurement of extracellular miRNA in archived plasma.

Author(s): Mitchell AJ, Gray WD, Hayek SS, Ko YA, Thomas S, Rooney K, Awad M, Roback JD, Quyyumi A, Searles CD

Publication: Sci Rep, 2016, Vol. 6, Page 32651

PubMed ID: 27623086 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of platelet contamination on miRNA levels and investigated the effects of freeze-thaw cycling of EDTA and citrate plasma before centrifugation to obtain PPP on platelet contamination, microparticle counts, and miRNA levels. To investigate the relationship between platelet counts and miRNA levels, fasting blood from 25 patients with peripheral artery disease and 27 case-matched controls was collected into EDTA tubes and centrifuged at 1800 x g f for 10 min to obtain plasma which was immediately frozen at -80°C. To investigate the effects of freeze-thawing plasma before isolation of PPP, blood was collected from six healthy patients into EDTA and citrate tubes and plasma was obtained by centrifugation at 1800 x g for 10 min. Half of each plasma specimen was immediately centrifuged at 1900 x g for 10 min to obtain PPP while the remainder was frozen at -80°C, thawed at 37°C, and then centrifuged to remove platelets.  miRNA was isolated using the miRNeasy Mini Kit and profiled using Exiqon’s Serum/Plasma Focus microRNA real-time PCR Panels. Microparticles were counted and characterized by flow cytometry. To investigate the effects of freeze-thaw on platelet membrane integrity, platelet pellets were resuspended in PBS, stored at room temperature or subjected to a single freeze-thaw cycle, centrifuged at 16100 x g for 20 min, and calcein was measured using a fluorescent-labelled antibody in a microplate reader. 

   Summary of Findings:

   Platelet counts were comparable between patients and controls but were modesty correlated with miRNA levels in plasma (R=0.47-0.60, P=0.00003-0.00216). PPP obtained from plasma after a freeze-thaw cycle had significantly higher numbers of microparticles as determined by calcein staining or sum of antibody staining than PPP obtained by centrifugation of fresh plasma (P= 0.001, both). Further investigation revealed an increase ­platelet (CD41+) microparticles (P=0.0007) but no change in leukocyte (CD45+), endothelial (CD146+), or RBC microparticle counts when plasma was freeze-thawed prior to the second centrifugation. PPP obtained by centrifugation of plasma after a freeze-thaw cycle instead of immediately also had higher levels of miR-21, miR-27b, and miR-425 (P<0.001, all), but levels of the RBC expressed miR-451 were unaffected. By comparing PPP to standard plasma it was determined that 99% of miR-27b, 89% of miR-425, 93% of miR-126-3p, and 96% of miR-21 was contributed to platelets, but only 1% of miR-451 was contributed by platelets.

   A single freeze-thaw cycle of plasma resulted in a 50% decrease in platelets and a consistent increase in platelet microparticles, but there was no effect of freeze-thaw cycling on PPP. Centrifugation of the thawed plasma removed most (not all) of the remaining platelets, but approximately 1/3 of platelet microparticles remained.  Similarly, freeze-thaw cycling of plasma from two patients resulted in increased miR-21 in the supernatant and microparticles but the magnitude of the increase depended on the subject with one subject showing a larger increase in miR-21 in the supernatant and the other in the microparticles.  A single-freeze thaw cycle of platelets suspended in PBS resulted in a large increase in calcein levels in the supernatant that the authors state was evidence of loss of membrane integrity.

   Although EDTA and citrated PPP had comparable levels of miRNA, EDTA PPP had more RBC-derived microparticles than citrate PPP. PPP prepared from freeze-thaw cycled EDTA plasma had much higher levels of platelet-derived miRNA (miR-27b, miR-21, and miR-126-5p) than PPP prepared from freeze-thaw cycled citrated plasma (P<0.001, P<0.0001, and P<0.01).  Importantly, levels of miRNA were not significantly different in citrated PPP obtained with or without freeze-thaw cycling of the plasma.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Cardiovascular Disease
   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Platelet-poor plasma
   Biospecimen Acquisition	Anticoagulant	EDTA Citrate
   Storage	Freeze/thaw cycling	0 cycles 1 cycle
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-21 miR-27b miR-126-3p miR-425 miR-451

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