Plasma Processing Conditions Substantially Influence Circulating microRNA Biomarker Levels.

Author(s): Cheng HH, Yi HS, Kim Y, Kroh EM, Chien JW, Eaton KD, Goodman MT, Tait JF, Tewari M, Pritchard CC

Publication: PLoS One, 2013, Vol. 8, Page e64795

PubMed ID: 23762257 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of plasma centrifugation steps, filtration of PPP and frozen storage of plasma prior to additional processing on platelet counts and levels of miRNA. K2EDTA plasma and serum were aliquoted and frozen at -80 degrees C before analysis. miRNA was extracted using miRNeasy and frozen at -80 degrees C until analysis using real-time qPCR arrays and individual real-time qPCR assays. Plasma and platelet rich plasma (PRP) were obtained by centrifugation at 3400 x g or 600 x g, respectively. Additional plasma specimens were frozen at -80 degrees C for up to 6 years before centrifugation at 1940 x g for 10 min to produce PPP.

   Summary of Findings:

   Centrifugation of plasma at 1940 x g for 10 min to produce PPP removed 80-90% of the platelets in fresh plasma or plasma that had been stored at -80 degrees C for up to 6 years before centrifugation. When PPP was then filtered, >99% of the platelets in the initial plasma were removed. PRP produced by centrifugation at 600 x g, instead of the 3400 x g used for standard plasma, had 13-19 fold more platelets than standard plasma. Along with platelet removal, a non-significant decline in microparticle counts was observed. The numbers of miRNAs detected in platelet concentrates, PRP, standard plasma, PPP, and filtered PPP were 315, 325, 277, 279, and 262, respectively. Of the 282 miRNAs detected in multiple specimen types, 72% were expressed differentially between specimen types, with 10% differing by 4-30 fold, 46% by 30-1000 fold and 15% by more than 1000 fold. miR-142-3p, let-7a, miR-233 and miR-16 were highly affected by plasma processing steps, but miR-451 and miR-122 were only minimally affected by plasma processing steps, and this was true regardless of whether or not plasma was stored for up to 6 years at -80 degrees C before additional centrifugation steps. Importantly, the rank order of miRNA expression was significantly different between standard plasma and filtered PPP (p<0.0001). The miRNAs most affected by plasma processing were moderately correlated with the miRNA most highly expressed in platelets (r=0.43, p<0.0001).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen
   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Coulter counter
   Cell count/volume	Hematology/ auto analyzer
   RNA	Real-time qRT-PCR
   RNA	Low density array

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Filtration	Unfiltered 0.22 uM filtered
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Platelet-poor plasma Platelet-rich plasma Platelets
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)
   Real-time qRT-PCR Specific	Targeted nucleic acid	let7a miR-16 miR-92a miR-122 miR-124 miR-233 miR-205 miR-210 miR-251
   Biospecimen Aliquots and Components	Centrifugation	Multiple durations compared Different number of centrifugation steps compared

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2. Study Purpose

   The purpose of this study was to determine the effects of filtration on platelet counts and levels of miRNA in serum and to compare miRNA profiles in serum and plasma. Serum and plasma were aliquoted and frozen at -80 degrees C before analysis. miRNA was extracted using miRNeasy and frozen at -80 degrees C until analysis using real-time qPCR arrays and individual real-time qPCR assays.

   Summary of Findings:

   Filtration removed microparticles from serum but only slightly decreased the platelet counts. 6% of miRNAs were differentially expressed by more than 4 fold between filtered and unfiltered serum. Specifically, the authors show miR-142-3p and let-7a were affected by filtration, and miR-223, miR-16, and miR-122 were unaffected by filtration of the serum. 6% of miRNAs were differentially expressed between serum and plasma.

   Biospecimens

   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Coulter counter
   Cell count/volume	Hematology/ auto analyzer
   RNA	Real-time qRT-PCR
   RNA	Low density array

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Filtration	Unfiltered 0.22 um filtered
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum
   Real-time qRT-PCR Specific	Targeted nucleic acid	let7a miR-16 miR-122 miR-142-3p miR-233 miR-451

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