title: Plasma processing conditions substantially influence circulating microRNA biomarker levels.
abstract: Circulating, cell-free microRNAs (miRNAs) are promising candidate biomarkers, but optimal conditions for processing blood specimens for miRNA measurement remain to be established. Our previous work showed that the majority of plasma miRNAs are likely blood cell-derived. In the course of profiling lung cancer cases versus healthy controls, we observed a broad increase in circulating miRNA levels in cases compared to controls and that higher miRNA expression correlated with higher platelet and particle counts. We therefore hypothesized that the quantity of residual platelets and microparticles remaining after plasma processing might impact miRNA measurements. To systematically investigate this, we subjected matched plasma from healthy individuals to stepwise processing with differential centrifugation and 0.22 µm filtration and performed miRNA profiling. We found a major effect on circulating miRNAs, with the majority (72%) of detectable miRNAs substantially affected by processing alone. Specifically, 10% of miRNAs showed 4-30x variation, 46% showed 30-1,000x variation, and 15% showed >1,000x variation in expression solely from processing. This was predominantly due to platelet contamination, which persisted despite using standard laboratory protocols. Importantly, we show that platelet contamination in archived samples could largely be eliminated by additional centrifugation, even in frozen samples stored for six years. To minimize confounding effects in microRNA biomarker studies, additional steps to limit platelet contamination for circulating miRNA biomarker studies are necessary. We provide specific practical recommendations to help minimize confounding variation attributable to plasma processing and platelet contamination.
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.
authors.forename: Heather H
authors.initials: HH
authors.lastname: Cheng
authors.forename: Hye Son
authors.initials: HS
authors.lastname: Yi
authors.forename: Yeonju
authors.initials: Y
authors.lastname: Kim
authors.forename: Evan M
authors.initials: EM
authors.lastname: Kroh
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chien
authors.forename: Keith D
authors.initials: KD
authors.lastname: Eaton
authors.forename: Marc T
authors.initials: MT
authors.lastname: Goodman
authors.forename: Jonathan F
authors.initials: JF
authors.lastname: Tait
authors.forename: Muneesh
authors.initials: M
authors.lastname: Tewari
authors.forename: Colin C
authors.initials: CC
authors.lastname: Pritchard
mesh_terms: D015415:Biomarkers / Q000097:blood; D001792:Blood Platelets / Q000378:metabolism; D001800:Blood Specimen Collection / Q000379:methods*; D016022:Case-Control Studies; D055252:Cell-Derived Microparticles / Q000378:metabolism; D005260:Female; D005374:Filtration; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood / Q000235:genetics; D035683:MicroRNAs / Q000097:blood*; D010949:Plasma / Q000378:metabolism*; D011786:Quality Control
chemical_list: D015415:Biomarkers; D035683:MicroRNAs