Platelets confound the measurement of extracellular miRNA in archived plasma

Abstract

Extracellular miRNAs are detectable in biofluids and represent a novel class of disease biomarker. Although many studies have utilized archived plasma for miRNA biomarker discovery, the effects of processing and storage have not been rigorously studied. Previous reports have suggested plasma samples are commonly contaminated by platelets, significantly confounding the measurement of extracellular miRNA, which was thought to be easily addressed by additional post-thaw plasma processing. In a case-control study of archived plasma, we noted a significant correlation between miRNA levels and platelet counts despite post-thaw processing. We thus examined the effects of a single freeze/thaw cycle on microparticles (MPs) and miRNA levels, and show that a single freeze/thaw cycle of plasma dramatically increases the number of platelet-derived MPs, contaminates the extracellular miRNA pool, and profoundly affects the levels of miRNAs detected. The measurement of extracellular miRNAs in archived samples is critically dependent on the removal of residual platelets prior to freezing plasma samples. Many previous clinical studies of extracellular miRNA in archived plasma should be interpreted with caution and future studies should avoid the effects of platelet contamination.

Circulating serum miR-223-3p and miR-16-5p as possible biomarkers of early rheumatoid arthritis

“…In contrast to our results, the concentrations of miR-223-3p were reported to be increased significantly in plasma of RA patients in another study [37]. It has been shown recently that plasma samples are commonly contaminated by platelets and a single freeze/thaw cycle of plasma increases the number of platelet-derived microparticles dramatically, contaminates the extracellular miRNA pool and affects the levels of miRNA detection [38]. It has been shown recently that plasma samples are commonly contaminated by platelets and a single freeze/thaw cycle of plasma increases the number of platelet-derived microparticles dramatically, contaminates the extracellular miRNA pool and affects the levels of miRNA detection [38].…”

Preanalytic processing of rat plasma influences thrombin generation and fibrinolysis assays

“…High residual platelet and PMP counts in PPP typically shorten lag time and enhance peak thrombin and ETP. Moreover, freeze–thaw cycling induces platelet activation and membrane fragmentation, and generates increased PMP in the thawed plasma that were not present after initial plasma centrifugation . Enhanced platelet and PMP‐mediated thrombin formation depend on reagent type, however, with TGA configured with “high” (ie, > 5 pM) tissue factor trigger reagents demonstrating more limited effects of sample processing .…”

Standardization procedure for plasma biomarker analysis in rat models of epileptogenesis: Focus on circulating microRNAs

“…At this speed, most platelets will be removed, because much lower centrifugation speeds (e.g., 600 × g ) are used to obtain platelets . This is important, as platelets have a large repertoire of miRNAs and are the major source of miRNAs in plasma, which can influence miRNA assessment. However, plasma may still contain residual platelets, and therefore, we recommend a second centrifugation (3000 × g , 10 min at 4°C) to remove residual platelets .…”

“…In case archived samples were frozen after the first centrifugation step, the second step can be applied after thawing, which effectively removes platelet contamination from these samples . However, it should be noted that the residual frozen platelets may have released miRNAs into plasma …”

“…13 This is important, as platelets have a large repertoire of miRNAs and are the major source of miRNAs in plasma, 14 which can influence miRNA assessment. However, plasma may still contain residual platelets, 13,14 and therefore, we recommend a second centrifugation (3000 9 g, 10 min at 4°C) to remove residual platelets. 14,15 In case archived samples were frozen after the first centrifugation step, the second step can be applied after thawing, which effectively removes platelet contamination from these samples.…”