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Published on: October 26, 2017
Platelets confound the measurement of extracellular miRNA in archived plasma
Adam J Mitchell1, Warren D Gray1, Salim S Hayek1
1Division of Cardiology, Department of Medicine, Emory University, Atlanta, GA, USA.
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.
Insights
Archived plasma samples can be contaminated by platelets during freeze-thaw cycles, affecting extracellular miRNA biomarker measurements. Proper platelet removal before freezing is crucial for accurate miRNA analysis in disease biomarker studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Biomarker Discovery
Background:
- Extracellular microRNAs (miRNAs) are promising disease biomarkers found in biofluids.
- Archived plasma is frequently used for miRNA biomarker discovery, but processing and storage effects are not well understood.
- Platelet contamination in plasma samples can confound extracellular miRNA measurements.
Purpose of the Study:
- To investigate the impact of freeze-thaw cycles on microparticle (MP) levels and extracellular miRNA concentrations in plasma.
- To determine if post-thaw processing effectively removes platelet contamination affecting miRNA detection.
- To assess the reliability of extracellular miRNA measurements in archived plasma samples.
Main Methods:
- Analysis of archived plasma samples using a case-control study design.
- Evaluation of microparticle (MP) counts and extracellular miRNA levels after a single freeze-thaw cycle.
- Correlation analysis between miRNA levels and platelet counts.
Main Results:
- A single freeze-thaw cycle significantly increases platelet-derived MPs in plasma.
- This increase in MPs contaminates the extracellular miRNA pool, altering detected miRNA levels.
- A correlation between miRNA levels and platelet counts persisted despite post-thaw processing.
Conclusions:
- Extracellular miRNA measurements in archived plasma are critically affected by platelet contamination from freeze-thaw cycles.
- Residual platelets must be removed before freezing plasma for accurate miRNA biomarker studies.
- Caution is advised when interpreting results from previous studies using archived plasma without stringent platelet removal protocols.
