Impact of Anticoagulation and Sample Processing on the Quantification of Human Blood-Derived microRNA Signatures.

Marion Mussbacher,Alice Assinger,Hans-Peter Holthoff,Teresa L Krammer,Patrick Starlinger,Matthias Hackl,Waltraud C Schrottmaier,Stephan Zeibig,David Pereyra,Stefan Heber

doi:10.3390/cells9081915

Abstract

Blood-derived microRNA signatures have emerged as powerful biomarkers for predicting and diagnosing cardiovascular disease, cancer, and metabolic disorders. Platelets and platelet-derived microvesicles are a major source of microRNAs. We have previously shown that the inappropriate anticoagulation and storage of blood samples causes substantial platelet activation that is associated with the release of platelet-stored molecules into the plasma. However, it is currently unclear if circulating microRNA levels are affected by artificial platelet activation due to suboptimal plasma preparation. To address this issue, we used a standardized RT-qPCR test for 12 microRNAs (thrombomiR®, TAmiRNA GmbH, Vienna, Austria) that have been associated with cardiovascular and thrombotic diseases and were detected in platelets and/other hematopoietic cells. Blood was prevented from coagulating with citrate–theophylline–adenosine–dipyridamole (CTAD), sodium citrate, or ethylenediaminetetraacetic acid (EDTA) and stored for different time periods either at room temperature or at 4 °C prior to plasma preparation and the subsequent quantification of microRNAs. We found that five microRNAs (miR-191-5p, miR-320a, miR-21-5p, miR-23a-3p, and miR-451a) were significantly increased in the EDTA plasma. Moreover, we observed a time-dependent increase in plasma microRNAs that was most pronounced in the EDTA blood stored at room temperature for 24 h. Furthermore, significant correlations between microRNA levels and plasma concentrations of platelet-stored molecules pointed towards in vitro platelet activation. Therefore, we strongly recommend to (i) use CTAD as an anticoagulant, (ii) process blood samples as quickly as possible, and (iii) store blood samples at 4 °C whenever immediate plasma preparation is not feasible to generate reliable data on blood-derived microRNA signatures.

Highlights

Over the last decade, circulating microRNAs have emerged as powerful biomarkers to predict and diagnose cardiovascular and thrombotic disease [1]
To assess whether the choice of anticoagulant during blood collection affected the plasma levels of miRNAs, we compared selected miRNAs that have been associated with alterations of platelet function between citrate–theophylline–adenosine–dipyridamole (CTAD), sodium citrate, and dipotassium ethylenediaminetetraacetic acid (EDTA) collection tubes (Figure 1)
When we analyzed the effect of temperature (RT vs. 4 ◦ C) on immediate plasma preparation (Figure S4), we found that temperature had a minor effect on circulating miRNA levels

Summary

Introduction

Over the last decade, circulating microRNAs (miRNAs) have emerged as powerful biomarkers to predict and diagnose cardiovascular and thrombotic disease [1]. Platelets are anucleate cells, they contain disproportionately high amounts of miRNAs relative to their low level of protein synthesis [2]. Platelets are currently estimated to contain about 750 different miRNAs [3]. These platelet-derived miRNAs can be detected in circulation where they significantly contribute to the pool of miRNAs in the plasma. The majority of plasma-derived miRNA is either bound to proteins or encapsulated in vesicles (e.g., extracellular vesicles, liposomes, and lipoproteins) and effectively protected from degradation by endogenous RNases [4]. Circulating miRNAs are considered biochemically stable and show superior performance as biomarkers when compared to mRNAs [5]

Methods

Results

Discussion

Conclusion