Haemolysis during Sample Preparation Alters microRNA Content of Plasma

Abstract

The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers.

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“…One issue that we would like to reiterate is the potential of contaminating cellular material, which has a higher concentration of miRNAs than plasma or serum, to confound miRNA quantitation (Kroh et al 2010). This contamination could arise from the presence of intact blood cells in serum/plasma or lysis of these cells (hemolysis) during sampling and processing (Duttagupta et al 2011, Kirschner et al 2011, McDonald et al 2011. Recent work found that an additional centrifuge step after plasma preparation efficiently removes intact blood cells and a significant fraction of contaminating cellular miRNAs (Duttagupta et al 2011).…”

“…Hemolysis, which frequently occurs at low levels during collection of serum/plasma, is a more difficult problem to circumvent. Two studies have highlighted the profound effect hemolysis can have on miRNA concentrations in blood fluids (Kirschner et al 2011, McDonald et al 2011. The extent of hemolysis can be estimated by the measurement of free hemoglobin and certain miRNAs (e.g.…”

“…The extent of hemolysis can be estimated by the measurement of free hemoglobin and certain miRNAs (e.g. miR-15b, miR-16, and miR-451), which may be necessary to determine whether a serum/plasma sample is suitable for analysis (Kirschner et al 2011, McDonald et al 2011. We believe that standardization of miRNA isolation from serum/plasma and other bodily fluids, taking into account factors such as phlebotomy protocol, sample processing, and miRNA extraction methodology, is one of the most urgent requirements for preclinical experimentation and will maximize the translatability of these studies.…”

“…However, there is currently no consensus on suitable small RNA reference genes in circulation (Kroh et al 2010). A number of studies have used miR-16 for normalization purposes (Lawrie et al 2008, but more recent work has found that this molecule is highly expressed in erythrocytes and that its levels in the cell-free content of blood can vary significantly due to hemolysis (Kirschner et al 2011, McDonald et al 2011. Our current preference is to process all samples from identical input volumes and then correct for technical variability of the RNA extraction using spiked-in control miRNAs from Caenorhabditis elegans that lack human homologs, as originally proposed by the Tewari Laboratory (Mitchell et al 2008).…”

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“…Hemolysis during sample preparation can alter the microRNA content of plasma or serum. 10 In addition, small differences in duration and centrifugal force can create differences in the presence of platelets in plasma, ranging from platelet rich to platelet poor or free plasma, which has an impact on the microRNA repertoire.…”