Overcoming haemolysis in the analysis of circulating miRNA expression

The development of molecular markers of papillary thyroid carcinoma (PTC) recurrence such as miRNAs has the potential to improve the clinical management of patients by assisting in risk stratification. Therefore, our aim was to identify specific circulating miRNAs that could be used as biomarkers of PTC recurrence. However, the quantification of miRNAs from plasma is difficult due to haemolysis which may have a substantial impact on detected miRNA level. Therefore, in this study we used different approaches to overcome haemolysis-associated inaccuracies in order to measure true circulating miRNA levels. We selected 3 miRNAs (miRNA- 146b, -222 and -21) which are overexpressed in PTC compared to healthy thyroid tissue and investigated the effect of haemolysis on the levels of these miRNAs by artificially introducing haemolysis in non-haemolysed plasma. We found out that miRNA-21 level depended greatly on haemolysis, while miRNA-146b and -222 were less affected. Then we measured miRNA levels in 65 blood plasma samples and found that using an absorbance at 414 nm of 0,25 as a cut-off to distinguish between the haemolysed and non-haemolysed plasma significantly decreased the variability in all three miRNAs levels (p < 0,05). Nine detected haemolysed samples were eliminated from further analysis and the rest 56 plasma samples were divided in 4 groups (samples collected before tumour removal, after removal, samples from patients with benign thyroid nodules and from healthy people). However, the levels of all analysed miRNAs were very similar between the groups. Hence, more research is needed to draw a conclusion about the potential of circulating miRNA- 146b, -222 and -21 as PTC biomarkers