Because of this, the results of all of the samples are presented together here. For the 16 identified miRNAs, we compared the miRNA plasma levels in samples from HBeAg positive, HBeAg negative, and healthy children. The miRNAs were consistently differentially expressed between the three groups (p<0.001). Interestingly, thorough levels of all miRNAs turned out to be significantly higher in samples from HBeAg positive children than from HBeAg negative children. All miRNAs had their lowest expression in healthy children. Results are shown in Figure 1. Figure 1 Levels of 16 identified miRNAs in plasma from HBeAg positive, HBeAg negative, and healthy children. Plasma levels of circulating miRNAs in HBeAg positive and HBeAg negative children respectively were calculated relative to healthy controls (Table 2).

Table 2 Plasma levels of circulating miRNAs in children with CHB relative to healthy controls. Blood samples from 35/60 children with CHB were not processed according to standard procedure. The samples were collected in EDTA tubes and immediately hereafter sent by mail to the Department of Clinical Biochemistry, Hvidovre Hospital, University of Copenhagen, Denmark for further processing. The freight implied a processing time of up to 48 hours from collection of blood samples to plasma isolation. To assess the impact of extended processing time on the plasma miRNA profile we compared the plasma levels of 16 miRNAs in 25 samples (16 HBeAg positive and 9 HBeAg negative) processed as generally recommended (within 4 hours of collection) and in 35 samples (18 HBeAg positive and 17 HBeAg negative) processed after a delay of up to 48 hours.

No difference was found (Table S3). The majority (53/60) of healthy controls submitted a blood sample immediately after initiation of anaesthesia (Thiopental). To investigate if the anaesthetic agents affected plasma miRNA levels, we compared the plasma levels of all 16 miRNAs in blood samples obtained before and after anaesthesia respectively. No difference was found (data not shown). Correlation between Circulating miRNAs and Clinical and Virological Parameters The HBeAg positive children were younger than the HBeAg negative children. We thus analysed the association between levels of circulating miRNAs and ages of children at sample date. The levels of miR-100, -122, and -122* were shown to correlate with age (p<0.001).

However, Carfilzomib this relationship was not confirmed when adjusted for HBeAg status (p=0.06, p=0.007, and p=0.06, respectively (Due to multiple testing only p<0.004 was regarded as statistically significant)). No correlation was found between plasma miRNA levels and gender or race (data not shown). We also investigated the relationship between circulating miRNAs and viral load and found a very strong positive correlation between plasma levels of all 16 miRNAs and HBV DNA (p<0.001). This correlation persisted when adjusted for age, gender, and ALT.