Relevance of pre-analytical blood management on the emerging cardiovascular protein biomarkers TWEAK and HMGB1 and on miRNA serum and plasma profiling.

BACKGROUND: Disease-independent sources of biomarker variability include pre-analytical, analytical and biological variance. The aim of the present study was to evaluate whether the pre-analytical phase has any impact on the emerging heart disease TWEAK and HMGB1 protein markers and miRNA biomarkers, and whether peptidome profiling allows the identification of pre-analytical quality markers. METHODS: An assessment was made of sample type (serum, EDTA-Plasma, Citrate-Plasma, ACD-plasma, Heparin-plasma), temperature of sample storage (room temperature or refrigerated), time of sample storage (0.5, 3, 6 and 9h) and centrifugation (one or two-step). Aliquots of all processed samples were immediately frozen (-80°C) before analysis. Proteins were assayed by ELISAs, miRNA expression profile by microarray and peptidome profiling by MALDI-TOF/MS. RESULTS: Temperature, time and centrifugation had no impact on TWEAK and HMGB1 results, which were significantly influenced by matrix type, TWEAK levels being significantly higher (F=194.7, p<0.0001), and HMGB1 levels significantly lower (F=36.32, p<0.0001) in serum than in any other plasma type. Unsuitable miRNA results were obtained using Heparin-plasma. Serum miRNA expression profiles depended mainly on temperature, while EDTA-plasma miRNA expression profiles were strongly affected by the centrifugation method used. MALDI-TOF/MS allowed the identification of seven features as indices of pre-analytical serum (m/z at 1206, 1350, 1865 and 2021) or EDTA-plasma (m/z 1897, 2740 and 2917) degradation. CONCLUSIONS: Serum and EDTA-plasma allow the analysis of both proteins and miRNA emerging biomarkers of heart diseases. Refrigerated storage prevents an altered miRNA expression profile also in cases of a prolonged time-interval between blood drawing and processing.

Multiple sclerosis: microRNA expression profiles accurately differentiate patients with relapsing-remitting disease from healthy controls.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system, which is heterogenous with respect to clinical manifestations and response to therapy. Identification of biomarkers appears desirable for an improved diagnosis of MS as well as for monitoring of disease activity and treatment response. MicroRNAs (miRNAs) are short non-coding RNAs, which have been shown to have the potential to serve as biomarkers for different human diseases, most notably cancer. Here, we analyzed the expression profiles of 866 human miRNAs. In detail, we investigated the miRNA expression in blood cells of 20 patients with relapsing-remitting MS (RRMS) and 19 healthy controls using a human miRNA microarray and the Geniom Real Time Analyzer (GRTA) platform. We identified 165 miRNAs that were significantly up- or downregulated in patients with RRMS as compared to healthy controls. The best single miRNA marker, hsa-miR-145, allowed discriminating MS from controls with a specificity of 89.5%, a sensitivity of 90.0%, and an accuracy of 89.7%. A set of 48 miRNAs that was evaluated by radial basis function kernel support vector machines and 10-fold cross validation yielded a specificity of 95%, a sensitivity of 97.6%, and an accuracy of 96.3%. While 43 of the 165 miRNAs deregulated in patients with MS have previously been related to other human diseases, the remaining 122 miRNAs are so far exclusively associated with MS. The implications of our study are twofold. The miRNA expression profiles in blood cells may serve as a biomarker for MS, and deregulation of miRNA expression may play a role in the pathogenesis of MS.