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.

Microsatellite (GT)(n) repeats and SNPs in the von Willebrand factor gene promoter do not influence circulating von Willebrand factor levels under normal conditions.

Von Willebrand factor (VWF) levels vary considerably in normal individuals, influenced by inherited and acquired modulators. ABO blood group is the major inherited determinant of VWF levels, but a role has also been attributed to the VWF gene promoter, haplotype 1 (-3268G/-2709C/-2661A/-2527G) being associated with higher VWF levels than haplotype 2 (-3268C/-2709T/-2661G/-2527A), and the polymorphic locus (GT)(n) modulating the shear stress-induced activation of the VWF promoter. We characterized the (GT)(n) of the VWF promoter in 394 healthy individuals and assessed whether its variable length influenced VWF levels in normal conditions. (GT)(n) proved highly polymorphic, with alleles from 15 to 24 repeats long. (GT)(21) and (GT)(19) were the most common variants (37.4% and 34.4%, respectively). Short GT repeats (15-19) segregated mainly with haplotype 1, long GT repeats (20-24) with haplotype 2 (p < 0.0001). The number of GT repeats did not correlate with VWF levels, nor did such levels correlate with haplotypes 1 and 2, considered alone or in association with the (GT)(n) locus. We conclude that (GT)(n) and -3268/-2709/-2661/-2527 loci are in strong linkage disequilibrium. This polymorphic region of the VWF promoter does not affect VWF levels under normal conditions, though it might represent an environmentally activable VWF regulation site.

Virus-inactivated plasma - Plasmasafe: a one-year experience.

BACKGROUND: Fresh-frozen plasma (FFP) is a widely used blood transfusion product. The transfusion safety of this product is ensured by legally obligatory tests, but can be further improved by using some technical procedures, such as methylene blue (MB) and solvent-detergent (SD) viral inactivation methods. Mainly organisational criteria led us to introduce the SD viral inactivation technique as a service activity. In this report we describe our first year of experience, following the introduction of the SD technique, and thus the use of SD-virally inactivated plasma (PlasmaSafe). MATERIALS AND METHODS: IN ORDER TO EVALUATE THE APPROPRIATE USE AND THE THERAPEUTIC EFFICACY OF PLASMASAFE IN OUR BLOOD TRANSFUSION UNIT, THE FOLLOWING PROGRAMME WAS PLANNED: quality control [prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen] of the FFP units (N=312); evaluation of the clinical effectiveness on 490 patients (879 transfusion events); pre- and post-treatment monitoring of indicators of coagulation (PT, aPTT, fibrinogen, proteins S and C, factor VIII) on 15 patients; treatment of three patients with thrombotic thrombocytopenic purpura (TTP) undergoing plasma-exchange; haemovigilance of adverse reactions provoked by SD-plasma. RESULTS: THE INDICATORS OF COAGULATION IN THE FFP UNITS VARIED GREATLY: the PT ranged from 50-120%, the aPTT from 24-41 seconds and the fibrinogen concentration from 1.42-6.84 g/L. Seventy-six percent of the patients responded to the plasma administration; moreover, two of 15 patients in whom protein S was assayed, showed no increase of this haemostatic protein. The TTP patients responded to plasma exchange treatment following four sessions of apheresis. During the observation period 8,422 PlasmaSafe units were transfused and no adverse reactions were recorded. CONCLUSION: PlasmaSafe, a pharmaceutical-like product with a standardised content of coagulation factors, was found to be effective at correcting coagulation defects and for treating TTP. No thrombotic complications or transfusion-related adverse reactions were recorded.