Background and aimThe potential of microRNAs (miRNA) as non-invasive diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, has recently been recognized. Previous studies have highlighted the importance of consistency in the methodology used, but to our knowledge, no study has described the methodology of sample preparation and storage systematically with respect to miRNAs as blood biomarkers. The aim of this study was to investigate the stability of miRNAs in blood under various relevant clinical and research conditions: different collection tubes, storage at different temperatures, physical disturbance, as well as serial freeze-thaw cycles.MethodsBlood samples were collected from 12 healthy donors into different collection tubes containing anticoagulants, including EDTA, citrate and lithium-heparin, as well as into serum collection tubes. MiRNA stability was evaluated by measuring expression changes of miR-1, miR-21 and miR-29b at different conditions: varying processing time of whole blood (up to 72 hours (h)), long-term storage (9 months at -80°C), physical disturbance (1 and 8 h), as well as in a series of freeze/thaw cycles (1 and 4 times).ResultsDifferent collection tubes revealed comparable concentrations of miR-1, miR-21 and miR-29b. Tubes with lithium-heparin were found unsuitable for miRNA quantification. MiRNA levels were stable for at least 24 h at room temperature in whole blood, while separated fractions did show alterations within 24 h. There were significant changes in the miR-21 and miR-29b levels after 72 h incubation of whole blood at room temperature (p<0.01 for both). Both miR-1 and miR-21 showed decreased levels after physical disturbance for 8 h in separated plasma and miR-1 in serum whole blood, while after 1 h of disturbance no changes were observed. Storage of samples at -80°C extended the miRNA stability remarkably, however, miRNA levels in long-term stored (9 months) whole blood samples were significantly changed, which is in contrast to the plasma samples, where miR-21 or miR-29b levels were found to be stable. Repetitive (n = 4) freeze-thaw cycles resulted in a significant reduction of miRNA concentration both in plasma and serum samples.ConclusionThis study highlights the importance of proper and systematic sample collection and preparation when measuring circulating miRNAs, e.g., in context of clinical trials. We demonstrated that the type of collection tubes, preparation, handling and storage of samples should be standardized to avoid confounding variables influencing the results.

BackgroundWe aimed to investigate the incidence and risk factors for ventricular fibrillation (VF) before primary percutaneous coronary intervention (PPCI) among patients with ST‐segment elevation myocardial infarction (STEMI) in a prospective nationwide setting.Methods and ResultsIn this case‐control study, patients presenting within the first 12 hours of first STEMI who survived to undergo angiography and subsequent PPCI were enrolled. Over 2 years, 219 cases presenting with VF before PPCI and 441 controls without preceding VF were enrolled. Of the 219 case patients, 182 (83%) had STEMI with out‐of‐hospital cardiac arrest due to VF, and 37 (17%) had cardiac arrest upon arrival to the emergency room. Medical history was collected by standardized interviews and by linkage to national electronic health records. The incidence of VF before PPCI among STEMI patients was 11.6%. Multivariable logistic regression analysis identified novel associations between atrial fibrillation and alcohol consumption with VF. Patients with a history of atrial fibrillation had a 2.80‐fold odds of experiencing VF before PPCI (95% CI 1.10 to 7.30). Compared with nondrinkers, patients who consumed 1 to 7 units, 8 to 14 units, or >15 units of alcohol per week had an odds ratio (OR) of 1.30 (95% CI, 0.80 to 2.20), 2.30 (95% CI, 1.20 to 4.20), or 3.30 (95% CI, 1.80 to 5.90), respectively, for VF. Previously reported associations for preinfarction angina (OR 0.46; 95% CI 0.32 to 0.67), age of <60 years (OR 1.75; 95% CI 1.20 to 2.60), anterior infarction (OR 2.10; 95% CI 1.40 to 3.00), preprocedural thrombolysis in myocardial infarction flow grade 0 (OR 1.65; 95% CI 1.14 to 2.40), and family history of sudden death (OR 1.60; 95% CI 1.10 to 2.40) were all associated with VF.ConclusionSeveral easily assessed risk factors were associated with VF occurring out‐of‐hospital or on arrival at the emergency room before PPCI in STEMI patients, thus providing potential avenues for investigation regarding improved identification and prevention of life‐threatening ventricular arrhythmias.