BackgroundPlasma extracellular vesicles (EVs), especially exosome-like vesicles (ELVs), are being increasingly explored as a source of potential noninvasive disease biomarkers. The discovery of blood-based biomarkers associated with ELVs requires methods that isolate high yields of these EVs without significant contamination with highly abundant plasma proteins and lipoproteins. The rising interest in blood-based EV-associated biomarkers has led to the rapid development of novel EV isolation methods. However, the field suffers from a lack of standardization and often, new techniques are used without critical evaluation. Size exclusion chromatography (SEC) has become the method of choice for rapid isolation of relatively pure EVs from plasma, yet it has technical limitations for certain downstream applications. The recently released exoEasy kit (Qiagen) is a new membrane affinity spin column method for the isolation of highly pure EVs from biofluids with the potential to overcome most of the limitations of SEC.MethodsBy using multiple complementary techniques we assessed the performance of the exoEasy kit in isolating ELVs from 2 ml of human plasma and compared it with the SEC qEV column (Izon Science).ResultsOur data show that exoEasy kit isolates a heterogenous mixture of particles with a larger median diameter, broader size range and a higher yield than the SEC qEV column. The exclusive presence of small RNAs in the particles and the total RNA yield were comparable to the SEC qEV column. Despite being less prone to low density lipoprotein contamination than the SEC qEV column, the overall purity of exoEasy kit EV preparations was suboptimal. The low particle-protein ratio, significant amount of albumin, very low levels of exosome-associated proteins and propensity to triglyceride-rich lipoprotein contamination suggest isolation of mainly non-ELVs and co-isolation of plasma proteins and certain lipoproteins by the exoEasy kit.ConclusionsWe demonstrate that performance of exoEasy kit for the isolation of ELVs for biomarker discovery is inferior to the SEC qEV column. This comprehensive evaluation of a novel EV isolation method contributes to the acceleration of the discovery of EV-associated biomarkers and the development of EV-based diagnostics.Electronic supplementary materialThe online version of this article (10.1186/s12967-017-1374-6) contains supplementary material, which is available to authorized users.

Belgium has a high burden of coronavirus disease 2019 (COVID-19), especially the region surrounding the Hospital East-Limburg, a tertiary care center. 1 Infection prevention measures were instituted in the hospital beginning March 4, 2020, including testing and contact tracing of all symptomatic patients and staff, changes in hospital operations, and provision of personal protective equipment (PPE). The first case was detected March 13 (Figure 1). We investigated the prevalence of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among hospital staff.

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Objectives: To evaluate the diagnostic performance of seven rapid IgG/IgM tests and the Euroimmun IgA/ IgG ELISA for antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in COVID-19 patients. Methods: Specificity was evaluated in 103 samples collected before January 2020. Sensitivity and time to seropositivity was evaluated in 167 samples from 94 patients with COVID-19 confirmed with RT-PCR on nasopharyngeal swab. Results: Specificity (confidence interval) of lateral flow assays (LFAs) was 91.3% (84.0e95.5) for IgM, 90.3% (82.9e94.8) for IgG, and 85.4% (77.2e91.1) for the combination IgM OR IgG. Specificity of the ELISA was 96.1% (90.1e98.8) for IgG and only 73.8% (64.5e81.4) for IgA. Sensitivity 14e25 days after the onset of symptoms was between 92.1% (78.5e98.0) and 100% (95.7e100) for IgG LFA compared to 89.5% (75.3e96.4) for IgG ELISA. Positivity of IgM OR IgG for LFA resulted in a decrease in specificity compared to IgG alone without a gain in diagnostic performance, except for VivaDiag. The results for IgM varied significantly between the LFAs with an average overall agreement of only 70% compared to 89% for IgG. The average dynamic trend to seropositivity for IgM was not shorter than for IgG. At the time of hospital admission the sensitivity of LFA was <60%. Conclusions: Sensitivity for the detection of IgG antibodies 14e25 days after the onset of symptoms was 92.1% for all seven LFAs compared to 89.5% for the IgG ELISA. The results for IgM varied significantly, and including IgM antibodies in addition to IgG for the interpretation of LFAs did not improve the diagnostic performance.

Background-Alterations in the balance of matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) are involved in left ventricular (LV) remodeling. Whether their expression is related to interstitial fibrosis or LV dysfunction in patients with chronic pressure overload-induced LV hypertrophy, however, is unknown. Methods and Results-Therefore, cardiac biopsies were taken in 36 patients with isolated aortic stenosis (AS) and in 29 control patients without LV hypertrophy. Microarray analysis revealed significantly increased mRNA expression of collagen types I, III, and IV and transcripts involved in collagen synthesis, including procollagen endopeptidase and lysine and proline hydroxylases, in AS compared with control patients. Collagen deposition was greater in AS than in control patients and was most pronounced in AS patients with severe diastolic dysfunction. Cardiac mRNA expression of TIMP-1 and TIMP-2 was significantly increased in AS compared with control patients (mRNA transcript levels normalized to GAPDH: TIMP-1, 0.67Ϯ0.1 in AS versus 0.37Ϯ0.08 in control patients; TIMP-2, 9.5Ϯ2.6 in AS versus 1.6Ϯ0.4 in control patients; PϽ0.05 for both) but did not differ significantly for MMP-1, -2, or -9. Cardiac TIMP-1 and -2 transcripts were significantly related to the degree of interstitial fibrosis and proportional to diastolic dysfunction in AS patients. Conclusions-Cardiac expression of TIMP-1 and TIMP-2 is significantly increased in chronic pressure-overloaded human hearts compared with controls and is related to the degree of interstitial fibrosis.