Concept: Blood plasma
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 2 years ago
Exosomes are nanoscale extracellular vesicles that play an important role in many biological processes, including intercellular communications, antigen presentation, and the transport of proteins, RNA, and other molecules. Recently there has been significant interest in exosome-related fundamental research, seeking new exosome-based biomarkers for health monitoring and disease diagnoses. Here, we report a separation method based on acoustofluidics (i.e., the integration of acoustics and microfluidics) to isolate exosomes directly from whole blood in a label-free and contact-free manner. This acoustofluidic platform consists of two modules: a microscale cell-removal module that first removes larger blood components, followed by extracellular vesicle subgroup separation in the exosome-isolation module. In the cell-removal module, we demonstrate the isolation of 110-nm particles from a mixture of micro- and nanosized particles with a yield greater than 99%. In the exosome-isolation module, we isolate exosomes from an extracellular vesicle mixture with a purity of 98.4%. Integrating the two acoustofluidic modules onto a single chip, we isolated exosomes from whole blood with a blood cell removal rate of over 99.999%. With its ability to perform rapid, biocompatible, label-free, contact-free, and continuous-flow exosome isolation, the integrated acoustofluidic device offers a unique approach to investigate the role of exosomes in the onset and progression of human diseases with potential applications in health monitoring, medical diagnosis, targeted drug delivery, and personalized medicine.
Previously, we reported strong influences of genetic variants on metabolic phenotypes, some of them with clinical relevance. Here we hypothesize that DNA methylation may have an important and potentially independent effect on human metabolism. To test this hypothesis we conducted what is to the best of our knowledge the first epigenome-wide association study (EWAS) between DNA methylation and metabolic traits (metabotypes) in human blood. We assess 649 blood metabolic traits from 1,814 participants of the KORA population study for association with methylation of 457,004 CpG sites, determined on the Infinium HumanMethylation450 BeadChip platform. Using the EWAS approach, we identified two types of methylome-metabotype associations. One type is driven by an underlying genetic effect; the other type is independent of genetic variation and potentially driven by common environmental and life-style dependent factors. We report eight CpG loci at genome-wide significance that have a genetic variant as confounder (p=3.9x10(-20) to 2.0x10(-108), r(2)=0.036 to 0.221). Seven loci display CpG-site-specific associations to metabotypes, but do not exhibit any underlying genetic signals (p=9.2x10(-14) to 2.7x10(-27), r(2)=0.008 to 0.107). We further identify several groups of CpG loci that associate with a same metabotype, such as 4-vinylphenol sulfate and 4-androsten-3beta,17beta-diol disulfate. In these cases the association between CpG-methylation and metabotype are likely the result of a common external environmental factor, including smoking. Our study shows that analysis of EWAS with large numbers of metabolic traits in large population cohorts are, in principle, feasible. Taken together, our data suggests that DNA methylation plays an important role in regulating human metabolism.
A highly sensitive reverse sandwich immunoassay for the detection of human cardiac myoglobin (cMb) in serum was designed utilizing a gold nanoparticle (AuNP)-enhanced surface plasmon resonance (SPR) biosensor. First, a monoclonal anti-cMb antibody (Mab1) was covalently immobilized on the sensor surface. AuNPs were covalently conjugated to the second monoclonal anti-cMb antibody (Mab2) to form an immuno-gold reagent (Mab2-AuNP). The reverse sandwich immunoassay consists of two steps: (1) mixing the serum sample with Mab2-AuNP and incubation for the formation of cMb/Mab2-AuNP complexes and (2) sample injection over the sensor surface and evaluation of the Mab1/cMb/Mab2-AuNP complex formation, with the subsequent calculation of the cMb concentration in the serum. The biosensor signal was amplified approximately 30-fold compared with the direct reaction of cMb with Mab1 on the sensor surface. The limit of detection of cMb in a human blood serum sample was found to be as low as 10pM (approx. 0.18ngmL(-1)), and the inter-assay coefficient of variation was less than 3%. Thus, the developed SPR-based reverse sandwich immunoassay has a sensitivity that is sufficient to measure cMb across a wide range of normal and pathological concentrations, allowing an adequate estimation of the disease severity and the monitoring of treatment.
Glycated hemoglobin (HbA1c) has been recently adopted as a diagnostic marker of type 2 diabetes. However, its usage is currently limited to fresh blood samples. To allow retrospective HbA1c measurement in blood banks developed in large epidemic studies, here, we contribute to validate HbA1c assessment in frozen versus fresh blood samples from a cohort of diabetic/nondiabetic adult subjects. HbA1c was measured by HPLC in 237 fresh whole blood samples and on the same samples after a 12-month storage and a further 6-month-refrozen storage. Mean HbA1c ± SD in fresh, frozen, and refrozen samples was 6.9 ± 1.2, 6.6 ± 1.1, and 6.4 ± 1.0 % for the Diabetes Control and Complications Trial and 52 ± 13, 49 ± 12, and 46 ± 11 mmol/mol for the International Federation of Clinical Chemistry and Laboratory Medicine reference, respectively. A significant correlation was found between fresh/frozen and fresh/refrozen (R = 0.994 and 0.993, P < 0.001) samples. HbA1c relative error ratio (%RER) between frozen/refrozen and fresh samples significantly correlated with HbA1c and depended on fresh value range, increasing in the five HbA1c classes (<6.0, 6.0-6.5, 6.5-7, 7-8, ≥8 %, corresponding to <42, 42-48, 48-53, 53-64, ≥64 mmol/mol, P < 0.001). In particular, the 6.5 % (48 mmol/mol) HbA1c diagnostic cutoff of fresh samples identified two classes reflecting significant differences in %RER (2.8 ± 2.0 and 3.3 ± 1.7; P < 0.05) between frozen and fresh samples. In conclusion, our results demonstrate a high correlation between data from fresh and frozen samples, with a very limited %RER between the two measurements, which increases with baseline HbA1c levels. Accordingly, when analyzing biobank frozen specimens for diagnostic purpose, the effect of the HbA1c range should be taken into account.
Immediate use of thawed fresh frozen plasma (FFP) when resuscitating hemorrhagic shock patients has become more common. According to the AABB (formerly known as American Association of Blood Banks), FFP is the preferred product that can be used up to 5 days after thawing. However, limited data exist on the clinical use and hemostatic profiles of Food and Drug Administration-approved liquid plasma (LQP), which can be stored at 1°C to 6°C for up to 26 days. We characterized changes in LQP hemostatic potential during 26 days of cold storage.
Adverse reactions after whole blood donation reduce the likelihood of a subsequent donation. Still, many donors return to give blood even after experiencing a reaction. Consequently, we evaluated the risk of recurrent syncope among returning donors.
The present investigation was carried out to evaluate the safety of hydro-ethanol extract of Bridelia ferruginea Benth (Euphorbiaceae) root bark. For acute toxicity study, a single dose of 2000 and 5000mg/kg of the B. ferruginea root bark extract was given orally to healthy male Wistar rats and Balb/c mice. The animals were observed for mortality and clinical signs for 3h and then daily for 14days. In the sub-chronic toxicity study, the extract was administered orally at doses of 250, 500 and 1000mg/kg/day for 28days to male Wistar rats. Animals were sacrificed to examine their organs, and urine and blood serum were analyzed. In the acute toxicity study, B. ferruginea root bark extract caused neither significant visible signs of toxicity, nor mortality in Wistar rats and Balb/c mice. In sub-chronic toxicity study, administration of the B. ferruginea root bark extract at 250, 500, and 1000mg/kg for 28 consecutive days to Wistar rats did not produce mortality. No significant differences were found in relative organ weights, biochemical studied parameters in treated groups compared to control group. No obvious histological changes were observed in organs of B. ferruginea extract treated animals compared to controls.
Acquired von Willebrand Syndrome (AvWS) is known as a frequent bleeding complication in patients on ventricular assist device (VAD) support. Clinicians demand that the requirements for VADs with regard to hemocompatibility should also include low susceptibility for AvWS. Clinical AvWS diagnosis is known to be a complex, high-price, and time-consuming analysis. This article investigates an easy-to-handle, time-efficient, and inexpensive method for comparative AvWS investigations in vitro. Von Willebrand Factor activity level (vWF : Ac) and von Willebrand Factor antigen level (vWF : Ag) were chosen from the complete set of clinically established parameters. Blood plasma (human and porcine) was exposed to an inhomogeneous shear field in a shear-inducing test set up for up to 4 h. Plasma samples were drawn after different load periods and analyzed for vWF : Ac and vWF : Ag. vWF multimer analysis of selected samples were used as reference for determination of high molecular weight multimer (HMWM) loss. AvWS was detected after 20 min of shear load via vWF : Ac/vWF : Ag ratio and multimer analysis. A good correlation between the vWF : Ac/vWF : Ag ratio and HMWM loss (multimer analysis) was found for human plasma. AvWS characteristics of human and porcine plasma for analyzed samples were comparable. A correlation between vWF : Ac/vWF : Ag ratio and HMWM in porcine plasma could not be found. Results gained in this study indicate that vWF : Ac/vWF : Ag ratio is sensitive enough for comparative AvWS investigations in vitro with human blood. The applicability of the method suggested in this article for AvWS characterization in porcine blood needs to be investigated in further studies. The selection of analysis kits promises a less cost- and labor-intensive, time-consuming, and complex method for comparative AvWS investigations in vitro compared with AvWS diagnosis in patients.
BACKGROUND: The suitability for omic analysis of biosamples collected in previous decades and currently stored in biobanks is not known. OBJECTIVES: We evaluated the influence of handling and storage conditions of blood-derived biosamples on transcriptomic, epigenomic (CpG methylation) and plasma metabolomic (UPLC-ToFMS) and wide-target proteomic profiles. METHODS: Initially we collected fresh blood samples without RNA preservative in heparin, EDTA or citrate and held them at room temperature for up to 24hr prior to fractionation into buffy coat, erythrocytes and plasma and freezing at -80°C or in liquid nitrogen. We developed methodology for RNA isolation from the buffy coats and conducted omic analyses. Finally, we analysed analogous samples from the EPIC-Italy and Northern Sweden Health and Disease Study biobanks. RESULTS: Microarray-quality RNA could be isolated from buffy coats (including most biobank samples) frozen within 8hr of blood collection, by thawing in RNA preservative. Different anticoagulants influenced the metabolomic, proteomic and, to a lesser extent, transcriptomic profiles. The latter were most affected by the delay (as little as 2hr) prior to blood fractionation, while storage temperature had minimal impact. Effects on metabolomic and proteomic profiles were noted in samples processed 8hr or more after collection, but none due to storage temperature. None of the variables examined significantly influenced the epigenomic profiles. No systematic influence of time-in-storage was observed in samples stored over a period of 13-17 years. CONCLUSIONS: Most samples currently stored in biobanks are amenable to meaningful omics analysis, provided that they satisfy collection and storage criteria defined in this study.
The ErySep system represents an alternative to centrifuge-based whole blood (WB) separation, using gravity and filtration through hollow-fibres (0·2 µm pore size) to produce red blood cell (RBC) and plasma components. The aim of this study was to characterise the quality of ErySep RBC and plasma units compared with standard products from WB held overnight.