Plasma 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.
By 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).
Our 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.
We 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.
This multicentre study was performed to evaluate the diagnostic accuracy of a wide spectrum of novel technologies nowadays available for detection of myeloperoxidase (MPO) and proteinase 3 ...(PR3)-antineutrophil cytoplasmic antibodies (ANCAs).
Sera (obtained at the time of diagnosis) from 251 patients with ANCA-associated vasculitis (AAV), including granulomatosis with polyangiitis and microscopic polyangiitis, and from 924 disease controls were tested for the presence of cytoplasmic pattern/perinuclear pattern and atypical ANCA (A-ANCA) by indirect immunofluorescence (IIF) (at two sites) and for the presence of PR3-ANCA and MPO-ANCA by eight different immunoassays.
The area under the curve (AUC) of the receiver operating characteristic curve to discriminate AAV from controls was 0.923 (95% CI 0.902 to 0.944) and 0.843 (95% CI 0.814 to 0.871) for the two IIF methods. For the antigen-specific immunoassays, the AUC varied between 0.936 (95% CI 0.912 to 0.960) and 0.959 (95% CI 0.941 to 0.976), except for one immunoassay for which the AUC was 0.919 (95% CI 0.892 to 0.945).
Our comparison of various ANCA detection methods showed (i) large variability between the two IIF methods tested and (ii) a high diagnostic performance of PR3-ANCA and MPO-ANCA by immunoassay to discriminate AAV from disease controls. Consequently, dual IIF/antigen-specific immunoassay testing of each sample is not necessary for maximal diagnostic accuracy. These results indicate that the current international consensus on ANCA testing for AAV needs revision.
•After two months SARS-CoV-2 anti-N IgG antibodies steadily decrease up to at least 8 months after positive PCR.•22.2 % of mild versus 2.6 % of severe COVID-19 patients never seroconverted.•Antibody ...levels are significantly higher in severe COVID-19 patients compared to mild COVID-19 patients.•70.6 % of severe compared to 38.9 % of mild COVID-19 patients remain seropostive up to 8 months after positive PCR.
Most SARS-CoV-2 infected patients develop IgG antibodies within 2–3 weeks after symptom onset. Antibody levels have been shown to gradually decrease in the first months after infection, but few data are available at six months or later.
A retrospective multi-center study was performed using 652 samples of 236 PCR-confirmed SARS-CoV-2 infected patients from 2 Belgian University hospitals. Patients were included if at least two samples were available (range 2–7 samples); including at least one sample collected 30 days or later after first positive PCR (range 0–240 days). Of those 236 patients, 19.1 % were classified as mild/asymptomatic (mild) and 80.9 % as moderate to critical (severe). IgG anti-nucleocapsid antibodies (anti-N) were measured using the Abbott Architect immunoassay.
22.2 % of mild and 2.6 % of severe COVID-19 cases never seroconverted (p < 0.001). Of the mild patients who seroconverted 0–59 days after PCR; 18.8 %, 40.0 % and 61.1 % were seronegative in the windows 60–119 days, 120–179 days and 180–240 days after PCR, respectively. In severe patients, these numbers were 1.9 %, 10.8 % and 29.4 % respectively (p < 0.05 each). Antibody levels were significantly higher in severe patients compared to mild patients in each 60 day window (p < 0.001 each).
SARS-CoV-2 anti-N IgG antibody levels steadily decreased after 2 months up to 8 months post PCR. Of severe COVID-19 patients, 70.6 % remained positive up to eight months after infection. Antibody levels were significantly lower in mild SARS-CoV-2 infected patients and 61.1 % became seronegative within 6 months after the first positive PCR.
Somatic ribosomal protein mutations have recently been described in cancer, yet their impact on cellular transcription and translation remains poorly understood. Here, we integrate mRNA sequencing, ...ribosome footprinting, polysomal RNA sequencing and mass spectrometry datasets from a mouse lymphoid cell model to characterize the T-cell acute lymphoblastic leukemia (T-ALL) associated ribosomal RPL10 R98S mutation. Surprisingly, RPL10 R98S induces changes in protein levels primarily through transcriptional rather than translation efficiency changes. Phosphoserine phosphatase (PSPH), encoding a key serine biosynthesis enzyme, was the only gene with elevated transcription and translation leading to protein overexpression. PSPH upregulation is a general phenomenon in T-ALL patient samples, associated with elevated serine and glycine levels in xenograft mice. Reduction of PSPH expression suppresses proliferation of T-ALL cell lines and their capacity to expand in mice. We identify ribosomal mutation driven induction of serine biosynthesis and provide evidence supporting dependence of T-ALL cells on PSPH.
This document provides a joint recommendation for venous blood sampling of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group for Preanalytical Phase (WG-PRE) ...and Latin American Working Group for Preanalytical Phase (WG-PRE-LATAM) of the Latin America Confederation of Clinical Biochemistry (COLABIOCLI). It offers guidance on the requirements for ensuring that blood collection is a safe and patient-centered procedure and provides practical guidance on how to successfully overcome potential barriers and obstacles to its widespread implementation. The target audience for this recommendation are healthcare staff members directly involved in blood collection. This recommendation applies to the use of a closed blood collection system and does not provide guidance for the blood collection with an open needle and syringe and catheter collections. Moreover, this document neither addresses patient consent, test ordering, sample handling and transport nor collection from children and unconscious patients. The recommended procedure is based on the best available evidence. Each step was graded using a system that scores the quality of the evidence and the strength of the recommendation. The process of grading was done at several face-to-face meetings involving the same mixture of stakeholders stated previously. The main parts of this recommendation are: 1) Pre-sampling procedures, 2) Sampling procedure, 3) Post-sampling procedures and 4) Implementation. A first draft of the recommendation was circulated to EFLM members for public consultation. WG-PRE-LATAM was also invited to comment the document. A revised version has been sent for voting on to all EFLM and COLABIOCLI members and has been officially endorsed by 33/40 EFLM and 21/21 COLABIOCLI members. We encourage professionals throughout Europe and Latin America to adopt and implement this recommendation to improve the quality of blood collection practices and increase patient and workers safety.
It is well recognized that nitrous oxide abuse can lead to vitamin B
deficiency presenting with neurological complications. Nevertheless, establishing this diagnosis can be challenging, and treatment ...guidelines are lacking.
In this paper, we present a case series of eight patients and discuss the diagnostic challenges and treatment options for vitamin B
deficiency due to nitrous oxide abuse presenting with neurologic complications.
Biochemical findings are not always straightforward and complementary testing is often necessary. Magnetic Resonance Imaging (MRI) revealed a longitudinally myelopathy extending over a long segment typically involving the dorsal columns of the cervical cord. To increase the lesion conspicuity, dedicated MRI sequences are needed. In our practice, we recommend the use of T2-weighted images (WI) with fat suppression (FS). Treatment consists of cessation of nitrous oxide abuse and supplementation with intramuscular injections of cobalamin. Due to a lack of treatment guidelines, we also describe the treatment schedule used in our neurology clinic and give a brief overview of treatment options suggested in the literature.
We described diagnostic steps en treatment plans in patients presenting with subacute neurological complications due to nitrous oxide abuse.
crea: creatinine; HCy: homocysteine; MCA: 2-methylcitric acid; MMA: methylmalonic acid; MRI: magnetic resonance imaging; SEP: somatosensory evoked potentials.
The International Organization for Standardization (ISO) 15189:2012 standard aims to improve quality in medical laboratories through standardization of all key elements in the total testing process, ...including the pre-analytical phase. It is hence essential that accreditation bodies, assessing laboratories against ISO15189:2012, pay sufficient attention to auditing pre-analytical activities. However, there are significant differences in how technical auditors interpret the pre-analytical requirements described in ISO15189:2012. In this consensus document, the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group for Pre-analytical Phase (WG-PRE) sets out to review pre-analytical requirements contained in ISO15189:2012 and provide guidance for laboratories on how to meet these requirements. The target audience for this consensus document is laboratory professionals who wish to improve the quality of the pre-analytical phase in their laboratory. For each of the ISO requirements described in ISO15189:2012, members of EFLM WG-PRE agreed by consensus on minimal recommendations and best-in-class solutions. The minimal consensus recommendation was defined as the minimal specification which laboratories should implement in their quality management system to adequately address the pre-analytical requirement described in ISO15189:2012. The best-in-class solution describes the current state-of-the-art in fulfilling a particular pre-analytical requirement in ISO15189:2012. We fully acknowledge that not every laboratory has the means to implement these best-in-class solutions, but we hope to challenge laboratories in critically evaluating and improving their current procedures by providing this expanded guidance.
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