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A prevalent challenge in isolating extracellular vesicles (EVs) from biological fluids is the reliable depletion of abundant contaminants-including free proteins and biomolecules, as well as nontarget vesicle subpopulations and other nanoparticulates-from the sample matrix while maximizing recovery. Sequential Filtration is a recently published approach for the size-based isolation of exosomes that is ideally suited for large-volume biofluid samples such as ascites , urine , lavage fluid, or cell-conditioned media. We describe a straightforward, three-step protocol comprising back-to-back steps of dead-end (normal) filtration, tangential-flow filtration, and track-etched membrane filtration that can be applied to yield a homogeneous population of exosome-sized extracellular vesicles. The approach is scalable and employs relatively gentle manipulation forces to fractionate and concentrate extracellular vesicles with good purity and functional integrity.

ABSTRACT

Background It is suspected that microbiome-derived trimethylamine N-oxide (TMAO) may enhance platelet responsiveness and accordingly be thrombophilic. The purpose of this prospective observational study is to evaluate TMAO in patients with subarachnoid hemorrhage (SAH) and compare it with a control group. A secondary aim was to investigate TMAO in the cerebrospinal fluid (CSF) from SAH patients. This should provide a better understanding of the role of TMAO in the pathogenesis of SAH and its thrombotic complications. Methods The study included patients with diagnosed spontaneous SAH recruited after initial treatment on admission and patients with nerve, nerve root, or plexus disorders serving as controls. Blood samples were gathered from all patients at recruitment. Additionally, sampling of SAH patients in the intensive care unit continued daily for 14 days. The CSF was collected out of existing external ventricular drains whenever possible. Results Thirty-four patients diagnosed with SAH, and 108 control patients participated in this study. Plasma TMAO levels at baseline were significantly lower in the SAH group (1.7 μmol/L) compared to the control group (2.9 μmol/L). TMAO was detectable in the CSF (0.4 μmol/L) and significantly lower than in plasma samples of the SAH group at baseline. Plasma and CSF TMAO levels correlated positively. The TMAO levels did not differ significantly during the observation period of 15 days. Conclusions Although we assumed that patients with higher TMAO levels were at higher risk for SAH a priori, plasma TMAO levels were lower in patients with SAH compared with control subjects with nerve, nerve root, or plexus disorders on admission to the hospital. A characteristic pattern of plasma TMAO levels in patients with SAH was not found.

Metabolic myopathies are heterogeneous hereditary diseases affecting skeletal muscle energy supply. Symptoms usually comprise pain, cramps, hypotonia, weakness, and myoglobinuria.We present a boy with recurrent myalgia and weakness after some minutes of exercise or during febrile infections since early infancy. First laboratory workup at the age of 9 years showed no abnormalities, apart from a slightly elevated creatine kinase. After exclusion of common structural and metabolic myopathies, next generation sequencing panel (4 years after the initial diagnostic metabolic workup) revealed two potentially pathogenic missense mutations in the CPT2 gene (c.149C > A (p.P50H) and c.1459G > A (p.E487K)).Our case underscores the clinical variability of muscle carnitine palmitoyltransferase II (CPT II) deficiency and illustrates a pitfall of diagnostic algorithms for metabolic myopathies. Myalgia following exercise of a few minutes duration would have argued for a carbohydrate and against a fatty acid metabolic defect. However, CPT II deficiency is the most common disorder of muscle fatty acid metabolism and should be considered even in atypical scenarios. Analyses of plasma acyl carnitine profile during acute metabolic crises may help to unmask biochemical markers which are often overlooked in dried-blood analyses.

ZusammenfassungDie Mukoviszidose oder Cystische Fibrose (CF) ist eine autosomal rezessiv vererbte Stoffwechselerkrankung und mit einer regional schwankenden Inzidenz von ca. 1:3.300–1:5.800 eine der häufigsten angeborenen Stoffwechselerkrankungen in Deutschland. Durch eine mutationsbedingte verminderte oder fehlende Funktion von Chloridkanälen kommt es hier zu einer Veränderung der Sekretzusammensetzung aller exokrinen Drüsen. Die mittlere Lebenserwartung von Mukoviszidose-Patienten konnte durch verbesserte Behandlungsstrategien auf mittlerweile über 40 Jahre erheblich gesteigert werden. Es hat sich dabei gezeigt, dass eine frühzeitige Diagnosestellung einen positiven Einfluss auf Krankheitsverlauf, Lebensqualität und Lebenserwartung der betroffenen Patienten hat. Diese Erkenntnis führte in den letzten 10 Jahren europaweit zur Aufnahme der Mukoviszidose in regionale und nationale Neugeborenenscreening-Programme. Mit dem Beschluss des Gemeinsamen Bundesausschusses zur Einführung des Mukoviszidosescreenings im August 2015 wurde Mukoviszidose nun auch in Deutschland als weitere Zielkrankheit in die Kinderrichtlinien aufgenommen und ist nach Veröffentlichung im Bundesanzeiger somit bundeseinheitlich als Bestandteil des deutschen Neugeborenenscreening-Programms vorgeschrieben. Das Procedere beinhaltet ein Stufenscreening mit der Kombination von Immunreaktivem Trypsin (IRT) und Pankreatitis-assoziiertem Protein (PAP) mit zusätzlicher Mutationsanalytik. Dank einer deutschlandweit früheren Diagnosestellung wird ein verbessertes Langzeitoutcome von Mukoviszidose-Patienten erwartet.

AbstractBackgroundUrinary ethyl glucuronide (EtG) has emerged as the biomarker of choice for alcohol abstinence monitoring in forensic toxicology and is now used in the listing decision process for liver transplantations (LTs) in the German transplant program. However, EtG analysis in this patient group is challenging due to severely impaired liver function, renal failure, co-morbidities and multidrug regimens. The aim of our study was to evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based EtG analysis for a precise abstinence monitoring in transplant candidates.MethodsEtG and ethyl sulfate (EtS) were analyzed by a commercial LC-MS/MS assay in 1787 spot urine samples of 807 patients (>85% from the Department of Hepatology) using a combination of quantifier and two qualifier mass transitions for each analyte. Influences of bacterial contamination, kidney and liver function were investigated.ResultsTwo hundred and sixty-four urine samples had elevated (≥0.5 mg/L) EtG concentrations when only analyzing one quantifier mass transition. Eleven results (4.2%) were found to be false positive after combining three mass transitions for EtG quantification and verification with parallel analysis of EtS. Decreased kidney function was associated with a significantly higher rate of positive EtG samples. One of the false positive results was caused by bacterial metabolism.ConclusionsMultimorbid pre-transplant patients have a high risk of individual analytical disturbances of EtG results obtained by LC-MS/MS. Therefore, EtG and EtS should always be measured by a combination of one quantifier and two qualifiers each and evaluated together.

Background: The so-called “liquid biopsy” has become a powerful tool for cancer research in the recent years, with circulating cell-free DNA (cfDNA) that originates from tumors as one of the most promising analytes. In contrast to plasma derived cfDNA, only a few studies were performed investigating urinary cfDNA. One reason might be quick degradation and hence relatively low concentrations of urinary cfDNA. This study focused on examining the stability of cfDNA in urine using different ways of preservation under various storage conditions. Methodology: To mimic patient samples, a pool of healthy male and female urine donors was spiked with a synthetic cfDNA reference standard (fragment size 170 bp) containing the T790M mutation in the EGFR gene and preserved with three different buffers and no buffer over four different storage periods (0; 4; 12; 24 h) at room temperature vs. fridge. Preservatives used were the Urinary Analyte Stabilizer (UAS, Novosanis), the Urine Conditioning Buffer (UCB, Zymo) and a self-prepared buffer called “AlloU”. The cfDNA was extracted with the QIAamp MinElute ccfDNA Mini Kit (Qiagen) and the concentration measured with the Qubit™ 4 fluorometer (Thermo Fisher Scientific). Droplet digital PCR (ddPCR) was used for detection and quantification of the T790M mutation (Bio-Rad) Results: From the model samples with no preservation buffer, almost no spiked cfDNA could be recovered and the T790M variant could not be detected using ddPCR, indicating that without preservation cfDNA was degraded below the detection limit by the nucleases present in urine. The most effective stabilizing buffer over all storage periods and at room as well as fridge temperature was the UAS, resulting in accurate detection of the T790M variant using ddPCR. Conclusion: From a technical point of view, urinary cfDNA might be useful for non-invasive disease monitoring when the samples are stabilized and stored adequately during clinical routine. Citation Format: Carolin Münch, Saikal Shamkeeva, Mitja Heinemann, Berend Isermann, Sabine Kasimir-Bauer, Bahriye Aktas, Ivonne Nel. Stability of urinary cell-free DNA and detection of T790M variant. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6693.