Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world‐wide survey to ISEV members to assess methods considered ‘best practices’ for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies.

The molecular heterogeneity of extracellular vesicles (EVs) and the co‐isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single‐EV and particle (siEVP) protein and RNA assay (^siEVPPRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The ^siEVPPRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single‐particle resolution, the ^siEVPPRA outperformed the sensitivities of bulk‐analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross‐validation. Despite the presence of single‐EV‐LP co‐isolates in serum, the ^siEVPPRA detected GBM‐associated vesicular RNA profiles in GBM patient siEVPs. The ^siEVPPRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.

Extracellular vesicles (EVs) of various types are released or shed from all cells. EVs carry proteins and contain additional protein and nucleic acid cargo that relates to their biogenesis and cell of origin. EV cargo in liquid biopsies is of widespread interest owing to its ability to provide a retrospective snapshot of cell state at the time of EV release. For the purposes of EV cargo analysis and repertoire profiling, multiplex assays are an essential tool in multiparametric analyte studies but are still being developed for high-parameter EV protein detection. Although bead-based EV multiplex analyses offer EV profiling capabilities with conventional flow cytometers, the utilization of EV multiplex assays has been limited by the lack of software analysis tools for such assays. To facilitate robust EV repertoire studies, we developed multiplex analysis post-acquisition analysis (MPA_PASS) open-source software for stitched multiplex analysis, EV database-compatible reporting, and visualization of EV repertoires.

Anti-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (anti-AMPAR) encephalitis manifests as limbic encephalitis in adults and is often associated with cancer. Although some reports suggest that it may occur in children, the clinical features in this population, as well as the prognostic factors and long-term outcomes in children and adults, are unknown. We performed a retrospective, international collaborative study of patients with anti-AMPAR encephalitis. Clinical information was reviewed, together with data from published pediatric patients. Clinical features of children and adults were compared with nonparametric tests. Survival rates (Kaplan-Meier curves) were compared using log-rank tests. Prognostic factors of poor outcome (modified Rankin Scale score >2) were identified using logistic regression models. A total of 115 patients were included, of whom 84 (71 adults, 13 children) had only AMPAR antibodies and 31 (27 adults, 4 children) had additional concurrent neural antibodies. Among patients with AMPAR antibodies alone, tumors were identified in 37 adults (56%) and none of the children (p < 0.0001). Children were more likely than adults to have behavioral/psychiatric symptoms (5/13, 39%, vs 8/71, 11%, p = 0.026) at onset, cerebellar dysfunction (6/13, 46%, vs 7/68, 10% p = 0.005) or movement disorders (5/13, 39%, vs 8/67, 12%, p = 0.032) during the disease course, and extratemporal brain MRI lesions (4/9, 44%, vs 5/44, 11%, p = 0.035). Among 34 patients with prolonged follow-up (>24 months), long-term neurocognitive sequelae were reported in 23 (68%), all adults. Failure to respond to first-line immunotherapy at multivariable analysis predicted a poor outcome (OR 8.0, 95% CI 1.1-59.2, p = 0.043). Among the 31 patients with concurrent neural autoantibodies, 22 (79%) had a tumor; those with high-risk antibodies had lower survival rates (p = 0.008). Children and adults with anti-AMPAR encephalitis show distinct clinical-radiologic features. At long-term follow-up, 68% of patients, all adults, have neurologic sequelae, with failure to respond to first-line immunotherapy being associated with worse outcomes.

Obesity is a growing global health concern, contributing to diseases such as cancer, autoimmune disorders, and neurodegenerative conditions. Adipose tissue dysfunction, characterized by abnormal adipokine secretion and chronic inflammation, plays a key role in these conditions. Adipose-derived extracellular vesicles (ADEVs) have emerged as critical mediators in obesity-related diseases. However, the study of mature adipocyte-derived EVs (mAdipo-EVs) is limited due to the short lifespan of mature adipocytes in culture, low EV yields, and the low abundance of these EV subpopulations in the circulation. Additionally, most studies rely on rodent models, which have differences in adipose tissue biology compared to humans. To overcome these challenges, we developed a standardized approach for differentiating human preadipocytes (preAdipos) into mature differentiated adipocytes (difAdipos), which produce high-yield, human adipocyte EVs (Adipo-EVs). Using visceral adipose tissue from bariatric surgical patients, we isolated the stromal vascular fraction (SVF) and differentiated preAdipos into difAdipos. Brightfield microscopy revealed that difAdipos exhibited morphological characteristics comparable to mature adipocytes (mAdipos) directly isolated from visceral adipose tissue, confirming their structural similarity. Additionally, qPCR analysis demonstrated decreased preadipocyte markers and increased mature adipocyte markers, further validating successful differentiation. Functionally, difAdipos exhibited lipolytic activity comparable to mAdipos, supporting their functional resemblance to native adipocytes. We then isolated preAdipo-EVs and difAdipo-EVs using tangential flow filtration and characterized them using bulk and single EV analysis. DifAdipo-EVs displayed classical EV and adipocyte-specific markers, with significant differences in biomarker expression compared to preAdipo-EVs. These findings demonstrate that difAdipos serve as a reliable surrogate for mature adipocytes, offering a consistent and scalable source of adipocyte-derived EVs for studying obesity and its associated disorders.