Enumeration of extracellular vesicles has clinical potential as a biomarker for disease. In biological samples, the smallest and largest vesicles typically differ 25-fold in size, 300,000-fold in ...concentration, 20,000-fold in volume, and 10,000,000-fold in scattered light. Because of this heterogeneity, the currently employed techniques detect concentrations ranging from 10(4) to 10(12) vesicles mL(-1) .
To investigate whether the large variation in the detected concentration of vesicles is caused by the minimum detectable vesicle size of five widely used techniques.
The size and concentration of vesicles and reference beads were measured with transmission electron microscopy (TEM), a conventional flow cytometer, a flow cytometer dedicated to detecting submicrometer particles, nanoparticle tracking analysis (NTA), and resistive pulse sensing (RPS).
Each technique gave a different size distribution and a different concentration for the same vesicle sample.
Differences between the detected vesicle concentrations are primarily caused by differences between the minimum detectable vesicle sizes. The minimum detectable vesicle sizes were 70-90 nm for NTA, 70-100 nm for RPS, 150-190 nm for dedicated flow cytometry, and 270-600 nm for conventional flow cytometry. TEM could detect the smallest vesicles present, albeit after adhesion on a surface. Dedicated flow cytometry was most accurate in determining the size of reference beads, but is expected to be less accurate on vesicles, owing to heterogeneity of the refractive index of vesicles. Nevertheless, dedicated flow cytometry is relatively fast and allows multiplex fluorescence detection, making it most applicable to clinical research.
Healthcare access and equity are human rights. Worldwide conflicts, violence, and persecution have increased the number of people from refugee or refugee-like backgrounds. Because urban areas are ...already densely populated, governments have aimed to increase refugee resettlement in rural and/or regional areas. Because of the complex healthcare needs of refugees, this creates challenges for healthcare service providers. Identifying barriers to accessing healthcare in rural areas is therefore important to better inform policy settings and programmes that will provide culturally appropriate patient-centred care to the refugee community.
This review scoped 22 papers written in English between 2018 and July 2023 from five countries (Australia, New Zealand, Germany, Bangladesh, and Lebanon) in order to provide an overview of the barriers and possible solutions to facilitate refugees' access to healthcare.
The reviewed literature summarised the perceptions of at least 3,561 different refugees and 259 rural health service providers and/or administrators and identified major challenges. These include communication (illiteracy in the resettlement country language and lack of a suitable interpreter), lack of cultural awareness of health services, discrimination, and access difficulties (transportation, availability of health specialist services, cost). As a consequence, it was identified that improving access to affordable housing, employment through credential recognition, competence-level education for children, facilitating language training, and adapting health information would increase resettlement and encourage access to healthcare.
Refugees face significant barriers to accessing and engaging with healthcare services. This impacts their integration into rural communities and increases the prevalence of psychosocial issues like feelings of loneliness, low self-esteem, a lack of autonomy, and a lack of empowerment over informed decision-making, especially for women, jobless men, and the elderly. These findings support the need for additional support for refugees and healthcare providers to improve language proficiency and cultural competency. Policymakers need to improve the availability and accessibility of employment, housing accessibility, and service mobility. Additionally, more research is needed to assess the efficacy of emerging innovative programmes that aim to close the gap by delivering culturally appropriate patient-centred care to refugee communities in rural areas.
Transmission electron microscopy (TEM) has nanometre resolution and can be used to distinguish single extracellular vesicles (EVs) from non-EV particles. TEM images of EVs are a result of operator ...image selection. To which extent operator image selection reflects the overall sample quality, and to which extent the images are comparable and reproducible, is unclear. In a first attempt to improve the comparability and reproducibility of TEM to visualise EVs, we compared operator image selection to images taken at predefined locations from the same grids, using four EV TEM preparation protocols, a single EV-containing sample and a single TEM instrument. Operator image selection leads to high-quality images that are more similar between the protocols. In contrast, images taken at predefined locations reveal differences between the protocols, for example in number of EVs per image and background quality. From the evaluated protocols, for only one protocol the operator image selection is comparable to the TEM images taken at predefined locations. Taken together, operator image selection can be used to demonstrate the presence of EVs in a sample, but seem less suitable to demonstrate the quality of a sample. Because images taken at predefined locations reflect the overall quality of the EV-containing sample rather than the presence of EVs alone, this is a first step to improve the comparability and reproducibility of TEM for monitoring the quality of EV-containing samples.
Presence of five or more circulating tumor cells (CTC) in patients with metastatic carcinomas is associated with poor survival. Although many objects positive for epithelial cell adhesion molecules ...and cytokeratin (EpCAM+CK+) are not counted as CTC, they may be an important predictor for survival. We evaluated the association between these objects and survival in patients with prostate cancer.
Included in this follow-up study were 179 patients with castration-resistant prostate cancer. CellSearch was used to isolate EpCAM+ objects and to stain DNA, cytokeratin and CD45. All EpCAM+CK+ objects were subdivided into seven classes on the basis of predefined morphological appearance in 63 independent samples. Association of each class with survival was studied using Kaplan–Meier and Cox regression analyses.
Each EpCAM+CK+CD45- class showed a strong association with overall survival (P < 0.001). This included small tumor microparticles (S-TMP), which did not require a nucleus and thus are unable to metastasize. A higher number of objects in any class was associated with decreased survival. A good prediction model included large tumor cell fragments (L-TCF), age, hemoglobin and lactate dehydrogenase. Models with S-TMP or CTC instead of L-TCF performed similarly.
EpCAM+CK+CD45- that do not meet strict definitions for CTC are strong prognostic markers for survival.
Cell-derived or extracellular vesicles, including microparticles and exosomes, are abundantly present in body fluids such as blood. Although such vesicles have gained strong clinical and scientific ...interest, their detection is difficult because many vesicles are extremely small with a diameter of less than 100 nm, and, moreover, these vesicles have a low refractive index and are heterogeneous in both size and composition. In this review, we focus on the relatively high throughput detection of vesicles in suspension by flow cytometry, resistive pulse sensing, and nanoparticle tracking analysis, and we will discuss their applicability and limitations. Finally, we discuss four methods that are not commercially available: Raman microspectroscopy, micro nuclear magnetic resonance, small-angle X-ray scattering (SAXS), and anomalous SAXS. These methods are currently being explored to study vesicles and are likely to offer novel information for future developments.
Essentials
Platelet extracellular vesicles (EVs) concentrations measured by flow cytometers are incomparable.
A model is applied to convert ambiguous scatter units to EV diameter in nanometer.
Most ...included flow cytometers lack the sensitivity to detect EVs of 600 nm and smaller.
The model outperforms polystyrene beads for comparability of platelet EV concentrations.
Summary
Background
Detection of extracellular vesicles (EVs) by flow cytometry has poor interlaboratory comparability, owing to differences in flow cytometer (FCM) sensitivity. Previous workshops distributed polystyrene beads to set a scatter‐based diameter gate in order to improve the comparability of EV concentration measurements. However, polystyrene beads provide limited insights into the diameter of detected EVs.
Objectives
To evaluate gates based on the estimated diameter of EVs instead of beads.
Methods
A calibration bead mixture and platelet EV samples were distributed to 33 participants. Beads and a light scattering model were used to set EV diameter gates in order to measure the concentration of CD61–phycoerythrin‐positive platelet EVs.
Results
Of the 46 evaluated FCMs, 21 FCMs detected the 600–1200‐nm EV diameter gate. The 1200–3000‐nm EV diameter gate was detected by 31 FCMs, with a measured EV concentration interlaboratory variability of 81% as compared with 139% with the bead diameter gate. Part of the variation in both approaches is caused by precipitation in some of the provided platelet EV samples. Flow rate calibration proved essential because systems configured to 60 μL min−1 differed six‐fold in measured flow rates between instruments. Conclusions
EV diameter gates improve the interlaboratory variability as compared with previous approaches. Of the evaluated FCMs, 24% could not detect 400‐nm polystyrene beads, and such instruments have limited utility for EV research. Finally, considerable differences were observed in sensitivity between optically similar instruments, indicating that maintenance and training affect the sensitivity.
FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of ...Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA- strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.
Essentials
Human salivary extracellular vesicles (EVs) expose coagulant tissue factor (TF).
Salivary EVs expose CD24, a ligand of P‐selectin.
CD24 and coagulant TF co‐localize on salivary EVs.
...TF+/CD24+ salivary EVs bind to activated platelets and trigger coagulation.
Summary
Background
Extracellular vesicles (EVs) from human saliva expose coagulant tissue factor (TF). Whether such TF‐exposing EVs contribute to hemostasis, however, is unknown. Recently, in a mice model, tumor cell‐derived EVs were shown to deliver coagulant TF to activated platelets at a site of vascular injury via interaction between P‐selectin glycoprotein ligand‐1 (PSGL‐1) and P‐selectin.
Objectives
We hypothesized that salivary EVs may deliver coagulant TF to activated platelets via interaction with P‐selectin.
Methods
We investigated the presence of two ligands of P‐selectin on salivary EVs, PSGL‐1 and CD24.
Results
Salivary EVs expose CD24 but PSGL‐1 was not detected. Immune depletion of CD24‐exposing EVs completely abolished the TF‐dependent coagulant activity of cell‐free saliva, showing that coagulant TF and CD24 co‐localize on salivary EVs. In a whole blood perfusion model, salivary EVs accumulated at the surface of activated platelets and promoted fibrin generation, which was abolished by an inhibitory antibody against human CD24.
Conclusions
A subset of EVs in human saliva expose coagulant TF and CD24, a ligand of P‐selectin, suggesting that such EVs may facilitate hemostasis at a site of skin injury where the wound is licked in a reflex action.
A validated assay for the enumeration of circulating melanoma cells (CMCs) may facilitate the development of more effective therapies for metastatic melanoma patients. In this study CD146+ cells were ...immunomagnetically enriched from 7.5 ml of blood. Isolated cells were fluorescently stained with DAPI, anti-molecular weight melanoma-associated antigen (HMW-MAA), anti-CD45 and CD34 and Ki67. CMCs were identified as CD146+, HMW-MAA+, CD45-, CD34-, Ki67-/+ cells. Eighty-eight percent of spiked SK-MEL28 cells in 7.5 ml blood were recovered. In all 55 healthy donors ≤1 CMCs were detected in 7.5 ml of blood. A retrospective analysis was conducted comparing CMC counts and overall survival in 79 blood samples from 44 melanoma patients. CMCs ranged from 0 to 8,042 per 7.5 ml. Two or more CMCs were detected in 18 (23%) of the patients and 30-100% (mean 84%) of the CMCs expressed the proliferation marker Ki67. Patients with ≥2 CMCs per 7.5 ml of whole blood, as compared with the group with <2 CMCs, had a shorter overall survival (2.0 months vs. 12.1 months, P=0.001).
Extracellular vesicles (EVs) in plasma are commonly identified by staining with antibodies and generic dyes, but the specificity of antibodies and dyes to stain EVs is often unknown. Previously, we ...showed that platelet-depleted platelet concentrate contains two populations of particles >200 nm, one population with a refractive index (RI) < 1.42 that included the majority of EVs, and a second population with an RI > 1.42, which was thought to include lipoproteins. In this study, we investigated whether EVs can be distinguished from lipoproteins by the RI and whether the RI can be used to determine the specificity of antibodies and generic dyes used to stain plasma EVs. EVs and lipoproteins present in platelet-depleted platelet concentrate were separated by density gradient centrifugation. The density fractions were analyzed by Western blot and transmission electron microscopy, the RI of particles was determined by Flow-SR. The RI was used to evaluate the staining specificity of an antibody against platelet glycoprotein IIIa (CD61) and the commonly used generic dyes calcein AM, calcein violet, di-8-ANEPPS, and lactadherin in plasma. After density gradient centrifugation, EV-enriched fractions (1.12 to 1.07 g/mL) contained the highest concentration of particles with an RI < 1.42, and the lipoprotein-enriched fractions (1.04 to 1.03 g/mL) contained the highest concentration of particles with an RI > 1.42. Application of the RI showed that CD61-APC had the highest staining specificity for EVs, followed by lactadherin and calcein violet. Di-8-ANEPPS stained mainly lipoproteins and calcein AM stained neither lipoproteins nor EVs. Taken together, the RI can be used to distinguish EVs and lipoproteins, and thus allows evaluation of the specificity of antibodies and generic dyes to stain EVs.
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