Extracellular vesicles and their therapeutic applications: a review article (part 2).

Extracellular vesicles (EVs) have emerged as a fascinating area of research in molecular biology, with diverse therapeutic applications. These small membrane-bound structures, released by cells into the extracellular space, play a crucial role in intercellular communication and hold great potential for advancing medical treatments. The aim of this study is to have a narrative review on the use and therapeutic applications of EVs. Their unique characteristics, including stability, biocompatibility, and the ability to traverse biological barriers, make them promising tools for targeted drug delivery. By engineering EVs to encapsulate specific cargo molecules, such as therapeutic proteins, small interfering RNA (siRNA), or anti-cancer drugs, researchers can enhance drug stability and improve targeted delivery to desired cells or tissues. This approach can minimize off-target effects and improve therapeutic efficacy. Based on our literature search, we found that EVs can be used as biomarkers to predict diseases. Although much progress has been made in understanding the biology and function of exosomes, there are still unanswered questions that require further research. This includes identifying appropriate and safe techniques for producing exosomes in large quantities, determining which types of cells are suitable for exosome donor cells for therapeutic purposes, and investigating the safety of exosomes in human studies. Overall, the use of exosomes in clinical therapeutic applications requires a strong understanding of molecular signaling cascades and exosome profiles, as well as the specificity and sensitivity of biomarker and drug delivery methods.

Emerging Roles of Circular RNA in Macrophage Activation and Inflammatory Lung Responses.

Circular RNA (circRNA) is a type of single-stranded RNA that forms a covalently closed continuous loop, unlike linear RNA. The expression of circRNAs in mammals is often conserved across species and shows tissue and cell specificity. Some circRNA serve as gene regulators. However, the biological function of most circRNAs is unclear. CircRNA does not have 5' or 3' ends. The unique structure of circRNAs provides them with a much longer half-life and more resistance to RNase R than linear RNAs. Inflammatory lung responses occur in the pathogenesis and recovery of many lung diseases. Macrophages form the first line of host defense/innate immune responses and initiate/mediate lung inflammation. For example, in bacterial pneumonia, upon pro-inflammatory activation, they release early response cytokines/chemokines that recruit neutrophils, macrophages, and lymphocytes to sites of infection and clear pathogens. The functional effects and mechanisms by which circRNAs exert physiological or pathological roles in macrophage activation and lung inflammation remain poorly understood. In this article, we will review the current understanding and progress of circRNA biogenesis, regulation, secretion, and degradation. Furthermore, we will review the current reports on the role of circRNAs in macrophage activation and polarization, as well as in the process of inflammatory lung responses.

Proteome of plasma extracellular vesicles as a source of colorectal cancer biomarkers.

The search for minimally invasive methods for diagnostics of colorectal cancer (CRC) is the most important task for early diagnostics of the disease and subsequent successful treatment. Human plasma represents the main type of biological material used in the clinical practice; however, the complex dynamic range of substances circulating in it complicates determination of CRC protein markers by the mass spectrometric (MS) method. Studying the proteome of extracellular vesicles (EVs) isolated from human plasma represents an attractive approach for the discovery of tissue-secreted CRC markers. We performed shotgun mass spectrometry analysis of EV samples obtained from plasma of CRC patients and healthy volunteers. This MS analysis resulted in identification of 370 proteins (which were registered by at least two peptides). Stable isotope-free relative quantitation identified 55 proteins with altered abundance in EV samples obtained from plasma samples of CRC patients as compared to healthy controls. Among the EV proteins isolated from blood plasma we found components involved in cell adhesion and the VEGFA-VEGFR2 signaling pathway (TLN1, HSPA8, VCL, MYH9, and others), as well as proteins expressed predominantly by gastrointestinal tissues (polymeric immunoglobulin receptor, PIGR). The data obtained using the shotgun proteomic profiling may be added to the panel for targeted MS analysis of EV-associated protein markers, previously developed using CRC cell models.

Extracellular Vesicles & Co.: scaring immune cells in the TME since ever.

The health tissue surrounding a solid tumor, namely the tumor microenvironment (TME), is an extremely complex universe of cells, extracellular matrix, and signals of various nature, that support and protect the growth of cancer cells. The interactions taking place between cancer cells and the TME are crucial not only for tumor growth, invasion, and metastasis but they also play a key role in modulating immune system responses to cancer, and vice-versa. Indeed, tumor-infiltrating immune cells (e.g., T lymphocytes and natural killers) activity is greatly affected by signals (mostly ligands/receptors and paracrine) they receive in the TME, which frequently generate an immunosuppressive milieu. In the last years, it has become evident that soluble and receptor signaling is not the only way of communication between cells in the TME, with extracellular vesicles, such as exosomes, playing a central role. Among the different new kind of vesicles recently discovered, migrasomes look like to be of extreme interest as they are not only different from the others, but also have been reported as able to deliver a very heterogeneous kind of messages, able to profoundly affect recipient cells' behavior. Indeed, the role played by the different classes of extracellular vesicles, especially in the TME, relies on their not-directional diffusion from the originating cells, while migrasomes released from migrating cells do have a directional effect. Migrasomes biology and their involvement in cancer progression, dissemination, and resistance to therapy is still a largely obscure field, but with promising development foreseen in the next future.

Advancements in extracellular vesicle targeted therapies for rheumatoid arthritis: insights into cellular origins, current perspectives, and emerging challenges.

Rheumatoid arthritis (RA) remains a challenging chronic autoimmune disorder characterized by persistent joint inflammation and damage. While modern regenerative strategies, encompassing cell/stem cell-based therapies, gene therapy, and tissue engineering, have advanced tissue repair efforts, a definitive cure for RA remains elusive. Consequently, there is growing interest in developing targeted therapies that directly address the underlying mechanisms driving RA pathogenesis, such as extracellular vesicles (EVs). These small membrane-bound particles can modulate immune responses within the inflammatory microenvironment of damaged cartilage. To launch the clinical potential of EVs, they can be isolated from various cell types through several techniques. EVs can carry various bioactive molecules and anti-inflammatory or pro-regenerative drugs, deliver them directly to the affected joints, and affect the behavior of injured cells, making them a compelling choice for targeted therapy and drug delivery in RA patients. However, there are still several challenges and limitations associated with EV-based therapy, including the absence of standardized protocols for EV isolation, characterization, and delivery. This review provides a comprehensive overview of the cellular sources of EVs in RA and delves into their therapeutic potential and the hurdles they must overcome.

Role of extracellular vesicle-associated proteins in the progression, diagnosis, and treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, characterized by difficulties in early diagnosis, prone to distant metastasis, and high recurrence rates following surgery. Extracellular vesicles (EVs) are a class of cell-derived particles, including exosomes, characterized by a phospholipid bilayer. They serve as effective carriers for intercellular communication cargo, including proteins and nucleic acids, and are widely involved in tumor progression. They are being explored as potential tumor biomarkers and novel therapeutic avenues. We provide a brief overview of the biogenesis and characteristics of EVs to better understand their classification standards. The focus of this review is on the research progress of EV-associated proteins in the field of HCC. EV-associated proteins are involved in tumor growth and regulation in HCC, participate in intercellular communication within the tumor microenvironment (TME), and are implicated in events including angiogenesis and epithelial-mesenchymal transition (EMT) during tumor metastasis. In addition, EV-associated proteins show promising diagnostic efficacy for HCC. For the treatment of HCC, they also demonstrate significant potential including enhancing the efficacy of tumor vaccines, and as targeting cargo anchors. Facing current challenges, we propose the future directions of research in this field. Above all, research on EV-associated proteins offers the potential to enhance our comprehension of HCC and offer novel insights for developing new treatment strategies.

Role of Extracellular Vesicles in the Progression of Brain Tumors.

Brain tumors, and, in particular, glioblastoma (GBM), are among the most aggressive forms of cancer. In spite of the advancement in the available therapies, both diagnosis and treatments are still unable to ensure pathology-free survival of the GBM patients for more than 12-15 months. At the basis of the still poor ability to cope with brain tumors, we can consider: (i) intra-tumor heterogeneity; (ii) heterogeneity of the tumor properties when we compare different patients; (iii) the blood-brain barrier (BBB), which makes difficult both isolation of tumor-specific biomarkers and delivering of therapeutic drugs to the brain. Recently, it is becoming increasingly clear that cancer cells release large amounts of extracellular vesicles (EVs) that transport metabolites, proteins, different classes of RNAs, DNA, and lipids. These structures are involved in the pathological process and characterize any particular form of cancer. Moreover, EVs are able to cross the BBB in both directions. Starting from these observations, researchers are now evaluating the possibility to use EVs purified from organic fluids (first of all, blood and saliva), in order to obtain, through non-invasive methods (liquid biopsy), tumor biomarkers, and, perhaps, also for obtaining nanocarriers for the targeted delivering of drugs.

Identification and Quantification of Extracellular Vesicles: Comparison of SDS-PAGE Analysis and Biosensor Analysis with QCM and IDT Chips.

This study presents and compares two methods for identifying the types of extracellular vesicles (EVs) from different cell lines. Through SDS-PAGE analysis, we discovered that the ratio of CD63 to CD81 in different EVs is consistent and distinct, making it a reliable characteristic for recognizing EVs secreted by cancer cells. However, the electrophoresis and imaging processes may introduce errors in the concentration values, especially at lower concentrations, rendering this method potentially less effective. An alternative approach involves the use of quartz crystal microbalance (QCM) and electroanalytical interdigitated electrode (IDT) biosensors for EV type identification and quantification. The QCM frequency shift caused by EVs is directly proportional to their concentration, while electroanalysis relies on measuring the curvature of the I-V curve as a distinguishing feature, which is also proportional to EV concentration. Linear regression lines for the QCM frequency shift and the electroanalysis curvature of various EV types are plotted separately, enabling the estimation of the corresponding concentration for an unknown EV type on the graphs. By intersecting the results from both biosensors, the unknown EV type can be identified. The biosensor analysis method proves to be an effective means of analyzing both the type and concentration of EVs from different cell lines.

Surface Engineered Osteoblast-Extracellular Vesicles Serve as an Efficient Carrier for Drug and Small RNA to Actively Target Osteosarcoma.

Osteosarcoma (OS) is a rare malignant tumor that affects soft tissue and has high rates of lung metastasis and mortality. The primary treatments for OS include preoperative chemotherapy, surgical resection of the lesion, and postoperative chemotherapy. However, OS chemotherapy presents critical challenges related to treatment toxicity and multiple drug resistance. To address these challenges, nanotechnology has developed nanosystems that release drugs directly to OS cells, reducing the drug's toxicity. Extracellular vesicles (EVs) are nanosized lipid-bilayer bound vesicles that act as cell-derived vehicles and drug delivery systems for several cancers. This study aims to utilize EVs for OS management by co-delivering Hdac1 siRNA and zoledronic acid (zol). The EVs' surface is modified with folic acid (FA) and their targeting ability is compared to that of native EVs. The results showed that the EVs' targeting ability depends on the parent cell source, and FA conjugation further enhanced it. Furthermore, EVs were used as the carrier for co-loading drug (zol) and small RNA (Hdac-1). This approach of using surface engineered EVs as carriers for cargo loading and delivery can be a promising strategy for osteosarcoma management.

The Role of Dental-derived Stem Cell-based Therapy and Their Derived Extracellular Vesicles in Post-COVID-19 Syndrome-induced Tissue Damage.

Long coronavirus disease 2019 (COVID-19) is linked to an increased risk of post-acute sequelae affecting the pulmonary and extrapulmonary organ systems. Up to 20% of COVID-19 patients may proceed to a more serious form, such as severe pneumonia, acute respiratory distress syndrome (ARDS), or pulmonary fibrosis. Still, the majority of patients may only have mild, self-limiting sickness. Of particular concern is the possibility of parenchymal fibrosis and lung dysfunction in long-term COVID-19 patients. Furthermore, it has been observed that up to 43% of individuals hospitalized with COVID-19 also had acute renal injury (AKI). Care for kidney, brain, lung, cardiovascular, liver, ocular, and tissue injuries should be included in post-acute COVID-19 treatment. As a powerful immunomodulatory tool in regenerative medicine, dental stem cells (DSCs) have drawn much interest. Numerous immune cells and cytokines are involved in the excessive inflammatory response, which also has a significant effect on tissue regeneration. A unique reservoir of stem cells (SCs) for treating acute lung injury (ALI), liver damage, neurological diseases, cardiovascular issues, and renal damage may be found in tooth tissue, according to much research. Moreover, a growing corpus of in vivo research is connecting DSC-derived extracellular vesicles (DSC-EVs), which are essential paracrine effectors, to the beneficial effects of DSCs. DSC-EVs, which contain bioactive components and therapeutic potential in certain disorders, have been shown as potentially effective therapies for tissue damage after COVID-19. Consequently, we explore the properties of DSCs in this work. Next, we'll look at how SARS-CoV-2 affects tissue damage. Lastly, we have looked at the use of DSCs and DSC-EVs in managing COVID-19 and chronic tissue damage, such as injury to the heart, brain, lung, and other tissues.

Lineage Mapping of Extracellular Vesicles: What Cells Do They Come from And Where Do They Go?

Significance: Release of extracellular vesicles (EVs) by various cell types has been shown to mediate the delivery of biologically active payloads from donor cells to recipient cells; however, it remains unclear what cell types these EVs come from. With a focus on fluorescent reporters to monitor the release of EVs, especially those under the control of cell type-specific promoters, we address the translational relevance of genetic tools in cultured cells, normal tissues, and in models of development, injury, cancer, and wound healing. Recent Advances: It is well established that EVs are released by many cell types in the body via fusion and release processes at the plasma membrane. Since there remains debate about what fraction of EVs are released through regulated endosomal trafficking pathways versus nonspecific mechanisms, the development and validation of novel molecular tools are important to address the cellular source of EVs. Critical Issues: There is a need to develop and characterize new cell type-specific reporter mouse models that build upon the examples detailed here to identify the cellular source of EVs with genetic approaches being useful in addressing these critical limitations. Future Directions: Advances in reporter systems will drive a better understanding of EV subsets to identify compartment-specific EV localization to guide the development of more translationally relevant models for the wound healing field.

The Evolution of Microfluidic-Based Drug-Loading Techniques for Cells and Their Derivatives.

Conventional drug delivery techniques face challenges related to targeting and adverse reactions. Recent years have witnessed significant advancements in nanoparticle-based drug carriers. Nevertheless, concerns persist regarding their safety and insufficient metabolism. Employing cells and their derivatives, such as cell membranes and extracellular vesicles (EVs), as drug carriers effectively addresses the challenges associated with nanoparticle carriers. However, an essential hurdle remains in efficiently loading drugs into these carriers. With the advancement of microfluidic technology and its advantages in precise manipulation at the micro- and nanoscales, as well as minimal sample loss, it has found extensive application in the loading of drugs using cells and their derivatives, thereby fostering the development of drug-loading techniques. This paper outlines the characteristics and benefits of utilizing cells and their derivatives as drug carriers and provides an overview of current drug-loading techniques, particularly those rooted in microfluidic technology. The significant potential for microfluidic technology in targeted disease therapy through drug delivery systems employing cells and their derivatives, is foreseen.

High-precision bioactive scaffold with dECM and extracellular vesicles targeting 4E-BP inhibition for cartilage injury repair.

The restoration of cartilage injuries remains a formidable challenge in orthopedics, chiefly attributed to the absence of vascularization and innervation in cartilage. Decellularized extracellular matrix (dECM) derived from cartilage, following antigenic removal through decellularization processes, has exhibited remarkable biocompatibility and bioactivity, rendering it a viable candidate for cartilage repair. Additionally, extracellular vesicles (EVs) generated from cartilage have demonstrated a synergistic effect when combined with dECM, potentially mitigating the inhibitory impact on protein synthesis by phosphorylating 4ebp, thereby promoting the synthesis of cartilage-related proteins such as collagen. In pursuit of this objective, we have innovated a novel bioink and repair scaffold characterized by exceptional biocompatibility, bioactivity, and biodegradability, establishing a tissue-specific microenvironment conducive to chondrogenesis. Within rat osteochondral defects, the biologically active scaffold successfully prompted the formation of transparent cartilage, featuring adequate mechanical strength, favorable elasticity, and dECM deposition indicative of cartilage. In summary, this study has effectively engineered a hydrogel bioink tailored for cartilage repair and devised a bioactive cartilage repair scaffold proficient in instigating cell differentiation and fostering cartilage repair.

Profiling of circulating extracellular vesicle microRNAs reveals diagnostic potential and pathways in non-obstructive and obstructive azoospermia.

The accurate diagnosis of non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) is crucial for selecting appropriate clinical treatments. This study aimed to investigate the pivotal role of miRNAs in circulating plasma extracellular vesicles (EVs) in distinguishing between NOA and OA, as well as uncovering the signaling pathways involved in azoospermia pathogenesis. In this study, differential expression of EV miR-513c-5p and miR-202-5p was observed between NOA and OA patients, while the selenocompound metabolism pathway could be affected in azoospermia through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. The predictive power of these microRNAs was evaluated using ROC-AUC analysis, demonstrating promising sensitivity, specificity, and area under the curve values. A binomial regression equation incorporating circulating plasma levels of EVs miR-202-5p and miR-513c-5p along with follicle-stimulating hormone was calculated to provide a clinically applicable method for diagnosing NOA and OA. This study presents a potentially non-invasive testing approach for distinguishing between NOA and OA, offering a possibly valuable tool for clinical practice.

Plasma Extracellular Vesicles Derived from Pediatric COVID-19 Patients Modulate Monocyte and T Cell Immune Responses Based on Disease Severity.

BACKGROUND: The COVID-19 pandemic has caused significant morbidity and mortality globally. The role of plasma-derived extracellular vesicles (EVs) in pediatric COVID-19 patients remains unclear. METHODS: We isolated EVs from healthy controls (n = 13) and pediatric COVID-19 patients (n = 104) with varying severity during acute and convalescent phases using serial ultracentrifugation. EV effects on healthy PBMCs, naïve CD4+ T cells, and monocytes were assessed through in vitro assays, flow cytometry, and ELISA. RESULTS: Our findings indicate that COVID-19 severity correlates with diverse immune responses. Severe acute cases exhibited increased cytokine levels, decreased IFNγ levels, and lower CD4+ T cell and monocyte counts, suggesting immunosuppression. EVs from severe acute patients stimulated healthy cells to express higher PDL1, increased Th2 and Treg cells, reduced IFNγ secretion, and altered Th1/Th17 ratios. Patient-derived EVs significantly reduced proinflammatory cytokine production by monocytes (p < .001 for mild, p = .0025 for severe cases) and decreased CD4+ T cell (p = .043) and monocyte (p = .033) populations in stimulated healthy PBMCs. CONCLUSION: This study reveals the complex relationship between immunological responses and EV-mediated effects, emphasizing the impact of COVID-19 severity. We highlight the potential role of plasma-derived EVs in early-stage immunosuppression in severe COVID-19 patients.

Exploring the Therapeutic Potential of Extracellular Vesicles Derived from Human Immature Dental Pulp Cells on Papillary Thyroid Cancer.

Mesenchymal stem-cell-derived extracellular vesicles (MSC-EVs) have been increasingly investigated for cancer therapy and drug delivery, and they offer an advanced cell-free therapeutic option. However, their overall effects and efficacy depend on various factors, including the MSC source and cargo content. In this study, we isolated EVs from the conditioned medium of human immature dental pulp stem cells (hIDPSC-EVs) and investigated their effects on two papillary thyroid cancer (PTC) cell lines (BCPAP and TPC1). We observed efficient uptake of hIDPSC-EVs by both PTC cell lines, with a notable impact on gene regulation, particularly in the Wnt signaling pathway in BCPAP cells. However, no significant effects on cell proliferation were observed. Conversely, hIDPSC-EVs significantly reduced the invasive capacity of both PTC cell lines after 120 h of treatment. These in vitro findings suggest the therapeutic potential of hIDPSC-EVs in cancer management and emphasize the need for further research to develop novel and effective treatment strategies. Furthermore, the successful internalization of hIDPSC-EVs by PTC cell lines underscores their potential use as nanocarriers for anti-cancer agents.

Tetrahydroisoquinoline reduces angiogenesis by interacting myeloma cells with HUVECs mediated by extracellular vesicles.

Multiple myeloma (MM) is a neoplastic condition resulting from the uncontrolled expansion of B-cell-derived plasma cells. The importance of angiogenesis in MM development has also been demonstrated. Extracellular vesicles (EVs) have vital functions in interactions between neighboring cells, such as angiogenesis. The objective of this in vitro study was to examine the transfection and angiogenesis effects of MM-EVs on endothelial cells (ECs) upon treatment with Tetrahydroisoquinoline (THIQ) as a bioactive organic compound derivative from isoquinoline. Following treatment of multiple myeloma cells (U266) with THIQ, MM-EVs were harvested and transmigrated to human umbilical vein endothelial cells (HUVEC) in a co-culture model. EVs transmigration was traced by flow cytometry. Correspondingly, the expression of angiogenic genes and/or proteins in U266 cells and HUVECs was measured by RT-PCR and ELISA methods. Likewise, the proliferation and migration of HUVECs treated with THIQ-treated MM-EVs were visualized and estimated by performing both tube formation and scratch wound healing methods. Surprisingly, the anti-angiogenic effect of THIQ-treated MM-EVs was evident by the decreased expression of CD34, VEGFR2, and IL-6 at the mRNA and/or protein levels after internalization of MM-EVs in HUVEC. Finally, tube formation and scratch wound healing experiments showed inhibition of HUVEC cell proliferation and migration by THIQ-treated MM-EVs compared to control MM-EVs. MM-EVs derived from THIQ-treated myeloma cells (U266) inhibited angiogenesis in HUVECs. This phenomenon is coordinated by the internalized THIQ-treated MM-EVs in HUVECs, and ultimately the reduction of angiogenic factors and inhibition of tube formation and scratch wound healing.

Evaluation of resazurin phenoxazine dye as a highly sensitive cell viability potency assay for natural killer cell-derived extracellular vesicle-based cancer biotherapeutics.

Natural killer cell-derived extracellular vesicles (NK-EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK-EVs' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine-based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK-EVs against leukaemia K562 cells (suspension model) and breast cancer MDA-MB-231 cells (adherent model) in vitro. The assay was evaluated based on common analytical parameters setforth by regulatory guidelines, including specificity, selectivity,accuracy, precision, linearity, range and stability. Our results revealed that this resazurin-based cell viability potency assay reliably and reproducibly measured a dose-response of NK-EVs' cytotoxic activity against both cancer models. The assay showed precision with 5% and 20% variation for intra-run and inter-run variability. The assay signal showed specificity and selectivity of NK-EVs against cancer target cells, as evidenced by the diminished viability of cancer cells following a 5-hour treatment with NK-EVs, without any detectable interference or background. The linearity analysis of target cancer cells revealed strong linearity for densities of 5000 K562 and 1000 MDA-MB-231 cells per test with a consistent range. Importantly, NK-EVs' dose-response for cytotoxicity showed a strong correlation (|ρ| ∼ 0.8) with the levels of known cytotoxic factors associated with the NK-EVs' corona (FasL, GNLY, GzmB, PFN and IFN-γ), thereby validating the accuracy of the assay. The assay also distinguished cytotoxicity changes in degraded NK-EVs, indicating the ability of the assay to detect the potential loss of sample integrity. Compared to other commonly reported bioassays (i.e., flow cytometry, cell counting, lactate dehydrogenase release assay, DNA-binding reporter assay and confluence assay), our results support this highly sensitive resazurin-based viability potency assay as a high-throughput and quantitative method for assessing NK-EVs' cytotoxicity against both suspension and adherent cancer models for evaluating NK-EVs' biotherapeutics.

Towards IVDR-compliance by implementing quality control steps in a quantitative extracellular vesicle-miRNA liquid biopsy assay for response monitoring in patients with classic Hodgkin lymphoma.

Previously, we showed that quantification of lymphoma-associated miRNAs miR-155-5p, -127-3p and let-7a-5p levels in plasma extracellular vesicles (EVs) report treatment response in patients with classic Hodgkin lymphoma (cHL). Prior to clinical implementation, quality control (QC) steps and validation are required to meet international regulatory standards. Most published EV-based diagnostic assays have yet to meet these requirements. In order to advance the assay towards regulatory compliance (e.g., IVDR 2017/746), we incorporated three QC steps in our experimental EV-miRNA quantitative real-time reverse-transcription PCR (q-RT-PCR) assay in an ISO-13485 certified quality-management system (QMS). Liposomes encapsulated with a synthetic (nematode-derived) miRNA spike-in controlled for EV isolation by automated size-exclusion chromatography (SEC). Additional miRNA spike-ins controlled for RNA isolation and cDNA conversion efficiency. After deciding on quality criteria, in total 107 out of 120 samples from 46 patients passed QC. Generalized linear mixed-effect modelling with bootstrapping determined the diagnostic performance of the quality-controlled data at an area under the curve (AUC) of 0.84 (confidence interval [CI]: 0.76-0.92) compared to an AUC of 0.87 (CI: 0.80-0.94) of the experimental assay. After the inclusion of QC steps, the accuracy of the assay was determined to be 78.5% in predicting active disease status in cHL patients during treatment. We demonstrate that a quality-controlled plasma EV-miRNA assay is technically robust, taking EV-miRNA as liquid biopsy assay an important step closer to clinical evaluation.