Hvidovre Hospital

Abstract Extracellular vesicles (EVs) hold significant promise as biomarkers, but their clinical translation is constrained by variability in pre-analytical handling and isolation. EV isolation methods directly shape which EV populations are captured and characterized, yet systematic method comparisons across multiple analytical dimensions are limited. We comprehensively evaluated eleven EV isolation methods to define their performance and applications. EVs were quantified by NanoFCM, profiled for tetraspanins (CD9, CD63, CD81) via MSD assays, and further characterized by LC-MS/MS proteomics. We show that different EV isolation methods recover different EV populations. Our data provide guidance on method selection based on downstream application needs and serve as a look-up tool if a protein of interest is detected. EV isolation methods broadened proteome coverage but showed divergent performance and recover different EV populations. While all methods captured EVs in the 50-150nm range, centrifugation and ultracentrifugation identified the broadest proteomes (up to 1093 proteins) driven by higher plasma protein carryover. Conversely, ExoEasy and qEV 70 isolated larger EVs and achieved stronger depletion of abundant plasma proteins but showed lower proteome coverage. A total of 117 proteins were detected across all isolation methods. Pre-clearing samples removed contaminants but at the cost of protein identifications. We demonstrate that method selection must align with the specific analytical goal: centrifugation for comprehensive proteome profiling, affinity/size-exclusion methods for contaminant-sensitive assays, and precipitation for high-throughput applications. This systematic characterization provides an evidence-based framework and look-up resource for matching isolation strategies to downstream applications and research questions. Graphical Abstract for Table of Contents This study evaluated 11 extracellular vesicle (EV) isolation methods which enriched distinct EV subpopulations with varying degrees of contaminants. No single approach optimized purity or proteome coverage; in this paper we present an Evidence-Based Framework to select plasma EV isolation methods based on downstream application needs.

To describe the epidemiology of adult anterior cruciate ligament (ACL) ruptures in Denmark from 2000 to 2018, focusing on: (1) overall ACL incidence rate (IR) per 100,000 inhabitants per year, (2) sports-specific ACL IR per 100,000 athletes per year and (3) ACL reconstruction (ACLR) IR per 100,000 inhabitants per year. This registry study utilized prospectively collected data from the Danish National Patient Registry (DNPR). Two ACL IRs were calculated: unadjusted IR based on registry data and adjusted IR corrected using patient self-reports to account for diagnostic misclassification. Sports-specific ACL IRs (2012-2018) were estimated by combining self-reported injury data with sports participation figures from the National Olympic Committee and Sports Confederation of Denmark. ACLR IRs were calculated using procedure codes from DNPR, with annual Danish population data as denominators. Between 2000 and 2018, 90,608 patients were identified: 40,691 completed questionnaires. Overall adjusted ACL IR remained stable between 107/100,000 (confidence interval [CI]: 104-111) in 2000 and 106/100,000 (CI: 103-109) in 2018, increasing among females from 81/100,000 (CI: 78-85) to 90/100,000 (CI: 87-94) and decreasing among males from 134/100,000 (CI: 129-140) to 122/100,000 (CI: 117-127). ACLR IR rose from 36/100,000 (CI: 34-38) to 43/100,000 (CI: 41-45), with increases in both sexes, particularly among younger (18-25 years) and older (>45 years) adults. Football was the leading cause of ACL injury overall and in men; women had more injuries in skiing and handball. The highest sports-specific ACL IR was in female handball players, peaking at 2880/100,000 athletes in 2017. Females showed higher IRs in pivoting sports, while males had higher rates in gymnastics. While overall ACL IRs remained stable, an increase among females and a decline in males were found. ACLR rates have risen in both sexes. Female handball players had by far the highest ACL IR. Level II.

BackgroundAssessment of subtle hip fractures on radiographs can be difficult, especially among less experienced emergency physicians, which may prolong the diagnosis and ultimately time to surgery. Clinical artificial intelligence (AI) decision support tools have shown great potential in assisting the detection of fractures on radiographs.PurposeTo investigate how a CE-marked AI fracture detection tool affects junior doctors' diagnostic accuracy in detecting hip fractures on radiographs.Material and MethodsEight junior doctors with affiliation to the Accident and Emergency (A&E) department read 246 hip radiographic examinations with and without AI support. The reference standard was determined by two musculoskeletal radiologists, to measure sensitivity and specificity for readers without and with support from the AI tool as well as the AI tool's standalone performance.ResultsMean sensitivity in detecting hip fractures increased significantly from 0.89 (95% confidence interval [CI] = 0.85-0.93) without AI support to 0.94 (95% CI = 0.92-0.97) (χ2 = 9.27; P = 0.002) with AI support and the false-negative cases was thereby reduced by 49%. There was no significant change in mean specificity 0.90 (95% CI = 0.86-0.93) to 0.91 (95% CI = 0.88-0.94) (χ2 = 0.34; P = 0.56). The AI standalone performance was 0.99 (95% CI = 0.99-1.00) and 0.73 (95% CI = 0.67-0.80) in sensitivity and specificity, respectively.ConclusionOut of eight junior doctors, seven detected more fractures with AI assistance than without. The applied performance gain for readers highlights the value of the product.

Asthma severity is influenced by complex immunologic and environmental factors. While allergic asthma is linked to increased susceptibility to respiratory infections, the combined role of allergy and antibiotic-treated infections in progression to severe asthma has not been fully evaluated. To evaluate whether allergic asthma and recurrent respiratory infections (RRI) requiring antibiotics are associated with increased risk of developing severe asthma. We conducted a registry-based cohort study using Swedish national registry data. Adults with mild-to-moderate asthma were identified in 2014 (baseline) based on prescription records and absence of severe disease indicators. During a two-year exposure window (2015-2016), RRI was defined as ≥ 2 antibiotic prescriptions for lower respiratory tract infections. The outcome was development of severe asthma during 2017-2019, based on ERS/ATS treatment criteria. Allergic asthma was defined by ≥ 2 prescriptions for anti-allergic medications at baseline. Among 113,393 patients, 24,692 (21.8%) had allergic asthma. RRI occurred more frequently in allergic versus non-allergic asthma (7.5% vs. 5.9%, p < 0.001). A total of 869 patients (0.77%) developed severe asthma. Incidence was higher in those with RRI and highest among patients with both allergic asthma and RRI (2.0%), corresponding to a relative risk of 3.47 (95% CI: 2.49-4.83) versus patients with neither exposure. Results were consistent after adjustment for age, sex and comorbidities. Allergic asthma and antibiotic-treated respiratory infections were independent and additive predictors of severe asthma progression. These findings support a clinically actionable risk profile and may inform targeted preventive strategies in asthma management.

Background/Objectives: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) or without NP (CRSsNP) are common upper airway diseases with major impact on healthcare utilization. Little is known about the overall national financial burden of CRS. We aimed to assess the excess financial burden of CRS from a countrywide perspective. Methods: Annual expenditure from healthcare, welfare transfers and foregone income was retrieved from national databases, annualized and compared to matched healthy comparators. Results: Of the 303,475 patients included with CRS (mean age 51, 55% female), 18,142 were subclassified as CRSsNP (24%) or CRSwNP (76%). For CRS patients, annual excess healthcare costs were €1315 (1296-1333) compared to comparators. Patients with CRS earned €1356 (1230-1479) less annually compared to comparators. Patients with CRS of working age (18-64 years) had excess welfare transfers (€816 (782-850) compared to comparators, driven by sick leave and disability. Increases in healthcare costs were seen for patients with CRSwNP (€5406 (4860-6012) annually) compared to CRSsNP (€4945 (4293-5696)) driven by increases in CRS-related costs. Total societal burden for the entire cohort was €686,052,898, of which systemic corticosteroid exposure-related conditions represented €20,332,825. Excess welfare transfers represented €174,394,048 annually. Conclusions: Chronic rhinosinusitis is associated with a significant financial burden, both in terms of societal healthcare and welfare expenditure and patients' personal finances due to lost income.

Poor appetite is a key contributor to malnutrition in older adults, partly due to reduced dietary intake. While nutritional deficits are well recognized, the biological mechanisms driving poor appetite remain incompletely understood. Growth Differentiation Factor-15 (GDF-15) and the Simplified Nutritional Appetite Questionnaire (SNAQ) may help identify patients at risk of insufficient intake. This study examines the association between GDF-15 and insufficient energy and protein intake and evaluates the predictive performance of GDF-15 and SNAQ - individually and in combination - to identify insufficient energy and protein intake (<75 % and <100 % of estimated requirements, respectively) in acutely admitted older adults. This exploratory study included 130 older adults (≥65 years) with or at risk of malnutrition, admitted for acute medical illness and assessed at baseline, and 8 and 16 weeks post-discharge (FW8 and FW16). GDF-15 plasma concentrations were measured from blood samples, SNAQ scores from validated questionnaire, and energy and protein intake were evaluated through 3-day dietary records. Associations were analyzed using regression models and predictive performance was evaluated using Receiver Operating Characteristic analysis. A doubling of GDF-15 showed a 6.73 % (-13.98-0.51) and 5.07 % (-12.18-2.05) lower baseline energy and protein intake, and results decreased after discharge. Using predefined cut-offs of ≥1500 pg/mL for GDF-15 and ≤14 for SNAQ, the highest positive predictive values (PPVs) were 86 (95 % CI: 0.83-0.89) and 90 (0.79-1.00) at baseline and FW8 for insufficient protein intake, respectively, with corresponding sensitivities of 84 (0.75-0.92) and 60 (0.44-0.74). For estimated cut-offs of 2095 pg/mL for GDF-15 at baseline and 18.5 for SNAQ at FW8, the highest PPVs were 89 (0.84-0.94) and 87 (0.86-0.87), with sensitivities of 73 (0.62-0.84) and 99 (0.96-1.00), both for insufficient protein intake, respectively. Combining GDF-15 and SNAQ yielded improved PPVs at the expense of reduced sensitivity in most models. All results were non-significant. Higher GDF-15 levels showed a non-significant trend toward lower baseline energy and protein intake. GDF-15 and SNAQ offer limited ability to identify insufficient energy and protein intake, based on predictive performance. These exploratory findings should therefore be interpreted cautiously. Larger studies are needed to validate and further refine the use of GDF-15 and SNAQ in clinical settings.

Tendinopathy represents a major musculoskeletal health problem, yet its pathogenesis remains poorly understood. Tendinopathy development is studied in humans with early (<3 months of symptoms, n=14) (ET) or chronic (>3 months, n=16) (CT) patellar tendinopathy and in healthy subjects (n=15) (CTRL). Pain increases, and function declines with tendinopathy duration and correlated with tendon size (3T and 7T MRI). Tendon blood flow (Doppler ultrasonography) increases gradually in ET and CT, while peritendinous blood flow only rose in CT. Microscopy-based mapping (immunofluorescence microscopy and Cell DIVE) of vasculature shows marked changes in CT only, indicating flow increases in existing vessels early in tendinopathy, whereas angiogenesis is a late phenomenon. Cell DIVE indicates perivascular cell recruitment and potential lymphatic expansion in tendinopathy. Further, proteomics reveals that most matrix regulation occurs late in tendinopathy. Data from a previous study from the lab demonstrate faster treatment effect in tendinopathy with shorter symptom duration, supporting that early tissue changes may be more receptive to treatment. It is concluded that early tendinopathy is dominated by pain correlating with tendon swelling and hyperperfusion, whereas chronic tendinopathy is characterized by neovascularization and matrisome changes. These findings suggest that targeting early tissue changes can lead to superior treatment effects in tendinopathy.