Methods Blood Samples Research Articles

Predicting susceptibility to COVID-19 infection in patients on maintenance hemodialysis by cross-coupling soluble ACE2 concentration with lymphocyte count: an algorithmic approach

IntroductionPatients on maintenance hemodialysis (MHD) were more vulnerable to and had a higher mortality during the COVID-19 pandemic. As angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine S1 member 2 (TMPRSS2) played crucial roles in viral entry into the human host cells, we therefore investigated in the MHD patients whether their plasma levels were associated with susceptibility to the COVID-19.MethodsBlood samples were collected from the patients in our then COVID-19 free center immediately upon lifting of the stringent quarantine measures in early December of 2022 and infection situation was observed within the following 2 weeks. Plasma levels of the soluble ACE2 (sACE2), ACE (sACE) and TMPRSS2 (sTMPRSS2) were measured with ELISA method. Data were stepwisely tested for independent effect, relevant role and synergistic action on the susceptibility by multiple logistic regression, receiver operating characteristic curve and multiple dimensionality reduction (MDR) method, respectively.ResultsAmong the 174 eligible patients, 95 (54.6%) turned COVID-19 positive with a male to female ratio of 1.57 during the observation period. Comparing with the uninfected, the infected had significantly higher sACE2 and lower sTMPRSS2 levels upon comparable sACE concentration. Besides the sACE2, factors associated with susceptibility were vintage and individual session time of the hemodialysis, smoking and comorbidity of hepatitis, whereas lymphocyte counts showed a tendency (p = 0.052). Patients simultaneously manifesting higher sACE2 level and lower lymphocyte counts had an increased infection risk as confirmed by the MDR method.ConclusionBy sorting out the susceptible ones expeditiously, this algorithmic approach may help the otherwise vulnerable MHD patients weather over future wave of COVID-19 variants or outbreak of other viral diseases.

Combined activation of complement and coagulation is associated with myocardial injury in ST-elevation myocardial infarction

Abstract Introduction Experimental data indicates that the complement system and the coagulation cascade can activate each other. The magnitude of this interaction in acute myocardial infarction is not known. We aimed to investigate a possible association between complement activation and coagulation activation in patients with acute ST-elevation myocardial infarction (STEMI), as well as how they relate to myocardial injury estimated by troponin T (TnT) and myocardial function assessed by echocardiography. Purpose To increase the understanding of the interaction between complement activation and coagulation activation in STEMI, and the clinical relevance to myocardial injury. Methods Blood samples were drawn after percutaneous coronary intervention (PCI) in 864 patients with STEMI. Myocardial function was measured as left ventricular ejection fraction (LVEF), estimated by visual approximation or Simpson biplane method within 3 months. Complement activation was assessed by ELISA of circulating levels of the terminal complement complex (TCC). Activation of coagulation was assessed by ELISA of prothrombin fragment 1+2 (F1+2) and D-dimer, and endogenous thrombin potential (ETP), analyzed by calibrated automated thrombogram assay. Results TCC was weakly correlated to F1+2 (r=0.086, p=0.012), D-dimer (r=0.176, p<0.001) and ETP (r=0.144, p<0.001). In univariate linear regression analysis, this association persisted for D-dimer and ETP. When adjusting for associated covariables, the association only persisted for ETP (Table 1). Patients with above-median TCC and D-dimer had significantly higher peak TnT (Figure 1). In logistic regression, having combined above-median TCC and D-dimer was predictive of an LVEF ≤40% (Table 2). Conclusion In this large STEMI cohort, complement activation by TCC was weakly associated with coagulation activation. Combined high levels of TCC and D-dimer were linked to higher peak TnT and lower LVEF, possibly reflecting greater myocardial injury. The interaction between complement and coagulation activation might be relevant for clinical outcomes in STEMI patients and warrants further investigation.

Health risk assessment of lead and cadmium exposure from food and snuff in Pakistani population

BackgroundSmokeless tobacco (SLT) is a tobacco-based product consumed without burning or smoking. Snuff, commonly known as naswar, is a widely used SLT product in Pakistan. This study is designed to investigate the concentrations of lead (Pb) and cadmium (Cd) in snuff and their associated health risks to consumers. MethodsBlood samples from snuff consumers and non-consumers were analyzed for Pb and Cd concentrations. Additionally, samples of drinking water and wheat flour were also analyzed for Pb and Cd. Health risk assessments were calculated using hazard quotient (HQ) and incremental lifetime cancer risk (ILCR) for Pb and Cd. ResultsSnuff consumers had significantly higher blood-Pb and blood-Cd levels compared to non-consumers. Approximately 38.5 % of snuff consumers were found to exceed blood-Pb levels from the safe limits (10 µg dL⁻¹) of the World Health Organization (WHO), while 48.9 % of snuff consumers were found to exceed blood-Cd levels from the WHO safe range of 0.03–0.12 µg dL⁻¹. Snuff samples contained Pb and Cd concentrations ranging from 5.3 to 22.4 µg g⁻¹ and 0.5–2.2 µg g⁻¹, respectively. Drinking water and wheat flour samples were found within the safe limits for Pb and Cd concentration, suggesting snuff as the primary source of exposure. Health risk assessments indicated elevated HQ values, for Pb (38.9 %) and Cd (14.3 %) detected in snuff, surpassing the safe HQ threshold of 1. Concerning ILCR from snuff, values for Pb were within the acceptable range, but alarmingly 100 % of samples for Cd exceeded the safe limits of USEPA (1.0×10⁻⁴), indicating a significant cancer risk in snuff users. ConclusionThis study highlights the exceeded Pb and Cd concentrations in snuff as well as also reporting significant carcinogenic and non-carcinogenic health risks to consumers. These findings emphasized the urgent need for regulatory bodies to ensure the safety of snuff before selling in the market. Public Health Professionals should also initiate educational campaigns to raise awareness among consumers and manufacturers about various health risks associated with the snuff.

A-070 Evaluation of Plain and Serum-Separator Blood Collection Tubes for the Measurement of Steroid Hormones by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Abstract Background Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) is the preferred method for quantifying steroids. Commonly measured steroids 11-deoxycortisol, androstenedione, testosterone, 17-hydroxyprogesterone and dehydroepiandrosterone (DHEA). Although serum separator blood collection tubes may not be recommended but their frequent use is attributed to the ease of serum separation. Moreover, the serum separator tube may be the only available option for analysis, without the need for additional sample collection. Under such conditions, from certain serum separator tubes, we observed occasional interference in LC-MS/MS steroid method. We pursued to evaluate the performance of blood collection tubes without and those with serum separators, for the quantification of specified steroids by LC-MS/MS. Methods Blood samples were collected from 10 volunteers using both plain and serum separator blood collection tubes procured from two major suppliers (Greiner and BD). The specified steroids were measured by LC-MS/MS method. The procedure involved addition of labeled internal standards to the samples and extraction of steroids in methyl tert-butyl ether. Subsequently, the extract dried under a stream of nitrogen, and the residue was reconstituted in the mobile phase. LC-MS/MS analysis involved multiple reaction monitoring (MRM) for the specified steroids and their internal standards. Results The figure shows a sample chromatogram from a subject with blood samples collected in a plain (A) and serum separator tubes from Greiner (B) and BD (C1 and C2, a zoomed of C1). In this sample, from the plain tube, the concentrations of 11-deoxycortisol, androstenedione, testosterone, 17-hydroxyprogesterone and DHEA were 27, 55, 690, 99 and 283 ng/mL respectively. No significant interference was observed in Greiner serum separator tube. Whereas BD serum separator tube exhibited several erroneous peaks making the quantitation of several steroids challenging or inaccurate. Conclusions In the described LC-MS/MS method, BD serum-separator tubes exhibited erroneous peaks. These peaks were not seen in Greiner serum-separator tubes.

B-323 Measurement of PIVIKA II Blood Level in Patients with Liver Diseases by Immunoassays

Abstract Background Liver diseases are the most common diseases globally and can be complicated to cirrhosis and cancer and lead to death if not managed. It is crucial to prevent patients with liver diseases to reach the level of complications in particular liver cancer or hepatocellular Carcinoma (HCC). There are many screening lab tests to monitor and evaluate patients with liver disease such as alpha fetoprotein (AFP), however, it lacks optimal sensitivity and specificity. Another promising screening test is Prothrombin Induced by Vitamin K deficiency or antagonist- II (PIVKA-II) to complements AFP in screening of HCC. The aim of this study was to evaluate two PIVKA-II immunoassays and compare it to AFP in blood samples of patients with liver diseases. Methods Blood samples were collected from 56 patients with liver diseases, chronic hepatitis B (n=14), chronic hepatitis C (n=18), liver cirrhosis (n=14), and HCC (n=10) at King Abdulaziz medical city, Riyadh, SA. Measurement of PIVKA-II levels were performed on all samples by Snibe and Abbott analyzers. Clinical data was collected from the HIS BestCare. AFP was performed on Abbott analyzer. Statistical analysis was carried by Excel sheet Microsoft and p value was set at 0.05. Results There was an excellent correlation (R2= 0.9327) between the PIVKA-II two immunoassays from Snibe and Abbott among patients with HCC (p=0.1384). The diagnostic sensitivity PIVKA-II for Abbott and Snibe were calculated to be 78% and 90% respectively among HCC patients at cutoff 32 mAU/mL with specificity of 16% and 44.4% respectively. This was corresponding to sensitivity of 90% and specificity of 67% on AFP at cutoff 10 ng/mL. Conclusions The two PIVKA-II two immunoassays were perfectly correlated among the samples of HCC patients. The sensitivity of both assays was high but with lower specificity. Snibe assay has superior sensitivity and specificity among patients with HCC.

B-290 Static Analytical Scopes and Lab Processes - An Existing Public Health Hazard

Abstract Background Most chromatographic-based procedures have defined analytical scopes. This allows for laboratories to include the most relevant analytes in test panels and for care providers to know exactly what is included and excluded in ordered tests. Analytical scopes can be developed based on drug trend data, consensus body recommendations, or mandatory guidelines. However, it is reasonable to conclude that strictly adhering to these scopes, or not having robust processes in place that permit for rapid scope changes, will result in the underreporting of drug use in a population or miss a relevant drug finding for a single patient. For example, SAMHSA’s minimum mandatory guidelines for urine workplace drug testing does not include fentanyl. While true the number of fentanyl positive cases that are missed in this population is speculative, a factual case in point is made by evaluating laboratory results for a major novel psychoactive substance (NPS) drug class - designer benzodiazepines (DB). We performed a retrospective review to study the frequency that designer benzodiazepines would have been missed in reporting without further intervention by laboratory staff. Methods Blood samples were screened by ELISA and LC-TOF/MS. Confirmation testing using two different LC-MS/MS panels was conducted on samples that screened presumptive positive for benzodiazepines. Analytical results were retrieved from the laboratory’s data management system using the following criteria: a) date range: Feb 2023 to Feb 2024, b) blood matrix, c) DUID testing cases where benzodiazepine screening was performed by ELISA and by LC-TOF/MS, and d) at least one DB was reported regardless of the presence of other drugs. Results Nine parent DB or their metabolites were identified in the 46 cases in this study: 8-aminoclonazolam (n=3), alpha-hydroxyetizolam (n=2), bromazolam (n=35), clonazolam (n=2), deschloroetizolam (n=1), etizolam (n=1), flubromazolam (n=7), flualprazolam (n=1), and pyrazolam (n=1). Benzodiazepine screening by ELISA and LC-TOF/MS was positive for 45 and 46 cases, respectively. In the 45 cases that screened positive by ELISA, 41 did not confirm positive for any classical benzodiazepine (CBZ) which included alprazolam, diazepam, lorazepam, clonazepam, temazepam, oxazepam, and the nordiazepam metabolite. Only 4 cases tested positive for both a DB and CBZ (alprazolam or clonazepam) simultaneously. Conclusions While it is understood that defined analytical scopes are a laboratory necessity, care providers, law enforcement, public policy decision makers, and communities at large need to understand the limitations and regulations surrounding analytical test procedures. For some testing, a laboratory is obligated to follow mandatory guidelines, but from a clinical or medicolegal perspective, the risk is two-fold. The first is that the causative drug producing any untoward effects goes unrecognized. The second is that symptoms displayed are attributed to the presence of other drugs. This work stresses the importance of sincere collaboration between laboratories and entities that are responsible for analytical scope development and providing funding to ensure the appropriateness of real-time drug surveillance and reporting efforts.

A-123 Comparative Assessment of Unmanned High-Speed Aerial Vehicles in Medical Logistics: Blood Sample Transportation Amidst Weather Variability

Abstract Background Drones, as an innovative technology, hold transformative potential in the field of medical logistics, especially in the transportation of sensitive medical materials like blood samples. This study aimed to assess and compare the impact of high-speed drone transportation with conventional car transportation on the integrity and preanalytics of blood samples, taking into account various blood materials and analytes. Methods Blood samples, including EDTA whole blood, serum, and plasma anticoagulated with Citrate and Lithium-Heparin transported as whole blood, were subjected to transportation via both drone and car. Drone flights were tested under diverse weather conditions (ranging between 4 and 20 degrees Celsius), and results were compared to samples stored without transportation. The maximum drone speed exceeded 100 km/h. A total of 28 analytes were tested on Serum, 20 on EDTA, 26 on Lithium-Heparin, and 5 on Citrate blood. Statistical analyses were performed using Passing Bablok and Bland-Altman methods. Results For Serum samples, the correlation coefficient (r) ranged from 0.830 to 1.000, and slopes varied from 0.913 to 1.111. Five analytes (total bilirubin, Calcium, Ferritin, Kalium, and Sodium) exhibited discrepancies between the negative control and the transported sample, with r lower than 0.800, slopes not between 0.8 and 1.2, and mean (%) not between +/- 10%. However, no significant differences were noted between drone and car transportation. Similar patterns emerged for EDTA, Lithium-Heparin, and Citrate samples, with r ranging from 0.829 to 0.997, 0.939 to 0.998, and 0.830 to 1.000, respectively. Slopes ranged from 0.956 to 1.051, 0.938 to 1.085, and 0.913 to 1.111. Despite identifying a few discrepancies in specific analytes, no significant differences were observed between transportation methods. Conclusions n comparison to transportation by car, drone transportation of blood samples did not lead to alterations in concentrations of commonly used analytes. Notably, instances of differences before and after transportation were attributed to the transportation process itself rather than the mode of transportation. This suggests that the use of drones for medical sample transport can be deemed comparable to, if not superior to, traditional car transportation methods.

Integrating multiomics and Single-Cell communication analysis to uncover Ankylosing spondylitis mechanisms

BackgroundAnkylosing spondylitis (AS) is a chronic inflammatory joint disorder, necessitating early diagnosis and effective treatment. The specific mechanism of action of Cassia twigs in the treatment of AS is not fully understood. MethodsBlood samples and clinical data from 28,458 individuals (6,101 with AS, 22,357 without AS) were collected. To construct a predictive model, we utilized logistic regressions and machine learning techniques to create a dynamic nomogram. Immune cell infiltration was evaluated using the GSE73754 dataset. Subsequently, we obtained vertebral bone marrow blood from AS patients for 10X single-cell sequencing. We also extracted and purified total RNA from hip joint ligament tissue samples from six AS patients and six non-AS patients. The genes related to the expression of AS and Cassia twigs were analyzed comprehensively, and the specific drug targets were identified by molecular docking. The interactions between immune cells through cell communication analysis were elucidated. ResultsWe developed a dynamic nomogram incorporating the neutrophil count (NEUT) and other variables. Neutrophil immune responses were confirmed through immune infiltration analysis utilizing GSE73754. We observed the early involvement of neutrophils in the pathology of AS. The CAT-expressing Cassia twigs gene could be used as a drug target for the treatment of AS. Moreover, comprehensive RNA analysis revealed notable CAT expression in neutrophils and various other immune cells. ConclusionsNeutrophils play dual roles in AS, regulating inflammation and initiating differentiation signals to other cells. The CAT gene, which is expressed in Cassia twigs, has emerged as a potential therapeutic target for AS treatment.

Non-enzymatic Determination of Glucose in Artificial Urine Using 3D-µPADs through Silver Nanoparticles Formation

Patients with diabetes often experience blood glucose fluctuations, making monitoring crucial. Traditional blood sampling methods pose risks of infection and pain. An alternative non-invasive approach using urine tests has been explored. Recent studies highlight microfluidic paper-based analytical devices (µPADs) as convenient, simple, and easily fabricated tools for non-invasive glucose measurement. This study aims to develop a concept of measuring glucose in artificial urine using 3D-µPADs in a non-enzymatic manner by utilizing glucose as a reducing agent for silver nanoparticle (AgNPs) formation. Embedding three-dimensional connectors in µPADs links the sample and detection zones to limit reagent mixing and improve glucose detection resolution. The optimal conditions were NaOH 10 M, starch 1%, and AgNO3 30 mM, with sample and detection zone volumes of 10 and 9 µL, respectively. The fifth reaction sequence involved AgNO3 in the detection zone and a solution of glucose, NaOH, and starch in the sample zone at 1:1:1 volume ratio. The reagent drying time was 15 min, with immobilization once and reaction time of 9 min. The method showed excellent linearity (R2 = 0.9905), precision (%RSD = 4.27%), accuracy (77.32–92.58%), and limit of detection (11.11 mg/dL).

Immunological responses to brain metastasis stereotactic radiosurgery in patient-matched longitudinal blood and tumour samples

BackgroundStereotactic radiosurgery (SRS) is highly effective as focal treatment for brain metastases (BrMs), but whether it can promote anti-tumour immune responses that synergise with immunotherapy remains unclear. We investigated this by examining blood samples from a clinical trial for HER2-amplified breast cancer (HER2-BC) BrMs, matched with longitudinal HER2-BC BrM samples resected from the same location in the same patient. MethodsBlood samples from 10 patients taken pre- and 7–14 days post-SRS were analysed by mass and flow cytometry. One patient received pre-operative SRS for a BrM that recurred 7 months after resection, followed by planned re-resection 8 days post-SRS. Pre- and post-SRS tumours from this patient were analysed by bulk RNAseq, multiplex immunohistochemistry (mIHC), and TCR sequencing. ResultsMonocytes, central memory CD8+ T and regulatory T cells were enriched in blood post-SRS, together with increased MHC-II expression on monocytes, conventional DCs, and monocytic MDSCs. In tumour, SRS upregulated antigen presentation, T cell proliferation and T cell co-stimulation signatures, alongside an influx of tumour-associated macrophages (TAMs) and CD4+ T cells. Specifically, TAMs and CD4+ T cells, but not CD8+ T cells, demonstrated spatial co-localisation post-SRS. These TAMs were lowly PD-L1 expressing, but CD4+ T cells showed increased PD-1 expression. A sizeable proportion of T cell clonotypes were retained post-SRS, and four clones demonstrated significant, non-stochastic expansion. ConclusionSystemic and local immunological changes in this homogenous patient cohort suggest that SRS may facilitate MHC-II-restricted T cell priming responses involving the monocyte-macrophage lineage and CD4+ T cells, which should be further explored.

BMP10 reflects pre-capillary pulmonary hemodynamics: association of biomarkers and hemodynamic parameters in pulmonary hypertension

Abstract Background and aims The role of biomarkers in diagnosing pulmonary hypertension (PH) and distinguishing between pre- and post-capillary PH remains poorly understood. We aimed to identify biomarkers with a strong association with mean pulmonary arterial pressure, mPAP (PH diagnosis) and pulmonary vascular resistance, PVR (pre-capillary component), but not with pulmonary arterial wedge pressure, PAWP (post-capillary component). Methods Blood samples were collected in patients undergoing right heart catheterization within a prospective cross-sectional study. Biomarkers measured included BMP10, NT-proBNP, ANG2, ESM1/endocan, FGF23, GDF15, IGFBP7, IL6, MyBPC3, proC3, and proC6/endotrophin. Primary outcomes were mPAP, PVR, and PAWP, while secondary outcomes included PH diagnosis (mPAP > 20 mmHg) and elevated PVR (> 2 Wood units). Multivariable linear and logistic regression models were used to assess the relationship between biomarkers and outcomes. Results Of the 127 patients included (age 66 ± 13 years, 54% female), 73% were diagnosed with PH. BMP10, NT-proBNP, ANG2, MyBPC3, and FGF23 showed a strong association with mPAP (p < 0.001). BMP10 and NT-proBNP were strongly associated with PVR (p < 0.001), while NT-proBNP and ANG2 were strongly associated with PAWP (p < 0.001). NT-proBNP had the strongest association with the diagnosis of PH (area under the curve = 0.76). BMP10 was the only biomarker associated with elevated PVR (OR 1.60, 95%CI 1.01–2.54, p = 0.04) but not with PAWP (p = 0.86). Conclusions Several biomarkers were strongly associated with mPAP, PAWP, and PVR. BMP10 was the only biomarker strongly associated with mPAP and PVR, but not with PAWP, thus reflecting the pre-capillary PH component. Measurement of BMP10 along with NT-proBNP may aid in diagnosing PH. Graphical abstract

Comprehensive biomarker assessment for predicting severe acute kidney injury and need of kidney replacement therapy in liver transplantation patients

Background Renal dysfunction is a common complication following liver transplantation (LT). This study aimed to determine whether a comprehensive assessment of kidney function using nineteen serum and urinary biomarkers (BMs) within the first 48 h post-LT could enhance the prediction of severe acute kidney injury (AKI) and the need of kidney replacement therapy (KRT) during the first postoperative week. Methods Blood and urine (U) samples were collected during the pre- and postoperative periods. Nineteen BMs were evaluated to assess kidney health in the first 48 h after LT. Classification and regression tree (CART) cross-validation identified key predictors to determine the best BM combination for predicting outcomes. Results Among 100 LT patients, 36 developed severe AKI, and 34 required KRT within the first postoperative week. Preoperative assessment of U neutrophil gelatinase-associated lipocalin (NGAL) and liver-type fatty acid-binding protein (L-FABP) predicted the need for KRT with 75% accuracy. The combined assessment of U osmolality (OSM), U kidney injury molecule 1 (KIM-1), and tissue inhibitor of metalloproteinase (TIMP-1) within 48 h post-LT predicted severe AKI with 80% accuracy. U-OSM alone, measured within 48 h post-LT, had an accuracy of 83% for predicting KRT need, outperforming any BM combination. Conclusions Combined BM analysis can accurately predict severe AKI and KRT needs in the perioperative period of LT. U-OSM alone proved to be an effective tool for monitoring the risk of severe AKI, available in most centers. Further studies are needed to assess its impact on AKI progression postoperatively. Registered at Clinical Trials (clinicaltrials.gov) in March 24th, 2014 by title ‘Acute Kidney Injury Biomarkers: Diagnosis and Application in Pre-operative Period of Liver Transplantation (AKIB)’ and identifier NCT02095431.

Rapid depletion of CD20+ B and T cells following ofatumumab therapy onset

BackgroundThe humanized monoclonal anti-CD20-antibody ofatumumab is highly effective in treating relapsing multiple sclerosis (MS). ObjectiveThis study aimed to investigate the immanent effect of ofatumumab on the peripheral immune system, particularly targeting B and T cells expressing CD20. MethodsBlood samples of 53 MS patients receiving ofatumumab were collected prior to first application and after one week, two weeks and three months. Multicolor flow cytometry was used to phenotype peripheral blood mononuclear cells, and immunoglobulin (Ig) concentrations were measured by nephelometry. ResultsAmong CD20+ lymphocytes, 13 % co-expressed CD3 (identifying them as CD3+CD20+ T lymphocytes), with a noticeable shift in the CD4/CD8-ratio towards CD8+ T cells. One week after administering ofatumumab, a significant reduction of CD20+ lymphocytes with complete depletion of CD3+CD20+ T lymphocytes was observed, persisting during the investigation period. During the treatment, IgM levels showed a slight but significant decrease, whereas IgA and IgG levels remained stable. ConclusionOfatumumab effectively depletes CD20+ lymphocytes already after the first administration. This depletion affects not only B cells, but also a small proportion of T cells (CD3+CD20+), affirming the hypothesis that the anti-inflammatory effects of CD20+ cell depletion might extend to the reduction of CD3+CD20+ T lymphocytes.

387. VALIDATION OF PROMARKERESO, A DIAGNOSTIC BLOOD TEST TO IDENTIFY ESOPHAGEAL ADENOCARCINOMA

Abstract Background Esophageal adenocarcinoma (EAC) has become the predominant type of esophageal cancer in Western countries. Early diagnosis and intervention of EAC significantly reduces the disease mortality. A previous study identified a panel of novel serum glycoprotein biomarkers that were associated with EAC, enabling the development of a prototype diagnostic blood test, PromarkerEso. The current study aimed to validate PromarkerEso in an independent cohort of patients with EAC. Methods Blood samples from an independent cohort of 165 participants (n=99 negative controls, n=66 diagnosed with EAC) were analyzed. Five candidate biomarkers were measured after lectin pulldown using a targeted mass spectrometry assay. Model performance was assessed by the area under the receiver operating characteristics curve (AUC-ROC) with sensitivity and specificity determined. Results After adjusting for clinical variables, diagnostic models showed good discrimination to distinguish participants with EAC from negative control participants. Conclusions This study validates a panel of serum glycoprotein biomarkers for the diagnosis of EAC. These biomarkers have potential utility as a simple blood test, PromarkerEso, that could be used for screening EAC in a large population.

Metabolomic biomarkers and altered phenylalanine metabolic pathway in preschool children with atopic dermatitis - A pilot study.

Background Atopic dermatitis (AD) has high prevalence in children. Current AD diagnosis and management focuses only on clinical phenotypes, but do not explore the endophenotypes, which are more important because they are a series of biomarkers linking clinical phenotype and genotype Aims Metabolomics can qualitatively and quantitatively capture real-time dynamic changes in a wide range of small molecule metabolites. This pilot study evaluated metabolomics biomarkers and altered metabolic pathways in preschool children with AD, aiming to explore the underlying molecular mechanisms and signalling pathways of the disease. Methods Blood samples of 23 preschool children with AD and 23 healthy children without AD or any other skin disease were collected. The untargeted metabolomic measurements were performed on a SCIEX-AD ultraperformance liquid chromatography system coupled with an AB SCIEX X500B QTOF system. Characteristics of small molecules in AD children were assessed and their associations with AD clinical index were evaluated. Altered metabolic pathways in AD children were also analysed using a comprehensive metabolomics platform. Results A total of 1,969 metabolites were identified, of which AD children exhibited 377 significantly altered metabolites. Multivariate statistical analysis demonstrated that the AD group and the control group could be clearly separated. Volcano plot analysis illustrated that 144 metabolites were up-regulated and 233 metabolites were down-regulated in AD children. The Severity Scoring of Atopic Dermatitis (SCORAD index) showed a moderate-to-strong association with estrogens, carotenes, leukotrienes, flavonols and keto acids in AD children (|r|=0.440-0.557). Several pathways, including the phenylalanine metabolism, were identified as altered in AD children. Limitations A small group of children was included in the study; the results need to be validated in larger sample sizes. Conclusion Results of this study illustrate potential alterations in metabolites and the phenylalanine metabolic pathway in preschool children with AD. Although this is a pilot study with a limited sample size, it may provide a new perspective for exploring the pathogenesis of AD, and for personalised treatment modalities.

Blood biomarkers for cardiac damage during and after radiotherapy for esophageal cancer: A prospective cohort study

PurposeThe aim of this study was to test the hypothesis that the levels of High Sensitive Troponin T (HS-TNT) and N-terminal Brain Natriuretic Peptide (NT-ProBNP) increase after radiation therapy in a dose dependent way and are predictive for clinical cardiac events. Materials and MethodsBlood samples during and after radiotherapy of 87 esophageal cancer patients were analysed regarding the course of HS-TNT and NT-ProBNP levels and their relationship with clinical toxicity endpoints and radiation dose volume parameters. ResultsHS-TNT values at the end of treatment correlated with the mean heart dose (p = 0.02), whereas the rise of NT-ProBNP correlated with the mean lung dose (p = 0.01). Furthermore, the course of both HS-TNT (p < 0.001) and NT-ProBNP (p < 0.01) levels were significantly different for patients who developed new cardiac events as opposed to those without new cardiac events. ConclusionSignificant correlations were found for both biomarkers with radiation dose and clinical toxicity endpoints after treatment. Therefore, these markers might be of additional value in NTCP models for cardiac events and might help us unravelling the mechanisms behind these toxicity endpoints.

Cellular immune response to a single dose of live attenuated hepatitis a virus vaccine in obese children and adolescents

BackgroundLimited data are currently available regarding the cellular immune response to a live attenuated hepatitis A virus (HAV) vaccine, especially in children with obesity. The objective of this retrospective cohort study was to compare the activation of antigen-specific interferon (IFN)-γ+ T cells in obese children and adolescents with healthy individuals before and after immunization with a single dose of live attenuated HAV vaccine. MethodsBlood samples were obtained from the 2021 study by Dumrisilp et al. investigating the immunogenicity of the live attenuated hepatitis A vaccine in children and young adults. Prior to enrollment, all 212 subjects had never received any HAV vaccine and tested negative for anti-HAV antibodies. The participants were vaccinated with a freeze-dried, live attenuated HAV vaccine of the H2 strain. In this study, we analyzed the stored peripheral blood mononuclear cells (PBMCs) obtained from a subgroup of 30 obese subjects and 30 normal-weight healthy controls of the same age and sex. PBMCs were collected before and 8–9 weeks after HAV vaccination for further analysis. These cells were stimulated with a recombinant antigen derived from HAV-VP3, and the immune response was evaluated using the IFN-γ enzyme-linked immunospot (ELISpot) assay. ResultsThe between-group analysis indicated that the T-cell response of obese participants was comparable to that of normal-weight controls both before and after vaccination. The change in IFN-γ production from before to after vaccination in the obese group was not significantly different from that of the control group. Additionally, in the obese group, no correlation was found between IFN-γ production and clinical characteristics such as sex, body mass index, waist circumference, and acanthosis nigricans. ConclusionTesting for cellular immune response provides a comprehensive understanding of the overall immune response to vaccination. This study, the first to explore this significant aspect, suggests that obesity does not affect the short-term cellular immune response to live attenuated HAV vaccination.

Significant hemolysis is present during irreversible electroporation of cardiomyocytes in vitro

BackgroundPulsed field ablation (PFA) of atrial fibrillation is a new method in clinical practice. Despite a favorable safety profile of PFA in atrial fibrillation ablation, rare cases of renal failure, probably due to hemolysis, have recently been reported. ObjectiveThe aim of this study was to determine the rate of hemolysis and cardiac cell death during in vitro PFA with different electric field intensities. MethodsBlood samples from healthy volunteers and mouse HL-1 cardiomyocyte cell lines were subjected to in vitro irreversible electroporation using 216 bipolar pulses, each lasting 2 μs with intervals of 5 μs, repeated 20 times at a frequency of 1 Hz. These pulses varied from 500 V to 1500 V. Cell-free hemoglobin levels were assessed spectrophotometrically, and red blood cell microparticles were evaluated by flow cytometry. Cardiomyocyte death was quantified with propidium iodide. ResultsPulsed field energy (1000 V/cm, 1250 V/cm, and 1500 V/cm) was associated with a significant increase in cell-free hemoglobin (0.32 ± 0.16 g/L, 2.2 ± 0.96 g/L, and 5.7 ± 0.39 g/L; P < .01) and similar increase in the concentration of red blood cell microparticles. Significant rates of cardiomyocyte death were observed at electric field strengths of 750 V/cm, 1000 V/cm, 1250 V/cm, and 1500 V/cm (26.5% ± 5.9%, 44.3% ± 6.2%, 55.5% ± 6.9%, and 74.5% ± 17.8% of cardiomyocytes; P < .01). ConclusionThe most effective induction of cell death in vitro was observed at 1500 V/cm. This intensity was also associated with a significant degree of hemolysis.

Vaccine‐induced humoral response of BNT162b2 and mRNA-1273 against BA.1, BA.5, and XBB.1.5. (sub)variants 6 months after a homologous booster: is immunogenicity equivalent?

IntroductionSome studies suggest that the monovalent mRNA-1273 vaccine is more effective than BNT162b2 in producing higher levels of antibodies. However, limited data are available, and the methods used are not directly comparable. Material and methodsBlood samples were obtained before the booster (third dose) and after 14, 90, and 180 days in two similar cohorts who received the original BNT162b2 or mRNA-1273 vaccine designed to target wild type SARS-CoV-2. The aim of our study is to compare their effectiveness by assessing the levels of binding and neutralizing antibodies specifically against each of the BA.1 variant, BA.5 variant, and the XBB.1.5 subvariant. ResultsOnce the peak was reached after two weeks, a drastic decline in binding and neutralizing antibodies was observed up to 6 months after the homologous booster administration. The humoral response was however more sustained with the mRNA-1273 booster, with half-lives of 167, 55, and 48 days for binding, BA.1, and BA.5 neutralizing antibodies compared to 144, 30, and 29 days for the BNT162b2 booster, respectively. Compared to the BA.1 variant, the neutralizing capacity was significantly decreased at 6 months with the BA.5 variant (fold-decrease: 1.67 to 3.20) and the XBB.1.5. subvariant (fold-decrease: 2.86 to 5.48). ConclusionAlthough the decrease in the humoral response was observed with both mRNA vaccines over time, a more sustained response was observed with the mRNA-1273 vaccine. Moreover, the emergence of Omicron-based variants causes a reduced neutralizing capacity, notably with the XBB.1.5. subvariant. The administration of subsequent boosters would therefore be needed to restore a sufficiently high neutralizing response.

Differential inflammatory profiles in carriers of reciprocal 22q11.2 copy number variants

BackgroundGenetic copy number variants (CNVs; i.e., a deletion or duplication) at the 22q11.2 locus confer increased risk of neuropsychiatric disorders and immune dysfunction. Inflammatory profiles of 22q11.2 CNV carriers can shed light on gene-immune relationships that may be related to neuropsychiatric symptoms. However, little is known about inflammation and its relationship to clinical phenotypes in 22q11.2 CNV carriers. Here, we investigate differences in peripheral inflammatory markers in 22q11.2 CNV carriers and explore their relationship with psychosis risk symptoms and sleep disturbance. MethodsBlood samples and clinical assessments were collected from 22q11.2 deletion (22qDel) carriers (n=45), 22q11.2 duplication (22qDup) carriers (n=29), and typically developing (TD) control participants (n=92). Blood plasma levels of pro-inflammatory cytokines, including interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), and anti-inflammatory cytokine interleukin-10 (IL-10) were measured using a MesoScale Discovery multiplex immunoassay. Plasma levels of C-reactive protein (CRP) were measured using Enzyme-linked Immunosorbent Assay (ELISA). Linear mixed effects models controlling for age, sex, and body mass index were used to: a) examine group differences in inflammatory markers between 22qDel, 22qDup, and TD controls, b) test differences in inflammatory markers between 22qDel carriers with psychosis risk symptoms (22qDelPS+) and those without (22qDelPS-), and c) conduct an exploratory analysis testing the effect of sleep disturbance on inflammation in 22qDel and 22qDup carriers. A false discovery rate correction was used to correct for multiple comparisons. Results22qDup carriers exhibited significantly elevated levels of IL-8 relative to TD controls (q<0.001) and marginally elevated IL-8 levels relative to 22qDel carriers (q=0.08). There were no other significant differences in inflammatory markers between the three groups (q>0.13). 22qDelPS+ exhibited increased levels of IL-8 relative to both 22qDelPS- (q=0.02) and TD controls (p=0.002). There were no relationships between sleep and inflammatory markers that survived FDR correction (q>0.14). ConclusionOur results suggest that CNVs at the 22q11.2 locus may have differential effects on inflammatory processes related to IL-8, a key mediator of inflammation produced by macrophages and microglia. Further, these IL-8-mediated inflammatory processes may be related to psychosis risk symptoms in 22qDel carriers. Additional research is required to understand the mechanisms contributing to these differential levels of IL-8 between 22q11.2 CNV carriers and IL-8’s association with psychosis risk.