Blood Assay Research Articles

Influences of Ethyl Acetate and Water Bulb Extracts of Crinum zeylanicum (Linn.) on Haematological Constituents of Rabbit Mediated Through Osteotomy

Crinum zeylanicum bulb is a traditional folk’s herb that has been widely accepted by most rural communities for the treatment of bone fracture and other illnesses. This study assessed the influences of C. zeylanicum bulb extracts on blood pictures of rabbits through close tibia osteotomy over the period of three months. Assay of the whole blood collected through cardiac puncture was done by determining the Packed Cell Volume (PCV%) (Micro-hematocrit method), White Blood Cell (WBC%), Red Blood Cell (RBC%) (Hemo- cytometer method), Haemoglobin (HB g/dl) (Cyanomoglobin method), Neutrophil (N%), Lymphocyte (L%), Monocyte (M %), Eosinophil (E%), and Basophil (B %) (Thin smear and Leishman’s Methods). The Mean Cell Volume (MCV%), Mean Corpuscular Hemoglobin (MCH %), and MCH-Concentration (MCHC %) were calculated according to standard methods. There was significant difference (p<0.05) recorded for MCV, MCH, N and L for treated-groups as compared with WBC, MCV and N for control group while the resultant increase of WBC by ethyl acetate extract (EAE) treated group than water extract (WE) and control groups was an indication that the plant has influence on the blood constituents. M, B and E were not dictated during the pre- and post-examinations. Hence, the use of C. zeylanicum can be monitored, modified, and/or regulated for medicinal uses as the characterization of the chemical components of the plant could be a better focus for further investigation.

Comparing endpoint and real-time PCR for forensic body fluid identification

ABSTRACT Identifying the body fluid source of stains can give an indication of activities, prioritize samples for DNA profiling, and provide probative evidence when the presence of DNA alone does not. Messenger RNA profiling assays using endpoint reverse-transcription polymerase-chain reaction (RT-PCR), were developed to address the issues with conventional body fluid identification and have since been used in forensic casework. However, endpoint RT-PCR has a narrow linear dynamic range (LDR), and there is currently no specific method for mRNA quantification to direct case strategy and optimize input template. Real-time reverse-transcription quantitative PCR (RT-qPCR) collects data during the amplification phase which reflects the starting quantity. The application of RT-qPCR assays as a quantification precursor to endpoint RT-PCR was investigated using previously described assays for circulatory blood, saliva, menstrual fluid, spermatozoa, seminal fluid, and vaginal material. While blood marker SLC4A1 and vaginal marker CYP2B7P showed a strong relationship, the remaining body fluid markers lacked a robust predictive relationship, likely due to differences in LDR. CYP2B7P and SLC4A1 showed high sensitivity when compared with other markers – resulting in a greater number of data points for estimation of the linear relationship. Compared with endpoint, the RT-qPCR assays had higher precision, LDR, and sensitivity.

Association of FoxP3<sup>+</sup> T regulatory lymphocytes with epicardial adipose tissue thickness in patients with coronary heart disease

Background. Increase ofthe epicardial adipose tissue (EAT) thickness isassociated with development of inflammation and cardiovascular complications, however, there is no data on the relationship between EAT thickening and the number of immunosuppressive regulatory T lymphocytes.The aim. To study the number of circulating T regulatory lymphocytes and nuclear translocation ofthe FoxP3 transcription factor in patients with stable coronary heart disease (CHD) depending on the epicardial adipose tissue thickness.Materials and methods. We examined 30 patients with chronic stable CHD. The EAT thickness was measured by echocardiography. Patients were divided into groups depending on the presence and absence of EAT thickening above 5 mm (groups 1 and 2, respectively). Imaging flow cytometry was used to determine the number of T regulatory lymphocytes and the level of FoxP3 nuclear translocation. The concentration of cytokines and high sensitivity C-reactive protein (hsCRP) was determined using enzyme-linked immunosorbent assay in blood serum.Results. Anthropometric indicators of obesity and the severity of atherosclerosis were comparable between groups. In group 2, there was an increase in low-density lipoprotein cholesterol concentration (p = 0.043), ratio of low-density lipoprotein cholesterol tohigh-density lipoprotein cholesterol (p = 0.017) and the concentration ofhsCRP (p = 0.044) andIL-1β (p = 0.005), adecrease in the number and relative count of Tregulatory lymphocytes (p = 0.020 andp = 0.026, respectively), aswellas thenumber of cells withFoxP3 nuclear translocation (p = 0.018) compared togroup1. According tomultiple logistic regression, the concentration ofhsCRP, IL-1β and T regulatory lymphocytes relative count in total were the predictors of EAT thickening (accuracy 80 %; sensitivity 75 %; specificity 84,6 %; AUC = 0.89).Conclusions. Thickening of epicardial adipose tissue inpatients withcoronary heart disease is associated with a decrease in the number of T regulatory lymphocytes andFoxP3 nuclear translocation inthem in presence of comparable anthropometric parameters of obesity and the severity of coronary atherosclerosis.

Detection of Islet Autoantibodies in Whole Blood by Antibody Detection by Agglutination-PCR (ADAP) Technology Is Sensitive and Suitable for General Population Screening Programs

Background. Detection of type 1 diabetes (T1D) at the preclinical stage is possible by detecting islet autoantibodies (IAs) years before the appearance of symptomatic diabetes. The Antibody Detection Israeli Research is a general population screening program searching for children with multiple IAs who are at risk of developing T1D. IAs are measured in capillary or venous whole blood (WB) samples using the novel ultrasensitive antibody detection by agglutination-PCR (ADAP) technology. Objective. To assess the accuracy and reliability of the ADAP assay in venous and capillary WB. Materials and Methods. In total, 50 children with T1D and 50 healthy controls participated in the study. Venous and capillary blood samples were drawn from participants with T1D, while only venous blood was drawn from the controls. The ADAP assay in venous and capillary blood was compared to the currently used assays in their ability to detect glutamic acid decarboxylase (GADA), islet antigen-2 (IA-2A), and insulin autoantibodies (IAAs). Results. The area under the curve using the receiver operating characteristic curves was comparable between the ADAP assay in WB and standard enzyme-linked immunosorbent assay (ELISA)/radioimmunoassay (RIA) for all three IAs GADA 0.946 (95% CI: 0.900–0.991) vs. 0.949 (0.906–0.992), P=0.873; IA-2A 0.747 (0.649–0.844) vs. 0.666 (0.587–0.744), P=0.106; IAA 1.000 (1.000–1.000) vs. 1.000 (1.000–1.000), P=1.000. The correlation between the levels of IA in venous and capillary WB using ADAP was R2 = 0.958 (P <0.01), R2 = 0.943 (P <0.01), and R2 = 0.711 (P <0.01) for GADA, IA-2A, and IAA, respectively. IA levels in venous and capillary WB using ADAP were comparable without a proportional bias in Bland–Altman’s plots of agreement, suggesting the two methods may be used interchangeably. Conclusions. The ADAP assay is reliable in detecting IA in venous and capillary WB samples with comparable performance to standard RIA and ELISA. These findings open avenues for widespread use of the ADAP assay in future general population screening programs to detect children at risk of developing T1D.

Plasma/Serum Proteomics based on Mass Spectrometry.

Human blood is a window of physiology and disease. Examination of biomarkers in blood is a common clinical procedure, which can be informative in diagnosis and prognosis of diseases, and in evaluating treatment effectiveness. There is still a huge demand on new blood biomarkers and assays for precision medicine nowadays, therefore plasma/serum proteomics has attracted increasing attention in recent years. How to effectively proceed with the biomarker discovery and clinical diagnostic assay development is a question raised to researchers who are interested in this area. In this review, we comprehensively introduce the background and advancement of technologies for blood proteomics, with a focus on mass spectrometry (MS). Analyzing existing blood biomarkers and newly-built diagnostic assays based on MS can shed light on developing new biomarkers and analytical methods. We summarize various protein analytes in plasma/serum which include total proteome, protein post-translational modifications, and extracellular vesicles, focusing on their corresponding sample preparation methods for MS analysis. We propose screening multiple protein analytes in the same set of blood samples in order to increase success rate for biomarker discovery. We also review the trends of MS techniques for blood tests including sample preparation automation, and further provide our perspectives on their future directions.

Age and chest computed tomography severity score are predictors of long-COVID.

About one-third of acute coronavirus disease 2019 (COVID-19) survivors have suffered from persisting symptoms called long-COVID. Clinical factors such as age and intensity (moderate or acute) of COVID-19 have been found to be associated with long-COVID. Many tissues might be damaged functionally or structurally during acute COVID-19 which can be detected by blood assays and chest computed tomography (CT). We aimed to evaluate the relationship between long-COVID and the initial findings of blood assays and chest CT as possible predictors. The study included patients with acute COVID-19. Laboratory tests and chest CT were obtained from each patient at the time of admission to the hospital. Chest CT was evaluated for pneumonic involvement and severity score. Multivariable regression model was created to find the factors that were independently associated with long-COVID. There were 60 (38.2%) patients with long-COVID and 97 (61.8%) without. Baseline demographic, laboratory and chest CT parameters were similar in both groups, except for age, chronic lung disease and chest CT severity score (46.9 ± 15.1 years vs 52.6 ± 15.9 years, p = 0.03; 11.7% vs 3.1%, p = 0.03 and 10.3 ± 9.6 vs 6.5 ± 7.6, p = 0.02, respectively). In multivariable model, chest CT severity score (OR: 1.059, 95% CI: 1.002-1.119, p = 0.04) and age (OR: 0.953, 95% CI: 0.928-0.979, p < 0.001) were independently associated with long-COVID. Chest CT severity score and age were independently associated with long-COVID and may be used to predict the future risk of long-COVID.

The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples

The expression of the HER2 (human epidermal growth factor receptor 2) protein in cancer cells is a well-established cancer marker used for diagnostic and therapeutic purposes in modern treatment protocols, especially in breast cancer. The gold-standard immunohistochemical diagnostic methods with the specific anti-HER2 antibodies are utilized in the clinic to measure expression level of the membrane-bound receptor. However, a soluble extracellular domain (ECD) of HER2 is released to the extracellular matrix, thus the blood assays for HER2 measurements present an attractive way for HER2 level determination. There is a need for accurate and validated assays that can be used to correlate the concentration of the circulating HER2 protein with disease clinical manifestations. Here we describe two monoclonal antibodies binding HER2 with a unique sequence of the complementarity-determining regions that recognize HER2 ECD. Development and validation of the sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the soluble HER2 in a variety of biological samples is also presented. The assay provides HER2 quantitation within a concentrations range from 1.56 to 100 ng/ml with sensitivity at the level of 0.5 ng/ml that meets the expectations for measurements of HER2 in the blood and tumor tissue samples. The method presents satisfactory intra- and inter-assay precision and accuracy for immunochemical quantification of biomarkers in biological samples. The utility of the generated monoclonal anti-HER2 antibodies has been confirmed for use in the precise measurement of HER2 (both cell-bound and soluble) in several types of biological material, including serum, solid tumor tissue, and cell culture medium. Additionally, the developed immunochemical tools have a potential for HER2 detection, not only in a wide range of sample types but also independently of the sample storage/pre-processing, allowing for comprehensive HER2 analysis in tissue (IHC), cultured cells (immunofluorescence) and blood (ELISA).

Antioxidant, Immunomodulatory and Fungicidal Potential of Different Extracts of Raphanus Raphanistrum L. Var. Caudatus

Background: Reactive oxygen species (ROS) have been implicated in the pathogenesis of various diseases, emphasizing the importance of antioxidants in maintaining cellular homeostasis. Raphanus raphanistrum, commonly known as wild radish, has been traditionally used for its medicinal properties, including antioxidant, immunomodulatory, and antifungal effects. The plant is rich in bioactive compounds such as flavonoids, isothiocyanates, and vitamins, which contribute to its therapeutic potential. Objective: This study aimed to evaluate the antioxidant, immunomodulatory, and antifungal activities of Raphanus raphanistrum extracts and to compare these activities with standard controls. Methods: Fresh Raphanus raphanistrum plants were collected, and extracts were prepared using methanol, chloroform, and water. The antioxidant activity was assessed using the DPPH radical scavenging assay. Immunomodulatory activity was evaluated through the luminol-enhanced chemiluminescence assay in whole blood and polymorphonuclear leukocytes (PMNS), measuring the oxidative burst. The antifungal activity was determined using the agar disk diffusion method against six pathogenic fungi. The statistical analysis was performed using SPSS version 25, with results presented as mean ± standard deviation for antioxidant and antifungal assays, and mean ± standard error for immunomodulatory activity. Results: The methanol extract exhibited significant antioxidant activity with DPPH scavenging percentages ranging from 56.55% at 150 µg/ml to 87.08% at 850 µg/ml. Immunomodulatory activity showed IC50 values of 37.8 µg/ml in whole blood and 18.9 µg/ml in PMNS for the methanol extract. The antifungal activity against Candida albicans showed zones of inhibition increasing from 15 mm at 150 µg/ml to 22 mm at 850 µg/ml. These activities were compared to standard controls, including ascorbic acid for antioxidant, ibuprofen for immunomodulatory, and miconazole for antifungal assays, demonstrating the extracts' competitive potential. Conclusion: Raphanus raphanistrum extracts showed promising antioxidant, immunomodulatory, and antifungal activities, indicating their potential as natural alternatives for the prevention and treatment of related diseases. Further in vivo studies and isolation of active compounds are recommended to fully understand the mechanisms and therapeutic applications.

SWOG S2200 (PAPMET2): A phase II randomized trial of cabozantinib with or without atezolizumab in patients with advanced papillary renal cell carcinoma (PRCC).

TPS493 Background: The role of immune therapy is not established in PRCC. The S1500 (PAPMET) clinical trial established single agent cabozantinib as the standard of care for PRCC (PMID 33592176) with a median progression free survival (PFS) of 9.0 months compared to 5.6 months with sunitinib. Trials have shown activity of PD-(L)1 antagonists as monotherapy (PMID 33529058) or in combination with targeted therapy (PMID 34491815). In a single arm study of cabozantinib/nivolumab the median PFS was 12.5 months (PMID 35298296). There are no prior randomized studies of immune therapy in PRCC. Single arm trials often overestimate the true effect size (PMID 31218346), highlighting the unmet clinical need for a randomized clinical trial in PRCC. We hypothesize that the combination will have higher clinical activity than single agent cabozantinib. Methods: This is a prospective randomized phase II clinical trial conducted through the NCTN and led by SWOG. The primary endpoint is a comparison of PFS between cabozantinib and cabozantinib/atezolizumab. Secondary endpoints include comparison of objective response rate, overall survival and safety. Patients are treated with cabozantinib 60mg/day versus cabozantinib 60mg/day + atezolizumab 1200 mg q3 weeks. 200 patients will be enrolled and randomized 1:1. Key inclusion criteria include a pathologic confirmation of PRCC; presence of metastasis; 0-1 prior systemic lines of therapy for metastatic disease; and measurable disease as defined by RECIST 1.1 criteria. Prior treatment with adjuvant pembrolizumab is allowed if completed greater than 6 months before enrollment. Key exclusion criteria include clinically significant autoimmune disease; ongoing use of strong CYP3A4 inhibitors, strong CYP3A4 inducers. Planned correlatives include stool microbiome testing and genomic/transcriptomic analysis from blood and baseline tissue assays. Clinical trial information: NCT05411081 .

P133 In vitro pharmacological profile of GLPG3667 suggests differentiation from the TYK2 inhibitors deucravacitinib and TAK-279 at their clinical dose regimens

Abstract Background Tyrosine kinase 2 (TYK2), a Janus kinase (JAK) family member, is attracting a lot of interest as a new target to treat patients with autoimmune and inflammatory diseases, including inflammatory bowel disease, and several inhibitors are currently in clinical development. In vitro pharmacological profiling of TYK2 inhibitors has been employed to evaluate potency and selectivity; however, such data are best viewed in relation to clinical exposure levels. We compared expected levels of inhibition of TYK2-dependent and -independent pathways for deucravacitinib, TAK-279 and GLPG3667 at their respective clinical dose regimens and demonstrate that relevant differences exist between these compounds. Methods Potency of the TYK2 inhibitors was compared for TYK2-dependent pathways (IFNα, IL-12), TYK2-independent pathways (IL-6, IL-2, GM-CSF) and the anti-inflammatory IL-10 pathway in in vitro human whole blood assays using STAT phosphorylation (flow cytometry) and cytokine release (ELISA). Sigmoidal Emax models were fitted to the concentration–inhibition data from healthy donors to estimate IC50, Emax and Hill slope for each assay and compound. The obtained relationships were subsequently coupled to the clinical pharmacokinetic (PK) profiles of the compounds (population PK model for GLPG3667, published PK metrics such as Cavg for the other compounds) to estimate the level of inhibition of the various pathways at clinically relevant dose regimens. Results Human whole blood assays performed for various JAK pathways demonstrated that all TYK2 inhibitors display selectivity for TYK2 over the JAK1–3 family members, with TAK-279 being the most selective. At exposure levels associated with its clinical dose of 150 mg once daily, GLPG3667 showed inhibition of the IFNα and IL-12 pathways, similar to the expected inhibition for deucravacitinib at its clinical dose regimens, without a measurable impact on TYK2-independent pathways. TAK-279 showed the most sustained inhibition of TYK2-dependent pathways. GLPG3667 showed no measurable inhibition of IL-10-mediated signalling up to the highest concentration tested (~10-fold above clinical concentrations), while inhibition was observed with deucravacitinib and TAK-279 at concentrations corresponding to the respective clinical dose regimens. Conclusion At concentrations corresponding to its highest anticipated clinical dose, GLPG3667 shows selective inhibition of TYK2-mediated signalling, with a level of inhibition similar to deucravacitinib clinical dose regimens. While GLPG3667 did not show any impact on IL-10-mediated signalling, inhibition of this anti-inflammatory pathway is expected for the allosteric TYK2 inhibitors at their clinical exposure levels.

Diagnostic accuracy of the IFN-γ release assay using RD1 immunodominant T-cell antigens for diagnosis of extrapulmonary tuberculosis.

The diagnosis of extrapulmonary tuberculosis (EPTB) poses a significant challenge, with controversies surrounding the accuracy of IFN-γ release assays (IGRAs). This study aimed to assess the diagnostic accuracy of RD1 immunodominant T-cell antigens, including ESAT-6, CFP-10, PE35, and PPE68 proteins, for immunodiagnosis of EPTB. Twenty-nine patients with EPTB were enrolled, and recombinant PE35, PPE68, ESAT-6, and CFP-10 proteins were evaluated in a 3-day Whole Blood Assay. IFN-γ levels were measured using a Human IFN-γ ELISA kit, and the QuantiFERON-TB Gold Plus (QFT-Plus) test was performed. Predominantly, the patients were of Afghan (62%, n=18) and Iranian (38%, n=11) nationalities. Eighteen individuals tested positive for QFT-Plus, accounting for 62% of the cases. The positivity rate for IGRA, using each distinct recombinant protein (ESAT-6, PPE68, PE35, and CFP-10), was 72% (n=21) for every protein tested. Specifically, among Afghan patients, the positivity rates for QFT-Plus and IGRA using ESAT-6, PPE68, PE35, and CFP-10 were 66.7%, 83.3%, 83.3%, 77.8%, and 88.9%, respectively. In contrast, among Iranian patients, the positivity rates for the same antigens were 54.5%, 54.5%, 54.5%, 63.6%, and 45.5%, respectively. In conclusion, our study highlights the potential of IGRA testing utilizing various proteins as a valuable diagnostic tool for EPTB. Further research is needed to elucidate the underlying factors contributing to these disparities and to optimize diagnostic strategies for EPTB in diverse populations.

Single extracellular vesicle surface protein-based blood assay identifies potential biomarkers for detection and screening of five cancers.

Extracellular vesicles (EVs) and EV proteins are promising biomarkers for cancer liquid biopsy. Herein, we designed a case-control study involving 100 controls and 100 patients with esophageal, stomach, colorectal, liver, or lung cancer to identify common and type-specific biomarkers of plasma-derived EV surface proteins for the five cancers. EV surface proteins were profiled using a sequencing-based proximity barcoding assay. In this study, five differentially expressed proteins (DEPs) and eight differentially expressed protein combinations (DEPCs) showed promising performance (area under curve, AUC > 0.900) in pan-cancer identification [e.g., TENM2 (AUC = 0.982), CD36 (AUC = 0.974), and CD36-ITGA1 (AUC = 0.971)]. Our classification model could properly discriminate between cancer patients and controls using DEPs (AUC = 0.981) or DEPCs (AUC = 0.965). When distinguishing one cancer from the other four, the accuracy of the classification model using DEPCs (85-92%) was higher than that using DEPs (78-84%). We validated the performance in an additional 14 cancer patients and 14 controls, and achieved an AUC value of 0.786 for DEPs and 0.622 for DEPCs, highlighting the necessity to recruit a larger cohort for further validation. When clustering EVs into subpopulations, we detected cluster-specific proteins highly expressed in immune-related tissues. In the context of colorectal cancer, we identified heterogeneous EV clusters enriched in cancer patients, correlating with tumor initiation and progression. These findings provide epidemiological and molecular evidence for the clinical application of EV proteins in cancer prediction, while also illuminating their functional roles in cancer physiopathology.

The Diagnostic Accuracy (DA) of ColonView Fecal Immunochemical Test (FIT) in Detecting Colorectal Adenoma (CRA) Can Be Improved by the Diagnostic Models (DM) that Include Triage and Risk Features of CRA.

This study assessed whether the diagnostic accuracy (DA) of ColonView (CV) fecal immunochemical test (FIT) in detecting colorectal adenoma (CRA) can be improved by the diagnostic models (DM) that include triage and risk features of CRA. A total of 5,090 participants of colorectal neoplasia (CRN) screening were recruited prospectively between January 2014 and December 2016. The CRN cohort of 486 patients included 222 CRA patients and 264 non-CRA patients of whom three consecutive fecal samples were analyzed by two fecal occult blood (FOB) assays (CV FIT test, HemoccultSENSA test). Hierarchical multilevel logistic models were used to test the DA of CV test and DMs, visualised as hierarchical summary receiving operating characteristic (HSROC) curves. In conventional receiving operating characteristic (ROC) analysis, the area under the curve (AUC) values of the age, height, weight, and body mass index (BMI) were 0.60, 0.57, 0.54, and 0.51, respectively. The AUC values for different DMs ranged from 0.69 (for DM without triage I/II and SENSA), and the highest AUC value of 0.70 was reached for DM with all variables included. In HSROC analysis, the AUC values for i) lowR variables, ii) highR variables, and ii) DMs were as follows: i) AUC=0.506, ii) AUC=0.566 and iii) AUC=0.732. The differences in AUC values were: between i) and ii) p=0.008; between i) and iii) p<0.0001; between ii) and iii) p<0.0001. The results demonstrated that DMs, particularly those including risk factors, significantly improved the DA of the CV FIT test in detecting CRA compared to traditional low-risk (lowR) and high-risk (highR) features alone. This study provides novel evidence supporting the enhanced diagnostic performance of DMs in combination with CV FIT testing for the detection of CRA.

ALLOSTATIC LOAD AND STATUS IN MID LIFE AND BEYOND

Abstract Objective This study explores how status and processes of cumulative dis/advantage shape allostatic load across the life course. Methods Data from these analyses are from a national sample of adults from the Biomarker Substudy of the Study of Midlife in the US (MIDUS), a longitudinal study of health and well-being in the United States (N=1186) aged 35-86 years old. Education level, multiple indicators of financial stability, and demographic data were used to construct a measure of status. Those in the Biomarker substudy completed a physiological assessment that included blood, urine, and saliva assays to assess functioning of the sympathetic and parasympathetic nervous systems, hypothalamic-pituitary-adrenal axis, cardiovascular, inflammatory immune activity, glucose metabolic system, and lipid metabolic system. These data were used to create an allostatic load index. An alternate allostatic load index was developed using reported medication to affect three of seven biological systems: cardiovascular and the glucose and lipid metabolic systems. Nested regression models were estimated to explain differences in allostatic load. Results Reported financial instability across the life course was associated with higher levels of allostatic load, and higher levels of education were associated with lower levels of allostatic load, both with and without reported medication usage. Conclusion These findings demonstrate that structural forces impact allostatic load, both with and without reported medication usage, and expand previous work on allostatic load and social status.

Developing a Tool for Differentiation Between Bacterial and Viral Respiratory Infections Using Myxovirus Resistance Protein A and C-Reactive Protein.

The aim was to assess the performance of a blood assay combining measurements of MxA (myxovirus resistance protein A) and CRP (C-reactive protein) to differentiate viral from bacterial respiratory infections. In a prospective study, MxA and CRP were measured in the blood by the AFIAS panel in adults admitted with respiratory infection. Patients were split into discovery and validation cohorts. Final diagnosis was adjudicated by a panel of experts. Microbiology-confirmed cases comprised the discovery cohort, and infections adjudicated as highly probable viral or bacterial comprised the validation cohort. A total of 537 patients were analyzed: 136 patients were adjudicated with definitive viral infections and 131 patients with definitive bacterial infections. Using logistic regression analysis, an equation was developed to calculate the probability for bacterial infection using the absolute value of MxA and CRP. Calculated probability≥0.5 and/or MxA to CRP ratio less than 2 applied as the diagnostic rule for bacterial infections. This rule provided 91.6% sensitivity and 90.4% negative predictive value for the diagnosis of bacterial infections. This diagnostic sensitivity was confirmed in the validation cohort. A MxA/CRP ratio less than 0.15 was associated with unfavorable outcome. The calculation of the probability for bacterial infection using MxA and CRP may efficiently discriminate between viral and bacterial respiratory infections.

Whole blood stimulation as a tool for studying the human immune system.

The human immune system is best accessible via tissues and organs not requiring major surgical intervention, such as blood. In many circumstances, circulating immune cells correlate with an individual's health state and give insight into physiological and pathophysiological processes. Stimulating whole blood ex vivo is a powerful tool to investigate immune responses. In the context of clinical research, the applications of whole blood stimulation include host immunity, disease characterization, diagnosis, treatment, and drug development. Here, we summarize different setups and readouts of whole blood assays and discuss applications for preclinical research and clinical practice. Finally, we propose combining whole blood stimulation with high-throughput technologies, such as single-cell RNA-sequencing, to comprehensively analyze the human immune system for the identification of biomarkers, therapeutic interventions as well as companion diagnostics.

Embedded macrophages induce intravascular coagulation in 3D blood vessel-on-chip

Macrophages are innate immune cells that prevent infections and help in wound healing and vascular inflammation. While these cells are natural helper cells, they also contribute to chronic diseases, e.g., by infiltrating the endothelial layer in early atherosclerosis and by promoting vascular inflammation. There is a crosstalk between inflammatory pathways and key players in thrombosis, such as platelets and endothelial cells – a phenomenon known as ‘thromboinflammation’. The role of the embedded macrophages in thromboinflammation in the context of vascular disease is incompletely understood. Blood vessels-on-chips, which are microfluidic vascular cell culture models, have been used extensively to study aspects of vascular disease, like permeability, immune cell adhesion and thrombosis. Blood perfusion assays in blood vessel-on-chip models benefit from multiple unique aspects of the models, such as control of microvessel structure and well-defined flow patterns, as well as the ability to perform live imaging. However, due to their simplified nature, blood vessels-on-chip models have not yet been used to capture the complex cellular crosstalk that is important in thromboinflammation. Using induced pluripotent stem cell-derived endothelial cells and polarized THP-1 monocytes, we have developed and systematically set up a 3D blood vessel-on-chip with embedded (lipid-laden) macrophages, which is created using sequential cell seeding in viscous finger patterned collagen hydrogels. We have set up a human whole blood perfusion assay for these 3D blood vessels-on-chip. An increased deposition of fibrin in the blood vessel-on-chip models containing lipid-laden macrophages was observed. We anticipate the future use of this advanced vascular in vitro model in drug development for early atherosclerosis or aspects of other vascular diseases.Graphical

ALZpath Dx: A novel accessible, validated, and scalable phosphorylated‐tau 217 (pTau217) assay in blood

AbstractBackgroundBlood‐based biomarkers for phosphorylated tau demonstrate strong diagnostic accuracy and compare favorably with current corresponding CSF and neuroimaging biomarkers. Of the four major pTau assays in use (pTau181, pTau217, and pTau231, Nfl), pTau217 appears to be the most robust marker, consistently outperforming plasma pTau 181, neurofilament light, and imaging markers. pTau217 has been shown to correlate with post‐mortem AD pathology, with the ability to differentiate AD from other types of dementia and predict future progression from both normal cognition and mild cognitive impairment to AD.(1,2) Additionally, pTau217 can be used to monitor AD progression because longitudinal increases in pTau217 is indicative of worsening of brain atrophy and cognition in AD.(3) pTau217, therefore, represents a cost‐effective and accessible blood‐based biomarker for tau pathophysiology, that can transform the AD diagnostic field.MethodALZpath has developed reagents for pTau217 and establish a robust and scalable fit for purpose plasma‐based ultra‐sensitive assay utilizing a proprietary monoclonal pTau217 antibody.ResultThe ultra‐sensitive blood‐based ELISA assay, using a peptide calibrator, has been developed on the semi‐automated single‐molecule array Simoa® platform. The assay has been launched for clinical use as laboratory‐developed test (LDT). Evaluation in independent clinical cohorts with multiple co‐morbidities is currently being established to advance to an in‐vitro diagnostic (IVD).ConclusionThe ALZpath pTau217 Simoa® assay is now a fit for purpose validated assay now available for commercial use. Clinical use of this assay is described in Snyder et al. Gouda et al. and Ashton et al. AAIC 2023.

Burden of cerebral small vessel disease modifies relationship between some plasma Abeta 42/40 assays, but not p‐tau181 assays, with amyloid PET

AbstractBackgroundWe aimed to determine if burden of cerebral small vessel disease (CSVD) modifies the relationship of plasma amyloid‐beta (Abeta) 42/40 ratio and/or plasma p‐tau 181 with amyloid PET.MethodWe studied N = 100 participants from the Alzheimer’s Disease Neuroimaging Initiative with 1) Abeta‐42/40 biomarker levels using Quanterix SIMOA (Quanterix), Washington University Mass Spectroscopy (WashU), Simoa HD‐X 4Plex, Roche Elecsys, Shimadzu Mass Spectroscopy, and Gothenburg Mass Spectroscopy, 2) p‐tau181 plasma biomarker levels using Lumipluse G, Roche Elecsys, Simoa p‐tau181V2 Advantage (Simoa Advntage), Simoa p‐tau181, 3) MRI brain, and 4) amyloid‐PET. We stratified by CSVD bruden tertile (calculated from total white matter FLAIR hyperintensity volume). Amyloid positivity was defined as standardized uptake value ratio ≥ 1.11 for florbetapir PET scans. We used box plots to visualize plasma Abeta‐42/40 and p‐tau181 levels stratified by Amyloid‐PET positivity and CSVD tertile. Using logistic regression models, we tested for an interaction between CSVD and plasma Abeta‐42/40 ratio or p‐tau181 on amyloid PET positivity in unadjusted models and models adjusted for age, Clinical Dementia Rating, and presence of ≥1 apolipoprotein E4 allele (ApoE4+).ResultThe cohort had an average age of 77 years, was 56% female, 50% cognitively normal, and 42% ApoE4+ (Table1). We found significant interactions between CSVD and plasma Abeta‐42/40 ratio on amyloid‐PET positivity for Quanterix (raw/adjusted: p = 0.035/p = 0.049), Simoa HD‐Plex (p = 0.047/p = 0.047), and Gothenburg assays (p = 0.026/p = 0.030), with worst discrimination between amyloid‐PET positivity among participants with highest CSVD burden (Figure1A). The Quanterix and Gothenburg assay demonstrated near total overlap between amyloid‐PET positive vs. negative groups at the highest tertile of CSVD. No significant interaction was found between CSVD and any plasma p‐tau181 assay on amyloid‐PET positivity (all p&gt;0.2, Figure1B).ConclusionOur findings suggest that individuals with a high burden of CSVD are at risk for incorrect Abeta‐42/40 ratio on certain assay platforms but not others. No interaction was found between CSVD and any plasma p‐tau181 assay on amyloid‐PET positivity. Further research in diverse cohorts with a high burden of CSVD is warranted to inform appropriate use of these blood assays and unravel mechanisms of this unexpected finding.

Triage Process at Endoscopy With ColonView Fecal Immunochemical Test (FIT) Will Enhance Diagnostic Accuracy (DA) of Colorectal Cancer Screening.

This study assessed the diagnostic accuracy (DA) of the predictive features of colorectal cancer (CRC, predictCRC), triage process (triage), and ColonView (CV) fecal immunochemical test (FIT) in a CRC screening setting. The diagnostic score models (DMs) including predictCRC with triage and CV test were also calculated. The study cohort of 544 patients included 58 CRC patients and 486 non-CRC patients who submitted three consecutive fecal samples for analysis, by two fecal occult blood (FOB) assays (CV FIT test, HemoccultSENSA test). Hierarchical multilevel logistic models were used to test the DA (for CRC) of each item of predictCRC (with triage I and II) and DMs, visualized as hierarchical summary receiving operating characteristic (HSROC) curves. The DA of the predictCRC location of neoplasm (Loc), triage I, and triage II showed 49%, 41%, and 93% sensitivity (Se), and 70%, 99.5%, and 88% specificity (Sp), respectively. The PPV+ of triage I (92%) was higher than that of Loc (22%) or triage II test (45%). In the conventional receiver operating characteristic (ROC) analysis, the area under the curve (AUC) values for the different DMs ranged from 0.880 (for DM without triage I and II), whereas the highest AUC value of 0.960 was reached for DM with triage I and II included in the formula. In the HSROC analysis, the AUC values were as follows: i) with all predictCRCs, AUC=0.717 and ii) with DMs, AUC=0.937. In the roccomp analysis, the difference in AUC values between i) and ii) was statistically significant (p<0.0001). In the detection of CRC, the DA of the new DMs with triage was far superior to that of DMs without triage. This is the first study to report evidence of improved DA in the detection of CRC using DMs including predictCRC with triage and CV FIT test.