Biomarker Assays Research Articles

Investigating Alzheimer’s Disease Resistance in Okinawan Centenarians’ Offspring: A Population Study in a Blue Zone

Alzheimer’s disease (AD) is a neurodegenerative condition characterized by cognitive decline and the accumulation of amyloid-β plaques and neurofibrillary tangles in the brain. Although developing the disease becomes increasingly common as people age, centenarians — individuals who reach the age of 100 — have shown an unexpected resilience to AD, and these individuals are more prevalent in regions known as blue zones, where people experience exceptional longevity and health. This study focuses on the offspring of centenarians in one of these blue zones, Okinawa, Japan, to investigate what potential protective factors they may possess against AD. By screening this unique population using a questionnaire and the Mini-Mental State Examination (MMSE) and tracking AD pathology through advanced biomarker assays following the A/T/N criteria, A: Aβ40/42 ratio, T: phosphorylated tau (pT217-tau), and N: neurofilament light chain (NfL) levels, we aim to uncover genetic, environmental, or lifestyle-related factors that contribute to the decreased incidence of AD in these populations. This study seeks to identify key factors contributing to their resilience by comparing the results of centenarian offspring with age-matched non-centenarians. The spouses of these offspring of centenarians will also be included in the study, acting as a control group of individuals who are likely to share many of the lifestyle and environmental factors with the centenarian offspring but with different genetics. These findings could provide valuable insights for the future development of public health strategies and approaches to mitigate AD risk in the broader population.

A semiautomated microfluidic ELISA for the detection of hemophagocytic lymphohistiocytosis biomarkers.

Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening condition characterized by a massive overactivation of the immune system. Because the clinical findings are nonspecific, the development of assays to facilitate rapid diagnosis is critical for patient care. The objectives of this study were to evaluate the performance of a microfluidic enzyme-linked immunosorbent assay (ELISA) for HLH biomarkers and investigate the impact of insourcing this testing on workflow, cost, and turnaround time in a tertiary-care cancer hospital. Trends in order volume were evaluated for C-X-C motif chemokine ligand 9 (CXCL9) and soluble interleukin 2 receptor ɑ (sIL2R), and a microfluidic ELISA was used to measure these analytes in serum samples. Analyte values, turnaround time, and costs were compared for this assay relative to reference laboratory testing. Test ordering has increased from 187 to 1030 requests annually over the past 5 years. Insourcing these analytes on a semiautomated ELISA can decrease time to result by approximately 2 days and generate a cost savings of roughly $140,000 annually within our laboratory. Using a semiautomated ELISA for sIL2R and CXCL9 may help physicians arrive at a diagnosis and monitor therapy for patients with HLH while decreasing turnaround time and costs within the clinical laboratory.

Multiplexed Glycan Immunofluorescence Identification of Pancreatic Cancer Cell Subpopulations in Both Tumor and Blood Samples.

Pancreatic ductal adenocarcinoma (PDAC) tumor heterogeneity impedes the development of biomarker assays suitable for early disease detection that would improve patient outcomes. The CA19-9 glycan is currently used as a standalone biomarker for PDAC. Furthermore, previous studies have shown that cancer cells may display aberrant membrane-associated glycans. We therefore hypothesized that PDAC cancer cell subpopulations could be distinguished by aberrant glycan signatures. We used multiplexed glycan immunofluorescence combined with pathologist annotation and automated image processing to distinguish between PDAC cancer cell subpopulations within tumor tissue. Using a training-set/test-set approach, we found that PDAC cancer cells may be identified by signatures comprising 4 aberrant glycans (VVL, CA19-9, sTRA, and GM2) and that there are three glycan-defined PDAC tumor types: sTRA type, CA19-9 type, and intermixed. To determine whether the aberrant glycan signatures could be detected in blood samples, we developed hybrid glycan sandwich assays for membrane-associated glycans. In both patient-matched tumor and blood samples, the proportion of aberrant glycans detected was consistent. Furthermore, our multiplexed glycan immunofluorescent approach proved to be more sensitive and more specific than CA19-9 alone. Our results provide proof of concept for a novel methodology to improve early PDAC detection and patient outcomes.

The Alzheimer's Association Global Biomarker Standardization Consortium (GBSC) plasma phospho-tau Round Robin study.

Phosphorylated tau (p-tau) is a specific blood biomarker for Alzheimer's disease (AD) pathology. Multiple p-tau biomarkers on several analytical platforms are poised for clinical use. The Alzheimer's Association Global Biomarker Standardisation Consortium plasma phospho-tau Round Robin study engaged assay developers in a blinded case-control study on plasma p-tau, aiming to learn which assays provide the largest fold-changes in AD compared to non-AD, have the strongest relationship between plasma and cerebrospinal fluid (CSF), and show the most consistent relationships between methods (commutability) in measuring both patient samples and candidate reference materials (CRM). Thirty-three different p-tau biomarker assays, built on eight different analytical platforms, were used to quantify paired plasma and CSF samples from 40 participants. AD biomarker status was categorised as "AD pathology" (n=25) and "non-AD pathology" (n=15) by CSF Aβ42/Aβ40 (US-FDA; CE-IVDR) and p-tau181 (CE-IVDR) methods. The commutability of four CRM, at three concentrations, was assessed across assays. Plasma p-tau217 consistently demonstrated higher fold-changes between AD and non-AD pathology groups, compared to other p-tau epitopes. Fujirebio LUMIPULSE G, UGOT IPMS, and Lilly MSD p-tau217 assays provided the highest median fold-changes. In CSF, p-tau217 assays also performed best, and exhibited substantially larger fold-changes than their plasma counterparts, despite similar diagnostic performance. P-tau217 showed the strongest correlations between plasma assays (rho=0.81 to 0.97). Plasma p-tau levels were weakly-to-moderately correlated with CSF p-tau, and correlations were non-significant within the AD group alone. The evaluated CRM were not commutable across assays. Plasma p-tau217 measures had larger fold-changes and discriminative accuracies for detecting AD pathology, and better agreement across platforms than other plasma p-tau variants. Plasma and CSF markers of p-tau, measured by immunoassays, are not substantially correlated, questioning the interchangeability of their continuous relationship. Further work is warranted to understand the pathophysiology underlying this dissociation, and to develop suitable reference materials facilitating cross-assay standardisation. Alzheimer's Association (#ADSF-24-1284328-C).

Establishment and performance analysis of a new multiplex detection method for influenza an and B virus antigen.

Influenza A and B virus detection is pivotal in epidemiological surveillance and disease management. Rapid and accurate diagnostic techniques are crucial for timely clinical intervention and outbreak prevention. Quantum dot-encoded microspheres have been widely used in immunodetection. The integration of quantum dot-encoded microspheres with flow cytometry is a well-established technique that enables rapid analysis. Thus, establishing a multiplex detection method for influenza A and B virus antigens based on flow cytometry quantum dot microspheres will help in disease diagnosis. To establish a codetection method of influenza A and B virus antigens based on flow cytometry quantum dot-encoded microsphere technology, which forms the foundation for the assays of multiple respiratory virus biomarkers. Different quantum dot-encoded microspheres were used to couple the monoclonal antibodies against influenza A and B. The known influenza A and B antigens were detected both separately and simultaneously on a flow cytometer, and the detection conditions were optimized to establish the influenza A and B antigen codetection method, which was utilized for their detection in clinical samples. The results were compared with the fluorescence quantitative polymerase chain reaction (PCR) method to validate the clinical performance of this method. The limits of detection of this method were 26.1 and 10.7 pg/mL for influenza A and B antigens, respectively, which both ranged from 15.6 to 250000 pg/mL. In the clinical sample evaluation, the proposed method well correlated with the fluorescent quantitative PCR method, with positive, negative, and overall compliance rates of 57.4%, 100%, and 71.6%, respectively. A multiplex assay for quantitative detection of influenza A and B virus antigens has been established, which is characterized by high sensitivity, good specificity, and a wide detection range and is promising for clinical applications.

Analytical Validation of a DNA Methylation Biomarker Test for the Diagnosis of Barrett's Esophagus and Esophageal Adenocarcinoma from Samples Collected Using EsoCheck®, a Non-Endoscopic Esophageal Cell Collection Device.

Barrett's esophagus (BE) is a known precursor to esophageal adenocarcinoma (EAC). Guidelines recommend BE screening in populations with multiple risk factors, for which non-endoscopic esophageal cell collection with biomarker testing is considered as an acceptable alternative to esophagogastroduodenoscopy (EGD). The aim of this study was to evaluate analytical performance characteristics of EsoGuard® (EG), a DNA methylation biomarker assay, as a laboratory-developed test (LDT) in esophageal samples collected with the swallowable EsoCheck® (EC) device. EG is a next-generation sequencing (NGS) assay that evaluates methylated vimentin (VIM) and cyclin A1 (CCNA1), clinically validated biomarkers for the detection of BE and EAC. The studies were conducted according to standards of College of American Pathology (CAP), Clinical Laboratory Improvement Amendments (CLIA), and New York (NY) state requirements for the analytical validation of molecular assays. Comparison to Sanger sequencing showed that EG was 100% accurate at all 31 CpG sites evaluated by the assay. The analytical sensitivity, specificity, and accuracy of the assay were 89%, 100%, and 96%, respectively. Intra- and inter-assay precision was 100%. The limit of detection (LOD) was 1 in 400 methylated cells, and the reference range was 84%. In summary, EsoGuard demonstrates high analytical accuracy, repeatability, and reproducibility in samples collected using the EsoCheck device.

Validation of an Epigenetic Prognostic Assay to Accurately Risk-Stratify Patients with Barrett's Esophagus.

INTRODUCTION: Esophageal adenocarcinoma (EAC) is the second-most lethal cancer in the United States, with Barrett esophagus (BE) being the strongest risk factor. Assessing the future risk of neoplastic progression in patients with BE is difficult; however, high-grade dysplasia (HGD) and early EAC are treatable by endoscopic eradication therapy (EET), with survival rates of 90%. Thus, it would be beneficial to develop a molecular assay to identify high-risk patients, who merit more frequent endoscopic surveillance or EET, as well as low-risk patients, who can avoid EET and undergo less frequent surveillance. METHODS: Deidentified endoscopic biopsies were acquired from 240 patients with BE at 6 centers and confirmed as future progressors or nonprogressors. Tissues were analyzed by a set of methylation-specific biomarker assays. Test performance was assessed in an independent validation set using 4 stratification levels: low risks, low-moderate risks, high-moderate risks, and high risks. RESULTS: Relative to patients in the low-risk group, high-risk patients were 15.2 times more likely to progress within 5 years to HGD or EAC. For patients in the high-risk category, the average risk of progressing to HGD or EAC within 5 years was 21.5%, 4-fold the BE population prevalence within 5 years, whereas low-risk patients had a progression risk of only 1.85%. DISCUSSION: This clinical assay, Esopredict, stratifies future neoplastic progression risk to identify higher-risk patients with BE who can benefit from EET or more frequent surveillance and lower-risk patients who can benefit from reduced surveillance.

CAPS: a simple clinical tool for β-amyloid positivity prediction in clinical Alzheimer syndrome.

With the advent of anti-β-amyloid therapies, clinical distinction between Aβ + and Aβ- in cognitive impairment is becoming increasingly important for stratifying referral and better utilization of biomarker assays. Cognitive profile, rate of decline, neuropsychiatric inventory questionnaire (NPI-Q), and imaging characteristics were collected from 52 subjects with possible/probable AD. Participants with Aβ+ status had lower baseline MMSE scores (24.50 vs. 26.85, p = 0.009) and higher total NPI-Q scores (2.73 vs. 1.18, p < 0.001). NPI-Q score was found to be the only independent predictor for β-amyloid positivity (p = 0.008). A simple scoring system, namely Clinical β-Amyloid Positivity Prediction Score (CAPS), was developed by using the following parameters: NPI-Q, rapidity of cognitive decline, and white matter microangiopathy. Data from 48 participants were included in the analysis of accuracy of CAPS. CAP Score of 3 or 4 successfully classified Aβ + individuals in 86.7% cases. Clinical β-Amyloid Positivity Prediction Score is a simple clinical tool for use in primary care and memory clinic settings to predict β-amyloid positivity in individuals with clinical Alzheimer Syndrome can potentially facilitate referral for Anti Aβ therapies.

PrecivityAD2™ Blood Test: Analytical Validation of an LC-MS/MS Assay for Quantifying Plasma Phospho-tau217 and Non-Phospho-tau217 Peptide Concentrations That Are Used with Plasma Amyloid-β42/40 in a Multianalyte Assay with Algorithmic Analysis for Detecting Brain Amyloid Pathology.

Alzheimer's disease (AD) is a progressive irreversible neurodegenerative disorder that represents a major global public health concern. Traditionally, AD is diagnosed using cerebrospinal fluid biomarker analysis or brain imaging modalities. Recently, less burdensome, more widely available blood biomarker (BBM) assays for amyloid-beta (Aβ42/40) and phosphorylated-tau concentrations have been found to accurately identify the presence/absence of brain amyloid plaques and tau tangles and have helped to streamline AD diagnosis. However, few BBMs have been rigorously analytically validated. Herein, we report the analytical validation of a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) multiplex method for quantifying plasma phosphorylated-tau217 (p-tau217) and non-phosphorylated-tau217 (np-tau217) peptide concentrations. We combined the p-tau217/np-tau217 concentrations ratio (%p-tau217) and the previously validated LC-MS/MS multiplex assay for plasma Aβ42/40 into a new multianalyte assay with algorithmic analysis (MAAA; PrecivityAD2™ test) that identifies brain amyloid status based on brain amyloid positron emission tomography. We found (a) the %p-tau217 assay is precise, accurate, sensitive, and linear over a wide analytical measurement range, and free from carryover and interference; (b) the pre-analytical specimen collection, processing, storage, and shipping conditions that maintain plasma tau peptide stability; and (c) using the measured analytical imprecision for plasma Aβ42/40 and p-tau217/np-tau217 levels in a worst-case scenario model, the PrecivityAD2 test algorithm for amyloid pathology classification changed for only 3.5% of participants from brain amyloid positive to negative, or from negative to positive. The plasma sample preparation and LC-MS/MS methods underlying the PrecivityAD2 test are suitable for use in the clinical laboratory and valid for the test's intended purpose: to aid in the diagnostic evaluation of individuals aged 55 and older with signs or symptoms of mild cognitive impairment or dementia.

A European Bioanalysis Forum recommendation for requiring a context-of-use statement for successful development and validation of biomarker assays.

The European Bioanalysis Forum, alongside key industry stakeholders, has been driving the discussions around the implementation of context-of use for biomarker assays to ensure that these assays are validated appropriately depending on their purpose. Insights into understanding why the implementation of context-of-use in assay strategies has also shown that the key stakeholder, or requester for the biomarker data, is responsible for providing the context-of-use statement for all biomarker assay requests. Expertsfrom across the industry haves repeatedly soughta cross-industry recommended format in which the context-of-use statement could be provided. In this manuscript, the European Bioanalysis Forum suggests a format for this.

62 Prognostic biomarker development using deep learning and spatial proteomics in papillary renal cell carcinoma

Abstract Background Papillary renal cell carcinoma (pRCC) is a prevalent but understudied kidney cancer pathology, constituting 15-20% of cases. While many patients achieve curative therapy using nephrectomy, others go on to develop progressive, metastatic disease with poor overall prognosis. In order to improve the current standards of care for pRCC patients, it is critical to identify new methods to identify high-risk patients likely to require more intense and risk-modulated follow-up. Our goals in this project are two-fold: (1) to identify prognostic patient clusters based on spatial architecture of tumor tissue, and (2) to identify a minimal subset of markers to accurately predict the defined clusters for clinical translation. Methods A tissue microarray of 100 pRCC patients was assayed using multiplex immunofluorescence with a 31-antibody panel with immune- and cancer-related proteins. We have developed novel algorithms to identify spatial neighborhoods and features. These features are then used to identify unsupervised clusters of patients. This new approach to classifying patients allows for a more specific and comprehensive description of each patient tumor’s spatial composition and association with diverse clinical outcomes. Next, we developed attention-based deep learning models capable of predicting these spatially-defined clusters directly from the underlying immunofluorescence images and subsequently profiled these for interpretability by retraining based on individual channels. We assessed each model iteratively on a validation set and a held-out test set for final model selection. We focussed on developing an accurate model with the minimal number of channels to enable development of a cost-efficient biomarker assay for the clinic. Results We performed a systematic analysis of the cellular and spatial phenotypes for each patient in our cohort and identified six (6) major spatial clusters. These clusters are based on the spatial neighborhood composition of the tumors (Fig 1A) and describe the composite effects of cell-cell interactions within a patient tumor. We identified one cluster in particular (cluster #6, mainly associated with spatial interaction of CD163+ macrophages) with a significantly worse prognosis than the other clusters. Using our advanced deep learning models, we have been able to achieve excellent validation accuracy in predicting a variety of clinical phenotypes and spatial features, including patient clusters and histological grade. We wished to then reduce the set of required features and began our efforts in predicting tumor vs. normal based on individual immunofluorescence channels and identified 6 channels with &amp;gt;75% individual predictive accuracy (Fig. 1B). Compositing all 6 markers further improves our accuracy to 90%. This success paves the way for exciting future prospects as we work to extend these efforts to identify a minimal clinically-applicable subset of protein markers. Conclusions The combination of computational spatial proteomics and deep learning models has the potential to identify new biomarkers specific to the spatial organization of patient tumors. We have identified a particular patient cluster with multiple associations with CD163+ macrophages demonstrating poor prognosis; selecting these patients may allow for tailored and more informed patient care. We are also working to enhance our deep learning models to circumvent the need for stepwise spatial analysis and, instead, allow for direct-from-image assessment of patients for particular spatial features indicative of various prognostic indicators. Our identified set of 6 markers serves as a basis for further refinements as we work to identify a minimal set of relevant markers able to predict various clinical outcomes from immunofluorescence imaging, offering a promising new avenue in the field of cancer pathology and biomarker development. DOD CDMRP Funding: yes

Cerebral ischemia biomarkers: Their roles in early diagnosis and prognosis with potential clinical applications

Cerebral ischemia, caused by a disruption in blood supply to the brain, remains a significant cause of morbidity and mortality worldwide. Identifying reliable biomarkers for the early diagnosis and prognosis of cerebral ischemia is crucial for timely intervention and improved patient outcomes. This review aims to provide a comprehensive overview of the current knowledge on cerebral ischemia biomarkers, focusing on their potential role in early detection and prediction of clinical outcomes. Specifically, we discuss the current advances in the field of cerebral ischemia biomarkers, which serve as essential tools for early diagnosis and monitoring of ischemic stroke. We examine several promising biomarkers, including omics data, genetic (FOXF2 and ATP5H), physiological and neuroinflammatory biomarkers, neuroimaging markers, blood-based biomarkers (proteins, microRNAs, and metabolites), and newer modalities such as exosomes, microvesicles, and cell-free deoxyribonucleic acid (cfDNA). In addition, we highlight the challenges and future directions in translating these biomarkers into clinical practice. Standardization and reproducibility, methodological limitations, and cost and accessibility are critical challenges in the translation of biomarkers into clinical practice. Addressing these challenges requires multi-stakeholder collaborations and coordinated efforts to establish standardized protocols, improve analytical methods, and develop cost-effective biomarker assays. The use of point-of-care testing devices or miniaturized lab-on-a-chip technologies can reduce costs and improve accessibility, particularly in low-resource settings. Furthermore, collaborations between academia, industry, and regulatory agencies can facilitate the translation of biomarkers by addressing regulatory and reimbursement hurdles that affect the affordability and availability of these tests.

Cost-Effectiveness of Hepatocellular Carcinoma Surveillance Strategies in Patients With Compensated Liver Cirrhosis in the United Kingdom

ObjectivesThis study aimed to evaluate the cost-effectiveness (CE) of 4 hepatocellular carcinoma (HCC) surveillance strategies in the United Kingdom, the gender and age with the biomarkers alpha-fetoprotein-L3, alpha-fetoprotein, and des-γ-carboxy prothrombin (GAAD) algorithm, which combines gender (biological sex) and age with Elecsys® biomarker assays, alpha-fetoprotein (AFP) and protein induced by vitamin K absence-II, ultrasound (US), US + AFP and GAAD + US. MethodsA de novo microsimulation state-transition Markov model was developed in Microsoft Excel® from the perspective of the UK National Health Service to calculate life-years, quality-adjusted life-years (QALYs), costs, incremental CE ratios, and net monetary benefits. Parameters were sourced from peer-reviewed published literature, national guidelines, and public cost databases. Sensitivity and scenario analyses were performed to evaluate the impact of parameter and structural uncertainty on the results. ResultsIn a simulated cohort of 100 000 patients, discounted costs and QALYs per patient were £8663 and 6·066 for US, £9095 and 6·076 for US + AFP, £8719 and 6·078 for GAAD alone, and £9114 and 6·086 for GAAD + US. At a CE threshold of £20 000/QALY, GAAD was the most cost-effective strategy; however, although most costly, GAAD + US was the most clinically effective. Sensitivity and scenario analyses indicated that HCC incidence along with costs associated with diagnostic performance influence CE. ConclusionConsidering the cost of US and low incidence of HCC in the United Kingdom, this study suggests that GAAD alone or in combination with US are cost-effective surveillance strategies compared with US and US + AFP. Although GAAD + US showed the highest QALY increase, GAAD alone is considered preferable regarding CE; however, better performance estimates for GAAD + US are needed to confirm.

Recommendations for Development and Validation of a Fit-For-Purpose Biomarker Assays Using Western Blotting; An-AAPS Sponsored Initiative to Harmonize Industry Practices.

Western blot (WB) assays are routinely used for detection and quantification of biomarkers. Although assay validation to measure biomarkers in complex matrices has become a mainstay process for ligand binding assays (LBA) and mass spectrometry (MS), no guidelines exist yet validate biomarker methods using WB techniques. In this cross-industry white paper, we outlined in detail the key steps for development and for validation of WB assays for protein biomarkers under different contexts of use (COU). In addition, we described how to determine the level of assay validation needed for biomarker assays using Western blotting. For simplicity, we described two paths of WB assay validation. The first path (Path 1) is for biomarkers being analyzed for exploratory research or for internal go- or no/go- decision making. The second path (Path 2) is for clinical decision making such as dose determination or drug response that need to be run in a regulated environment. This work is supported through AAPS Biomarkers and Precision Medicine subteam and represents AAPS members opinion.

Efficacy of stem cells versus microvesicles in ameliorating chronic renal injury in rats (histological and biochemical study)

Chronic exposure to heavy metals as aluminum chloride (AlCl3) could result in severe health hazards such as chronic renal injury. The present study aimed to evaluate the therapeutic potential of adipose tissue-derived stem cells (ASCs) in comparison to their microvesicles (MV) in AlCl3-induced chronic renal injury. Forty-eight adult male Wistar rats were divided into four groups: Control group, AlCl3-treated group, AlCl3/ASC-treated group, and AlCl3/MV-treated group. Biochemical studies included estimation of serum urea and creatinine levels, oxidative biomarkers assay, antioxidant biomarkers, serum cytokines (IL-1β, IL-8, IL-10, and IL-33), real time-PCR analysis of renal tissue MALT1, TNF-α, IL-6, and serum miR-150-5p expression levels. Histopathological studies included light and electron microscopes examination of renal tissue, Mallory trichrome stain for fibrosis, Periodic acid Schiff (PAS) stain for histochemical detection of carbohydrates, and immunohistochemical detection of Caspase-3 as apoptosis marker, IL-1B as a proinflammatory cytokine and CD40 as a marker of MVs. AlCl3 significantly deteriorated kidney function, enhanced renal MDA and TOS, and serum cytokines concentrations while decreased the antioxidant parameters (SOD, GSH, and TAC). Moreover, serum IL-10, TNF-α, miR-150-5p, and renal MALT1 expression values were significantly higher than other groups. Kidney sections showed marked histopathological damage in both renal cortex and medulla in addition to enhanced apoptosis and increased inflammatory cytokines immunoexpression than other groups. Both ASCs and MVs administration ameliorated the previous parameters levels with more improvement was detected in MVs-treated group. In conclusion: ASCs-derived MVs have a promising ameliorating effect on chronic kidney disease.

Miniaturizing Nanotoxicity Assays in Daphnids.

The rapid progress of the modern world has resulted in new materials and products created at an accelerating pace. As such, nanoparticles have widespread applications and often find their way into the aquatic ecosystem. In the case of freshwater ecosystems, one of the commonly used bioindicators species used for pollution assessment is Daphnid magna. The Organization for Economic Co-operation and Development (OECD), and other organizations such as the European Chemicals Agency (ECHA) and Environmental Protection Agency (EPA), have set guidelines for acute toxicity testing in daphnids that are severely lacking in terms of information on the characteristics of the exposure vessel when studying the adverse effects of nanoparticles (NPs). Understanding the toxicity mechanisms of nanomaterials is imperative given the scarcity of information on their adverse effects. Furthermore, miniaturization of nanotoxicity assays can reduce the number of daphnids used, as well as the cost and nanomaterial waste, and provide results even at the individual animal level with enhanced reproducibility of testing. In this study, the impact of the exposure vessel on the observed physiological changes of daphnids was investigated for a silver nano ink. Exposures in eleven commercially available vessels; nine made of plastic and two made of glass were compared for 24 h. The effect of surface to volume ratio of the exposure vessel and the animal number or "crowding" during exposure was investigated in the context of miniaturizing biomarker assays as alternatives to traditional experimental setups in Daphnid magna. Toxicity curves showed differences depending on the vessel used, while a novel feeding rate assay and the activity of key enzymes were assessed as physiology endpoints.

High variability in assays of blood markers of collagen turnover in cardiovascular disease: Implications for research and clinical practice.

Fibrosis is a common feature of many chronic diseases, including heart failure, which can have deleterious effects on cardiac structure and function that are associated with adverse outcomes. By-products of collagen synthesis and degradation, such as carboxy- and amino-terminal pro- or telo-peptides of collagen type I and III (PICP, PINP, PIIINP, and CITP) have been extensively investigated as markers of fibrosis. Although the majority of studies report on the reproducibility of their assay results, there is no a comparison of biomarker assays across studies. Therefore, we conducted a systematic review adhering to PRISMA guidelines. The search terms employed in Medline were: 'collagen AND cardiac' or 'collagen AND heart'. This query yielded a total of 1049 articles. Thereafter, specific search criteria were applied: (i) original English-language papers; (ii) human studies; (iii) in-vivo investigations; and (iv) blood/serum/plasma samples. Overall, 89 studies were identified (42 on PIIINP, 32 on PICP, 29 on CITP, and 17 on PINP). The range of reported values for PIIINP was between 0.06 to 11 800 μg/l; for PICP 0.006 to 1265 μg/l; for CITP 0.3 to 5450 μg/l; for PINP 0.15 to 80 μg/l. Extreme variations in values for fibrosis biomarkers were observed across studies, especially when different assays were used, but also with the same assays. Our findings show that it is challenging to ascertain normal ranges or compare studies for the measurement of fibrosis biomarkers. Given the potential implications for clinical practice and current lack of awareness of these issues, this subject warrants comprehensive acknowledgement and understanding.

Head-to-head comparison of leading blood tests for Alzheimer's disease pathology.

Blood tests have the potential to improve the accuracy of Alzheimer disease (AD) clinical diagnosis, which will enable greater access to AD-specific treatments. This study compared leading commercial blood tests for amyloid pathology and other AD-related outcomes. Plasma samples from the Alzheimers Disease Neuroimaging Initiative were assayed with AD blood tests from C2N Diagnostics, Fujirebio Diagnostics, ALZPath, Janssen, Roche Diagnostics, and Quanterix. Outcomes measures were amyloid positron emission tomography (PET), tau PET, cortical thickness, and dementia severity. Logistic regression models assessed the classification accuracies of individual or combined plasma biomarkers for binarized outcomes, and Spearman correlations evaluated continuous relationships between individual plasma biomarkers and continuous outcomes. Measures of plasma p-tau217, either individually or in combination with other plasma biomarkers, had the strongest relationships with all AD outcomes. This study identified the plasma biomarker analytes and assays that most accurately classified amyloid pathology and other AD-related outcomes.

C-Reactive Protein as an Early Marker of Severity and Outcome in Patients with SARS-CoV-2 Infection

Abstract Background SARS CoV-2 infection is a major cause of morbidity and mortality worldwide. The incidence of SARS CoV-2 infection is rising with millions of people getting infected daily. Mortality rate varies from a country to the other(0.3-6%), most of which are attributed to respiratory failure. Several studies have been performed to identify biomarkers for use in the early identification of patients at risk. Aim of the work This study was done to estimate and evaluate the plasma levels of CRP in patients with SARS CoV-2 and to study the association of CRP levels with severity of disease progression and prognosis of those patients. Patients and Methods This retrospective cohort study was conducted on 100 patients admitted to El-Obour Ain Shams University Specialized Hospital for Isolation during the period between January and October 2020. Results Regarding history patients whom were old or have any of the following comorbid conditons as hypertension, Diabetes mellitus, chronic liver disease and active cancer were statistically more prone to not surviving. Regarding complete blood picture, there was significant decrease in haemoglobin and or increase in total leukocytic count in those who did not survive. Serum Ferritin and D-Dimer were significantly high in those who did not survive. As regard CRP, it was significantly higher in the non-survivor group, Also it correlated with severity as ICU admission and need of respiratory support. Regarding the ROC curve result the best cut-off of CRP to predict mortality was &amp;gt;129mg/l, with Area under the curve = 0.981, Sensitivity = 90.91%, Specificity= 97.75%, positive predictive value = 83.3% and negative predictive value =98.9%. Regarding the ROC curve result the best cut-off of CRP to predict need of respiratory support was &amp;gt;55.3mg/L, with Area under the curve = 0.976, Sensitivity = 100%, Specificity= 86.21%, positive predictive value = 52.0% and negative predictive value =100.0%. Regarding the ROC curve result the best cut-off of CRP to predict need of ICU admission was &amp;gt;55.3mg/L, with Area under the curve = 0.987, Sensitivity = 100.0%, Specificity= 93.75%, positive predictive value = 80.0% and negative predictive value =100.0%. Conclusion Laboratory and clinical medicine groups should actively collaborate and optimize their individual expertise to potentially prove standardized biomarkers assays that will optimize patient care.

Development of a Cellular Assay as a Personalized Model for Testing Chronic Wound Therapeutics

Exudates of non-healing wounds contain drivers of pathogenicity. We utilized >800 exudates from non-healing and healing wounds of diverse etiologies, collected by three different methods, to develop a wound-specific, cell-based functional biomarker assay. Human dermal fibroblast proliferation served as readout to a) to differentiate between healing and non-healing wounds, b) follow the healing process of individual patients, and c) assess the effects of therapeutics for chronic wounds ex vivo. We observed a strong correlation between wound chronicity and inhibitory effects of individual exudates on fibroblast proliferation, with good diagnostic sensitivity (76-90%, depending on the sample collection method). Transition of a clinically non-healing to a healing phenotype restored fibroblast proliferation and extracellular matrix formation while reducing inflammatory cytokine production. Transcriptional analysis of fibroblasts exposed to ex vivo non-healing wound exudates revealed an induction of inflammatory cytokine- and chemokine pathways and the unfolded protein response, indicating that these changes may contribute to the pathology of non-healing wounds. Testing the wound therapeutics platelet derived growth factor and silver sulfadiazine yielded responses in line with clinical experience and indicate the usefulness of the assay to search for and profile new therapeutics.