Validation Of Assay Research Articles

Saliva-based point-of-care assay to measure the concentration of pyrazinamide using a mobile UV spectrophotometer

Abstract Introduction Pyrazinamide, one of the first-line antituberculosis drugs, displays variability in drug exposure that is associated with treatment response. A simple, low-cost assay may be helpful to optimize treatment. This study aimed to develop and validate a point-of-care assay to quantify the concentration of pyrazinamide in saliva. Methods All measurements were conducted using the nano-volume drop function on the mobile ultraviolet (UV) spectrophotometer (NP80, Implen, Germany). Assay development involved applying second derivative spectroscopy in combination with the Savitzky–Golay filter between wavelengths of 200–300 nm to increase spectral resolution. Assay validation included assessing selectivity, linearity, accuracy, precision, carry-over and matrix effects. Specificity was also analysed by evaluating the impact of co-administered medications on pyrazinamide results. Sample stability was measured at various temperatures up to 40°C. Results The calibration curve (7.5–200 mg/L) was linear (R2 = 0.9991). The overall accuracy (bias%) and precision (CV%) ranged from −0.66% to 5.15%, and 0.56% to 4.95%, respectively. Carry-over and matrix effects were both acceptable with a bias% of <±4% and CV% of <7.5%. Commonly co-administered medications displayed negligible interferences. Levofloxacin displayed analytical interference (bias% = −10.21%) at pyrazinamide concentrations < 25 mg/L, but this will have little clinical implications. Pyrazinamide was considered stable in saliva after 7 days in all storage conditions with a CV% of <6.5% and bias% of <±10.5% for both low- and high-quality control concentrations. Conclusions A saliva-based assay for pyrazinamide has been successfully developed and validated using the mobile UV spectrophotometer.

Multicenter validation of an RNA-based assay to predict anti-PD-1 disease control in patients with recurrent or metastatic head and neck squamous cell carcinoma: the PREDAPT study.

Despite advances in cancer care and detection, >65% of patients with squamous cell cancer of the head and neck (HNSCC) will develop recurrent and/or metastatic disease. The prognosis for these patients is poor with a 5-year overall survival of 39%. Recent treatment advances in immunotherapy, including immune checkpoint inhibitors like pembrolizumab and nivolumab, have resulted in clinical benefit in a subset of patients. There is a critical clinical need to identify patients who benefit from these antiprogrammed cell death protein 1 (anti-PD-1) immune checkpoint inhibitors. Here, we report findings from a multicenter observational study, PREDicting immunotherapy efficacy from Analysis of Pre-treatment Tumor biopsies (PREDAPT), conducted across 17 US healthcare systems. PREDAPT aimed to validate OncoPrism-HNSCC, a clinical biomarker assay predictive of disease control in patients with recurrent or metastatic HNSCC treated with anti-PD-1 immune checkpoint inhibitors as a single agent (monotherapy) and in combination with chemotherapy (chemo-immunotherapy). The test used RNA-sequencing data and machine learning models to score each patient and place them into groups of low, medium, or high. The OncoPrism-HNSCC prediction significantly correlated with disease control in both the monotherapy cohort (n=62, p=0.004) and the chemo-immunotherapy cohort (n=50, p=0.01). OncoPrism-HNSCC also significantly predicted progression-free survival in both cohorts (p=0.015 and p=0.037, respectively). OncoPrism-HNSCC had more than threefold higher specificity than programmed death-ligand 1 combined positive score and nearly fourfold higher sensitivity than tumor mutational burden for predicting disease control. Here, we demonstrate the clinical validity of the OncoPrism-HNSCC assay in identifying patients with disease control in response to anti-PD-1 immune checkpoint inhibitors. NCT04510129.

EZH2 Promotes Glioma Cell Proliferation, Invasion, and Migration via Mir-142-3p/KCNQ1OT1/HMGB3 Axis : Running Title: EZH2 Promotes Glioma cell Malignant Behaviors.

This study investigates the role and molecular mechanism of EZH2 in glioma cell proliferation, invasion, and migration. EZH2, miR-142-3p, lncRNA KCNQ1OT1, LIN28B, and HMGB3 expressions in glioma tissues and cells were determined using qRT-PCR or Western blot, followed by CCK-8 assay detection of cell viability, Transwell detection of invasion and migration, ChIP analysis of the enrichment of EZH2 and H3K27me3 on miR-142-3p promoter, dual-luciferase reporter assay and RIP validation of the binding of miR-142-3p-KCNQ1OT1 and KCNQ1OT1-LIN28B, and actinomycin D detection of KCNQ1OT1 and HMGB3 mRNA stability. A nude mouse xenograft model and a lung metastasis model were established. EZH2, KCNQ1OT1, LIN28B, and HMGB3 were highly expressed while miR-142-3p was poorly expressed in gliomas. EZH2 silencing restrained glioma cell proliferation, invasion, and migration. EZH2 repressed miR-142-3p expression by elevating the H3K27me3 level. miR-142-3p targeted KCNQ1OT1 expression, and KCNQ1OT1 bound to LIN28B to stabilize HMGB3 mRNA, thereby promoting its protein expression. EZH2 silencing depressed tumor growth and metastasis in nude mice via the miR-142-3p/KCNQ1OT1/HMGB3 axis. In conclusion, EZH2 curbed miR-142-3p expression, thereby relieving the inhibition of KCNQ1OT1 expression by miR-142-3p, enhancing the binding of KCNQ1OT1 to LIN28B, elevating HMGB3 expression, and ultimately accelerating glioma cell proliferation, invasion, and migration.

Analytical measuring interval, linearity, and precision of serology assays for detection of SARS-CoV-2 antibodies according to CLSI guidelines.

Reliable and validated serology assays are of increasing importance as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus continues to evolve and cause outbreaks. Validation of serology assays along with calibration to the International and National Standards (such as anti-SARS-CoV-2 Immunoglobulin WHO International Standard 20/136 or Frederick National Laboratory for Cancer Research's National Serology Standard COVID-NS01097) is critical to ensuring that results from clinical studies are reliable and comparable among various assays and laboratories. We describe the design and execution of a comprehensive study that established the analytical measuring intervals, linearity, precision, and repeatability of four in-house developed serology enzyme-linked immunosorbent assays (SARS-CoV-2 anti-Spike immunoglobin G [IgG] and immunoglobin M [IgM] and anti-Nucleocapsid IgG and IgM) following applicable Clinical and Laboratory Standards Institute (CLSI) guidelines. Overall, this study provides practical guidance on experimental design strategies and data analysis techniques, pertaining to the validation of COVID-19 serology assays according to CLSI guidelines, for use in clinical research studies.

Clinical validation of an RSV neutralization assay and analysis of cross-sectional sera associated with 2021-2023 RSV outbreaks to investigate the immunity debt hypothesis.

Population surveillance studies of serum-neutralizing activity against RSV are crucial for evaluating RSV vaccine efficacy and vulnerabilities to new strains. Here, we designed and validated a high-throughput assay for assessing anti-RSV neutralizing activity, standardized its measurements for comparison with other methodologies, and demonstrated its applicability to real-world samples. Our assay is precise, linear, and yields measurements consistent with other standardized assays, offering a methodology useful for large-scale studies of RSV immunity. We also find no significant difference in neutralizing titers among adults between those taken before and after large RSV outbreaks associated with the latter stages of the coronavirus disease of 2019 (COVID-19) public health emergency, underlining the need for a greater understanding of the dynamics of serological responses to RSV infection.

From bench to bedside and back to bench: investigating the role of platelet heterogeneity in coronary artery disease

Abstract Background/Introduction Platelets exhibit considerable heterogeneity in RNA content, size, and thrombogenicity. Our preliminary investigation of the transcriptome of the RNA-rich reticulated platelets(RPs) in healthy donors revealed an enrichment of prothrombotic transcripts compared to mature platelets(MPs). Recently, we validated the prognostic role of elevated RPs at clinical level in a modern cohort of ACS patients treated with potent P2Y12 inhibitors. However, the pathophysiology of RP hyperreactivity remains unclear, particularly its correlation with adverse events and cardiovascular death. Purpose To study the biology of RPs in patients with chronic coronary syndrome(CCS) and to elucidate their role in disease progression. Methods RPs were isolated from peripheral blood of CCS patients(N=20) and compared with MPs. Cell viability and reactivity of RPs were quantified by FACS. Deep transcriptomic profiling was performed using post-sorting bulk RNA sequencing of RPs and MPs and analyzed with ad-hoc bioinformatic pipelines. Proteomic profiling using mass cytometry(CyTOF) was used to validate the transcriptomic findings with single cell resolution. Results FACS analysis confirmed RPs hyperreactivity showing increased P-Selectin expression upon activation with TRAP, ADP and Collagen(Fig. 1A). Total RNA-sequencing detected 2213 differentially regulated genes with an enrichment in RPs of key functional transcript such as, the von Willebrand Factor VWF(p=3.06*10-33), the thromboxane receptor TBXA2R(p=2.07*10-29) and the collagen receptor GP6(p=1.29*10-38, Fig. 1B-C). Gene ontology and gene set enrichment analyses detected an upregulation of relevant categories as "Regulation of platelet activation" and "Platelet aggregation"(Fig. 1D-F). In addition, we detected a novel alternative splicing event on the collagen receptor transcript GP6 upregulated in RPs(Fig. 1G). We detected 33 differentially regulated miRNAs, including miR-409, miR-134 and miR-432, which have been already linked to prothrombotic phenotypes(Fig. 1H). Additionally, we detected an enrichment of circular RNAs in RPs compared to MPs with several circular RNA upregulated in RPs, that have not been described before(Fig. 1I-K). Mass cytometry detected a significant upregulation in RPs of relevant proteins including the collagen receptor GPVI(p=8.98*10-12, Fig.2) and the thrombin receptor PAR1(p=5.21*10-11). Validation assays in an independent cohort detected an upregulation of the PI3K/AKT pathway in RPs which is downstream of the receptor PAR1 and GP6. Conclusion This study represents the first comprehensive multiomic characterization of RPs in CCS patients, providing insights into their prothrombotic phenotype and into their correlation with cardiovascular events. These findings suggest potential avenues for tailored therapeutic interventions targeting the identified upregulated pathways in patients with elevated RPs, warranting further investigation at the clinical level.

A microphysiological assay for studying T-cell chemotaxis, trafficking and tumor killing

Tumors in patients non-responsive to immunotherapy harbor a series of barriers that impede the efficacy of effector T-cells. Consequently, therapeutically modulating the chemotaxis machinery to enable effector T cell infiltration and function in the tumor could result in more successful therapeutic outcomes. Complexin-vitromodels allow re-creation ofin-vivotumor complexities in anin-vitrosetting, allowing improved translatability to patient biology at the laboratory scale. We identified a gap in available industrial scale microphysiological (MPS) assays for faster validation of targets and strategies that enable T-cell chemotaxis and effector function within tumor microenvironments. Using a commercially available, 96-chip 2-lane microfluidic assay system, we present a novel, scalable, complexin vitroMPS assay to study 3D T-cell chemotaxis and function within native, extracellular matrix (ECM)-rich multicellular tumor environments. Activated or naïve CD3+ T-cells stained with far-red nuclear stain responded to the chemokine gradients generated within the matrigel-collagen ECM by migrating into the microfluidic channel (∼5 mm horizontal window), in a concentration- and cell type-dependent manner. Furthermore, we observed and tracked chemotaxis and cancer cell killing function of antigen-specific CD4.CD8. chimeric antigen receptor (CAR)-T cells that responded to CXCR3 agonist gradient built through the expansive 5 mm of cancer cell colony containing stroma. The 2-lane assay system yielded useful information regarding donor and dose-dependent differences in CAR-T cell chemotaxis and tumor killing. The scalable assay system allows a granular window into immune cell migration and function in tissue spaces beyond endothelium, addressing a missing gap in studying tissue-specific immune cell chemotaxis and function to bring forward advancements in cancer immunotherapy.

Potential and challenges of clinical high-dimensional flow cytometry: A call to action.

Clinical biomarker strategies increasingly integrate translational research to gain new insights into disease mechanisms or to define better biomarkers in clinical trials. High-dimensional flow cytometry (HDFCM) holds the promise to enhance the exploratory potential beyond traditional, targeted biomarker strategies. However, the increased complexity of HDFCM poses several challenges, which need to be addressed in order to fully leverage its potential and to align with current regulatory requirements in clinical flow cytometry. These challenges include among others extended timelines for assay development and validation, the necessity for extensive knowledge in HDFCM, and sophisticated data analysis strategies. However, no guidelines exist on how to manage such challenges in adopting clinical HDFCM. Our CYTO 2024 workshop "Potential and challenges of clinical high-dimensional flow cytometry" aimed to find consensus across the pharmaceutical industry and broader scientific community on the overall benefits and most urgent challenges of HDFCM in clinical trials. Here, we summarize the insights we gained from our workshop. While this report does not provide a blueprint, it is a first step in defining and summarizing the most pressing challenges in implementing HDFCM in clinical trials. Furthermore, we compile current efforts with the goal to overcome some of these challenges. As such we bring the scientific community and health authorities together to build solutions, which will accelerate and simplify the full adoption of HDFCM in clinical trials.

Strategies to reduce the culture medium costs for a high‐yield and high‐selectivity bio‐based 2,3‐butanediol production

AbstractBio‐based 2,3‐butanediol (2,3‐BDO) production on a large scale depends on critical factors, such as culture medium, oxygen supply, pH and biosafety. In this study, three strategies for reducing culture medium costs were investigated: carbon/nitrogen (C/N) ratio; low‐cost nitrogen sources (crude yeast extract, brewer's yeast extract, corn steep liquor, urea, sodium nitrate, ammonium chloride, ammonium sulfate and dibasic ammonium phosphate); and microbial pH autoregulation. Batch fermentations were performed in a microaerobic environment using wild‐type and safe Paenibacillus peoriae NRRL BD 62. The yield and selectivity of 2,3‐BDO were used as control variables. A ratio between 2,3‐BDO production and glucose consumption (YP/S) of almost 80% and an optical purity of 87% levo‐2,3‐BDO, with no acetoin accumulation, were achieved in an NH4Cl‐based medium at C/N = 8.5 g/g and without external pH control, considering an initial glucose of 10 g/L. Based on Free on Board prices, a 63% savings in culture medium costs was achieved by replacing commercial yeast extract at pH 5. Validation assays with higher initial glucose concentrations showed a YP/S of 0.40 g/g and an optical levo‐/meso‐2,3‐BDO ratio of 1:0.8, with negligible acetoin accumulation. Therefore, the NH4Cl‐based medium at C/N = 8.5 g/g and without pH control was considered economically promising for high‐yield and high‐selectivity bio‐based 2,3‐BDO production.

Exploring You-gui Pill for the Treatment of Diabetic Erectile Dysfunction: Data Mining Analysis, Network Pharmacology and Experiments In Vitro.

The You-gui pill (YGP) is a classical compound used for treating antidiabetic erectile dysfunction (DMED). However, the specific active ingredients responsible for its effects on DMED and their mechanisms remain unclear. In this paper, we used data mining techniques to analyze high-frequency herbs and herb combinations used in Chinese medicine for the treatment of DMED based on existing literature. Using network pharmacology to study the active components and mechanism of action of YGP against DMED, molecular docking was used to analyze the interactions of the active components with major structural proteins, nonstructural proteins, and mutants. Also, the therapeutic effect of YGP on hyperglycemic modelling and its underlying mechanisms were experimentally validated in CCEC cells by analyzing the expression of its relevant target mRNAs. Network pharmacological analysis identified the three core components of YGP as quercetin, kaempferol, and β-sitosterol, and constructed a PPI network map of common targets of YGP and DMED, which included HIF-1α, ALB, Bcl-2, INS, IL-1β, IL-6, TNF-α, CASP3, and TP53. Combined with molecular docking results, these targets had a strong binding affinity between them and the active ingredient compounds, with the highest affinity for HIF-1α and TNF-α. During the in vitro cellular assay validation, the HIF-1α, ALB, Bcl-2, TNF-α, and IL-6 mRNA in CCECs cells showed positive regulation after YGP intervention. The combination of "data mining - network pharmacology - molecular docking - experimental validation" provides a powerful methodological basis for the study of the main active components and mechanism of action of YGP against DMED, as well as the development and application of the drug.

Validation of a breast cancer assay for radiotherapy omission: an individual participant data meta-analysis.

There are currently no molecular tests to identify individual breast cancers where radiotherapy (RT) offers no benefit. Profile for the Omission of Local Adjuvant Radiotherapy (POLAR) is a 16-gene molecular signature developed to identify low risk cancers where RT will not further reduce recurrence rates. An individual participant data meta-analysis was performed in 623 cases of node-negative ER+/HER2-negative early breast cancer enrolled in three RT randomized trials for whom primary tumor material was available for analysis. A Cox proportional hazards model on time to locoregional recurrence (LRR) was used to test the interaction between POLAR score and RT. 429 (69%) patients' tumors had a high POLAR score and 194 (31%) had a low score. Patients with high POLAR score had, in the absence of RT, a 10-year cumulative incidence of LRR: 20% (15%-26%) vs 5% (2%-11%) for those with a low score. Patients with a high POLAR score had a large benefit from RT (hazard ratio [HR] for RT vs no RT: 0.37 [0.23-0.60], p < .001). In contrast, there was no evidence of benefit from RT for patients with a low POLAR score (HR: 0.92 [0.42-2.02], p = .832). The test for interaction between RT and POLAR was statistically significant (p = .022). POLAR is not only prognostic for locoregional recurrence but also predictive of benefit from radiotherapy in selected patients. Patients ≥ 50 years with ER+/HER2-negative disease and a low POLAR score could consider omitting adjuvant RT. Further validation in contemporary clinical cohorts is required.

Establishment and validation of a real-time fluorescent PCR freeze-dried type assay for 11 sheep and goats pathogens

Small ruminants are economic mainstay in developing countries because of its direct contribution to food security, but the occurrence of epidemics poses a continuous threat. Early diagnosis can strengthen the prevention of zoonosis. Therefore, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens in this study. The results showed that there was no non-specific amplification in systems containing different pathogen positive controls; The areas under the ROC curves were all in the range of 0.99 to 1.00; The LOD counted by Digital PCR were 194, 96, 84, 40, 265, 68, 208, 236, 118, 53 and 723 copies/mL for M. ovis, Pathogenic Leptospira, C. burnetii, BTV, PPRV, Brucella, exJSRV, C. abortus, ORFV, FMDV and CPV, respectively. The coefficients of variation of both intra-group and inter-group replicate tests were less than 5.00%; The accelerated thermostatic lyophilization test was used to predict the validity period; The LOD of each positive pathogen was still detected after 6 days at 56 ℃, suggesting that the validity period can be at least 567 days at room temperature. In this study, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens, which were Mycoplasma ovis (M. ovis), Pathogenic Leptospira, Coxiella burnetii (C. burnetii), Bluetongue virus (BTV), Peste des pestis ruminants virus (PPRV), Brucella, exogenous jaagsiekte sheep retrovirus (exJSRV), Chlamydia abortus (C. abortus), Orf virus (ORFV), Foot-and-mouth disease virus (FMDV), and Capripox virus (CPV).

Validation of Serum Tetrahydrocannabinol Assay and Its Clinical Application

Abstract Background The widespread use of marijuana across all age groups in the United States, driven by the legalization of recreational and medical cannabis, has underscored the need for accurate measurement of tetrahydrocannabinol (THC) and its metabolites in blood specimens. This requirement spans both legal and medical contexts, prompting the development of precise diagnostic tools. Methods We developed a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, utilizing a straightforward protein precipitation extraction method to quantify THC and its metabolites: THC, 11-OH-THC, THC-COOH, cannabidiol (CBD), and THC-COOH glucuronide. The assay was rigorously validated for precision, linearity, stability, carryover, and quality assurance to ensure its reliability and accuracy for clinical use. Results Precision studies demonstrated a coefficient of variation ranging from 1.7% to 4.9%. The analytical measurement range was established at 1-100 ng/mL for THC, CBD, and 11-OH-THC, 5-500 ng/mL for THC-COOH, and 10-1000 ng/mL for THC glucuronide, accommodating the broad spectrum of THC/metabolite concentrations observed in our patient cohort. Carryover from all analytes were negligible (&amp;lt;0.01%). Sample stability was confirmed for up to two weeks at -20°C and through three freeze-thaw cycles. Despite a median THC concentration of 5 ng/mL in a limited sample size (N=44), high concentrations of THC and its metabolites correlated with trauma-related incidents and a history of polysubstance abuse in emergency department admissions. Conclusions The LC-MS/MS assay offers superior sensitivity and specificity compared to traditional immunoassays for cannabinoid detection, effectively distinguishing between THC and its metabolites. Its implementation will offer an advanced approach for analyzing the prevalence and clinical implications of THC and metabolite levels in various patient populations, including trauma, pediatric patients, and organ transplant candidates.

TGF-β-mediated crosstalk between TIGIT+ Tregs and CD226+CD8+ T cells in the progression and remission of type 1 diabetes

Type 1 diabetes (T1D) is a chronic autoimmune condition characterized by hyperglycemia resulting from the destruction of insulin-producing β-cells that is traditionally deemed irreversible, but partial remission (PR) with temporary reversal of hyperglycemia is sometimes observed. Here we use single-cell RNA sequencing to delineate the immune cell landscape across patients in different T1D stages. Together with cohort validation and functional assays, we observe dynamic changes in TIGIT+CCR7− Tregs and CD226+CCR7−CD8+ cytotoxic T cells during the peri-remission phase. Machine learning algorithms further identify TIGIT+CCR7− Tregs and CD226+CD8+ T cells as biomarkers for β-cell function decline in a predictive model, while cell communication analysis and in vitro assays suggest that TIGIT+CCR7− Tregs may inhibit CD226+CCR7−CD8+ T cells via TGF-β signaling. Lastly, in both cyclophosphamide-induced and streptozotocin (STZ)-induced mouse diabetes models, CD226 inhibition postpones insulitis onset and reduces hyperglycemia severity. Our results thus identify two interrelated immune cell subsets that may serve as biomarkers for monitoring disease progression and targets for therapeutic intervention of T1D.

Deep Learning-Enhanced Paper-Based Vertical Flow Assay for High-Sensitivity Troponin Detection Using Nanoparticle Amplification.

Successful integration of point-of-care testing (POCT) into clinical settings requires improved assay sensitivity and precision to match laboratory standards. Here, we show how innovations in amplified biosensing, imaging, and data processing, coupled with deep learning, can help improve POCT. To demonstrate the performance of our approach, we present a rapid and cost-effective paper-based high-sensitivity vertical flow assay (hs-VFA) for quantitative measurement of cardiac troponin I (cTnI), a biomarker widely used for measuring acute cardiac damage and assessing cardiovascular risk. The hs-VFA includes a colorimetric paper-based sensor, a portable reader with time-lapse imaging, and computational algorithms for digital assay validation and outlier detection. Operating at the level of a rapid at-home test, the hs-VFA enabled the accurate quantification of cTnI using 50 μL of serum within 15 min per test and achieved a detection limit of 0.2 pg/mL, enabled by gold ion amplification chemistry and time-lapse imaging. It also achieved high precision with a coefficient of variation of <7% and a very large dynamic range, covering cTnI concentrations over 6 orders of magnitude, up to 100 ng/mL, satisfying clinical requirements. In blinded testing, this computational hs-VFA platform accurately quantified cTnI levels in patient samples and showed a strong correlation with the ground truth values obtained by a benchtop clinical analyzer. This nanoparticle amplification-based computational hs-VFA platform can democratize access to high-sensitivity point-of-care diagnostics and provide a cost-effective alternative to laboratory-based biomarker testing.

Copy Number Analysis of Whole Exome Sequencing Data

Abstract Background Genetic alterations implicated in constitutional disorders range in size from single nucleotide substitutions to losses and gains of millions of bases. Whole exome sequencing (WES) captures many clinically relevant variants, but an analytic pipeline optimized for detection of small substitutions, insertions, and deletions may miss pathogenic copy number variants (CNVs). Historically, our laboratory performed separate cytogenetic tests to evaluate for larger genomic imbalances. Addition of copy number analysis to our WES pipeline could improve the assay’s diagnostic yield. This study describes part of our strategy to validate copy number analysis of our constitutional whole exome sequencing data. Methods We identified 18 CNV specimens that underwent both chromosomal microarray analysis (CMA) and WES between 2017 and 2023. Variants included 13 deletions (size range 14Kb-2Mb) and 5 duplications (size range 325Kb-10Mb). The standard method of constitutional copy number analysis in our clinical laboratory is CMA using the Affymetrix Cytoscan HD Array and analysis software. CNVs identified on CMA were reviewed with reference to the Database of Genomic Variants and multiple academic hospital databases. WES was performed on an Illumina sequencing system following capture-based library enrichment with the Integrated DNA Technologies xGen Exome Research, CNV Backbone, Human mtDNA Research, and Human ID Research Panel probes. CNVs identified on WES were called using the Broad Institute’s Genomic Analysis Toolkit (GATK) with a quality score cutoff of 120. Whole exome sequencing data was visualized on the Integrative Genomics Viewer. Results 14 out of 18 CNVs reported on CMA were also detected on WES with a quality score at or above 120 (78%). This includes 10 out of 13 deletions (77%) and 3 out of 5 duplications (60%). For both deletions and duplications, the size ranges of detected variants at or above the cutoff score and undetected variants overlapped. One duplication reported on CMA was detected on WES with a quality score below 120. Visualization of whole exome sequencing data showed variation in probe density that may have affected CNV calling. Conclusions The majority of CNVs reported after CMA (14/18, 78%) were also detected on WES data using a GATK quality score cutoff of 120. The data does not suggest an association between CNV size and detection by WES, although interpretation is limited by the small number of cases. Data visualization may be required to identify some CNVs. Future steps to characterize the limitations of copy number analysis of WES data include assessments of variance in probe density and read depth across the exome. Copy number analysis of WES data from cases with normal CMA results will also be performed for assay validation.

Characterization of the Biological Variability of the Angiome Biomarkers over Time in Healthy Subjects.

Biomarker analyses are an integral part of cancer research. Despite the intense efforts to identify and characterize biomarkers in cancer patients, little is known regarding the natural variation of biomarkers in healthy populations. Here we conducted a clinical study to evaluate the natural variability of biomarkers over time in healthy participants. The angiome multiplex array, a panel of 25 circulating protein biomarkers, was assessed in 28 healthy participants across 8 timepoints over the span of 60 days. We utilized the intraclass correlation coefficient (ICC) to quantify the reliability of the biomarkers. Adjusted ICC values were calculated under the framework of a linear mixed-effects model, taking into consideration age, sex, body mass index (BMI), fasting status, and sampling factors. ICC was calculated to determine the reliability of each biomarker. HGF was the most stable marker (ICC=0.973), while PDGF-BB was the most variable marker (ICC=0.167). In total, ICC analyses revealed that 22 out of 25 measured biomarkers display good (≥0.4) to excellent (>0.75) ICC values. Three markers (PDGF-BB, TGF-1, PDGF-AA) had ICC values <0.4. Greater age was associated with higher IL-6 (p=0.0114). Higher BMI was associated with higher levels of IL-6 (p=0.0003) and VEGF-R3 (p=0.0045). Of the 25 protein biomarkers measured over this short time period, 22 markers were found to have good or excellent ICC values, providing additional validation for this biomarker assay. This data further supports the validation of the angiome biomarker assay and its application as an integrated biomarker in clinical trial testing.

Nonclinical immunogenicity assessment of E3112, a recombinant human hepatocyte growth factor, and its impact on pharmacokinetics in rats and monkeys

E3112 is a recombinant human hepatocyte growth factor currently in development for the treatment of acute liver failure. The assessment of immunogenicity is crucial in the development of biotherapeutics. Consequently, a semi-quantitative assay of anti-drug antibody (ADA) was developed in rat and monkey serum using a ligand binding assay with electrochemiluminescence detection. A standard tiered approach was employed for the immunogenicity assessment, comprising a screening assay and a subsequent confirmatory assay. In the assay validation studies, selectivity, sensitivity, prozone effects, reproducibility, drug tolerance, and stability were evaluated. These assessments were conducted using a surrogate positive control of ADA. The accuracy and precision of the surrogate ADA were within ± 20 % and 20 %, respectively. The stability of ADA was also confirmed under a variety of conditions. The developed assays were successfully employed for the assessment of immunogenicity in rats and monkeys following the administration of a repeated dose of E3112. The administration of E3112 resulted in an increase in ADA levels, with higher levels observed in rats than in monkeys. Systemic exposures of E3112 in rats with higher ADA levels were lower than those with lower ADA, confirming the utility of nonclinical immunogenicity in interpreting pharmacokinetics and its inter-individual variability.

Anti-SARS-CoV-2 IgM Antibody Levels Measured by an In-House ELISA in a Convalescent Latin Population Persist over Time and Exhibit Neutralizing Capacity to Several Variants of Concern.

The coronavirus, SARS-CoV-2, is the causative agent for COVID-19, first registered in Wuhan, China and responsible for more than 6 million deaths worldwide. Currently, RT-PCR is the gold-standard method for diagnosing COVID-19. However, serological tests are needed for screening acute disease diagnosis and screening large populations during the COVID-19 outbreak. Herein, we described the development and validation of an in-house enzyme-linked immunosorbent assay (ELISA) for detecting the levels of anti-spike-1-RBD IgM antibody (CovIgM-ELISA) in well-defined serum/plasma panel for screening and identifying subjects infected with SARS-CoV-2 in a Latin population. In-house CovIgM-ELISA has the format of an indirect ELISA. It was optimized by checkerboard titration using recombinant SARS-CoV-2 spike-S1-RBD protein as an antigen. We found that, compared to the RT-PCR as the standard method, the in-house CovIgM-ELISA displayed sensitivities of 96.15% and 93.22% for samples collected up to 30 or 60 days after infection, respectively, as well as 95.59% specificity with 97.3% accuracy. The agreement kappa value (κ) of our CovIgM-ELISA was substantial when compared to RT-PCR (κ = 0.873) and the anti-SARS-CoV-2 IgM ELISA (InBios Int) (κ = 0.684). The IgM levels detected in the population positively correlated with the neutralizing activity against the wild-type, Alpha and Delta variants of concern, but failed to neutralize Omicron. These data indicate that our in-house CovIgM-ELISA is a compatible performing assay for the detection of SARS-CoV-2 infection.

B-177 Development of a Contrived Fecal Matrix for Clinical Validation of a LC-MS/MS Assay to Quantify Microbiota-derived Metabolites in Stool Specimens

Abstract Background The gut microbiota plays a critical role in health and its disruption can impact clinical outcomes in a variety of circumstances, including graft-versus-host disease following hematopoietic stem cell transplantation, Clostridioides difficile infections, and liver disease. Metagenomic and metabolomic analyses of fecal material is frequently performed in the research setting; however, evaluation of the gut microbiota is not yet incorporated into routine patient care. One barrier to its implementation is the lack of matrix-appropriate materials for validation of clinical assays. While surrogate fecal matrices are being developed for metagenomic sequencing, matrices specific for targeted metabolomics are currently unavailable. In this study, we designed a contrived fecal matrix to validate a new LC-MS/MS method for measurement of microbiota-derived metabolites from stool specimens. Methods Common food products and mucin were individually analyzed by LC-MS/MS for quantities of three microbiota-derived metabolites - butyrate, deoxycholic acid (DCA) and taurocholic acid (TCA). Food items with the lowest endogenous metabolite concentrations were selected to create a contrived fecal matrix with a consistency similar to human stool specimens. Calibrators and quality controls (QC) were then created by spiking butyrate, DCA, and TCA into either the contrived fecal matrix or 50% methanol (MeOH). The internal standards (ISTD) used were d7-butyrate, d4-DCA, and d4-TCA, respectively. Three human stool specimens were selected and spiked at a range of metabolite concentrations to assess matrix effects. All samples, calibrators and controls were homogenized in 80% MeOH extraction solvent containing the ISTDs using beadruptor tubes. The suspension was centrifuged and 25 µL of supernatant was derivatized using 12.5 µL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 12.5 µL 3-nitrophenylhydrazine at 40°C for 30 minutes. For optimal separation of isomers, 2 µL of sample was injected and separated on a CORTECS T3 column (1.6 µm, 2.1 x 100 mm, Waters) at 45°C with a flow rate of 0.350 mL/min and run-time of 20 min using the Exion LC AD (Sciex). Analytes were detected using a Sciex QTRAP 6500 mass spectrometer with an electrospray ionization source. Results A contrived fecal matrix containing a mix of avocado, black beans, gluten-free flour, and cornstarch blended in water and mucin was created. The contrived matrix was confirmed to contain minimal to no endogenous levels of the 3 metabolites of interest: butyrate, DCA, and TCA. Validation of the LC-MS/MS assay using the contrived fecal matrix showed a linear analytical measurement range from 25-2000 µM for butyrate, 1-60 µM for DCA, and 0.5-640 µM for TCA. Intra-assay precision at 7 QC levels for all analytes was &amp;lt;10%. The matrix effects from use of the contrived fecal matrix compared to patient fecal samples were 140-148% for d7-butyrate and 101-112% for d4-DCA across multiple spiked analyte concentrations. Conclusions Our LC-MS/MS method quantifies butyrate, deoxycholic acid, and taurocholic acid, metabolites which serve as indicators of microbiota health in liver disease patients. The contrived fecal matrix provides an additional tool for the development and validation of clinical assays to assess the gut microbiota.