Validation Of Assay Research Articles

Harmonization of Vaccine Ligand Binding Assays Validation.

The urgency and importance of organizing a global effort to harmonize clinical assay validation specific to the vaccine industry was identified during the drafting of the 2020 White Paper in Bioanalysis due to the lack of clarity and regulatory guidance/guidelines in vaccine immunoassay validation. Indeed, the Workshop on Recent Issues in Bioanalysis (WRIB) issues the White Paper in Bioanalysis yearly, which is one of the high-profile articles of the Bioanalysis Journal focused on detailed discussions and recommendations on vaccine assay validation. Since 2017, participation in the WRIB working groups by vaccine assay validation experts and regulators has rapidly increased due to its unique format where industry leaders and regulators can meet and exchange ideas on topics of interest to both groups. In early 2021, Vaccine manufacturers approached WRIB for sponsoring/supporting the authorship and publication of an overarching vaccine assay validation document based on the 2017-2020 discussions and consensus starting from immunogenicity assays first and followed by future papers on molecular and cell-based assay validation. Using industry and WRIB vaccine network, a vaccine immunogenicity assay validation working group was assembled consisting of 16 companies. The work on the first white paper started officially in April 2021 focusing on Vaccine LBA Validation (Part 1), and the drafting of Vaccine LBA Development (Part 2) and Vaccine LBA Monitoring & Transfer (Part 3) are presently ongoing and expected to be published shortly after this paper. Moreover, recommendations on Vaccine Cell-Based Assays Validation (ELISpot and Flow cytometry) and Vaccine Molecular Assays Validation (PCR, NGS, NanoString) are also on the WRIB publications agenda and the drafting is planned to start in mid-2024. For too long, vaccine scientists have not had a clear validation guidance for clinical vaccine immunogenicity assays. We hope that this common effort will help close this regulatory gap.

Abstract A012: Analytical validation of a cfDNA-based fragmentomic profiling assay for the detection and quantification of 14 HPV types in cervical cancer

Abstract Purpose: Human papillomavirus (HPV) is the primary cause of cervical cancer, one of the most common cancers affecting women globally. Despite advances in prevention and screening, there remains a critical need for reliable, non-invasive assays to detect and monitor HPV-driven cervical cancers. Liquid biopsy offers a promising solution to meet this need. Here, we report the analytical validation of a plasma-based fragmentomic profiling assay designed to detect and characterize circulating tumor tissue modified viral (TTMV)-HPV DNA from 14 HPV types associated with cervical cancer. Methods: A high-complexity, laboratory-developed test was developed that employs 68 primers and 34 probes to target 14 HPV types via droplet digital PCR (ddPCR). A quantitative algorithm was applied to integrate fragment counts to generate a TTMV-HPV DNA Score, reflective of the quantity of circulating tumor-derived HPV DNA. Analytical validation was performed by determining the limit of blank (LOB), limit of quantitation (LOQ), and limit of detection (LOD) for each HPV type. To measure the limits of assay performance, engineered samples were used for reference interval studies. Synthesized target-specific DNA regions of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68 were diluted to varying concentrations to perform assay validation. Results: The assay demonstrated excellent specificity, with LOBs ranging from 0 to 0.07 copies/µL across the 14 HPV types. LODs ranged from 0.20 to 1.02 copies/µL, indicating a high level of sensitivity. The assay also showed high accuracy and precision, with coefficients of variation (CV) below 20% for all HPV types across both intra- and inter-assay precision studies. Regression analysis confirmed strong linearity (R2 > 0.99) across a wide range of analyte concentrations. Conclusion: These results demonstrate that TTMV-HPV DNA assay accurately and reproducibly detects circulating HPV DNA for 14 HPV types commonly associated with cervical cancer, offering a valuable tool for both diagnosis and ongoing surveillance in clinical settings. Future studies will present the clinical validation in order to establish the prognostic utility of the assay in predicting the likelihood of HPV-driven cervical cancer in patients. Citation Format: Sunil Kumar, Joshua Hutcheson, David Conway, Michael Horan, Syandan Chakraborty, Chloe Wiseman, Alicia Gunning, Evgeny Skyrypkin, Cassin Williams, Catherine Del Vecchio Fitz, Piyush B Gupta. Analytical validation of a cfDNA-based fragmentomic profiling assay for the detection and quantification of 14 HPV types in cervical cancer [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A012.

Abstract B049: Validation of the PGDx elio™ plasma complete for comprehensive genomic profiling of solid tumors through liquid biopsy with Epic Sciences

Abstract Introduction: To help guide treatment decisions and enable clinical trial matching for cancer patients, tissue-based comprehensive genomic profiling (CGP) is an essential precision oncology tool. Liquid biopsy may be used as a complementary approach to assess tumor-specific DNA alterations circulating in the blood when tissue is limited or unavailable. PGDx’s elio™ plasma complete is a next-generation-sequencing (NGS), cell-free DNA (cfDNA) CGP assay that identifies clinically significant variants from the plasma of patients with advanced and metastatic solid cancers. Methods: The PGDx elio plasma complete is a hybrid-capture, DNA-based kitted solution, optimized for 25 ng of cfDNA input with a targeted panel covering coding exons for 521 genes, and is designed to detect single nucleotide variants (SNVs), small insertions and deletions (indels), copy number amplifications (CNAs), and translocations. It also evaluates blood MSI and tumor mutational burden (bTMB) to guide immunotherapy selection. This study presents the validation of the PGDx elio plasma complete assay to enable the processing of patient blood samples in Epic Sciences' CAP/CLIA-certified laboratory. In the validated configuration, patient plasma will be received and isolated at Epic Sciences and subsequently shipped to PGDx/Labcorp Oncology for cfDNA extraction, library preparation, sequencing, and bioinformatics analysis. Finally, clinical report generation will be carried out by Velsera’s CGW software. Analytical validation was assessed using 105 replicates of SeraCare® (n=21) and Horizon (n=3) reference materials to establish analytical sensitivity, accuracy, and reproducibility and repeatability. Specificity was assessed using 20 non-cancerous plasma samples that were also tested orthogonally. For clinical validation a cohort of >150 cancer patient plasma samples were evaluated, including lung, breast, colorectal, head and neck, pancreatic, and prostate cancers, and compared to Illumina’s TSO500 ctDNA or Labcorp Plasma Focus results. Results: Analytical sensitivity and specificity were evaluated for SNVs, indels, CNAs, fusions, MSI and bTMB. Across all variant types, sensitivity was ≥90%, and specificity was ≥95% at LoDs. The reproducibility and repeatability study resulted in ≥95% agreement for all variant types. Analytical sensitivity was calculated using vendor-verified variants in each control. SeraCare® controls and Horizon reference materials were used to assess variant concordance with an aggregate analytical performance across all control samples with ≥95% PPA and NPA for all variant types. Conclusions: The validation of the PGDx elio plasma complete liquid biopsy assay as part of the Epic Sciences' CAP/CLIA diagnostic workflow for cfDNA-based NGS demonstrates that the liquid biopsy approach is highly sensitive, specific, accurate, reproducible, and robust for comprehensive genomic profiling to complement tissue- based testing and inform clinical decision making. Citation Format: Cynthia Maddox, Kenneth C Valkenburg, Cesar Nalvarte, Amanda Harvey, Amy Greer, Ellen L Verner, Renee L Sears, Martin Blankford, Eric A Severson, Taylor J Jensen, Marcia Eisenberg, Shakti Ramkissoon. Validation of the PGDx elio™ plasma complete for comprehensive genomic profiling of solid tumors through liquid biopsy with Epic Sciences [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B049.

Abstract B066: Analytical validation of a multiplexed CTC-mRNA assay for androgen receptor pathway inhibitors

Abstract Introduction: Androgen receptor pathway inhibitors (ARPIs) have been shown to improve survival for men with metastatic prostate cancer, however, the use of 2nd line ARPIs has limited efficacy. There is a critical need to develop biomarker-driven treatment strategies for this clinical need. We have previously reported on a novel circulating tumor cell (CTC) multiplexed mRNA biomarker that has prognostic and predictive capability in clinical utility trials to detect AR- driven resistance and Neuroendocrine Prostate Cancer (NEPC). However, before these biomarkers can be tested in prospective clinical validation trials and clinical care, they must undergo thorough analytical validation. We report a novel approach to the analytical validation of rare cell assays for gene expression analysis and the testing of this CTC mRNA assay. Methods: Analytical validation for assays with single-cell level detection is particularly challenging in that results may be categorical as well as quantitative: sometimes measured at higher or lower levels, and other times resulting in no detectable signal, precluding the use of standard measures of precision. The inter-metastatic cellular heterogeneity composition of a liquid biopsy further complicates efforts to demonstrate clinical-grade precision at the single-cell level. To address this, we applied logistic regression to replicates of patient samples, cell lines and unique DNA and mRNA oligonucleotides to define the minimum amount of analyte to achieve high precision for the underlying PCR and exclusion-based sample preparation (ESP) mRNA extraction technologies. Results: We demonstrated clinical-grade precision at the single-cell level, comparable to the sensitivity previously demonstrated for qPCR, and analyte quantities of 15 total transcripts. Equivalent analyte yield was obtained from the extraction of single-cell quantities of cell line cells with or without a maximum of 10,000 background cells, demonstrating robustness to a range of sample purities. Extractions from 10 healthy donors demonstrate analytical specificity of the assay, with a synthetic mRNA spike-in control used to confirm successful extractions. Replicates of cell line extractions at different concentrations were compared to 44 replicate patient samples to delineate technical precision from biological heterogeneity. Discussion: The automated CTC-mRNA extraction technology demonstrates high precision equivalent to PCR with single-cell sensitivity. We can further reproducibly detect as low as 15 transcripts. We have achieved a semi-qualitative assay read-out that maximizes the analytical sensitivity of the assay, allowing the clinical reporting of multiplexed results for clinical utility in prostate cancer. This validation approach also serves as a model for clinical implementation of other single-cell liquid biopsy assays. Currently, this assay is being prospectively evaluated in the ARCTIC trial for patients with prostate cancer treated with an ARPI (NCT06141993). Citation Format: Jennifer L Schehr, Rory M Bade, Anupama Singh, Jacob C Caceres, Nasya A Ramli, Matthew C Mannino, Diego Eyzaguirre, Alyssa M Hintz, Manushi N Vatani, Serena K Wolfe, Kayo Miyazaki, Elizabeth Haas, Joshua Faulkes, Dana E Rathkopf, Susan Halabi, Andrew J Armstrong, Shuang G Zhao, Joshua M Lang. Analytical validation of a multiplexed CTC-mRNA assay for androgen receptor pathway inhibitors [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B066.

Abstract A015: Validation of a cancer early-detection assay on liquid biopsies using DNA methylation

Abstract There is growing awareness of the benefits of early cancer detection. Early intervention can improve patient outcomes, leading to a reduction in the health care burden associated with cancer, and can also provide critical information for personalized treatments. Liquid biopsies enable quick, easy, non-invasive, and real-time testing that is suitable for screening and increases accessibility, particularly in developing countries. The use of epigenomic techniques, such as DNA methylation profiling, enhances the depth and breadth of screening beyond what standard genetic analysis can offer. Universal Diagnostics (UDx), a respected pioneer in the field of early cancer detection, has partnered with Hologic Diagenode, which brings 20 years of experience in epigenomics, to develop a customized early detection workflow for colorectal cancer (CRC). This analysis relies on DNA methylation biomarkers identified within cell-free DNA (cfDNA) from plasma. The UDx assay provides not only easy screening but also a more comprehensive analysis than that possible with conventional markers. Genome-wide methylation profiles from healthy individuals and cancer patients were used to identify biomarkers. These biomarker candidates were then validated for performance; the results of which are presented in this poster. Citation Format: Ekaterina Gracheva. Validation of a cancer early-detection assay on liquid biopsies using DNA methylation [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A015.

Dual Assay Validation of Rosmarinus officinalis Extract as an Inhibitor of SARS-CoV-2 Spike Protein: Combining Pseudovirus Testing, Yeast Two-Hybrid, and UPLC-Q Exactive Orbitrap-MS Profiling.

This study evaluates the effectiveness of Traditional Chinese Medicine (TCM) extracts in blocking the interaction between the SARS-CoV-2 Spike protein and human ACE2 receptor, utilizing a dual-method approach to explore the antiviral potential of natural compounds. This work aims to evaluate the capability of TCM extracts in inhibiting the SARS-CoV-2 Spike protein and ACE2 receptor interaction using advanced biochemical assays. A dual-method screening approach was utilized, beginning with a pseudovirus assay to assess the inhibition capabilities of TCM extracts invitro, followed by a split-ubiquitin yeast two-hybrid (Y2H) system to validate interactions in live cells. Active compounds were characterized and quantified using UPLC-Q-Exactive-Orbitrap-MS. Among the 91 TCM extracts tested, Rosmarinus officinalis exhibited the most potent inhibition in both pseudovirus and Y2H assays, significantly reducing viral entry and disrupting the Spike-ACE2 interaction. Comprehensive chemical profiling via UPLC-Q-Exactive-Orbitrap-MS identified 132 compounds, including phenolics, flavonoids, and terpenoids. This research validates the use of TCM extracts in viral inhibition strategies, demonstrating the utility of integrating traditional remedies with modern scientific approaches to discover new therapeutic agents.

Laboratory validation of a clinical metagenomic next-generation sequencing assay for respiratory virus detection and discovery.

Tools for rapid identification of novel and/or emerging viruses are urgently needed for clinical diagnosis of unexplained infections and pandemic preparedness. Here we developed and clinically validated a largely automated metagenomic next-generation sequencing (mNGS) assay for agnostic detection of respiratory viral pathogens from upper respiratory swab and bronchoalveolar lavage samples in <24 h. The mNGS assay achieved mean limits of detection of 543 copies/mL, viral load quantification with 100% linearity, and 93.6% sensitivity, 93.8% specificity, and 93.7% accuracy compared to gold-standard clinical multiplex RT-PCR testing. Performance increased to 97.9% overall predictive agreement after discrepancy testing and clinical adjudication, which was superior to that of RT-PCR (95.0% agreement). To enable discovery of novel, sequence-divergent human viruses with pandemic potential, de novo assembly and translated nucleotide algorithms were incorporated into the automated SURPI+ computational pipeline used by the mNGS assay for pathogen detection. Using in silico analysis, we showed that after removal of all human viral sequences from the reference database, 70 (100%) of 70 representative human viral pathogens could still be identified based on homology to related animal or plant viruses. Our assay, which was granted breakthrough device designation from the US Food and Drug Administration (FDA) in August of 2023, demonstrates the feasibility of routine mNGS testing in clinical and public health laboratories, thus facilitating a robust and rapid response to the next viral pandemic.

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

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. 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. 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. A saliva-based assay for pyrazinamide has been successfully developed and validated using the mobile UV spectrophotometer.

Design, development, and validation of a strand-specific RT-qPCR assay to differentiate replicating versus nonreplicating Rabies virus.

The Rabies virus, a single-strand RNA virus with a negative-sense polarity, is responsible for causing encephalitis and is a zoonotic disease. If not promptly treated after infection, it has a close to 100% fatality rate. Similar to other negative-sense polarity single-strand RNA viruses, the Rabies virus requires the creation of a positive-strand RNA intermediate for replication. One approach to identify this replication activity is to detect the complementary strand of the viral RNA genome in suspected infected cells or tissues. The reported Rabies virus RT-qPCR detection methods are designed to detect total viral load in samples without distinguishing between positive- and negative-strand for RNA viruses. As such, in this study, a sensitive Taqman-based strand-specific RT-qPCR assay has been developed to quantitatively detect both the positive- and negative-strand of the Rabies virus. This method demonstrates good reproducibility across a wide dynamic range and exhibits linearity of 8 logs with a lower limit of detection of 103 copies/μL for the positive-strand and 9 logs with a lower limit of detection of 102 copies/μL for the negative-strand. Notably, it can accurately detect a specific viral RNA strand even in the presence of high levels of the opposite strand, confirming the method’s specificity. In summary, a reliable strand-specific RT-qPCR assay has been developed and validated to differentiate replicating from non-replicating Rabies virus.

Validation of a molecular multiplex assay for the simultaneous detection and differentiation of bluetongue virus and epizootic haemorrhagic disease virus in biological samples.

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are Culicoides-transmitted viruses, circulating in multiple serotypes, that cause two relevant WOAH-listed diseases of ruminants. Following its first identification in Tunisia in 2021, a novel EHDV strain belonging to serotype 8 has been detected in cattle showing BTV-like symptoms in Italy and Andalusia, Spain in 2022, and soon after in Portugal, and France. These are European regions with recurrent circulations of different BTV serotypes. Hence, in this study we describe the validation of a TaqMan RT-qPCR pan-BTV/pan-EHDV assay, based on well-established primers and probes sets, able to simultaneously detect and distinguish between BTV and EHDV. The implemented assay, characterized by high sensitivity and specificity and good reproducibility, can be successfully applied for the rapid and affordable diagnosis needed in the current epidemiological situation, and can be a powerful tool to be employed in surveillance and control strategies with a significant reduction of costs.

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.

Letter to the Editor regarding “Clinical validation of a tissue-agnostic genome-wide methylome enrichment molecular residual disease assay for head and neck malignancies” by G Liu et al

Letter to the Editor regarding “Clinical validation of a tissue-agnostic genome-wide methylome enrichment molecular residual disease assay for head and neck malignancies” by G Liu et al

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.