贝克曼库尔特

Abstract Background Plasma phosphorylated tau at threonine 217 (p-Tau217) is a leading blood-based biomarker for Alzheimer’s disease (AD). Robust analytical characterization on high-throughput platforms is essential for research use and clinical translation. Objective To evaluate the analytical performance of an automated plasma p-Tau217 immunoassay and characterize its discrimination of PET-defined amyloid status. Methods We performed analytical validation of the Access Research Use Only (RUO) plasma p-Tau217 immunoassay on the Beckman Coulter DxI 9000 Access Immunoassay Analyzer and evaluated biomarker discrimination of PET-defined amyloid pathology in a subset of the Bio-Hermes-001 cohort spanning the symptomatic cognitive continuum (mild cognitive impairment or mild AD dementia; cognitively unimpaired participants excluded; n = 449). Analytical precision, sensitivity, linearity, specificity, interference, and sample stability were assessed per Clinical and Laboratory Standards Institute guidelines. Discrimination of PET-defined amyloid status was evaluated using receiver operating characteristic curve and indeterminate zone analyses. Results The assay demonstrated high precision (within-laboratory CV ≤7.1%), excellent sensitivity (limit of detection 0.018–0.021 pg/mL), linearity across the analytical measuring range (R² > 0.99), strong epitope specificity (≤1.0% cross-reactivity with other tau phosphoisoforms), and minimal interference from over 60 endogenous and exogenous substances. In 449 research participants plasma p-Tau217 showed strong discrimination between amyloid-positive and amyloid-negative groups (AUC 0.881; 95% CI 0.846–0.915). Application of indeterminate zones systematically improved classification metrics at the cost of fewer definitive classifications. Conclusions These findings support the Access p-Tau217 (RUO) assay as a robust, high-throughput assay for plasma biomarker-based discrimination of PET-defined amyloid pathology in AD applications.

Abstract Introduction Lipid nanoparticles (LNPs) have emerged as nanoscale carriers for therapeutic agents, including mRNA vaccines and gene therapies. Accurate methods for characterizing nucleic acid encapsulation within these particles are essential for LNP development and quality control. This study presents an optimized flow cytometric protocol that uses SYTO 9 Green, a nucleic acid-binding dye, to differentiate and quantify nucleic acid-loaded versus empty LNPs with high sensitivity using the CytoFLEX nano Flow Cytometer. Methods We optimized a SYTO 9 Green staining protocol and applied it to empty and loaded LNP samples (Cytiva-sourced LNP1/LNP2 and UBC-sourced siRNA-LNPs). A sequential gating strategy was used to ensure single-particle detection by excluding debris and doublets. Dye titration experiments (ranging from 25 µM down to 3.125 µM SYTO 9 Green) were performed, along with critical controls, including unstained LNPs and SYTO 9 Green dye-only samples. Results Our results demonstrate that SYTO 9 Green staining enabled clear, concentration-dependent discrimination between loaded and empty LNPs. Loaded LNPs exhibited strong fluorescence signals, with positive staining efficiency peaking at optimal dye concentrations. Staining efficiency varies across concentrations, reflecting a complex interplay between dye-to-target binding, nucleic acid content, and potential quenching effects. In contrast, empty LNPs consistently showed minimal background staining (< 1% positive events). Unstained LNPs and dye-only controls presented negligible positive events, confirming that the observed signals are attributable to nucleic acid targets. Conclusion These findings highlight the combined use of the CytoFLEX nano Flow Cytometer and optimized SYTO 9 Green staining as a sensitive, robust approach for precise, multiparametric characterization of nucleic acid encapsulation in LNPs. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)

Abstract Introduction High-throughput single-cell analysis requires strategies that minimize reagent use while enabling multiplexed detection of intracellular targets. Protein barcoding using spectrally distinct dyes offers a scalable approach to simultaneously analyze multiple samples under identical staining conditions. Here, we describe a workflow employing Pacific Blue and DyLight800 dyes for barcoding fixed and permeabilized cells, enabling multiplexed immunostaining and spectral unmixing on the CytoFLEX mosaic Spectral Detection module. Methods Cells were treated, fixed, and permeabilized prior to barcoding with 16 unique dye combinations of Pacific Blue and DyLight800. Barcoded samples were pooled and stained with antibodies targeting phosphorylated nuclear proteins conjugated to PE and APC. Enhanced green fluorescent protein (eGFP) transfection served as a reporter for transfection efficiency. Data acquisition was performed using the CytoFLEX mosaic Spectral Detection module, followed by computational unmixing and gating to resolve individual barcoded populations. Results Spectral profiles confirmed distinct intensity signatures for each barcoded combination, allowing accurate discrimination of up to 16 samples per well. Integration with plate-based workflows enabled processing of 1,536 samples per plate. Barcoding did not interfere with antibody binding or eGFP detection Conclusion Multiplexed analysis revealed consistent staining patterns across pooled samples, validating the approach for high-throughput intracellular signaling studies. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)

INTRODUCTION:Plasma phosphorylated tau (p-tau), particularly p-tau217, is a highly specific biomarker of Alzheimer's disease (AD) pathology. However, plasma p-tau217 can be elevated in rare non-AD conditions. Brain-derived (BD) p-tau217 may reduce these off-target effects, but its performance against neuropathology has not been evaluated. METHODS:We compared p-tau217, BD p-tau217, their amyloid beta 42 (Aβ42) ratios, BD p-tau217/p-tau217, and BD p-tau217/BD tau in end-of-life plasma from 288 neuropathologically characterized participants using a fully automated immunoassay. Biomarkers were assessed against National Institute on Aging-Alzheimer's Association (NIA-AA) classification, Thal phase, Braak stage, cognitive decline, and tau-PET (positron emission tomography). RESULTS:All markers tracked neuropathological severity, with BD p-tau217 having larger fold-changes than p-tau217 but BD p-tau217/Aβ42 enhancing this further. BD p-tau217/BD tau achieved the highest area under the curve (AUC) for distinguishing Intermediate/High from Not/Low AD neuropathological change (ADNC) (0.89 vs 0.82 for p-tau217). Although BD p-tau217/p-tau217 showed smaller fold-changes, it had the strongest association with continuous tangle burden in AD (R2 = 0.68) and best predicted Clinical Dementia Rating Sum of Boxes (CDR-SB decline) (R2 = 0.26). DISCUSSION:BD p-tau217 and BD-based ratios enhance dynamic range and prognostic performance while maintaining diagnostic accuracy, supporting further clinical evaluation.

The study of bacteriophages (phages) and effects on their microenvironments expanded exponentially within the last decade. While there are multiple described methods for phage quantitation, there is still a need for a rapid, label-free method. To this end, we established a procedure for rapid phage quantitation through novel use of a particle size analyzer with Polarization Intensity Differential (PIDS) technology and eliminated the need for labels or knowledge of bacterial host. We validated the procedure and analysis method, termed PhageFOTO (Fast Optical Tallying of Objects) using several physiologically different phages ranging from ~6 nm capsid width (Inoviridae) to ~90 nm capsid width (Caudoviricetes). PhageFOTO demonstrated 89 ± 4.3%, 98 ± 1.7%, and 94 ± 2.7% accuracy for quantitating PhiX, M13, and T4 phages/mL respectively as compared to the gold standard plaque assay with limit of detection for particle concentration occurring around 107 phages/mL. PhageFOTO proved to be a novel, rapid, label free method for phage counting that does not rely on knowledge of the bacterial host presenting unique capability for quantitation of phage samples.