Flow-based Assay Research Articles

Novel ultrasensitive automated kinetic exclusion assay for measurement of plasma levels of soluble PD-L1, the predictive and prognostic biomarker in cancer patients treated with immune checkpoint inhibitors

Recently, the blood plasma or serum levels of soluble programmed death protein 1 (PD-L1), but not tissue PD-L1 expression level, have been proposed as an effective predictive and prognostic biomarker in patients treated with immune checkpoint inhibitors for different types of cancers. The quantification of soluble PD-L1 in blood will provide a quick evaluation of patients’ immune status; however, the available assays have limitations in their sensitivity, reproducibility, and accuracy for use in clinical settings. To overcome these problems, this study was dedicated to developing an ultrasensitive automated flow-based kinetic exclusion assay (KinExA) for the accurate and precise measurement of soluble PD-L1 in plasma. The assay was developed with the assistance of KinExA™ 3200 biosensor. In this assay, PD-L1 in its calibrator or plasma sample solution was pre-equilibrated with anti-PD-L1 monoclonal antibody. The equilibrated mixture solution was then passed rapidly over PD-L1 protein that has been coated onto polymethylmethacrylate beads consolidated as a microcolumn in the observation cell of the KinExA™ biosensor. The free anti- PD-L1 antibody was bound to the immobilized PD-L1, however, the unbound molecules were removed from the beads microcolumn by flushing the system with phosphate-buffered saline. Fluorescein-labeled secondary antibody was passed rapidly over the beads, and the fluorescence signals were monitored during the flow of the labeled antibody through the beads. The calibration curve was generated by plotting the binding percentages as a function of PD-L1 concentrations in its sample solution. The working range of the assay with very a good correlation coefficient on a 4-parameter equation (r = 0.9992) was 0.5 − 100 pg mL─1. The assay limit of detection and quantitation were 0.15 and 0.5 pg mL─1, respectively. The recovery values of plasma-spiked PD-L1 were in the range of 96.4–104.3 % (±3.7–6.2 %). The precision of the assay was satisfactory; the values of the coefficient of variations did not exceed 6.2 % for both intra- and inter-day precision. The automated analysis by the proposed KinExA facilitates the processing of many specimens in clinical settings. The overall performance of the proposed KinExA is superior to the available assays for plasma levels of soluble PD-L1. The proposed assay is anticipated to have a great value in the measurement of PD-L1 where a more confident result is needed.

Impact of a positive crossmatch on pediatric heart transplant outcomes

BackgroundPediatric heart transplant (HT) candidates experience high waitlist mortality due to a limited donor pool that is constrained in part by anti-HLA sensitization. We evaluated the impact of CDC and Flow donor-specific crossmatch (XM) results on pediatric HT outcomes. MethodsAll pediatric HT between 1999 and 2019 in the OPTN database were included. Donor-specific XM results were sub-categorized based on CDC and Flow results. Primary outcomes were treated rejection in the first year and time to death or allograft loss. Propensity scores were utilized to adjust for differences in baseline characteristics. ResultsA total of 4695 pediatric HT patients with T-cell XM data were included. After propensity score adjustment, a positive T-cell CDC-XM was associated with two times higher odds of treated rejection (OR 2.29 (1.56,3.37)) and shorter time to death/allograft loss (HR 1.50 (1.19,1.88)) compared to a negative Flow-XM. HT recipients who were Flow-XM positive with negative/unknown CDC-XM did not have higher odds of rejection or shorter time to death/allograft loss. An isolated positive B-cell XM was also not associated with worse outcomes. Over the study period XM testing shifted from CDC- to Flow-based assays. ConclusionsA positive donor-specific T-cell CDC-XM was associated with rejection and death/allograft loss following pediatric HT. This association was not observed with a positive T-cell Flow-XM or B-cell XM result alone. The shift away from performing the CDC-XM may result in loss of important prognostic information unless the clinical relevance of quantitative Flow-XM results on heart transplant outcomes is systematically studied.

Point-of-care potentials of lateral flow-based field screening for Mycoplasma bovis infections: a literature review.

Point-of-care (POC) field screening for tools for Mycoplasma bovis (M. bovis) is still lacking due to the requirement for a simple, robust field-applicable test that does not entail specialized laboratory equipment. In accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines, this review identifies the methodologies that were retrieved based on our search strategy that have been reported for the diagnosis of m. bovis infection between 2014 and diagnostics. A search criterion was generated to curate 103 articles, which were reduced in number (to 46), following the screening guidelines of PRISMA. The 43 articles included in the study present 25 different assay methods. The assay methods were grouped as microbiological culture, serological assay, PCR-based assay, LAMP-based assay, NGS-based assay, or lateral flow assay. We, however, focus our discussion on the three lateral flow-based assays relative to others, highlighting the advantages they present above the other techniques and their potential applicability as a POC diagnostic test for M. bovis infections. We therefore call for further research on developing a lateral flow-based screening tool that could revolutionize the diagnosis of M. bovis infection.

Discovery and Development of Cyclic Peptide Proteasome Stimulators.

The proteasome degrades proteins, which is essential for cellular homeostasis. Ubiquitin independent proteolysis degrades highly disordered and misfolded proteins. A decline of proteasomal activity has been associated with multiple neurodegenerative diseases due to the accumulation of misfolded proteins. In this work, cyclic peptide proteasome stimulators (CyPPSs) that enhance the clearance of misfolded proteins were discovered. In the initial screen of predicted natural products (pNPs), several cyclic peptides were found to stimulate the 20S core particle (20S CP). Development of a robust structural activity relationship led to the identification of potent, cell permeable CyPPSs. In vitro assays revealed that CyPPSs stimulate degradation of highly disordered and misfolded proteins without affecting ordered proteins. Furthermore, using a novel flow-based assay for proteasome activity, several CyPPSs were found to stimulate the 20S CP in cellulo. Overall, this work describes the development of CyPPSs as chemical tools capable of stimulating the proteasome and provides strong support for proteasome stimulation as a therapeutic strategy for neurodegenerative diseases.

EXTH-02. A TERT-SPECIFIC TCR-LIKE ANTIBODY CAR T CELL APPROACH FACILITATES KILLING OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) is a difficult form of brain cancer to treat as GBM cells utilize various strategies to evade host immune responses. Prior studies have shown autologous cytotoxic T cells (CTL) modified to express chimeric antigen receptors (CAR) facilitating host CTL responses against cancers. This study investigates the ability of T-cell receptor (TCR)-like antibody clones against TERT to facilitate specific CAR T cell mediated killing of GBM. Lentiviral vectors were created for five TCR-like antibody CAR constructs recognizing an HLA-A*02:01-restricted TERT-derived antigen. Nur77-GFP Jurkat reporter cell lines and 3 healthy donor-derived CTLs transduced with these vectors were used in flow-based and in vitro killing assays to assess specificity and functionality. K562 cells that did, and did not, express HLA-A*02:01 were targets in the flow-based assay. Flow cytometry captured activation of transduced Jurkat cells via GFP and CD69 upregulation. Three luciferase-transduced GBM cell lines were targets in the cytotoxic killing assay and visualized via bioluminescence imaging (BLI). Increased GFP and CD69 expression was seen on Jurkat cells transduced with clones 3A12 and 3H2 only when co-cultured with TERT antigen and A*02 positive K562 suggesting specific recognition of the TERT:HLA complex. Furthermore, healthy donor-derived CTL transduced with 3A12 and 3H2 CAR constructs demonstrated in vitro killing of tumor cells by BLI. Interestingly, the 3H2 construct only showed cytotoxicity in the presence of exogenous TERT antigen. However, when the heavy and light chains of the 3H2 clone were flipped in the lentiviral construct (3H2F), specificity to the TERT:HLA complex was preserved but the CAR T cells now demonstrated cytotoxicity of GBM without needing exogeneous peptide. TCR-like antibody clones 3A12 and 3H2F specifically recognize an HLA-A*02:01-restricted TERT-derived antigen, facilitating effective in vitro cytotoxicity of GBM cells. Ongoing experiments will evaluate the in vivo activity of these TERT-specific CAR constructs and assess potential off-target toxicity.

Stathmin-1 Regulates Hematopoietic Stem Cell Mitochondrial and Cellular Function

Mitochondrial function is important to control HSC quiescence and self-renewal. HSCs are known to have low oxidative phosphorylation (OXPHOS), and low mitochondrial reactive oxygen species, however, studies have shown that HSCs have high mitochondrial membrane potential (MMP), which supports their function (Hinge et al 2020; Mansell et al 2021). Microtubules are important for normal mitochondrial function, acting as the main pathway through which mitochondria transport occurs. Stathmin 1 (Stmn1) is a cytoplasmic phosphoprotein that regulates microtubule dynamics via the promotion of microtubule catastrophe or the sequestration of free alpha/beta-tubulin dimers. It is highly expressed in normal hematopoietic stem cells (HSCs) and neural tissues. Also known as oncoprotein 18 (OP18) or leukemia-associated protein 18 (LAP18), Stmn1 is overexpressed in multiple cancers, including both myeloid and lymphoid leukemias. Despite its robust expression in normal HSCs and its elevated expression in leukemias, little is known about Stmn1's role in either normal or malignant HSCs. Herein, we found that Stmn1 is a critical regulator of HSC mitochondrial and cellular function. To determine the role of Stmn1 in HSCs, we first assessed HSC numbers and function in Stmn1 -/-mice. We determined HSC and progenitor numbers, as well as peripheral blood lineages, in young adult Stmn1 -/-mice and WT littermates. Compared to WT, Stmn1 -/- mice have mild anemia and thrombocytopenia, however, HSC numbers are similar. Despite normal HSC numbers, Stmn1 -/-HSCs had markedly impaired repopulating activity in competitive transplantation experiments (10% vs. 60% chimerism at 24 weeks p<0.0001). Similar defects in repopulating activity were found in non-competitive transplants and competitive intra-tibial transplants (done to circumvent any potential homing defects). In addition, Stmn1 -/- HSCs formed fewer colonies when plated in methocult, and were not capable of serial replating. Finally, Stmn1 -/- mice had delayed hematopoietic recovery following a single dose of the chemotherapeutic agent 5-fluorouracil (5-FU). We next assessed the cycling and apoptosis of HSCs. Stmn1 -/- HSCs are hyper-quiescent at baseline, although they can be stimulated to cycle in response to cytokine stimulation (1.3% vs. 3.4% S/G 2/M phase at baseline and 3.4% vs. 7% S/G 2/M phase following stimulation). In addition, they have elevated levels of apoptosis compared to WT (18.5% vs. 9.90%). Consistent with its role as a microtubule destabilizer, Stmn1 expression in HSCs coincides with tubulin expression by confocal immunofluorescent microscopy, and Stmn1 -/- HSCs have decreased tubulin compared to WT HSCs. To gain further insight into their impaired function, we next performed RNA-seq on sorted Stmn1 -/- and WT HSCs. Differential pathway analysis showed alterations in genes associated with cellular metabolism, particularly mitochondrial function (OXPHOS pathway) in the Stmn1 -/- HSCs compared to WT. Indeed, measurement of mitochondrial function using the Seahorse analyzer showed that Stmn1 -/- HSCs and progenitors have impaired maximal respiratory capacity compared to WT (251.29 vs 411.84 pmol/min). In addition, Stmn1 -/- HSCs showed reduced mitochondrial membrane potential and increased mtROS as assessed by flow-based assays. Finally, the mitochondria of Stmn1 -/- HSCs showed markedly abnormal cristae morphology by TEM, consistent with their impaired function, and Stmn1 -/- HSCs have decreased mitochondria number compared to WT (medium 12.8 vs 17.3 per 2D slice cell; p<0.0250). As elevated ROS can impair HSC function, we assessed Stmn1 -/- HSCs following treatment with MitoTEMPO, a specific scavenger of mitochondrial superoxide. Treatment of Stmn1 -/- mice with 4mg/Kg of MitoTEMPO or vehicle control for two weeks resulted in improved HSC colony formation and serial replating capacity in Methocult. Together, these data identify Stmn1 as a critical regulator of HSC function and suggest a model whereby Stmn1 promotes normal microtubule dynamics and mitochondrial health in HSCs. Ongoing experiments are aimed at understanding Stmn1's role in regulating mitochondrial quality control processes such as mitophagy and fission in both normal and malignant HSCs, and in elucidating the effects of Stmn1 loss on leukemic cell growth and viability.

Multiple Doses of Cnty-101, an iPSC-Derived Allogeneic CD19 Targeting CAR-NK Product, Are Safe and Result in Tumor Microenvironment Changes Associated with Response: A Case Study

Introduction Despite recent approvals of autologous cell therapy products for relapsed, refractory (R/R) non-Hodgkin lymphoma, there remains a clear unmet need for safe, efficacious, and affordable off-the-shelf products that can deliver durable responses. CNTY-101 is an allogeneic iPSC-derived chimeric antigen receptor NK (CAR-NK) cell product with a CD19-targeting CAR, secreted IL-15 to enhance persistence, EGFR safety switch, and a suite of Alloevasion TM edits (B2M and CIITA knock outs, and HLA-E knock in) intended to reduce allo-rejection in the recipient, thereby allowing for repeated dosing. Here we describe a case-study of the first subject dosed in ELiPSE-1, a first-in-human phase 1, multicenter, open-label study of CNTY-101 in subjects with R/R CD19-Positive B-cell malignancies, which is currently enrolling patients (NCT05336409). Methods A 63-year-old subject with R/R POD 24 (progression of disease within 2 years) follicular lymphoma, treated with 4 prior lines of therapy was enrolled at dose level 1 (DL1), 100 million cells CNTY-101. To date, the patient has received four 28-day cycles of a single infusion of CNTY-101 at DL1 with day 1 of each cycle being approximately 6 weeks apart (Table 1). Cycles 1 and 2 included three days of standard Fludarabine and Cyclophosphamide lymphodepletion (LDC), cycles 3 and 4 were given with no LDC. In cycles 2-4, low dose (3 million units) subcutaneous interleukin-2 (IL-2) was administered on days 1-8 of each cycle. Each cycle of CNTY-101 beyond cycle 1 required FDA approval. The described schedule facilitated evaluation of feasibility, safety, biomarker and efficacy evaluations after each infusion, +/- IL-2, and +/- LDC. Biomarker assessments were performed on blood and serum collected pre- and post- each infusion, and on tumor biopsies collected pre- and post-1 st infusion only. Pharmacokinetic (PK) assessments were performed using ddPCR, serum cytokines were measured using Meso Scale Discovery technology, serum anti-drug-antibodies (ADA) were measured using flow-based assays to detect direct IgG and IgM binding to the cell product or assays specifically measuring ADA targeting the CAR construct. Functional ADA were evaluated using an assay to detect complement-dependent cytotoxicity (CDC) function of bound ADA. Tumor biopsies at baseline and day 8 were interrogated for changes in the immune microenvironment using custom Vectra multiplexed immunofluorescence panels and RNA-ISH methodology. Results All doses of CNTY 101 with and without IL-2 or LDC demonstrated acceptable safety and clinical benefit (i.e. stable disease or better per Lugano 2014 criteria). No concerted changes in inflammatory cytokines and mediators associated with cytokine release syndrome or neurotoxicity were detected. The IL-2 dosing regimen resulted in continuous measurable bioavailability of IL-2 during the period of dosing, including trough periods. PK measurements showed that CNTY-101 cells were detected after each infusion with comparable kinetics, with a limited duration in circulation. Serum assessments after the 1 st three cycles showed that, despite administration of two cycles with and one cycle without LDC, no evidence of functional (CDC-inducing) pre-existing or induced ADA were observed. Importantly, tumor microenvironment analyses demonstrated a dramatic increase in T cells within 8 days of the 1 st CNTY-101 cell infusion. Specific increases in proliferating cytotoxic T cells, intratumoral production of TNFα and IFNγ, and key checkpoint molecules were observed, suggestive of induction of adaptive immune responses within the tumor. These responses were associated with tumor shrinkage and ongoing CR of a duration of 5 months since the first CNTY-101 infusion. Conclusions The presented case study demonstrates the potential and feasibility of safely delivering up to 4 cycles of CNTY-101 +/- IL-2 and +/- LDC, at the 100 million dose level. No measurable induction of CDC-inducing functional ADA was detected after the 1 st 3 cycles. Additionally, tumor studies have provided early insight into the mechanism of action. The treatment was associated with changes in tumor microenvironment within 8 days post-infusion, augmentation of adaptive T cell responses, and tumor shrinkage. Updated data will be presented at the conference.

Gravity-driven and rotation-controlled microfluidic chip for point-of-care nucleic acid detection in the fully closed environment

Point-of-care nucleic acid detection is essential for diagnosis and food safety, especially in resource-limited areas. This study reports a gravity-driven and rotation-controlled (GR) chip-coupled lateral flow-based assay (LFA) for point-of-care nucleic acid detection. The sample solution is added to the inlet of the GR chip and flows into the loop-medicated isothermal amplification (LAMP) chamber by the action of gravity. After the LAMP reaction, the GR chip is manually rotated to allow the amplified solution to flow into the LFA chamber for result readout. The GR chip integrates the LAMP reaction and LFA in a fully closed environment, avoiding the aerosol contamination problem. The GR chip's simple and ingenious structure simplifies the fabrication and reduces its cost, making it possible for point-of-care and even home self-testing.

The senescent secretome drives PLVAP expression in cultured human hepatic endothelial cells to promote monocyte transmigration

Liver sinusoidal endothelial cells (LSEC) undergo significant phenotypic change in chronic liver disease (CLD), and yet the factors that drive this process and the impact on their function as a vascular barrier and gatekeeper for immune cell recruitment are poorly understood. Plasmalemma-vesicle-associated protein (PLVAP) has been characterized as a marker of LSEC in CLD; notably we found that PLVAP upregulation strongly correlated with markers of tissue senescence. Furthermore, exposure of human LSEC to the senescence-associated secretory phenotype (SASP) led to a significant upregulation of PLVAP. Flow-based assays demonstrated that SASP-driven leukocyte recruitment was characterized by paracellular transmigration of monocytes while the majority of lymphocytes migrated transcellularly. Knockdown studies confirmed that PLVAP selectively supported monocyte transmigration mediated through PLVAP's impact on LSEC permeability by regulating phospho-VE-cadherin expression and endothelial gap formation. PLVAP may therefore represent an endothelial target that selectively shapes the senescence-mediated immune microenvironment in liver disease.

The modulation of platelet function by growth hormone in growth hormone deficient Hypopituitary patients

BackgroundGrowth hormone deficiency (GHD) has been implicated in increased cardiovascular and cerebrovascular disease risk seen in hypopituitarism, however the mechanism remains speculative. We hypothesise that platelet abnormalities may play a contributory role. Herein we examined platelet behaviour in GHD hypopituitary patients, pre- and post-growth hormone (GH) replacement.MethodsThis study utilizes a physiological flow-based assay to quantify platelet function in whole blood from patient cohorts under arterial shear. Thirteen GH Naïve hypopituitary adults with GHD and thirteen healthy matched controls were studied. Patients were assessed before and after GH treatment. All other pituitary replacements were optimised before the study. In addition to a full endocrine profile, whole blood was labelled and perfused over immobilised von Willibrand factor (vWF). Seven parameters of dynamic platelet-vWF interactions were recorded using digital image microscopy and analysed by customised platelet tracking software.ResultsWe found a significantly altered profile of platelet-vWF interactions in GHD individuals compared to healthy controls. Specifically, we observed a marked increase in platelets shown to form associations such as tethering, rolling and adherence to immobilized vWF, which were reduced post GH treatment. Speed and distance platelets travelled across vWF was similar between controls and pre-therapy GHD patients, however, this was considerably increased post treatment. This may indicate reduced platelet signaling resulting in less stable adhesion of platelets post GH treatment.ConclusionsTaken together observed differences in platelet behaviour may contribute to an increased risk of thrombosis in GHD which can in part be reversed by GH therapy.

Molecular Diagnosis and Haemato-biochemical Alteration in Canine Parvoviral Infection in Dogs

The present study was undertaken to diagnose and assess the haematological & biochemical alteration in parvoviral gastroenteritis in dogs. Total of 12 dogs suspected for parvovirus infection were diagnosed by using lateral flow-based immunochromatographic assay rapid canine parvovirus (CPV) antigen test kit and polymerase chain reaction (PCR) technique in the faeces of affected dogs. All 12 dogs were found positive by a rapid antigen test kit and VP2 gene of CPV PCR assay using the CPV-2ab primer pair and expected size of 681 bp. The haemato-biochemical study revealed a decrease in Hb, TEC, serum total protein, albumin and globulin, whereas the increase in absolute neutrophil count and BUN in parvovirus-infected dogs, indicated anemia and hypoproteinemia in parvovirus infected dogs.

Single-cell imaging and transcriptomic analyses of firm adhesion between patient-derived cancer and endothelial cells under shear stress

Adhesion between cancer cells and endothelial cells, lining the blood vessels, is a key event during tumour progression and metastasis formation. However, the analysis of its underlying cellular and molecular mechanisms is largely limited by the intrinsic difficulties to study the interactions between circulating cancer cells and endothelial cells in vivo, and in vitro under conditions that mimic the in vivo blood flow. Here, we developed a method to study cell:cell firm adhesion under shear-stress conditions coupled to high-content live-cell imaging, and single-cell RNAseq analysis. As the model system, we used cancer cells freshly isolated from patient-derived xenografts (PDXs) and human primary endothelial cells. Breast cancer is the most common cancer in women worldwide and the leading cause of cancer-related deaths among women. Therefore, we set up protocols for breast cancer PDX tumour dissociation, isolation and purification to obtain freshly isolated PDX-derived human cancer single cell suspension. We then implemented an in vitro assay to study cancer to endothelial cells firm adhesion under shear-stress, using an all–human microfluidic model coupled to time-lapse and live-cell imaging. Finally, we developed a method to successfully retrieve, separate and enrich alive endothelial and cancer cells from the flow-based firm adhesion assay. Most notably, we used retrieved cells for single-cell RNAseq analysis and showed that samples quality, number of cells and transcripts per cell were consistent and optimal for downstream discovery analyses. In conclusion, we developed a workflow method that can provide insights into the mechanisms of cancer adhesion to endothelial cells, and identify new targets for personalized treatments development for the clinic to prevent and/or treat breast cancer metastasis formation.

New Complement Dependent Cytotoxicity Assay and its application to COVID-19 patient sera harboring anti-lymphocyte antibodies.

Abstract Complement dependent cytotoxicity (CDC) is one of the ways IgM and IgG antibodies can mediate the lysis of target cells, being IgM the antibody (Ab) isotype with the highest CDC activity. In a cohort of hospitalized COVID-19 patients we identified that 27% carried IgM anti-lymphocyte Ab in their sera. To quantify the sera potential lytic activity against healthy donor (HD) PBMC we developed a flow-based CDC assay with an internal negative control. The assay allows for normalization of the results and therefore inter-experiment data comparison without the need of using counting beads. In short, after CFSE labelling, 2 million PBMC are incubated with either complete media (CM) or with the different experimental conditions (Ab, sera, plasma), then they are washed thoroughly before adding them to equal numbers of unlabeled PBMC (internal negative control). This mixture of hot CFSE+ and cold CFSE-targets is then incubated with rabbit complement for an hour, stained and flow. A “ratio tube” containing the mixture with the CM incubated CFSE cells, is left without rabbit complement at 4 degrees and used to calculate the initial CFSE+/CFSE− ratio. We calculate the percentage of CDC lysis based on the CFSE+/CFSE− ratio in each tube after normalizing it to the ratio tube. Deploying this assay, we found CDC activity against B and T cells in five and two COVID-19 patients’ sera, respectively, out of 23 patients tested. This improved CDC assay shows high reproducibility and allows for quick, reliable, and easy test of multiple conditions. This work was supported by the Divisions of Intramural Research of NIAID, NIH

Benzenesulfonamide derivatives as Vibrio cholerae carbonic anhydrases inhibitors: a computational-aided insight in the structural rigidity-activity relationships

Vibrio cholerae causes life-threatening infections in low-income countries due to the rise of antibacterial resistance. Innovative pharmacological targets have been investigated and carbonic anhydrases (CAs, EC: 4.2.1.1) encoded by V. cholerae (VchCAs) emerged as a valuable option. Recently, we developed a large library of para- and meta-benzenesulfonamides characterised by moieties with a different flexibility degree as CAs inhibitors. Stopped flow-based enzymatic assays showed strong inhibition of VchαCA for this library, while lower affinity was detected against the other isoforms. In particular, cyclic urea 9c emerged for a nanomolar inhibition of VchαCA (KI = 4.7 nM) and high selectivity with respect to human isoenzymes (SI≥ 90). Computational studies revealed the influence of moiety flexibility on inhibitory activity and isoform selectivity and allowed accurate SARs. However, although VchCAs are involved in the bacterium virulence and not in its survival, we evaluated the antibacterial activity of such compounds, resulting in no direct activity.

Effects of extended transport on cryopreserved allogeneic hematopoietic progenitor cell (HPC) product quality and optimal methods to assess HPC stability.

Since the beginning of the COVID-19 pandemic, cryopreservation of hematopoietic progenitor cell (HPC) products has been increasingly used to ensure allogeneic donor graft availability prior to recipient conditioning for transplantation. However, in addition to variables such as graft transport duration and storage conditions, the cryopreservation process itself may adversely affect graft quality. Furthermore, the optimal methods to assess graft quality have not yet been determined. A retrospective review was performed on all cryopreserved HPCs processed and thawed at our facility from 2007 to 2020, including both those collected onsite and by the National Marrow Donor Program (NMDP). HPC viability studies were also performed on fresh products, retention vials, and corresponding final thawed products by staining for 7-AAD (flow cytometry), AO/PI (Cellometer), and trypan blue (manual microscopy). Comparisons were made using the Mann-Whitney test. For HPC products collected by apheresis (HPC(A)), pre-cryopreservation and post-thaw viabilities, as well as total nucleated cell recoveries were lower for products collected by the NMDP compared to those collected onsite. However, there were no differences seen in CD34+ cell recoveries. Greater variation in viability testing was observed using image-based assays compared to flow-based assays, and on cryo-thawed versus fresh samples. No significant differences were observed between viability measurements obtained on retention vials versus corresponding final thawed product bags. Our studies suggest extended transport may contribute to lower post-thaw viabilities, but without affecting CD34+ cell recoveries. To assess HPC viability prior to thaw, testing of retention vials offers predictive utility, particularly when automated analyzers are used.

Metformin increases natural killer cell functions in head and neck squamous cell carcinoma through CXCL1 inhibition

BackgroundMetformin slows tumor growth and progression in vitro, and in combination with chemoradiotherapy, resulted in high overall survival in patients with head and neck cancer squamous cell carcinoma (HNSCC) in...

To evaluate the feasibility of cadmium/tellurium (Cd/Te) quantum dots for developing N-terminal Natriuretic Peptide (NT-proBNP) in-vitro diagnostics

ABSTRACT Quantum dots have been widely used for biomedical applications like imaging, targeted drug delivery, and in-vitro diagnostics for better sensitivity. In-vitro diagnostic, lateral flow-based assay systems are gaining attention in the field of biomarker analysis mainly due to ease of test and quick availability of results. In the study, the potential of water-soluble carboxylic (-COOH) functionalized photoluminescent Cadmium Telluride Quantum Dots (CdTe) nanoparticles for lateral flow-based detection of N-terminal Natriuretic Peptide (NT-proBNP) biomarker (for heart failure) detection has been evaluated. Monoclonal antibodies were conjugated with COOH functionalized CdTe with EDC-NHS coupling chemistry, and conjugation was confirmed using FTIR. The CdTe nanoparticle exhibited an emission maximum at 715 nm when it is excited with 375 nm. The COOH functionalized CdTe showed an antigen concentration-dependent linearity in the lateral flow applications when the dye was prepared freshly and used. However, a relative reduction in CdTe quantum dot fluorescence intensity with time was observed. Factors such as low stability could be due to the quenching of the fluorescence of CdTe. This limits its commercial viability as an in-vitro diagnostic tool; thus, modifications of the quantum dots are required to have a stable preparation for its commercial potential for quantifications.

P618: SECOND-GENERATION CD19 TARGETING TRI-SPECIFIC KILLER ENGAGER DRIVES ROBUST NK CELL FUNCTION AGAINST B CELL MALIGNANCIES

Background: Chronic Lymphocytic Leukemia (CLL) involves uncontrollable clonal expansion of CD5+/CD19+ B lymphocytes. While patients can sometimes coexist with CLL for years, eventually there is progression to bulky adenopathy and pancytopenia from marrow suppression requiring therapy. In recent years, targeted therapies, including utilization of the inhibitors for B cell receptor (BCR) signaling and for B cell lymphoma 2 (Bcl-2) protein, have been effective treatment options. However, these treatments are associated with limitations such as development of drug resistance and the lower treatment efficacy ratio in high-risk patients. Meanwhile, CAR-T cell immunotherapy failed to produce effective treatment outcomes against CLL, mostly due to defects in the effector T cells leading to product failures, as well as being associated with high levels of off-target cytotoxicity. Aims: Thus, approaches to CLL treatment are necessary and NK cell immunotherapy presents a viable and non-toxic option to this problem. Methods: Our group previously demonstrated the immunotherapeutic potential of utilizing first-generation CD19-targeting 161519 Tri-Specific Killer Engager (TriKE®) to drive natural killer (NK) cell cytotoxicity against B-cell malignancies. We have now produced a novel second-generation CD19 TriKE, ‘CAM161519’, utilizing a humanized camelid anti-CD16 VHH single domain antibody (sdAb) ‘CAM16’, a wild-type IL-15 component, and anti-CD19 tumor antigen scFv all linked via short peptide linkers. The TriKE primarily works by generating a cytolytic bridge between NK cells and CD19 expressing B cell malignancies while also providing an expansion and survival signal to the NK cells. Using flow-based assays and imaging based cytolytic assays we explored the capability of CAM161519 to target a variety of B cell lines (Raji, Daudi, and Nalm6) and primary B cell malignancies from patients with CLL and B cell acute lymphoblastic leukemia (B-ALL). Expanded NK cells were incubated with primary CLL targets from 6 different patients and no treatment (NT), rhIL-15 (30 nM), Rituximab (10 ug/mL), or CAM16159 (30 nM). NK cell degranulation and IFNg production was evaluated after 5 hours of co-culture (Figure). CLL killing was evaluated in a similar co-culture, but with a staining mix containing CD5 and CD19, to identify CLL tumor cells in order to determine the percent CLL targets killed normalized to incubation of CLL cells with NK cells alone (Figure Image). Results: The CAM161519 robustly activated NK cell degranulation (CD107a) and inflammatory cytokine production (IFNg) against B cell lines. The activation was specific as it was strongly reduced against CD19 knockout Nalm6 cells. The TriKE also induced specific NK cell proliferation. IncuCyte imaging based cytolytic assays demonstrated strong killing activity against Raji targets. When evaluating activity against primary targets, CAM161519 induced enhanced NK cell degranulation, cytokine production, and cytotoxicity using flow cytometry-based assays against primary CLL (Figure Image) and B-ALL targets when compared to IL-15 treatment. CAM161519 also rescued functionality of NK cells from CLL patients against their autologous CLL cells. Image:Summary/Conclusion: Taken together, these data are indicative of the potential for clinical translation of the CAM161519 TriKE in the CLL and B-ALL settings alone or in combination with cellular products.

Design of Gold Nanoparticle Vertical Flow Assays for Point-of-Care Testing.

Vertical flow assays (VFAs) or flow-through assays have emerged as an alternate type of paper-based assay due to their faster detection time, larger sample volume capacity, and significantly higher multiplexing capabilities. They have been successfully employed to detect several different targets (polysaccharides, protein, and nucleic acids), although in a limited number of samples (serum, whole blood, plasma) compared to the more commonly known lateral flow assays (LFAs). The operation of a VFA relies mainly on gravity, coupled with capillary action or external force to help the sample flow through layers of stacked pads. With recent developments in this field, multiple layers of pads and signal readers have been optimized for more user-friendly operation, and VFAs have achieved a lower limit of detection for various analytes than the gold-standard methods. Thus, compared to the more widely used LFA, the VFA demonstrates certain advantages and is becoming an increasingly popular platform for obtaining qualitative and quantitative results in low-resource settings. Considering the wide application of gold nanoparticles (GNPs) in VFAs, we will mostly discuss (1) the design of GNP-based VFA along with its associated advantages/disadvantages, (2) fabrication and optimization of GNP-based VFAs for applications, and (3) the future outlook of flow-based assays for point-of-care testing (POCT) diagnostics.

Clinical Performance of a Lateral Flow SARS-CoV-2 Total Antibody Assay.

BackgroundSerological assays for SARS-CoV-2 are important tools for diagnosis in patients with negative RT-PCR testing, pediatric patients with multisystem inflammatory syndrome, and serosurveillance studies. However, lateral flow-based serological assays have previously demonstrated poor analytical and clinical performance, limiting their utility.MethodsWe assessed the ADEXUSDx COVID-19 lateral flow assay for agreement with diagnostic RT-PCR testing using 120 specimens from RT-PCR–positive patients, 77 specimens from symptomatic RT-PCR–negative patients, and 47 specimens obtained prepandemic. Specimens collected <14 days from symptom onset in RT-PCR–positive patients were compared relative to the Abbott SARS-CoV-2 IgG assay.ResultsThe ADEXUSDx COVID-19 Test yielded an overall positive percent agreement (PPA) of 92.5% (95%CI 85.8 to 96.3) and negative percent agreement of 99.2% (95% CI 94.9–100.0) relative to RT-PCR and in prepandemic specimens. Relative to days from symptom onset, the PPA after 13 days was 100% (95% CI 94.2–100); from 7 to 13 days, 89.7 (95% CI 71.5–97.2); and from 0 to 7 days, 53.8 (95% CI 26.1–79.6). The overall agreement between the Abbott and ADEXUSDx assays was 80.9%. Twenty-five specimens were positive by both assays, 9 specimens were negative by both assays, and 8 specimens were positive by only the ADEXUSDx assay.ConclusionsWe demonstrate high PPA and negative percent agreement of the ADEXUSDx COVID-19 assay and diagnostic testing by RT-PCR, with PPA approximately 90% by 7 days following symptom onset. The use of waived testing for antibodies to SARS-CoV-2 with high sensitivity and specificity provide a further tool for combatting the COVID-19 pandemic.