Multiparametric Assay Research Articles

OMIP-109: 45-color full spectrum flow cytometry panel for deep immunophenotyping of the major lineages present in human peripheral blood mononuclear cells with emphasis on the T cell memory compartment.

The need for more in-depth exploration of the human immune system has moved the flow cytometry field forward with advances in instrumentation, reagent development and availability, and user-friendly implementation of data analysis methods. We developed a high-quality human 45-color panel, for comprehensive characterization of major cell lineages present in circulation including T cells, γδ T cells, NKT-like cells, B cells, NK cells, monocytes, basophils, dendritic cells, and ILCs. Assay optimization steps are described in detail to ensure that each marker in the panel was optimally resolved. In addition, we highlight the outstanding discernment of cell activation, exhaustion, memory, and differentiation states of CD4+ and CD8+ T cells using this 45-color panel. The panel enabled an in-depth description of very distinct phenotypes associated with the complexity of the T cell memory response. Furthermore, we present how this panel can be effectively used for cell sorting on instruments with a similar optical layout to achieve the same level of resolution. Functional evaluation of sorted specific rare cell subsets demonstrated significantly different patterns of immunological responses to stimulation, supporting functional and phenotypic differences within the T cell memory subsets. In summary, the combination of full spectrum profiling technology and careful assay design and optimization results in a high resolution multiparametric 45-color assay. This panel offers the opportunity to fully characterize immunological profiles present in peripheral blood in the context of infectious diseases, autoimmunity, neurodegeneration, immunotherapy, and biomarker discovery.

CD56briCD38+ as a novel neutrophil-specific marker in chronic myeloid leukemia

BackgroundChronic myeloid leukemia (CML) is classified as a subtype of myeloproliferative neoplasm, and bone marrow flow cytometry does not reveal any specific immunophenotype. Interestingly, aberrant expression of cluster of differentiation (CD) markers such as CD56 and CD38 has been observed on neutrophils in CML patients. Therefore, we investigated the abnormal expression of CD56 and CD38 in CML neutrophils to explore their diagnostic value in identifying CML through flow cytometry. MethodsWe have developed a multi-parameter flow cytometry assay to identify aberrant immunophenotypes in CML neutrophils among bone marrow nucleated cells. ResultsCompared to healthy donors and patients with a reactive neutrophilia or other hematological malignancies, the percentage of CD56briCD38+ neutrophil subsets in CML patients exhibits a distinctive increase (cut-off value, 2.0%). The specificity and sensitivity associated with the learning cohort (168 samples) were 90.8% and 84.9%, respectively, while in the validation cohort (194 samples), they were 90.7% and 84.7%. The accumulation of CD56briCD38+ neutrophil subsets, which demonstrate are abnormal characteristics, is independent of the neutrophil count, BCR/ABL1 fusions and risk stratification but associated with blast cells immunophenotype. Moreover, this increase disappears in CML patients after treatment with tyrosine kinase inhibitors when the curative effect was satisfactory. ConclusionsWe conclude that an increase in the proportion of CD56briCD38+ neutrophil subsets exceeding 2.0% of total neutrophils serves as a highly sensitive and specific flow cytometry marker, enabling rapid and accurate identification of CML.

High-Throughput Assay for Predicting Diarrhea Risk Using a 2D Human Intestinal Stem Cell-Derived Model.

Gastrointestinal toxicities (GITs) are the most prevalent adverse events (AE) reported in clinical trials, often resulting in dose-limitations that reduce drug efficacy and delay development and treatment optimization. Preclinical animal models do not accurately replicate human GI physiology, leaving few options for early detection of GI side effects prior to human studies. Development of an accurate model that predicts GIT earlier in drug discovery programs would better support successful clinical trial outcomes. Chemotherapeutics, which exhibit high rates of clinical GIT, frequently target mitotic cells. Therefore, we hypothesized that a model utilizing proliferative cell populations derived from human intestinal crypts would predict the occurrence of clinical GITs with high accuracy. Here, we describe the development of a multiparametric assay utilizing the RepliGut® Planar system, an intestinal stem cell-derived platform cultured in an accessible high throughput Transwell™ format. This assay addresses key physiological elements of GIT by assessing cell proliferation (EdU incorporation), cell abundance (DAPI quantification), and barrier function (TEER). Using this approach, we demonstrate that primary proliferative cell populations reproducibly respond to marketed chemotherapeutics at physiologic concentrations. To determine the ability of this model to predict clinical diarrhea risk, we evaluated a set of 30 drugs with known clinical diarrhea incidence in three human donors, comparing results to known plasma drug concentrations. This resulted in highly accurate predictions of diarrhea potential for each endpoint (balanced accuracy of 91% for DAPI, 90% for EdU, 88% for TEER) with minimal variation across human donors. In vitro toxicity screening using primary proliferative cells may enable improved safety evaluations, reducing the risk of AEs in clinical trials and ultimately lead to safer and more effective treatments for patients.

Multiparametric Assays Capture Sex- and Environment-Dependent Modifiers of Behavioral Phenotypes in Autism Mouse Models

BackgroundCurrent phenotyping approaches for murine autism models often focus on one selected behavioral feature, making the translation onto a spectrum of autistic characteristics in humans challenging. Furthermore, sex and environmental factors are rarely considered. Here, we aimed to capture the full spectrum of behavioral manifestations in 3 autism mouse models to develop a “behavioral fingerprint” that takes environmental and sex influences under consideration. MethodsTo this end, we employed a wide range of classical standardized behavioral tests and 2 multiparametric behavioral assays—the Live Mouse Tracker and Motion Sequencing—on male and female Shank2, Tsc1, and Purkinje cell–specific Tsc1 mutant mice raised in standard or enriched environments. Our aim was to integrate our high dimensional data into one single platform to classify differences in all experimental groups along dimensions with maximum discriminative power. ResultsMultiparametric behavioral assays enabled a more accurate classification of experimental groups than classical tests, and dimensionality reduction analysis demonstrated significant additional gains in classification accuracy, highlighting the presence of sex, environmental, and genotype differences in our experimental groups. ConclusionsTogether, our results provide a complete phenotypic description of all tested groups, suggesting that multiparametric assays can capture the entire spectrum of the heterogeneous phenotype in autism mouse models.

Monitoring of Immune Memory by Phenotypical Lymphocyte Subsets Identikit: An Observational Study in a Blood Donors' Cohort.

The cross-talk between the innate and adaptive immune response represents the first defense weapon against the threat of pathogens. Substantial evidence has shown a relationship between immune phenotype lymphocytes and COVID-19 disease severity and/or implication in susceptibility to SARS-CoV-2 infection. Recently, belonging to ABO blood groups has been investigated as a correlation factor to COVID-19 disease. This pilot study investigated lymphocyte typing in a cohort of blood donors to understand the underlying mechanism in SARS-CoV-2 infection linked to the blood group. The study cohort consisted of 20-64-year-old subjects, without comorbidities, from both sexes, who were COVID-19 vaccinated with previous or no infection history. Whole blood samples, collected at A.O.R.N. Sant'Anna and San Sebastiano Hospital (Campania Region), were processed by multiparametric cytofluorimetric assay, to characterize CD4+ helper and CD8+ cytotoxic T cell CD3+ subpopulations. The CD45RA, CCR7, CD27, CD28, CD57 and PD-1 markers were investigated to delineate the peripheral T-cell maturation stages. Differences were detected in ABO blood types in CD3+, CD4+ gated on CD3+, CD8+ and CD8+ gated on CD3+ percentage. These results contribute to identifying a memory cell "identikit" profile in COVID-19 disease, thus leading to a useful tool in precision medicine.

Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro.

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30µM (R2=0.9932±0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.

An automated, high-resolution phenotypic assay for adult Brugia malayi and microfilaria

Brugia malayi are thread-like parasitic worms and one of the etiological agents of Lymphatic filariasis (LF). Existing anthelmintic drugs to treat LF are effective in reducing the larval microfilaria (mf) counts in human bloodstream but are less effective on adult parasites. To test potential drug candidates, we report a multi-parameter phenotypic assay based on tracking the motility of adult B. malayi and mf in vitro. For adult B. malayi, motility is characterized by the centroid velocity, path curvature, angular velocity, eccentricity, extent, and Euler Number. These parameters are evaluated in experiments with three anthelmintic drugs. For B. malayi mf, motility is extracted from the evolving body skeleton to yield positional data and bending angles at 74 key point. We achieved high-fidelity tracking of complex worm postures (self-occlusions, omega turns, body bending, and reversals) while providing a visual representation of pose estimates and behavioral attributes in both space and time scales.

Evaluation of a New Multiparametric Microdot Array-Based Immunoassay Panel for Systemic Autoimmune Disease Diagnosis.

The early reliable detection and quantification of autoantibodies play an important role in autoimmune disease diagnosis and in disease-course monitoring. New technologies, such as the multiplexed determination of autoantibodies, have recently been introduced and are being adopted more frequently. The aim of this study was to evaluate the ability of a new microdot array-based multiparametric assay (ZENIT AMiDot CTD panel, A. Menarini Diagnostics, Firenze, Italy) to correctly classify patients with autoimmune rheumatic diseases (ARDs) and compare it to a fluorescence enzyme immunoassay (FEIA) for the detection of anti-ENAs. The study included 69 consecutive samples from patients with ARDs that were analyzed using two different methods (FEIA and AMiDot) to detect anti-CENP B and six anti-ENA antibodies: anti-Scl-70, anti-SSB/La, anti-Jo-1, anti-U1-RNP, anti-Ro52, and anti-Ro60. The control group sera came from sixty-eight blood donors. Tests were run on the automated slide processor ZENIT FLOW, and then the slides were imaged and analyzed using ZENIT fast. Since the samples were selected for at least one antibody positivity with an ARD diagnosis, we did not calculate clinical sensitivity but only specificity, which was 98.53%, ranging from 90% for anti-SSB/La antibodies to 100% for anti-CENP B ones. Mean agreement among the methods assessed by Cohen's kappa was 0.816 ± 0.240. The assay demonstrated good clinical performance and may be considered a valuable aid in detecting ARD patients, offering an alternative to methods such as FEIA which are largely in use today.

Coarsened Mixtures of Hierarchical Skew Normal Kernels for Flow and Mass Cytometry Analyses

Cytometry is the standard multi-parameter assay for measuring single cell phenotype and functionality. It is commonly used for quantifying the relative frequencies of cell subsets in blood and disaggregated tissues. A typical analysis of cytometry data involves cell classification—that is, the identification of cell subgroups in the sample—and comparisons of the cell subgroups across samples or conditions. While modern experiments often necessitate the collection and processing of samples in multiple batches, analysis of cytometry data across batches is challenging because differences across samples may occur due to either true biological variation or technical reasons such as antibody lot effects or instrument optics across batches. Thus a critical step in comparative analyses of multi-sample cytometry data—yet missing in existing automated methods for analyzing such data—is cross-sample calibration, whose goal is to align corresponding cell subsets across multiple samples in the presence of technical variations, so that biological variations can be meaningfully compared. We introduce a Bayesian nonparametric hierarchical modeling approach for accomplishing both calibration and cell classification simultaneously in a unified probabilistic manner. Three important features of our method make it particularly effective for analyzing multi-sample cytometry data: a nonparametric mixture avoids prespecifying the number of cell clusters; a hierarchical skew normal kernel that allows flexibility in the shapes of the cell subsets and cross-sample variation in their locations; and finally the “coarsening” strategy makes inference robust to departures from the model not captured by the skew normal kernels. We demonstrate the merits of our approach in simulated examples and carry out a case study in the analysis of a multi-sample cytometry data set. We provide an R package for our method.

Dynamic bTMB combined with residual ctDNA improves survival prediction in locally advanced NSCLC patients with chemoradiotherapy and consolidation immunotherapy

BackgroundLiquid biopsy-based biomarkers, including circulating tumor DNA (ctDNA) and blood tumor mutational burden (bTMB), are recognized as promising predictors of prognoses and responses to immune checkpoint inhibitors (ICIs), despite insufficient sensitivity of single biomarker detection. This research aims to determine whether the combinatorial utility of longitudinal ctDNA with bTMB analysis could improve the prognostic and predictive effects. MethodsThis prospective two-center cohort trial, consisting of discovery and validation datasets, enrolled unresectable locally advanced non-small-cell lung cancer (LA-NSCLC) patients and assigned them to chemoradiotherapy (CRT) or CRT + consolidation ICI cohorts from 2018 to 2022. Blood specimens were collected pretreatment, 4 weeks post-CRT, and at progression to assess bTMB and ctDNA using 486-gene next-generation sequencing. Dynamic ∆bTMB was calculated as post-CRT bTMB minus baseline bTMB levels. Decision curve analyses were performed to calculate Concordance index (C-index). ResultsOne hundred twenty-eight patients were enrolled. In the discovery dataset (n = 73), patients treated with CRT and consolidation ICI had significantly longer overall survival (OS; median not reached [NR] vs 20.2 months; P < 0.001) and progression-free survival (PFS; median 25.2 vs 11.4 months; P = 0.011) than those without ICI. Longitudinal analysis demonstrated a significant decrease in ctDNA abundance post-CRT (P < 0.001) but a relative increase with disease progression. Post-CRT detectable residual ctDNA correlated with significantly shorter OS (median 18.3 months vs NR; P = 0.001) and PFS (median 7.3 vs 25.2 months; P < 0.001). For patients with residual ctDNA, consolidation ICI brought significantly greater OS (median NR vs 14.8 months; P = 0.005) and PFS (median 13.8 vs 6.2 months; P = 0.028) benefit, but no significant difference for patients with ctDNA clearance. Dynamic ∆bTMB was predictive of prognosis. Patients with residual ctDNA and increased ∆bTMB (∆bTMB > 0) had significantly worse OS (median 9.0 vs 23.0 months vs NR; P < 0.001) and PFS (median 3.4 vs 7.3 vs 25.2 months; P < 0.001). The combinatorial model integrating post-CRT ctDNA with ∆bTMB had optimal predictive effects on OS (C-index = 0.723) and PFS (C-index = 0.693), outperforming individual features. In the independent validation set, we confirmed residual ctDNA predicted poorer PFS (median 50.8 vs 14.3 months; P = 0.026) but identified more consolidation ICI benefit (median NR vs 8.3 months; P = 0.039). The combined model exhibited a stable predictive advantage (C-index = 0.742 for PFS). ConclusionsThe multiparameter assay integrating qualitative residual ctDNA testing with quantitative ∆bTMB dynamics improves patient prognostic risk stratification and efficacy predictions, allowing for personalized consolidation therapy for LA-NSCLC.

Abstract 6409: A multiparametric flow cytometry assay to monitor regeneration of B-cells following anti B-cell therapy in cancer and autoimmune patients

Abstract Purpose Functional dysregulation of B-cells leads to a variety of autoimmune disorders and blood cancers. Multiple B-cell depleting therapies (e.g., BTK inhibitors, antibodies or CAR-Ts) are currently in active clinical trial investigation. Following therapeutic elimination of pathogenic B-cells, it is critical to monitor the productive regeneration of fully functional B-cells as they mediate innate and adaptive immune responses. To this end, we developed a multiparametric flow cytometry assay for exquisite monitoring of early stages of B cell development, maturation, and antibody production to correlate it with treatment outcomes and determine if continuous depletion of B-cells is necessary to maintain clinical benefits for patients. Study Design We designed a high throughput 12-parameter flow cytometry panel incorporating primary differentiation markers namely, CD45, CD3, CD38, CD10, CD27, CD19, CD20, IgD, Lambda &amp; Kappa Light Chains required for identification of different developmental stages of B-cells in peripheral blood using an in vitro diagnostic grade flow cytometry system (BD FACSLyric). The optimized method underwent robust fit-for-purpose analytical validation per CLSI H62 guidelines with emphasis on establishing assay sensitivity, specificity, and reproducibility across experimental days, operators, and instruments. Results We were able to reliably identify different stages of B-cell development, namely, Naïve (CD19+CD20+IgD+CD27-), Unswitched/Marginal (CD19+CD20+IgD+CD27+), Memory (CD19+CD20+IgD-CD27+), Transitional (CD19+CD20+IgD+CD27-CD10+), Antibody Secreting Cells (CD19+CD20-IgD-CD27+CD38hi) as well as clonality (CD19+CD20+Lambda+ or Kappa+). The lower limit of quantitation was determined to be around 60 events, based upon analysis of 90 data points with good reproducibility (CV range: 0-11.7%) across experimental days, operators and instruments demonstrating sensitive detection and robust performance. Conclusion In summary, we have developed and validated a 12-parameter flow cytometry assay focusing on monitoring the regeneration of different subsets of B-cells in various B-cell malignancies (Non-Hodgkin’s Lymphoma, B CLL) and autoimmune disorders (Multiple Sclerosis, Rheumatoid Arthritis, Systemic Lupus Erythematosus). The high reproducibility/robustness of the assay demonstrates its suitability for use in clinical trials as a valuable tool for safety monitoring and disease evaluation enabling development of novel therapeutic agents. Citation Format: Amit Kumar Mehta, Ruby Tandon, Anil Pahuja, Jane Gao, Kevin Nguyen, Naveen Dakappagari, Zeni Alfonso. A multiparametric flow cytometry assay to monitor regeneration of B-cells following anti B-cell therapy in cancer and autoimmune patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6409.

Abstract 6090: Integration of innate and adaptive immune signatures for early detection of cancer

Abstract Up to 50% of cancer patients are diagnosed at a late stage with tumors that are often unresectable, leading to intensive treatments and a preventable loss of life. Whilst multiple assays have been developed to tackle the issue of disease mortality, these approaches have serious limitations. For example, circulating tumor DNA (ctDNA) preferentially detects hard-to-treat tumors and micrometastatic disease, with limited sensitivity and specificity for early-stage malignancy, underscoring an urgent need for novel early detection strategies. Substantial evidence exists to demonstrate that signals related to innate (e.g. pro-inflammatory cytokines) and adaptive (e.g. antibodies and T cell receptors [TCRs]) immune engagement arise early in the development of cancer. The immune system therefore offers potential as an exquisitely sensitive and highly specific intrinsic early detection system for cancer. Here we propose the development of a pan-cancer novel early detection assay, using peripheral blood to measure three key cancer-specific components of the anti-tumor immune response: i) T cell receptor (TCR) sequences, ii) antibody signatures, iii) cytokine markers. Our preliminary data demonstrates that an age and smoking-matched cohort of lung cancer patients can be distinguished from healthy donors using TCR sequencing data processed through a published machine learning approach (p&amp;lt;0.01). In support of this, a pilot study on a custom peptide microarray comprised of lung cancer associated antigens and recurrent neoantigens demonstrated that lung cancer patient samples show significantly higher antibody intensities than healthy donors. Thirdly, using data from a prospective cohort study, we have demonstrated that inflammatory markers can be leveraged to predict future cancer diagnosis, and identified key protein markers for future work. These data provide proof-of-principle for the use of these analytes as a tool for early detection. We are actively curating a unique cohort of samples for this work from a prospective study, Nodule Immunophenotyping Biomarker for Lung Cancer Early Diagnosis (NIMBLE) (NCT05432739), which recruits patients with indeterminate lung nodules that may represent early cancers (&amp;gt;280 patients to date). This malignancy has high mortality rates and a significant need for early detection strategies. Through this, we aim to understand the early immunobiological response to cancer and leverage this information to design a novel multi-parametric immunopredictor assay for early detection of cancer. Citation Format: Evelyn Fitzsimons, Alexander Coulton, Hongchang Fu, Marcellus Augustine, Richard Lee, James Reading, Kevin Litchfield. Integration of innate and adaptive immune signatures for early detection of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6090.

Immunoglobulin E-Mediated Food Sensitization in a Moroccan Pediatric Population with Celiac Disease

Introduction: Celiac disease is a chronic autoimmune disorder that occurs following the ingestion of gluten, in genetically predisposed individuals. Patients with celiac disease, especially children, are likely prone to develop allergic reactions to different food allergens. However, the relationship between food allergy and celiac disease remains not elucidated. The aim of this pioneering study was to evaluate the prevalence of allergic food sensitization in children with celiac disease in Morocco. Methods: A total of 57 children with confirmed celiac disease, including 25 males and 32 females with a mean age of 8.6 ± 4.4 years, underwent a food allergen-specific immunoglobulin E (IgE) screening. This screening was conducted using a multiparametric immunodot assay (Euroline Food “Maghreb,” Euroimmun). Statistical analysis was performed using R software. Results: Among the 57 cases tested, the overall rate of IgE-mediated sensitization to food allergens was found to be 48% (27/57), dominated by chicken, with 51.9% (14/27), followed by almond, 40.7% (11/27), sesame, 40.7% (11/27), potato 33.3% (9/27), and apple 18.5% (5/27). Of the s-IgE positive cases, 74% were sensitized at least to one allergen, 37% (10/27) were sensitized to both chicken and almond allergens. A significant correlation was observed between almond, sesame, chicken, and potato. Conclusion: The current study highlighted a high prevalence of food allergen sensitization in children with celiac disease. This underlines the potential benefit in screening for food allergy in celiac patients.

Multiparametric Analysis of Apoptosis by Flow Cytometry.

Flow cytometry remains the most widely used method for detecting and quantifying apoptosis and related forms of cell death in mammalian cells. The multiparametric nature of flow cytometry allows multiple apoptotic characteristics to be labeled and analyzed in a single sample, making it a powerful tool for analyzing the complex progression of apoptotic death. This chapter provides methods for combining assays for single apoptotic characteristics like caspase activation, annexin V binding, and cell membrane permeability into multiparametric assays that provide deeper insights into the cell death process. This approach to analyzing multiple apoptotic characteristics simultaneously yields far more information than single-parameter assays. While more informative than single-parameter assays, these multicolor methods can still be analyzed on relatively simple flow cytometers, making them widely accessible.

Evaluation of Antimicrobial Activity and Cytotoxicity of an Epoxy Resin-Based Endodontic Sealer Containing Nanoparticles Amorphous Calcium Phosphate

Background. The main cause of posttreatment disease in endodontics is the remaining of microorganisms within the root canal walls after endodontic therapy. Therefore, it is essential to use root canal sealers with potent antibacterial properties. These sealers play a vital role in eradicating any remaining microorganisms and preventing recontamination, especially in situations where there might be microleakage. Aim. The objective of this research was to examine the antimicrobial properties of epoxy root canal sealers containing nanoparticle amorphous calcium phosphate (NACP) against planktonic Enterococcus faecalis in a controlled laboratory environment. Furthermore, the study aimed to assess the potential cytotoxic effects of these sealers. Method. In order to determine the antimicrobial activity, the epoxy resin sealer (AH Plus, Dentsply, Germany) was supplemented with NACP from Sigma–Aldrich, at a concentration of 3wt.%, as per the previous flowability tests. The agar well diffusion assay method was employed to evaluate the antibacterial efficacy. For this, cultured plates (n = 8) were prepared, with each plate containing three wells: one with AH Plus, one with AH Plus + NACP, and one with NACP alone. Subsequently, the plates were sited at 37°C in an incubator and allowed to incubate overnight. The width of the inhibition zones was then analyzed and recorded by the SPSS statistical software package (Version 20.0 for Windows, SPSS, Chicago, IL, USA). The cytotoxicity of the NACP incorporated AH Plus and AH Plus sealers were tested indirectly by MTT assay and directly by the multiparametric high content screening toxicology assay using fibroblast-like cells as cell lines. Results. AH Plus + NACP showed a higher antimicrobial activity than AH Plus with significant difference ( P &lt; 0.0001 ). Both materials showed nonsignificant difference compared to negative control, which indicated lower cytotoxicities. For AH Plus, P = 0.3599 , 0.5959 , and 0.4071 , with time intervals 24, 72, and 168 hr, respectively. For AH Plus + NACP, P = 0.4386 , 0.6182 , and 0.2080 , with 24, 72, and 168 hr, respectively. Conclusions. NACP incorporation with epoxy resin sealer AH PLUS had a higher antimicrobial activity with lower cytotoxic effect indicating their potential therapeutic values.

Ixazomib-Based Consolidation and Maintenance Prolongs Progression-Free Survival after Salvage Autologous Stem Cell Transplantation (sASCT): Results from Interim Analysis of UK-MRA Myeloma XII (ACCoRD)

Introduction: sASCT in multiple myeloma (MM) induces superior durability of response over non-transplant consolidation 1. Currently there is no prospective evidence for post-sASCT consolidation and maintenance. This is important given that most patients relapsing after first ASCT (ASCT1) are lenalidomide refractory (Len-Ref) following maintenance. The role of a proteasome inhibitor (PI) containing post-sASCT consolidation and maintenance strategy is explored in UK-MRA Myeloma XII (ACCoRD) trial using the second-generation oral PI, ixazomib. The results of a planned interim analysis (IA) of efficacy and safety are presented here. Patients and Methods: ACCoRD enrolled patients who relapsed, requiring treatment more than 12m after ASCT1, delivering an oral PI/IMID re-induction regimen (ixazomib, thalidomide and dexamethasone; ITD) prior to randomization between sASCT conditioned with high-dose melphalan (HDM) vs. ixazomib-augmented ( iMel). The second randomization between observation (standard of care in sASCT; OBS) and post-transplant ITD consolidation and ixazomib maintenance (CON/MAINT), was conducted at D100 post-sASCT. The primary endpoint was progression-free survival (PFS). Responses were assessed in accordance with IMWG criteria with MRD defined by the limit of the multi-parameter flow assay (&amp;lt;10 -5). All patients underwent genetic risk categorization by a central laboratory with high-risk (HiR) classified as presence of t(4;14), t(14;16), t(14;20), del(17p) or gain(1q) and ultra-high risk (UHiR) the presence of more than one lesion. Pre-planned subgroup analysis included: genetic risk status (SR/HiR/UHiR) and previous treatment received (PI exposure/Len-Ref). Results: ACCoRD recruited between March 2017 and June 2022, with database lock for IA in May 2023. 496 patients were enrolled and 206 patients randomized in this comparison (103 CON/MAINT, 103 OBS). Median age was 62.5y (range 34, 78) with 35.7% patients &amp;gt;65y. The median observed TTP from ASCT1 was 32m (range 2, 212), with 12.5% patients relapsing after lenalidomide maintenance (Len-Ref). The proportion of patients with an ASCT1 TTP &amp;lt;18m, 18-24m and &amp;gt;24m was 13.5%, 15.1% and 71.5%, respectively. Of those with complete genetic results at trial entry, 58.3% had standard risk, 31.1% had HiR and 10.7% had UHiR disease. 61.5% of patients were PI-exposed. The median time from sASCT to CON/MAINT was 4.3m (3.4, 6.7). The ORR following sASCT was 83.3%, with ³VGPR in 50.5% and MRD negative in 19.5%. After median follow-up of 27.3m (IQR 13.1, 38.4), 139 patients had progressed or died (CON/MAINT 62, OBS 77). ITT analysis showed CON/MAINT was associated with longer PFS than OBS (HR 0.54, 95% CI 0.38, 0.76, median PFS CON/MAINT 21m vs. OBS 13m, p=0.0004, Figure 1A). Subgroup analyses (Figure 1B) suggested that there were similar benefits for PFS in terms of baseline characteristics: age, sex, ISS and performance status. There was similar benefit for patients by previous treatment received (PI/Len). However, patients with the shortest remission after ASCT1 achieved the greatest benefit with CON/MAINT (p=0.023). Patients achieving poor response (&amp;lt;VGPR) after ITD re-induction benefitted most from CON/MAINT (P=0.008). 45 patients had a PFS2 event (18 CON/MAINT, 27 OBS). CON/MAINT showed a non-significant improvement with longer PFS2 than OBS (HR 0.56, 95%CI 0.30, 1.01, median PFS2 CON/MAINT NR vs. OBS 44m, p=0.055). 25 patients have died (9 CON/MAINT, 16 OBS). Similarly, CON/MAINT showed a non-significant improvement with longer overall survival (OS) than OBS (HR 0.48, 95%CI 0.21, 1.09, 3y OS: CON/MAINT 81.9% vs. OBS 70.4%). Interpretation of PFS2 and OS are limited by short follow-up and will be updated at final analysis. CON/MAINT was well tolerated with no significant additional toxicity. 87.4% of patients on the CON/MAINT arm completed CON treatment. For those who completed CON, MAINT was administered for median 15 cycles (range 1, 57). The most common grade 3+ adverse events in CON/MAINT were upper respiratory infection (6.8% of patients) thrombocytopenia (5.8%), lung infections (2.9%), lymphopenia (2.9%) and neutropenia (2.9%). Conclusion: This IA demonstrates a clear advantage in durability of response when sASCT is followed by consolidation and maintenance which is consistent across subgroups. The impact on PFS2, OS and quality of life remains to be clarified. 1.Cook G, et al The Lancet Oncology 15(8), 874-885.

268. Human Mesenchymal Stem Cells Augment Neutrophil Function to the Human Fungal Pathogen, Candida albicans

Abstract Background Neutrophils are critical innate immune cells that play a crucial role in protection against invading pathogens. Mesenchymal stem cells (MSC) are multipotent progenitors that can differentiate into osteoblasts, chondrocytes, and adipocytes and possess immunomodulatory effects. MSC are the focus of clinical investigation in various comorbidities, including diabetes, cancer, and invasive infections. Methods Human MSC were derived from bone marrow and have been characterized to meet all criteria of MSC (ISCT guideline). Cells were seeded at 100,000 cells per well in fibronectin (50ug/mL)-coated 24-well plate and incubated for 24 hours at 37°C, 5% CO2. The next day, 0.5 mL of whole blood from three healthy donors was added for another 24 hours, after which the neutrophils were isolated, viability measured, and a high throughput multiparametric neutrophil function assay was performed, compared to non-MSC exposed neutrophils (control neutrophils). Functions tested included ROS (dihydrorhodamine 123 (DHR123), phagocytosis of iRFP-expressing Candida albicans, ectodomain shedding (loss of CD62L), and degranulation (increased expression of CD66b). Multiparametric assays were measured on a BD FACS Celesta 4 laser flow cytometer and data was analyzed on FlowJo software. Results Neutrophils co-cultured with MSC had an average viability of 96.3% compared to control neutrophils at 92.15%. Using the multiparametric neutrophil functional assay, neutrophils exposed to MSC ex vivo had increased functional responses to the fungal pathogen Candida albicans. MSC-conditioned neutrophils exhibited a 33.2% increase in degranulation, a 13.4% increase in ROS generation, and 16.7% additional phagocytosis compared to non-MSC neutrophil (control). Compared to fresh neutrophils isolated the day of testing, MSC-conditioned neutrophils had an average increase in phagocytosis, degranulation, shedding, and ROS generation of 36.9%, 62.7%, 50.3%, and 37.8%, respectively. Conclusion MSC increase the functional effector activity of neutrophils in healthy controls, specifically ROS generation, phagocytosis, ectodomain shedding, and degranulation in response to C. albicans. These data raise the possibility of MSC as a novel therapeutic intervention for patients with invasive infections. Disclosures Michael Mansour, MD, PhD, Thermofisher: Grant/Research Support

Autoantibody status according to multiparametric assay accurately estimates connective tissue disease classification and identifies clinically relevant disease clusters

ObjectiveAssessment of circulating autoantibodies represents one of the earliest diagnostic procedures in patients with suspected connective tissue disease (CTD), providing important information for disease diagnosis, identification and prediction of potential...

Comparative analysis between zebrafish and an automated live-cell assay to classify developmental neurotoxicant chemicals

Modern toxicology's throughput has dramatically increased due to alternative models, laboratory automation, and machine learning. This has enabled comparative studies across species and assays to prioritize chemical hazard potential and to understand how different model systems might complement one another. However, such comparative studies of high-throughput data are still in their infancy, with more groundwork needed to firmly establish the approach. Therefore, this study aimed to compare the bioactivity of the NIEHS Division of Translational Toxicology's (DTT) 87-compound developmental neurotoxicant (DNT) library in zebrafish and an in vitro high-throughput cell culture system. The early life-stage zebrafish provided a whole animal approach to developmental toxicity assessment. Chemical hits for abnormalities in embryonic zebrafish morphology, mortality, and behavior (ZBEscreen™) were compared with chemicals classified as high-risk by the Cell Health Index (CHI™), which is an outcome class probability from a machine learning classifier using 12 parameters from the SYSTEMETRIC® Cell Health Screen (CHS). The CHS was developed to assess human toxicity risk using supervised machine learning to classify acute cell stress phenotypes in a human leukemia cell line (HL60 cells) following a 4-h exposure to a chemical of interest. Due to the design of the screen, the zebrafish assays were more exhaustive, yielding 86 total bioactive hits, whereas the SYSTEMETRIC® CHS focusing on acute toxicity identified 20 chemicals as potentially toxic. The zebrafish embryonic and larval photomotor response assays (EPR and LPR, respectively) detected 40 of the 47 chemicals not found by the zebrafish morphological screen and CHS. Collectively, these results illustrate the advantages of using two alternative models in tandem for rapid hazard assessment and chemical prioritization and the effectiveness of CHI™ in identifying toxicity within a single multiparametric assay.

Profiling alveolar macrophage responses to inhaled compounds using in vitro high content image analysis

One of the main hurdles in the development of new inhaled medicines is the frequent observation of foamy macrophage (FM) responses in non-clinical studies in experimental animals, which raises safety concerns and hinders progress into clinical trials. We have investigated the potential of a novel multi-parameter high content image analysis (HCIA) assay as an in vitro safety screening tool to predict drug induced FM.Rat (NR8383) and human U937-derived alveolar macrophages were exposed in vitro to a panel of model compounds with different biological activity, including inhaled bronchodilators, inhaled corticosteroids (ICS), phospholipidosis inducers and proapoptotic agents. An HCIA was utilized to produce drug-induced cell response profiles based on individual cell health, morphology and lipid content parameters.The profiles of both rat and human macrophage cell lines differentiated between cell responses to marketed inhaled drugs and compounds known to induce phospholipidosis and apoptosis. Hierarchical clustering of the aggregated data allowed identification of distinct cell profiles in response to exposure to phospholipidosis and apoptosis inducers. Additionally, in NR8383 cell responses formed two distinct clusters, associated with increased vacuolation with or without lipid accumulation. U937 cells presented a similar trend but appeared less sensitive to drug exposure and presented a narrower range of responses.These results indicate that our multi-parameter HCIA assay is suitable to generate characteristic drug-induced macrophage response profiles, thus enabling differentiation of foamy macrophage phenotypes associated with phospholipidosis and apoptosis. This approach shows great potential as pre-clinical in vitro screening tool for safety assessment of candidate inhaled medicines.