Healthy Human Blood Research Articles

Single-cell atlas of healthy human blood unveils age-related loss of NKG2C+GZMB−CD8+ memory T cells and accumulation of type 2 memory T cells

Extensive, large-scale single-cell profiling of healthy human blood at different ages is one of the critical pending tasks required to establish a framework for the systematic understanding of human aging. Here, using single-cell RNA/T cell receptor (TCR)/BCR-seq with protein feature barcoding, we profiled 317 samples from 166 healthy individuals aged 25-85 years old. From this, we generated a dataset from ∼2 million cells that described 55 subpopulations of blood immune cells. Twelve subpopulations changed with age, including the accumulation of GZMK+CD8+ Tcells and HLA-DR+CD4+ Tcells. In contrast to other Tcell memory subsets, transcriptionally distinct NKG2C+GZMB-CD8+ Tcells counterintuitively decreased with age. Furthermore, we found a concerted age-associated increase in type 2/interleukin (IL)4-expressing memory subpopulations across CD4+ and CD8+ Tcell compartments (CCR4+CD8+ Tcm and Th2 CD4+ Tmem), suggesting a systematic functional shift in immune homeostasis with age. Our work provides novel insights into healthy human aging and a comprehensive annotated resource.

WU-NK-101 (W-NK), a Memory-like (ML) NK Cell, Naturally Overcomes Tumor Microenvironment (TME) Metabolic Challenges, Retaining Anti-Tumor Potency

Cellular engineering revolutionized adoptive cell therapy (ACT) through improved antigen recognition and cellular activation. Cancer cells are metabolically active and extensively consume nutrients from the TME, rendering it nutrient-deprived, acidic, and hypoxic, and resulting in intratumoral metabolic demands which negatively impact ACT antitumor activity. This is central to T cells, however, some of these challenges can be overcome through engineering, an ideal solution would be to have a cellular product that is inherently adept at overcoming these challenges. W-NK is a cytokine-reprogrammed, expanded, cryopreserved, ML-NK cell product derived from healthy human peripheral blood mononuclear cells; it comes with naturally effective potency towards tumor cell killing, and importantly can overcome metabolic challenges present in the TME. We aim at deciphering what metabolic features make W-NK particularly potent in adverse TME. W-NK characterization of metabolic fitness/adaptability was with transcriptomics (sc-RNA-seq; 10X Genomics) flow cytometry, bioenergetics (Seahorse Real-Time Cell Metabolic Analysis; Agilent), and proteomic (Sciex Zeno TOF 7600 tandem-mass spectrometry). The aforementioned assays evaluated the effect of differing media/TME on W-NK: 1) conventional (N) media (pH 7, glucose 11 mM); 2) TME-aligned media (pH 5.9, glucose 6 mM, immune suppressive agents, e.g., PGE2, TGFb1); and 3) ascites derived from patients with malignancy. Media composition was analyzed biochemically using clinical assays (Core Lab Clinical Studies at Washington University, St. Louis, USA), and by immune secretome profiling (Nomic Bio, Quebec, CA). At baseline, W-NK cells have a unique metabolic phenotype, with higher expression of cell surface nutrient transporters, e.g., GLUT1, CD98, ASCT2, MCT1 and PiT1, compared to conventional NK cells (cNK); additionally, W-NK metabolism was consistent with aerobic (“Warburg”) glycolysis, with ~80% of ATP coming from glycolysis vs. ~30% for cNK. In TME/ascites media, W-NK surface nutrient receptor expression adapted to meet metabolic demand. For example, transporters for amino acids, lactate and pyruvate are upregulated in these hypoglycemic medias. This coincided with a 50% shift in ATP manufacturing from glycolysis to mitochondria, suggesting that nutrients bypassed glycolysis to enter tricarboxylic acid cycle and oxidative phosphorylation. Proteomic analyses of W-NK cells cultured with either TME-aligned media or ascites supported this hypothesis, with up-regulation of metabolic pathways (ShinyGO) capturing amino-acid and lipid metabolism, and mitochondrial function. Overall, W-NK retained cytotoxic function in TME and ascites, as well as in native TME-aligned 3D assays from primary surgical tumor samples. Conversely, cNKs did not adapt to TME media, whether in nutrient transporter expression or ATP manufacturing, and demonstrated reduced survival and function, including cytotoxicity. W-NK has enhanced metabolic fitness, flexibility, and plasticity. This enables W-NK to utilize diverse macronutrients, engage different metabolic pathways, and maintain optimal ATP manufacturing. Overall, W-NK inherently survives and maintains function in the TME, a limiting factor for immune cell-based ACT. These data herald the promise of NK cell therapy; a Phase 1 clinical study of W-NK in acute myeloid leukemia is currently open and enrolling patients (NCT# 05470140).

IgG from patients with mild or severe COVID‑19 reduces the frequency and modulates the function of peripheral mucosal-associated invariant T cells in PBMCs from healthy individuals.

Lower levels of peripheral mucosal-associated invariant T (MAIT) cells have been observed in the peripheral blood of patients with severe coronavirus disease 2019 (COVID-19). Following on from previous research into the effect of the IgG repertoire on human lymphocytes, the present study aimed to evaluate if immunoglobulin G (IgG) antibodies obtained from patients with mild or severe COVID-19 contribute to these effects on MAIT cells. Culture experiments were performed using healthy human peripheral blood mononuclear cells (PBMCs) and different repertoires of IgG obtained from patients with COVID-19 as a mild or severe disease and compared with mock, healthy control or therapeutic IgG conditions. The results indicate that the IgG repertoire induced during the development of mild and severe COVID-19 has, per se, the in vitro potential to reduce the frequency of MAIT cells and the production of IFN-γ by the MAIT cell population in PBMCs from healthy individuals. In conclusion, the results of the present study indicate that IgG in patients with severe COVID-19 may participate in the reduction of peripheral MAIT cell frequency and hinder the antiviral activity of these cells.

Reconditioned monocytes are immunomodulatory and regulate inflammatory environment in sepsis

Sepsis is caused by dysregulated immune response to severe infection and hyper inflammation plays a central role in worsening the disease. The immunomodulatory properties of mesenchymal stem cells (MSCs) have been evaluated as a therapeutic candidate for sepsis. Reconditioned monocytes (RM), generated from healthy human peripheral blood mononuclear cells (PBMCs) exhibit both macrophage and MSCs-like properties. RM were administered at different stages of sepsis in a mouse model. It reduced serum levels of IL6, MCP-1, IL-10, improved hypothermia, increased survival, and recovery from 0 to 66% when combined with antibiotics in the mouse model. The reduced human leucocyte antigen DR molecules expression on RM enables their co-culture with PBMCs of sepsis patients which resulted in reduced ROS production, and up-regulated TGF-β while down-regulating IL6, IL8, and IL-10 in-vitro. RM are potentially immunomodulatory, enhance survival in sepsis mouse model and modulate inflammatory behaviour of sepsis patient’s PBMCs.

Modeling of time-dependent elliptic contact parameters for biological nanoparticles with common geometries: Human red blood cell

Extracting mechanical properties of living cells by Atomic Force Microscope (AFM) can cause cell destruction due to uncontrolled forces. In this regard, the reaction of healthy (MCF-7) and cancerous (MCF-10A) breast cells and human Red Blood Cells (RBCs) to contact force was simulated by using different contact theories and applying time-dependent mechanical models. To validate the simulation results, the topography test and nanomanipulation of MCF-7 and MCF-10A using AFM was implemented. Experimental data showed that the average maximum diameter of MCF-7 and MCF-10A is 25.24 and 12.06 μm, respectively. Furthermore, the elastic modulus of the MCF-7 and MCF-10A cells was calculated in the range of 1.23 ± 0.02 kPa, and 1.17 ± 0.02 kPa, respectively. In addition, the contact theories were developed for application in the case of different geometries, and utilized for modeling the contact mechanics of human RBCs. The results showed that Hertz and DMT theories accurately calculate the contact behavior of living cells in small deformations; However, in case of large contact deformations, Tatara and Chang's theories provide more accurate results. Furthermore, the effect of RBC geometry on the contact parameters is negligible due to the significant difference in the dimensions of RBCs in comparison with AFM-tip.

In-Depth Proteomic Map of Innate Lymphoid Cells from Healthy Human Skin and Blood

Innate lymphoid cells (ILCs) are essential players in the skin-associated immune system, nevertheless little is known about their proteomes and proteomic diversity. In this study, we describe about 6,600 proteins constitutively expressed by ILC2s and ILC3s from healthy human skin and blood using state-of-the-art proteomics. Although the vast majority of proteins was expressed by both ILC subsets and in both compartments, the skin ILC2s and ILC3s were more distinct than their counterparts in blood. Only skin ILC3s expressed uniquely detected proteins. Our in-depth proteomic datasetallowed us to define the cluster of differentiation marker profiles of the ILC subsets, explore distribution and abundance of proteins known to have immunological functions, as well as identify subset-specific proteins that have not previously been implicated in ILC biology. Taken together, our analyses substantially expand understanding of the protein expression signatures of ILC subsets. Going forward, these proteomic datasets will serve as valuable resources for future studies of ILC biology.

Design of a Low-Frequency Dielectrophoresis-Based Arc Microfluidic Chip for Multigroup Cell Sorting.

Dielectrophoresis technology is applied to microfluidic chips to achieve microscopic control of cells. Currently, microfluidic chips based on dielectrophoresis have certain limitations in terms of cell sorting species, in order to explore a microfluidic chip with excellent performance and high versatility. In this paper, we designed a microfluidic chip that can be used for continuous cell sorting, with the structural design of a curved channel and curved double side electrodes. CM factors were calculated for eight human healthy blood cells and cancerous cells using the software MyDEP, the simulation of various blood cells sorting and the simulation of the joule heat effect of the microfluidic chip were completed using the software COMSOL Multiphysics. The effect of voltage and inlet flow velocity on the simulation results was discussed using the control variables method. We found feasible parameters from simulation results under different voltages and inlet flow velocities, and the feasibility of the design was verified from multiple perspectives by measuring cell movement trajectories, cell recovery rate and separation purity. This paper provides a universal method for cell, particle and even protein sorting.

Anatomical structures, cell types, and biomarkers of the healthy human blood vasculature

More than 150 scientists from 17 consortia are collaborating on an international project to build a Human Reference Atlas, which maps all 37 trillion cells in the healthy adult human body. The initial release of this atlas provided hierarchical lists of the anatomical structures, cell types, and biomarkers in 11 organs. Here, we describe the methods we used as part of this initiative to build the first open, computer-readable, and comprehensive database of the adult human blood vasculature, called the Human Reference Atlas-Vasculature Common Coordinate Framework (HRA-VCCF). It includes 993 vessels and their branching connections, 10 cell types, and 10 biomarkers. With this paper we are releasing additional details on vessel types and subtypes, branching sequence, anastomoses, portal systems, microvasculature, functional tissue units, mappings to regions vessels supply or drain, geometric properties of vessels, and links to 3D reference objects. Future versions will add variants and connections to the lymph vasculature; and, it will iteratively expand and improve the database as additional experimental data become available through the participating consortia.

A review on rheological models and mathematical problem formulations for blood flows

A review on constitutive equations proposed for mathematical modeling of laminar and turbulent flows of blood as a concentrated suspension of soft particles is given. The rheological models of blood as a uniform Newtonian fluid, non-Newtonian shear-thinning, viscoplastic, viscoelastic, tixotropic and micromorphic fluids are discussed. According to the experimental data presented, the adequate rheological model must describe shear-thinning tixotropic behavior with concentration-dependent viscoelastic properties which are proper to healthy human blood. Those properties can be studied on the corresponding mathematical problem formulations for the blood flows through the tudes or ducts. The corresponding systems of equations and boundary conditions for each of the proposed rheological models are discussed. Exact solutions for steady laminar flows between the parallel plates and through the circular tubes have been obtained and analyzed for the Ostwald, Hershel-Bulkley, and Bingham shear-thinning fluids. The influence of the model parameters on the velocity profiles has been studied for each model. It is shown, certain sets of fluid parameters lead to flattening of the velocity profile while others produce its sharpening around the axis of the channel. It is shown, the second-order terms in the viscoelastic models give the partial derivative differential equations with high orders in time and mixed space-time derivatives. The corresponding problem formulations for the generalized rhelogical laws are derived. Their analytical solutions in the form of a normal mode are obtained. It is shown, the dispersion equations produce an additional set for the speed of sound (so called second sound) in the fluid. It is concluded, the most general rheological model must include shear-thinning, concentration and second sound phenomena

Metatranscriptome analysis of blood in healthy individuals and irritable bowel syndrome patients.

Introduction. Although the presence of micro-organisms in the blood of healthy humans is a relatively new concept, there is a growing amount of evidence that blood might have its own microbiome.Gap Statement. Previous research has targeted the taxonomic composition of the blood microbiome using DNA-based sequencing methods, while little information is known about the presence of microbial transcripts obtained from the blood and their relation to conditions connected with increased gut permeability.Aim. To detect potentially alive and active micro-organisms and investigate differences in taxonomic composition between healthy people and patients with irritable bowel syndrome (IBS), we used the metatranscriptomics approach.Methodology. We collected blood samples from 23 IBS patients and 26 volunteers from the general population, and performed RNAseq on the isolated RNA. Reads corresponding to microbial genomes were identified with Kraken 2's standard plus protozoa and fungi database, and re-estimated at genus level with Bracken 2.7. We looked for trends in the taxonomic composition, making a comparison between the IBS and control groups, accounting for other different factors.Results. The dominant genera in the blood microbiome were found to be Cutibacterium, Bradyrhizobium, Escherichia, Pseudomonas, Micrococcus, Delftia, Mediterraneibacter, Staphylococcus, Stutzerimonas and Ralstonia. Some of these are typical environmental bacteria and could partially represent contamination. However, analysis of sequences from the negative controls suggested that some genera which are characteristic of the gut microbiome (Mediterraneibacter, Blautia, Collinsella, Klebsiella, Coprococcus, Dysosmobacter, Anaerostipes, Faecalibacterium, Dorea, Simiaoa, Bifidobacterium, Alistipes, Prevotella, Ruminococcus) are less likely to be a result of contamination. Differential analysis of microbes between groups showed that some taxa associated with the gut microbiome (Blautia, Faecalibacterium, Dorea, Bifidobacterium, Clostridium, Christensenella) are more prevalent in IBS patients compared to the general population. No significant correlations with any other factors were identified.Conclusion. Our findings support the existence of the blood microbiome and suggest the gut and possibly the oral microbiome as its origin, while the skin microbiome is a possible but less certain source. The blood microbiome is likely influenced by states of increased gut permeability such as IBS.

Mechanisms of glabridin inhibition of integrin αIIbβ3 inside-out signals and NF-κB activation in human platelets

BackgroundPlatelets play a crucial role in cardiovascular diseases (CVDs) and are activated by endogenous agonists like collagen. These agonists initiate signal transduction through specific platelet receptors, resulting in platelet aggregation. Glabridin, a prenylated isoflavonoid found in licorice root, is known for its significance in metabolic abnormalities. Glabridin has been observed to inhibit collagen-induced platelet aggregation, but the precise mechanisms, specifically concerning NF-κB activation and integrin αIIbβ3 signaling, are not yet fully understood.MethodsIn this study, platelet suspensions were prepared from healthy human blood donors, and the aggregation ability was observed using a lumi-aggregometer. The inhibitory mechanisms of glabridin in human platelets were evaluated through immunoblotting and confocal microscopy. The anti-thrombotic effects of glabridin were assessed by histological analysis of lung sections in acute pulmonary thromboembolism and by examining fluorescein-induced platelet plug formation in mesenteric microvessels in mice.ResultsGlabridin inhibited integrin αIIbβ3 inside-out signals such as Lyn, Fyn, Syk, and integrin β3 activation and NF-κB-mediated signal events, with similar potency to classical inhibitors BAY11-7082 and Ro106-9920. Glabridin and BAY11-7082 inhibited IKK, IκBα, and p65 phosphorylation and reversed IκBα degradation, while Ro106-9920 only reduced p65 phosphorylation and reversed IκBα degradation. BAY11-7082 reduced Lyn, Fyn, Syk, integrin β3, phospholipase Cγ2 and protein kinase C activation. Glabridin reduced platelet plug formation in mesenteric microvessels and occluded vessels in thromboembolic lungs of mice.ConclusionOur study revealed a new pathway for activating integrin αIIbβ3 inside-out signals and NF-κB, which contributes to the antiplatelet aggregation effect of glabridin. Glabridin could be a valuable prophylactic or clinical treatment option for CVDs.

Production of an interleukin-10 blocking antibody by genetically engineered macrophages increases cancer cell death in human gastrointestinal tumor slice cultures.

Although it can promote effector T-cell function, the summative effect of interleukin-10 (IL-10) in the tumor microenvironment (TME) appears to be suppressive; therefore, blocking this critical regulatory cytokine has therapeutic potential to enhance antitumor immune function. As macrophages efficiently localize to the TME, we hypothesized that they could be used as a delivery vehicle for drugs designed to block this pathway. To test our hypothesis, we created and evaluated genetically engineered macrophages (GEMs) that produce an IL-10-blocking antibody (αIL-10). Healthy donor human peripheral blood mononuclear cells were differentiated and transduced with a novel lentivirus (LV) encoding BT-063, a humanized αIL-10 antibody. The efficacy of αIL-10 GEMs was assessed in human gastrointestinal tumor slice culture models developed from resected specimens of pancreatic ductal adenocarcinoma primary tumors and colorectal cancer liver metastases. LV transduction led to sustained production of BT-063 by αIL-10 GEMs for at least 21 days. Transduction did not alter GEM phenotype as evaluated by flow cytometry, but αIL-10 GEMs produced measurable quantities of BT-063 in the TME that was associated with an ~5-fold higher rate of tumor cell apoptosis than control.

OMIP-94: Twenty-four-color (thirty-marker) panel for deep immunophenotyping of immune cells in human peripheral blood.

This newly established 24-color (30-marker) panel focuses on the characterization of the main human immune cell subtypes and was optimized for the analysis of human whole blood using a full spectrum flow cytometer. The panel covers all main leukocyte populations: neutrophils, eosinophils and basophils, monocytes (with additional subsets), dendritic cells, innate lymphoid cells and lymphocytes. As for lymphocytes, this panel includes CD4+ T helper, Treg cells, and CD8+ cytotoxic T cells. Further T cells subsets are included with special focus on invariant T cells: γδ T cells (including δ2TCR variant), invariant NKT cells and MAIT (mucosal-associated invariant T cells) cells. Additionally, total B cells (including Bregs and plasmocytes), NK cells, and NKT cells are included. For the overall check of activation status of the analyzed immune cells we used HLA-DR, CD38, CD57, CD69, PD-1, and CD94. In addition, we used CD62L, CD45RA, CD27, and CD39 to describe the differentiation status of these cells. The panel was designed to maximize the information that can be obtained from surface markers in order to avoid the need for fixation and permeabilization steps. The presented multimarker panel offers the possibility to discover new immune cell subtypes which in patients and in cohort studies may lead to the identification of altered immune phenotypes and might give a link to immune system based or to certain other diseases. This panel was developed for a full spectrum flow cytometer equipped with a minimum of three lasers. We developed this panel using healthy human fresh blood, however it was also successfully used for staining of isolated human peripheral blood mononuclear cells (PBMC).

A Review of Machine Learning Methods Recently Applied to FTIR Spectroscopy Data for the Analysis of Human Blood Cells.

Machine learning (ML) is a broad term encompassing several methods that allow us to learn from data. These methods may permit large real-world databases to be more rapidly translated to applications to inform patient-provider decision-making. This paper presents a review of articles that discuss the use of Fourier transform infrared (FTIR) spectroscopy and ML for human blood analysis between the years 2019-2023. The literature review was conducted to identify published research of employed ML linked with FTIR for distinction between pathological and healthy human blood cells. The articles' search strategy was implemented and studies meeting the eligibility criteria were evaluated. Relevant data related to the study design, statistical methods, and strengths and limitations were identified. A total of 39 publications in the last 5 years (2019-2023) were identified and evaluated for this review. Diverse methods, statistical packages, and approaches were used across the identified studies. The most common methods included support vector machine (SVM) and principal component analysis (PCA) approaches. Most studies applied internal validation and employed more than one algorithm, while only four studies applied one ML algorithm to the data. A wide variety of approaches, algorithms, statistical software, and validation strategies were employed in the application of ML methods. There is a need to ensure that multiple ML approaches are used, the model selection strategy is clearly defined, and both internal and external validation are necessary to be sure that the discrimination of human blood cells is being made with the highest efficient evidence.

Effect of Sodium Hypochlorite 0.05% on MMP-9 Extracellular Release in Chronic Wounds.

In chronic wounds, high concentrations of matrix metalloproteinases (MMPs) can cause excessive proteolysis and slow wound healing. Consequently, restoring a proper MMP balance can help reduce the risk of a chronic wound. An antiseptic solution containing 0.05% sodium hypochlorite (Amukine Med 0.05%, Angelini S.p.A.; hereafter termed NaClO solution) is available on the market. The NaClO solution was proven effective and safe in managing infected skin wounds. To further characterize its activity, this study evaluated the in vitro activity of the NaClO solution on the monocyte release of MMPs. Human monocytic THP-1 (ATCC® TIB-202™) cell lines were differentiated into macrophages and treated with different concentrations of NaClO (from 0.05% to 5 × 10-7%). In addition, the THP-1 cell line was stimulated with wound fluid (WF) from patients with active venous leg ulcers in the inflammatory phase. The effect of NaClO (0.025-0.0062%) was also evaluated on healthy human peripheral blood serum samples. The effects of treatments on the gelatinolytic activity of MMP-9 were evaluated by gelatin zymography. The effects on MMPs release were evaluated through the Pro™ Human MMP 9-plex Assay. An exploratory scratch wound healing assay was also performed. The NaClO solution reduced the gelatinolytic activity of MMP-9 and its activated form. The downregulation of MMP-9 gelatinolytic activity was also observed in peripheral blood serum. The MMPs profile showed a reduction in MMP-1 release (p < 0.05) and a slight reduction of the release of MMP-9 and MMP-12 after the treatment with LPS and the NaClO solution. A slight improvement in wound healing was observed after macrophage activation and treatment with the NaClO solution. The results obtained suggest a possible ability of the NaClO solution to modulate the proteolytic pathways in the wound microenvironment, further characterizing its activity and use in clinical practice during wound care.

Evidence for EpCAM and Cytokeratin Expressing Epithelial Cells in Normal Human and Murine Blood and Bone Marrow.

Epithelial cells have been identified in the blood and bone marrow of patients with cancer and other diseases. However, the presence of normal epithelial cells in the blood and bone marrow of healthy individuals has yet to be identified in a consistent way. Presented here is a reproducible method for isolating epithelial cells from healthy human and murine blood and bone marrow (BM) using flow cytometry and immunofluorescence (IF) microscopy. Epithelial cells in healthy individuals were first identified and isolated via flow cytometry using epithelial cell adhesion molecule (EpCAM). These EpCAM+ cells were confirmed to express keratin using immunofluorescence microscopy in Krt1-14;mTmG transgenic mice. Human blood samples had 0.18% ± 0.0004 EpCAM+ cells (SEM; n=7 biological replicates, 4 experimental replicates). In human BM, 3.53% ± 0.006 (SEM; n=3 biological replicates, 4 experimental replicates) of mononuclear cells were EpCAM+. In mouse blood, EpCAM+ cells constituted 0.45% ± 0.0006 (SEM; n=2 biological replicates, 4 experimental replicates), and in mouse BM, 5.17% ± 0.001 (SEM; n=3 biological replicates, 4 experimental replicates) were EpCAM+. In mice, all the EpCAM+ cells were immunoreactive to pan-cytokeratin, as determined by IF microscopy. Results were confirmed using Krt1-14;mTmG transgenic mice, with low (8.6 native GFP+ cells per 106 cells analyzed; 0.085% of viable cells), but significant numbers (p < 0.0005) of GFP+ cells present in normal murine BM, that were not the result of randomness compared with multiple negative controls. Further, EpCAM+ cells in mouse blood were more heterogeneous than CD45+ cells (0.58% in BM; 0.13% in blood). These observations conclude that cells expressing cytokeratin proteins are reproducibly detectable among mononuclear cells from human and murine blood and BM. We demonstrate a method of tissue harvesting, flow cytometry, and immunostaining that can be used to identify and determine the function of these pan-cytokeratin epithelial cells in healthy individuals.

Abstract 780: Digital PCR-based EGFR T790M mutation detection from human blood samples collected in PAXgene Blood ccfDNA Tubes

Abstract Introduction: Circulating cell-free DNA (ccfDNA) is used as an analyte in liquid biopsy-based cancer research with promising applications in the fields of cancer screening, therapeutic decision making and monitoring and minimal residual disease control. However, despite the multitude of future applications, challenges regarding the sensitivity of analytical tests remain, thereby limiting the routine use of ccfDNA within the above mentioned fields. In this study, we investigated the compatibility of the PAXgene® Blood ccfDNA Tube and the QIAcuity® Digital PCR System as an optimal “sample to insight” workflow with regard to the detection of EGFR T790M mutations spiked into apparently healthy human donor blood specimens. Methods: Blood from 22 apparently healthy consented donors was collected into PAXgene Blood ccfDNA Tubes* (PreAnalytiX). Within 2 h of blood draw, blood samples were either spiked with an EGFR-Multiplex 5% AF cfDNA standard (SensID) containing an EGFR T790M mutation or left unspiked as a control. Tubes were incubated for 3 d at room temperature to simulate transport over a weekend and plasma was generated by double centrifugation. Plasma pools were generated from spiked and unspiked samples and ccfDNA was isolated with the QIAamp® Circulating Nucleic Acid Kit (QIAGEN). CcfDNA quantity was measured by Qubit® dsDNA HS Assay (Thermo Fisher Scientific) and fragment size distribution was determined by TapeStation® Cell-free DNA ScreenTape® (Agilent Technologies). EGFR T790M mutation detection was performed using the dPCR Mutation Assay EGFR 6240 Human (QIAGEN) with a 26K 24-well Nanoplate (QIAGEN) on the QIAcuity Digital PCR System (QIAGEN). Data analysis was conducted using the QIAcuity Software Suite (QIsAGEN). Results: CcfDNA isolated from PAXgene Blood ccfDNA Tubes had a fragment size distribution profile with distinct mononucleosomal peaks at ~180 bps without high-molecular weight DNA, indicating successful nucleated cell stabilization over the simulated transport duration and extraction of high-quality ccfDNA. Digital PCR analysis of extracted ccfDNA revealed positive detection of EGFR T790M mutations in spiked blood samples at a relative mutation fraction of approximately 2% in the investigated samples. Conclusion: PAXgene Blood ccfDNA Tubes enabled high-quality ccfDNA isolation by stabilizing nucleated cells during simulated transport condition. Spiked EGFR T790M mutations were successfully detected by dPCR following sample storage. This proof of principle study shows the combined usability of both systems for mutation detection in human blood specimens, highlighting the potential of both technologies for a broader application in the liquid biopsy field to detect oncogenic driver as well as tumor suppressor gene mutations in a multitude of tumor types. *The PAXgene Blood ccfDNA Tube is for Research Use Only in the US. Not for use in diagnostic procedures. Citation Format: Christian Neander, Andrea Huxhold, Maike Schoenborn, Thorsten Voss. Digital PCR-based EGFR T790M mutation detection from human blood samples collected in PAXgene Blood ccfDNA Tubes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 780.

Abstract 2294: The development of 3D deformable microfilter with a DNA aptamer for detecting circulating tumor cell

Abstract Introduction: The analysis of blood for circulating tumor cells (CTCs) has opened new avenues for cancer diagnostics, including early detection, risk assessment and staging. However, research about CTCs has been challenging due to the rarity of CTCs in bloodstream. We develop a three-dimensional deformable microfilter with a EpCAM aptamer for detecting CTCs and conduct a pilot study to evaluate the efficacy of our novel detection devise. Methods: The cancer cell suspension with blood from healthy volunteers and human blood sample from cancer patients are passed through the microfilter and counted by fluorescence microscope. We also countered the number of cancer cells by CellSearch eq \o\ac(○,R)R. The peripheral blood sample from upper gastrointestinal cancer patients are also passed through the microfilter. Results: Even in the presence of 100 times normal cell, cancer cells were captured on the substrate successfully from cell suspensions with several mixing ratios. We can capture cancer cells suspended with PB from healthy volunteer more effectively compared with CellSerach®. A total of 15 patients and 40 samples were enrolled (Stage I: II: III: IV: recurrence=1:1:3:4:6). The average number of CTCs was 3.1 cells/ml (0-6.3 cells). Among the 15 patients with GC, CTCs were detected by our system in 13 patients. Even in GC patients suspected with peritoneal dissemination without definitive mass by imaging, CTCs were successfully detected. For the patients who were measured multiple times with clinical time course, we found a close correlation between CTC number and CEA level or image assessment. Conclusion: We successfully detected CTCs in the patients with upper gastrointestinal cancer patients. Although our pilot study indicates the potential of a novel devise to capture CTC effectively, a large scale of validation is needed to confirm the clinical utility. Citation Format: Masaaki Iwatsuki, Yuta Nakashima, Yusuke Kitamura, Keiichiro Yasuda, Hideo Baba. The development of 3D deformable microfilter with a DNA aptamer for detecting circulating tumor cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2294.

Derivatization of Dihydropyrrolidone-Thiadiazole Heterocyclic Compounds and an Evaluation of their Antibacterial and Anti-Biofilm Activities

AbstractDihydropyrrolidone-thiadiazole inhibitors targeting YycG histidine kinase have been designed, synthesized and evaluated for their antibacterial, bactericidal, anti-biofilm, cytotoxic and hemolytic activities, and for their ability to promote autophosphorylation. 4-(Benzofuran-2-carbonyl)-1-(5-(benzylthio)-1,3,4-thiadiazol-2-yl)-3-hydroxy-5-(2-hydroxyphenyl)-1,5-dihydro-2H-pyrrol-2-one exhibits the best bacteriostatic activity against Gram-positive bacteria such as S. epidermidis SE1457, MSSA SA113, and E. faecalis FB1 (MIC = 3.13–25 μM). Its antibacterial activity against methicillin-sensitive Staphylococcus aureus (MSSA) SA113 is comparable to that of linezolid. Most of the products exhibit good inhibitory effects against the biofilms of the tested strains. Among the products, three show strong inhibitory effects on the biofilm formation of S. epidermidis SE1457, MSSA SA113, and E. faecalis FB1, and their inhibition rates reach more than 90% at 6.25 μM. Cytotoxicity and hemolytic activity tests suggest that all the synthesized compounds have little effect on the growth of mammalian cells (Vero cells) and exhibit no hemolytic activity toward healthy human red blood cells.

Platelet Rich Plasma's Use on Recovering Achilles Tendon Ruptures of Wistar Strain White Mouse

Achilles tendon is the most common site of lower extremity tendon rupture. Current treatment has not been able to restore the biological and mechanical properties of the tendon to pre-injure condition. One of the treatments used to improve Achilles tendon healing is the application of PRP. The research would like to determine the effect of using Platelet Rich Plasma (PRP) on the recovery of tendon rupture in animals model (wistar strain white rats). This is an experimental study with a post-test only control group design using animals model (white rats). This study was conducted at the INA Lab Laboratory, Padang in November 2022. The samples were divided into two groups, the control group and the treatment group where both of these groups were injured and sutured to the tendons. The control group was not given PRP, while the treatment group was given 0.1 cc of PRP. The PRP used was heterologous from healthy human blood. Observations were made after 1 week both groups were maintained and treated. There were 12 white rats which were divided into the PRP group and the control group. The PRP group had a lower histopathological score (5.83) than the control group (10.50). In this study, there was a significant difference between the recovery of tendon ruptures in animal model (white rats) that were given Human Platelet Rich Plasma (PRP) and experimental animals (rats) that were not given PRP (p value 0.007). PRP improves healing in Achilles tendon rupture.