Whole Blood Cells Research Articles

Human Supplementation with AM3, Spermidine, and Hesperidin Enhances Immune Function, Decreases Biological Age, and Improves Oxidative–Inflammatory State: A Randomized Controlled Trial

The positive effect of AM3, spermidine, and hesperidin, which have antioxidant and anti-inflammatory properties, on immunity is known, but their effect on the rate of aging, known as biological age (BA), is unclear. This work aims to test if the intake of a blend of AM3 (150 mg), spermidine (0.6 mg), and hesperidin (50 mg) for 2 months could decrease BA and improve immunity, redox, and inflammatory states. For this, 41 participants (30–63 years) were randomly divided into placebo and supplement groups. The supplement group took two capsules daily with AM3, spermidine, and hesperidin for two months, while the placebo group took capsules containing only calcium phosphate and talcum powder. Before and after the treatment, peripheral blood was collected. Immune function was assessed in leukocytes, redox state in whole-blood cells, erythrocytes, and plasma, and cytokine concentration in both mononuclear cell cultures and plasma. Finally, the Immunity Clock model was applied to determine BA. The results show that the intake of this blend improves the immune functions that constitute the Immunity Clock, decreasing BA by 11 years and reducing the oxidative–inflammatory state of the participants. Therefore, this supplement can be proposed as a strategy to rejuvenate BA and achieve healthy aging.

Valuable Contributions and Lessons Learned from Proteomics and Metabolomics Studies of COVID-19.

The COVID-19 pandemic caused by the SARS-CoV-2 virus infected more than 775,686,716 humans and was responsible for the death of more than 7,054,093 individuals. COVID-19 has taught us that the development of vaccines, repurposing of drugs, and understanding the mechanism of a disease can be done within a short time. The COVID-19 proteomics and metabolomics has contributed to its diagnosis, understanding of its progression, host-virus interaction, disease mechanism, and also in the search of suitable anti-COVID therapeutics. Mass spectrometry based proteomics was used to find the potential biomarkers of different stages of COVID-19 including severe and nonsevere cases in the blood serum. Notably, protein-protein interaction techniques to understand host-virus interactions were also significantly useful. The single-cell proteomics studies were carried out to ascertain the changes in immune cell composition and its activation in mild COVID-19 patients versus severe COVID-19 patients using whole-blood and peripheral-blood mononuclear cells. Modern technologies were helpful to deal with the pandemic; however, there is still scope for further development. Further, attempts were made to understand the protein-protein, metabolite-metabolite, and protein-metabolite interactomes, derived from proteins and metabolite fingerprints of COVID-19 patients by reanalysis of COVID-19 public mass spectrometry based proteomics and metabolomics studies. Further, some of these interactions were supported by the literature as validations in the COVID-19 studies.

Numerical-experimental estimation of the deformability of human red blood cells from rheometrical data

The deformability of human red blood cells (RBCs), which comprise almost 99% of the cells in whole blood, is largely related not only to pathophysiological blood flow but also to the levels of intracellular compounds. Therefore, statistical estimates of the deformability of individual RBCs are of paramount importance in the clinical diagnosis of blood diseases. Although the microscale hydrodynamic interactions of individual RBCs lead to non-Newtonian blood rheology, there is no established method to estimate individual RBC deformability from the rheological data of RBC suspensions, and the possibility of this estimation has not been proven. To address this issue, we conducted an integrated analysis of a model of the rheology of RBC suspensions, coupled with macrorheological data of human RBCs suspended in plasma. Assuming a nonlinear curve of the relative viscosity of the suspensions as a function of the cell volume fraction, the statistical average of the membrane shear elasticity was estimated for individual intact RBCs or hardened RBCs. Both estimated values reproduced well the experimentally observed shear-thinning non-Newtonian behavior in these suspensions. We hereby conclude that our complementary approach makes it possible to estimate the statistical average of individual RBC deformability from macrorheological data obtained with usual rheometric tests.

68Ga Radiolabeling of NODASA-Functionalized Phage Display-Derived Peptides for Prospective Assessment as Tuberculosis-Specific PET Radiotracers.

This research presents the development of positron emission tomography (PET) radiotracers for detecting Mycobacterium tuberculosis (MTB) for the diagnosis and monitoring of tuberculosis. Two phage display-derived peptides with proven selective binding to MTB were identified for development into PET radiopharmaceuticals: H8 (linear peptide) and PH1 (cyclic peptide). We sought to functionalize H8/PH1 with NODASA, a bifunctional chelator that allows complexation of PET-compatible radiometals such as gallium-68. Herein, we report on the chelator functionalization, optimized radiosynthesis, and assessment of the radiopharmaceutical properties of [68Ga]Ga-NODASA-H8 and [68Ga]Ga-NODASA-PH1. Robust radiolabeling was achieved using the established routine method, indicating consistent production of a radiochemically pure product (RCP ≥ 99.6%). For respective [68Ga]Ga-NODASA-H8 and [68Ga]Ga-NODASA-PH1, relatively high levels of decay-corrected radiochemical yield (91.2% ± 2.3%, 86.7% ± 4.0%) and apparent molar activity (Am, 3.9 ± 0.8 and 34.0 ± 5.3 GBq/μmol) were reliably achieved within 42 min, suitable for imaging purposes. Notably, [68Ga]Ga-NODASA-PH1 remained stable in blood plasma for up to 2 h, while [68Ga]Ga-NODASA-H8 degraded within 30 min. For both 68Ga peptides, minimal whole-blood cell binding and plasma protein binding were observed, indicating a favorable pharmaceutical behavior. [68Ga]Ga-NODASA-PH1 is a promising candidate for further invitro/in vivo evaluation as a tuberculosis-specific infection imaging agent.

Effects of hematocrit and non-Newtonian blood rheology on pulsatile wall shear stress distributions in vascular anomalies: A multiple relaxation time lattice Boltzmann approach

In the present study, we investigate the flow dynamics of non-Newtonian blood, focusing on the distribution of wall shear stress (WSS) and hematocrit levels, which is the volume percentage of red blood cells in whole blood. We analyze these factors under pulsatile conditions, in vascular anomalies such as stent channels and intracranial aneurysms. To achieve this, a three-dimensional computational approach based on the lattice Boltzmann method (LBM) with a multiple relaxation time (MRT) collision operator is employed. To represent the blood's shear-thinning properties, we developed a constitutive model inspired by the Carreau–Yasuda model. This model considers the variability in blood viscosity with shear rate correlated with hematocrit levels based on experimental data documented in the literature. The accuracy of the employed MRT-LBM is demonstrated by the consistency of results with analytical solutions for steady state and experimental data for pulsatile WSS distributions in non-Newtonian and Newtonian fluids. Results indicate that, in areas narrowed by stenosis or expanded by aneurysms, hematocrit levels affect flow dynamics. Higher hematocrit levels intensify pulsatile flow through stenotic regions, increasing WSS cyclic variations. We derived a density distribution function to demonstrate how shear rates vary in vascular anomalies, revealing blood viscosity changes and non-Newtonian properties. These properties complicate flow patterns, resulting in non-linear WSS distributions, which are essential for understanding endothelial cell reactions and disease pathways. Pulsatile blood flow and altered rheological properties due to increased hematocrit affect saccular aneurysm fluid dynamics over time and space, causing vorticities to change shape, size, and intensity.

Harmonic Imaging of Stem Cells in Whole Blood at GHz Pixel Rate.

The pre-clinical validation of cell therapies requires monitoring the biodistribution of transplanted cells in tissues of host organisms. Real-time detection of these cells in the circulatory system and identification of their aggregation state is a crucial piece of information, but necessitates deep penetration and fast imaging with high selectivity, subcellular resolution, and high throughput. In this study, multiphoton-based in-flow detection of human stem cells in whole, unfiltered blood is demonstrated in a microfluidic channel. The approach relies on a multiphoton microscope with diffractive scanning in the direction perpendicular to the flow via a rapidly wavelength-swept laser. Stem cells are labeled with metal oxide harmonic nanoparticles. Thanks to their strong and quasi-instantaneous second harmonic generation (SHG), an imaging rate in excess of 10 000 frames per second is achieved with pixel dwell times of 1 ns, a duration shorter than typical fluorescence lifetimes yet compatible with SHG. Through automated cell identification and segmentation, morphological features of each individual detected event are extracted and cell aggregates are distinguished from isolated cells. This combination of high-speed multiphoton microscopy and high-sensitivity SHG nanoparticle labeling in turbid media promises the detection of rare cells in the bloodstream for assessing novel cell-basedtherapies.

A microfluidic platform for automatic continuous flow layer‐by‐layer assembly of polymer multilayered nanofilms

AbstractCapillary flow layer‐by‐layer assembly is a microfluidic platform for high throughput preparation and screening of multilayered polymer nanofilms. However, it is time‐consuming and labor‐intensive, since the switching of polyelectrolyte solutions is done manually. As the emergence of flow chemistry and microdevices for artificial intelligence‐based lab automation, an automatic system that can switch polymer solutions and washing liquids is urgently needed for building functional LbL films inside microfluidic devices. Herein, we construct a continuous flow (ConF)‐layer‐by‐layer (LbL) device that can automatically fabricate polymer nanofilms inside a microfluidic device. By including a flow separator and a mixer (a model polyelectrolyte pair), chitosan and carboxymethyl cellulose can be deposited inside a microfluidic device using the ConF‐LbL system. Resulting nanofilms are studied by investigating thickness and surface roughness of the nanofilms and comparing with the other conventional LbL methods. The acid dissolution method was used to compare the differences in stability between nanofilms made from ConF‐LbL and static immersive method in similar conditions. Furthermore, by using one optimized film formulation for coating on the inside walls of a microfluidic chip, we successfully demonstrated the ability of this device to capture cancer cells in whole blood. This ConF‐LbL platform is expected to be a convenient tool to coat functional nanofilms inside microdevices for biomedical and sensor applications.

Gum Arabic Increases Phagocytosis of Escherichia coli by Blood Leukocytes of Young and Old Healthy Volunteers.

Gum arabic, a polysaccharide exudate from Acacia senegal (L.) Willdenow trees, has already been used by African native people in natural medicine. Using whole-blood samples from young (20-35 years) and older (>80 years) healthy volunteers (each group n = 10), the effect of an aqueous solution of GA on phagocytosis of Escherichia coli was examined with a gentamicin protection assay. Whole-blood samples of each volunteer were stimulated with GA and as a control with CpG oligodeoxynucleotides (Toll-like receptor -9 agonists) for 2 h, then co-incubated with E. coli for 30 min and thereafter treated with gentamicin for up to 240 min to kill extracellular bacteria. Then, whole-blood cells were lysed with distilled water, and colony-forming units were counted by quantitative plating. Cytokine enzyme-linked immunosorbent assay for the detection of TNF-α and IL-6 was performed using the blood supernatant. The GA concentration tested (20 mg/mL) did not affect the viability of eukaryotic cells. Phagocytosis of E. coli by whole-blood leukocytes derived from young (p = 0.008) and older (p = 0.004) healthy volunteers was increased by 120.8% (young) and 39.2% (old) after stimulation with GA. In contrast, CpG only stimulated the bacterial phagocytosis by cells derived from young volunteers (p = 0.004). Stimulation of whole blood with GA increased the intracellular killing of E. coli in young (p = 0.045) and older volunteers (p = 0.008) and induced a TNF-α release in whole blood collected from older volunteers but not from younger ones (p = 0.008). These data encourage the isolation of active compounds of GA and the initiation of clinical trials addressing the preventive effect of GA on bacterial infections.

Acute Exposure to High-Altitude Results in Changes to Immune Cell Populations

High altitude is a challenging environment due to the reduced atmospheric pressure and oxygen availability, increased ultraviolet radiation, and reduced temperature relative to sea level. Previous studies in individuals who travel to high altitude demonstrate differential expression of genes involved in inflammation and innate immune system activation. This study aims to extend these findings by defining changes in immune cell population distributions and cell phenotypes during travel to high altitude. We hypothesized that acute high-altitude exposure would lead to shifts in immune cell populations toward a pro-inflammatory phenotype due to cellular stress caused by hypoxemia and tissue hypoxia. Participants (N=17) traveled to Barcroft Station in the White Mountain Research Center (3800 m). Whole blood was collected during fasting at sea level on the morning of ascent and each morning at high altitude for three days. Cells in whole blood were fixed and stained within 1-2 hours of collection and were processed via flow cytometry upon return to sea level. We observed a decrease in classical monocytes on the first (p=0.010) and second (p=0.003) mornings of high-altitude acclimatization to sea-level baseline, and an increase in intermediate monocytes on the first (p=0.013) and second (p=0.014) mornings at high altitude. We also observed an increase in B cells on the second (p=0.004) and third (p=0.004) mornings at high altitude compared to sea-level baseline (mean: 3.53%, SD: ±1.82). These results indicate that acute high altitude exposure results in a shift toward a pro-inflammatory monocyte phenotype and supports prior research suggesting a significant role of hypoxia in modulating B cell function. This research was supported through funding provided by a private donor. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Precision Phenotypic Profiling and Capture of Circulating Tumor Cells via a Vertical Laminar Flow-Stacked Microfluidic Chip.

The heterogeneity of circulating tumor cells has a significant impact on the diagnosis, treatment, and monitoring of cancer. Research on the subtypes of circulating tumor cells can bring better treatment outcomes for cancer patients. Here, we proposed a microfluidic chip for the magnetic capture of subtypes of circulating tumor cells from the whole blood and phenotypic profiling by stacking laminar flow vertically. Circulating tumor cells were sorted and captured by the three-dimensional regulation of both magnetic fields in the vertical direction and flow fields in the lateral direction. Using EpCAM-magnetic beads, we achieved sorting and sectional capture of target cells in whole blood and analyzed the surface expression levels of the captured cells, confirming the functionality of the microfluidic chip in sorting and capturing subtypes of circulating tumor cells. This microfluidic chip can also aid in the subsequent subtype analysis of other rare cells.

Abstract 7485: Microfluidic detection of dual positive cells in whole blood of metastatic breast cancer patients

Abstract Background. Dual positive cells (DPCs) that express both epithelial markers and leukocytic markers have been observed in peripheral blood of breast cancer patients. DPCs acquire functional behaviors from both cell types which may drive tumor progression. However, the lack of reliable methods to isolate and identify DPCs has hindered the investigation of their origin and clinical implications in breast cancer patients. While microfluidic devices have been widely used for the detection of circulating tumor cells (CTCs) by liquid biopsy, DPC detection is underexplored in these devices. Herein, we demonstrate rapid onchip isolation and detection of DPCs in whole blood samples from metastatic breast cancer (MBC) patients using an integrated microfluidic device. Methods. Our microfluidic device consists of a unique preconditioning component that concentrates targets in whole blood and a downstream cell immobilization component that captures target cells. We included 7 female patients with MBC, of which 29% are white (2), 57% are African American (4), and 14% are Latina (1). The median age is 50y (34y -75y). All patients were undergoing systemic breast cancer therapy at the time of blood collection. A total of 9 samples (4 mL each) were collected from either an antecubital vein (peripheral vein) or the subcutaneous port catheter (central vein) of these patients. All samples were processed within 5 hours of blood drawn in our novel microfluidic biochip. CTCs (EpCAM+/CK+, CD45-, DAPI+) and DPCs (EpCAM+/CK+, CD45+/CD66b+, DAPI+) were identified by immunofluorescence after cells were fixed. Results. Processing of each sample was completed within 40 minutes using our microfluidic device. Both DPCs and CTCs were detected in all the samples (9/9). The average DPC count was 3 DPCs per 4 mL whole blood with a standard deviation of 1.9 cells. The average CTC count was 9 with a standard deviation of 5 per 4 mL whole blood. 89% of the samples (8/9) had > 5 CTCs after the sample volume is scaled to the standard 7.5 mL. No correlation was found between DPC count and CTC count (Pearson correlation coefficient r=0.08). While the blood collection location (central vs peripheral veins) had a significant impact on the CTC count (12.6 vs 4 on average with p<0.01), there was no statistical difference in DPC counts in the samples collected from central and peripheral veins. Conclusions. Both DPCs and CTCs can be rapidly isolated and detected from MBC patient whole blood in our microfluidic device. Our preliminary results indicate no correlation between DPCs and CTCs, suggesting that DPCs might not originate in blood circulation of MBC patients. More work with a larger patient cohort will be needed to further investigate the clinical implications of DPCs on MBC patients. Citation Format: Jian Zhou, Qiyue Luan, Celine Macaraniag, Arielle B. Guzman, Maria Mantice, Oana Danciu, Kent F. Hoskins, Ian Papautsky. Microfluidic detection of dual positive cells in whole blood of metastatic breast cancer 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 7485.

Abstract 79: Enabling fast and convenient immune profiling of fresh and long-term stabilized human whole blood samples with CyTOF

Abstract Accurate phenotyping of immune cells in whole blood (WB) from patients with cancer is critical for making disease prognoses and monitoring clinical efficacy of immunotherapies. Fresh WB must be analyzed within 24 hours of collection to minimize changes in cellular composition. However, WB collection and cytometric analysis are often performed at different sites, which can result in sample processing delays. Several WB preservation reagents have been developed to address this challenge, including PROT1 (Smart Tube Inc.) and Cytodelics Whole Blood Cell Stabilizer (Cytodelics). However, not all antibody panels are compatible with these reagents. A CyTOF® panel was developed to be compatible with these commercial WB stabilizers and for use in pharmaceutical and clinical research. CyTOF flow cytometry uses metal-tagged antibodies to identify cellular and functional phenotypes. Advantages and features of CyTOF technology enable rapid design and application of 50-plus-marker panels and convenient workflows in which samples can be stained and acquired in a single tube. Compensation is not required since CyTOF flow cytometry has low signal spillover and no autofluorescence. Moreover, antibody cocktails and stained samples can be frozen for later use and acquisition. Thus, CyTOF technology overcomes major hurdles of fluorescence-based cytometry and provides a streamlined and flexible workflow in clinical research. The CyTOF panel contains 20 antibodies to identify over 30 immune cell populations. For easy customization, there are more than 30 additional open channels to analyze markers of interest. The panel works with fresh and stabilized WB samples and is amenable to different staining and acquisition workflows. To show the flexibility of sample staining and stabilization, WB from three healthy donors was assessed using two stabilization workflows. First, fresh WB samples were stained with the antibody panel, followed by PROT1 or Cytodelics stabilization and storage at -80 °C. The second workflow involved immediate stabilization/fixation of WB with PROT1 or Cytodelics and storage at -80 °C. Subsequently, the samples for the second workflow were thawed, surface stained, and acquired. To reduce technical variability from staining, the antibodies were pooled together and frozen at -80 °C as single-use aliquots. Furthermore, all samples were barcoded and acquired as a single tube to reduce variability from sample acquisition. The CyTOF panel developed for broad immune profiling is compatible with WB stabilizers, which overcomes traditional and logistical challenges with WB processing and acquisition. Furthermore, freezing antibody cocktails is a unique feature of CyTOF flow cytometry, ensuring batch-to-batch consistency in clinical research. Thus, CyTOF workflows enable swift and convenient analysis of WB samples. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Michael Cohen, Shakir Hasan, Stephen Li, Christina Loh. Enabling fast and convenient immune profiling of fresh and long-term stabilized human whole blood samples with CyTOF [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 79.

Abstract 7501: Gene expression profiling of rare cells captured from liquid biopsy using the genesis cell isolation system

Abstract Background: Targeting rare cell types such as circulating tumor cells (CTCs), considered to be metastatic precursors in cancer progression, may provide long-term clinical benefits by enhancing patient outcomes. The study of CTCs comes with technical challenges due to their heterogeneity and low cell numbers, leading to difficulties in genomic characterization. This study aims to overcome these technical challenges by developing a comprehensive gene expression profiling workflow using qPCR and RNA sequencing (RNA-Seq) on rare cells enriched with Genesis System with Celselect SlidesTM. Methods: To mimic CTCs in blood, we created a rare cell model sample by spiking A549 non-small cell lung cancer cells at concentrations of 50, 100, 500 and 1000 cells/ml in whole blood from healthy donors. The Genesis System, which can capture rare cells of 8µm-30µm, isolated 83.73% of spiked A549 cells in whole blood. Using the same blood samples without A549 spiked in, we enriched cells using the Genesis and it was used as a baseline for gene expression controls. High quality RNA was extracted from each of the enriched samples and used for both qPCR and RNA-Seq to detect the gene expression signature of the non-small cell lung cancer cells. Results: Our analyses revealed significant differential expression of cancer-related genes (p < 0.05) in all A549-spiked samples when compared to the blood-only controls. Notably, in the 50 cells/ml dilution, RNA-Seq analysis identified over 3,000 differentially expressed genes, encompassing protein-coding genes, long non-coding RNAs, and small non-coding RNAs. Among these, 759 genes were consistently present across all enriched samples, suggesting a common expression pattern. To investigate the potential utility of these genes as non-small cell lung cancer biomarkers, we constructed a custom qPCR array comprising 38 of these genes and pre-amplified their targets using 50pg of RNA from the enriched cell samples. Notably, six genes from this panel (MET, TOP2A, NF1, SPP1, GAPDH, and ANXA5) exhibited significant differential expression (+/- 4-fold, p<0.05) in the enriched samples. MET and TOP2A, in particular, are well-known biomarkers for non-small cell lung cancer. Conclusion: Our liquid biopsy workflow demonstrates the recovery of high-quality RNA from enriched rare cells, including CTCs. It enables comprehensive profiling of the tumor transcriptome, encompassing RNAs of various sizes, including miRNAs. Our approach has the potential to facilitate the identification of patient-specific biomarkers, enabling swift monitoring using preamplification and qPCR in advancement of targeted oncology therapy and research. Citation Format: Srikanth Perike, Angelica Olcott, Cissy Jiang, Nish Kumar, Nathan Knapp, Jennifer Placek, Candice Cox, Yoon-Tae Kang, Linda Lingelbach, Elizabeth Dreskin. Gene expression profiling of rare cells captured from liquid biopsy using the genesis cell isolation system [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 7501.

Abstract 3750: Dual isolation and profiling of circulating tumor cells and circulating tumor DNA from a single liquid sample using the Genesis Cell Isolation System and Droplet Digital PCR

Abstract Introduction: Recent advances in technology support the use of liquid biopsy as a noninvasive method for cancer monitoring and diagnosis. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are both regarded as key liquid biopsy biomarkers, offering significant disease-related molecular insights. Despite their potential, dual isolation and profiling have been challenged in a clinical setting by the lack of established platforms and optimized protocols that support this approach. The Genesis System with Celselect SlidesTM captures CTCs based on size and deformability, then the enriched cells can be recovered for subsequent downstream analysis. Concurrently, Droplet Digital PCR (ddPCRTM) can be utilized to interrogate cell-free DNA (cfDNA) to determine the presence of a range of DNA aberrations present in ctDNA. This study, using a model lung CTC-ctDNA sample, illustrates the feasibility of this dual-biomarker approach to detect cancer-associated nucleic acid mutations simultaneously with CTC enumeration, using commercially available kits and instruments. Methods: To model lung cancer samples, blood samples were prepared by spiking in two model circulating markers, cultured cancer cells (model CTCs, human ㅣlung cancer cell line A549) and fragmented genomic DNA containing EGFR mutations (model ctDNA, multiplexed 23 ctDNA [~145 bp band > 95%]), into whole blood from a healthy donor prior to enrichment/extraction. Plasma isolation from the whole blood model samples was performed using either two-step centrifugation or Ficoll-Paque PLUS solution for plasma/buffy coat separation. The extracted plasma layer was used for ctDNA separation, while the remaining cell layer was used for CTC isolation on the Genesis System with a modified protocol. Results: The Genesis System isolated 78-79% of spiked cancer cells in whole blood at a concentration of 40 cells/ml. Despite additional plasma separation steps and liquid handling, the system isolated CTCs spiked in with minimal cell loss (<6%). With an optimized ctDNA extraction, more than 70% of spiked ctDNA in blood samples in the size range of 120-220 bp was extracted. Using the Bio-Rad QX200 ddPCR System and ddPCR EGFR T790M Mutation Detection Assay, blood samples with the model ctDNA spiked in showed positive droplets (17 copies) for EGFR T790M while control blood samples without model ctDNA spiked in showed no positive droplets. Discussion and Conclusions: This work illustrates an optimized protocol for the dual isolation and profiling of CTCs and ctDNA from a single blood sample by using an automated CTC isolation platform and high-sensitivity ddPCR based ctDNA mutation detection. This protocol enables comprehensive liquid biopsy studies using a single blood sample from cancer patients with readily available instruments and kits, which can be applied to many other cancers. Citation Format: Yoon-Tae Kang, Abiodun Bodunrin, Errile Pusod, Kris Simonyi, Adam Corner, David Coe, Michelle Racey, Elizabeth J. Dreskin. Dual isolation and profiling of circulating tumor cells and circulating tumor DNA from a single liquid sample using the Genesis Cell Isolation System and Droplet Digital PCR [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 3750.

Cytotoxic CX3CR1+ T cells drive vascular inflammation in giant cell arteritis but not in Takayasu's arteritis.

To compare the involvement of cytotoxic CX3CR1+ T cell subsets between giant cell arteritis (GCA) and Takayasu's arteritis (TAK). We examined the proportions of CX3CR1+ CD4+ and CD8+ T cells in whole blood freshly obtained from 30 treatment-naive patients with active large vessel vasculitis (GCA, n=22 and TAK, n=8) and 16 healthy controls (HC). Infiltration of CX3CR1+ T cells into the affected arteries was assessed using immunohistochemical staining. Furthermore, CX3CR1+ CD4+ and CD8+ T cells were followed up after glucocorticoid treatment for longitudinal assessment of both diseases. The proportion of CX3CR1+ CD4+ T cells was significantly higher in GCA than in HC but not in TAK. No differences were observed in the proportions of CX3CR1+ CD8+ T cells among the GCA, TAK, and HC groups. The increased proportion of CX3CR1+ CD4+ T cells in GCA strongly correlated with the severity of systemic inflammation, whereas no significant correlation was found in TAK. Compared to TAK, CX3CR1+ CD4+ T cells from GCA patients showed significantly higher expression of granzyme B and perforin. The inflamed temporal arterial tissues of the GCA were infiltrated by numerous CX3CR1+ T cells, contributing to inflammation, disruption of the elastic lamina, and intimal hyperplasia. In contrast, no infiltration of CX3CR1+ T cells was observed in the aortitis lesions of TAK. Longitudinal analysis of post-glucocorticoid treatment showed a reduction in CX3CR1+ T cells in GCA, whereas no significant change was observed in TAK. Differences in immune mechanisms between GCA and TAK highlight cytotoxic CX3CR1+ T cells as potential drivers for GCA-related inflammation and vessel damage but not for TAK.

Abstract 7239: Eradication of B cell lymphoma in a mouse model through in vivo transduction of T cells with an aCD19-CAR utilizing a CD3-targeted lentiviral vector

Abstract One of the challenges in CAR-T cell therapies is the need for ex vivo processing of autologous T cells. The generation of CAR-T cells directly within the patient has therefore become an attractive alternative. Here, we pseudotyped lentiviral vectors with a VSV-G protein engineered to de-target the LDL receptor and to bind to CD3 via a displayed CD3-specific single-chain antibody (G-CD3), allowing for targeted recognition of T cells. In ex vivo experiments, we demonstrated that engineered G-CD3-LV specifically recognizes and transduces T cells in PBMC and whole blood without apparent off-target transduction of other cells. Furthermore, unlike G-WT-LV, which necessitates T cell activation for transduction, G-CD3-LV does not require pre-activation or the presence of cytokines for successful T cell transduction. The presence of CD3 antibodies on the surface of G-CD3-LV alone was sufficient to activate T cells, as evidenced by CD25 expression and the induction of T cell proliferation. For in vivo evaluation of retargeted LV efficiency NSG mice implanted with NALM6 B-lymphoma were treated with G-CD3 LV encoding αCD19-CAR/GFP (G-CD3/LV-CAR). The tumor was established by tail vein injection of NALM6/F-Luc cells, and 3 or 10 days later animals were humanized via intraperitoneal (IP) injection of human PBMC. Four hours after PBMC transfer, G-CD3/LV-CAR was delivered by IP route. Blood was drawn every week to monitor the generation of CAR-T cells, cytokine levels, and vector copy numbers. Tumor burden was monitored by bioluminescence imaging. Treatment with G-CD3/LV-CAR at both time points resulted in complete tumor eradication. Subsequently, significant levels of CAR-T cells were observed in the blood through Flow analysis, peaking at two weeks after LV injection. Importantly, mice treated on day 10 after tumor establishment reached higher levels of CAR-T cells compared to those treated on day 3 (CAR-T levels were 38±6.5% vs. 10±2.38% of hCD45+ cells, respectively). These results suggest that CAR-T cells proliferated proportionally in response to the level of tumor antigen. With the eradication of the tumor, CAR-T levels in the blood diminished. We also demonstrated that the levels of CAR-T cells correlated well with vector copy numbers. Throughout the treatment, mice did not exhibit any signs of toxicity or body weight loss. Additionally, by measuring cytokines in the blood produced by human cells, we found only a moderate increase in hIFNγ (0.852±0.681 ng/ml) at the peak of CAR-T cell levels in the blood, while the levels of hIL-2 and hTNFα were below the detection limit. In summary, these data highlight the efficiency and potential applicability of fully retargeted, serum stable LV vectors for in vivo CAR-T generation, offering promising prospects to optimize CAR-T cells therapy applications. Citation Format: Karina Krotova, Gopal Naik Nenavath, Nandakumar Packiriswamy, Rianna Vandergaast, Yumeng McDaniel, Melissa Moy, Zachary Larson, Luke Schnebeck, Chia-Hsuan Chin, Kyle Gromer, Kah-Whye Peng, Stephen J. Russell. Eradication of B cell lymphoma in a mouse model through in vivo transduction of T cells with an aCD19-CAR utilizing a CD3-targeted lentiviral vector [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 7239.

Soetomo COVID-19 Prognostic Score: A Multi-Parametric Model for Early Prediction of Disease Severity of COVID-19 in Tertiery -Resource Hospital

Objective: Coronavirus disease 2019 (COVID-19) became a global pandemic, with high mortality in severely ill patients. This study aimed to develop a novel scoring system to prognosticate disease severity in COVID-19 patients that is effective and widely available in tertiary medical resource settings.Material and Methods: Laboratory-confirmed COVID-19 patients were enrolled in this retrospective cohort, divided into severe and non-severe groups. We randomly assigned 70% of the subjects to establish a novel scoring system, while the remaining 30% was used for internal validation. The model was constructed by multivariate logistic regression using the first clinical, laboratory, and radiological finding of statistically analysis of group patients. receiver operating characteristic (ROC) and cross-tabulation were used to evaluate the performance of our score and compare it with other models.Results: A total of 599 patients were included. The Soetomo COVID-19 prognostic score predictors included age, fever, specific comorbidities (diabetes, hypertension, cardiac disease, lung tuberculosis), respiratory rate, heart rate, SF ratio, whole blood cell (WBC) count, neutrophil lympocyte ratio (NLR), blood urea nitrogen (BUN), and a RALE score. The area under the ROC of the model indicated an excellent discriminatory ability (training datasets 0.715 [95% CI 0.664-0.767, p-value<0.001]; testing datasets 0.720 [95% CI 0.638-0.802, p-value<0.001]). Our scoring system was superior to both qSOFA and MEWS regarding predictive value. The sensitivity and specificity were 60.6% and 82.5%, respectively.Conclusion: The developed scoring system accurately predicted a significant proportion of severe disease in COVID-19 patients.

Cancer cell membrane biomimetic composites of cisplatin prodrug for targeted eradication of circulating tumor cells in whole blood

The dissemination of circulating tumor cells in the peripheral blood circulation system plays a fundamental role in cancer recurrence and metastasis. Therefore, it is a pivotal scientific requirement to eradicate circulating tumor cells in whole blood system effectively. In this work, we provide a pH and GSH responsive drug delivery and release composites using cancer cell membrane biomimetic nanoparticles loaded with Pt(IV) prodrug (CM-Pt(IV) NPs), which exhibits high selectivity and biocompatibility. Cancer cells tend to form homogeneous aggregates by interacting with tumor-specific binding proteins on their surface. Thus, the cancer cell membrane coating possesses a homologous targeting ability to circulating tumor cells. The core nano-carrier composites loaded with Platinum(IV) agents were prepared by combining PLGA, Hyaluronic acid, and Cystamine. Within the nano-carrier composites, the S–S bond was reduced to S–H due to the high concentration of GSH present in cancer cells under an environment with a pH of 6.8. Consequently, this facilitates the release of platinum drugs that can specifically bind to cell DNA and effectively eradicate cancer cells. This highly selective delivery and precise release composites enable CM-Pt(IV) NPs to eliminate circulating tumor cells in the peripheral blood system effectively. This study provides an efficient drug delivery strategy for treating metastatic cancer.

Soluble Urokinase Plasminogen Activator Receptor (SuPAR) Analysis for Diagnosis of Periprosthetic Joint Infection.

Soluble urokinase plasminogen activator receptors (suPARs) are a biomarker for inflammatory diseases. This study aims to investigate its diagnostic properties regarding periprosthetic joint infections (PJI). This retrospective cohort study included adult patients who underwent joint puncture for suspected PJI. The presence of PJI was determined according to the criteria of the European Bone and Joint Infection Society (EBJIS). Laboratory study analyses included the determination of white blood cells (WBC) in whole blood, C-reactive protein (CRP) in blood plasma, and suPAR in both blood plasma and synovial fluid. Appropriate diagnostic cut-off values were identified utilizing Youden's J, and their diagnostic performance was determined by calculating the positive (PPV) and negative predictive value (NPV) for each marker. Sixty-seven cases were included in the final analysis. Forty-three samples (64%) were identified as periprosthetic joint infection (PJI) and twenty-four specimen (36%) were PJI negative cases. The PPV and NPV were 0.80 and 0.70 for synovial suPAR, 0.86 and 0.55 for CRP, 0.84 and 0.31 for WBC and 1.00 and 0.31 for plasma suPAR. Synovial suPAR showed a solid diagnostic performance in this study and has the potential to be an alternative or complementary biomarker for PJI. Further investigations in larger patient collectives are indicated.

OMIP-101: 27-color flow cytometry panel for immunophenotyping of major leukocyte populations in fixed whole blood.

This 27-color flow cytometry antibody panel allows broad immune-profiling of major leukocyte subsets in human whole blood (WB). It includes lineage markers to identify myeloid and lymphoid cell populations including granulocytes, monocytes, myeloid dendritic cells (mDCs), natural killer (NK) cells, NKT-like cells, B cells, conventional CD4 and CD8 T cells, γδ T cells, mucosa-associated invariant T (MAIT) cells and innate lymphoid cells (ILC). To further characterize each of these populations, markers defining stages of cell differentiation (CCR7, CD27, CD45RA, CD127, CD57), cytotoxic potential (perforin, granzyme B) and cell activation/proliferation (HLA-DR, CD38, Ki-67) were included. This panel was developed for quantifying absolute counts and phenotyping major leukocyte populations in cryopreserved, fixed WB collected from participants enrolled in large multi-site tuberculosis (TB) vaccine clinical trials. This antibody panel can be applied to profile major leukocyte subsets in other sample types such as fresh WB or peripheral blood mononuclear cells (PBMCs) with only minor additional optimization.