Principal Cells Research Articles

Abstract A051: Identifying patterns of cell-free DNA associated with elevated risk of diffuse large B-cell lymphoma in adult dogs

Abstract Background: Nonrandom patterns of cell-free DNA (cfDNA) are closely associated with malignancy. We hypothesize that they are also found in the systemic microenvironment that gives rise to cancer, and that such signatures can inform a risk stratification test. To achieve this, we have initiated the Lymphoma Risk Assessment (LyRA) study, using machine learning on a training set of dogs with known health status and a test set of otherwise healthy adult pet dogs to identify cfDNA signatures that are associated with elevated risk of developing diffuse large B- cell lymphoma (DLBCL). We will validate the predictive accuracy of these signatures, including the persistence of the risk environment and the interval between risk detection and development of clinical disease. Since cfDNA is the primary input of the LyRA test, we first performed a pilot study to assess how sample characteristics affect cfDNA quality and subsequent low-pass whole genome sequencing (WGS) data. Methods: Blood samples were collected from pet dogs with the owner’s informed consent. Paired samples were collected into potassium (K3)EDTA tubes and additive-free red top tubes (n = 5 dogs) or K3EDTA tubes and Cell-free DNA BCT® Streck Tubes (n = 6 dogs). Additional serum samples (n=3) were generously provided by the Morris Animal Foundation Golden Retriever Lifetime Study. cfDNA was isolated using the QIAamp® Circulating Nucleic Acid kit. Total cfDNA concentration was measured by PicoGreen™ and fragment distribution by Agilent High Sensitivity D500 ScreenTape® Assay, with comparisons made by starting material (plasma vs. serum) and collection tubes. Results: Serum had both higher total concentrations of cfDNA (PicoGreen™) and calibrated concentrations of long fragment cfDNA (515-658 bp Agilent traces) compared to plasma. In addition, total cfDNA concentrations increased with malignancy, following the pattern: no cancer < cancer < hematological cancer. Median fragment lengths of Agilent peaks also increased with malignancy for short (125-181 bp) and medium (340-425 bp) fragments, in both serum and plasma, but decreased for long fragments. In these pilot data the trends seen were consistent with our hypothesis and with prior studies, although due to sample size, no statistical testing was performed. Conclusions: The pilot study demonstrated that the effects of sample parameters on cfDNA quality are consistent with existing literature, validating our methods and supporting the rationale for the LyRA study. These features may prove valuable when integrated with genomic data to identify high-risk microenvironments. Recent research has shown that a majority of cfDNA in tumor patients originates from non-malignant cells and current methods often overlook potentially important patterns in this so-called 'discard cfDNA.' By focusing on these overlooked patterns, with an emphasis on risk stratification, the LyRA test could shift clinical management to a prevention-focused approach, serving as a model of testing and interception for DLBCL and other cancers in dogs, and potentially in humans. Citation Format: Lauren E Burt, Amy E Treeful, Mitzi Lewellen, Kimberly Demos-Davies, Andrea Chehadeh, Sara Pracht, Ashley J Schulte, Caitlin Feiock, Julia Medland, Davis Seelig, Kelly M Makielski, Ali Khammanivong, Antonella Borgatti, Michael S Henson, Michael Linden, Daniel Vallera, Gary R Cutter, Aaron L Sarver, Jaime F Modiano. Identifying patterns of cell-free DNA associated with elevated risk of diffuse large B-cell lymphoma in adult dogs [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A051.

Abstract A013: Quality control of the biobanked matched tissue: Blood samples for the development of diverse liquid based molecular assays

Abstract The availability of high-quality matched biological samples (tumor-blood) is the major limiting factor for research and development of novel, liquid biopsy-based assays in oncology. Frequently, ongoing specifically timed collections of blood samples are required. De-identified tissue blocks (formalin-fixed, paraffin embedded (FFPE) or flash frozen) matched with patient's blood samples collected in fixative-containing (Streck) of EDTA containing tubes, were collected at worldwide geographic locations and transferred to biobanking facility (Reference Medicine, Phoenix, AZ). Additional blood samples were collected at various times regarding the cancer surgery. Plasma and buffy coat samples were locally prepared and shipped to the biobank. A subset of blood samples without matched tissues from healthy donors were also collected. All samples were shipped under strict transport conditions. Quality control performed at the biobanking facility included pathology review and nucleic acids quality assessment. A total of over 3,000 cases were collected and provided to end-users working on various assays based on cfDNA (e.g. monitoring for minimal residual disease or multi-cancer early detection). Immuno-reactivity to various antigens in samples collected in Streck and EDTA tubes (from 50 patients) was assessed by peptide microarray binding assay. Feedback information on the quality of samples was shared in 900 cases. Approximately 4% of cases had failed internal pathology review, due to poor preservation, lack of adequate tissue composition, or low tumor volume and 11% failed internal nucleic acid quality assessment (DNA concetration and integrity). The blood collection tubes had no significant impact on serologic testing using peptide microarary assay. Customer feedback data showed that overall success rate exceeded 85%. Type of blood collection tubes (Streck vs EDTA) had no impact on serologic testing and studies looking at predicted neoantigens based on NGS data can be confirmed using peptide epitope arrays. Blood collected for NGS works without compromise on peptide arrays. Future validation of other collection tubes for blood or other types of fluids is needed to confirm their adequacy for specific assays. Citation Format: Zoran Gatalica, Phillip Stafford, Chris Diehnelt, Inga Rose. Quality control of the biobanked matched tissue: Blood samples for the development of diverse liquid based molecular assays [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A013.

Abstract A038: Investigating PD-L1 status in circulating tumor cells isolated from blood samples of lung cancer patients

Abstract Background: This research study aimed to assess the performance of a research use only (RUO) immunofluorescence (IF) assay targeting programmed death-ligand 1 (PD-L1) evolution in epithelial and/or mesenchymal circulating tumor cells (CTCs) isolated from blood using Parsortix® microfluidic technology. Upregulation of PD-L1 enables cancer cells to evade the host immune response. Assessment of PD-L1 status in the tumor, as determined by traditional tissue biopsy, can indicate if immunotherapy has the potential to be an effective treatment. However, PD-L1 expression in tumor biopsies may not reflect metastatic sites in their entirety and can become outdated during tumor evolution, as the process cannot be repeated due its invasive nature. Liquid biopsy offers a minimally invasive option for dynamic testing of PD-L1 in CTCs as patients undergo treatment. Methods: Analytical linearity, and specificity and sensitivity were assessed by spiking HCC1954 and Hs 578T cancer cell lines into the blood samples of 34 healthy volunteers. The assay was then evaluated in samples from 47 metastatic lung cancer patients, among which 32 had a known PD-L1 tissue status (19 positive, 13 negative). Up to six successive draws were taken for each patient, collected into Streck Cell-Free DNA blood collection tubes. ANGLE’s Parsortix® instruments were used to isolate spiked cancer cells or CTCs from blood samples based on their size and deformability. The CTC- enriched harvests were processed onto ANGLE’s CellKeepTM slides and IF stained with ANGLE’s Portrait® PD-L1 RUO assay for PD-L1 identification on CTCs. Slides were imaged on a BioView automated imaging system. Results: Analytical specificity of PD-L1 was 98% and analytical sensitivity was 81%. Analytical linearity (R2=0.91) was shown over a 0–500 cells range. In metastatic lung cancer patients, ≥1 CTC was identified in at least one draw for 91% of donors, with 55% of those donors exhibiting ≥1 PD-L1 positive CTC. A majority (81%) of donors exhibited a positive correlation between PD-L1 tissue status and CTC expression. However, the remaining 19% of donors exhibited discordance with a PD-L1 negative tissue status but PD-L1 positivity identified in ≥1 CTC. Of the PD-L1 positive CTCs identified, 98% expressed mesenchymal markers. Dynamic change was shown in all but one donor, with PD-L1 status of the CTCs changing over time. Conclusions: This study demonstrated the ability to determine PD-L1 status in CTCs from the blood of metastatic lung cancer patients and, subject to further study, the future potential to develop dynamic PD-L1 testing for advancement of more personalized cancer treatments. Patients with a PD-L1 negative tissue status exhibited PD-L1 positive CTCs, demonstrating the feasibility of a more repeatable and accurate assessment of PD-L1 status than can be achieved through a traditional tissue biopsy. Citation Format: Morgan Spode, Chloe Goodwin, Aarabhi Varatharajah, Aaron Cottingham, Elisha Duhig, Laura Kaja, David Greaves, Mariacristina Ciccioli, Anne-Sophie Pailhes-Jimenez. Investigating PD-L1 status in circulating tumor cells isolated from blood samples of lung cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A038.

Abstract A024: Evaluating the effect of plasma-related preanalytical variables on the robustness of the liquid biopsy early CRC detection Signal-C assay

Abstract Liquid biopsies and cell-free DNA (cfDNA) analysis have emerged as novel non-invasive diagnostic tools in oncology. Analysis of cfDNA methylation status has been proposed as promising approach for several diagnostic purposes, and robust methylation-detecting technologies are of primary importance for the clinical implementation of novel diagnostic assays. Signal-C is an accurate NGS-based methyl-sensitive assay detecting colorectal cancer (CRC) currently under clinical validation. In this study we assessed the influence of different preanalytical variables influencing plasma stability, as the storage time of plasma samples and the tube type used for blood collection, on Signal-C assay results to determine its robustness. 958 samples with 3ml to 4ml plasma were used for cfDNA extraction and run through Signal-C assay following UDX protocol. In brief, 8 to 20ng cfDNA were used as an input for methylation- specific library preparation, which was run according to manufacturer’s instructions. Hybrid- capture was performed to enrich libraries for genomic regions included in Signal-C assay and sequenced on Illumina Novaseq machines. Relevant sequencing QC metrics, such as depth filtered, read yield from raw to filtered and PCR duplicate proportion were calculated, together with cancer predictions according to Signal-C test. Pearson coefficient and Pearson's Chi- squared test with Yates' continuity correction have been used to assess correlations, and Wilcoxon test has been used to measure the significance of the differences between quality metrics with respect to the tube type. We have analyzed the influence of different plasma-related preanalytical variables on sequencing metrics and cancer predictions of Signal-C assay. Plasma age, calculated as the time elapsed between blood collection and cfDNA quantification, shows a small negative correlation with YIELD_RAW_FILTERED (R = - 0.08, p=0.013) and a small positive correlation with PCR_DUP_PROP (R = 0.098, p=0.0024), while it doesn't influence DEPTH_FILTERED values (p>0.05). Use of different blood collection tubes (EDTA or Streck tubes) influenced in a small but significative manner the YIELD_RAW_FILTERED and the PCR_DUP_PROP (p<0.001), while it doesn’t affect the DEPTH_FILTERED values (p>0.05). Despite the sequencing QC metrics being subject to small but statistically significant differences, the overall cancer classification performance of Signal-C assay was not affected by the changes in plasma age (Pearson's product-moment correlation, sensitivity p=0.87, specificity p=0.091) nor in tube type used (Pearson's Chi-squared test with Yates' continuity correction, p=0.219). Here we evaluated the effect of plasma’s storage time and the type of blood collection tube on Signal-C assay performance. While signal processing features show small differences between the tested variables, this does not affect overall clinical performance of the test, supporting its robustness. Additional studies to analyze the effect of other preanalytical variables on Signal-C performance are underway to establish its robustness. Citation Format: Fernando Trincado Alonso, Pol Canal Noguer, Kristi Kruusmaa, Arianna Bertossi. Evaluating the effect of plasma-related preanalytical variables on the robustness of the liquid biopsy early CRC detection Signal-C assay [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A024.

Abstract A036: Next gen liquid biopsy: Comprehensive analysis from a single tube of blood

Abstract Introduction There remains a largely untapped potential for utilizing liquid biopsy as a non- invasive, real-time window into tumor biology. In this study, we report on the use of the high- multiplex protein quantitation capability of the OrionTM spatial biology platform (RareCyte®, Inc) and cell picking capability of the CyteFinder® rare cell platform, to perform a comprehensive liquid biopsy analysis of whole blood that provides a deeper understanding of samples. This novel technology was used to characterize circulating tumor cells (CTCs), perform immune profiling of white blood cells (WBCs), and perform targeted mutation profiling of CTCs and cell-free DNA (cfDNA). All of these analytes can be measured from one 7.5 mL draw of blood. This array of tests was applied to cancer samples to demonstrate clinical feasibility. MethodsBlood samples were collected into AccuCyte® blood collection tubes. Buffy coats were processed to microscope slides using AccuCyte® blood processing kits. Slides were fixed and stained with an 18-plex immuno-oncology panel and imaged using the Orion Spatial Biology platform to evaluate protein biomarker expression on CTCs and to profile WBCs. Individual CTCs and WBCs from clinical samples were isolated using the CytePicker® for downstream molecular analysis. cfDNA was isolated from plasma collected during the AccuCyte process. Picked CTCs, WBCs, and cfDNA were sequenced using the CleanPlex® OncoZoom® Cancer Hotspot Kit. ResultsCancer patient blood was tested with a high-plex immunofluorescence assay using the Orion spatial biology system. CTCs were identified and phenotypically characterized in multiple cancer types. On the same slides, immunophenotyping of WBCs was performed. T- cell, B-cell, and macrophage populations were quantified. Additional sub-populations of T-cells (regulatory and memory) and proliferating cells (Ki67+) were enumerated. Expression of checkpoint-specific markers PD-L1 and PD-1 was measured on CTCs and WBCs. Sequencing results from individual picked CTCs showed evidence of minor clones representative of tumor heterogeneity that were not detectable in the cfDNA analysis. Conclusions This novel application of a high-plex spatial biology imaging system to liquid biopsies, in conjunction with the proven capabilities of rare cell detection and genomic analysis of single cells and cfDNA, has the potential to revolutionize liquid biopsy testing. From a single blood sample, this approach allows one to ask important questions about the complex interactions between the immune system and the tumor and to follow these interactions longitudinally in real time over the course of the disease. The goal is to leverage this unprecedented depth of analysis to improve patient outcomes and better understand the biological complexity of disease. Future directions of this work are to study rare types of WBCs that indicate immune activation and response to the tumor, and the detection and longitudinal monitoring of cell therapies such as CAR T cells after infusion. Citation Format: Arturo B Ramirez, Jon Ladd, Erin Bayer, Brock Bartels, Rachel Ponting, Arista Tischner. Next gen liquid biopsy: Comprehensive analysis from a single tube of blood [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A036.

Abstract A032: Performance evaluation of a new protein stabilizing blood collection tube with a novel whole blood separation device for clinical plasma proteomics

Abstract Introduction: We have developed a novel blood separation device (BCD) which separates plasma from cellular components by lateral flow and stabilizes the sample for transport to a testing laboratory. Patient blood is drawn into an anticoagulant blood collection tube (BCT) and then applied to the separation device. There is outstanding interest in the field of clinical proteomics regarding the formulation in BCTs and whether they would also additionally the plasma proteome. This is especially critical where hemoglobin may provide interference to the analyte being measured. We evaluated the performance of a newly available plasma protein stabilizing BCT in combination with the BCD and other currently available K2EDTA BCT (no stabilization). Methods: Whole blood from normal healthy donors was drawn into protein stabilizing BCT and a comparator anticoagulant BCT. At baseline, 24-, 48-, 72- and 120-hour time points, a 250 μL aliquot of whole blood from each tube was spotted on to the BCD to allow separation of the cellular components and plasma. All of the test specimens on the spotted devices were allowed to dry overnight to replicate time in transit. After drying, proteins were subjected to MALDI-TOF MS (mass spectrometry) after extraction with water and co- crystallization with sinapinic acid. Spectra were acquired in triplicate and m/z feature intensities for hemoglobin were assessed in R. Results: Visual inspection of the new protein stabilizing and K2EDTA BCTs showed the steady accumulation of hemoglobin over time in the plasma layer in the latter tubes. After spotting on the BCD and MALDI-TOF analysis, plasma from the protein- stabilizing BCT showed no significant increases in either the hemoglobin α or β subunits over the five (5) day period. This was consistent across all donors (heme α p = 0.7933, heme β p = 0.6425) and at all timepoints assessed. In the K2EDTA BCT, both the α and β hemoglobin subunits increased significantly over time and across all donors (heme α p < 0.0001, heme β p = 0.0232). The hemoglobin α subunit accumulated in the plasma component to a greater degree than did the β subunit (heme α slope = 4.863E-5, heme β slope = 1.398E-5). These data suggest that the formulation in the protein stabilizing BCT may attenuate red blood cell hemolysis to a greater degree as compared to conventional K2EDTA blood collection tubes. Additionally, the mean reproducibility for both hemoglobin subunits was lower when the protein stabilizing BCT was used than when the anticoagulant BCT was used across all donors at all time points (protein stabilizing BCT x ̅ CV = 8.5%, anticoagulant BCT x ̅ CV = 8.9%). Conclusions: The use of the newly available protein stabilizing BCT with the BCD reduces the accumulation of hemoglobin in the plasma component while improving reproducibility when compared to the currently used anticoagulant BCT. Interference by heme was significantly attenuated as measured by a sensitive MALDI-ToF assay. Citation Format: Colin McDowell, Nylev Vargas, Gary A Pestano. Performance evaluation of a new protein stabilizing blood collection tube with a novel whole blood separation device for clinical plasma proteomics [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A032.

Autologous Serum Tear Production in Anticoagulated Patients.

Autologous serum tears (ASTs) are produced from serum obtained by blood draw and used to treat severe dry eye diseases. Multiple cases of failed AST production occurred due to serum becoming a solidified, gel-like substance after centrifugation. The aim of this study was to identify risk factors associated with inability to compound AST. From January 2019 to December 2022, 99 patients received AST produced at Mayo Clinic in Arizona. Patient demographics, medical diagnoses, medications, laboratory results, and events surrounding blood draws were obtained through an IRB-approved retrospective chart review. The laboratory and pharmacy protocol procedures were observed in person after the occurrence of the last production issue. Ten total clotted serum episodes occurred in 4 patients, each with multiple occurrences. The indication for AST was chronic ocular graft-vs-host disease (n = 3) and Sjögren syndrome (n = 1). Eighty percent of episodes were associated with either a systemic infection (n = 7) or pulmonary embolism (n = 1) within the prior month. At the time of blood draw, all 4 patients were treated with antiplatelet and/or anticoagulation medications, including rivaroxaban, apixaban, aspirin, and low-molecular-weight heparin. The AST protocol was increased from 30 to 60 minutes of resting time to allow clotting before centrifugation. Inadequate clotting during this stasis period likely resulted in fibrinogen remaining suspended in the serum, leading to solidified plasma instead of liquid serum. We hypothesize risk of inadequate clotting would be increased in patients taking anticoagulation or antiplatelet medications. Our recommendations include ensuring 60-minute collection tube resting time for all patients, including those on anticoagulation medication.

Thick Ascending Limb Specific Inactivation of Myh9 and Myh10 Myosin Motors Results in Progressive Kidney Disease and Drives Sex-specific Cellular Adaptation in the Distal Nephron and Collecting Duct.

Our previous work established a role for actin associated myosin motor proteins MYH9 and MYH10 in the trafficking of thick ascending limb (TAL) specific cargoes, uromodulin (UMOD) and Na+K+2Cl- cotransporter (NKCC2). Here, we have generated a TAL-specific Myh9&10 conditional knockout (Myh9&10 TAL-cKO) mouse model to determine the cell autonomous roles for MYH9&10 proteins in TAL cargo transport and to understand the consequence of TAL dysfunction in the adult kidney. Myh9&10 TAL-cKO mice develop progressive kidney disease with pathological tubular injury confirmed by histological changes, tubular injury markers, upregulation of ER stress/unfolded protein response pathway, and higher blood urea nitrogen and serum creatinine. However, male mice survive twice as long as female mice. We determined that the sexual dimorphism in morbidity is due to adaptation of the distal nephron and the collecting ducts in response to TAL dysfunction and significantly lower NKCC2 expression. We demonstrate that this triggers a compensatory mechanism involving sex-specific cellular adaptation within the distal tubules and collecting ducts to boost sodium reabsorption. While both sexes overcompensate by activating ENaC expression in the medullary collecting ducts resulting in hypernatremia, this is subdued in male Myh9&10 TAL-cKO mice as they initially promote higher sodium chloride cotransporter (NCC) expression within the distal nephron. Our results indicate that compromised TAL function results in maladaptation of medullary collecting duct cells, which acquire cortical-like properties, including ENaC expression. This work further confirms a cell autonomous role for myosin motor proteins MYH9&10 in the maintenance of NKCC2 expression in the TAL and uncover adaptive mechanisms of the distal nephron and the collecting duct segments in response to TAL dysfunction.

Restoring Compromised Cl- in D2 Neurons of a HD Mouse Model Rescues Motor Disability.

Huntington's disease (HD) is a progressive neurodegenerative disorder with no cure, characterized by significant neurodegeneration of striatal GABAergic medium spiny neurons (MSNs). Early stages of the disease are characterized by the loss of dopamine 2 receptor-expressing MSNs (D2 MSNs) followed by degeneration of dopamine 1 receptor-expressing MSNs (D1 MSNs), leading to aberrant basal ganglia signaling. While the early degeneration of D2 MSNs and impaired GABAergic transmission are well-documented, potassium chloride cotransporter 2 (KCC2), a key regulator of intracellular chloride (Cl-), and therefore GABAergic signaling, has not been characterized in D1 and D2 MSNs in HD. We aimed to investigate whether Cl- regulation was differentially altered in D1 and D2 MSNs and may contribute to the early degeneration of D2 MSNs in male and female symptomatic R6/2 mice. We used electrophysiology to record the reversal potential for GABAA receptors (EGABA), a read-out for the efficacy of Cl- regulation, in striatal D1 and D2 MSNs and their corresponding output structures. During the early symptomatic phase (P55-P65), Cl- impairments were observed in D2 MSNs in R6/2 mice, with no change in D1 MSNs. Cl- regulation was also dysfunctional in the globus pallidus externa, resulting in GABA-mediated excitation. When we overexpressed KCC2 in D2 MSNs using AAV-mediated delivery, we delayed the onset of motor impairments in R6/2 mice. We demonstrate that Cl- homeostasis is differentially altered in D1 and D2 MSNs and may contribute to the enhanced susceptibility of D2 MSNs during HD progression.Significance Statement Huntington's Disease is an inherited neurodegenerative disease caused by a repeat expansion in the Huntingtin gene and characterized by the sequential loss of dopamine 2 and dopamine 1 receptor-expressing medium spiny neurons (D2 and D1 MSNs) of the striatum. MSNs release GABA, which depends on proper Cl- regulation for inhibition. We asked whether Cl- homeostasis is differentially altered in D1 and D2 MSNs and their output structures, and whether this altered expression contributes to the pattern of degeneration between these two principal striatal cell types. Using electrophysiology, biochemistry, and fluorescence imaging, we determined that Cl- regulation was impaired in D2 MSNs in R6/2 mice, with no change in D1 MSNs. Cl- was also dysregulated in the globus pallidus externa resulting in excitatory GABA.

Activation of 5-HT7 receptors in the mouse dentate gyrus does not affect theta-burst-induced plasticity at the perforant path synapse.

The study examined the effects of 5-HT7 receptor activation on GABAergic transmission within the dentate gyrus and plasticity at the glutamatergic perforant path input. Immunofluorescence imaging was performed using transverse hippocampal slices from transgenic mice expressing green fluorescent protein (GFP) under the Htr7 promoter. This was followed by whole-cell patch clamp electrophysiological recordings assessing the effects of pharmacologically activating 5-HT7 receptors on spontaneous inhibitory postsynaptic currents recorded from dentate granule cells and hilar mossy cells-two glutamatergic neuron types present in the dentate gyrus. Extracellular recordings of field excitatory postsynaptic potentials were then performed to assess whether 5-HT7 receptor activation influenced theta-burst stimulation-evoked plasticity of the perforant path synaptic input. It was found that parvalbumin and somatostatin interneurons in the dentate gyrus expressed GFP, which suggests they express 5-HT7 receptors. However, activation of 5-HT7 receptors had no effect on GABAergic transmission targeting mossy cells or granule cells. There was also no effect of 5-HT7 receptor activation on perforant path plasticity either with intact or blocked GABAA receptor signaling. The presence of 5-HT7 receptors in a subset of parvalbumin and somatostatin interneurons in the mouse dentate gyrus could mean that they are involved in the inhibitory control of dentate gyrus activity. However, this potential effect was not evident in slice recordings of inhibitory transmission targeting principal cells and did not affect perforant path plasticity. Further experiments are needed to fully elucidate the functional role of these receptors in the dentate gyrus.

Single Nucleus RNA Sequencing Reveals Cellular and Molecular Responses to Vanadium Exposure in Duck Kidneys

Vanadium (V) exposure is known to induce renal toxicity, yet its specific effects on renal cell types and molecular mechanisms remain incompletely understood. We used single nucleus RNA sequencing (snRNA-seq) to characterize the impact of V on duck kidney cells at a cellular resolution. Following a 44-day exposure, immunofluorescence analysis revealed a significant increase in α-SMC expression in the renal interstitium, indicative of fibrotic response. SnRNA-seq identified 12 major cell types organized into 19 clusters within the kidney. Significant changes in cell composition were observed, notably an increase in proximal tubule cells (PT2 subtype), glomerular endothelial cells, principal cells, and alterations in immune cell proportions, while collecting duct intercalated cells (CD-IC) and thick ascending limb showed decreased percentages. Differential gene expression analysis highlighted pathways implicated in V toxicity across different cell types. Changes in drug metabolism-cytochrome P450, butanoate metabolism, and actin cytoskeleton regulation were exhibited by PT cells. Alterations in collecting duct secretion, oxidative phosphorylation, and bicarbonate reclamation pathways were shown in CD-IC cells. Furthermore, immune cells displayed changes in T cell receptor and chemokine signaling pathways, indicative of altered immune responses. Taken together, these findings contribute to a better shedding light on the pathogenic mechanisms of V induced renal injury.

COMPARISON OF YIELD AND QUALITY OF CIRCULATING CELL-FREE DNA FROM K2EDTA AND K3EDTA COLLECTIONS IN HEALTHY SUBJECTS

Background: Circulating cell-free DNA (cfDNA) is a biomarker for various clinical applications, including detecting and monitoring cancer. However, blood collection tubes can affect the yield and quality of cfDNA. Since specific cfDNA collection tubes are costly, K2EDTA and K3EDTA anticoagulant tubes are alternatives in routine clinical laboratories. Objectives: This study aimed to compare the efficiency of cfDNA extraction from plasma collected in K2EDTA and K3EDTA tubes and evaluate implementation for molecular diagnostics. Methods: Blood samples from 38 healthy subjects were collected in K2EDTA and K3EDTA tubes that were processed within 2 hours. The extracted cfDNA was measured and performed using SYBR Green-based qPCR for three endogenous reference genes (GAPDH, HPRT1, TFRC). The cfDNA yield and the amplification efficiency of these genes were compared between K2EDTA and K3EDTA tubes using the Mann-Whitney U test. Results: There were no significant differences in cfDNA concentration between K2EDTA and K3EDTA tubes (p=0.051). However, qPCR analysis revealed significantly higher copy numbers of TFRC and HPRT1 in K2EDTA tubes than in K3EDTA tubes (p<0.05). No significant difference was found for GAPDH. Conclusion: The results indicate that K2EDTA and K3EDTA tubes are an alternative option for cfDNA analysis if samples are processed quickly after a blood draw, which offers flexibility and cost savings in resource-limited areas.

Comparison of a two-step Tempus600 hub solution single-tube vs. container-based, one-step pneumatic transport system.

Efficient and timely transportation of clinical samples is pivotal to ensure accurate diagnoses and effective patient care. During the transportation process, preservation of sample integrity is crucial to avoid pre-analytical aberrations on laboratory results. Here, we present a comparative analysis between a two-step Tempus600 hub solution single-tube and a one-step, container-based pneumatic transport system (PTS) from Airco, for the in-house transportation of blood samples. Ten blood samples from healthy volunteers were split in 10 mL collection tubes filled at full or half capacity for transportation with the two PTS (about 250 m). To compare the impact of transportation, markers of hemolysis such as lactate dehydrogenase (LDH), potassium (K+), and the hemolysis index (HI), were determined. Additionally, differences in HI in routine samples and repeated transportation was investigated. To assess and compare the mechanistic impact profiles, we recorded the acceleration profiles of the two PTS using a shock data logger. Transportation using the Tempus600 hub solution resulted in 49 and 46 % higher HI with samples filled to total or half capacity, respectively. Routine samples transported with the Tempus600 hub solution showed a higher median HI by 23 and 33 %. Additionally, shock logger analysis showed an elevated amount of shocks (6.5 fold) and shock intensities (1.8 fold). The Tempus600 hub solution caused an increased number of unreportable LDH or K+ results based on the hemolysis index. However, it was only statistically significant for LDH (p<0.01 and p<0.08)- while the comparisons for K+ were not statistically significant (p<0.28 and p<0.56).

The Impact of Tube Type, Centrifugation Conditions, and Hemolysis on Plasma Circulating MicroRNAs.

In recent years, liquid biopsy has emerged as a promising tool for the diagnosis and prognosis of numerous diseases, including cancer. Among the biomolecules analyzed in liquid biopsies are plasma circulating microRNAs (miRNAs), small non-coding RNAs that have proven to be crucial in the regulation of gene expression and the pathobiology of different health conditions, making them useful as biomarkers. However, variations in preanalytical conditions during biospecimen collection and processing can affect the analytical results. Herein, we determined how the type of collection tube, the number of centrifugations, and the degree of hemolysis can affect plasma circulating miRNA levels. A cohort of 11 healthy donors was included. Whole blood was collected and handled in three different conditions, and miRNAs levels were analyzed using quantitative RT-PCR. Our results show that the differences in the type of preservative tubes influence hemolysis, measured as OD at 414 nm. Moreover, the number of centrifugations performed also altered miRNAs levels, increasing or decreasing them depending on the miRNA analyzed. Hence, our study shows that alterations in preanalytical conditions affect miRNAs levels, particularly the number of centrifugations and the type of collection tubes. In our work, we highlight the importance of registering the preanalytical conditions in a standardized way that might be considered when analytical results are obtained.

The biology of water homeostasis.

Water homeostasis is controlled by a brain-kidney axis that consists of central osmoreceptors, synthesis and secretion of arginine vasopressin (AVP) and AVP-responsive aquaporin-2 (AQP2) water channels in kidney collecting duct principal cells that facilitate water reabsorption. In addition to AVP, thirst represents a second line of defense to maintain water balance. Water balance disorders arise because of deficiency, resistance or inappropriate secretion of AVP or disturbances in thirst sensation (hypodipsia, polydipsia). People with water balance disorders are prone to develop hyponatremia or hypernatremia, which expose cells to osmotic stress and activate cell volume regulation mechanisms. This review covers several recent insights that have expanded our understanding of central osmoregulation, AQP2 regulation and cell volume regulation. This includes the role of with-no-lysine kinase 1 (WNK1) as a putative central osmolality sensor and, more generally, as an intracellular crowding sensor that coordinates the cell volume rescue response by activating sodium and potassium cotransporters. Furthermore, several new regulators of AQP2 have been identified, including for AVP-dependent AQP2 regulation (yes-associated protein, nuclear factor of activated T-cells, microRNAs) and AVP-independent AQP2 regulation (epidermal growth factor receptor, fluconazole, prostaglandin E2). It is also becoming increasingly clear that long-term cell volume adaptation to chronic hypotonicity through release of organic osmolytes comes at the expense of compromised organ function. This potentially explains the complications of chronic hyponatremia, including cognitive impairment, bone loss and vascular calcification. This review will illustrate why these new insights derived from basic science are also relevant for developing new approaches to treat water balance disorders.

Regulation of the gap junction interplay during postnatal development in the rat epididymis.

During postnatal development of the rat epididymis, a change in the expression of gap junction proteins, or connexins (Cxs), occurs, in which Gjb2 (Cx26) and Gja1 (Cx43) levels in the proximal epididymis are decreased, while Gjb1 (Cx32), Gjb4 (Cx30.3) and Gjb5 (Cx31.1) levels increase. The mechanism(s) responsible for the switch in Cx expression is unknown. The aim of this study is to identify the mechanisms responsible for the decrease in GJB2 protein levels and the increase in other Cxs during postnatal development. Results indicate that decreased Gjb2 expression for 48h does not alter the expression of other Cxs in RCE-1 principal cells, suggesting a lack of compensatory expression. Sequence analysis of both Gjb2 and Gjb1 promoters identified common multiple response elements to steroid hormones. Using RCE-1 cells, we observed that dexamethasone increased Gjb2 mRNA levels by twofold after 48h, while estradiol had no effect. Orchidectomy in rats resulted in a significant increase in GJB2 and decreased GJB1 in the caput and corpus epididymidis. Changes in Cxs protein levels were prevented by testosterone in orchidectomized rats. Similar results were observed in the prostate, another androgen-receptive organ. LNCaP cells, which are androgen-responsive, showed that exogenous dihydrotestosterone (DHT) decreased Gjb2 mRNA levels by approximately 50% concomitant with a 1.5-fold increase in Gjb1 levels. Using a GJB1 promoter construct we showed that DHT could induce transactivation of the luciferase transgene, while transactivation of two GJB2 promoters were unaltered. Results indicate that androgens and glucocorticoids regulate the expression of epididymal Cxs.

Case-Based Training on Best Practices in Effective Test Utilization for Clinical Laboratories: A Comparison of Two Educational Formats

Abstract Introduction/Objective While a survey of ASCP membership showed improvements being made in effective test utilization (ETU), there is an ongoing need for formal education on best practices. Case-based education drawn from real-world scenarios provides an opportunity to address this need, aimed at increasing the understanding of evidence- based guidelines to inform clinical test ordering and reduce overconsumption of supplies without compromising patient care. The poster presents two examples of case-based education presented in an innovative microlearning format and an eLearning format designed for clinical laboratory professionals. Methods/Case Report The activities presented 9 - 10 practical, real-world scenarios as educational cases along with assessment questions addressing a range of topics that included ETU for pancreatitis and cardiac injury, blood collection tube consumption and conservation, strategies for reducing repeat daily lab test ordering, and others. Each case was designed for learners to be able to review the content and answer the assessment questions in 5 minutes. In the microlearning activity, learners received the cases and questions via email on a scheduled frequency and completed them via mobile device. The questions were repeated up to 2 times to reinforce learning. Participants in the eLearning activity accessed the same cases and assessment questions via a different learning platform (with no staggered delivery). Results (if a Case Study enter NA) More than 2,500 learners have participated in one or both ETU activities, and feedback has been highly positive. The assessment results show proficiency gains for a range of laboratory personnel, including laboratory managers/supervisors, histotechnologists, medical laboratory scientists, laboratory technicians, pathologists, and other laboratory roles. Conclusion The results also show how case-based scenarios can help promote best practices in ETU, which may be particularly beneficial for laboratory decision-makers. In addition to showing increased understanding of ETU strategies, the results also pose lessons learned for designing similar types of case-based education.

On-Demand Seizures Facilitate Rapid Screening of Therapeutics for Epilepsy.

Animal models of epilepsy are critical in drug development and therapeutic testing, but dominant methods for pharmaceutical evaluation face a tradeoff between higher throughput and etiological relevance. For example, in temporal lobe epilepsy, a type of epilepsy where seizures originate from limbic structures like the hippocampus, the main screening models are either based on acutely induced seizures in wild type, naïve animals or spontaneous seizures in chronically epileptic animals. Both types have their disadvantages - the acute convulsant or kindling induced seizures do not account for the myriad neuropathological changes in the diseased, epileptic brains, and spontaneous behavioral seizures are sparse in the chronically epileptic models, making it time-intensive to sufficiently power experiments. In this study, we took a mechanistic approach to precipitate seizures "on demand" in chronically epileptic mice. We briefly synchronized principal cells in the CA1 region of the diseased hippocampus to reliably induce stereotyped on-demand behavioral seizures. These induced seizures resembled naturally occurring spontaneous seizures in the epileptic animals and could be stopped by commonly prescribed anti-seizure medications such as levetiracetam and diazepam. Furthermore, we showed that seizures induced in chronically epileptic animals differed from those in naïve animals, highlighting the importance of evaluating therapeutics in the diseased circuit. Taken together, we envision our model to advance the speed at which both pharmacological and closed loop interventions for temporal lobe epilepsy are evaluated.

Proteomic analysis of CD29+ Müller cells reveals metabolic reprogramming in rabbit myopia model

The prevalence of myopia is rapidly increasing, significantly impacting the quality of life of affected individuals. Prior research by our group revealed reactive gliosis in Müller cells within myopic retina, prompting further investigation of their role in myopia, which remains unclear. In this study, we analyzed protein expression changes in CD29+ Müller cells isolated from a form deprivation-induced rabbit model of myopia using magnetic activated cell sorting to investigate the role of these cells in myopia. As the principal glial cells in the retina, Müller cells exhibited significant alterations in the components of metabolic pathways, particularly glycolysis and angiogenesis, including the upregulation of glycolytic enzymes, such as lactate dehydrogenase A and pyruvate kinase, implicated in the adaptation to increased metabolic demands under myopic stress. Additionally, a decrease in the expression of proteins associated with oxygen transport suggested enhanced vulnerability to oxidative stress. These findings highlight the proactive role of CD29+ Müller cells in modifying the retinal environment in response to myopic stress and provide valuable insights into mechanisms that could help mitigate myopia progression.

Hierarchical Classification of Factors Associated With Noninvasive Prenatal Testing Failures and Its Impact on Pregnancy Outcomes

Abstract Objective To conduct a hierarchical classification analysis of the nonreportable results of noninvasive prenatal testing in an attempt to reduce failure rates and provide pregnant women with accurate information to alleviate their anxiety. Methods In this study, 30,039 singleton pregnancies who underwent noninvasive prenatal testing in a single center from May 2019 to April 2022 were collected, and 811 samples with initial noninvasive prenatal testing failure were retrospectively analyzed. Grouping was based on the reasons for initial test failure; tracking the noninvasive prenatal testing results and prenatal diagnosis results (if any) of the “z-scores in the gray area” group and analyzing the possible influencing factors of the “low fetal fraction” group in the pre-experimental and experimental period by using one-way analysis of variance, Mann-Whitney U test, and χ2 test; and tracking the pregnancy outcomes of the test failures samples to analyze the risk of perinatal complications and adverse pregnancy outcomes of the different types of test failures. Results None of the samples' initial nonresults because of z-scores in the gray area were ultimately found to have chromosomal aneuploidy. However, pregnancy complications (P = 0.018) and a high likelihood of adverse pregnancy outcomes (P = 0.048) may still occur. Maternal gestational age (P &lt; 0.001), body mass index (P &lt; 0.001), library concentration (P &lt; 0.001), and fetal gender (P &lt; 0.001) were considered to be the associated factors for the initial low fetal fraction results. This may be associated with pregnancy complications (P &lt; 0.001) and a high likelihood of adverse pregnancy outcomes (P = 0.034). The body mass index (P = 0.015) and time between draws (P = 0.001) were associated with the second test’s success. The incidence of low fetal fraction samples was more frequent with blood collection tubes of the G type than with the K type (P &lt; 0.001). Conclusion Initial nonresults because of z-scores in the gray area did not imply an increased risk of aneuploidy, but vigilance is needed for an increased risk of pregnancy complications and adverse pregnancy outcomes. Because of the low fetal fraction, the initial absence of results may be related to the assay method, as well as the effect of blood collection tubes and the need to be alert to the risk of pregnancy complications and adverse pregnancy outcomes.