Quality Of Samples Research Articles

Enhancing Paddy Rice Drying Quality with Non-Destructive LPG Refrigeration System in a Fixed-Bed Dryer

High relative air humidity in humid climates slows the process of paddy drying and leads to rehydration. The use of a liquefied petroleum gas (LPG) refrigeration system for dehumidifying ambient air effectively reduces drying time and improves product quality in a non-destructive manner. This environmentally friendly method enhances both the quality of the rice samples and the efficiency of the drying process. This study examined the impact of dehumidification on Hashemi rice quality and moisture reduction, using the Response Surface Method to optimize drying conditions at three levels of temperature (40, 45, and 50 °C), air velocity (1.7, 2.2, and 2.7 m/s), and relative humidity (70, 80, and 90%). The results showed that dehumidification led to a moisture content of 6% and the lowest cracking rate of 2%. Temperature was the most influential factor, affecting responses by 73.09% under dehumidified conditions. Optimal conditions of 40 °C, 80% RH, and 1.7 m/s air velocity resulted in a cracking rate of 2% and a desirability index of 0.958. The Response Surface Method showed an excellent fit for the cracking index (R² = 0.98). These results highlight the LPG refrigeration system as an effective, non-destructive, and environmentally friendly method for drying paddy in humid regions, making it suitable for industrial applications.

The Changes in Color and Image Parameters and Sensory Attributes of Freeze-Dried Clones and a Cultivar of Red-Fleshed Apples

The target of breeding red-fleshed apples is to increase their potential health benefits related to red flesh coloration and consumer acceptance. The objective of this study was to determine the usefulness of four clones (90, 120, 156, and 158) of red-fleshed apples for freeze-drying compared to the cultivar ‘Trinity’. Red-fleshed apples were dried in the form of slices using a laboratory freeze-dryer. The changes in color features and image texture parameters after drying and the sensory quality of freeze-dried samples were assessed. Trends of increase in the value of the L* parameter and decrease in the a* and b* parameters after freeze-drying were observed. Furthermore, freeze-drying caused statistically significant changes in analyzed image textures named XHMean, RHMean, SHMean, VHMean, LHMean, and UHMean. Machine learning models developed based on the color parameters L*, a*, and b* distinguished raw and freeze-dried red-fleshed apples with an average accuracy of 84% for clone 90 up to 99.0% for clone 156, and models based on twenty selected image textures exhibited an accuracy of 98.5% for clone 156 to 100% for clones 90 and 158 and the cultivar ‘Trinity’. The very attractive external appearance, medium-intense fruity smell, crunchiness, and intense fruity taste of all the apple slices were revealed. The innovative aspect of this study included the comparison of the drying behavior and sensory quality of the new clones and a standard cultivar of red-fleshed apples. Moreover, innovative methods and results were used to determine the effect of freeze-drying on red-fleshed apple quality, considering novel models involving thousands of image textures and machine learning algorithms.

Evaluation of an alternative centrifugation protocol for reducing total turnaround time

Abstract Objectives Centrifugation is a key process that should be controlled to ensure an adequate sample quality. To achieve rapid, standardized, and consistent sample centrifugation, we aimed to evaluate an alternative centrifugation protocol and its impact on the results of 20 biochemical determinations in serum. Methods The study included 45 ambulatory patients. Two serum-separating tubes were collected from each patient (Becton Dickinson (BD) Vacutainer® SST™ II Advance, 8.5 mL Ref. 366468). One of the tubes was centrifuged at 2530 × g for 10 min (control method), while the other tube was centrifuged under alternative centrifugation conditions, namely 2530 × g for 7 min. Results The analysis of results revealed that calcium, total bilirubin, and magnesium exhibited a constant and proportional systematic bias. However, considering the proportional mean difference, all analytes met the desirable bias performance established by our laboratory, except for magnesium, which met the minimum bias criteria. Conclusions Our study demonstrates that both centrifugation protocols are interchangeable for measuring the studied analytes, thereby ensuring adequate sample quality.

Multivariate Moment Least-Squares Variance Estimators for Reversible Markov Chains

Markov chain Monte Carlo (MCMC) is a commonly used method for approximating expectations with respect to probability distributions. Uncertainty assessment for MCMC estimators is essential in practical applications. Moreover, for multivariate functions of a Markov chain, it is important to estimate not only the auto-correlation for each component but also to estimate cross-correlations, in order to better assess sample quality, improve estimates of effective sample size, and use more effective stopping rules. Berg and Song introduced the moment least squares (momentLS) estimator, a shape-constrained estimator for the autocovariance sequence from a reversible Markov chain, for univariate functions of the Markov chain. Based on this sequence estimator, they proposed an estimator of the asymptotic variance of the sample mean from MCMC samples. In this study, we propose novel autocovariance sequence and asymptotic variance estimators for Markov chain functions with multiple components, based on the univariate momentLS estimators from Berg and Song. We demonstrate strong consistency of the proposed auto(cross)-covariance sequence and asymptotic variance matrix estimators. We conduct empirical comparisons of our method with other state-of-the-art approaches on simulated and real-data examples, using popular samplers including the random-walk Metropolis sampler and the No-U-Turn sampler from STAN. Supplemental materials for this article are available online.

Microcrystal Growth Pathways Investigated with Machine Learning Segmentation and Classification in Scanning Electron Microscopy.

Advances in electron microscopy have revolutionized material characterization on the nano- and microscales, providing important insights into local ordering, structure, and size and quality distributions. While shape and size can be rigorously quantified through microscopy, it is often limited to local structure analysis and fails to describe bulk sample quality. Herein, a flexible machine learning (ML) tool is described that can segment and classify faceted crystals in scanning electron microscopy (SEM) micrographs to determine sample quality through the crystal size and product distribution. As a case study, this tool was applied to investigate crystal growth pathways (classical nucleation and growth compared to nonclassical growth) in DNA-mediated nanoparticle assembly through size and product (single crystal, fused crystal, or noncrystal) distribution of samples containing over 13000 colloidal crystal products. Strong DNA bond strengths (controlled by DNA sequence) lead to fast nucleation that exhausts the monomer concentration, resulting in smaller colloidal crystals. Alternatively, increased thermal energy and crystallization time lead to nonclassical crystallization pathways (coalescence) that result in larger colloidal crystals. This tool is useful since experimental conditions can now be deliberately identified to control colloidal crystal size and size distribution, important considerations for researchers interested in designing and synthesizing colloidal crystal metamaterials.

Wearable threads for monitoring sanitizer quality using dye displacement assay.

This study employs zero-cost (≈0.01 $) and durable thread-based devices to evaluate the quality of simulated and commercial sanitizer samples through dye displacement assay (DDA). A diverse range of sanitizer compositions, including ethanol concentrations of 55%, 75%, and 95% (v/v), were analysed. This investigation encompasses an assessment of the marker type (Doms and Hauser brands) on the migration distance of the dye region marked on thread devices. Our results demonstrate a proportional increase in the migration distance of the dye with increasing ethanol concentrations due to a decrease in the coefficient of viscosity and solvation power of ethanol on dye molecules. Additionally, a field trial for the thorough assessment of commercial sanitizer quality using thread-based devices further underscores the efficacy of this methodology. A calibration plot for a braided thread with Doms marker dye provides a reliable means to quantitatively assess the ethanol content in different commercial sanitizer compositions. Our findings collectively highlight the significance of this innovative method as a valuable tool for quality control and assessment for public health and hygiene as well as for preparing us for another pandemic.

Role of macroscopic on-site evaluation of endoscopic ultrasound-guided fine-needle aspiration/biopsy: Results of a multicentric prospective study.

The concept of macroscopic on-site evaluation (MOSE) was introduced in 2015 when the endoscopist observed better diagnostic yield when the macroscopically visible core on MOSE was superior to 4 mm. Recent studies suggest that MOSE by the endoscopist may be an excellent alternative to rapid on-site evaluation, and some classifications have been published. Few studies have assessed the adequacy of histologic cores in MOSE during endoscopic ultrasound-guided fine-needle aspiration/biopsy (EUS-FNA/FNB). To evaluate the performance of MOSE during EUS-FNA/FNB. This multicentric prospective study was conducted in 16 centers in 3 countries (Egypt, Iraq, and Morocco) and included 1108 patients with pancreatic, biliary, or gastrointestinal pathology who were referred for EUS examination. We prospectively analyzed the MOSE in 1008 patients with available histopathological reports according to 2 classifications to determine the adequacy of the histological core samples. Data management and analysis were performed using a Statistical Package for Social Sciences (SPSS) version 27. A total of 1074 solid lesions were biopsied in 1008 patients with available cytopathological reports. Mean age was 59 years, and 509 patients (50.5%) were male. The mean lesion size was 38 mm. The most frequently utilized needles were FNB-Franseen (74.5%) and 22 G (93.4%), with a median of 2 passes. According to 2 classifications, 618 non-bloody cores (61.3%) and 964 good samples (95.6%) were adequate for histological evaluation. The overall diagnostic yield of cytopathology was 95.5%. The cytological examination confirmed the diagnosis of malignancy in 861 patients (85.4%), while 45 samples (4.5%) were inconclusive. Post-procedural adverse events occurred in 33 patients (3.3%). Statistical analysis showed a difference between needle types (P = 0.035) with a high sensitivity of FNB (97%). The analysis of the relationship between the MOSE-score and the final diagnosis showed a significant difference between the different scores of the MOSE (P < 0.001). MOSE is a simple method that allows endoscopists to increase needle passes to improve sample quality. There is significantly higher FNB sensitivity and cytopathology diagnostic yield with good MOSE cores.

Characterization of poly(A) and poly(T) tail lengths in plasmid DNA by liquid chromatography high-resolution mass spectrometry.

Rapid advances in messenger RNA (mRNA) technology necessitate effective analytical methods. This study describes the development of a novel in vitro method using ion-pair reversed-phase liquid chromatography coupled to high-resolution mass spectrometry (IP-RP-LC-HRMS) for assessing the poly(A) and complementary poly(T) tail lengths directly into the DNA template used to manufacture mRNA. Briefly, after the validation of poly(A) tail length in the plasmid by Sanger sequencing, double-stranded DNA fragments containing these tails in the plasmid of interest were amplified by thepolymerase chain reaction (PCR), purified on silica column, and digested with restriction enzymes ClaI and HindIII. Gel or capillary electrophoresis confirmed sample quality and enzymatic digestion efficiency. Subsequently, poly(A) and complementary poly(T) tails were extracted and analyzed by LC-MS to determine their length and heterogeneity at a single-nucleotide resolution. Three DNA templates containing poly(A) tail lengths of 60A-G, 95A, or 108A were studied. LC-MS results correlated well with Sanger sequencing, identifying major populations of 60A-G, 95A, or 108A. Surprisingly, unlike Sanger sequencing, LC-MS analysis revealed minor poly(A) populations with lengths longer or shorter than the theoretically encoded poly(A) tail length. This finding could be explained by (i) the slippage of bacterial DNA polymerase I during plasmid replication in bacterial culture, which occurs on repeat mononucleotide sequences, or (ii) the slippage of Q5® High-Fidelity DNA Polymerase during PCR amplification. In conclusion, the method is easy, rapid, and accurate and could replace Sanger sequencing to assess the poly(A) and complementary poly(T) tail lengths in plasmid DNA.

QUALIDADE PARA GARANTIR A CONFIABILIDADE DOS RESULTADOS DOS TESTES DA COVID 19

Objective: To analyze and identify the main quality criteria essential to ensure the reliability of COVID-19 test results performed in clinical laboratories. Literature Review: Ensuring the quality of COVID-19 testing is vital for the effectiveness of pandemic control strategies. This involves meticulous care with the selection and implementation of tests, the formation and training of teams, and adherence to standards and good practices. Only with an ongoing commitment to quality and accuracy can we ensure that test results reflect the true situation of COVID-19 infection and aid in public health decision-making.Conclusion: To ensure the reliability of COVID-19 test results, it is essential to take a comprehensive approach that encompasses test accuracy, sample quality, rigorous validation, staff qualification, and transparency.

Host-Guest Binding Free Energies à la Carte: An Automated OneOPES Protocol.

Estimating absolute binding free energies from molecular simulations is a key step in computer-aided drug design pipelines, but the agreement between computational results and experiments is still very inconsistent. Both the accuracy of the computational model and the quality of the statistical sampling contribute to this discrepancy, yet disentangling the two remains a challenge. In this study, we present an automated protocol based on OneOPES, an enhanced sampling method that exploits replica exchange and can accelerate several collective variables to address the sampling problem. We apply this protocol to 37 host-guest systems. The simplicity of setting up the simulations and producing well-converged binding free energy estimates without the need to optimize simulation parameters provides a reliable solution to the sampling problem. This, in turn, allows for a systematic force field comparison and ranking according to the correlation between simulations and experiments, which can inform the selection of an appropriate model. The protocol can be readily adapted to test more force field combinations and study more complex protein-ligand systems, where the choice of an appropriate physical model is often based on heuristic considerations rather than systematic optimization.

Strategic insights into the cultivation of pancreatic cancer organoids from endoscopic ultrasonography-guided biopsy tissue

BACKGROUND The frequent suboptimal efficacy of endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) to culture pancreatic cancer (PC) organoids (PCOs) poses a major challenge in the advancement of personalized medicine for advanced PC. AIM To explore how to obtain appropriate puncture tissues from EUS-FNB and optimize the strategy for efficiently constructing PCOs, providing an efficient tool for the advancement of personalized medicine. METHODS Patients who underwent EUS-FNB for the diagnosis of PC tissue were prospectively enrolled. We refined the endoscopic biopsy procedures and organoid cultivation techniques. All tissue specimens verified by on-site pathological assessment were cultured in a semi-suspended medium in a microfluidic environment. We assessed differences in PCOs cultured beyond and below five generations examining patient demographics, specimen and organoid attributes, and the sensitivity of organoids to a panel of clinical drugs through cell viability assays. RESULTS In this study, 16 patients with PC were recruited, one sample was excluded because onsite cytopathology showed no tumor cells. Successful organoid generation occurred in 93.3% (14 of 15) of the EUS-FNB specimens, with 60% (9 of 15) sustaining over five generations. Among these patients, those with a history of diabetes, familial cancer, or larger tumors exhibited enhanced PCO expandability. The key factors influencing long-term PCOs expansion included initial needle sample quality (P = 0.005), rapid initiation of organoid culture post-isolation (P ≤ 0.001), and high organoid activity (P = 0.031). Drug sensitivity analysis revealed a partial response in two patients following therapeutic intervention and surgery and stable disease in four patients, indicating a moderate correlation between organoid response and clinical outcomes. CONCLUSION Optimal initial needle sampling, rapid and precise biopsy sample processing, process isolated samples as soon as possible, and sufficient cellular material are crucial for successful cultivating PCOs. High organoid activity is an important factor in maintaining their long-term expansion, which is essential for shortening the time of drug sensitivity analysis and is the basis of PC research.

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.

Next generation tissue biobanking: quality assured, financed, and integrated?

Biobanks are essential for biomedical research, particularly in the era of personalized medicine. In Germany, 36biobanks have been established over the past decade that are connected under the German Biobank Alliance (GBA). These biobanks store high-quality biological samples along with clinical data to support research projects. Biobanks can be integrated, handling both tissue and liquid samples, or set up as separate entities depending on specific requirements.Tissue biobanking is especially complex due to the invasive nature of tissue collection and the non-replicability of the samples. Close collaboration between clinics, pathologists, IT specialists, and biobank managers is crucial to ensure the quality of samples and promote interdisciplinary research.The integration of pathology and biobanking is key, both organizationally and technically. Shared IT systems, standardized protocols, and collaborative governance structures are vital for efficient data management. Quality assurance, ethical guidelines, and data protection are critical to maintaining public trust and legal compliance.Long-term financial models are needed to ensure the sustainability of biobanks. The GBA supports emerging biobanks through its "Starterkit" initiative, offering guidance and best practices to help new biobanks develop.Tissue biobanks are indispensable for advancing the understanding of diseases and developing new therapies. However, they must adhere to strict ethical and legal standards to maximize their scientific and societal value.

Abstract A069: Effect of different voided urine sample storage time and temperature, and preservatives, on analysis with the multiplex bead-based Oncuria® bladder cancer immunoassay

Abstract Urinalysis accuracy requires reliable sample stability that is dependent on the chosen collection and storage conditions. If urine sample storage at room temperature (20°C) does not affect sample quality, this may reduce laboratory handling time and expense. This study evaluated whether different voided urine sample collection and storage parameters affected subsequent biomarker analysis by the multiplex Oncuria® bladder cancer immunoassay. The Oncuria® immunoassay simultaneously quantifies 10 protein analytes in urine to generate a bladder cancer diagnostic signature. Samples were stored at varied temperatures (20°C, 4°C, -80°C) for up to 1 month. The effects of adding two commercial urine sample stabilizers and antibiotics (trimethoprim) were also assessed. Subsequently, multiple potential biospecimen stabilizers were added to urine samples and evaluated with Oncuria® in hopes of allowing the urine sample to remain at room temperature for extended periods of time. First, it was demonstrated that voided urine samples stored at room temperate without such stabilizers had different levels of the 10 analytes associated with the Oncuria® test compared to voided urine samples stored at 4°C. Next, we evaluated the effects of commercially available biospecimen stabilizers. Despite the addition of these stabilizers, the levels of the 10 analytes were altered when the samples were stored at room temperature for prolonged periods of time. Therefore, we could not identify a suitable biospecimen stabilizer that would not require sample refrigeration. In conclusion, to minimize sample degradation/alteration after collection, voided urine samples should be refrigerated until analyzed with Oncuria® as the refrigeration is advantageous for the storage and the transport of these urine samples. Citation Format: Sunao Tanaka, Kaoru Murakami, Toru Sakatani, Riko Lee, Wayne Hogrefe, Fernando Siguencia, Steven M Smith, Charles J Rosser, Hideki Furuya. Effect of different voided urine sample storage time and temperature, and preservatives, on analysis with the multiplex bead-based Oncuria® bladder cancer immunoassay [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 A069.

Effect of sustained elevated temperature on compression and split-tensile properties of concrete made with waste foundry sand

PurposeThe aim of the current study is to inspect the influence of high temperatures on the compressive and split-tensile-strength (STS) of concrete mixtures produced by replacing natural river sand with waste-foundry sand (WFS) at 25%, 50%, 75% and 100%. When the experimental findings and the projected outcomes were compared by IS:456-2000 code equations, the STS results predicted by the suggested mathematical equations exhibit lower variations. It is proposed to employ the presented mathematical formulas to evaluate the STS of concrete cylindrical specimens at higher temperatures.Design/methodology/approachAfter fabricating, concrete mixtures were allowed to cure for 28 days. For the purpose of avoiding explosive spalling during the heating process, concrete samples are taken out from the curing chamber after 28 days and allowed to dry for two days. The manufactured concrete specimen is exposed to 100 °C, 200 °C, 300 °C, 400 °C, 500 °C and 600 °C temperatures for a duration of 2 h. After the specimens have cool down to room temperature (RT), the physical test, ultrasonic-pulse-velocity (UPV) test, compressive strength test and STS test are carried out.FindingsWith an increase in WFS content, concrete specimens' residue compressive-strength and STS decreases. The STS of samples declines as the WFS content rises with increase in temperature interval. According to the UPV test, the concrete samples quality is “good” up to 400 °C; after 500 °C, it ranges from “doubtful to poor.” The UPV values of various mixes declined as the temperature increased. Mass losses increase with exposure to greater temperatures and with an increase in the proportions of WFS in concrete specimens. For mixtures MWFS-0, MWFS-1, MWFS-2, MWFS-3 and MWFS-4 (0%, 25%, 50%, 75% and 100% WFS content), no cracks were present on any of the samples below 400 °C. Concrete surfaces start to show cracks whenever the intervals of temperature increase above 400 °C.Originality/valueIn this investigation, WFS elements are totally substituted for natural sand in concrete mixtures. The residue strength properties, including residual compressive strength and residual STS, were found to be lower after exposures to greater temperature when comparisons were made to referral mixtures. When comparing specimens’ compressive strength, higher temperatures have more effects on the STS of samples with higher WFS contents.

Abstract 4120763: Predicting Disease Risk Among Variants in Cardiomyopathy-Associated Genes Across Diverse Genetic Ancestries

Background: Many cases of cardiomyopathy (CM) are genetically inherited, and the expansion of broad genomic sequencing has opened the door for predicting those at risk for developing disease. However, genetic tools that help determine variant pathogenicity employ genetic data from predominantly European genetic ancestries. Understanding the full diversity of genetic variants in cardiomyopathy genes is central to identifying which variants will cause disease. Hypothesis: Participants of non-European genetic ancestry will have a higher burden of cardiomyopathy gene variants with "VUS" designation in ClinVar but will have equivalent/less penetrance of clinical cardiomyopathy compared to a European ancestry group. Methods: Participants in the AllofUs database with exome sequencing (ES) were queried and a cohort of 230,013 participants were retained after sample quality control filters. Participant genetic ancestry was based on the designated ancestry group determined via AllofUs as previously described (1). Participants were queried for variants in genes with definitive or moderate evidence of hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic cardiomyopathy (ACM) in ClinGen. Variants were filtered by ClinVar status (1* and above P/LP, 1* and above B/LB, and VUS). Participant CM disease status was determined via ICD-9/10 code. Results: For each ancestry group, prevalence of cardiomyopathy ranged from 0.34% in the Latino/admixed American ancestry group to 1.09% in the African group, with an average prevalence of 0.91% across all groups. The European group had a statistically significant lower percentage of ClinVar VUS variants at 26%, while all other non-European ancestry groups had a combined VUS prevalence rate of 35% (****p&lt;0.0001). The penetrance of CM in participants with CM VUS variants was lower in combined non-European ancestry groups (0.84%) than European ancestry (1.02%) (*p=0.0156). Conclusion: Prevalence of ClinVar VUS variants varies across different genetic ancestry groups, with European ancestry having a lower burden of VUS variants compared to non-European groups. However, non-European ancestry groups with increased burden of VUS variants have lower disease penetrance. Citations: (1) The All of Us Research Program Genomics Investigators. Genomic data in the All of Us Research Program. Nature 627 , 340–346 (2024). https://doi.org/10.1038/s41586-023-06957-x

EPCO-16. METHOD TO MODEL AND CORRECT NOISE IN SINGLE-NUCLEI RNA-SEQUENCING DATA FROM POST-MORTEM BRAIN

Abstract Post-mortem brain samples are crucial for neuropathological research. For brain cancers, they provide insights about disease progression, microenvironmental factors, and treatment resistance. In some brain diseases, brain tissue may only be available post-mortem. Establishing a reference atlas of healthy brain also requires post-mortem donations. Beyond logistical and ethical challenges of post-mortem samples, there are also post-mortem factors that mask biological signal. In this study, we specifically aim to model post-mortem gene expression changes in single-nuclei RNA-sequencing (snRNA-seq) data. We analyze a public snRNA-seq dataset of pediatric high-grade glioma (pHGG) that has matched samples at initial resection and autopsy. We find a clear gene signature, composed of mitochondrial genes, stress-related genes, and metabolism-related genes, that is upregulated post-mortem. This signature also appears in post-mortem snRNA-seq data from other brain diseases and healthy donors. We classify it as “post-mortem noise”. We model the post-mortem noise as a combination of technical and biological artifact. The technical artifact is correlated with the percentage of mitochondrial reads and is a general measure of sample quality. The biological artifact is correlated with post-mortem interval and represents gene expression changes due to post-mortem stress. We apply our model to the pHGG data by regressing out the technical and biological artifact. Removing the post-mortem noise results in an improvement in malignant versus normal cell prediction, cell type classification, and the uncovering of pathways related to glioma progression. We confidently identify several normal cell types and predict their interactions with cancer cells, highlighting the utility of wider possible margins post-mortem. Therefore, after removing post-mortem noise, we analyze glioma progression at an unparalleled timepoint and characterize late-stage changes in the tumor microenvironment. In conclusion, we develop a method that models post-mortem noise in snRNA-seq brain data. Our method can be broadly applied to future brain cancer studies using post-mortem tissue.

EXTH-38. EVALUATION OF STORAGE CONDITIONS ON SERUM-DERIVED EXTRACELLULAR VESICLES IN BRAIN TUMOR SAMPLES

Abstract Tumor-derived Extracellular Vesicles (EVs) are emerging as potential liquid biopsy tool in the field of cancer diagnosis and therapeutic targets. The functionality of EVs depends on its diverse cargo, which is known to alter host-receipient cells. Nevertheless, extracting EVs while preserving its integrity and functionality of its contents is challenging, specifically in Low-middle Income countries (LMIC) countries. Our study focussed on extraction of serum derived EVs analysis from brain-tumor samples encompassing Oligodendrogliomas, Astrocytomas and Glioblastomas. The stability of EVs were examined in serum stored for 2 years at -80°C to understand the effect of preservation and storage in different conditions, that were left exposed to room temperature for variable periods of time before freezing. To explore the optimal suitable conditions, EVs were isolated from serum samples using IZON column size-exclusion based-method, and evaluated for structural integrity and stability through nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) technology. Interestingly, the majority of isolated vesicles exhibited round, intact structure with bilayer membrane of size varying between 30 - 250nm. Moreover, protein content of EVs positive markers (CD63, CD81 and CD9) was also detected with minimal contaminations evidenced by weak bands for negative marker (ApoB), as determined by Western blot analysis. Further, column-based EV-derived RNA demonstrated optimal yield and purity was confirmed by RNA integrity number (RIN) using Qubit. While cryopreservation had minimum detrimental effects on mRNA sample quality, leaving it exposed to room temperature before freezing had a significant effect on EV isolation, as samples were often haemolyzed. This will provide insights in optimizing pre-analytical and analytical procedures for more effective outcomes in the clinical setting

QLTI-09. CLINICAL TRIALS IN NEURO-ONCOLOGY: DESIGN, LOGISTICS, CHALLENGES, AND PRACTICAL CONSIDERATIONS

Abstract Timely, secure and reliable transportation, precise handling and storage of biological specimens are critical for the success of clinical research. Research in neuro-oncology often involves complex assays such as next-generation sequencing where the integrityof brain tumor specimens is paramount. These challenges are pronounced in most Lower Middle-Income Countries (LMIC), due to climatic factors and infrastructure limitations, necessitating stringent measures to preserve sample quality and their genetic data integrity for downstream analysis. In this study, we conducted extensive cohort analysis on 80 tissues randomly picked samples of the 3600 samples collected across India to assess the impact of collection protocols, transportation logistics, and long-term storage on the genetic material of tissues. The quality and quantity of the isolated nucleic acids was determined by BioAnalyzer and Qubit parameters. Tissue samples were collected and stored at -80°C in RNAstable® within 4-12 hrs of collection. The DNA and RNA obtained from tissue samples stored for over one year was found to be of good quality with corresponding DNA Integrity Number (DIN) and RNA Integrity Number (RIN)between 7-9. Also, optimum quantity of DNA and RNA was obtained for whole exome and transcriptome sequencing. Upon sequencing, these samples showed good read coverage of 100X or more. These findings provide critical insights into the measures necessary for optimizing sample preservation and transportation to ensure accurate research outcomes. The results indicate that appropriate sample collection, transportation, and long-term storage of bio-specimens can be used effectively for future research. Additionally, the outcome underscores the potential for long-term studies in neuro-oncology using archived biological samples without compromising data integrity.

Research on positive and negative sample sampling strategies in contrastive learning

Abstract. Contrastive learning is an important technique in the field of machine learning for learning data representations. In the field of self-supervised visual representation learning, the strategy of positive and negative sample selection is key to improving the efficiency and effectiveness of model learning. Traditional self-supervised learning methods often employ random sampling strategies to select positive and negative samples, but this approach may result in uneven sample quality when dealing with complex datasets, thereby affecting the learning outcomes. To alleviate this problem, this study is dedicated to exploring more effective strategies for positive and negative sample selection and processing to optimize the self-supervised learning process. To this end, we propose an improved method of self-supervised learning called contrastive learning with enhanced diversity. On the one hand, this method utilizes the weight parameters of the DINO pre-trained model to initialize the feature extraction network of SimCLR, providing more accurate calculations of feature similarity. On the other hand, by setting a threshold on the feature similarity matrix and penalizing (subtracting 0.5 from) similarity scores that do not exceed this threshold, we reduce the excessive impact of high similarity scores on model training, thereby helping the model better distinguish between positive and negative samples. In downstream image classification tasks, we conducted detailed evaluations of the improved model, specifically including fine-tuning and linear evaluation aspects. Experiments show that the proposed approach improves the performance of the loss functions and improves the accuracy of the proposed SimCLR model.