Serial Sections Research Articles

Abstract 446: Integrated spatial transcriptomics and lipidomics analyses of intraductal papillary mucinous neoplasm identifies enrichment of long chain sulfatide as an early metabolic alteration

Abstract Purpose: To define the metabolic alterations associated with malignant progression of intraductal papillary mucinous neoplasm (IPMN). Experimental Procedures: Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) was conducted for spatial characterization of lipid profiles on frozen tissue sections from resected human IPMN patients. The specimen set consisted of either low-grade dysplasia (LG) (n=15), high-grade (HG) dysplasia (n=4), or with an associated pancreatic ductal adenocarcinoma (PDAC) (n=4) as well as 15 PDAC tissues. Utilizing serial sections to the same human IPMN cases analyzed with MALDI-MS imaging, we used the Visium Spatial Gene Expression technology platform to characterize the spatiomolecular underpinnings of LG and HG IPMN. H&E images of the same tissue section used for the Visium workflow, were used to annotate the spots in the dataset covering the epithelial lining of the IPMN lesions (“epilesional” areas) and the spots covering the adjacent microenvironment surrounding the lesion (“perilesiopecific” area). Findings were compared with lipidomic analyses of cystic fluid from 89 IPMN patients. The anti-cancer efficacy of UGT8-IN-1, a selective small molecule inhibitor of ceramide galactosyltransferase (CGT, also known as UGT8), was assessed in vitro and in mutant Kras;Gnas allografts. Results: MALDI/MS-based spatial imaging of human resected IPMN tissues and pancreata from a mutant Kras;Gnas mouse model of IPMN revealed long-chain hydroxylated sulfatides, particularly the C24:0(OH) and C24:1(OH) species, to be selectively enriched in the IPMN and PDAC neoplastic epithelium. Visium spatial transcriptomics data confirmed that the cognate transcripts engaged in sulfatide biosynthesis, including UGT8, Gal3St1, and FA2H, were co-localized with areas of sulfatide enrichment. Lipidomic analyses of cystic fluid from 89 patients with IPMN identified several sulfatide species to be significantly elevated in patients with IPMN/PDAC compared to those with low-grade IPMN. Inhibition of sulfatide metabolism via UGT8-IN-1 reduced viability of Kras;Gnas murine cell lines in vitro and attenuated tumor growth in an allograft IPMN model. UGT8-IN-1 treatment resulted in reduced levels of several sulfatide species, accompanied by a concomitant increase in a subset of quantified sulfatides precursor ceramides, accumulation of mitochondrial mass and reactive oxygen species and time-dependent increases in autophagy/mitophagy markers p62/SQSTM1 and LC-3B, suggesting impaired mitophagy. Conclusion: Enhanced sulfatide metabolism is an early alteration in cystic pre-cancerous lesions of the pancreas that persists through invasive neoplasia. Targeting sulfatide biosynthesis might represent an actionable vulnerability for cancer interception. Citation Format: Yihui Chen, Marta Sans, Fredrik Thege, Rongzhang Dou, Jimin Min, Michele Yip-Schneider, Jianjun Zhang, Ranran Wu, Ehsan Irajizad, Yuki Makino, Kimal Rajapakshe, Mark Hurd, Ricardo León-Letelier, Jody Vykoukal, Jennifer Dennison, Kim-Anh Do, Robert Wolff, Paola Guerrero, Michael Kim, Max Schmidt, Anirban Maitra, Samir Hanash, Johannes Fahrmann. Integrated spatial transcriptomics and lipidomics analyses of intraductal papillary mucinous neoplasm identifies enrichment of long chain sulfatide as an early metabolic alteration [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 446.

Abstract 1044: Low-cost, high-sensitivity InSituPlex® mIF assays on the Parhelia Omni-Stainer™

Abstract Background: Multiplex immunofluorescence (mIF) is an important tool for extracting complex phenotypic information from normal or diseased tissues, and provides accurate and predictive insights on the tissue microenvironment faster. For patient samples in translational and clinical research, high sensitivity, specificity, and reproducibility are critical; however, throughput and cost considerations are also important in designing clinical studies. Enabling an easy-to-use high-performance assay such as InSituPlex® on robust and affordable auto-stainers like the Parhelia Omni-Stainer™ can allow researchers to gain a deeper understanding of the tissue micro-environment through high-quality multiplexed biomarker detection in a fraction of the time and cost. Methods: We performed Ultivue InSituPlex® assays with OmniVUE™ panels using the Parhelia Omni-Stainer™, a low-cost, high-throughput tissue auto-stainer with reduced requirements for reagents with a minimal dead volume. We compared the whole-slide staining performance across replicates using human tonsil and tumor tissue arrays across multiple independent runs over multiple days. The stained slides were scanned using the Zeiss AxioScan.Z1 high-throughput imaging system, and image batches were processed using Ultivue’s UltiStacker.AI and UltiAnalyzer.AI software. Results: Tighter temperature control of the slide in the Omni-Stainer supports optimal InSituPlex® signal amplification. Intra-run and inter-day analysis on serial sections of human tonsil and tumor tissue arrays demonstrated a high degree of consistency in InSituPlex® assay performance, specifically with marker-specific signal intensities and cell densities. The Omni-Stainer facilitates the use of low reagent volumes at each step of the assay due to its efficient reagent use, thus lowering the cost of reagents per slide. Conclusions: This study demonstrated a fast, simple and robust workflow to perform high-performance InSituPlex® mIF assays on the low-cost, high-throughput Parhelia Omni-Stainer™. Ultivue’s configurable OmniVUE™ mIF panels can be run on the Omni-Stainer with reduced associated reagent costs per slide. This workflow can support larger clinical and translational studies in a more cost-effective fashion, leading to in-depth analysis of tissue microenvironment through highly precise multiplexed biomarker detection. Citation Format: Kevin Hwang, Lauren Duro, Alex Veith, Nelson Wedin, Nikolay Samusik, Je H. Lee. Low-cost, high-sensitivity InSituPlex® mIF assays on the Parhelia Omni-Stainer™ [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 1044.

Abstract 2352: Development of an AI-based algorithm to quantify eosinophils in H&E images from colorectal cancer (CRC) tissue sections guided by biomarker staining using multiplex immunofluorescence imaging

Abstract Background: Eosinophils are innate immune granulocytes that migrate to areas of inflammation to combat against infection and disease. Best known for their detrimental role in asthma and allergic disorders, there is growing interest in the involvement of eosinophils in cancer. Eosinophils are routinely observed in the tumor microenvironment (TME) and, depending on the cancer type, have been shown to drive other immune cells to either suppress or promote tumor growth - In colorectal cancer (CRC), eosinophil infiltration into the TME has been linked to a favorable prognosis. However, the behavior of eosinophils and their effect on associated immune mediators in the TME remains poorly understood. Currently, eosinophils are primarily identified from H&E stained tissue sections based on morphological features by a pathologist, but it can be challenging to reliably and efficiently identify all eosinophils based on morphology alone. Here, we present a novel image analysis workflow that established an AI-based cell classifier which can accurately quantify eosinophils in H&E stained CRC tissue sections by leveraging biomarker staining of eosinophils using multiplex immunofluorescence (mIF) imaging in conjunction with the morphological characteristics of eosinophils observed by H&E. Methods: CRC tissue sections were labeled with a 5plex panel of eosinophil-specific markers and imaged by mIF using the PhenoImager HT platform (Akoya). Biomarker fluorescence was then quenched, sections stained by H&E, and reimaged using the PhenoImager. mIF and H&E images were then imported into the HALO® platform for algorithm development. Halo AI cell classification of eosinophils was trained on the morphological features of eosinophil staining in H&E images, guided by eosinophil-specific labeling in the 5plex mIF images. H&E staining was also performed on a serial section of each specimen. Morphological identification of eosinophils in H&E images was performed by a pathologist. Results: Correlation analysis was performed to evaluate the relationship, per high power field (HPF), between manual eosinophil counts by a pathologist and AI algorithm derived eosinophils counts. The results showed the two methods are highly correlated, demonstrating reliable algorithm performance. Conclusion: The AI-based eosinophil detection algorithm established here enables high-throughput analysis and quantification of eosinophils from H&E stained CRC tissue specimens and facilitates morphology diagnosis. Citation Format: Arezoo Hanifi, Elizabeth Blain, James Hargrove, Jeff Lock, Nam Tran, Vladislav Chizhevsky, Qingyan Au. Development of an AI-based algorithm to quantify eosinophils in H&E images from colorectal cancer (CRC) tissue sections guided by biomarker staining using multiplex immunofluorescence imaging [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 2352.

Abstract 67: Predicting colorectal patient prognoses by functional characterisation of heterogeneous cell types and their spatial interaction using a new technique: Whole slide imaging mass cytometry

Abstract Immunotherapies stimulate T cell function in the tumor microenvironment (TME). CD3+ and CD8+ T cell infiltration can predict disease recurrence in colorectal cancer (CRC) patients and has been validated as Immunoscore® (IS). The TME comprises multiple heterogeneous populations of immune cells, including T cells and cancer-associated fibroblasts (CAFs); CRC tumors are also morphologically diverse. Standard BioTools™ (SBI) has developed a novel whole slide Imaging Mass Cytometry™ technique that allows complete analysis of cell population heterogeneity in the TME using >40 markers. We hypothesised there are multiple heterogeneous subtypes of CAFs within CRC tumors and that they modulate T cell infiltration and patient outcome. In collaboration with SBI, we studied a cohort of CRC patients stratified based on T cell infiltrate and disease recurrence. We identified multiple CAF populations that interact with tumor-infiltrating T cell populations. Slides from 10 CRC patients with high or low CD3+CD8+ T cell infiltrate (IS) were stained with a panel encompassing markers for CAF and T cell heterogeneity, function, and metabolism. Slides were imaged using the Hyperion XTi™ Imaging System at SBI. Using a scan mode called Preview Mode, the whole slide was imaged in minutes to identify CAF and T cell phenotypic regions of interest. Next, the same slide was imaged at 1 μm resolution using Cell Mode (CM). Finally, for each sample, an additional serial section was imaged using a slightly lower resolution called Tissue Mode (TM). TM allowed us to image the samples in several hours instead of days while capturing the full heterogeneity of the entire tissue sample. We used clustering to identify phenotype clusters and neighbourhood analysis to study spatial enrichment. We also compared results between CM and TM. We report: Identification of heterogeneous CAF subtypes in CRC patients with high or low IS; determination of CAF populations that spatially interact with T cells in patients with high or low IS; and examination of consistent spatial interactions between CAF and T cells in CRC, and whether these interactions differ in patients with high or low IS. TM revealed several heterogeneous cellular populations that differed between patients with high or low IS; importantly, low IS tumors had more podoplanin+αSMA+ CAF populations and fewer podoplanin+αSMA- cell populations compared with high IS tumors. High IS tumors had more PD-1+CD8+ T cells compared with low IS tumors; these PD-1+CD8+ T cells spatially interacted with podoplanin+αSMA- CAF populations in high IS tumors. CAF-PD-1+CD8+ T cell interactions were absent from low IS tumors. Thus, CAF-PD-1+CD8+ T cell interactions are important in CRC patients with high IS, and these cellular interactions require further investigation. Citation Format: Rory M. Costello, Sonya Fenton, Helen McGuire, Liang Lim, David Howell, Qanber Raza, Jyh Yun Chwee, Roslyn Kemp. Predicting colorectal patient prognoses by functional characterisation of heterogeneous cell types and their spatial interaction using a new technique: Whole slide imaging mass cytometry [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 67.

Abstract 6169: High throughput in-situ spatial sequencing of proteins and RNA in FFPE tissue

Abstract We have developed an high throughput high resolution in situ multi-omic platform to enable studies in the rapidly evolving landscape of oncology and immunology research. Despite the potential of existing tools, the speed and throughput of current methodologies pose significant limitations. Here, we introduce a novel spatial sequencing platform employing a rapid 4-color SBS chemistry with sub-micron resolution imaging and an ultra-high throughput capacity. The platform simultaneously profiles RNA transcripts and proteins within formalin-fixed paraffin-embedded (FFPE) tissues. With up to 40 cm^2 of tissue area imaging within a 24-hour timeframe, the platform can power extensive, multi-omic studies in cancer research. To evaluate the performance of the platform, we prepared 5 um serial sections from healthy and tumor kidney FFPE block and used a novel tool to precisely position multiple tissue sections on a slide. Following probe binding and amplification, we used SBS readout to profile 105 transcripts and 6 proteins (via DNA-conjugated antibodies). Images were analyzed using a custom pipeline for feature detection and decoding, and cell segmentation using Cellpose. Results showed strong correlation to single-cell transcription profiles and cell type signatures defined by scRNA-Seq. Additionally, we see good agreement between single cell transcript and protein profiles. Existing spatial biology platforms are known to struggle with degraded and challenging clinical sample types (e.g. decalcified bone marrow), due to RNA fragmentation. We successfully profiled immune cells in FFPE-embedded bone marrow biopsies, measuring 143 transcript targets and 8 proteins. We believe this represents a significant step forward for in-situ analysis of bone marrow samples, and could be valuable for studying the pathology and response to treatment in blood cancers such as AML and CML. In-situ sequencing of antibody genes and T-cell receptors provides a deeper view of cellular immune response. As a step towards this goal, we demonstrated in-situ sequencing of antibody heavy and light chains within a B cell line, resulting in accurate readout of 28 bases within the CDR3 regions. In conclusion, we present an SBS-based approach for in-situ detection of targeted gene transcripts and proteins at an unprecedented throughput, and its application to the analysis of human kidney and bone marrow FFPE tissue samples. The capability to deeply profile tissues at high throughput could enable larger translational studies, and assembly of 3D maps of gene and protein expression at sub-cellular resolution. Citation Format: Michael Lawson, Yuji Ishitsuka, Andrew Pawlowski, Zhenmin Hong, Jake Koh, Kenneth Gouin, Nathan Ing, Richard Que, Yeoan Youn, Sara Ding, Tony Facchini, Ryan Costello, Katelyn Nelson, Jamie Stover, Howon Lee, Sarthak Duggal, Taylor Plaziak, Kaitlin Cameron, Mimi Abdu, Daan Witters, Martin Fabani, Eli Glezer. High throughput in-situ spatial sequencing of proteins and RNA in FFPE tissue [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 6169.

Abstract 2911: Prevalence of potential antibody drug conjugate targets and co-expression with PD-L1 in breast cancer and lung cancer patients with clinical response information

Abstract Breast and non-small cell lung cancer (NSCLC) are two of the most important diseases for antibody drug conjugate (ADC) development. ADCs targeting HER2 and TROP2 are currently approved in select breast and lung subgroups with ADC’s to many other tumor targets currently in clinical development. Based on TCGA mRNA expression data, ADC tumor targets currently under investigation for treatment of solid tumors have variable overlap in breast and lung cancers, however little is known about the overlap of ADC targets with PD-L1 which is a predictive biomarker in TNBC and for pembrolizumab monotherapy. In the current study, the prevalence and co-expression of ADC targets (HER2, TROP2, Nectin-4, B7-H3, B7-H4, HER3, FOLR1) were investigated in the context of PD-L1 status and clinical response data for standard-of-care (SOC) and immunotherapy, in breast and NSCLC cancer subtypes. Tissue microarrays (TMAs) comprising treatment naïve tumor samples (1mm cores) and including TNBC (26 patients), ER+ BC (90 patients), and NSCLC (adeno & squamous subtypes; 146 patients), all with full clinical follow-up information, were examined by immunohistochemistry (IHC) in serial sections for each ADC target and PD-L1, applying widely used and well optimized IHC assays on the Ventana and Dako Link 48 platforms. Established scoring methods were employed for analysis of PD-L1 and HER2, except for the addition of an exploratory ultra-low score for HER2. All other ADC targets were scored by digital image analysis to deliver percentage positive tumor expression at high (3+), moderate (2+), weak (1+) and negative (0) intensities. Taken together, this analysis revealed variable prevalence for each target in breast and lung cancer histologies by subtype, including differing overlap between ADC targets within tumor indications. In addition, prevalence of each ADC target in breast cancer characterized by HR status and HER2 subgroups (HER2-positive, low, ultra-low, negative) indicated potential opportunities for ADC clinical development in these subgroups. Co-prevalence of the selected ADC targets with PD-L1 CPS>10 (predictive for Pembrolizumab response in TNBC) and PD-L1 CPS categories of interest for NSCLC revealed patterns of expression in support of combination therapies. Furthermore, expression and co-prevalence data for each ADC target +/- PD-L1 was characterized in association with response to immunotherapy or other SOC. In summary, these data provide better understanding of which breast and lung cancer subtypes express the selected ADC targets uniquely and competitively which may be key to future clinical and diagnostic development. Citation Format: Marie Cumberbatch, Woo Ho Kim, Milan Bhagat, John Cogswell. Prevalence of potential antibody drug conjugate targets and co-expression with PD-L1 in breast cancer and lung cancer patients with clinical response information [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 2911.

Abstract 6876: Cyclic immunofluorescent analysis of the tumor microenvironment across human brain cancer subtypes

Abstract Introduction: The tumor microenvironment (TME) has been the focus of a myriad of studies over the last decade and advancements in the field of cancer research have grown as a result. Improvements in the number of biomarkers that can be screened has increased across a variety of methods and the field of spatial profiling has largely adapted to keep pace with this. However, many challenges persist: 1) tissue microarrays can be limited by tumor heterogeneity and 2) newer technology can be cost-prohibitive. Brain tumors are of significant importance, especially those of glial origin, as they swiftly progress and result in a high fatality rate. As such, the need for more diverse therapeutic approaches is growing with many already pioneering new IHC-based strategies. The aim of this study was to address both challenges addressed above in the neurobiology space. Methods: Biomarkers of importance in brain cancer tissues were identified and three distinct panels were designed. The first panel consisted of CD68, CTLA-4, p16ink4A, p14ARF, and p53. The second included PDPN, Calretinin, MAGE-D2, PTEN, and Vimentin. The third panel included IDH2, BRAF, GFAP, VEGF, and BOP1. Staining was achieved using Bethyl Laboratories IHC-validated primary and secondary antibodies and Akoya Opal™ Polaris 7-color IHC kit fluorophores (Akoya Biosciences [NEL861001KT]). Optimization was achieved using FFPE tissue microarrays containing normal neural tissue and brain cancer subtypes. The final optimized order was then tested on whole serial sections of meningioma, astrocytoma, and glioblastoma in six-color mIF. Images were generated using the PhenoImager HT® and subsequently merged and analyzed by Pathomics. Results: Phenotyping and total cell counts for each marker were identified for each serially stained tissue set. Expression levels of MAGE-D2 and BRAF were high across malignant tissues of variable gradations. Conversely, in tissues with high expression of MAGE-D2, p53 levels in these samples were lower. This is consistent with findings that suggested that MAGE-D2 is a potential dissociator of p53 by impairing transcriptional activity. Macrophage expression appeared to correlate with CTLA4 and overall tumor infiltration. Conclusions: Utilizing several unique biomarkers to understand the spatial relationships and phenotypes within the tumor microenvironment is becoming a more common practice among researchers. In using this method of serially staining tissue sections and merging the resulting images, more researchers will have access to spatial information which may have been cost-prohibitive otherwise. Additionally, in utilizing whole sections of difficult-to-treat cancerous tissue rather than microarrays, phenotypic information is available across the entire tumor microenvironment enabling a more comprehensive assessment of the TME. Citation Format: Danielle Fails, Michael Spencer. Cyclic immunofluorescent analysis of the tumor microenvironment across human brain cancer subtypes [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 6876.

Re-Evaluation of Vascular Histogenesis in the Root Tips of Selected Species in the Poaceae Using New Methods: Analysis of the Plerome, Vascular Initials, Pericycle and Late-Maturing Metaxylem Vessels

Serial sectioning and 3D image reconstruction methods were applied to elucidate the structures of the apices of root vascular cylinders (VCs) in taxa of the Poaceae: Zea mays “Honey Bantam”, Z. mays ssp. mexicana, Hordeum vulgare and Oryza sativa. The primary and nodal roots were investigated. Observations were performed using high-quality sectioning and 3D image-processing techniques improved and developed by the authors. We found that a quiescent uniseriate plerome was located at the most distal part of each VC. Vascular initials were located immediately basipetally to the plerome as a specific uniseriate layer that could be classified into central and peripheral initials that produced all the cells in the VC. No supplying of cells from the plerome to the vascular initials was observed. Numerical analysis revealed a “boundary point” along the root axis where the rate of increase of the vascular cell number markedly declined, and the VC diameter, number of vascular cells, and number of late-maturing metaxylem vessels (LMXs) at that point showed a similar relationship among the taxa and the types of roots examined (primary vs. nodal). The plerome and vascular initials layer can be considered independent after seed germination in these taxa. A boundary point at which procambial cell proliferation sharply declined was identified. The diameters of the VCs, number of LMXs, and number of vascular cells at the boundary point were found to be strongly related to each other.

Brain atlas of the annual Garcialebias charrua fish.

Annual fish have become attractive study models for a wide range of disciplines, including neurobiology. These fish have developed different survival strategies. As a result, their nervous system is under considerable selective pressure when facing extreme environmental situations. Fish from the Austrolebias group exhibit rapid neurogenesis in different brain regions, possibly as a result of the demanding conditions of a changing habitat. Knowledge of cerebral histology is essential for detecting ontogenic, anatomical, or cytoarchitectonic changes in the brain during the short lifespan of these fish, such as those reflecting functional adaptive plasticity in different systems, including sensory structures. The generation of an atlas of Garcialebias charrua (previously known as Austrolebias charrua) establishes its anatomical basis as a representative of a large group of fish that share similarities in their way of life. In this work, we present a detailed study of both gross anatomy and microscopic anatomy obtained through serial sections stained with the Nissl technique in three orientations: transverse, horizontal, and parasagittal planes. This atlas includes accurate drawings of the entire adult brain of the male fish Garcialebias charrua, showing dorsal, ventral, and lateral views, including where emergence and origin of cranial nerves. This brain atlas allows us to understand histoarchitecture as well as the location of neural structures that change during adult neurogenesis, enabling comparisons within the genus. Simultaneously, this atlas constitutes a valuable tool for comparing the brains of other fish species with different behaviors and neuroecologies.

Raman microspectroscopy for label‐free diagnosis of amyloid light‐chain amyloidosis in various organs

AbstractSystemic amyloidosis is a group of diseases in which misfolded proteins aggregate as fibrous amyloid proteins with a β‐sheet structure and deposit in organs, resulting in organ failure. Most types of amyloidosis have a poor prognosis, and prompt diagnosis is essential for treatment. Systemic immunoglobulin light‐chain (AL) amyloidosis is a type of amyloidosis that occurs when abnormal immunoglobulin light‐chain proteins are deposited in various organs and tissues. The deposition of amyloid proteins in tissues has traditionally been confirmed using Congo red staining and polarised light microscopy, which show apple‐green birefringence. In this study, we aimed to verify whether amyloid deposition in the heart, kidney, rectum, duodenum and skin can be detected using Raman microspectroscopy. Serial sections were prepared from formalin‐fixed paraffin‐embedded tissue biopsy samples obtained from patients with systemic amyloidosis. One of the serial sections was stained with Congo red to confirm the deposition of amyloid proteins using polarised light microscopy, whereas the other was left unstained for Raman microspectroscopy. A characteristic peak at Raman shift of 1665–1680 cm−1, which may represent a β‐sheet structure of amyloid proteins, was recorded in the area where the amyloid deposition had been confirmed by Congo red staining. Based on the peak at 1640–1680 cm−1, a colour map was obtained to detect amyloid protein‐positive regions. Thus, amyloid protein detection using Raman microspectroscopy may be useful for rapid diagnosis of amyloidosis.

Resveratrol ameliorates DSS-induced ulcerative colitis by acting on mouse gut microbiota.

Ulcerative colitis (UC) is a multi-faceted, recurrent immune disorder caused by dextran sulfate sodium (DSS). The intestinal microbiota has multiple functions in the host, so UC requires long-term potent medication. The effect of resveratrol (RSV) has seldom been reported, and this study researched that. Herein, the effect of RSV and Grape seed oil that anti-inflammatory ability in experimental mice was explored, also why RSV altered Gut Microbiota has been researched. In this experiment, the effects of experimental drugs on colon length in mice with DSS-induced colitis were compared. H&E Staining was performed on serial sections of colon tissues and histological scores were determined for all groups. The expression of cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α) in the colon tissue of mice was detected by immunohistochemical staining. In the end, the α-diversity index, sobs index, and rarefaction curve of the cecal and colon microbiota of different groups of mice were measured. Bray-Curtis-based Venn diagram of PCoA (principal coordinate analysis) and OTUs distribution in mouse gut microbiota were obtained. The results showed that the use of 40mg/kg RSV (high dose) significantly reduced the severity of UC. The use of 10mg/kg RSV (low dose) significantly reduced the effect of shortened colon length in DSS mice. Compared with the DSS-treated group, the levels of COX-2 and TNF-α in the colon tissues of RSV + DSS-treated mice were significantly decreased. According to this experiment, 19 mouse gut microbiota species had a relative abundance greater than 0.1%, with Beerella, Bacteroides, Helicobacter, Oscillator, and cecum pylori being more abundant in the colon than in the colon. A higher relative abundance of Lachnospira NK4A136 was observed in DSS and RSV groups compared with the control group, whereas the opposite was observed for Alloprevotella. This proves that resveratrol increases the uniformity and diversity of gut microbes to a certain extent, and has a protective effect on the gut.

Spleen white pulp structural and cellular composition in experimental furosemide-­induced hypomagnesemia

Relevance. Magnesium deficiency in the blood (hypomagnesemia) is due to many reasons, among which loop diuretics (furosemide) occupy acertain place. The role of the spleen in this process has not been determined. The aim of the work was to elucidate the effect of furosemide-­induced hypomagnesemia on the immune structures of the white pulp of rat spleen. Materials and Methods. Furosemide (Lasix® Aventis Pharma Ltd, India) was injected daily intraperitoneally at adose of 30 mg/kg to the experimental group of white outbred rats for 6 days, animals of the control group received an injection of 0.9% NaCl. Investigated: blood serum for the content of magnesium, calcium, sodium and iron; serial sections of the white pulp of the spleen after staining with hematoxylin and eosin to assess the structure and azure-­II-eosin to assess the cellular composition. With amicroscope magnification of 280 times, the ratio (in%) of primary (PLNS) and secondary lymphoid nodules of the spleen (SLNS) was calculated, the following were measured (µm): the diameter of the germinal center (GC), the width of the mantle and marginal zone, the diameter of the periarteriolar lymphoid sheath (PALS). In GC, the peripheral zone of lymphoid nodules, PALS at amagnification of 1500 times per field of view (100 μm2) was counted and presented as apercentage of the number of lymphocytes; macrophages; cells, mitotic and apoptotic elements. Morphometric analysis was carried out using Image ProPlus6.0 software (Media Cybernetics, USA). Statistical processing was carried out using the Statistica 10.0 software package with the determination of the arithmetic mean (M) and its error (m). Results and Discussion. The administration of furosemide led to adecrease in magnesium in the blood serum by 1.6 times (p0.05). In the white pulp of the spleen of animals of the experimental group, the proportion of SLNS decreased by 18.14%, the number of SLNS increased by 42.5% (p0.05). The diameter of SLNS increased insignificantly, the diameter of GC and the width of the marginal zone significantly increased by 27.1 and 24.8%, respectively. The proportion of macrophages increased by 20.6% in GC SLNS, and by 17.0% in PALS. The highest increase in the proportion of cells with signs of apoptosis was found in the periarteriolar lymphoid sheath of experimental animals - 34.6% (p0.05). Conclusion. Furosemide loading causes the development of dyselementosis, with the most significant loss of magnesium (hypomagnesemia) and has apronounced effect on the immune parameters of the spleen, represented by white pulp structures. Therefore, correction of the elemental status and monitoring of the state of the spleen in hypomagnesemia caused by the use of loop diuretics is anecessary element in the prevention of complications associated with the use of diuretic drugs.

Drusen in the macula and parapapillary region.

To examine histological characteristics and differences between drusen beneath the retinal pigment epithelium (small hard drusen) located in the macula and located in the parapapillary region. We histomorphometrically examined human eyes enucleated due to uveal melanomas or secondary angle-closure glaucoma. The study included 106 eyes (age, 62.6 ± 15.2years) with macular drusen (n = 7 globes) or parapapillary drusen (n = 29 eyes) and 70 eyes without drusen. In all drusen, periodic-acid-Schiff-positive material was located between the RPE basal membrane and the inner collagenous layer of Bruch's membrane (BM). Macular drusen as compared with parapapillary drusen had lower height (15.2 ± 10.1µm versus 34.3 ± 19.8µm; P = 0.003), while both groups did not differ significantly in basal drusen width (74.0 ± 36.3µm versus 108.7 ± 101.0µm; P = 0.95). Eyes with macular drusen and eyes without drusen did not differ significantly in BM thickness (2.74 ± 0.44µm versus 2.55 ± 0.88µm; P = 0.57) or in RPE cell density (35.4 ± 10.4 cells/480µm versus 32.8 ± 7.5 cells/480µm; P = 0.53), neither in the drusen region nor in the drusen vicinity, while BM thickness (4.60 ± 1.490µm; P < 0.001) and RPE cell density (56.9 ± 26.8 cells/480µm; P = 0.005) were higher at the parapapillary drusen. Eyes with macular drusen, eyes with parapapillary drusen, and eyes without drusen did not differ significantly in choriocapillaris density (all P > 0.10) and thickness (all P > 0.35). Limitations of the study, among others, were a small number and size of drusen examined, diseases leading to enucleation, lack of serial sections, limited resolution of light microscopy, and enucleation-related and histological preparation-associated artefacts. The findings of this study, also taking into account its methodological limitations, suggest that macular drusen and parapapillary drusen shared the morphological feature of periodic-acid-Schiff-positive material between the RPE basal membrane and BM and that they did not vary significantly in choriocapillaris thickness and density. RPE cell density and BM thickness were higher in parapapillary drusen than in macular drusen.

Integrating X-ray phase-contrast imaging and histology for comparative evaluation of breast tissue malignancies in virtual histology analysis

Detecting breast tissue alterations is essential for cancer diagnosis. However, inherent bidimensionality limits histological procedures’ effectiveness in identifying these changes. Our study applies a 3D virtual histology method based on X-ray phase-contrast microtomography (PhC μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT), performed at a synchrotron facility, to investigate breast tissue samples including different types of lesions, namely intraductal papilloma, micropapillary intracystic carcinoma, and invasive lobular carcinoma. One-to-one comparisons of X-ray and histological images explore the clinical potential of 3D X-ray virtual histology. Results show that PhC μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT technique provides high spatial resolution and soft tissue sensitivity, while being non-destructive, not requiring a dedicated sample processing and being compatible with conventional histology. PhC μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT can enhance the visualization of morphological characteristics such as stromal tissue, fibrovascular core, terminal duct lobular unit, stromal/epithelium interface, basement membrane, and adipocytes. Despite not reaching the (sub) cellular level, the three-dimensionality of PhC μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT images allows to depict in-depth alterations of the breast tissues, potentially revealing pathologically relevant details missed by a single histological section. Compared to serial sectioning, PhC μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT allows the virtual investigation of the sample volume along any orientation, possibly guiding the pathologist in the choice of the most suitable cutting plane. Overall, PhC μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT virtual histology holds great promise as a tool adding to conventional histology for improving efficiency, accessibility, and diagnostic accuracy of pathological evaluation.

Anatomy of muscle connections in the male urethra and anorectal canal.

To elucidate the male urethral muscular structure and its relationship with the anorectal canal muscles, as establishing an anatomical foundation for urethral function will contribute to the prevention, diagnosis, and treatment of urinary incontinence. Eight male cadavers were used. Using a multifaceted approach, we performed macroscopic anatomical examination, histological analysis of wide-range serial sectioning and immunostaining, and three-dimensional (3D) reconstruction from histological sections. In the macroscopic anatomical examination, pelvic halves were meticulously dissected in layers from the medial aspect. In the histological analysis, the tissue, including the urethra and anorectal canal, was serially sectioned in the horizontal plane. The muscular structures were reconstructed and visualised in 3D. The membranous portion of the urethra had three muscle layers: the longitudinal and circular muscles (smooth muscle) and the external urethral sphincter (skeletal muscle). The circular muscle was connected posteriorly to the longitudinal rectal muscle. The external urethral sphincter had a horseshoe shape, with its posterior ends continuing to the external anal sphincter, forming a 3D ring-like sphincter. This study revealed skeletal and smooth muscle connections between the male urethra and anorectal canal, enabling urethral compression and closure. These anatomical muscle connections suggest a functional linkage between them.

Effective alignment method using a diamond notch knife for correlative array tomography.

Correlative array tomography, combining light and electron microscopy via serial sections, plays a crucial role in the three-dimensional ultrastructural visualization and molecular distribution analysis in biological structures. To address the challenges of aligning fluorescence and electron microscopy images and aligning serial sections of irregularly shaped biological specimens, we developed a diamond notch knife, a new tool for puncturing holes using a diamond needle. The diamond needle featured a triangular and right-angled tip, enabling the drilling of deep holes upon insertion into the polished block face. This study describes the application of the diamond notch knife in correlative array tomography.

REVIEW OF COMPLETENESS OF DEATH CERTIFICATE FILLING AT BANTEN

Background: Filling in the Death Certificate at RSU Banten is the part that gets attention in this study. The Death Certificate form was found to be incomplete in patient identification, completeness of important reports, author authentication and good records. The Medical Certificate of Cause of Death (SMPK) is the main source of mortality data. Death information is usually obtained from health practitioners or in cases of death due to accidents, violence and other diseases. Based on a preliminary study in March 2020 at Banten General Hospital, it was found that out of 20 death certificate forms there were 10 incomplete death certificate forms, including the incomplete identification component of 7.75 (39%) such as in the death serial number section, the main number population and population number. 9 (45%) important report components were incomplete, such as the type of examination, ICD code, method and location of burial. The incomplete authentication component of 10 (50%) is as in the reporting signature and reporting name. An incomplete record component of 6.6 (35%) is like a lot of blanks. The purpose of the study was to find out the Completeness of Filling in Death Certificates at RSU Banten and the results of this study were expected to be useful for hospitals as input and consideration in addressing the issue of completeness of medical certificates causing death. and the approach used in this research is to use a comparative approach, namely through a checklist and interviews at RSU Banten. The research method used is quantitative research. The population in January- February 2020 was 233 death certificate forms at RSU Banten, while the subject population was 12 officers who were responsible for completing death certificates. The sample object taken for this research is a Death Certificate form as many as 77 samples. While the sample of subjects in this study were 2 officers who filled out death certificates. Sampling Technique Subjects in this study used non-random (non-probability sampling) with purposive sampling. The results showed that the SOP (Standard Operating Procedure) already exists and is complete which regulates the Filling of Death Certificates at RSU Banten in the installation section of the funeral procession (IPJ), but it is still not fully implemented because there are still many incomplete forms, from the 5 SOP items it is known that those who are complete with SOP guidelines are (100%) while those that are incomplete with SOP guidelines are (0%). The results of completeness in patient identification with a complete percentage are found in the sub-components of medical record numbers and names, which are 77 (100%), while the incomplete percentages are found in the sub-components of death serial number, population identification number and population identification number, which is 77 (100% , Important reports on death certificates that are filled out completely are found in the time of death sub-component, which is 77 (100%), while the completeness of filling out important reports that are incomplete on the death serial number is 77 (100%).Authentication Author on death certificates which is filled in completely is contained in the sub-components of the doctor's TTD sign and the doctor's name, which is 77 (100%), while the incomplete completeness of author authentication on the reporting blood pressure, the name of the reporter, the identity card number of the reporting person, the address of the reporter is 77 (100%). Both the death certificate that is filled in completely, there are sub-components, there are no scribbles and there is no type - ex, which is 77 (100%). while the completeness of incomplete filling on a good record is found in the sub-components, there are no empty parts, namely 77 (100%). Data collection was carried out by survey, namely the researcher conducted direct interviews with respondents using a questionnaire. The research results obtained the following data; There were97 toddlers who had pneumonia or 26.5%, almost all of them 94.5% of mothers had good knowledge about pneumonia, 46.7% of mothers had sufficient behavior and 49.5% of mothers had sufficient attitudes and more than half of them 70.2% of mothers had not been exposed to information about pneumonia. The Bivariate results found that attitude and information exposure had a significant relationship with the incidence of pneumonia, while behavior and knowledge had no significant relationship with the incidence of pneumonia among toddlers in the city of Cilegon. From the results of this study, it is hoped that the Cilegon City Health Office can increase socialization and counseling about pneumonia through cooperation with related agencies so that it can improve the degree of public health.

Comparative Characterization of Human Meibomian Glands, Free Sebaceous Glands, and Hair-Associated Sebaceous Glands Based on Biomarkers, Analysis of Secretion Composition, and Gland Morphology.

Meibomian gland dysfunction (MGD) is one of the main causes of dry eye disease. To better understand the physiological functions of human meibomian glands (MGs), the present study compared MGs with free sebaceous glands (SGs) and hair-associated SGs of humans using morphological, immunohistochemical, and liquid chromatography-mass spectrometry (LCMS)-based lipidomic approaches. Eyelids with MGs, nostrils, lips, and external auditory canals with free SGs, and scalp with hair-associated SGs of body donors were probed with antibodies against cytokeratins (CK) 1, 8, 10, and 14, stem cell markers keratin 15 and N-cadherin, cell-cell contact markers desmoglein 1 (Dsg1), desmocollin 3 (Dsc3), desmoplakin (Dp), plakoglobin (Pg), and E-cadherin, and the tight junction protein claudin 5. In addition, Oil Red O staining (ORO) was performed in cryosections. Secretions of MGs as well as of SGs of nostrils, external auditory canals, and scalps were collected from healthy volunteers, analyzed by LCMS, and the data were processed using various multivariate statistical analysis approaches. Serial sections of MGs, free SGs, and hair-associated SGs were 3D reconstructed and compared. CK1 was expressed differently in hair-associated SGs than in MGs and other free SGs. The expression levels of CK8, CK10, and CK14 in MGs were different from those in hair-associated SGs and other free SGs. KRT15 was expressed differently in hair-associated SGs, whereas N-cadherin was expressed equally in all types of glands. The cell-cell contact markers Dsg1, Dp, Dsc3, Pg, and E-cadherin revealed no differences. ORO staining showed that lipids in MGs were more highly dispersed and had larger lipid droplets than lipids in other free SGs. Hair-associated SGs had a smaller number of lipid droplets. LCMS revealed that the lipid composition of meibum was distinctively different from that of the sebum of the nostrils, external auditory canals, and scalp. The 3D reconstructions of the different glands revealed different morphologies of the SGs compared with MGs which are by far the largest type of glands. In humans, MGs differ in their morphology and secretory composition and show major differences from free and hair-associated SGs. The composition of meibum differs significantly from that of sebum from free SGs and from hair-associated SGs. Therefore, the MG can be considered as a highly specialized type of holocrine gland that exhibits all the histological characteristics of SGs, but is significantly different from them in terms of morphology and lipid composition.

Retroperitoneal fasciae as barriers for nerve and arterial passages connecting the retroperitoneal region to the peritoneal organs.

The fascia of the pancreatic head is referred to as the retropancreatic fascia of Treitz, and that of the body and tail of the pancreas is named the retropancreatic fascia of Toldt. However, the spatial relationship between the nerves, fascia, and the distribution of the fascia on the dorsal side of the pancreas remains unclear. Therefore, this study aimed to explore the distribution of these fasciae and elucidate the spatial relationship between the nerves and arteries connecting the retroperitoneal space and the peritoneal organs by studying eight cadavers using macroscopic anatomical examination, wide-range serial sectioning, and three-dimensional reconstruction. The fasciae of Treitz and Toldt converge caudally to the root of the superior mesenteric artery (SMA), forming a narrower gap around the roots of the celiac trunk and SMA than in the celiac plexus. The fasciae eventually get closer to each other, and the boundary between them becomes obscured, providing coverage to the anterior surface of the aorta between the SMA and the inferior mesenteric artery. The celiac plexus does not penetrate the fascia but converges before spreading into the pancreas. Similarly, the arteries pass through this gap in the fasciae. Our findings suggest that the retroperitoneal space and peritoneal organs are connected through a narrow no-fascia area, with the distribution of the fascia relating to nervous and vascular pathways. Our findings reveal that the distribution of the avascular plane may provide a crucial anatomical foundation for abdominal digestive organ surgery by reducing bleeding volume and determining the dissection region.

GAUSS-EM, guided accumulation of ultrathin serial sections with a static magnetic field for volume electron microscopy

Serial sectioning electron microscopy (EM) of millimeter-scale three-dimensional (3D) anatomical volumes requires the collection of thousands of ultrathin sections. Here, we report a high-throughput automated approach, GAUSS-EM (guided accumulation of ultrathin serial sections-EM), utilizing a static magnetic field to collect and densely pack thousands of sections onto individual silicon wafers. The method is capable of sectioning hundreds of microns of tissue per day at section thicknesses down to 35nm. Relative to other automated volume EM approaches, GAUSS-EM democratizes the ability to collect large 3D EM volumes because it is simple and inexpensive to implement. We present two exemplar EM volumes of a zebrafish eye and mouse olfactory bulb collected with the method.