Serial Sections Research Articles

Programming-free Image-Analysis Workflows for IHC, IF and Spatial Proteomics: More than just Cell Counting

Quantitative analysis of cells using a specific protein marker is one of the most frequently performed tasks in both preclinical and clinical histopathology. The primary alternatives include immunohistochemistry and immunofluorescence. This article will briefly shed some light on the pros and cons of both methods, the main ones being that immunohistochemistry is widely available, but in many laboratories, it is limited to only one marker per image. Utilizing more than one marker can be achieved through methods such as establishing duplex or triplex stains, serial sections, or re-staining. However, immunofluorescence excels where a deep multi-marker characterization of single cells is required. Spatial proteomics systems have recently increased the plexity to as many as 100 parallel markers, allowing advanced co-expression, rare cells, and spatial neighbourhood analyses. Bioinformatic analysis aspects for both modalities are discussed, outlining two generic workflows as they are realised in a professional image analysis software used by biomedical researchers. While cell segmentation and typing are at the core, a number of pre- and post-processing steps, such as tissue detection, comparison of ROIs, hotspot search, spatial clustering or neighbourhood analysis should be performed to provide more comprehensive read-outs.

Gender Differences in the Impact of a High-Fat, High-Sugar Diet in Skeletal Muscles of Young Female and Male Mice

In the context of the increasing number of obese individuals, a major problem is represented by obesity and malnutrition in children. This condition is mainly ascribable to unbalanced diets characterized by high intakes of fat and sugar. Childhood obesity and malnutrition are not only associated with concurrent pathologies but potentially compromise adult life. Considering the strict correlation among systemic metabolism, obesity, and skeletal muscle health, we wanted to study the impact of juvenile malnutrition on the adult skeletal muscle. To this aim, 3-week-old C56BL/6 female and male mice were fed for 20 weeks on a high-fat. high-sugar diet, and their muscles were subjected to a histological evaluation. MyHCs expression, glycogen content, intramyocellular lipids, mitochondrial activity, and capillary density were analyzed on serial sections to obtain the metabolic profile. Our observations indicate that a high-fat, high-sugar diet alters the metabolic profile of skeletal muscles in a sex-dependent way and induces the increase in type II fibers, mitochondrial activity, and lipid content in males, while reducing the capillary density in females. These data highlight the sex-dependent response to nutrition, calling for the development of specific strategies and for a systematic inclusion of female subjects in basic and applied research in this field.

Evaluating the Quality of Serial EM Sections with Deep Learning.

Automated image acquisition can significantly improve the throughput of serial section scanning electron microscopy (ssSEM). However, image quality can vary from image to image depending on autofocusing and beam stigmation. Automatically evaluating the quality of images is, therefore, important for efficiently generating high-quality serial section scanning electron microscopy (ssSEM) datasets. We tested several convolutional neural networks for their ability to reproduce user-generated evaluations of ssSEM image quality. We found that a modification of ResNet-50 that we term quality evaluation Network (QEN) reliably predicts user-generated quality scores. Running QEN in parallel to ssSEM image acquisition therefore allows users to quickly identify imaging problems and flag images for retaking. We have publicly shared the Python code for evaluating images with QEN, the code for training QEN, and the training dataset.

Abstract PS16-10: SPATIAL IMMUNE CELL DISTRIBUTION CAN REFINE PROGNOSIS IN EARLY-STAGE ER+/HER2-/LN- BREAST CANCER

Abstract Lymphocytic infiltrate is a known prognostic biomarker in estrogen receptor(ER)-negative breast cancers (BCs). Comparatively ER+ disease is putatively cold, however, there exists an infiltrate-rich subset of ER+ tumours with significant spatial heterogeneity and unknown clinical impact. Using serial sections taken from early-stage, ER+/HER2- breast tumours of Irish patients enrolled in the TAILORx trial (n=450), we aimed to investigate the prognostic potential of markers encompassing tumour architecture. Immunohistochemistry for Ki67 and CD45 (leukocyte common-antigen), and staining for haematoxylin and eosin was applied to all sections [1]. Classifiers for stromal fraction (SF), stromal-infiltrate (sTIL), Ki67-LI, and their spatial relationships within the tumour were generated using QuPath [2] and R Studio. Trained marker classifiers were validated against an expert pathologist (R2 of M-Score to: CD45%: 0.968, Ki67-LI: 0.814, sTIL: 0.864) to define observed lymphocytes as tumour or stromal-infiltrating, retain only tumour Ki67+ density, and to investigate SF. Cohort mean SF was 67.69% (range 16.35 – 98.29%), with marginally significant differences in recurrence prediction for cohort high v low SF by mean (c-index = 0.58, p< 0.032), and luminal A disease (p=0.037) only. sTILs did not differ significantly between high/low mean SF (Mann-Whitney p=0.6201), though sTIL was prognostic in the OncotypeDx intermediate Recurrence Score (RS) category overall (p=0.0076). This trend appeared strongest in intermediate RS patients receiving chemo-endocrine therapy (HT+CT) (p < 0.0001) vs endocrine therapy (HT) alone (p=0.86). Investigating the effects of prescribed therapy on sTIL-derived recurrence risk also revealed significant trends in those patients receiving HT+CT only (p < 0.00001) vs HT alone (p=0.26). Spatial analysis of sTILs suggest that tumours become more immune excluded as Oncotype Dx RS increases - particularly from intermediate to high RS (Wilcoxon Intermediate v High: p=0.0077), with significant differences in survival for high/low tumour-immune hotspots observed in Intermediate RS, HT+CT treated patients (p=0.0052). Further spatial analysis suggests that high normalised frequency of infiltrate [method adapted from 3] (within 7um of tumour cells) have negative prognostic impact for premenopausal patients (p=0.017), and inter-sectional analysis identifies niche tumour environments where high Ki67-LI colocalize with immune infiltrates[methods adapted from 4, 5], most notable in Intermediate RS tumours.. Overall these data suggest immune-spatial subsets can be used to refine risk stratification of ER+ breast cancer.

Abstract PO3-13-11: Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies

Abstract Emerging antibody drug conjugate (ADC) therapies targeting human trophoblast cell-surface antigen (TROP2) and human epidermal growth factor receptor 2 (HER2) are transforming the treatment landscape for breast cancer. Sacituzumab govitecan (SG) and trastuzumab deruxtecan (T-DXd) have gained approval for an overlapping set of "HER2-low" metastatic breast cancers, including hormone receptor (HR)-positive HER2 non-amplified and "triple-negative" subtypes. Nevertheless, the optimal selection of patients and treatment sequencing for these ADC therapies remains a clinical challenge. Clinical trial objective response rates to SG are approximately 30%, compared to 30-90% for T-DXd depending on HER2 expression levels. While both drugs are thought to be targeted therapies, the value of measuring the target and the best methods to do so are still not established. We believe that quantitative measurement of TROP2 and HER2 antigen expression levels could establish thresholds for responders, enabling more effective patient selection for ADC therapies. Here, we present a TROP2, high-sensitivity HER2, and cytokeratin (CK) quantitative immunofluorescence (QIF) multiplex assay. Using a ten-cell line standard array and proteomic mass spectrometry, we can convert tumor compartment QIF intensity to protein concentration in fmol/mm2 for tissue specimens. Anti-HER2, anti-TROP2 antibodies, and fluorescence detection systems were titrated and combined to maximize signal-to-noise ratio on our cell line standard array and breast cancer tissue microarrays (TMA). The multiplex assay was designed for automated slide stainers (Leica BOND Rx) and fluorescence slide scanning (Rarecyte CyteFinder II HT). We perform our analyses in QuPath using an image processing plugin developed for automated QIF/IHC analysis (Qymia). Reproducible TROP2 and HER2 QIF scoring (R² > 0.95) was achieved across multiple staining batches using serial sections of breast cancer TMAs and cell standard arrays. This assay has a TROP2 linear range between 0.63 - 9.17 fmol/mm2 (about 1 million TROP2 receptors/cell) and HER2 linear range between 0.09 - 0.565 fmol/mm2 (about 60,000 HER2 receptors/cell). We then applied this multiplex assay to two serial retrospective primary breast cancer cohorts from Yale University to quantitatively measure TROP2 and HER2 expression (338 clinical cases). We find a weak negative correlation between TROP2 and HER2 expression in our breast cancer cohorts (Pearson r = -0.14, p = 0.0097, n = 338). TROP2 expression levels were above the limit of detection (LOD) in 90.2% of cases, with 4.1% exceeding the limit of linearity (LOL), and a mean TROP2 expression of 4.05 fmol/mm2. For HER2, 67.2% of cases were above the LOD, with 7.1% exceeding the LOL, and a mean HER2 expression of 0.186 fmol/mm2. Both TROP2 and HER2 were below the LOD in 3.0% of cases, which we define as “negative”. We found 29.9% expressed TROP2 and were HER2-negative, and 6.8% expressed HER2 and were TROP2-negative. Our future studies will aim to quantitatively define expression thresholds for T-DXd and SG response with the goal to produce a clinical grade assay for ADC patient selection and determine the value of the assay to help select which ADC to give first. HER2 and TROP2 protein expression summary in Yale breast cancer cohort Table 1: Summary of HER2 and TROP2 protein expression levels in serial retrospective primary breast cancer cohort of 338 cases using our high-sensitivity HER2 and TROP2 multiplex immunofluorescent assay Citation Format: Charles Robbins, Mengni He, Revekka Khaimova, Katherine Bates, Nay Nwe Nyein Chan, Daniel Liebler, Regan Fulton, David Rimm. Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-11.

Abstract PO5-13-11: Quantitative Assessment of HER2 Expression in Intraductal Neoplasms of the Breast

Abstract HER2 (ERBB2) is an established prognostic and predictive marker for patients with invasive breast cancer and used in HER2-targeted therapy. The clinical and biological significance of expression of HER2 protein in patients with intraductal neoplasms of the breast is not well characterized. In this study, we used our HS-HER2 (High Sensitivity-HER2) which is a CAP/CLIA certified laboratory derived test (LDT) using quantitative immunofluorescence (QIF). We evaluated HER2 expression in DCIS (ductal carcinoma in situ), LCIS (lobular carcinoma in situ) and benign lesions to correlate with their clinicopathologic characteristics. Thirty- eight cases of DCIS, and fourteen cases of LCIS and thirty benign cases were selected from the Yale Pathology department archives and 252 ROIs (regions of interest) were annotated by a board-certified pathologist. DCIS is classified according to the three-tier nuclear grading system: low, intermediate, and high-nuclear grade. LCIS is classified as PLCIS (pleomorphic variant) and CLCIS (classic LCIS) according to the WHO classification of breast tumors. Serial sections of FFPE tumor specimens were used to accurately quantify the HER2 expression by the HS-HER2 assay and the acquisition by QuPath v.04 with Qymia extension. According to the LOD (limit of detection,3 amol/mm2), LOQ (limit of quantification, 9 amol/mm2) and LOL (limit of linearity, 23 amol/mm2) of the optimized HS-HER2 assay as performed in our CLIA lab shows a broad range of HER2 expression in both DCIS and LCIS lesions, ranging from 0.7 amol/mm2 to 111.4 amol/mm2 in DCIS and 3.6 amol/mm2 to 49.9 amol/mm2 in LCIS. High grade DCIS lesions express higher average HER2 levels (111.4 + 0.7 amol/mm2) than combined low and intermediate grade DCIS (77.9 +1.9 amol/mm2) with no significant difference between low/intermediate and high-grade DCIS. CLCIS lesions express higher average HER2 level (49.9 + 3.6 amol/mm2) than PLCIS (35.1 + 5.4amol/mm2) but again this is not statistically significant. HER protein expression is relatively low in benign lesions ranging from 0.8 to 8.4 amol/mm2. Using the HS-HER2 assay, our results show quantitative level HER2 levels in DCIS and LCIS breast lesions. These findings may be important as HER2 targeted therapies that work in gene-unamplified cancer (HER2 conjugated ADCs) gain broader usage in the clinic. Citation Format: Nay Nwe Nyein Chan, Haiying Zhan, Revekka Khaimova, Thazin Aung, Charles Robbins, Patricia Gaule, David Rimm. Quantitative Assessment of HER2 Expression in Intraductal Neoplasms of the Breast [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-11.

Ameliorative Mechanism of Dietary Vitamin D and Magnesium on Newborn’s Pulmonary Toxicity Induced by Cadmium

Cadmium (Cd) exposure in mothers can cause respiratory issues in newborns, but the exact toxicity mechanisms are not fully understood. Vitamin D deficiency in Cd-exposed rats is associated with increased cadmium accumulation in tissues. Finding a cost-effective medication that is vital for the body while also reducing the effects of poisoning is crucial in treating poisonings. To investigate the mechanisms of Cd-induced lung toxicity, we examined the impact of prolonged Cd exposure in female rats before pregnancy on newborn lung health, focusing on sera TNF-α level, lung P53, Foxo1 mRNA, and lung VEGF, and BMP-4 protein level. A total of 50 rats were divided into control, Cd, Cd+Vitamin D, Cd+Mg, and Cd + Vitamin D+Mg groups. Cd exposure resulted in higher serum TNF-α levels and a significant rise in P53 mRNA levels. Additionally, the occurrence of hemorrhage, inflammatory cell infiltration, and thickening of alveolar walls decreased following treatment with vitamin D + Mg. Although Cd did not affect the newborns' body weight, it did impair their lung function. These findings suggest that the Cd-induced increase in the P53 gene expression could be alleviated by vitamin D and Mg, along with the elevation of VEGF and BMP-4 proteins and Foxo1 gene expression. The study revealed that environmental toxins can sometimes harm molecules and proteins, leading to damage in critical fetal tissues. However, these issues can be mitigated through essential supplements. Structured AbstractThe increasing role of Cd in the erratic behavior of numerous biological and molecular entities, notably the development of fetal lung tissue, has made it beneficial to investigate the possible adverse effects of Cd exposure in pregnant mothers and fetal organ development, where instinctive molecular events occur. Researchers are encouraged to create new aspects of medications to reduce clinical symptoms and improve the quality of life due to exposure to metal toxins, particularly in industrialized countries. The present study aimed to evaluate histopathological and molecular modifications of fetal lungs caused by maternal Cd toxicosis and evaluate the possible ameliorating effects of vitamin D and Mg alone and in combination with fetal lung developmental abnormalities, followed by maternal toxin induction, which can be generalized to humans.Fifty female Wistar rats were purchased from the Pasteur Institute of Iran. To induce the model, cadmium at a dose of 2mg/kg body weight was injected intraperitoneally into the female rats over 28 days before mating (5 days after injection in a week). Afterward, the female rats were randomly divided into type IV polycarbonate cages and mated with healthy male rats. The pregnancy was confirmed by observation of the vaginal plaque, which was subsequently observed, and the number of days of embryo formation was calculated. Subsequently, the pregnant rats were assigned to the following groups and received PBS, vitamin D, Mg, or vitamin D + Mg. At the end of the nine-day treatment period (the 6th day of pregnancy to the 14th day), the neonates were born vaginally, and their body weight and mortality were recorded. The P53 and Foxo1 gene expression levels in the left and right lobes of the homogenized lungs of the newborns in each group were assessed. TNF-alpha was detected in the sera collected from the newborns by ELISA. The isolated left and right lung tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer and the superior phase was collected to determine the total protein content by Lowry's method and VEGF and BMP-4 protein levels. The obtained lung samples from newborn rats were fixed in a 10% formalin solution for tissue processing. The fixed samples were embedded in paraffin, and serial paraffin sections were prepared for hematoxylin and eosin staining. This study is the first to examine how maternal Cd exposure affects fetal lung development and to estimate the impact of prescribing Mg and vitamin D during pregnancy. The present study assessed the effects of a repeated dose of Cd for 4 weeks before pregnancy on the lung development of newborn rats born to mothers treated with vitamin D and Mg. The results showed that the P53 gene was overexpressed in the model group, while Foxo1 gene expression was downregulated, negatively impacting the lung structure and developmental indices of the fetuses. Therefore, the intake of vitamin D and Mg may contribute to improving the various stages of Cd-induced lung injury by modulating lung inflammation and mucosal secretion while also positively influencing the number of surviving offspring.

Increased tumor-infiltrating plasmacytoid dendritic cells express high levels of PD-L2 and affect CD8+ T lymphocyte infiltration in human laryngeal squamous cell carcinoma

The infiltration and prognostic significance of tumor-infiltrating plasmacytoid dendritic cells (TI-pDC) have been elucidated in various human solid cancers. However, the infiltrating patterns and functional importance of TI-pDC in laryngeal squamous cell carcinoma (LSCC) remain unknown. In this study, flow cytometric analyses were conducted to characterize the infiltration of dendritic cells and T lymphocytes, along with their respective subgroups in tumor tissues (TT), para-carcinoma tissues (PT), and peripheral blood (PB) from LSCC patients. Immunohistochemical staining for CD4 and CD8, as well as immunofluorescence staining for CD123, were performed on serial tissue sections to investigate the co-localization of TI-pDC and tumor-infiltrating T lymphocytes (TIL) within the tumor microenvironment (TME). Our results demonstrated significantly lower percentages of all three DC subsets in PB compared to TT and PT. Notably, the pDC percentage was markedly higher in TT than in PT. Moreover, TI-pDC percentage was significantly elevated in N+ stage patients compared to those with N0 stage. The results of survival analysis consistently demonstrated that high levels of TI-pDC infiltration were indicative of a poor prognosis. Further investigation revealed a significant negative correlation between TI-pDC and CD8+ TILs; notably, pDCs expressed an inhibitory surface molecule PD-L2 rather than PD-L1 within PT. Collectively, our findings suggest that increased TI-pDC is associated with adverse outcomes in LSCC patients while exhibiting an inhibitory phenotype that may play a crucial role in suppressing CD8+ TILs within LSCC tumors. These results highlight the potential therapeutic strategy targeting PD-L2+ pDCs for immunotherapies against LSCC.

An Extensive Study Regarding the Microscopic Anatomy of the Early Fetal Human Optic Nerve

The development of the optic nerve and its surrounding tissues during the early fetal period is a convoluted period because it spans both the organogenesis period and the fetal period. This study details the microscopic anatomy and histoembryology of the optic nerve in embryos during the early fetal period, including the second half of the first trimester of pregnancy. Serial sections through the orbit of variously aged embryos allowed us to analyze the nerve in both longitudinal and transverse aspects. A histological assessment and description of the structures surrounding and inside the nerve were performed, highlighting the cellular subtypes involved. By employing immunohistochemical techniques, we could characterize the presence and distribution of astrocytes within the optic nerve. Our findings suggest that by the 8th gestational week (WG) the structures are homologs to all the adult ones but with an early appearance so that maturation processes take place afterward. By this age, the axons forming the nerve are definitive adult axons. The glial cells do not yet exhibit adult phenotype, but their aspect becomes adult toward the 13th week. During its development the optic nerve increases in size then, at 14 weeks, it shrinks considerably, possibly through its neural maturation process. The morphological primordium of the blood–nerve barrier can be first noted at 10 WG and at 13 WG the morphological blood–nerve barrier is definitive. The meningeal primordium can be first noted as a layer of agglomerated fibroblasts, later toward 13 WG splitting in pachymeninx and leptomeninges and leaving space for intrinsic blood vessels.

Perspective Article: Space-time dynamics of genome replication studied with super-resolved microscopy

Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids. We illustrate this problem with EdU pulse-labeling (10 min) and chase experiments (80 min) performed with mouse myeloblast cells. Following light optical serial sectioning of nuclei with 3D structured illumination microscopy (SIM), seven DNA intensity classes were distinguished as proxies for increasing DNA compaction. In nuclei of cells fixed immediately after the pulse-label, we observed a relative under-representation of EdU-labeled DNA in low DNA density classes, representing the active nuclear compartment (ANC), and an over-representation in high density classes representing the inactive nuclear compartment (INC). Cells fixed after the chase revealed an even more pronounced shift to high DNA intensity classes. This finding contrasts with previous studies of the transcriptional topography demonstrating an under-representation of epigenetic signatures for active chromatin and RNAPII in high DNA intensity classes and their over-representation in low density classes. We discuss these findings in the light of current models viewing CDs either as structural chromatin frameworks or as phase-separated droplets, as well as methodological limitations that currently prevent an integration of this contrasting evidence for the spatial nuclear topography of replication and transcription into a common framework of the dynamic nuclear architecture.

RegWSI: Whole slide image registration using combined deep feature- and intensity-based methods: Winner of the ACROBAT 2023 challenge

Background and objectiveThe automatic registration of differently stained whole slide images (WSIs) is crucial for improving diagnosis and prognosis by fusing complementary information emerging from different visible structures. It is also useful to quickly transfer annotations between consecutive or restained slides, thus significantly reducing the annotation time and associated costs. Nevertheless, the slide preparation is different for each stain and the tissue undergoes complex and large deformations. Therefore, a robust, efficient, and accurate registration method is highly desired by the scientific community and hospitals specializing in digital pathology. MethodsWe propose a two-step hybrid method consisting of (i) deep learning- and feature-based initial alignment algorithm, and (ii) intensity-based nonrigid registration using the instance optimization. The proposed method does not require any fine-tuning to a particular dataset and can be used directly for any desired tissue type and stain. The registration time is low, allowing one to perform efficient registration even for large datasets. The method was proposed for the ACROBAT 2023 challenge organized during the MICCAI 2023 conference and scored 1st place. The method is released as open-source software. ResultsThe proposed method is evaluated using three open datasets: (i) Automatic Nonrigid Histological Image Registration Dataset (ANHIR), (ii) Automatic Registration of Breast Cancer Tissue Dataset (ACROBAT), and (iii) Hybrid Restained and Consecutive Histological Serial Sections Dataset (HyReCo). The target registration error (TRE) is used as the evaluation metric. We compare the proposed algorithm to other state-of-the-art solutions, showing considerable improvement. Additionally, we perform several ablation studies concerning the resolution used for registration and the initial alignment robustness and stability. The method achieves the most accurate results for the ACROBAT dataset, the cell-level registration accuracy for the restained slides from the HyReCo dataset, and is among the best methods evaluated on the ANHIR dataset. ConclusionsThe article presents an automatic and robust registration method that outperforms other state-of-the-art solutions. The method does not require any fine-tuning to a particular dataset and can be used out-of-the-box for numerous types of microscopic images. The method is incorporated into the DeeperHistReg framework, allowing others to directly use it to register, transform, and save the WSIs at any desired pyramid level (resolution up to 220k x 220k). We provide free access to the software. The results are fully and easily reproducible. The proposed method is a significant contribution to improving the WSI registration quality, thus advancing the field of digital pathology.

Development and validation of a deep learning-based microsatellite instability predictor from prostate cancer whole-slide images

Microsatellite instability-high (MSI-H) is a tumor-agnostic biomarker for immune checkpoint inhibitor therapy. However, MSI status is not routinely tested in prostate cancer, in part due to low prevalence and assay cost. As such, prediction of MSI status from hematoxylin and eosin (H&E) stained whole-slide images (WSIs) could identify prostate cancer patients most likely to benefit from confirmatory testing to evaluate their eligibility for immunotherapy and need for Lynch syndrome testing. Prostate biopsies and surgical resections from prostate cancer patients referred to our institution were analyzed. MSI status was determined by next-generation sequencing. Patients sequenced before a cutoff date formed an algorithm development set (n = 4015, MSI-H 1.8%) and a paired validation set (n = 173, MSI-H 19.7%) that consisted of two serial sections from each sample, one stained and scanned internally and the other at an external site. Patients sequenced after the cutoff date formed a temporally independent validation set (n = 1350, MSI-H 2.3%). Attention-based multiple instance learning models were trained to predict MSI-H from H&E WSIs. The predictor achieved area under the receiver operating characteristic curve values of 0.78 (95% CI [0.69–0.86]), 0.72 (95% CI [0.63–0.81]), and 0.72 (95% CI [0.62–0.82]) on the internally prepared, externally prepared, and temporal validation sets, respectively, showing effective predictability and generalization to both external staining/scanning processes and temporally independent samples. While MSI-H status is significantly correlated with Gleason score, the model remained predictive within each Gleason score subgroup.

An effective method for preparing histological sections of blow fly intra-puparial stages for minimum PMI estimations.

Blow flies (Diptera: Calliphoridae) are generally early colonisers of fresh cadavers, enabling the estimation of a minimum post-mortem interval (minPMI) based on an accurate aging of the oldest immature stages associated with a cadaver. In blow flies, the pupal stage and the subsequent development of the adult take place inside a protective case, the puparium, formed from the hardened and darkened cuticle of the third instar larva. Because the puparium is an opaque structure that shows virtually no external changes, qualitative analyses of the internal tissues can be very informative for determining reliable age-specific morphological markers. Those analyses can be performed using either non-invasive but expensive and not widely accessible techniques, or traditional histological methods, which are invasive as they require the serial sectioning of the sample. Histological methods are often readily available for forensic researchers and practitioners; however, the histological study of blow fly intra-puparial stages has traditionally been hampered by the poor paraffin infiltration of tissues due to the abundance of fat bodies, resulting in usually fragmented sections and the subsequent loss of relevant information. We present here an effective method for the preparation of histological sections of blow fly intra-puparial stages, maximising the paraffin infiltration while enabling the production of clean and entire sections that allow for the use of reliable age-specific morphological markers, thus improving the accuracy of minPMI estimations when access to more costly techniques is not feasible.

HISTOCHEMICAL AND IMMUNOHISTOCHEMICAL FEATURES OF DIFFERENTIATED TROPHOBLAST IN CHORIONIC VILLI OF THE PLACENTA IN PRETERM LABOR

The importance of studying the histochemical and immunohistochemical features of the diff erentiated trophoblast in chorionic villi of the placenta in preterm labor is determined by the threatening nature of this condition for both mother and fetus, as well as its high prevalence and serious consequences for the health of both. The understanding of the damage to the trophoblast of chorionic villi in the placenta can be expanded by histochemical and immunohistochemical methods, which allow the assessment of the concentration of specifi c marker molecules in one way or another.Objective. To determine certain histochemical and immunohistochemical characteristics of proteins in the trophoblast of theintermediate and terminal chorionic villi of the placenta in preterm labor.Material and Methods. The obtained material (30 placentas from preterm deliveries and 30 placentas from normalpregnancies) was fi xed for 20-24 hours in 10 % neutral formalin solution buff ered in Lilly’s phosphate buff er. After tissue removal, the placental tissue was dehydrated in an ascending ethanol series and embedded in paraffi n at a temperature of approximately 58 °C. Serial histologic sections were cut at 5.0 μm thickness using an MS-2 sliding microtome. After deparaffi nization, histological sections were stained with hematoxylin and eosin, histochemical methods for total protein with bromophenol blue according to Bonhéme, and immunohistochemical techniques according to the manufacturer’s protocols (Dako, Denmark). In particular, immunohistochemical reactions were performed with monoclonal antibodies against trophoblast hormone- placental lactogen and placental alkaline phosphatase. Visualization of primary antibodies was performed using the Dako polymer visualization system with diaminobenzidine as chromogen (resulting in brown staining of the sites of studied antigens). In addition to the descriptive method of histopathologic research, computer morphometry of digital microphotographs of histologic sections was performed using a Delta Optical Evolution 100 microscope and an Olympus SP550UZ digital camera. Digital copies of the images were processed using a legitimate copy of the ImageJ v1.52f computer program developed for histometric studies (National Institutes of Health, USA). Specifi cally, the evaluation of staining intensity (optical density) was performed on digital microphotographs using the method of computer microdensitometry. For this purpose, a microprobe method was used to obtain a computer brightness value in an 8-bit analysis system with 256 gray levels – from black (0) to white (255).The obtained values were then transformed into relative optical density values (r.OD) by logarithmic transformation (naturallogarithm method). The relative optical density value ranges from 0 (absolute transparency of the object) to 1 (absolute opacity of the object). The obtained digital data were processed using statistical analysis methods. A legitimate copy of the statistical analysis computer program PAST v4.14 was used, with a preliminary check for normal distribution using the Shapiro- Wilk test. Since, according to this test, the hypothesis of normal distribution was not rejected for the statistical samples studied (at p=0.05), parametric methods of statistical analysis were applied: calculation of the mean and its standard error, Student’s t-test (two-tailed, unpaired). In addition, the non-parametric Mann- Whitney test was used for the reliability of the conclusions. The research was conducted as part of the scientifi c research project «Preservation and Restoration of Reproductive Health in Women and Girls with Obstetric and Gynecological Pathology» at the Department of Obstetrics and Gynecology of Bukovinian State Medical University (state registration number 0121U110020, duration 2021-2025).Results. Microscopic examination of histological sections stained with hematoxylin and eosin did not reveal any diff erencesin the structure of chorionic villus trophoblast in the placenta of preterm labor compared to normal pregnancy. However,histochemical and immunohistochemical methods of investigation revealed a number of features in the trophoblast of chorionic villi, where fundamental events related to substance exchange occur – intermediate immature, intermediate mature, terminal villi, including terminal «specialized villi».Conclusions. According to the obtained histochemical and immunohistochemical data, in preterm labor, compared to normalpregnancy, no changes are observed in the trophoblast of intermediate immature villi, while in intermediate mature and terminal villi, there is a decrease in histochemical staining for total protein and immunohistochemical staining for specifi c trophoblast proteins – placental lactogen hormone and placental alkaline phosphatase enzyme.

Three-dimensional ultrastructural and anatomical analysis of prostatic neuroendocrine cells in mice.

A few studies have examined the ultrastructure of prostatic neuroendocrine cells (NECs), and no study has focused on their ultrastructure in three dimensions. In this study, three-dimensional ultrastructural analysis of mouse prostatic NECs was performed to clarify their anatomical characteristics. Three 13-week-old male C57BL/6 mice were deeply anesthetized, perfused with physiological saline and 2% paraformaldehyde, and then placed in 2.5% glutaraldehyde in 0.1 M cacodylate (pH 7.3) buffer for electron microscopy. After perfusion, the lower urinary tract, which included the bladder, prostate, coagulation gland, seminal vesicle, upper vas deferens, and urethra, was removed, and the specimen was cut into small cubes and subjected to postfixation and en bloc staining. Three-dimensional ultrastructural analysis was performed on NECs, the surrounding cells, tissues, and nerves using focused ion beam/scanning electron microscope tomography. Twenty-seven serial sections were used in the present study, and 32 mouse prostatic NECs were analyzed. Morphologically, the NECs could be classified into three types: flask, flat, and closed. Closed-shaped NECs were always adjacent to flask-shaped cells. The flask-shaped and flat NECs were in direct contact with the ductal lumen and always had microvilli at their contact points. Many of the NECs had accompanying nerves, some of which terminated on the surface in contact with the NEC. Three-dimensional ultrastructural analysis of mouse prostatic NECs was performed. These cells can be classified into three types based on shape. Novel findings include the presence of microvilli at their points of contact with the ductal lumen and the presence of accompanying nerves.

Cytoarchitectonic Analysis and 3D Maps of the Mesial Piriform Region in the Human Brain

The mesial piriform region plays a central role in olfaction. Its small size and complex geometry, however, make it a difficult target in functional neuroimaging studies, while histological maps often represent schematic drawings, which are not compatible with requirements for modern imaging. To bridge this gap, cytoarchitectonic analysis and mapping of the region was performed in serial histological sections over their full extent in 10 postmortem brains. The temporobasal areas PirTBd and PirTBv and temporal areas PirTu and PirTit were identified and analyzed. Probabilistic cytoarchitectonic maps of the piriform areas in MNI reference space and high-resolution maps of the amygdala-piriform region on the BigBrain model were calculated as part of the Julich-Brain. Differences in the cytoarchitectonic “texture” of the region were quantified based on the Gray Level Co-Occurrence Matrix. Results showed that allocortical areas were not consistently associated with the rostral Limen insulae, although it was often suggested as a landmark in neuroimaging protocols. PirTu was associated with the uncal tip. PirTit was the largest area, reaching to the temporal pole, with a “temporal” (caudal) and a “temporopolar” (rostral) part having complex neighborhood relationships. The probabilistic maps reflect interindividual variability; they are openly available via the digital EBRAINS platform to serve as an anatomical reference for studies related to olfaction.

Crystallographic faceting of bulk tungsten surfaces observed during in situ heating in an environmental scanning electron microscope

The surface faceting of metals has been studied for several decades, typically using experimental methods such as low-energy electron diffraction. In the current paper, surface faceting of tungsten pellets was studied during in situ heating inside an environmental scanning electron microscope (ESEM), which allowed for direct, high-fidelity observation of morphological changes in response to high-temperature annealing under a moderate vacuum (0.8 Torr). Additionally, a second set of tungsten samples was annealed in an ultrahigh vacuum chamber (10−8 Torr), albeit without direct observation via ESEM. This study revealed that oxygen plays a crucial role in tungsten surface faceting and in a morphological transition to vertex rounding. Furthermore, this paper discusses relevant techniques for identifying the crystallographic indices of surface facets. It is demonstrated that a combination of electron backscatter diffraction, tilted-sample imaging from multiple angles, and serial sectioning in a focused ion beam system can be effectively deployed to determine the geometry of faceting, which in turn can be verified with the aid of software for generating Wulff shapes. The influence of processing environment on tungsten surface faceting enables the possibility of designing and controlling the morphology and crystallographic facets of tungsten surfaces.

A semantic segmentation algorithm for automated rapid melt pool identification from cross-sectional micrographs

Melt pool contours are an important empirical process measurement for metallic additive manufacturing. Melt pool boundaries indicate the maximal extent of the melting temperature contour. Melt pool boundaries characterize the effects of process parameters on melt pool size and variability and provide calibration and validation data for process simulations predicting thermal fields and microstructure. Serial cross sectioning of samples can produce numerous micrographs valuable for building robust melt pool contour datasets. However, the acquisition of contours from micrographs by hand is error prone and prohibitively time consuming for datasets large enough to capture variation within individual scan tracks and across a range of process conditions. In this work, we propose a semantic segmentation algorithm for automating melt pool contour acquisition from micrographs. The proposed algorithm is demonstrated on datasets from two different single scan track experiments conducted on a Powder Bed Fusion-Laser Beam platform using a bare Ti-6Al-4 V plate and an Inconel 718 plate with a 40 μm powder layer at different preheat temperatures. The algorithm was successfully trained and predicted the melt pool boundaries in the 300 image Ti-6Al-4 V dataset with 95% accuracy and an estimated reduction of 91% in processing time compared to data acquisition by hand. For the 1201 image Inconel 718 dataset, the algorithm achieved 89% accuracy and an estimated 99% time savings compared to hand measurements. The results for both datasets are demonstrated with a discussion about observed variability in the melt pool characteristics.

MORPHOMETRIC PARAMETERS OF MATURITY OF PLACENTAL CHORINAL VILLI IN PREMATURE LABOR

Aim – to establish some morphometric parameters of placental villi in premature birth in comparison with physiological pregnancy.Material and methods. The material (30 placentas in premature births and 30 placentas in physiological pregnancies) was fixed for 22-24 hours in a neutral buffered 10% formalin solution. Next, the material was dehydrated in an ascending battery of alcohols and embedded into paraffin at 580С. Serial histological sections 5 μm thick were made on a sled microtome. After deparaffinization, serial histological sections were stained with hematoxylin and eosin, and with a chromotrope - water blue according to N.Z.Slinchenko. In addition to the descriptive method of histopathological examination, computer morphometry was performed on previously obtained digital copies of optical microscopic images of the tissue (Delta Optical Evolution 100 microscope {planachromatic lenses according to the required optical magnification} and Olympus SP550UZ digital camera). Digital copies of the image were analyzed using a freeware copy of the ImageJ v1.52 computer program specialized for digital histometric studies. In particular, the percentage of different types of chorionic villi was calculated on digital copies of optical images using the unrepeatable score test method. The obtained digital data were processed statistically. In particular, with the help of a copy of the free (freeware) computer program for statistical calculations PAST v 4.14, a preliminary test for the normality of the distribution was applied according to the Wilkie-Hahn-Shapiro test. For all studied statistical samples, according to this criterion, the hypothesis about the normality of the distribution was not rejected (at p=0.05), therefore, parametric methods of statistical analysis were used: calculation of the arithmetic mean and its error, the odd two-sided Student's test. Along with the Student's test, the non-parametric Mann-Whitney U test was also used. Results. It was considered most important to start assessing the state of the placenta according to the percentage ratios between different types of villous formations. In particular, according to modern ideas about different types of chorionic villi in the II and III trimesters of pregnancy, the following types of villous formations of the placenta were distinguished: stem villi ("early" and late"), intermediate villi (immature and mature), terminal villi, terminal "specialized" villi, mesenchymal villi, embryonic villi, trophoblastic and villous processes. The total amount of the specified villi should be equal to 100%. The division of villi into primary, secondary, and tertiary villi is relevant only for the 1st trimester of pregnancy, therefore, it is not suitable for studying the morphological features of premature birth.Conclusions. According to the obtained morphometric data, the immaturity of the chorionic tree is noted in premature birth compared to physiological pregnancy. This can be seen from a noticeable increase in the percentage of stem "early" villi, intermediate immature and mature villi, mesenchymal and embryonic villi, trophoblastic and villous processes, but with a significant decrease in the percentage of terminal villi.

Quantification of follicles in human ovarian tissue using image processing software and trained artificial intelligence.

Cancer survival rates in prepubertal girls and young women have risen in recent decades due to increasingly efficient treatments. However, many such treatments are gonadotoxic, causing premature ovarian insufficiency (POI), loss of fertility and ovarian endocrine function. Implantation of donor ovarian tissue encapsulated in immune-isolating capsules is a promising method to restore physiological endocrine function without immunosuppression or risk of reintroducing cancer cells harbored by the tissue. The success of this approach is largely determined by follicle density (FD) in the implanted ovarian tissue, which is analyzed manually from histologic sections and necessitates specialized, time-consuming labor. To address this limitation, we developed a fully automated method to quantify FD that does not require additional coding. We first analyzed ovarian tissue from 12 human donors between 16 to 37years old using semi-automated image processing with manual follicle annotation and then trained artificial intelligence program based on follicle identification and object classification. One operator manually analyzed 102 whole slide images (WSIs) from serial histologic sections. Of those, 77 images were assessed by a second manual operator, followed with an automated method utilizing artificial intelligence (AI). Of the 1181 follicles the control operator counted, the comparison operator counted 1178, and the AI counted 927 follicles with 80% of those being correctly identified as follicles. The three-stage AI pipeline finished 33% faster than manual annotation. Collectively, this report supports the use of artificial intelligence and automation to select tissue donors and grafts with the greatest FD to ensure graft longevity for POI treatment.