The first session of the 2025 European Society of Toxicologic Pathology (ESTP) Congress reviewed routine and specialized methods for microscopic evaluation of neural tissues during nonclinical studies. Three longer presentations reviewed brain sampling approaches in safety assessments, including an example to accentuate topographical analysis and integration of toxicology data; specific brain and spinal cord sampling for molecular and protein analyses; and an overview of technical aspects of intraparenchymal drug delivery. Four shorter talks discussed the uses, advantages, disadvantages, and interpretation of several special neurohistological techniques (stains and immunohistochemical markers) for assessing test item-associated responses. Common special methods used (when warranted) for nonclinical studies include Fluoro-Jade or silver stains for detecting neuronal death, Luxol fast blue (LFB) for examining myelin, anti-glial fibrillary acidic protein (GFAP) to demonstrate reactive astrocytes, and anti-ionized calcium-binding adaptor molecule 1 (IBA1) to highlight reactive microglia and macrophages, though alternatives methods were described. The last presentation discussed artificial intelligence as an aid in detecting subtle toxicant-induced lesions during digital pathology analyses (using the Olney lesion [acute neuronal vacuolation and necrosis in the cerebral cortex] as an example). Taken together, talks in this session provided a cohesive overview of traditional and innovative approaches to facilitate microscopic evaluation for potential neurotoxicity in nonclinical studies.

Hematoxylin and eosin (H&E) staining is a suitable approach for detecting substantial structural changes in neural tissues but is less sensitive for identifying subtle alterations to subcellular structures and various chemical constituents, including myelin. Neurohistological methods to better evaluate myelin integrity by light microscopy include acidophilic dyes (eg, eriochrome cyanine R, toluidine blue [used with hard plastic sections]); lipoprotein-binding dyes (eg, Luxol fast blue [LFB], Weil's iron hematoxylin); lipid impregnation with metals (eg, Marchi's, which uses osmium tetroxide for en bloc staining before embedding); and immunohistochemical (IHC) methods to highlight various antigens (eg, myelin basic protein [MBP] and peripheral myelin protein 22 [PMP22]). Some IHC methods reveal enhanced marker expression in damaged myelin (eg, matrix metalloproteinase-9 [MMP9], S100). In neuropathology investigations, H&E is the first-tier screening method, whereas myelin stains (often LFB alone or in combination with dyes that highlight other structural elements) are second-tier procedures performed in combination with other neurohistological procedures to examine neuroaxonal injury and/or glial responses. The choice of myelin method depends on such considerations as cost, institutional preference, the procedure (fixation and embedding medium), and the study objective.

Due to the prevalence of hematoxylin and eosin (H&E) staining in routine histological preparations, understanding the factors that impact stain color characteristics is vital to attain consistently high-quality stains. In the last decade, increased use of digital pathology and image analysis (specifically by optical density [OD] measures) has provided new ways of assessing staining precision. This paper combines data from two studies that tracked H&E staining quality in both nuclear and cytoplasmic components of 12 tissues (11 human and 1 porcine) by OD after overuse of H&E staining reagents from 5 vendors. Both studies showed a decrease in eosin stain intensity by OD and visual inspection (by a histologist) with reagent overuse. This trend varied in degree by tissue type and reagent vendor. Nonetheless, staining quality for H&E staining from all vendors and for all organs remained acceptable (but not always optimal) for microscopic evaluation by the College of American Pathologists (CAP) and National Society for Histotechnology (NSH) staining criteria when stained sections were reviewed by a board-certified veterinary pathologist.

The central tenet of scientific research is the rigorous application of the scientific method to experimental design, analysis, interpretation, and reporting of results. In order to confer validity to a hypothesis, experimental details must be transparent and results must be reproducible. Failure to achieve this minimum indicates a deficiency in rationale, design, and/or execution, necessitating further experimental refinement or hypothesis reformulation. More importantly, rigorous application of the scientific method advances scientific knowledge by enabling others to identify weaknesses or gaps that can be exploited by new ideas or technology that inevitably extend, improve, or refine a hypothesis. Experimental details, described in manuscript materials and methods, are the principal vehicle used to communicate procedures, techniques, and resources necessary for experimental reproducibility. Recent examination of the biomedical literature has shown that many published articles lack sufficiently detailed methodological information to reproduce experiments. There are few broadly established practice guidelines and quality assurance standards in basic biomedical research. The current paper provides a framework of best practices to address the lack of reporting of detailed materials and methods that is pervasive in histological slide-based assays. Our goal is to establish a structured framework that highlights the key factors necessary for thorough collection of metadata and reporting of slide-based assays.

Staining quality and reproducibility are essential factors to monitor laboratory quality assurance. In the last decade, there has been an increase in the use of digital pathology and image analysis. While the adoption of these tools provides a potential means to track staining precision by optical density (OD), it also presents challenges. Results from image analysis are more sensitive to variations in staining than microscopic evaluation by a pathologist. There are two goals with this study. The first was to track the precision of hematoxylin and eosin (H&E) staining, in both nuclear and cytoplasmic components by OD. The second was to determine the impact of different pre-analytical and analytical variables on the OD results. Specifically, the endpoints investigated were quality parameters including impacts of section thickness, protocol manipulation, expired hematoxylin on staining precision and reproducibility of staining over time. Our results show that image analysis of H&E-stained tissue sections is a viable tool for assessing and verifying staining quality. We also show that OD analysis results for H&E-stained sections are affected by changing pre-analytical and/or reagent variables. These authors chose a graphical rather than fully statistical analysis of the results to highlight the utility of visual aids in demonstrating H&E staining reproducibility.

The biodistribution of AAVHSC7, AAVHSC15, and AAVHSC17 following systemic delivery was assessed in cynomolgus macaques (Macaca fascicularis). Animals received a single intravenous (IV) injection of a self-complementary AAVHSC-enhanced green fluorescent protein (eGFP) vector and tissues were harvested at two weeks post-dose for anti-eGFP immunohistochemistry and vector genome analyses. IV delivery of AAVHSC vectors produced widespread distribution of eGFP staining in glial cells throughout the central nervous system, with the highest levels seen in the pons and lateral geniculate nuclei (LGN). eGFP-positive neurons were also observed throughout the central and peripheral nervous systems for all three AAVHSC vectors including brain, spinal cord, and dorsal root ganglia (DRG) with staining evident in neuronal cell bodies, axons and dendritic arborizations. Co-labeling of sections from brain, spinal cord, and DRG with anti-eGFP antibodies and cell-specific markers confirmed eGFP-staining in neurons and glia, including protoplasmic and fibrous astrocytes and oligodendrocytes. For all capsids tested, 50 to 70% of glial cells (S100-β+) and on average 8% of neurons (NeuroTrace+) in the LGN were positive for eGFP expression. In the DRG, 45 to 62% of neurons and 8 to 12% of satellite cells were eGFP-positive for the capsids tested. eGFP staining was also observed in peripheral tissues with abundant staining in hepatocytes, skeletal- and cardio-myocytes and in acinar cells of the pancreas. Biodistribution of AAVHSC vector genomes in the central and peripheral organs generally correlated with eGFP staining and were highest in the liver for all AAVHSC vectors tested. These data demonstrate that AAVHSCs have broad tissue tropism and cross the blood-nerve and blood-brain-barriers following systemic delivery in nonhuman primates, making them suitable gene editing or gene transfer vectors for therapeutic application in human genetic diseases.

Context.— Advancements in genomic, computing, and imaging technology have spurred new opportunities to use quantitative image analysis (QIA) for diagnostic testing. Objective.— To develop evidence-based recommendations to improve accuracy, precision, and reproducibility in the interpretation of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) for breast cancer where QIA is used. Design.— The College of American Pathologists (CAP) convened a panel of pathologists, histotechnologists, and computer scientists with expertise in image analysis, immunohistochemistry, quality management, and breast pathology to develop recommendations for QIA of HER2 IHC in breast cancer. A systematic review of the literature was conducted to address 5 key questions. Final recommendations were derived from strength of evidence, open comment feedback, expert panel consensus, and advisory panel review. Results.— Eleven recommendations were drafted: 7 based on CAP laboratory accreditation requirements and 4 based on expert consensus opinions. A 3-week open comment period received 180 comments from more than 150 participants. Conclusions.— To improve accurate, precise, and reproducible interpretation of HER2 IHC results for breast cancer, QIA and procedures must be validated before implementation, followed by regular maintenance and ongoing evaluation of quality control and quality assurance. HER2 QIA performance, interpretation, and reporting should be supervised by pathologists with expertise in QIA.

This symposium will provide participants with a broad introductory overview of the field of digital pathology (DP), including: 1) Applications, benefits and limitations of DP. 2) DP technology issues (primarily whole slide imaging). 3) Regulatory, workflow and financial considerations. 4) Hands-on session focused on basic digital slide viewing using a variety of digital slides if possible. We will also cover the recently updated guidelines from the American Telemedicine Association on Telepathology, with particular reference to their impact on use of digital pathology. This symposium will provide participants with a broad introductory overview of the field of digital pathology (DP), including: 1) Applications, benefits and limitations of DP. 2) DP technology issues (primarily whole slide imaging). 3) Regulatory, workflow and financial considerations. 4) Hands-on session focused on basic digital slide viewing using a variety of digital slides if possible. We will also cover the recently updated guidelines from the American Telemedicine Association on Telepathology, with particular reference to their impact on use of digital pathology.

Digital Pathology Systems (DPS) are dynamic, image-based computer systems that enable the acquisition, management, and interpretation of pathology information generated from digitized glass slides. This article provides a roadmap for (1) qualification of a whole slide scanner (WSS) during a validation project, (2) validation of software required to generate the whole slide image (WSI), and (3) an introduction to visual digital image evaluation and image analysis. It describes a validation approach that can be utilized when validating a DPS. It is not the intent of this article to provide guidance on when validation of DPS is required. Rather, the article focuses on technical aspects of validation of the WSS system (WSS, IT infrastructure, and associated software) portion of a DPS and covers the processes of setting up the WSS for scanning a glass slide through saving a WSI on a server. Validation of a computerized system, such as a DPS, for use in a regulated nonclinical environment is governed by Code of Federal Regulations (CFR) Title 21 part 11: Electronic Records; Electronic Signature and predicate rules associated with Good Laboratory Practices documents including 21 CFR part 58. Similar regulation and predicate rules apply in the European Union and Japan.

Digital pathology is a dynamic, image-based environment that enables the acquisition, management and interpretation of pathology information generated from a digitized glass slide. The Digital Pathology System (DPS) includes a whole slide scanner (WSS), image acquisition software, image viewing and database software, image analysis software, and the necessary IT infrastructure to support the DPS. Validation is an ongoing process to establish documented evidence that provides a high degree of assurance, that a process or system will consistently perform according to predetermined specifications and quality attributes. To date, efforts to validate digital pathology systems in a clinical healthcare environment have been very limited. Therefore the strengths and weaknesses of validation are essentially unknown. Documentation from governing organizations such as the Food and Drug Administration (FDA), Centers for Medicare and Medicaid (CMS), and the College of American Pathologists (CAP) around validation practices is scarce. Attempts at validation of digital pathology systems are likely limited due to a lack of understanding on how to efficiently conduct a validation and how to navigate regulations that could have an impact on laboratory accreditation and healthcare compliance. Validation of a digital pathology system (DPS) is necessary for clinical use to ensure laboratory compliance for CLIA, State regulations, The Joint Commission, and CAP accreditations, to protect patient safety and confidentiality, to assure digital pathology data is accurate, and to maximize the value of a DPS. This white paper will not provide a step by step approach to validation. It will serve as a high level overview of what should be considered, while aligning with other resources available to the pathology community including CAP Checklists and the recently published CAP draft guidelines for “Validating Whole Slide Imaging Systems for Diagnostic Purposes in Pathology.” (1) This white paper will use a key code to help readers naviagate the information based on their role in validation.