As the name indicates, the field of toxicologic pathology integrates the disciplines of pathology and toxicology. In the preclinical field, toxicologic pathologists integrate these two fields in an experimental setting to evaluate the safety of different therapeutic modalities, ranging (but not limited to) from both veterinary and human pharmaceuticals, (agro)chemicals, medical devices, vaccines to small molecules as well as advanced therapy medicinal products (ATMPs).
As such, the histopathological assessment of tissue and organ samples represents one of the key endpoints of nonclinical toxicology and safety studies. The thorough assessment of any structural changes or abnormalities in tissues and organs caused by the exposure to the test compound provides crucial information about the potential toxic effects at cellular and tissue levels.
Histopathology evaluation of organs and relevant tissues is typically performed, but not limited, to the following types of toxicology studies:
- General toxicology/ tolerability studies
- Dose-range finding studies
- Repeated dose toxicity
- Acute/ subacute/ subchronic/ chronic
- Reproductive toxicity
- Inhalation/ injection/ oral toxicity studies
- Carcinogenetic studies
- Medical device biocompatibility studies
Assessing the preclinical in vivo toxicology of novel therapeutic modalities oftentimes means that a considerable number of large-scale studies are being performed, with toxicology assessments taking place across multiple species. As toxicology studies are the crucial endpoint required for determining a novel medicinal product’s safety, the timelines for obtaining data in timely manner are often tight.
Therefore, outsourcing the histopathology part of your toxicology studies can be highly relevant, especially if you, as a service provider, receive an overwhelming number of requests that exceeds your internal pathology capacities. In line, outsourcing your toxicologic pathology assessments allows you take on more studies to help your clients accelerate access to their data while ensuring confidence in the obtained results.
In this article, we further discuss how you can get the most value out of your histopathology portion of toxicology studies outsourced to a histopathology service provider.
Knowledge of inter-species histologic variations
Obtaining a highly accurate pathological evaluation of toxicity studies depends on the pathologist’s knowledge of background lesion histopathology i.e., how familiar are they with incidental findings in species beyond the commonly used laboratory animals?
Background or incidental lesion are characterised as tissue-level variations that are thought of as a change in tissue morphology but can still fall into the accepted reference range (1). Background changes can be congenital or hereditary, normal variations of findings that are unique to the histology of an animal species, related to trauma or normal aging, and physiologic or hormonal changes (1).
Although proportionally the biggest number of toxicology studies is carried out in common laboratory animals (namely rats and mice), in order fully demonstrate safety, the findings need to be confirmed across other species (namely larger lab animals like dogs and non-human primates [NHPs] etc,). Moreover, if common toxicology animals are unsuitable due to pharmacodynamic, pharmacokinetic, and/or pathophysiological differences then hamsters, gerbils, guineapigs, pigs and mini pigs are used as well.
The knowledge and experience with working different species is important because each animal species used in preclinical research has specific histologic characteristics which need to be taken into account when evaluating a toxicity of a novel therapeutic product. As such, inter-species histologic variations prove to be the key to understanding toxicity effects of your novel compound enabling the differentiation of normal vs abnormal tissue architecture. For instance, what is considered pathological in rats, may be a background lesion in NHPs, and vice versa. In addition to that, the knowledge of species-specific pathology should be combined with severity changes in and differently dosed animals and control groups.
Therefore, when choosing your partner for toxicologic pathology assessments, it is important to discuss how familiar the pathologist is with recognising histological background of spontaneous lesions from actual compound-induced toxicity effects across multiple species. This issue may be further exacerbated by the fact that most veterinary and toxicological pathology textbooks have readily available reference information for disease pathology in rodents but not in NHPs (2). As an example, initiatives where pathologists publish comprehensive summaries of the most common incidental findings in NHPs such as cynomolgus monkeys (Macaca Fascicularis) are published (2), there is still a lack of available resources, further highlighting the reliance on the pathologists’ own knowledge and experience in working with less commonly used laboratory species.
By working with Connected-Pathology you can obtain high-quality toxicology assessment results thanks to our veterinary pathologists that have the necessary expertise of working with variety of common laboratory species like mouse, rat as well as dogs and NHPs.
High-volume sample processing and staining capabilities
Toxicologic pathology assessment oftentimes necessitates the evaluation of toxicity of the test compound across multiple organs, meaning that histopathology service providers need to be able to provide large-volume sample processing and staining capacities to accommodate the hundreds and thousands of slides that need to be evaluated.
Haematoxylin and Eosin (H&E) staining is the standard histological staining technique used in toxicologic pathology. Accurate and sensitive interpretation of H&E slides can be ensured by utilizing auto-staining machines that provide unmatched staining consistency across all of the study slides that need to be analysed. H&E provides crucial information into various types of lesions like apoptosis, necrosis, inflammation and inflammatory cell infiltration, fibrosis, collagen deposition, vacuolation and so on.
Depending on the identified lesions, a pathologist will provide further recommendations to perform other staining in order to fully characterise the nature of the observed lesion. Immunohistochemsistry (IHC) can be used to visualise specific proteins of interest that may be involved in mediating the lesions that were identified, or additionally special stains can be performed to provide insight into the origin of the lesion. Although not commonly performed over the years, the Special Interest Group of the European Society of Toxicology Pathology (ESTP) has recently reported on the use and recommendation of in situ hybridization (ISH) in preclinical toxicologic pathology as well (3).
At Connected-Pathology we work with fully automatised and digitalized laboratory workflows thanks to automated H&E Dako stainer and Roche’s BenchMark ULTRA IHC/ISH and DISCOVERY ULTRA systems that enable us to perform your large scale studies with shorter turn-around times, whilst maintaining high quality of staining.
Depending on the objective of the study, different scoring and assessment systems can be applied. For toxicity studies, for example, the INHAND scoring scheme (Table 1) is used, whereas medical devices studies are scored according to the ISO guidelines and so on.
INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) guidelines are a standardized set of guidelines developed by a collaborative effort between international toxicologic pathologists. These guidelines provide a systematic and uniform approach for characterizing and classifying microscopic lesions (abnormalities or changes) in preclinical animals used in toxicology studies. Overall, the INHAND guidelines play a pivotal role in ensuring the quality and reliability of toxicology studies, which in turn supports the safety evaluation of pharmaceuticals, chemicals, and other substances. They are considered a cornerstone in the field of toxicologic pathology and are widely adopted by the scientific and regulatory communities worldwide.
TABLE 1. INHAND Scoring Scheme applied in our toxicologic pathology assessment studies across all species.
|0||Within normal limits||Tissue considered to be normal, under the conditions of the study and considering the age, sex, and strain of the animal concerned. Alterations may be present, which, under other circumstances, would be considered deviations from normal.|
|1||Minimal||The amount of change present barely exceeds that which is considered to be within normal limits.|
|2||Slight||In general, the lesion is easily identified but of limited severity.|
|3||Moderate||The lesion is prominent, but there is significant potential for increased severity.|
|4||Severe||The degree of change is as complete as possible (occupies the majority of the organ).|
At Connected-Pathology, the format of the requested report and lesion scoring is thoroughly discussed beforehand, ensuring that you obtain the most suitable toxicologic pathology assessment and reports to match your research needs.
Advances in Toxicologic Pathology
The intersection of artificial intelligence (AI) and histopathology represents a dynamic and rapidly evolving field with significant implications for preclinical research. This paradigm shift highlights the exciting potential of utilizing AI for evaluating histopathology images. Although still far from being widely applied in research, nowadays many histopathology service providers are slowly preparing for the shift toward more and more AI-automated analyses.
Recently, the society of Toxicologic Pathology have provided an opinion article detailing the application of AI and machine learning in toxicologic pathology (4). Toxicologic pathology remains manual labour heavy as a pathologist usually needs to review and analyse hundreds of slides per study, especially considering that most tissues and organs lack any compound test-related changes. In addition, species-specific background lesions need to be also recognised and noted. AI has been shown to be consistently superior to humans at completing repetitive, detailed tasks rapidly and accurately and algorithm performance has been shown to be comparable with that of an expert pathologist (5). This further suggests that the field will see a development of various smart tools to help the pathologist reach conclusions more efficiently and precisely than ever before. Given these many waves of change, toxicologic pathologists will be expected to be at least very familiar with, if not fluent in AI and ML technologies (4).
At Connected-Pathology, we are continuously developing and training novel algorithms to help deliver results in a more efficient way. To date, Connected-Pathology has developed various AI-driven proprietary software. Our proprietary web-based viewer, I-Connect, allows you to view your high-resolution histopathology images as soon as they are scanned, from the comfort of your web browser. Tx-Connect puts you in control with a centralized system that seamlessly integrates the functions of a laboratory informatics system together with a comprehensive digital pathology workflow.