Pathology for Toxicologists E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Non-pathologists, such as toxicologists and study personnel, can find it difficult to understand the data they receive from pathologists. Toxicological pathologists write long, detailed and highly technical reports. Study personnel are under daily pressure to decide whether lesions described in pathology reports are treatment-related and thus important to the pharmaceutical company or whether the lesions are background changes and thus of little significance.
Written by experienced toxicological pathologists, Pathology for Toxicologists: Principles and Practices of Laboratory Animal Pathology for Study Personnel serves to bridge the gap in the understanding of pathology data, enabling non-pathologists to more easily comprehend pathology reports, better integrate pathology data into final study reports and ask pathologists relevant questions about the test compound.
This succinct, fully referenced, full colour book is suitable for toxicologists at all stages of their training or career who want to know more about the pathology encountered in laboratory animals used in safety studies. Key features include important chapters on spontaneous and target organ lesions in rats, mice, non-human primates, mini pigs, rabbits and beagle dogs as well as information on general pathology, macroscopic target organ lesions, ancillary pathology techniques, haematology, biochemistry and adversity.
Pathology for Toxicologists: Principles and Practices of Laboratory Animal Pathology for Study Personnel includes:
- Colour diagrams explaining how lesions are caused by either external compounds or spontaneously
- The anatomic variations and background lesions of laboratory animals
- Advice on sampling tissues, necropsy, ancillary pathology techniques and recording data
- A chapter on the haematology and biochemistry of laboratory animals
- Full colour photographs of common macroscopic lesions encountered in laboratory animals
- A comprehensive glossary
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Veröffentlichungsjahr: 2017
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Table of Contents
Title Page
copyright
List of Contributors
Preface
Chapter 1: An Introduction to Pathology Techniques
1.1 Animal Considerations
1.2 Necropsy
1.3 Lung Inflation with Fixative
1.4 Fixation
1.5 Making Glass Slides
1.6 Special Histochemical Stains
1.7 Decalcification
1.8 Immunohistochemistry
1.9 Tissue Crossreactivity Studies
1.10 Electron Microscopy
1.11 In Situ Hybridisation
1.12 Laser Capture Microscopy
1.13 Confocal Microscopy
1.14 Image Analysis
1.15 Digital Imaging
1.16 Spermatocyte Analysis
1.17 Good Laboratory Practice
1.18 Inhalation Studies
1.19 Continuous-Infusion Studies
1.20 Carcinogenicity
1.21 Biologicals
1.22 The Pathology Report
1.23 Conclusion
References
Chapter 2: Recording Pathology Data
2.1 What is a Pathology Finding?
2.2 Standardisation of Pathology Findings
2.3 ‘Inconsistencies’ in Pathology Recording
2.4 Blind Review
2.5 Historical Control Data: Pros and Cons
2.6 The Use of Peer Review in Pathology
References
Chapter 3: General Pathology and the Terminology of Basic Pathology
3.1 Cellular Responses to Insults
3.2 Inflammation
3.3 Circulatory Disturbances
3.4 Disorders of Tissue Growth
3.5 Tissue Repair and Healing
3.6 Neoplasia
3.7 Immune System
References
Chapter 4: Common Spontaneous and Background Lesions in Laboratory Animals
4.1 Rats
4.2 Mice
4.3 Dogs
4.4 Minipigs
4.5 Non-Human Primates
4.6 Rabbits
4.7 Experimental Procedures
4.8 Causes of Death in Rats and Mice
4.9 Conclusion
References
Chapter 5: Target Organ Pathology
5.1 Skin
5.2 Eye
5.3 Gastrointestinal Tract
5.4 Liver
5.5 Respiratory System
5.6 Urinary System
5.7 Lymphoreticular System
5.8 Musculoskeletal System
5.9 Cardiovascular System
5.10 Endocrine System
5.11 Reproductive System
5.12 Central and Peripheral Nervous System
5.13 Ear
References
Chapter 6: Clinical Pathology
6.1 Clinical Pathology in Study Phases and Good Laboratory Practice
6.2 What is Measured in Clinical Pathology?
6.3 Haematology
6.4 Coagulation
6.5 Clinical Chemistry
6.6 Urinalysis
6.7 Acute-Phase Proteins
6.8 The Biomarker Concept
6.9 Reference Intervals
6.10 Instrumentation, Validation and Quality Control
6.11 Data Analysis and Interpretation
6.12 Reporting
6.13 Food Consumption and Body Weight (Gain)
6.14 Organ Weights
6.15 Examples of Typical Clinical Pathology Profile Changes in Toxicologic Clinical Pathology
6.16 Microsampling
6.17 Conclusion
Acknowledgments
References
Chapter 7: Adversity: A Pathologist's Perspective
7.1 LOAEL, NOEL and NOAEL: Definition
7.2 Adversity
7.3 Determining Adversity using Pathology Findings: Factors to Consider
7.4 Communicating NOAEL in Toxicity Studies
7.5 Conclusion
References
Chapter 8: Limitations of Pathology and Animal Models
8.1 Limitations of In Vivo Animal Models
8.2 Efficacy/Disease Models as Toxicology Models
8.3 Limitations of Efficacy/Disease Models as Toxicology Models
8.4 Limitations of Pathology within In Vivo Toxicology Models
8.5 Managing Risk Associated with Subjectivity and the Potential for Pathologist Error
References
Glossary
Index
End User License Agreement
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Guide
cover
Table of Contents
Preface
Begin Reading
List of Illustrations
Chapter 1: An Introduction to Pathology Techniques
Figure 1.1 Necropsies or post mortem examinations on experimental animals such as this mouse are a fundamental part of toxicological pathology.
Figure 1.2 Post mortem imbibition of bile pigment in the mesenteric fat tissue adjacent to the gall bladder. Photograph taken from a bovine necropsy.
Figure 1.3 Artefacts induced at necropsy include inclusions of foreign material into the issue, such as plant material (*) during brain removal.
Figure 1.4 Production of glass slides suitable for histopathological analysis.
Figure 1.5 Tissue trimming and placement in a cassette.
Figure 1.6 The blocking sheet.
Figure 1.7 Small pieces of tissue (*) from a previous cassette superimposed on another tissue.
Figure 1.8 Machine for tissue dehydration, clearing and impregnation with paraffin wax.
Figure 1.9 Rotary microtome.
Figure 1.10 H&E staining.
Figure 1.11 Quality control.
Figure 1.12 Phosphotungstic acid haematoxylin (PTAH).
Figure 1.13 Growth-hormone IHC stains positive cells in pituitary.
Figure 1.14 Phospholipidosis is characterised by lamellar bodies (*), which are visible in this electron micrograph of a rat heart.
Chapter 2: Recording Pathology Data
Figure 2.1 How a pathology term is created.
Figure 2.2 Chronic progressive nephropathy (CPN) is a common and well-recognised background finding in rats. The lesion progresses from occasional basophilic tubules in the early stages to multiple different morphological findings in the later (chronic) stages. A pathologist who lumps this lesion will record different grades of CPN throughout the time course of the lesion whereas a splitter will describe the different findings seen at each stage.
Chapter 3: General Pathology and the Terminology of Basic Pathology
Figure 3.1 Severe cell injury is not difficult to recognise. Here, the pus exuding from this ruminant brain clearly indicates that the tissue is not normal. This is called ‘purulent meningitis’.
Figure 3.2 Yellow fatty change in an enlarged ruminant liver.
Figure 3.3 Infarcts (*) in a dog kidney, showing a sharp demarcation between normal (red-brown) tissue and necrotic (yellow) tissue.
Figure 3.4 Coagulative necrosis (*) in a ruminant kidney.
Figure 3.5 Liquefactive necrosis in a ruminant brain.
Figure 3.6 Caseous necrosis in the centres of ruminant liver abscesses.
Figure 3.7 Fat necrosis in ruminant mesenteric fat tissue.
Figure 3.8 Dry gangrene in the black ear tips of a pig.
Figure 3.9 Fibrinoid necrosis in beagle pain syndrome, showing bright pink material in the blood vessel wall.
Figure 3.10 Apoptosis (*) in a mouse lymph node, showing cell shrinkage, chromatin condensation, cytoplasmic blebs and phagocytosis of apoptotic bodies by adjacent macrophages.
Figure 3.11 Clinical signs of inflammation.
Figure 3.12 Major actions of the exudative phase of acute inflammation.
Figure 3.13 Cells involved in inflammation: neutrophils, macrophages, eosinophils, mast cells and basophils.
Figure 3.14 The complement system.
Figure 3.15 Types of inflammation.
Figure 3.16 Fibrinous inflammation around a ruminant heart.
Figure 3.17 Formation of fibrous tissue (scar tissue) (*) in the scars of old infarcts in a dog kidney.
Figure 3.18 Simultaneous tissue destruction and repair in inflammation.
Figure 3.19 Multinucleate giant cell.
Figure 3.20 Functions of inflammation.
Figure 3.21 Hyperaemia on the surface of a ruminant brain.
Figure 3.22 Petechiae on the surface of a canine heart.
Figure 3.23 Hydrothorax is the accumulation of fluid in the thoracic cavity.
Figure 3.24 Conversion of fibrinogen to fibrin during clotting (haemostasis).
Figure 3.25 Thrombosis in a canine large blood vessel.
Figure 3.26 Epistaxis in a dog.
Figure 3.27 Cell growth responses.
Figure 3.28 Thickened whitish scar at the edge of an ulcerative lesion.
Figure 3.29 Interrelationship between different systems in inflammation.
Chapter 4: Common Spontaneous and Background Lesions in Laboratory Animals
Figure 4.1 Squamous cyst (*) in a mouse spinal cord.
Figure 4.2 Bluish thymus within the thyroid follicles of a mouse.
Figure 4.3 Haematopoiesis (*) in the liver.
Figure 4.4 Thickening of the glomerular basement membranes and periglomerular fibrosis (*) in an ageing mouse, indicative of CPN.
Figure 4.5 Thickened mucosa filled with dilated gastric glands, indicating adenomatous hyperplasia of the glandular stomach.
Figure 4.6 Enlarged nuclei (*) in the cells of the liver.
Figure 4.7 Enlarged mouse spleen (*) at necropsy.
Figure 4.8 Loss of hair and ulcerative skin lesions in a C57Bl/6 mouse.
Figure 4.9 Lymphoma, manifesting in enlarged submandibular lymph nodes and enlarged thymus.
Figure 4.10 Pituitary tumour in a female Sprague Dawley rat.
Chapter 5: Target Organ Pathology
Figure 5.1 Fur/hair loss or thinning (alopecia) in a mouse.
Figure 5.2 Acanthosis (epidermal thickening) in a young rat pup.
Figure 5.3 Necrosis, erosion and ulceration of the skin in the preputial gland area of a male mouse.
Figure 5.4 Petechiae on the skin of a nude mice.
Figure 5.5 Squamous cell carcinoma in the dorsal skin above the scapula in a rat.
Figure 5.6 Conjunctivitis in a mouse.
Figure 5.7 Whitish opacification of the eye due to the presence of a cataract.
Figure 5.8 Squamous papilloma in the mouth of a rat.
Figure 5.9 Squamous cell carcinoma in the stomach of a rodent.
Figure 5.10 Chronic colitis (*) in a mouse.
Figure 5.11 Polyp in the colon of a rat.
Figure 5.12 Leiomyosarcoma extending from the ileum outer surface in a rat.
Figure 5.13 Yellow areas (*) on the reddish-brown surface of the liver, indicating liver necrosis and inflammation.
Figure 5.14 Jaundice/icterus in a mouse, visible as a generalised yellow colour of the abdominal muscles.
Figure 5.15 Hepatocellular adenoma (*) in the liver of a mouse.
Figure 5.16 Distended, air-filled murine stomach and intestines, due to obstruction of the airflow due to inflammation of the nasal turbinates.
Figure 5.17 Slightly raised white foci on the lung surface, indicating the presence of macrophage aggregates.
Figure 5.18 Bronchiolaveolar adenoma (*) in the lung of a mouse.
Figure 5.19 Shrunken, wrinkled, pale white rat kidneys at necropsy, indicating fibrosis and chronic interstitial nephritis.
Figure 5.20 Yellow left kidney with raised abscesses (*), indicating pyelonephritis in a mouse.
Figure 5.21 Urinary stones in the bladder of a mouse.
Figure 5.22 Hydronephrosis in both kidneys of a mouse.
Figure 5.23 Haematuria (*) in a mouse urinary bladder.
Figure 5.24 Severe enlargement of the spleen (*) due to lymphoma in a mouse.
Figure 5.25 Severely enlarged submandibular, axillary and inguinal lymph nodes in lymphoma in a mouse.
Figure 5.26 Enlarged parathyroid gland (*) in renal failure in a dog.
Figure 5.27 Small red raised areas in the lung of a dog, indicating the spread of a haemangiosarcoma from the spleen.
Figure 5.28 Pituitary adenoma (large, dark red mass at the base of the cranial cavity) (*) in a rat.
Figure 5.29 Enlargement of the thyroid (*) in a rat.
Figure 5.30 Phaemochromocytoma in an adrenal adjacent to the kidney in a rat.
Figure 5.31 Testicular atrophy (*) in the left testis of a mouse.
Figure 5.32 Ovarian cyst (*) in a mouse.
Figure 5.33 Mammary gland fibroadenoma in a rat.
Chapter 6: Clinical Pathology
Figure 6.1 The three phases in clinical pathology.
Figure 6.2 The main materials for toxicologic clinical pathology testing are anticoagulated whole blood for haematology (e.g. in EDTA), serum collected after centrifugation of clotted whole blood for clinical chemistry and Wright–Giemsa-stained blood smears for microscopic evaluation and validation of quantitative and qualitative aspects of red and white blood cells and platelets. The arrow points to the feathered edge, where platelet clumps or atypical cells can preferentially be seen.
Figure 6.3 Microhaematocrit tubes with haemolysis, lipaemia (small fat layer after centrifugation; arrow) and marked icterus in the plasma layer. Such discolourations can be accidental or treatment-related and can interfere with analytical measurements.
Figure 6.4 Increased numbers of reticulocytes indicate active haematopoiesis in the bone marrow, a physiologic response to blood loss due to moderate or excessive blood sampling, haemorrhage or haemolysis. (a) Traditionally, reticulocytes are visualised by vital stains, such as new methylene blue, which make the reticular pattern of remnant ribosomes and RNA visible. (b) In the haematology analyser, reticulocytes are identified by their remnant RNA (in the absence of a nucleus) (
x
-axis), with most immature reticulocytes on the far-right side. (c) In a Wright–Giemsa-stained blood smear, younger red blood cells are characterised by a larger diameter (anisocytosis) and the lack of a central pallor, as well as a slightly bluish tinge, termed ‘polychromasia’. (d) In the analyser, these cells typically have a higher volume (
y
-axis) and a lower haemoglobin concentration (
x
-axis). In a very pronounced response, there can actually be two populations of red blood cells, with normal and lower haemoglobin concentrations (
x
-axis).
Figure 6.5 (a–e) dog; (f) cat. Leukocytes and platelets in canine blood smear. Wright–Giemsa, x100 objective (a–e). (a) Segmented neutrophil with pale cytoplasm and an irregular convoluted and knobby condensed nucleus (left); monocyte with slightly basophilic/bluish cytoplasm and an irregular shaped nucleus with a reticulated chromatin pattern (right). (b) Eosinophil with large eosinophilic cytoplasmic granules and an irregular ribbon-shaped nucleus, (c) Basophil with numerous purple cytoplasmic granules and an irregular lobulated nucleus. (d) Lymphocyte with a small rim of deeply basophilic/blue cytoplasm and a dense bean-shaped nucleus. (e) Segmented (right) and band (left) neutrophil with pale cytoplasm and the typical band-shaped nucleus. (f) Platelet aggregates ×20 objective.
Figure 6.6 Coagulation in the classic cascade model, including (a) the intrinsic and extrinsic systems and the common pathway, (b) the main cellular contributors to clot formation, endothelial cells and platelets and (c) thrombotic clot formation with the participation of activated platelets and crosslinked fibrin strands in haemostasis following disruption of a vessel wall. The cell-based model for coagulation can be seen in Hoffman and Monroe (2001).
Figure 6.7 Clinical pathology data-interpretation algorithm.
Chapter 7: Adversity: A Pathologist's Perspective
Figure 7.1 Approach to defining the adversity of a pathological finding.
Chapter 8: Limitations of Pathology and Animal Models
Figure 8.1 Sinus dilatation in the mouse lymph node – also called ‘cystic degeneration’, ‘cystic ectasis’, ‘lymphangiectasis’ and ‘lymphatic cysts’.
List of Tables
Chapter 1: An Introduction to Pathology Techniques
Table 1.1 Special histochemical stains.
Chapter 2: Recording Pathology Data
Table 2.1 Comparison of data types.
Table 2.2 Examples of linear and nonlinear grading schemes that could be used for semiquantitative analysis of non-neoplastic lesions in laboratory animal tissues.
Source
: Scudamore (2014). Reprinted with permision from Wiley-Blackwell.
Chapter 3: General Pathology and the Terminology of Basic Pathology
Table 3.1 Differences between benign and malignant tumours.
Chapter 6: Clinical Pathology
Table 6.1 Recommended haematology variables for routine toxicology studies
Table 6.4 Recommended urinalysis variables for routine toxicology studies
Table 6.2 Recommended coagulation variables for routine toxicology studies
Table 6.3 Recommended serum chemistry analytes for routine toxicology studies
Table 6.5 Acute-phase proteins that show measurable changes in different laboratory animal species (Honjo et al., 2010; Heegaard et al., 2013; Christensen et al., 2014).
Table 6.6 Common nonspecific change patterns in toxicologic clinical pathology.
Table 6.7 Common patterns in chemistry profiles in toxicologic clinical pathology.
Chapter 7: Adversity: A Pathologist's Perspective
Table 7.1 Selected definitions of ‘adverse effect’ from the published literature.
Pathology for Toxicologists
Principles and Practices of Laboratory Animal Pathology for Study Personnel
Edited by Elizabeth McInnes
This edition first published 2017 © 2017 John Wiley & Sons Ltd
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List of Contributors
Elizabeth McInnes
Cerberus SciencesThebarton, SAAustralia
Natasha Neef
Vertex PharmaceuticalsBoston, MAUSA
Cheryl L. Scudamore
MRC Harwell, Harwell Science and Innovation CampusOxfordshireUK
Bhanu Singh
Discovery SciencesJanssen Research & DevelopmentSpring House, PAUSA
Barbara von Beust
Independent consultantWinterthurSwitzerland
Preface
Seemingly minor differences in opinion between the study pathologist and the study director or vice president of safety assessment (positions often filled by toxicologists) can escalate to cause real problems with the development of a test article in the pharmaceutical and agrochemical industries. Pathology is an imprecise science that relies on the observation of subtle variations in patterns of cellular arrangement and the tinctorial affinity of certain cells for staining procedures. Non-pathologists, such as toxicologists, can find it difficult to understand the data they receive from pathologists, partly due to the subjectivity of the discipline and the variations between pathologists, and partly due to the terminology that pathologists use.
There is a lack of pathological texts aimed at study personnel. This book has been written for toxicologists at all stages of their training or career who want to know more about the pathology encountered in laboratory animals, including study directors, study monitors, undergraduate and postgraduate toxicology students, toxicology report reviewers and research scientists employed in the pharmaceutical industry. The aim is to help study personnel bridge the gap in the understanding of pathology data. The book will enable them to understand the pathology reports they receive and the common pathologies encountered, so that they can more easily integrate pathology data into their final study report and ask pathologists relevant questions where there are gaps in understanding.
We have attempted to make the book user-friendly and easy to understand. Important lesions in rats, mice, non-human primates, mini pigs and beagle dogs, the most common laboratory animals used in the industry, are discussed. The compound-induced pathology in all the major organ systems is covered, as are clinical pathology, adversity and the limitations of pathology. There is also a glossary, which should help all non-pathologists understand the language of toxicological pathology. The aim is to demystify such terms as “chronic focal hepatic hypertrophy with Ito cell tumor.”
This book is intended to give study personnel an insight into the uncertainties encountered by the pathologist when reading studies and to provide them with explanations for why pathologists cannot always make up their minds. We trust it will improve communication and understanding between pathologists, toxicologists and study directors, so that a more succinct and helpful toxicologist report can be written.
Elizabeth McInnes
Chapter 1An Introduction to Pathology Techniques
Elizabeth McInnes
Cerberus Sciences, Thebarton, SA, Australia
Learning Objectives
Understand the role of study personnel in necropsies.
Understand the various steps involved in producing glass slides from harvested tissues.
Understand the ancillary techniques used in toxicological pathology.
Discover what carcinogenicity, inhalation and crossreactivity studies entail.
This book is aimed at all study personnel – including study monitors, study directors and toxicologists – who are exposed to pathology reports, necropsies, peer review, haematology and biochemistry results and adversity on a regular basis. The secret to an informative, relevant and useful pathology report is an open and collegial relationship between the study director and the study pathologist (Keane, 2014). This chapter aims to describe the various stages of the pathological process (e.g. necropsy, fixation of tissues, cutting of slides) in order to demonstrate where crucial errors which can cause problems at a later stage, may arise. In addition, it includes a brief overview of ancillary techniques that pathologists sometimes use (e.g. electron microscopy). Finally, it discusses carcinogenicity studies, digital pathology, biological drugs and crossreactivity studies, and their impact on study personnel. Throughout the chapter, the client is referred to as the ‘sponsor’ of a particular pharmaceutical study.
Pathology is the study of disease, particularly the structural and functional changes in tissues and organs. Toxicological pathology is concerned predominantly with cell and tissue injury in animals treated with introduced chemical compounds or biological drugs. Studies are regulated by international bodies such as the Organisation for Economic Co-operation and Development (OECD), the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Animal testing to determine the safety of pharmaceuticals, medical devices and food/colour additives is required by the FDA before it will give approval to begin clinical trials in humans. Pathology data may be quantitative (haematology, chemistry data, organ weights) or qualitative (microscopic diagnoses), and the toxicological pathology report is divided into macroscopic and microscopic findings. Study personnel are ultimately responsible for the study report, including the pathology report and data. Thus, study personnel need to understand what the pathology report means and how it has been generated. This chapter aims to help the study director understand the processes involved in a study, from harvesting tissues from the animal to generating glass slides to producing a pathology report.
1.1 Animal Considerations
The main species of animal used in the pharmaceutical industry are rats, mice, dogs, non-human primates, minipigs and rabbits. Occasionally, farm animals, hamsters, cats and gerbils are also used. There are no absolute reasons for selecting a particular animal species for systemic toxicity, but for acute oral, intravenous, dermal and inhalation studies and studies of medical devices, the mouse or rat is preferred, with the option of the rabbit in the case of dermal and implantation studies. Non-rodent species may also need to be considered for testing, although a number of factors might dictate the number and choice of species. Carcinogenicity studies generally use rats and mice.
All animal studies must be conducted according to the animal welfare laws of the country in which they are based, and in general studies may use protected animals only if there are no other reasonable, practicable choices for achieving a satisfactory result. Laboratory animals may only be used in minimal numbers, where they have the lowest possible degree of neurophysiological sensitivity and where the study causes minimal pain, suffering, distress and lasting harm. Animal suffering must be balanced against the likely benefits for humanity, other animals and the environment. In general, in the planning of all preclinical studies, due consideration must be given to reduction, refinement and replacement (the ‘3 Rs’) (Tannenbaum and Bennett, 2015).
Generally, only healthy purpose-bred young adult animals of known origin and with defined microbiological health status should be used in pathology studies (i.e. health monitoring of sentinel animals must be performed before the study starts). Health monitoring involves testing for the various bacteria, viruses, parasites and protozoa that may infect experimental animals and compromise study results (McInnes et al., 2011). The weight variation within a sex should not exceed 20% of the mean weight, and when female animals are used, they should be non-pregnant and should never have borne young.
Laboratory animals should be given a short acclimatisation period at the start of the study. Control of environmental conditions and diet and proper animal care techniques are necessary throughout the study in order to produce meaningful results. The number of animals needed per dose group depends on the purpose of the study. Group sizes should increase with the duration of treatment, such that at the end of the study sufficient animals are available in every group for a thorough biological evaluation and statistical analysis.
1.2 Necropsy
Necropsies or post mortem examinations (Figure 1.1) on experimental animals are a fundamental part of toxicological pathology (Fiette and Slaoui, 2011). They generally take place at the end of a study, but are also conducted if an animal dies early. The necropsy and pathology data are the single most important aspect of the pathology process, and study personnel get only one chance to retrieve them: once the tissues have been discarded, potentially valuable information is lost forever. At the necropsy, all macroscopic findings and abnormalities visible to the naked eye (e.g. enlarged liver, ulcerated skin, the presence of diarrhoea) are noted and recorded. In addition, tissues are collected for examination under the microscope. The pathologist, necropsy supervisor, prosector, phlebotomist and weighing assistant are all responsible for the recording the macroscopic data (Keane, 2014). Some studies collect all tissues (a full tissue list is indicated in the study plan or protocol), while others harvest only a limited list. A copy of the study plan should be available in the necropsy room to ensure that study personnel collect the correct tissues. Sometimes, all the tissues are collected into formalin, but slides are only cut if the sponsor decides there is a need later on. Harderian glands and draining lymph nodes are examples of tissues that are not always collected: study personnel should check the study plan before beginning the necropsy.
Figure 1.1 Necropsies or post mortem examinations on experimental animals such as this mouse are a fundamental part of toxicological pathology.
Study directors and toxicologists are often required to attend the necropsies of the animals on their studies. Although not directly involved in the necropsy process, it is nevertheless important that study personnel understand the process and are able to provide advice and management, particularly if unusual tissues are to be collected, severe test article-related findings are observed in high-dose animals, or deviations from standard operating procedures (SOP) occur. The study pathologist may be consulted if there is an unusually high rate of unscheduled deaths in the study or it is difficult to characterise macroscopic findings (such as very white teeth noted when treatment causes defects in enamel formation) (Keane, 2014).
Carbon dioxide asphyxiation provides a rapid form of euthanasia for mice (Seymour et al., 2004) and rats, but it can cause severe lung haemorrhage, which may make microscopic examination of the lungs difficult. Barbiturate overdose is an effective form of euthanasia, but it requires the use of pentobarbital sodium (Seymour et al., 2004). Larger animals (e.g. rabbits, non-human primates and dogs) are euthanised by an overdose of sodium pentobarbitone, which may cause congestion of some organs and is highly irritant if injected into the tissues around the vein.
Control and treated animals should always be necropsied by the same team of technicians, and the animal numbers and order of examination should be randomised. The identity of an animal is given in a tattoo, ear notch or microchip and should be recorded on all necropsy storage containers in indelible ink.
Organs should be weighed at necropsy; increases and decreases in organ weight can often be correlated with the microscopic findings observed by the pathologist. To ensure meaningful organ weights are recorded, the organs should be taken from an exsanguinated animal (if possible), and excess moisture and adipose tissue should be removed. Guidelines on the weighing of organs are outlined in various papers (Michael et al., 2007; Sellers et al., 2007).
The macroscopic lesions observed at necropsy may be the only pathological data generated from certain studies and must be presented in the form of an incidence table. Consequently, lesions should be described consistently throughout the necropsy process, and standardised terms should be used. The use of an agreed, standardised macroscopic glossary will help to reduce the incidence of different personnel using different terms to describe the same lesion (Scudamore, 2014). In studies in which histopathology will be performed, the macroscopic lesions observed at necropsy are very important to the pathologist, as they often correlate with the lesions observed under the light microscope (e.g. an enlarged yellow liver at necropsy will often have lipid vacuolation in haepatocytes revealed under light microscopy).
Macroscopic lesions at necropsy should simply be described: no attempt at interpretation or diagnosis should be made at this stage (e.g. necropsy staff should not describe an enlarged, mottled liver as ‘hepatitis’ or a yellow tissue colour should not be described as jaundice). This is because once signed, the anatomic pathology report cannot be easily reinterpreted. All macroscopic lesions observed at necropsy should be described in terms of size and distribution (focal, multifocal and diffuse), colour and consistency (soft, friable, firm, hard, fluid filled, gritty, etc.). The location, size and number of a mass or lesion should be recorded. A standard diagram of the dorsal and ventral aspects of the animal is useful for recording the exact locations of lesions and masses. All measurements should be made in millimetres, and terms such as ‘enlarged’, ‘pale’ and ‘small’ should be avoided or should be accompanied by an actual measurement or colour. In particular studies, it may be useful to photograph certain lesions in order to illustrate their exact nature and severity to future study personnel. However, although photographs are a good record of macroscopic lesions observed at necropsy, there may be Good Laboratory Practice (GLP) and legal issues to contend with (Suvarna and Ansary, 2001).
Autolysis occurs within 10 minutes of the death of an animal (Pearson and Logan, 1978), so necropsy should be performed as quickly and efficiently as possible, with limited tissue handling, squeezing and tissue damage. Post mortem change occurs as a result of autolysis (action of enzymes from the ruptured cells on the dead animal's cells) and putrefaction (degradation of tissue by the invasion of certain microorganisms); changes include rigor mortis (stiffening of limbs and carcase), clotting of the blood, hypostatic congestion (pooling of blood into the dependent side of the carcase, termed ‘livor mortis’), imbibition of blood (or bile pigment; Figure 1.2) and gaseous distension of the alimentary tract. In addition, pseudomelanosis (the greenish or blackish discolouration of tissues due to ferrous sulphide) tends to occur in organs that lie adjacent to the intestines, such as the liver. Most of these changes will be visible if an animal dies during the night or on the weekend, and every effort should be made to store the carcase in a fridge and to perform a necropsy as soon as possible thereafter.
Figure 1.2 Post mortem imbibition of bile pigment in the mesenteric fat tissue adjacent to the gall bladder. Photograph taken from a bovine necropsy.
1.3 Lung Inflation with Fixative
Tracheal instillation of the lungs with fixative at necropsy is required to improve the histology of the pulmonary architecture in mice and rats, and is recommended for all rodent studies. Tracheal instillation of fixative may be performed either after removing the lungs from the thoracic cavity or with the lungs in situ (Braber et al., 2010). It may sometimes be easier to inflate only one lung lobe, using a needle and syringe to inject formalin (Knoblaugh et al., 2011).
1.4 Fixation
In general, fixation of tissues maintains cellular integrity and slows the breakdown of tissues by autolysis. The most common fixative is 10% neutral buffered formalin, which ensures rapid tissue penetration, is easy to use and is inexpensive. However, formalin is highly toxic and carcinogenic and may have effects on the immune system (Costa et al., 2013). Tissues should be fixed at a 1 : 10 or 20 ratio of fixative to tissue for at least 48 hours. Modified Davidson's is the recommended fixative for eyes and testes, as it prevents retinal detachment in the eye and separation of cells lining the seminiferous tubules in the testes. Glutaraldehyde or osmium tetraoxide is used for the fixation of tissues intended for electron microscopy. Artefacts which occur at necropsy include inclusions of foreign material into the tissue (e.g. plant material during brain removal (Figure 1.3) and the incorporation of sharp shafts of hair into soft tissues) and pressure and pinch effects (from forceps) (McInnes, 2011). These can be confused with lesions by an inexperienced pathologist.
Figure 1.3 Artefacts induced at necropsy include inclusions of foreign material into the issue, such as plant material (*) during brain removal.
1.5 Making Glass Slides
The production of glass slides suitable for histopathological analysis involves a number of steps performed in the histology laboratory (Figure 1.4).
Figure 1.4 Production of glass slides suitable for histopathological analysis.
1.5.1 Trimming
In the first step, formalin-fixed tissues collected during the necropsy are further cut up in order to fit into the embedding cassettes (Knoblaugh et al., 2011). Two steps in the pathology phase of the study cannot be repeated: the necropsy and the macroscopic tissue trimming. This is because if tissues are discarded after the necropsy or at trimming, it is impossible to return to them. Great care should thus be exercised at the trimming stage. All tissues should be trimmed in the same way, from the same area in the organ, and all described gross lesions must be identified and included in the cassette (Figure 1.5). The cassette lid will cause impression marks on the tissue surface if the tissue is too big for the cassette (Knoblaugh et al., 2011). Excellent trimming and blocking patterns indicating how to section each tissue and which tissues should be placed together in a cassette are contained in Ruehl-Fehlert et al. (2003), Kittel et al. (2004) and Morawietz et al. (2004). The staff involved in trimming should be aware that variations in the incidence of certain lesions (such as thyroid C-cell findings and thyroid tumours) can be associated with the type of section taken (i.e. transverse compared to longitudinal).
Figure 1.5 Tissue trimming and placement in a cassette.
It is essential that the cassette be marked correctly with the animal's identification number, sex and group, and with either the tissue name(s) or a number indicating which tissues are always trimmed into that particular cassette (Figure 1.6). The blocking sheet (Figure 1.6) indicates which tissue has been processed in which cassette. Multiple tissues may be grouped in one cassette (e.g. different tissues from the gastrointestinal tract), but certain tissues (e.g. adrenal and bone) should not be grouped together, since differences in consistency will cause problems during the microtoming of the wax blocks. The collection and histological examination of heart valves was overlooked in the past, but with the introduction of reporting of valvular lesions associated with the use of antiobesity drugs (fenfuramine-fentermine) (Connolly et al., 1997), these tissues must now be examined. Thus, histology technicians now endeavour to cut the heart into sections, which allows for the visualisation of the major aortic and pulmonic heart valves. Artefacts introduced at this stage include small pieces of tissue from the previous cassette superimposed on another tissue (due to failure to clean the knife between tissues and between animals) (Figure 1.7) (McInnes, 2011).
Figure 1.6 The blocking sheet.
Figure 1.7 Small pieces of tissue (*) from a previous cassette superimposed on another tissue.
1.5.2 Tissue Processing
After trimming, the cassettes are placed in a machine that allows the tissues to undergo a series of steps which include tissue dehydration, clearing and impregnation with paraffin wax (Figure 1.8). Paraffin wax serves to keep tissue firm and intact, and in the correct orientation for sectioning.
Figure 1.8 Machine for tissue dehydration, clearing and impregnation with paraffin wax.
1.5.3 Embedding
During embedding, a trained technician places the paraffin wax-infiltrated tissues and additional wax into a mould, which is chilled to produce tissue blocks.
1.5.4 Microtoming
During microtoming, thin sections (~4–6 µm) are cut from the wax block using a rotary microtome (Figure 1.9). The operation of this device is based upon the rotary action of a hand wheel, which advances the specimen (wax block) towards a rigidly held blade. The thin wax sections are then floated in a water bath, and appropriately identified glass slides are used to scoop them out of the water. The glass slides are placed in an oven to melt off the wax, leaving only the unstained tissue. Histology technicians require fine motor skills in order to ensure that all of the anatomic features of small tissues such as the adrenals and pituitary (e.g. cortex and medulla of adrenal) are displayed on a single slide. The formalin fixed wet tissues may be discarded after the study is finalised, however the wax tissue blocks and the glass slides are archived according to the protocol (Keane, 2014).
Figure 1.9 Rotary microtome.
1.5.5 Staining
Before staining, all tissues are transparent, and it is hard to make out any cellular detail under the microscope. For this reason, stains are bound to different parts of the tissue, allowing these components to be visualised. Histochemical stains are made up of chemical dyes that bind to tissues by the same mechanism as a chemical interaction. The most commonly used stain is haematoxylin and eosin (H&E), which stains acidic tissue components pink (the eosin binds to the cytoplasm, making it pink) and basic tissues blue (the haematoxylin binds to the nuclei of the cells, making them blue) (Figure 1.10).
Figure 1.10 H&E staining.
After staining, the section is mounted with a coverslip to prevent the tissue from drying out, prevent surface damage to the tissue and improve tissue transparency. If the pathologist discovers that a tissue is missing, all attempts must be made to find the missing tissue (by going back to the wet tissues, or by cutting deeper into the original block if no wet tissue remains); if a tissue is inadequate, it must be improved (by re-embedding). Occasional missing or inadequate tissues are acceptable in a study, but large numbers will compromise its completeness, and the study may have to be repeated – at great cost.
1.5.6 Quality Control
The final step in slide processing is to carefully check the glass slide for artefacts, to make sure that the information on the block is the same as that on the slide and to ensure that the slides have been arranged in the correct order according to the blocking sheet (which pertains to one animal) (Figure 1.11).
Figure 1.11 Quality control.
1.6 Special Histochemical Stains
Stains with specific affinities for different tissue components (e.g. calcium, fat) can be used to confirm the identity of these tissues or substances (see Table 1.1). Examples include phosphotungstic acid haematoxylin (PTAH) (Figure 1.12), which demonstrates muscle striations; periodic acid–Schiff (PAS), which stains glycogen and carbohydrate; Congo red, which stains amyloid; and Oil Red O, which stains lipid (can only be used on fresh-frozen tissue that has not been fixed in formalin). Pathologists may ask for special stains after examining a slide (e.g. to confirm the presence of collagen and thus the diagnosis of a fibrosarcoma) and this will necessitate a protocol amendment (Keane, 2014). Alternatively, special stains may be incorporated into the study plan from its inception (e.g. when a sponsor requests special staining of all fat tissue in order to assess the effect of compounds such as peroxisome proliferator-activated receptor (PPARs) agonists on white and brown fat; Long et al., 2009).
Table 1.1 Special histochemical stains.
Substance
Tissue seen in
Special stain used
Bile
Liver
Fouchet's
Lipofuscin
Various (generally older animals)
PAS, Sudan black B, Schmorl's, Long Ziehl-Neelsen technique
Glycogen
Liver, muscle
PAS with diastase
Haemosiderin (golden brown)
Spleen, others
Perl's Prussian blue
Formalin pigment (artefact)
Blood-rich tissues, large areas of haemorrhage
- (need to extract it from sections using picric acid)
Melanin (more dark brown–black)
Lung, muscle, others
By exclusion
Fat
Liver
Oil Red O on frozen non-formalin fixed tissue
Collagen
Scar tissue, tumour
Masson's trichome
Figure 1.12 Phosphotungstic acid haematoxylin (PTAH).
1.7 Decalcification
Tissues which contain high levels of calcium slats such as bone and teeth are difficult to cut so they require decalcification with solutions such as formic acid to remove the calcium salts, soften the tissue and make it less brittle and easier to cut. Artefacts associated with excessive periods of tissue decalcification have been described (McInnes, 2011).
1.8 Immunohistochemistry
Immunohistochemistry (IHC) is used when H&E staining does not give sufficient information about the cell type of interest. Immunohistochemistry is requested by the pathologist when he or she wants definite confirmation or detection of a specific cell type. For instance, H&E staining cannot distinguish between T- and B-cells but IHC using antibodies against CD3 and CD19 can; thus, IHC is a method of detecting a molecule or epitope on a cell, in situ, in a tissue section, using an antibody to that molecule and a visible label.
Immunohistochemistry is useful in the identification of unknown tumours and in the detection of infectious agents. Toxicological pathologists may need to know the exact identity of a cell that displays test article-related changes; markers such as glial fibrillary acid protein (GFAP) (which stains reactive astrocytes), synaptophysin and chromagranin (which stain neuroendocrine cells), growth hormone in pituitary cells (Figure 1.13) and LAMP-2(+) for hepatic phopholipidosis are thus all useful. Other common antibodies include proliferating cell nuclear antigen (PCNA), which stains cells in active proliferation (G1, G2, S and M phases), and the TUNEL and caspase 3 antibodies, which stain cells in apoptosis.
Figure 1.13 Growth-hormone IHC stains positive cells in pituitary.
Fresh tissue samples frozen in OCT compound (Tissue Tek, UK) are optimal for IHC or in situ hybridisation, but formalin fixed tissue may also be used provided various techniques are applied to break down the formalin bonds (such as microwaving the tissue in citrate buffer, which is called ‘antigen retrieval’). Cell morphology is generally poorer in fresh-frozen sections than in paraffin wax-embedded sections, but more antibodies tend to work on fresh-frozen tissue.
Mast cells are usually fairly easy to detect, due to their prominent granulation, but they can be highlighted, if required, using the histochemical stain toluidine blue or by using IHC with the antibody against CD117. Mononuclear immune cells such as macrophages and lymphocytes in rodents may be identified using a basic panel of IHC markers for macrophages (F4/80, Mac2), T-cells (CD3, CD4, CD8) and B-cells (CD19, CD23) (Ward et al., 2006).
Challenges in IHC include the fact that most antibodies are developed against human antigens, and thus toxicological pathologists are never sure whether a given antibody will work in rodent tissues (good positive and negative controls are essential); the difficulty of cutting suitable cryostat sections from frozen tissues; the problems involved in unmasking antigens (antigen retrieval) in formalin-fixed tissue; and high levels of background staining. Polyclonal antibodies bind to several different epitopes and are thus more sensitive but less specific than monoclonal antibodies, which bind to a single epitope.
