An Introduction to Pathology Techniques
Cerberus Sciences, Thebarton, SA, Australia
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.
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.
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.
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.
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).
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.
The production of glass slides suitable for histopathological analysis involves a number of steps performed in the histology laboratory (Figure 1.4).
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).
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