2.1 Sample selection and trimming patterns
In any experimental system there is a necessity for controls and defined protocols to ensure repeatable and valid results. Histology is no different from any other laboratory technique in this respect and standardization of tissue sampling, trimming and sectioning greatly improves the quality and reproducibility of data that can be obtained from tissue sections. Consistent presentation of tissue sections on slides also reduces the amount of time taken to read the slides, reduces pathologist fatigue and reduces the risk of lesions being missed. For any given study a subset of tissues of interest will usually be identified in advance and harvested systematically at necropsy (Chapter 1). Once adequately fixed, tissue samples must then be selected, trimmed and embedded prior to sectioning. Standardization of the site of tissue to be sampled, amount of tissue to be evaluated and orientation of the tissue to be sectioned all help to improve consistency. The specific requirements of the study will determine what tissue type is examined but in general the probability of observing a specific lesions relates to how much tissue is examined. In practice the amount of tissue that is examined is inevitably constrained by the costs of histology and pathologist time and so, in practice, it is useful to have standardized techniques that maximize the chances of identifying lesions while minimizing the cost. Published guides for sampling and trimming most mouse tissues are available (Ruehl-Fehlert et al. 2003; Kittel et al. 2004; Morawietz et al. 2004) but it may be useful to produce in-house guides that detail the requirements of a specific study (Figure 2.1).
Figure 2.1 Example of a pictorial guide for identification of organ at necropsy and sampling of tissue for histology.
A tissue trimming guide should include:
- tissue to be sampled;
- site of sampling—may specify region, such as kidney cortex or liver lobe;
- number of samples—for example, single section, multiple sections from different lobes/areas;
- plane of sampling—cross section or longitudinal;
- size of sample;
- orientation of tissue in block—for example, which tissue or region is closest to slide label or which face of the tissue (up or down) is sectioned of the tissue;
- whether left or right samples should be identified separately—for example, unilateral lesions;
- separation of tissues that look the same when fixed/processed—for example, identification of the individual lymph nodes.
There is often a temptation to try to reduce costs by presenting as many tissues as possible in a single wax block. This is often a false economy as tissues may be lost or poorly orientated and the blocks may be more difficult for histologists to produce. The numbers of wax blocks that are needed can be reduced by grouping tissues of similar density into a single block—for example, liver, kidney, spleen and thymus (an example is given in Table 2.2). For complex or very small tissues, such as eyes, ovary, pituitary and adrenal glands, it may be difficult to orientate tissues to get representative sections of all the tissue components if they are blocked with other tissues, for example to achieve a good section of adrenal cortex and medulla it is best to place the adrenals in a separate cassette. Specific regions of intestine may be difficult to identify, particularly when modified by pathological changes and so the embedding schedule should be organized to aid identification. This can be achieved through a number of approaches, including placing the different sections of small or large intestinal regions that have histological similarities in separate blocks (see Table 2.2), using cassette dividers to separate tissues or ensuring that tissues are always orientated in a set pattern on the slide. Hard tissues which have been decalcified will also be better in a separate block as it is hard to section tissues of different densities without creating artefacts such as folds and wrinkles (McInnes 2011b).
Table 2.2 Example of a blocking pattern for full phenotypic evaluation of mouse tissues. Blocks 1–15 give a good overview of tissues and 16–19 are advisable for completeness. LS = longitudinal section. TS = transverse section (Kittel et al. 2004; Moraweitz et al. 2004; Ruehl-Fehlert et al. 2004). More detailed guidance for specific investigations of individual organs is provided in the relevant chapters.
| Block number | Tissues | Orientation/comments |
| 1 | Thymus, mesenteric lymph node, salivary glands | Thymus—representative LS sample Mesenteric lymph node—representative LS sample Salivary glands—LS to include all three major glands |
| 2 | Heart, skeletal muscle | Heart—LS section through all four chambers Muscle—TS of gastrocnemius |
| 3 | Liver, kidney, spleen | Liver—TS from left lateral and TS left and right medial lobes including gall bladder Kidney—TS from each organ or TS from left and LS from right (ensuring papilla included in both sections) Spleen—TS from largest part of organ |
| 4 | Lung | Lung—LS of four large lung lobes (remove accessory lobe) Inflate with fixative |
| 5 | Thyroid, parathyroid, aorta, oesophagus | Aorta—TS from thoracic aorta (brown fat often around aorta) Oesophagus—TS Thyroid, parathyroid—TS through thyroid and trachea Trachea—separate TS from below thyroid if needed |
| 6 | Adrenals | Adrenal glands—LS embed on any surface Ensure medulla is present |
| 7 | Brain | 3 × TS sections Can include spinal cord |
| 8 | Ovaries or testes and epididymides | Ovary—if possible LS to include ovary, oviduct and tip of uterine horn Testes—TS from both or one TS and one LS Epididymides- LS from both |
| 9 | Urinary bladder, uterus and vagina or prostate and seminal vesicles | Urinary bladder—LS Uterus—TS both horns Vagina—LS through vagina, cervix and uterine body Seminal vesicle/coagulating gland—TS both sides Prostate—LS after careful positioning of dorsal and ventral lobes |
| 10 | Stomach, duodenum, jejunum, colon, pancreas | Stomach—LS sections through greater and lesser curvature including glandular and nonglandular Intestines—TS of each region Pancreas—LS left lobe |
| 11 | Ileum, caecum, rectum | Intestines—TS of each region |
| 12 | Skin, mammary | LS—from thoracic area to maximize chances of mammary tissue |
| 13 | Eyes (with optic nerve), Harderian gland | Eyes—orientate to include optic nerve Harderian gland and lens—LS and lens |
| 14 | Pituitary | Pituitary – LS |
| 15 | Sternum (includes bone marrow) | Sternum—LS to include marrow |
| 16 | Femur and stifle joint | Femur—LS to include stifle joint |
| 17 | Spinal cord | Spinal cord—following decalcification TS of cervical, thoracic and lumbar regions |
| 18 | Peripheral nerve (usually sciatic) | Nerve—LS |
| 19 | Brown and white fat | Brown fat—TS from dorsal shoulder fat pad White fat—LS perigenital fat pad |
It is important that the expectations for the histopathology results are discussed with the pathologist before tissue blocks and sections are made so that the selection and trimming of tissues can be optimized before embedding. Although it is possible to reorientate embedded tissues by melting the paraffin wax and re-embedding it is inevitably more difficult to re-orientate small pieces of trimmed tissue at this stage and impossible to change the sample or retrieve other parts of tissues which have been disposed of after necropsy.
2.2 Controls
Control animals and proper experimental design (Zeiss et al. 2012) are an essential part of the evaluation of macroscopic and microscopic pathology, as they are for any other scientific technique. As demonstrated in this book and others (McInnes 2011b), mouse tissues rarely present with the unaltered hypothetical histology seen in textbooks because the tissue is usually affected by the appearance of common background lesions, histological or anatomical variations and artefacts. Background lesions will vary with strain, age of mouse, laboratory, health status, environmental and nutritional conditions. Controls are therefore vital to allow comparison of these background changes (in the specific laboratory/animal house) and any induced changes in the mouse populations and to distinguish these from genuine treatment or experimentally induced effects including those induced by genetic modification. Although historical control data may be useful it cannot account for changes that may occur during the time course of an experiment in a particular laboratory. Examples of this type of variation include the potential effect of high fat diets on liver histology and clinical chemistry parameters and the effect of a subclinical infection in the colony on affected tissues. Reduction in numbers or absence of controls may seem attractive from a 3Rs (reduce, replace, refine) perspective (Wells 2011) or to reduce the costs of histological and pathological analysis but may be unjustifiable if an experiment ultimately needs to be repeated because of uninterpretable or equivocal data. Historical control data has limited statistical validity and absent or limited numbers of concurrent control animals compared to treated animals may also limit the validity of any statistical evaluation of the data.
2.3 Standardizing terminology
The use of glossaries of standardized terms can be helpful in generating incidence data for lesions encountered in pathology. Glossaries can be developed for macroscopic and microscopic findings and can be specific to an individual laboratory or part of a more widely recognized nomenclature allowing for easier comparison between analyses performed in different laboratories.
2.3.1 Macroscopic terminology
Observation of changes at necropsy may be the end point in a study if no further microscopic analysis is to be performed. In this situation, information gathered during the dissection may form the basis of incidence data from the experiment. However, in many instances the observations made at necropsy may not only provide a data set in themselves but may be useful pointer to/ or correlate with the microscopic data.
Unless observations at necropsy are the result of a known intervention, for example measuring the size of a xenograft tumour and counting metastatic nodules, no assumption should be made about the pathological basis of any changes observed. Lesions of similar appearance may have very different underlying pathology and aetiologies—for example an enlarged mouse spleen may be due to lymphoma, other neoplasm types, increased extramedullary haematopoiesis, lymphoid hyperplasia or congestion (Chapter 9). Other ‘apparent’ abnormalities may relate to physiological/agonal changes such as hypostatic congestion, post-mortem change, artefacts or even normal anatomical features such as Peyer’s patches in the intestine. This may be confusing to the novice. It is therefore safer to describe what is seen during the necropsy rather than make a judgement about the pathology it may represent. A ‘wrong’ macroscopic diagnosis may be misleading if there is no histological follow up or may need to be explained in subsequent reports if microscopic follow up refutes the observational ‘guess’. In summary, the use of diagnostic terms should be avoided and macroscopic changes recorded should be limited to descriptive terms.
The extent of data recording that is required should be considered before the time of necropsy and should be detailed in a protocol or standard operating procedure. It may be useful to have a tick sheet to ensure that all tissues are taken, weighed and described as necessary (Chapter 1). An agreed lexicon of terms should be followed, which should take into account the anatomical location of any lesions their size, shape, distribution and colour (King et al. 2006). The use of standardized terminology will help to reduce interobserver variability if multiple people are involved in necropsy of animals from a specific study or set of studies. To ensure systematic recording of lesions it may be useful to have an illustrated guide (Figure 2.1), particularly for the anatomy of major organs of interest. In addition it is useful to be aware of the terminology used to describe the axial location of lesions (Figure 2.2). It may be appropriate to record size based on measurements (in millimetres) or as a proportion of the organ or area affected. Terms such as enlarged or reduced in size should be avoided unless they are justified for example by a measurable change in organ dimensions or weight.
Figure 2.2 Anatomical land mark descriptors. (a) Cranial view of a mouse. (b) Caudal view of a mouse. (c) Lateral view of mouse. (d) Standing mouse.
An example of a glossary of terms for macroscopic descriptions is given below (Table 2.3) with an illustration of distribution terms in Figure 2.3. If a number of different people are involved in recording necropsy images it may be useful to create an illustrated glossary with photographic examples of each term that is to be used to improve consistency.
Table 2.3 Example of a glossary of macroscopic terms.
| Feature | Terms | Comments and examples |
| Anatomical location | Organ and subdivision | For example, liver, specific lobe Axial location, e.g. cranial/caudal |
| Distribution | Unilateral or bilateral Focal Multifocal Coalescing Locally extensive Diffuse Segmental | See Figure 2.3 A single affected area Many affected areas Affected foci merge together Large focal lesion Whole organ or site affected Section of tubular organ affected |
| Size | Measured in mm or cm Extent of lesion—percentage affected Increased weight Number of lesions | Measurements preferred. Ensure scale is used if photographing an image |
| Shape | Circular Ovoid Nodular Raised Laminated Punctate Depressed | |
| Colour | Red White Black Brown Green Yellow Pale Dark Stippled/streaked/mottled | Keep to simple primary colours. Additional simple descriptors Usually accumulation of blood Multiple possibilities connective tissue, exudates Blood, melanin, other pigments Pigments Bile or putrefactive change Usually bilirubin accumulation—icterus Use in conjunction with a colour term Use in conjunction with a colour term Use in conjunction with a colour term |
| Consistency/texture | Hard Firm Soft Fluctuant Friable Gas or fluid filled Gritty | Like bone Like tendon Like brain Like filled urinary bladder |
| Contents | Volume Clear Opaque White Yellow Red Frothy Viscous Gas | Keep separate samples of hollow organ or body cavity fluids if required |
Figure 2.3 Examples of the use of terminology for describing macroscopic lesions. (a) and (b) could be described as (unilateral) focal lesions affecting one kidney or lung lobe respectively, (c) as multifocal to coalescing lesions, (d) as bilateral multifocal lesions, (e) as multifocal lesions affecting all lung lobes and (f) as locally extensive or diffuse lesions affecting left, right cranial and right middle lung lobes.
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