Cytology uses cells to try and diagnose many diseases including cancer, requiring:

 Good-quality samples

 Confidence that the sample is representative

 Detailed knowledge of cell morphology

 Detailed knowledge of disease processes

 Diagnosis consistent with the presentation

Good-Quality Samples

One of the commonest pitfalls is sample artefact, often in the form of cell damage. Nuclei may remain intact, but without the cytoplasm, they are difficult if not impossible to interpret. Contamination can be adjacent tissues that complicate interpretation, especially if unknown, such as aspirating pulmonary masses through the liver. Excessive blood that is not dispersed, or is clotting, contracts and distorts the diagnostic nucleated harvest. Alternatively it may be exogenous contaminant, ultrasound-guided aspirates having too much gel obscuring the cells, or exposure to formalin, even fumes, preventing most routine stains being used effectively. Optimal sampling and preservation cannot be overemphasised. Cells cannot just be expressed on to a slide in droplets. Whilst cell damage may be less, they are too thick, precluding examination of cell detail vital to make a cytological diagnosis. Once the material is pooled on a slide, it must be smeared gently to create a monolayer. This smearing can be done in several ways requiring practice to allow consistent well preserved smears. The most universally applicable is the 'squash' preparation, but using a technique and style that suits the sample (and the person) is more important, selecting the most appropriate for that situation.

Confidence That the Sample is Representative

Representation and therefore diagnostic confidence is very difficult to assess. The more cells we have, the more likely we are to make the correct diagnosis, but the correct diagnosis can be made with experience using only very few cells in a known context. The more experienced and specialised the cytopathologist, the more likely a correct diagnosis is to be made and also as much useful information as possible is obtained.

Consistently producing very good samples requires not only experience in sampling but also adaptation to the tissue and the lesion in question. As a general rule, 'needle-only' sampling is the best starting method. The history and appearance of the lesion are very important, especially if sampling is performed under ultrasound guidance. A large heterogeneous mass that has only one smear made is unlikely to be entirely represented in that preparation. Using palpation and/or echogenic guidance allows us to sample multiple areas, both at the periphery and centre of the mass to maximise our chances of making an accurate diagnosis. If sampled well like this, cytology can provide more accurate information than trucut biopsy specimens. Tumour centres can be necrotic but should be sampled as it can provide information on the grade of the tumour. Suction can be required for firmer lesions but it is important to remember that suction is only to draw the tissue on to the needle end for 'to and fro'/'cheese core' sampling rather than actually pulling the tissue cells out/ off; suction otherwise increasing the likelihood of haemic artefact. Conversely in small masses it can allow the needle to exit the mass and sample adjacent tissue. This is especially true with only one person sampling as it is difficult to immobilise a mass, guide the needle and provide suction without exiting it. The combination of 'abnormal' and 'normal' tissue that is then sampled can make a diagnosis more difficult, if not impossible.

Detailed Knowledge of Cell Morphology and Disease Processes

Specific cells, organs and processes are too numerous to describe in a single lecture. However, all cells have basic features that should always be examined in conjunction, especially as we are trying to distinguish neoplasia from 'tissue reaction', and if neoplastic, discriminate benign from malignant cancerous lesions.

Fundamentally neoplasia is the identification of 'inappropriate cellular proliferation' so we must first cytologically exclude (where possible) a physiological reason for cellular proliferation. This often hinges on excluding an inflammatory/infectious process as well as only finding one type of tissue cell. Cancer is 'uncontrolled and anarchic or ectopic cellular proliferation'. Cytologically we identify cancer not from a mass-like effect with tissue invasion or vascular/lymphatic involvement but from comparing cell features and inferred interactions such that malignancy equates to cellular variation and pleomorphism most commonly. However, leukaemias and other 'immune system cancer' are to a great extent the opposite, as here we strive to identify clonal proliferation as too many cells of similar morphology. We must apply the correct criteria appropriate to the tissue process or cell of origin.

Malignant features, so-called, are a misnomer as there are no objective cellular features that if seen identify malignancy. If used in isolation without appreciation, misdiagnosis is likely. Cancer cytology actually relies on identifying features of cellular 'activation' and (uncommitted) 'proliferation', with variation in these features indicating malignancy. Veterinary literature somewhat arbitrarily quotes 'three or more features of malignancy' should be found to diagnose cancer and this is a good starting point. Some clinician talk of 'significant' features, but which are significant is an unknown, quoted features varying slightly from book to book. The crucial point is identifying significant variation between cells, in whatever form it takes.

Commonly used cytological stains give a similar pattern of cellular features but the texturing and colours vary considerably and often detrimentally if not done well. The commonest specialised veterinary cytologist's stain is either a modified Wrights or May-Grünwald-Giemsa. These provide the broadest but most detailed textural colouring that can be applied to blood smears and aspirates. Rapid stains most commonly used in practice can be fine but the staining times must be varied to accommodate the varying preparations and prevent under- or over-staining. Whilst all stains have inherent limitations, rapid stains, especially if not used appropriately, will miss important nuclear and cellular features that could provide diagnostic or important clinical information.

Nucleus

This typically stains purple. Simplistically it is composed of DNA - heterochromatin (dark 'inactive' DNA) and euchromatin (light 'active' DNA). The varying combination of these gives the cell nuclei characteristic textures (e.g., stippled or reticular). Cells in many tissues are 'resting' and so are mainly heterochromatin - nuclei appear smoother and darker purple. As the nucleus becomes 'active', whiter areas (euchromatin) are seen. Proliferative tissue cells therefore have more 'open' chromatin, with larger whiter and darker areas. This activity can be great enough to show as a focal 'hyperactive' point - the nucleolus, where DNA is actually being 'read and translated'. Very 'active' cells have prominent and possibly large or multiple nucleoli. The nuclear membrane is often oval to circular, but can have characteristic shapes. Cancer cells have variation in these features.

Cytoplasm

This is surrounded by an invisible membrane. However, it forms certain shapes that are very useful and whether this shape is discrete and very clear, or poorly demarcated is also crucial. As compared to the nucleus, resting or non-proliferative cells have paler smooth cytoplasm. 'Active' cells have darker blue cytoplasm. If the cell is 'productive' or phagocytic, material can be seen as distinctly coloured granules, indistinct smudges, as a diffuse hue, or in the form of clear white vacuoles.

Populations

No tissue has only one type of cell in it. Therefore the combination and arrangement of cells can be very important, not only in identifying the tissue/organ, but also whether any expected populations are absent or increased. Certain populations, such as inflammatory cells, can be part of the disease process. Many tissue types have cellular arrangements that are characteristic.

Background

The appearance of the background is often affected by contamination but can be very useful. For example, joint fluid is lightly pink and stippled, whereas lymph nodes are lightly basophilic. The background may identify significant processes, such as haemorrhagic organisation or necrosis, that are vital when making a comprehensive interpretation/diagnosis. Inflammation is one of the most significant, as this inherently creates cytological variation that can be very misleading and allow an incorrect diagnosis of cancer.

All of these features will be present in some form in every single sample examined. 'Normal' tissue is uniform, with cells appearing very similar and regularly arranged. Benign proliferation can be almost identical to this. Malignant tissue will have many similar features, but crucially there will be more variation from cell to cell, in size, shape and interactions because the proliferation is now uncontrolled and excessive.

Diagnosis Consistent with the Presentation

Whenever you make a cytological diagnosis, it should be made within the context of the history etc. so the cytological diagnosis is 'not at odds' with the clinical history, presuming it is known. This not only gives more confidence in the representation, but is also an inherent 'check'. If the context is unknown, the cytological interpretation/diagnosis is likely to be more uncertain. However, it is also very easy to use this information to 'over-diagnose', providing the wrong diagnosis when actually there are too few (intact) cells or there are other differential diagnoses for the same populations. This judgment relies in part on self-confidence, which comes with experience and specialisation.