Congress on Demand 2021: Diagnostics

Common mistakes in sampling: Optimal cytology smears, correct sample handling and contextualisation of the findings with the clinical history are all essential steps for achieving an accurate cytological diagnosis. Good quality cytology smears provide excellent morphologic details of cells and infectious agents, often allowing to differentiate between inflammatory and neoplastic processes, identify the tumour type and its behaviour (benign or malignant). When performing a fine-needle aspirate (FNA), the aim is to produce a monolayer of cells with minimal cell rupture. An incorrect technique can produce unsuitable samples precluding adequate evaluation and identification of the cells. Another common pre-analytical mistake in cytology is to collect a single aspirate, especially from larger masses. A single mass may contain areas of necrosis, inflammation, neoplasia or normal tissue cells and a single slide is unlikely to be representative of the entire lesion. If a mass is fluid-filled, collection of fluid and adjacent solid areas would be recommended, as fluid cytology alone rarely reveals the nature of the surrounding mass. Labelling of the slides with patient name and origin of the FNAs is another crucial step. The importance of sample handling before processing and staining should not be underestimated. Fluid samples should be collected in the correct tubes and adequately stored; unstained cytology slides should not be exposed to formalin fumes. Finally, adequate staining procedures are essential to guarantee and highlight the cellular details that are required for the diagnosis. Taking care of all these simple steps will prevent the most common sampling mistakes, increasing the diagnostic power of cytology.

Common mistakes in interpreting: When interpreting cytology, it is vital to consider the clinical history and appearance of the lesion as well as the cytological appearance and to have likely differential diagnoses in mind. Organisms may not be visible in infected lesions if antibiotics are given before sampling. Bacteria are rarely seen in septic arthritis. Fungi and mycobacteria can be difficult to see with routine stains. The lesion may have mixed pathology such as focal areas of necrosis or inflammation within a tumour and sometimes the fine needle aspirate may harvest only some of these components and not be wholly representative. Hence if neoplasia is suspected but only inflammation is seen, resampling different areas would be recommended. We are familiar with looking for criteria of malignancy to make a diagnosis of neoplasia. However, hyperplastic or dysplastic cells can sometimes be impossible to distinguish from neoplastic cells, since all three can show criteria of malignancy. This is a particular problem of mesenchymal cells because the fibroblasts in granulation tissue or in inflammatory lesions can resemble the neoplastic cells seen in soft tissue sarcomas. The history and appearance may help distinguish these although biopsy will often be required. Just as non-neoplastic cells can look malignant, the converse is also true. Some malignant tumours consist of cells which do not display marked criteria of malignancy. Examples include haemophagocytic histiocytic sarcoma, some malignant melanomas and thyroid carcinoma. Knowledge of the clinical presentation and expected pathology will help minimise errors in interpretation. Cytology should never be performed in isolation.

This case-based session explores and discusses common but still challenging cytology cases using live cytology slide examination, enabling you to see the step by step process of how we examine a slide and how the findings lead us to a diagnosis or differential diagnoses.

How does the cytologist do it? Although there are some exceptions, benign tumours consist of a uniform population of cells that resemble their normal non-neoplastic counterpart whilst malignant tumours generally show variability. In benign tumours cells are small and uniform, with small nuclei and a low nuclear:cytoplasmic (N:C) ratio. Nucleoli may be absent or nuclei may contain 1-2 small nucleoli. When in aggregates the cell arrangement is orderly and neat. Malignant tumours are recognised by identifying cellular, nuclear and cytoplasmic criteria of malignancy: Abnormal location for that cell type e.g. metastatic carcinoma cells should not be present in a lymph node; macrocytosis and karyomegaly with anisocytosis and anisokaryosis; cell clusters may be chaotic and disordered with cell or nuclear moulding; increased N:C ratio; large nucleus and sparse; bi- and multinucleation – anisokaryosis within one cell is especially significant; multiple nucleoli or a single large nucleolus; coarsely stippled to clumped nuclear chromatin; frequent/ abnormal mitoses; increased cytoplasmic basophilia and/or abnormal cytoplasmic vacuolation or granulation, or excessive secretory product. The shape and arrangement of cells will help identify the ‘family’ of cells: Epithelial cells are columnar, cuboidal, roundish or polygonal and in cohesive clusters. Mesenchymal cells are oval to spindle shaped and seen individually or in loose aggregates, sometimes with a swirling pattern, with poorly defined cell borders. Round cells are discrete. The quantity and appearance of the cytoplasm distinguishes lymphoid cells, plasma cells, histiocytic cells and mast cells.

What else does the oncologist need to know (TNM)? Staging determines the extent of disease in cancer patients, to inform treatment decisions. Recommended staging is strongly influenced by the diagnosis and likely behaviour of the tumour: a diagnosis is essential. Full staging is most appropriate for high grade tumours, and in older patients (identifying comorbidities) or before invasive/expensive treatments. Cytology is particularly useful for superficial masses and those accessible by ultrasound guidance. Carcinomas and round cell tumours tend to exfoliate well, sarcomas not. Primary tumour extent is assessed clinically and by imaging. Carcinomas, mast cell tumours, and malignant melanomas tend to metastasise by the lymphatic route, requiring assessment of locoregional lymph nodes. The closest node (moving from peripheral to central) is often likely to be the draining node, but lymphangiography can identify unexpected draining nodes in high grade tumours. Identifying and sampling these nodes leads to better staging. Imaging of retropharyngeal, axillary, medial iliac and inguinal nodes by ultrasound or CT is useful: CT allows imaging of sacral nodes e.g. in anal sac adenocarcinoma patients. FNA has a variable rate of false negatives in different tumours: in particular, FNA may be insensitive to oral melanoma metastases. For distant metastases, cytology is especially useful for assessing splenic and hepatic nodules.