An experienced cytologist examining cytological preparations can sometimes make a diagnosis in a couple of seconds. With his experience he makes use of pattern recognition. With difficult preparations, however, a more systematic approach will lead to results sooner. This is certainly true for the inexperienced cytologist. In this paper a guideline is given for examining cytological preparations obtained by aspiration biopsy or exfoliative cytology. It is not possible in just one paper to give a complete overview of all cytological diagnoses. Those wishing to become better qualified in cytology after reading this text can pursue the subject in the references that are provided.

This paper only concerns the examination of cytological preparations. It should be realized, however, that for good diagnosis the quality of the preparation is of decisive importance. "In cytology the specimen is everything: Garbage in -Garbage out." (V. Perman, 1984). Good biopsy technique, good fixation of the smears, and good staining technique are essential. In addition, a proper use of a good microscope is of course of essence!

Apart from the possibility of poor quality of the biopsy, cytology has another limiting factor as mentioned earlier: a negative (e.g., tumor-negative) report is less reliable than a positive report. Severe inflammation in a tumor, necrosis of tissue in a tumor, or inadequate biopsies can result in a "false negative" report. One should always take this limitation into account. As with all diagnostic techniques it is also advisable to consider the probability of the cytological diagnosis in terms of the probability of the clinical diagnosis, and not to see it as an independent certainty.

The examination of a preparation goes through three phases. The first is determining the tissue of origin, the second is differentiating among inflammation, hyperplasia/benign tumor and malignant tumor, and the third is classifying the inflammatory process or the tumor.

Tissue of Origin

Because the cytological characteristics of hyperplasia, inflammation, and malignancy can differ in different tissues, it is important to first determine the most frequently occurring type of cell in the cytological preparation. The following three tissues can be distinguished.

I. Hematopoietic Tissue

These are cells originating from blood, bone marrow, lymph glands, spleen, and thymus. Mast cells, histiocytes, and macrophages also belong to this category. Cells of hematopoietic origin are individual cells, mostly round in shape, and have a distinct cytoplasmic membrane. The smears are usually rich in cells. Preparations that have not been streaked out can incorrectly suggest a tissue organization, but this will be found to disappear along the edges of the smear. The cell size can vary from small to medium-sized. Usually they contain little cytoplasm.

II. Epithelial Glandular Tissue

Cells of this type of tissue have the tendency to be connected to each other. They can form two- or three-dimensional clusters, but also monolayers or acini or duct structures. Sometimes there is a stromal component present. Individual cells can also be present. The cellularity is usually slightly less than in preparations with hematopoietic cells. Benign epithelial cells do also have a round nucleus. The cytoplasm is often abundant and the cell borders are distinct. Sometimes, for example in endocrine tumors, the cytoplasm is stripped from the nucleus when the smear is streaked out and there are mostly naked nuclei. The cell size can vary from small (basal cells) to very large (squamous epithelial cells).

III. Mesenchymal Tissue

Within this type are included the cells arising from connective tissue, muscle, cartilage, and bone. Because of the strong attachment between the cells it is difficult to obtain material. Smears of this type of tissue are thus also characterized by a low cellularity and the cells are mostly separate from each other, although groups of cells can also be found. The amount of cytoplasm can vary markedly and the cell borders are often rather vague. The shape of the nucleus can differ but is often oval. The cells can have characteristic morphologic forms, of which the spindle-shaped cell is the best known. Synovial cells, osteoblasts, and chondroblasts can have an egg-shaped outline in which the nucleus is eccentric and sometimes lies halfway outside the outline of the cytoplasm. Fat cells can be very large and can already be recognized at a low magnification. These cells are generally connected together as a tissue, have distinct cell margins, and have an oval nucleus that lies in the middle of the light, transparent cytoplasm.

Benign Pathologic Processes

Inflammation

If primarily inflammatory cells are encountered in a preparation, the process may be an acute or subacute inflammation or a more chronic and sometimes granulomatous inflammation. But it can also be a secondary inflammation in a neoplastic process. It is thus important even in the presence of inflammatory cells to continue looking for other types of cells. It is then sometimes difficult to differentiate among dysplastic, reactive, and neoplastic cells.

Inflammatory processes can be differentiated on the basis of duration (acute versus chronic), the type (purulent, pyogranulomatous, granulomatous, and eosinophilic), and the etiology (bacterial, parasitic, fungal, allergic, foreign-body reaction, etc.).

In a purulent inflammation the majority of the cells are neutrophilic polymorphonuclear leukocytes. They can account for than 85% of the cells present in an acute inflammation and decrease to 30-50% in a more chronic inflammation. In a purulent inflammation there can also be macrophages. Macrophages can be present in the inflammatory process within a couple of hours and thus they are no indication of chronicity. This is in contrast to the presence of plasma cells and lymphocytes. These cells only arrive at the site of the inflammation after 1-2 weeks. The inflammatory process can further be characterized by the presence or absence of degenerative changes in the inflammatory cells. Karyolysis is characterized by swelling of the nucleus, less intense staining, and then disintegration of the nucleus. This is especially seen in a severe inflammation with the release of many toxic materials and proliferation of bacteria. If these characteristics are seen, a special effort must be made to look for infectious agents. In contrast, pyknosis and karyorrhexis are characteristics of a slower cell death. Pyknosis causes the nucleus to shrink markedly and to lose its chromatin pattern. The nucleus becomes a uniformly darkly stained mass. If this pyknotic nucleus disintegrates, the process is called karyorrhexis.

In a granulomatous inflammation there are fewer polymorphonuclear cells than in a purulent inflammation. In contrast, there are more macrophages and epithelioid cells. The macrophages usually show fewer signs of phagocytosis than in a purulent inflammation. There can also be multinucleated giant cells. If the infection becomes chronic, lymphocytes and plasma cells are encountered again. This type of inflammation should lead to consideration of infection by fungi, infection by actinomyces or similar bacteria, and reactions to a foreign body.

Eosinophilic inflammations contain large numbers of eosinophilic granulocytes. This type of inflammation occurs in the eosinophilic granuloma in the cat, the lick granuloma in the dog, and also in parasitic infections and allergic reactions. Eosinophilic granulocytic infiltrates are also a characteristic finding in many mast cell tumors.

Tissue Regeneration

Many morphological changes related to an increased metabolic status of the cell may be present e.g., basophilic cytoplasm (high RNA- and protein content), prominent or multiple nucleoli, and increased numbers of mitoses. Differentiation with malignancy may be difficult. Usually a normal maturation of the cells can be found. Scar tissue may be attended with young connective tissue cells and blood capillaries. Some inflammatory cells can also be found.

Neoplastic Processes

For differentiation between malignant tumors and benign tumors or hyperplasia, both general criteria and cell-related (cytoplasmic and nuclear) criteria can be used. In the following paragraphs the different types of criteria of malignancy will be discussed. It must be realized that no single criterion is in itself indicative of malignancy. The nuclear criteria weigh the most heavily. In addition, the criteria are not identical for different types of cells and locations. The malignancy criteria are most often used for the epithelial tumors. In mesenchymal tumors they are less predictive, and the hematopoietic tumors are usually diagnosed on different criteria.

With regard to the general criteria, the appearance of the entire population of cells is examined. The absence of inflammatory cells, the presence of a uniform cell population (i.e., one cell type) of pleomorphic cells (i.e., with variable shapes) are indications of malignancy. A high cellularity and abnormal cellular relationships (e.g., cluster forming) can also occur with malignancy. Cluster formation means three-dimensional cell growth without organized cell relationships. In contrast, monolayers and formation of acini and ducts indicate better differentiation of the cell population. The occurrence of a type of cell where it does not usually occur makes the lesion immediately suspicious. A good example of this is the presence of epithelial cells in a lymph node.

Table 1. Summary of cytoplasmic and nuclear characteristics of malignancy.

The cytoplasmic criteria of malignancy include strong basophilia of the cytoplasm and even marked variability in basophilia of cells immediately adjacent to each other. This basophilia is related to the amount of RNA and protein synthesis in the cytoplasm and is thus a measure of the cell activity. Reactive cells can, however, also be strongly basophilic. Anisocytosis (marked variation in cell size) and macrocytosis (abnormally large cells) are more often seen in malignant than in benign tumors. Other cytoplasmic criteria of malignancy are the occurrence of abnormal cytoplasmic inclusions (especially cannibalism of other cells), atypical vacuolization, and a high nuclear/cytoplasmic (N/C) ratio (relatively little cytoplasm) and a marked variation in the N/C ratio.

The most important criteria of malignancy are, however, the nuclear criteria. Giant nuclei, anisokaryosis (difference in nuclear size), multiple nuclei per cell (especially if they also differ in size and are not 2n), abnormal nuclear shapes and marked variation in shapes, irregular and thickened nuclear membranes (especially easily seen in Papanicolaou staining), and an irregular, coarse chromatin pattern are nuclear characteristics that point to malignancy. An increased mitotic index and especially abnormal mitotic forms are other nuclear criteria of malignancy. The malignancy criteria of the nucleoli are multiple nucleoli, abnormal large nucleoli, anisonucleolisis and abnormally shaped nucleoli. Some authors have proposed that the presence of 3-4 of the above nuclear criteria of malignancy should be considered indicative of malignancy, for this number of many malignancy criteria should not occur in hyperplasia. In practice, however, it can still be difficult to differentiate between a malignant and a hyperplastic reaction, especially in mesenchymal proliferations. An overview of the most important of the above-mentioned criteria of malignancy is given in Table 1.

References

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9.  Tvedten H. Cytology of neoplastic and inflammatory masses. In: Willard, Tvedten, Turnwald (eds), WB Saunders Co, Philadelphia, 1989, pp 327-348.