ASPIRATION CYTOLOGY

In order to make a preparation using the aspiration technique, a 10 ml syringe, 21–25 G cannulas, a slide and a glass cover (60×24 mm) are needed.

Usually, there is no need to shave or disinfect the skin before sampling. The cannula will be inserted through the tissue and advanced in several directions into the tumor while aspiration is performed repeatedly. An aspiration pistol is helpful because it allows aspiration using one hand, while the free hand can be used to set volume. The size of the cannula depends on the increase in the volume to be aspirated. For the aspiration of skin or subcutaneous tissue volume, 22 G cannulas are indicated, for intrathoracic and intraabdominal tumors. In the case of neoplasias that are prone to hemorrhage, smaller gauge cannulas are recommended (23–25 G). Aspiration is completed when blood and tissue have accumulated in the cone of the cannula. When the cannula is extracted from the tissue, the creation of vacuum is no longer needed, so that the research material does not enter the syringe body. Only the material in the cannula will be investigated. After the material has been sprayed on a slide, a second slide or a cover glass will be placed over the first one. When due to the surface tension between the two slides, the aspirated material has doubled its diameter, the two slides will be separated without being pressed against one another. After being air-dried, the smears will be stained with a rapidly staining method.

SCRAPINGS

This method is most adequate for an intraoperative diagnosis of a suspect tissue and for surface lesions. The tissue from the cut edge of a surgically prepared sample will be removed by tamponing. Then, the almost dry portion of the cut tissue will be carefully placed on a slide. Pressure, as well as rotation and smoothing movements should be avoided as they may damage the cells. The cellular material can also be obtained by scraping the cut portion with a scalpel blade or a fine brush. The study material will be placed directly on the slide and after being air dried, it will be stained.

EXAMINATION OF MATERIAL

In principle, the examination of a cytological sample should respect the following diagram:

Diagram of a cytological preparation (according to KESSLER, 1999)

First, the quality of the smear is checked, only the preparations containing a high number of cells will be used. Artifacts and ruptured cells are recognized because of their chromatin, which appears effaced in the direction in which the smear has been prepared. Insufficient material or material that cannot be examined/interpreted can be the result of an inadequate processing technique or incomplete exfoliation of tumor cells.

At a low magnification, the diagnosing and search of tumor cells that are frequently found at the periphery of the smear in aspiration samples will be initiated, then the cells will be examined at a higher magnification (400–1000x). The size scale is given by erythrocytes, which are frequently found in high numbers. Certain criteria are used for the differentiation of malignant cells from benign cells. The most important feature of malignant cells is polymorphism, which is seen in the various changes of the cytoplasm and cell nucleus. The cells of a malignant neoplasm frequently show a size that differs from normal. In addition, the cells almost invariably possess a nuclear polymorphism. The most important nuclear changes are: different sizes and shapes, alterations in the structure of chromatin and nucleoli. There are also changes in the cytoplasm, which are less important than the changes in the nucleus. In order to diagnose a malignant tumor, three of the malignancy criteria of the Table “Criteria for Malignancy” should be met.

Malignant tumors in cytology are divided, according to their origin and morphology, in three groups: epithelial tumors, mesenchymal tumors (carcinomas and sarcomas), and round cell tumors.

EPITHELIAL TUMORS

Epithelial cells show good exfoliation from tissues and frequently result in cell-rich preparations. The characteristic criterion is the appearance in cell clusters with relatively well distinguished cell borders. The basic shape of the cell varies from round to polygonal. It has a round nucleus and abundant cytoplasm, but there are important differences in the amount, color of granulations and presence of vacuoles. Compared to mesenchymal cells, epithelial tumor cells show a more marked tendency to basophilia.

MESENCHYMAL TUMORS

Mesenchymal tissue is less prone to cell exfoliation. This is shown by the fact that the cell collection in an aspirate or scraping usually contains a smaller amount of cells compared to epithelial tissue cells. Poorly differentiated tumors produce more smears with a high number of cells compared to well differentiated tumors. In addition, there is a correlation between the number of cells obtained and the degree of differentiation of the tumor. When a well differentiated mesenchymal neoplasm is suspected, a second aspiration is performed for correction, with a larger gauge cannula (22 G). The cytological picture of mesenchymal tumors frequently includes small polygonal to spindle shaped individual cells or cell clusters, with unclear cell granulations, blurred cytoplasm and oval nucleus.

ROUND CELL TUMORS

Round cell tumors designate a group of neoplasms which are histogenetically differentiated, but have common cytological properties. Here are included the tumors of the hematopoietic system, histiocytomas, mast cell tumors, melanomas and Sticker sarcoma. As a characteristic feature, the aspirates from these tumors do not contain cell clusters but individual cells, which are round, and cell granulations are visible. Usually, preparations are cell rich [18].

CYTOLOGY OF MALIGNANT COLLECTIONS FROM THE BODY CAVITIES (according to HIRSCHBERGER, 1999)

Body cavity collections are triggered in the first place by four pathogenic mechanisms:

1. Disturbances in the hydrostatic pressure
2. Disturbances in oncotic pressure
3. Increase in capillary permeability
4. Limited lymphatic drainage

The filtered fluid will not be all physiologically reabsorbed in the blood, part of it will flow through the lymph channels back to circulation. The hydrostatic pressure of capillaries drives blood (fluid) to tissues or body cavities. In addition to cardiopathies, hepatopathies, organ torsion, hernia and inflammation, tumors can also raise hydrostatic pressure by compression on blood vessels. The accumulation of a fluid in a body cavity can also be due to low oncotic pressure. This may occur through a diminished water binding capacity in the capillaries conditioned by hypoproteinemia, but can also be induced by increased intracavitary oncotic pressure.

The exchange of plasma and interstitial fluid is influenced by the following factors: capillary permeability, intra- and extravasal oncotic pressure, and lymph circulation.

The following factors may be responsible for the increase in intracavitary oncotic pressure: metabolic exchange products, cell decomposition products in the body cavities (inflammation cells, mesothelial cells and tumor cells), as well as the marked penetration of some substances from the blood system into the body cavities. The last mentioned are triggered by a high capillary permeability that can also be conditioned by tumors and inflammation. Deregulations of lymph circulation are frequently a cause of cavity collections. Their etiology can be tumoral lymph nodes or inflammation and altered lymph vessels.

Obtaining and preparation of collection content from body cavities:

Paracentesis can be performed with an extremely fine cannula and when the maneuver is faultless, it does not pose any risk to the patient. A sonographic control of the puncture is extremely useful. A “blind” but careful puncture is practicable at certain sites. After the puncture site has been shaved and disinfected, cannulas 0.9–1.2 mm in diameter and 60 mm long will be used. The fluid will be aspirated with a 10 ml syringe and will be transferred to containers that have been initially prepared.

For a clinical research, 1–2 ml puncture fluid are sufficient. In order to perform a cytological research and to define the number of cells, up to 10 ml puncture fluid will be introduced in a container filled with 10 ml EDTA (1 mg EDTA/ml puncture fluid). When an inflammatory bacterial process is suspected, puncture fluid will be taken and introduced in a tube with broth for bacteriological researches.

The cytological processing of the EDTA puncture fluid will be initiated within an hour. It is not possible to send the puncture fluid by post, moreover, stained and unstained smears should be prepared. After the cellular concentration has been established, the EDTA puncture fluid will be centrifuged and the resulting cellular sediment will be resuspended with the same amount of residual fluid. If a centrifuge is not available, the fluid can be left to sediment for half an hour and the fluid that has formed at the surface can be pipetted. One drop of 10 μl of the sediment will be placed on the slide and will be spread like a blood drop. In rare cases, it is possible to make smears of substances extremely rich in cells from pus or even with pure blood.

If there are suspended particles with a flake appearance, the puncture fluid should be given special attention. These mixtures frequently contain very important parts for the diagnosing of a body cavity collection – bacterial colonies and tumor cell clusters.

Paracenteses in the dog and cat (according to HIRSCHBERGER, 1999)

Interpretation of results:

The smear should extend over half the slide and end with a brush-shaped train. In the middle of the smear, cells are frequently flattened, adjacent to one another, while at the end of the smear, cell dendrites, large cells (macrophages, tumor cells, cell aggregates) gather. These two portions of the smear should be analyzed.

In order to interpret the cytological results of a cavity collection, the number of cells is extremely important. If the smear contains only inflammatory cells, the presence of an inflammatory process can be supported only when the number of cells is also high, about 500 nucleated cells/μl. In the case of an unknown cellular content, only a differentiated picture can be described, which cannot be interpreted with reference to diagnosis. The nucleate cell content (erythrocytes and thrombocytes) can be determined with a trained cell counter or with a hemocytometer. In the case of blood punctures, the hematocrit can also be determined. The blood content will always be macroscopically overestimated. Puncture samples that at first sight only contain blood frequently have a hematocrit of less than 10%.

The cytological examination of a puncture sample results in an ordering of what has been found according to 3 hydropic causes:

1. tumor
2. inflammation
3. hemorrhage

TUMORS CARCINOMAS/SARCOMAS

Cell populations from body cavity collections that are classified according to malignancy criteria as malignant should present at least 5 malignancy criteria (not at least 4, like in the case of tissue punctures), in order to be considered malignant. An extremely important restriction is that these 5 malignancy criteria should belong to the nucleus. The malignancy criteria of the cytoplasm are not taken into consideration. In contrast to sarcoma cells, carcinoma cells become easily exfoliated and are easy to discover in smears. In about 2/3 of all body cavity collections conditioned by carcinomas, tumor cells from puncture fluids are found. Round cell tumors (malignant lymphomas, mast cell tumors, histiocytomas) can be recognized in most cases from punctures. The sensitivity of the cytology of collections in the diagnosis of malignant tumors is 63%, specificity about 100%.

MESOTHELIAL CELLS/MALIGNANT MESOTHELIOMA

Mesothelial cells are relatively large cells that appear as individual cells or in clusters. Under different stimuli, (e.g. traumas, inflammation, neoplasia), mesothelial cells can proliferate rapidly and show certain malignancy criteria. These reactive blast-like mesothelial cells may be sometimes confused with malignant cells. Mesothelial cells are called “body cavity chimeras” because they can be similar in appearance to other cell populations. Good experience is necessary in the evaluation of body cavity collections in order to differentiate reactive mesothelial cells from other cells. Inflammatory processes reinforce this effect so that in the presence of cytological indications of a marked inflammation, the diagnosis of malignancy should be established extremely carefully.

Malignant mesotheliomas are a special challenge for cytology. The differentiation of mesothelial cells, which are highly reactive, with a blastic characteristic, and grow in cell clusters, from malignant mesothelial cells is extremely difficult even for specialists and requires histological evaluation. Malignant mesotheliomas are frequently so poorly differentiated that they are no longer phenotypically similar to mesothelial cells and are confused with carcinomas.

MALIGNANT LYMPHOMA

Malignant lymphomas are composed in the dog and cat in the first place of large “non-matured” cells (lymphoblasts and prolymphoblasts). These cells can be found in malignant lymphomas and in collections. Small mature lymphocytes can also represent a tumor cell population, incidence being lower. Small circulating lymphocytes gather in congestion (cardiac congestion, lymphadenopathy) in the body cavity, this process occurring more frequently in the pleura than in the peritoneal cavity. Because of the long, physiologically conditioned life duration, lymphocytes exist in a more than proportional concentration compared to neutrophilic granulocytes.

INFLAMMATION

Inflammations are designated according to the dominant cell population. Neutrophilic inflammations have a proportion of at least 80% neutrophilic granulocytes, macrophagocytic inflammations at least 50% macrophages. Neutrophilic-macrophagocytic inflammations come in between these two. Neutrophilic inflammations appear especially in acute inflammations and bacterial infections and cells degenerate depending on the toxicity of the inflammation process. Purely pneumonic inflammations do not degenerate. Neutrophilic granulocytes chemotactically move into the pleural cavity, in the absence of microorganisms at that site.

Macrophagocytic inflammations develop in chronic inflammations and less toxic inflammations. Macrophages always appear where there is cellular necrosis or microbial infections persist. Mycoses, mycobacterioses and feline infectious peritonitis have typical causes.

Even fats in chilothorax are capable, as less toxic agents, to attract macrophages and neutrophilic granulocytes, which can phagocytose chilomicrons.

Feline infectious peritonitis cytologically demonstrates the picture of a macrophagocytic inflammation or of a neutrophilic-macrophagocytic inflammation. The puncture sample shows along with macrophages and neutrophilic granulocytes, many lymphocytes and some plasma cells. The cell content is highly variable and ranges between 300–1200 nucleated cells/μl [19].

After having been applied on slides directly from the sample or by using a brush soaked in physiological serum, smears will be fixed by drying or in a fixing fluid. Fixation is performed by the immersion of the wet slides in 95° ethyl alcohol, or in a polyethylene glycol and ethyl alcohol mixture, especially for Papanicolaou staining, but other fixing mixtures are not excluded, when other stainings or histochemical reactions are used for scientific purposes.

Usual stainings are May-Grumwald-Giemsa and Papanicolaou, then Romanowsky and Wright; a rapid staining method that has given good results is with polychrome blue. In some special cases, the PAS reaction and alcian blue staining are recommended.

Cytological and/or histopathological examinations aim to establish a diagnosis of certainty, which cannot be achieved for each case. Prognosis and the establishment of an adequate therapeutic approach for each individual patient should be considered.

All procedures applied for diagnostic purposes initially aim to establish a pathological condition, then its nature (cancerous, inflammatory or other) and, finally, to make a diagnosis of certainty. The establishment of a positive diagnosis requires the correlation of the results of clinical and laboratory investigations with cytological examination.

In order to determine the neoplastic nature of a lesion, cytological examination will take into consideration some cellular criteria, which allow the identification of a neoplastic process [15]. Cytological examination concerns:

• – the cytoplasm: basophilia, vacuoles or foamy appearance, density, irregular shape, size;
• – the nucleus: size, homogeneous or reticular appearance;
• – mitoses: mitotic number or index (absent, rare, moderate or numerous);
• – nucleoli: large, obvious, multiple, irregular shapes;
• – the nucleocytoplasmic ratio: usually high;
• – cell size: large;
• – cell number: frequently high in tumors with distinct cells and in carcinomas; occasional high number in sarcomas;
• – intercellular relationships: indistinct borders (carcinomas); nuclear disaggregate agglomerates; subaggregates or acini-like structures;
• – cell activity: elaboration of cellular products;
• – cell position: aberrant.

Cytologic examination will assess: whether cells are in a proliferation activity; the presence of undifferentiated or poorly differentiated cells, which are independent or not from the tissue from which they have been harvested. Hematopoietic tissue blastocytes as undifferentiated cells have a basophilic cytoplasm, large nuclei and smooth homogeneous or finely reticular nuclear chromatin. Nuclei are large, with a nucleocytoplasmic ratio in favor of the nucleus, and nucleoli are distinct. These aspects are found in the cells of proliferated tissues, whether proliferation is normal or neoplastic, which shows that the described picture is not sufficient to make a neoplasia diagnosis [14].

The major difference between non-neoplastic and neoplastic changes consists of the fact that normal proliferation represents a continuous development of cells along a differentiation line, depending on the tissue type. Minimal differentiation is present in neoplastic proliferation, neoplastic cells being almost always pleomorphic. These are variable in size and staining, and nuclei are polymorphic in terms of size, shape and staining. Neoplastic cells can have multiple nuclei, extremely large nucleoli and irregular shapes.

Numerous cells can be obtained on smears from carcinomas and discrete tumors, while an inconsistent number of cells, sometimes insufficient for diagnosis, is obtained from sarcomas by aspiration. When a sarcoma scrape is available, the smear allows the establishment of positive diagnosis.

A certain indicator of malignancy is represented by the presence of immature pleomorphic cells, located in an aberrant region or tissue, which also allows the identification of metastases.

Cytologically, three types of malignant tumors can be identified from tumor tissues: discrete cell tumors, carcinomas and sarcomas.

1. Discrete (hidden) cell tumors are formed by cells that may function independently from other cells, which can be considered as individual cells in foreign tissues. This category includes malignant leukocytes, mastocytomas, melanomas and venereal round cell sarcomas in dogs. These tumor types are easy to identify on smears performed from aspirated material or by impression. The cellular characteristics of each mentioned neoplasm are sufficiently particularized to establish positive diagnosis.

Carcinoma is characterized by the presence of a high number of cells, each being extremely poorly delimited, and cells are grouped in large clusters. Intercellular relations can be identified in ductal or acinar arrangements, in smears obtained from adenocarcinomas. Occasionally, adenocarcinoma cells present large vacuoles or clear granular structures with secretory products.

Sarcoma is derived from cells of mesenchymal origin. Aspiration frequently fails to produce a sufficient number of cells for a cytological diagnosis. This is why surgical biopsy is used to obtain a sufficiently large sample for evaluation. In the smear, cells are individual and are frequently fusiform.

Mastocytoma is relatively easy to identify by cytologic examination. Primary mastocytoma develops in the skin, and it can subsequently invade the abdominal cavity organs (spleen, intestine), bone marrow and blood (mast cell leukemia). Cytoplasmic granules are easy to evidence in the stained smear (with polychrome blue they stain violet). There are variations between different mastocytomas (from one subject to another and from one tissue to another), which are remarkable in terms of granulations (staining intensity, number in the cytoplasm, size, etc.).

Usually, nuclei are round, nucleoli are not identified, and mitoses are absent. Sometimes, nuclei can be lobated, with fissures in their membranes. Nucleoli or mitotic figures are exceptional. Mast cell pleomorphism is a poor prognostic factor, and survival will be significantly diminished, compared to tumors with homogeneous cells, with rare pleomorphic cells. Eosinophils and/or macrophages can also be identified in mastocytoma [10,12].

Histiocytoma appears as a benign cutaneous tumor, specific for dogs. Macroscopically, it appears as a well outlined cutaneous bulge, being considered by some specialists a proliferative inflammatory lesion. The lesion is found in young dogs, under 2 years of age. It usually appears as a solitary, more rarely multiple tumor. The examination of smears detects well individualized cells, with round, oval or dented nuclei and slightly colored cytoplasm, which can occasionally be foamy or vacuolated. Mitoses can be present, but other malignancy criteria are insignificant.

Venereal round cell sarcoma in dogs frequently occurs in the genital tract mucosae, but it has also been identified in other locations (head, trunk and oral cavity). The smear contains a high number of individually displayed, well distinct cells, or clusters of small cells. The cell cytoplasm is moderately basophilic, sometimes it presents vacuoles, nuclei are large and round or oval, eccentric, with homogeneous karyoplasm. Mitoses are numerous, especially in young expanding tumors, while regressing tumors present a moderate number of lymphocytes, plasmacytes and macrophages.

Melanoma in the smear is formed by individual cells, and malignancy as well as the form of primary tumor or metastasis are difficult to establish. Melanomas are common in white or gray horses, as well as in dogs, and amelanotic melanomas may also develop, especially in dogs. In smears, malignant cells appear as round or fusiform, with eccentric nuclei, but dendritic and multinucleated giant cells can also be identified. Cell cytoplasms contain small pigmented granulations, of dark brown to black color, which may also be detected outside of cells, being derived from cells detached and damaged during the performance of the smear.

Lymphoma is rich in cells, which determines the appearance of numerous cells in the smear. Locations can be multiple, in the lymph nodes, bone marrow, and thoracic or abdominal effusions. Kidney, liver and skin lymphoma in smears has a lower number of cells.

Malignant lymphocytes are large, with a large nucleus and scant cytoplasm. The karyoplasm is homogeneous, but marked irregular condensations may occur in some cells, which suggests that these are mature cells. Some lymphocytes have multiple large nucleoli. The cytoplasm is in a low amount, slightly or intensely basophilic. Numerous cells are varied in terms of mitotic figures and stages.

In thoracic and peritoneal fluids, malignant lymphoid cells are much more variable than in lymph nodes. Some cells have features of mature cells, others are blastic and pleomorphic. Cells have a moderate amount of basophilic cytoplasm, cytoplasmic membranes are irregular, with fissures, and nuclei are irregular, with fissures or multiple projections.

In horses, lymphoma is formed by poorly or non-pleomorphic cells, many of which are mature, but with abnormal location, e.g. in the thorax. In cattle, malignant lymphocytes are extremely pleomorphic, sometimes difficult to recognize as lymphoid cells.

The cytological examination of bone marrow indicates a maturation of cells from a specific cellular line, but abnormal cells are present, even if they are not numerous.

2. Carcinomas in smears are characterized by exfoliated cells, present in large groups, with round or oval shapes, and eccentric nucleoli. Cellular limits, cytoplasmic membranes, are frequently indistinct. Cells may have an acinar or ductal arrangement around a central lumen, suggesting adenocarcinoma. In general, epithelia are highly susceptible to become hyperplastic, which makes this phenomenon of hyperplasia and neoplastic proliferation much similar. These aspects of similarity make difficult the diagnosis of epithelial tumors.

Squamous cell carcinoma is more frequently located in the skin, conjunctiva in cattle, ear in cats, mucosae, etc. Squamous tumor cells are large, and the nucleoplasmic ratio is low. In the smear, cells are isolated, more rarely in small clusters. Squamous cells are easy to recognize, they are large, nuclei are intensely chromatic, some are pyknotic, the cytoplasm is moderately basophilic. Special attention should be given to ulcerative lesions in which hyperplastic squamous cells, similar to neoplastic cells, appear. In the case of unusual locations, such as in the lymph nodes or peritoneal fluid (in horses, in stomach squamous cell carcinoma), the presence of squamous cells facilitates the carcinoma diagnosis.

Uroepithelial neoplasms can be located in any segment of the urinary tract, frequently in the urinary bladder. In the smear, exfoliated cells appear as solitary or in pleomorphic cell agglomerations, in the urinary sediment. Cells present extremely wide variations in size, nuclei are well evidenced, with distinct irregular nucleoli. The cell cytoplasm is moderate to abundant and moderately basophilic. Urinary type epithelium can undergo squamous metaplasia. The diagnosis of neoplasm is not difficult to make, but there are some difficulties in defining location.

Adenocarcinomas can be mammary, pancreatic, gastric, salivary, prostatic, etc. Smears with numerous cells are performed by aspiration. Cells appear in the smear under the form of large clusters, sometimes with ductal or acinar arrangement, with peripheral nuclei and the cytoplasm oriented towards the center of the cluster.

Cells are poorly delimited, highly pleomorphic and intensely basophilic. Cytoplasmic vacuoles are frequent in gastric and pancreatic adenocarcinomas, and foamy cells appear occasionally. Some cells have a signet ring appearance, due to a large vacuole that moves the nucleus towards the periphery. In general, the nuclei of adenocarcinomas are polymorphic, with large variations in size and staining intensity, having a homogeneous or reticular karyoplasm. Single or multiple nucleoli are obvious and mitoses may be present.

Thyroid carcinoma is difficult to diagnose in smears performed by aspiration. Likewise, perianal gland carcinomas are not easy to diagnose in smears.

3. Sarcomas cytologically occur on smears with generally few, individual, fusiform cells; this is why the tumor scraping method is used, in order to obtain a cell-rich smear. Free nuclei are frequently found, and sometimes multiple nuclei, with obvious large nucleoli. The cytoplasm is moderately basophilic and sometimes vacuolized. The difficulty of diagnosing may be in the case of granulation tissue, where non-pleomorphic hyperplastic fibroblasts appear, with the almost constant presence of inflammatory cells, such as neutrophil granulocytes, macrophages and mast cells.

Hemangiopericytoma can cytologically be very similar to fibrosarcoma, but cells are smaller, of conical shape, gathered in small groups (5–10 cells), with the predominance of individual cells.

Leiomyosarcoma and rhabdomyosarcoma: they are cytologically similar to fibrosarcomas. Cells are individual, fusiform, with moderately basophilic cytoplasm, and the nucleocytoplasmic ratio is moderate to high. Sometimes, few cells with multiple nuclei appear, occasionally nucleoli being large.

Chondrosarcoma and osteosarcoma: cytologically, they have round, slightly fusiform cells, almost all cells showing a slightly conical cytoplasm. Amorphous azurophilic material frequently occurs intercellularly. The cell cytoplasm is moderately basophilic, and the nucleoplasmic ratio is from moderate to high. Nuclei are eccentric, cells with 2–4 nuclei being rarer.

Hemangiosarcomas: cytologically, they appear intensely hemorrhagic, with the presence of fusiform cells, a basophilic, foamy or vacuolated cytoplasm. The nucleoplasmic ratio is moderate, nuclei are vesicular, with obvious nucleoli. The cytoplasm is basophilic, from moderate to rich, foamy or vacuolated.

The cytology of cancer effusions is a complementary examination in positive diagnosing. Following clinical examination and history, the cytologic examination of various effusions is required, in the case of the suspicion of:

• – mediastinal lymphoma in felines;
• – detection of an effusion in patients with mammary or ovarian neoplasms, since these are known to have a high metastatic potential;
• – detection of an isolated cavitary effusion, without a precise clinical explanation, such as a pleural effusion that has evolved asymptomatically;
• – routine cytologic examinations of any effusions [6].

According to the cited authors, the methodology of fluid sampling requires some rules that need to be respected: absolute sterilization conditions, no anesthesia, needles of 10–12/10, 40 mm, EDTA tubes and dry tubes.

Pericardial effusions are harvested: with the animal in left lateral decubitus or standing; by right side puncture, intercostal space 3–5, lower 1/3 – middle 1/3. If possible, ultrasonographic guidance will be performed, sometimes anesthesia being required [9,13].

Pleural effusions are harvested with the animal standing, on the right or left side, in the intercostal space 6–7, in the lower 1/3 – middle 1/3.

Abdominal effusions are harvested with the animal in lateral decubitus, on the white line, 2 cm behind the umbilicus.

The fluids obtained, without being treated with preserving fluids, will be used for smears as soon as possible. The fluid can be stored for 24 hours at +4°C, without any cellular changes. In order to obtain a cellular concentrate, the fluid is submitted to centrifugation, with the disadvantage of displacing cells from their natural connections. This disadvantage can be avoided by the introduction of a fixative in the fluid, such as 50° ethyl alcohol or polyethylene glycol fixing agents.

The smear will be dried immediately, in order to avoid cellular retractions and distortions. Drying can be performed at a not too high temperature. Dried smears can be kept for a certain period without being stained. The fixation of the wet smear by the introduction in a 95° ethyl alcohol bath or by using polyethylene glycol and ethyl alcohol fixatives is indicated for Papanicolaou staining.

Diagnostic laboratories use routine stainings, May-Grunwald-Giemsa as a basic staining, and Papanicolaou staining for fluids that have been preserved. We consider polychrome blue, tannic acid staining, a routine method that has given good results. These stainings can be completed with the PAS reaction and alcian blue staining.

For a positive diagnosis, the cytologist should collect history data, the results of clinical and paraclinical investigations, as well as the presumed clinical diagnosis. The cytologist will evaluate the macroscopic appearance of the fluid (serohemorrhagic or total hemorrhagic, clear, unclear, etc.).

Nucleated cells of various origins and belonging to different cell lines are identified in the fluids obtained. In time, these cells may undergo important morphological changes. The presence of three categories of cells can be found in a smear:

• – cells derived from the serosae, mesothelium, or mesothelial cells;
• – normal local cells or cells migrated from the blood (polynucleated cells, lymphocytes, monocytes, histiocytes, mastocytes and plasmacytes);
• – malignant cells, the most important for a metastatic process and very rarely, for a primary process, except for mesothelioma [3].

Malignant tumors pathologically underlie the development of an effusion in two cases:

1. Invasion of serosae:
   • – either by implantation metastasis (ovarian or mammary carcinoma), desquamation of malignant cells in the effusion fluid, which are frequently massive;
   • – or by contiguity of serous tumors, tumor cells that are distributed in a highly variable number in body cavities.
2. Secondary circulatory or inflammatory disorders, associated with the transudate concerned (bleeding, mesothelial hyperplasia or submesothelial lymphangitis) will cause the absence of malignant cells from the effusion, and cytological diagnosis will be negative [5].

If a positive result confirms the existence of a cancer process, a negative result does not allow its exclusion.

The examination of a smear will take into consideration: the differentiation of some categories of cells, which involves an extremely precise knowledge of morphological features and variability; the assessment of the cells present in the smear, in order to understand the context of the development and/or evolution of lesions; the analysis of elements suspected to be malignant, in this context. Interpretation of the results of these observations in the context of data provided by the clinician.

Mesothelioma is a malignant tumor that is formed by a unistratified pavimentous structure, on a basal membrane, being found on a relatively thick connective-elastic and vascular chorion. In hyperplastic mesothelioma, mesoepithelial cells multiply, becoming globular and desquamated under the form of isolated cells or islands. In the cavity fluid, mesothelial cells change their appearance, becoming macerated. They are capable of multiplying, in which case mitoses are noted.

Basophilic mesothelial cells are the first slightly changed desquamated mesothelial cells. They are frequently found in effusions, isolated or grouped in islands. The shape of these cells is round or oval, outline is clear, the nucleolus is central or slightly eccentric, 15–40 μm in diameter. The cytoplasm is highly basophilic with fine granulations, having microvilli at the periphery, cellular limits are indistinct, forming an acidophilic halo. The nucleus is always dense, and the nucleolus is difficult to detect. Mitoses, binucleated cells and extremely rare polynuclear cells with syncytial appearance can be noted.

Degenerated mesothelial cells result from the transformation of basophilic mesothelial cells when mesothelial proliferation is accompanied by an inflammatory process, which leads to an extremely polymorphic morphological picture. The nucleus has folds or is dented, chromatin is dispersed, and the cytoplasm contains granulations and vacuoles that seem to form true phagosomes. These macrophagic cells can be grouped into three morphological types:

• – basophilic macrophagic mesothelial cells, with a round or reniform nucleus and poorly vacuolized bluish cytoplasm;
• – vacuolized (microvacuolar) macrophagic mesothelial cells, of large sizes, with irregular outline, bluish or gray cytoplasm, containing numerous vacuoles, being poorly differentiated from histiocytes;
• – macrovacuolar signet ring cells, which are large, cytoplasms contain voluminous vacuoles that are optically devoid of content, moving the nucleus towards the cell periphery [1, 7, 8].

Polynuclear cells are present in high numbers in inflammatory effusions. Degenerative images indicate a purulent transformation of the effusion.

Monocytes and macrophages or histiomonocytes may be derived from transformations in effusions of blood monocytes or histiocytes from submesothelial connective spaces. These cells are voluminous, with highly irregular nuclei compared to mesothelial cells, the cytoplasm is bluish-pale, gray, vacuolated, with irregular outline. They may contain phagocytic particles (erythrocytes, degenerated leukocytes, bacteria, pigments, etc.).

Lymphocytes and plasmacytes are present concomitantly with polynuclear cells and macrophages, in inflammatory effusions, tubercular effusions, in lymphomas with nucleo-cytoplasmic anomalies characteristic of malignant transformations.

General cytological criteria of malignancy. The difficulty of a cytological diagnosis of certainty consists in the absence of consistent cytological criteria and the differentiation between the reaction of an irritating process and a malignant lesion. However, some clarifications need to be made.

The general aspect of a cancerous cell population is characterized by:

• – abnormal monomorphism, in contrast with the background polymorphism of the smear, associated with anisocytosis and always anisokaryosis that creates the impression of the same line, but with an anarchic evolution;
• – tendency to form voluminous cell plaques and tridimensional “bullas”;
• – numerous, frequently aberrant mitoses. Cellular characteristics, in progressive evolution, are considered: nuclear abnormalities; size of cells and cytoplasmic changes. Nuclear characteristics are evaluated by: the nucleoplasmic ratio; the high variability in shape; a frequently aberrant configuration; a massive peripheral distribution of chromatin and the presence of prominent nucleoli of variable sizes, sometimes true nuclei in nuclei;
• – global increase in cell diameters, up to giant sizes;
• – cytoplasmic changes, which are less characteristic, but are frequently noted: exaggerated basophilia, vacuolization that can result in the formation of signet ring cells, presence of atypical granulations.

The essential difficulty of this examination consists in the similarity between malignant cells and transformed mesothelial cells.

Some clarifications are required, regarding diagnosis by cytologic examination: positive diagnosis in some cases poses no difficulties; some effusion generating cancers are always negative in the smear; border cases have maximal difficulties.

Lymphoma with mediastinal location is the most frequent and best known, the main morphological changes consisting in large sized cells, nucleus with scant chromatin and basophilic cytoplasm. It should be emphasized that lymphoid cells from effusions undergo relatively rapid degenerative changes, which makes difficult accurate diagnosing.

Myeloid leukemias are quite rarely found with localized forms, myelomas. However, they can be accompanied by effusions in which granulocytic cells are recognized, with malignant elements and numerous eosinophilic polynuclear cells that fill the smear.

The main tumors in carnivores, which can generate effusions, are presented in what follows:

Ovarian and mammary carcinomas can produce abdominal and thoracic effusions. These lesions are characterized by: massive desquamation and plaques or large ballooning cells; vacuolized cytoplasms with ring cell formations, PAS-positive reaction or alcian blue with intracytoplasmic secretion.

Digestive tract and adnexal gland carcinomas can produce abdominal effusions. Abdominal effusions in the case of these tumors contain few characteristic cells, based on which positive diagnosis might be established.

Primitive lung adenocarcinomas are rarer in carnivores, being at the origin of most pleural effusions with massive, cytologically significantly positive desquamation.

Hemangiosarcomas are frequently responsible for sanguinolent effusions with cavitary hemorrhages. Cytological diagnosis is more rarely used, although desquamated tumor cells are found in the blood mass.

Mesotheliomas are little reported and known in carnivores, but they are a concern for cytologists. Mesothelioma and its desquamated cells are found at the border between atypical hyperplasia and the malignant process. This makes difficult to establish a cytological diagnosis of certainty.

Different tumors can produce effusions, having a variable cell content: seminomas, osteosarcomas, thymomas and malignant fibrous histiocytomas.

The cytological examination of an effusion can establish the following situations:

• – negative, absence of abnormal cells;
• – suspect - cells with poorly defined character among normal cells;
• – positive, absence of cells with malignant character [6].

The cytological examination of cavitary effusions is or can be, due to its easiness and rapidity to perform, extremely useful for the clinician.