Cytology for the practitioner (Proceedings)

Cytology is a critical tool not only for the oncologist, but also for veterinarians in general practice. Cytology is easy, safe, fast, relatively inexpensive, and is often diagnostic. Cytology can be used to evaluate lymph nodes and masses (external or internal), and also fluids such as effusions and discharges. Results may change further diagnostic plans, treatment recommendations, or what a client chooses to do. For example, studies comparing evaluation of lymph nodes for metastatic disease via physical examination (PE) versus cytology showed 20-40% of patients felt to have normal or slightly enlarged lymph nodes on PE had metastatic disease found with cytology. Conversely, only 50-60% of patients with moderately to severely enlarged lymph nodes on PE had metastatic disease. Thus, PE only of lymph nodes has poor sensitivity and specificity for metastatic evaluation. Cytology is of great benefit in these cases.

Any lump or bump should be aspirated!! Mast cell tumors, for example, can feel just like lipomas. We routinely "map" all of our patients. This procedure includes measuring and aspirating all masses. The masses are numbered on a body chart, and the fine needle aspiration results are recorded. Once a mapping has been accomplished, it stays in the animal's chart and can be referenced if an owner thinks a mass is growing or has identified a new mass. Thus, veterinarians unfamiliar with the case will not need to go through testing that has already been performed.

Sample collection and preparation for cytology is simple and requires little equipment. Needles (25, 22, 20, or 19 gauge), 6 cc syringes, clean glass slides, and occasionally a scalpel blade for scraping are all that is needed. In vivo collection technique can be performed with a needle alone or with a syringe attached to the needle. Before obtaining the sample, make sure the slides are set out (at least 4-6) and the syringe is ready. Stabilize the mass in your non-dominant hand. For the needle alone technique, direct the needle into the mass; withdraw (yet stay within skin) and push back into the mass several times rapidly. Draw several cc air into the syringe, connect it to needle and expel needle contents onto a slide (make sure the bevel is down and the contents come out near the frosted end). For the needle plus syringe procedure, connect the needle and syringe, direct the needle into the mass, draw back plunger and release several times, with pressure released withdraw the needle from mass, disconnect the needle and draw several cc air into syringe, reconnect and expel needle contents onto the slide.

Slide preparation is key for good cytology results. The samples must be smeared out quickly to prevent clotting and need to be in a thin layer for best cellular evaluation. Even if only a small dot of sample is expelled onto the slide, smearing it is important. Cells that dry in a little round "puddle" of fluid maintain more of a 3-dimensional appearance, do not spread out their cytoplasm, and are difficult to assess. After the contents are expelled onto a slide, place a second slide onto the first cross-wise to the first slide) and pick up a SMALL amount of material. Pick up a third slide, gently place the second slide across the third slide to squash the sample flat and smear the sample by pulling the second slide away from the frosted edge of the third slide while maintaining slide contact with gentle pressure. The second slide needs to stay FLAT on the third slide (no edge to catch cells and drag them). Now, the second slide can be used again to pick up another small bit of sample from slide 1, and can be smeared onto a 4th slide, etc. When just a small amount of sample remains on slide 1, use slide 2 to smear it as well. Most aspirates will yield 3-6 good slides, allowing for in-house staining and evaluation of one slide while leaving several unstained for laboratory evaluation. Also, by making multiple smears, the samples are more thinly spread out which improves diagnostic ability. This is particularly helpful with very cellular aspirates.

Cytology can also be of benefit even when a sample is being obtained and sent for histopathology. In some tumor types, particularly round cell tumors, cytology can provide a more precise diagnosis that histopathology. Thus, one may want to collect a cytologic sample in vitro, or after tissue has been removed from the patient. This can be done via impression smears or scrapes. Impression smears are best for round cell or epithelial tissues – tissues that exfoliate more easily. Without disturbing surgical margins, use a blade to cut a fresh section of tissue (maximum 1x1 cm). Gently blot the cut face on gauze or paper to rid it of excess moisture and blood. Press the blotted area onto the slide. Make several rows of impressions. If cellular yield is low with this approach, the scrape technique can be used. This may be best for mesenchymal tissues. Begin in the same manner as with the impression technique, but after blotting the sample, use the blade to scrape cells from the cut surface. Gently smear the collected cells from the blade across the slide in one direction.

Preparation and staining of the slides is as follows. Air dry the slides. Standard hematologic stains are preferred for most purposes (Wright's, Wright's-Giemsa, Diff Quick, etc.) If submitting slides to an outside laboratory, submit them air-dried and unfixed, or fixed and stained. Do not submit them fixed and unstained or cell quality will diminish. Do not have cytology slides near formalin. Even the fumes can distort cytologic quality. Smears should be made and kept away from biopsy formalin jars.

Once the slide is stained it is ready for microscopic examination. Scan the slide on low power (10x) first to get an overall feel for the cellularity of the sample and then go to higher power to evaluate the cell types and individual cytologic features of the cells. The two most important questions as a practitioner to answer when looking at a slide are 1. Did I hit the intended tissue? (salivary gland vs. lymph node, blood vs. bone marrow) and 2. Is it a good sample? (cells not all ruptured, enough cells for evaluation). After assessing these points, evaluate if the cells are from normal tissue. Note if inflammation (see cell types below) is present. If so, caution must be used in interpreting other cells on the slide. Inflammation can induce cells to look "malignant" when they are actually just reactive. Next ask if there are cells in abnormal places (non-lymphoid cells in lymph node, non-hepatic cells in liver). Finally, note the predominant cell type (as follows).

Inflammatory processes usually have a mixed population of cells including inflammatory and tissue cells. The inflammatory process is characterized by the cell types present. When neutrophils predominate (>85%), this is considered a suppurative response. The character of the neutrophils should be closely evaluated. If there is a bacterial infection, the neutrophils will often undergo karyolysis and the nucleus will appear pale and swollen. These cells are often called degenerative neutrophils. Also the cells should be closely examined for intracytoplasmic microorganisms. A pyogranulomatous response consists of a mixture of neutrophils, epithelioid macrophages and multinucleated giant cells. This type of inflammatory response is commonly observed with fungal infections or responses to foreign material. Granulomatous inflammation consists primarily of macrophages with multinucleated giant cells and lesser numbers of other inflammatory cells. This type of inflammatory response does not always exfoliate well and may be of low cellularity. Eosinophilic inflammation is more subtle. Cytologic specimens containing greater than 12% eosinophils are considered eosinophilic inflammatory lesions. Common causes include allergic or hypersensitivity response, parasitic infestation or paraneoplastic response.

Tissue cells are classified as epithelial, mesenchymal (connective tissue) or round cells

Epithelial

Epithelial/glandular cells are found in the linings of tracts (respiratory, digestive/oral, urogenital), in skin and mucous membranes, and in glands (pancreas, thyroid, adrenal, prostate, mammary, etc.). Benign epithelial lesions are adenomas, while malignant versions are carcinomas. The cells are generally round and the sample is often highly cellular. The major defining feature of this cell population is clustering, or clumping, of cells. This feature can be observed on low power evaluation. Note that any very cellular aspirate can give the appearance that the cells of the sample are clustered together, even with mesenchymal masses. Assess less cellular regions to ascertain if in these areas cells are still bound together with other cells or if they do appear individually, as mesenchymal cells will. Round cells tend to be pressed together, as they exfoliate very well and thus provide cellular samples, but they do not tightly adhere to each other or swirl together to form 3-dimensional structures. Small spaces can often be seen between non-epithelial cells.

Neuroendocrine tissues such as thyroid and adrenal organs, while in the epithelial category, do have a few distinctive features. These cells tend to rupture very easily. Thus, samples from these tissues may have fields of naked nuclei in a smooth bluish background representative of the lost cytoplasm. Additionally, while most neuroendocrine tumors are malignant (thyroid carcinomas, insulinomas, etc), the cytologic appearance of these tumors is often very uniform with few features of malignancy. In these cases, knowing the site of origin of the mass and the fact that the tissue appears neuroendocrine is more important for predicting biologic behavior than are the malignant features of the cells.

Mesenchymal

Mesenchymal cells are found in connective tissues such as cartilage, bone, smooth and striated muscle, blood vessels, fibroblasts, etc. When benign, the masses are categorized as the cell of origin and then "oma" – e.g. fibroma. When malignant, the masses are termed sarcomas. Mesenchymal cells may not exfoliate as well as epithelial tissue; however, most mesenchymal masses will provide enough cells for cytologic evaluation, and some exfoliate very well. In benign lesions, it is not uncommon to see only 1-2 cells on a slide. The cells are commonly spindle shaped with wispy cytoplasmic borders. The nuclei are often oval in shape. Their cell shape, indistinct cytoplasmic borders, and the fact that they occur as single cells in the preparations characterize mesenchymal cells. At times mesenchymal masses have a round cell appearance, most notably with osteosarcoma. Malignant osteoblasts are notable for their "plasmacytoid" appearance in some cases.

Round cell

Round cells are called such based on their cytologic appearance: discrete, round cells with distinct cytoplasmic margins. This category includes lymphoid tissue, plasma cells, mast cells, histiocytomas, transmissible venereal tumors (TVT), and melanoma (which can have a round or mesenchymal appearance). Often the specific cell type can be cytologically determined in round cell tumors, unlike in epithelial or mesenchymal masses. The round cells will have round to bean-shaped nuclei, lacy to smudged chromatin and are generally small to medium sized cells. Individual features of the listed round cell types can help to provide a diagnosis. Lymphoblasts have a high nuclear:cytoplasmic (N:C) ratio, are as large or larger than neutrophils, have basophilic cytoplasm and open chromatin, and often have multiple nucleoli of varying sizes. Plasma cells have round, eccentrically located nuclei with ropy or clumped chromatin. ?The cytoplasm is more abundant than in lymphocytes and has a dark blue/grey hue. A perinuclear clear zone is often apparent. The most notable feature of mast cells is the presence of pink/purple-staining cytoplasmic granules. The granules may be scant or so abundant that the cell nuclei are completely obscured. Often granules litter the background of the slide and may be mistaken for cocci. Mast cells are large round cells with a centrally located round nucleus ("fried egg" appearance). Mast cells secrete chemoattractant factors for eosinophils and occasionally fibroblasts. Some mast cell populations have granules which fail to take up Diff Quick stains; these granules will often stain with other stains. If agranular cells with a "fried egg" appearance and eosinophils are noted on a mass aspirate, unstained slides should be sent to a laboratory that will use a Wright's stain to assess for granules. Histiocytomas are large cells with a moderate amount of blue-grey cytoplasm with clearing at the periphery of the cytoplasm. The cells can show extreme pleomorphism despite the tumor's benign behavior. Histiocytomas can be difficult to distinguish from some plasma cell tumors cytologically. The clinical appearance of the lesion is supportive of the diagnosis in these situations. TVTs also have a large amount of cytoplasm, usually grey-blue to blue in color, and can be quite pleomorphic. The defining feature of the TVT (other than the clinical presentation!) are unevenly distributed vacuoles in the cytoplasm. Melanoma can have a round cell appearance – assess for fine black/blue/green cytoplasmic granules. Amelanotic melanoma can be difficult to diagnose, yet even amelanotic masses will often have a few cells with pigment. Malignant melanomas are highly pleomorphic tumors with large cells that contain prominent and bizarre nucleoli.

Features of malignancy?

Once the cell type has been characterized, the next step is to determine if the process is hyperplastic or neoplastic and if it is neoplastic, is it benign or malignant. It is important to evaluate for features of malignancy in epithelial or mesenchymal cells. The biologic behavior of the round cell tumors is based on cell type and site of the mass rather than specific cytologic features. Malignant criteria include: prominent and multiple nucleoli, anisocytosis, anisokaryosis, multiple nuclei, mitotic figures and nuclear molding. There are others, but these are the most common. When these criteria are present it is fairly easy to identify a malignant tumor; however, not all malignancies express these criteria. Therefore, cytology cannot always rule out a malignant process. Histopathology may be needed for diagnosis of malignancy. For example, soft tissue sarcomas are locally very aggressive yet may have a benign cytologic appearance. If 3 features of malignancy are present, then the lesion is most likely malignant. If malignant features are lacking, the lesion may still have a malignant behavior and a biopsy is indicated.

Background

The final step in cytologic evaluation is examination of the material in the background. Proteinaceous material is eosinophilic and granular and is usually observed evenly distributed throughout the sample. Granules from ruptured mast cells or melanocytes must be distinguished from bacteria. Most cytologic samples contain some level of peripheral blood contamination. With the red blood cells come white blood cells. It is important to remember this when trying to distinguish inflammation from peripheral blood contamination. It may be necessary to do a CBC on the patient to determine the WBC in situations where increased numbers of WBC are noted on a cytology slide.