Kristen R. Friedrichs and Karen M. Young

Sample Collection

Proper collection and preparation techniques are prerequisites to obtaining diagnostic samples of high quality. Supplies necessary for collecting cytologic samples from a variety of tissues, body cavities, and mucosal surfaces are available in most clinics. These include hypodermic needles and syringes, scalpel blades and handles, propylene urinary catheters, bone marrow aspiration needles, cotton swabs, clean glass slides, marking pencils, and collection vials and tubes (tubes with ethylenediaminetetraacetic acid [EDTA] and plain sterile tubes). For aspiration of internal lesions, obtained by guidance with ultrasonography or computed tomography (CT), longer spinal needles and butterfly catheters (used to connect the spinal needle to the aspirating syringe) are useful. Cytologic specimens also can be made from tissues collected during biopsy (see Chapter 9). All supplies should be assembled in one location for ready access. Although life-threatening situations are rarely encountered when collecting cytologic specimens, supplies and medications should be available to control bleeding and to treat anaphylaxis. The latter occurs rarely when aspirating mast cell tumors because of release of histamine.

For external or easily accessible lesions, such as cutaneous and subcutaneous masses or enlarged lymph nodes, aspiration simply requires stabilization of the mass and consideration of underlying structures, such as large vessels and nerves. Some large abdominal masses can be aspirated blindly if they can be stabilized and if they are unlikely to be highly vascular or an abscess, aspiration of which may result in hemorrhage or dissemination of infection, respectively. Aspiration of intrathoracic and intraabdominal lesions is typically accomplished with guidance by imaging, either by ultrasonography or by CT, to aid in targeting the lesion and avoiding large vessels and other sensitive areas. Defects in cortical bone also can be identified with imaging, which can facilitate needle placement for aspiration of bone lesions. Cavity effusions are collected easily without imaging if fluid volume is significant; however, imaging can target smaller accumulations of fluid and provide a measure of safety. If there is particular concern for hemorrhage following aspiration, imaging can be repeated to look for evidence of bleeding at the aspiration site. Collection of cytologic specimens from the eye, brain, and lung requires special consideration and expertise.

Collection Techniques

Fine-needle aspiration (FNA) is by far the most common method for collecting cytologic specimens. Small-gauge needles (22 to 25 g) are sufficient for smaller lesions and result in less hemorrhage. Large-gauge needles (18 to 20 g) may be required to collect sufficient material from masses containing abundant matrix (i.e., firm masses and sarcomas), but specimens may contain more blood. Medium-sized syringes (12 to 15 cc) yield more vacuum for aspiration than smaller syringes (3 to 6 cc). The intent of aspiration is to draw cells into the needle shaft, not to fill the syringe with material unless the lesion is fluid-filled. After the needle is inserted into the lesion, vacuum is maintained in the syringe while the needle is redirected into the tissue several times to collect a broad representation of cells. This is especially important when aspirating lymph nodes to search for metastasis. Following aspiration, vacuum is released prior to removing the needle from the tissue, the needle is removed from the tissue and then from the syringe, the syringe is filled with air and reattached to the needle, and the cells are expelled onto a glass slide. An alternative technique is to obtain cells without aspiration by holding the needle by the hub between the thumb and middle finger while covering the hub opening with the forefinger (to prevent blood or other fluids from escaping) and rapidly and repeatedly inserting the needle into the lesion with redirection until cells are packed into the needle shaft.¹ This method often yields as much cellular material as the aspiration technique and produces less hemorrhage and patient discomfort. Similar to the aspiration technique, a syringe is used to expel the material in the needle onto a glass slide. A second clean slide is then placed on top of the sample and the two slides are pulled apart in parallel, taking care not to exert pressure on the sample. The aim is to obtain a monolayer of intact cells. Failure to spread the specimen immediately leads to a sample that is too thick to interpret; conversely, aggressive pressure on the sample may rupture many if not all cells, also leading to a nondiagnostic specimen.

Cytologic material may be collected from mucosal surfaces such as the respiratory, gastrointestinal, and genital tracts by saline washes or with a brush or biopsy forceps inserted through an endoscope. Cytologic materials collected using an endoscopic brush are gently rolled onto a glass slide and often result in highly cellular smears. In contrast, rolling a cotton swab over the surface of a lesion is only moderately successful at collecting sufficient material for cytologic evaluation of tumors. Traumatic catheterization is the best method for collecting material from bladder masses because of the risk of seeding tumor cells when transitional cell carcinomas (TCCs) are aspirated transabdominally. Traumatic catheterization is accomplished with an open-ended polypropylene urinary catheter attached to a large (50 to 60 cc) syringe. The catheter is inserted into the urethra and bounced off the bladder wall in the region of the lesion (typically using ultrasound guidance), taking care not to perforate the bladder wall. Saline can be flushed into the bladder to facilitate collection of cells and cellular particles, some of which may be large enough to process for histologic evaluation.

Imprinting and scraping are excellent means of preparing cytologic specimens from biopsied tissues. When making imprints, a fresh surface should be exposed on the piece of tissue using a scalpel blade and then gently blotted on absorbent paper until little blood or tissue fluid appears on the paper. The tissue is held with forceps, and the fresh surface is gently pressed repeatedly onto the glass slide, using slightly different pressure with each imprint. The final specimen will contain a row of imprints of varying thickness, one or more of which should be suitable for evaluation. Common mistakes when preparing imprints include insufficient blotting and application of too much pressure, resulting in excessive blood or cellular disruption, respectively. Sometimes mucosal or connective tissue is obtained instead of tumor cells if the incorrect surface is imprinted onto the slide. When tumors such as sarcomas contain abundant matrix, imprinting will often not yield sufficient numbers of cells for evaluation. The surface of these lesions should be cross-hatched with a scalpel blade and imprinted without blotting; this may liberate cells embedded in matrix. Alternatively, the surface of firm lesions can be scraped several times in one direction with a scalpel blade held at 45 degrees to the tissue. The material on the edge of the blade is then gently spread on a glass slide. When using biopsy tissue to prepare cytologic specimens, care must be taken not to disrupt surfaces or margins important for histologic evaluation, especially for excisional biopsies in which assessment of tumor margins is fundamental to the evaluation.

Tissue particles or mucus collected by saline washes or by traumatic catheterization can be retrieved with a pipette and gently pressed between two glass slides. If washes or cavity fluids are cell-poor, the cells in the fluid must be concentrated to prepare slides of sufficient cellularity. Collection fluid can be centrifuged, the supernatant decanted, and the cell pellet or sediment resuspended in a small amount of remaining fluid and then spread on a glass slide. Similar to preparation of blood smears, the feathered edge of the fluid should be included on the slide because nucleated cells will accumulate there and may be best evaluated at the edge. Alternatively, when spreading the suspended cell pellet fluid on a glass slide, the spreader slide can be abruptly lifted off the slide, leaving a line of fluid—and concentrated cells—on the slide instead of a feathered edge. The best method to concentrate cells in cell-poor fluid samples is to use a cytocentrifuge, but most practices lack this equipment.

Cytologic Stains

A variety of quick stains are available for immediate examination of cytologic specimens and include quick Romanowsky stains, such as Diff-Quik. A specific set of staining jars should be kept exclusively for cytologic specimens and not used concurrently for dermatologic specimens. The jars containing the stain components should be capped between uses to prevent evaporation and contamination of the fixative and stains. Maintenance, including scheduled replacement of stain components, is important to avoid artifacts such as stain precipitate and contamination with organisms or debris that might be misinterpreted. Slides should be completely air-dried prior to fixation in the methanol fixative. Stains must thoroughly penetrate the smear, and in well-stained smears nuclei should be purple (Figure 7-1, A). A thick sample requires more contact time with the stains; understained slides (Figure 7-1, B) can be restained for a longer period of time, and overstained slides can be destained with methanol and restained for a shorter period of time. If the slides will be sent to an outside diagnostic laboratory, clinicians are encouraged to stain a slide to ensure that sufficient material was collected and that cells are intact prior to submitting additional unstained slides for evaluation. Additional specimens should be collected if only noncellular material is present or if all the cells are lysed. For some lesions, the first slide prepared may be the only slide that contains cellular material. It is best to send this slide unstained to the diagnostic laboratory, but, if it is stained, be sure to include it with the other slides.

Quick Romanowsky stains provide good nuclear detail and usually sufficient cytoplasmic detail for cytologic interpretation. Mast cell granules occasionally fail to stain with aqueous quick stains (Figure 7-2, A). Wright-Giemsa and modified Wright stains provide a broader palette of colors and excellent staining of cytoplasmic granules (Figure 7-2, B) but require more steps and longer staining times or use of an automated stainer. Fixation of wet smears is required for Papanicolaou staining, which is not frequently used in veterinary cytology. Heat fixation is not required or recommended for cytologic specimens.

Cytochemical and immunocytochemical staining may be necessary to determine the specific tumor type. A complete list of available special stains and antibodies is beyond the scope of this chapter; consultation with a veterinary cytopathologist is recommended when considering the necessity and use of these stains. Cytochemical stains identify specific chemical compounds or structures within the cytoplasm or nucleus and include stains such as Prussian blue for iron, periodic acid-Schiff (PAS) for carbohydrates, alkaline phosphatase for identifying osteoblasts,² and a wide variety of leukocyte markers, including Sudan black B, peroxidase, chloracetate esterase, and nonspecific esterases. Immunocytochemical staining procedures utilize antibodies to identify specific proteins or peptides within or on the surface of the cells. Common antibodies used in veterinary oncology include those directed against CD3 (T-cells), CD79a and CD20 (B-cells), cytokeratin (epithelial cells), vimentin (mesenchymal cells), and Melan A (melanocytes). Use of a single stain or antibody is discouraged, as cell lineage is rarely identified and aberrant expression by neoplastic cells may lead to an erroneous diagnosis if a single marker is evaluated; a panel of stains or antibodies usually is necessary for complete identification.

Examination and Description of Cytologic Specimens

A good microscope, ideally equipped with a digital camera to document cytologic findings for the medical record or for consultation, should be used for examining cytologic specimens. The 4×, 10×, and 20× objectives are useful for scanning the slide and assessing cellular arrangements and general cell shape, whereas the 40× (“high dry”) and 50× or 100× (oil-immersion) objectives are required for examining cellular detail. To improve clarity the 40× objective requires an additional optical interface, which can be provided by applying a drop of immersion oil or permanent mounting medium to the slide followed by a coverslip. As a note of warning, the 40× objective lens is easily coated with oil applied to the slide for viewing the specimen with oil-immersion objectives; if this occurs, the lens should be cleaned immediately with glass cleaner and lens paper to prevent accumulation of oil inside the objective lens. Proper use, including correct placement of the condenser for viewing stained and unstained specimens, and maintenance of the microscope are essential to adequate examination of cytologic specimens.

Consider the following when examining the slide preparation: (1) Is the specimen of sufficient quality to permit a clinically useful interpretation? Clinical decisions should not be made from specimens that are poorly cellular or have too many ruptured cells. (2) Based on the tissue sampled, do the cells represent the expected population, an abnormal population, or both? It is important to become familiar with the cytologic appearance of “normal” cells in frequently aspirated tissues, such as lymph node and liver. (3) Does the abnormal population represent inflammation or a neoplasm? Whenever inflammation is found in a lesion suspected to be a tumor, caution is advised in making a definitive diagnosis of neoplasia. Although some tumors are accompanied by neutrophilic inflammation, experienced cytopathologists recognize that primary inflammatory lesions can convincingly mimic neoplastic lesions. (4) If neoplasia is likely, what is the tissue of origin and is the tumor benign or malignant? These questions can sometimes be answered by cytologic evaluation of the tumor but often require confirmation with histologic examination.

Specimens of Diagnostic Quality

What constitutes adequate cellularity depends on the type of tumor. Aspirates of mesenchymal tumors, which often contain extracellular matrix, tend to be less cellular than those of epithelial and discrete round cell tumors. The degree of cellularity also has an impact on the level of confidence expressed in the interpretation, and diagnostic opinions are often qualified with “possible” or “probable” for poorly cellular specimens compared with “diagnostic for” or “consistent with” for highly cellular specimens. All cytologic specimens contain some ruptured cells, but to render a meaningful interpretation the majority of cells should be intact. Material from ruptured cells is recognized as stringy strands of chromatin or swollen magenta nuclei, often with obvious nucleoli, and free cytoplasmic fragments (see Figure 7-1, A). Large lymphocytes and cells from endocrine tumors are highly susceptible to lysis, and extra care should be taken not to exert pressure on the cells when preparing cytologic specimens from these lesions.

Nonneoplastic Cells and Noncellular Material Found in Cytologic Specimens

The submandibular salivary gland occasionally is aspirated instead of the mandibular lymph node and is recognized by clusters of foamy cells in a background of mucin and blood. When tissue containing a metastatic tumor is aspirated, the specimen may contain only neoplastic cells or may contain normal cells from the tissue (e.g., lymphoid populations in a lymph node), which helps confirm location of the tumor. Necrosis can be found in tumors that have outgrown their blood supply. Necrotic cells lack detail and consist of gray-pink, indistinct cytoplasm and amorphous nuclei (Figure 7-3); they should not be confused with apoptotic or pyknotic cells, which retain distinct cytoplasmic borders that surround condensed nuclear fragments.

Aspiration usually results in some degree of sampling hemorrhage leading to the presence of few or many erythrocytes admixed with nucleated cells. Aspiration of splenic lesions and thyroid and vascular tumors may result in pronounced hemorrhage and abundant blood in the cytologic specimen. Preexisting intralesional hemorrhage is indicated by the presence of macrophages containing erythrocytes or hemosiderin. Small numbers of peripheral leukocytes, primarily neutrophils, will accompany hemorrhage, but the presence of neutrophils in numbers greater than their proportion in blood is supportive of inflammation. Neutrophilic inflammation may accompany tumors, most notably squamous cell carcinoma and large tumors with necrotic centers; however, inflammation can induce criteria of malignancy in nonneoplastic populations, especially fibroblasts and squamous cells, and biopsy may be required to confirm suspected neoplasia when inflammation is prominent. Some tumors are associated with infiltration of specific inflammatory cells (e.g., eosinophils in mast cell tumors).

For tumors that produce ground substance(s), such as sarcomas, or that elicit a scirrhous response, such as some carcinomas, extracellular matrix may be observed in cytologic specimens. Collagen and osteoid consist of collections of smooth or fibrillar magenta material, whereas chondroid matrix typically forms larger lakes of bright pink-to-purple material. Mucin may be secreted by a variety of tumors, including salivary, biliary, and intestinal carcinomas and synovial and myxomatous sarcomas. Mucin is pale blue to pink, and cells surrounded by mucin are often aligned in rows. Ultrasound gel may be a contaminant of slides prepared from ultrasound-guided aspirates if the needle is not cleaned prior to expelling cells onto the slide. Ultrasound gel appears as granular, bright magenta material when stained with cytologic stains and, if abundant, may impair cytologic examination.

Description of Neoplastic Populations

Determination of the number of cells exfoliated and the shape and arrangement of cells early in the cytologic evaluation aids in formulating an initial list of differential diagnoses, permitting placement of tumors in three broad categories: epithelial, mesenchymal, and discrete round cell tumors. Briefly, cells from epithelial tumors exfoliate well and are round, cuboidal, columnar, or polygonal cells arranged in cohesive sheets or clusters; cells from mesenchymal tumors exfoliate poorly and are spindle-shaped, stellate, or oval cells arranged individually or in noncohesive aggregates; and cells from discrete round cell tumors exfoliate well and are individualized round cells that are arranged in a monolayer. Cellular arrangements observed in cytologic specimens and their associated histologic correlates and tissue types have been described.³

Proper terminology should be used to succinctly describe cell populations and convey important information. The terms homogeneous and heterogeneous describe cell populations (Figures 7-4 and 7-5). Homogeneous denotes a population of one cell type (excluding erythrocytes and associated leukocytes), which is typical of most tumors. Heterogeneous refers to mixed populations of cells, which are commonly found in aspirates of inflammatory lesions; however, some neoplasms will contain heterogeneous populations of cells (e.g., mast cell tumors accompanied by eosinophils and fibroblasts [see Figure 7-5] and squamous cell carcinomas with associated neutrophilic inflammation [see later]). The terms monomorphic and pleomorphic describe the morphologic appearance of cells within a single population. Monomorphic describes cells of a single lineage in which the cells have a uniform morphologic appearance (see Figure 7-4, A). Monomorphic features typically are associated with benign tumors, but a number of malignant tumors are cytologically monomorphic. In contrast, pleomorphic is used to describe cells of a single lineage that have variable morphologic features (see Figure 7-4, B). Pleomorphic features comprise a set of criteria of malignancy and suggest malignant behavior, but can be observed in nonneoplastic cells found in primary inflammatory lesions.

Criteria of malignancy are cellular features within a single population that suggest malignant behavior, with greater emphasis placed on nuclear criteria. The more criteria observed, the more likely the tumor is malignant. Cellular and cytoplasmic criteria of malignancy include variation in cell size (anisocytosis), abnormal cellular arrangement (3-dimensional [3D] clusters instead of a monolayer), cells that are smaller or larger than their normal counterpart, variable nuclear-to-cytoplasmic (N : C) ratios or N : C ratios that differ from what is expected for the cell type, intensely basophilic cytoplasm (hyperchromasia), abnormal vacuolation or granulation, and aberrant phagocytic activity. The nucleus is the most important component of the cell when determining the biologic behavior of a neoplasm. Nuclear criteria of malignancy include variation in nuclear size (anisokaryosis), unusual nuclear shape, multinuclearity, variation in nuclear size within the same multinucleated cell, nuclear fragments, multiple nucleoli that vary in size and shape within the same nucleus or among cells, increased mitoses, and nonsymmetric mitoses (Figure 7-6). When Papanicolaou stain is used, additional nuclear features such as irregular thickening of the nuclear membrane can be evaluated. Cellular gigantism (cell >10 times the diameter of an erythrocyte) and the presence of macronuclei (>5 times the diameter of an erythrocyte) or macronucleoli (larger than an erythrocyte) are particularly disturbing criteria of malignancy. In nonneoplastic cells the chromatin pattern is finely stippled in replicating or metabolically active cells and condensed in mature quiescent cells. Finely stippled chromatin is also common in rapidly proliferating neoplastic cells, and chromatin that is irregularly clumped or ropy is unusual and suggestive of a neoplastic process. Some nonneoplastic cells, including mesothelial cells, fibroblasts, and squamous epithelial cells, may have criteria of malignancy when they are highly proliferative in the presence of inflammation. Conversely, some malignant tumors such as apocrine gland tumors of the anal sac have few criteria of malignancy.

Sending Cytologic Samples to a Diagnostic Laboratory

When using a referral diagnostic laboratory, two to four unstained smears should be sent. If a highly cellular smear has been stained and examined by the oncologist for confirmation of sample quality and the cellularity of the remaining unstained smears is in question, send the stained smear in addition to the unstained smears. Pack all slides in rigid slide containers to prevent breakage during shipment. For shipment by commercial mail services, place slide holders in a cardboard box with sufficient padding; padded envelopes are not recommended because these may not provide sufficient protection. Slides should not be refrigerated prior to or during shipment. Exposure to formalin or formalin fumes should be avoided during the preparation and shipment of cytologic specimens because this will permanently alter staining characteristics and render the sample nondiagnostic; biopsy specimens preserved in formalin should be sent separately from cytologic specimens, or each type of sample should be sealed in separate plastic bags. If cavity fluids or mucosal washes are submitted, include two freshly made unstained smears along with the fluid (in EDTA) or wash (sealed container). Plain tubes (red top) or sterile vials are required for specimens that may be cultured. For all submitted glass slides, indicate how the slides were prepared and whether a concentration method was used for cavity effusions.

Interpretation of Cytologic Specimens

The final interpretation of a cytologic specimen should be based not only on the cytologic findings but also on signalment, history, clinicopathologic findings, and imaging results. This information should be provided in a concise but complete summary to the individual evaluating the sample. When submitting samples to a cytopathologist, the exact location of the lesion should be clearly described because “thoracic mass” could indicate a mass located in the skin, subcutis, body wall, mediastinum, thoracic cavity, or pulmonary parenchyma; the differential diagnoses will be different for different locations. For clinicians who perform an initial evaluation of the cytologic specimen, observational and interpretative skills can be developed by comparing your findings with those described in the cytopathologist’s complete report and by considering the information obtained from other diagnostic tests.

Confidence in cytologic interpretation is based on the quality of the specimen, the completeness of the clinical information provided, and the experience of the cytopathologist. Terms that express the degree of certainty, such as “consistent with,” “diagnostic for,” “cannot rule out,” “probable,” and “possible,” may be used and interpreted differently by cytopathologists and clinicians, respectively.⁴ If the certainty of an interpretation or diagnosis is unclear, the clinician should consult the cytopathologist. Correlations between cytologic and histologic interpretations or diagnoses are highly variable, depending on tissue types, disease processes, and methods of collection and preparation.

Epithelial, Mesenchymal, and Discrete Round Cell Tumors

The ability to identify specific tumor types by cytologic evaluation can aid in treatment planning and prognostication. Even if a specific diagnosis cannot be made, classification of the tumor as an epithelial, mesenchymal, or discrete round cell neoplasm can provide sufficient information to formulate a differential diagnosis and plan additional diagnostic procedures.

Tumors of Epithelial Tissues

Tumors derived from epithelial tissue comprise the largest category of neoplasms and include tumors of epithelial surfaces, such as the skin and respiratory, gastrointestinal, and urogenital tracts, as well as tumors of glands and organs. Given their diverse origin, the cytomorphologic appearance of these neoplasms can be highly variable; however, some features are shared by most epithelial tumors. Epithelial cells have intercellular junctions that connect the cells to each other and do not elaborate extracellular matrix. Therefore the cells exfoliate well, resulting in highly cellular specimens, and are arranged in cohesive sheets or clusters in cytologic smears (Figure 7-7). The cytoplasmic borders of individual cells typically are distinct, but this can vary in certain types of tumors. Poorly differentiated epithelial tumors have few or no identifying features and tend to be round cells with moderate-to-high N : C ratios and basophilic cytoplasm. In some cases, the cells no longer have intercellular junctions and appear as discrete round cells (Figure 7-8). Determining the tissue of origin in these cases is difficult, and histologic evaluation, with or without immunohistochemical analysis, is necessary to define the specific tumor type.

Figure 7-7 Fine-needle aspirate of a basal cell tumor (epithelioma). Note the monomorphic population of cohesive cells aligned in rows.

Figure 7-8 Fine-needle aspirate of an anaplastic colonic carcinoma. The tumor cells have high N : C ratios and are sometimes individualized.

Tumors of Hair Follicles and Sebaceous Glands

Differentiating among adnexal tumors of skin by cytologic evaluation may be difficult when identifying features are absent or when multiple cell types are present. Many adnexal tumors have a large component of basilar cells that are small cuboidal or round cells with high N : C ratios and are arranged in tightly cohesive sheets or in palisading rows (see Figure 7-7). Nuclei are uniformly round with condensed to reticular chromatin, and nucleoli are indistinct or appear as a small single nucleolus. The cytoplasm is lightly basophilic and may contain black melanin granules (Figure 7-9). Tumors of hair follicle origin and matrical cysts often have a central cystic space filled with mature squamous cells, keratin flakes, or keratin debris, and this material may be aspirated when the mass is sampled. Tumors with sebaceous differentiation contain clusters of large round cells filled with oily-appearing vacuoles that partially obscure the small central nucleus (Figure 7-10). Tumors of basal cell origin (cutaneous basilar epithelial neoplasms) include trichoblastoma, pilomatricoma, basal cell epithelioma, sebaceous epithelioma, and others, and histologic examination is usually required to identify the specific type. Fortunately, the majority of adnexal tumors are benign; malignant types can occur and typically have pleomorphic features that predict their biologic behavior.

Figure 7-9 Fine-needle aspirate of a basal cell tumor in which the cells are heavily pigmented.

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