Chapter 2

Cell Types and Criteria of Malignancy

James H. Meinkoth, Rick L. Cowell and Ron D. Tyler

Examining cytologic preparations often presents a potentially confusing array of different cell types as well as a potentially endless variety of cell debris and contaminants. With experience, most of the common lesions are recognized quickly. In case of a lesion or sample site not previously encountered, it is helpful to keep in mind certain fundamental questions that need to be considered when evaluating a sample. Cells can usually be classified into one of a few basic categories, based on common features shared by different cells in that category.1–4 Recognizing the common features of the different basic cell types sometimes makes it possible to classify cells that are not obvious at first.

An orderly approach to examining slides and answering certain questions in a logical order will reduce the chances of missing important information or misdiagnosing or overdiagnosing the sample.

Are Sufficient Numbers of Well-Stained, Well-Preserved, Intact Cells Present to be Evaluated?

A basic premise of cytology is that interpretations are generally based on entire populations of cells, not on low numbers of individual cells. Any one cell or few cells from a lesion may show features that are atypical or unusual. This is especially true if cells are coming from a tissue in which the cells are not well preserved or exposed to injurious stimuli. Cells coming from inflammatory reactions or areas of tissue repair often show cellular atypia that is the result of dysplasia.

In addition to atypia seen with inflammation and tissue repair, cells that undergo aging changes may be difficult to interpret. Cells present in fluid samples (e.g., thoracocentesis, abdominocentesis) may undergo morphologic changes over time if slides are not made immediately after collection. These changes can range from subtle alterations such as cellular or nuclear swelling and altered staining characteristics to overt pyknosis or lysis. Even if slides are prepared immediately after collection, cells may have undergone in vivo aging. Cells from the fluid portion of cystic lesions (such as some mammary tumors) or cells that have been in prolonged contact with urine (such as urine sediment preparations or urethral or bladder samples collected by traumatic catheterization) often have significant artifacts that must not be misinterpreted as criteria of malignancy.

Interpretations based on inadequately cellular specimens that may not contain a representative sample of the lesion could give a false impression of normalcy or, even worse, may result in a false impression of neoplasia that is not really present. Although no easily defined limit to the question, “How many cells are enough?” exists, slides should have many cells per field across a large portion of the slide. It has been said that if the question “Is the specimen adequately cellular or not?” even comes to mind when looking at the slide, it probably is not.³ When a large lesion is being evaluated, several smears of good cellularity collected from different areas of the lesion should be evaluated to assess any variability that may be present within different parts of the lesion. Large neoplasms may have areas of inflammation or necrosis that will yield markedly different cell populations compared with adjacent areas.

The cellularity of the smear is often evident the moment the slides are stained. A slide containing high numbers of nucleated cells is visibly blue after staining. Slides that are perfectly clear after staining probably have low numbers of nucleated cells, although sufficient cells may still be present for a diagnosis. Therefore, cellularity must be confirmed by looking at the slide under the microscope. Even for practitioners who do not have the time or desire to evaluate cytology preparations themselves, it is beneficial to stain one or two smears and determine that an adequately cellular specimen is being sent off for evaluation.

When examined microscopically, the slides should first be scanned using low power (10× to 20×) to assess the cellularity of the slide and find the area(s) containing the highest number of well-stained, well-spread-out, intact cells for evaluation (Figure 2-1). Cells are often unevenly distributed across the slides, especially with impression smears or aspirates of solid tissue lesions. Even with slides made from fluid samples, large cells may all be pulled out to the feathered edge, where they can be easily overlooked if the whole slide is not scanned first (Figure 2-2).

Figure 2-1 Low-power image of different areas of a slide.
The top image shows an area that although highly cellular, is well spread out and the cells are well stained. Although cell detail is difficult to evaluate at this magnification, it is possible to see the characteristic nuclear shape of some well-spread-out neutrophils and to differentiate the nucleus and cytoplasm of mononuclear cells (arrows). The lower image shows another area from the same slide where the cells are not well spread out and thus have not stained well. Most of the area above and to the right of the red line is too thick to evaluate. A few well-spread-out cells are seen in the lower left (arrows).

Figure 2-2 Images from a slide made from hemorrhagic pleural effusion showing uneven distribution of cells on the slide.
A, Numerous large clusters of cells (arrows) have all been pulled out to the edge of the smear. Some of these show marked atypia allowing for a diagnosis of neoplastic effusion (carcinoma). Inset shows higher magnification of atypical cell cluster indicated by the red arrow. B, The majority of the smear contained predominantly erythrocytes with a few small nuclei (neutrophils and macrophages) visible. Scanning on low power quickly allowed the atypical cells to be found at the edge of the smear. These could have easily been overlooked if the observer started out at high magnification in the body of the smear.

After locating the cellular areas of the slides, it is important to determine whether the cells are sufficiently spread out for evaluation, intact and well stained. This can usually also be done on low power (10× to 20×), which will allow a greater portion of the slide to be evaluated in a short time. Inexperienced cytologists often frustrate themselves by spending an inordinate amount of time trying to identify cells that cannot be interpreted because they are not spread out, are poorly stained, or are ruptured. Intact, well-stained, well-spread cells should have a clearly evident demarcation between the nucleus and the cytoplasm (Figure 2-3). The nucleus may be irregularly shaped (particularly in neoplastic cells), but the nuclear outline should be smooth and distinct. A fuzzy appearance around the outline of the nucleus generally indicates that the cell has been minimally traumatized during sample collection, preparation, or both. More significant cell trauma can result in the nucleus appearing fragmented or full of holes (see Figure 2-3). Severely traumatized cells will often appear as strands of light pink nuclear chromatin (Figure 2-4). Some traumatized or ruptured cells will be present in virtually any cytologic specimen. The sample is usually still interpretable if the majority of the cells are intact; however, the traumatized cells are not evaluated. This is particularly important when determining criteria of malignancy because nuclei may appear enlarged and nucleoli may appear more prominent in traumatized cells. If the majority of the cells are traumatized or ruptured, additional samples usually need to be collected.

Figure 2-3 Lymph node aspirate from a dog showing well-spread-out cells.
A clear distinction can be seen between the nucleus and the cytoplasm (arrowheads). Several traumatized cells are also present. Note that their nuclear chromatin appears excessively fragmented (arrows).

Figure 2-4 Severely traumatized cells.
The streaks of material (arrows) represent smeared-out nuclear chromatin from ruptured cells.

Cells that are not well spread out often stain diffusely dark, and it is difficult to recognize the line of demarcation and color distinction between the cytoplasm and the nucleus (Figure 2-5). Usually, the contrast between the blue cytoplasm and the purple (or pink-purple) nucleus can be seen at relatively low magnification if the cells are well spread out. Poorly spread, poorly stained cells often appear only as different shades of blue. This allows the observer to scan large areas of the slide quickly at low magnification to find areas of the slide worth examining at higher magnification.

Figure 2-5 Poorly spread-out cells from a different area of the same slide shown in Figure 2-3.
The cells are diffusely dark with the nuclei and cytoplasm staining different shades of blue. Compare this with the purple color of the well-spread-out nuclei in Figure 2-3. Also, it is difficult to discern the demarcation between the nucleus and the cytoplasm.

Nucleoli will often be more prominent than usual in understained cells, and this can result in a false impression of malignancy if this artifact is not recognized. The nuclei of well-stained cells are usually dark purple, generally more intensely and deeply stained than the surrounding cytoplasm. Some variation will exist in the intensity and pattern of nuclear staining between cell types. In case of uncertainty as to whether light staining of nuclei is a characteristic of the cell or the result of understaining, it may be helpful to evaluate the staining of more familiar cells. Usually, some neutrophils will be present as the result of peripheral blood contamination or inflammation within the lesion and make a good reference to evaluate how well cells are stained (and spread out).

In most specimens, significant variation in cellularity, degree of cell spreading, and, hence, staining quality from area to area on the slide will exist. This is particularly true of highly cellular specimens such as lymph node aspirates, which may have areas that are of diagnostic quality even if the majority of the slide is thick and understained. Diligent scanning of the slides on low power is necessary to find these areas before attempting to evaluate the cells at higher magnification. If the entire slide is found to be understained, restaining the slide before immersion oil is added may improve the staining quality and result in a diagnostic sample.

Are All of the Cells on the Smear Inflammatory Cells?

A good initial decision, particularly for the beginning cytologist, is to determine if the smear is composed entirely of inflammatory cells. In most general practices, inflammatory lesions are more commonly sampled compared with neoplastic lesions. Also, most clinicians initially feel more comfortable recognizing inflammatory cells because they are more familiar with the morphology of these cells, having viewed them many times in peripheral blood smears. Many clinicians choose to screen their cytology specimens, interpreting inflammatory lesions in-house and submitting those composed of tissue cells to an outside agency. Although inflammatory cells in tissues often look the same as they do in peripheral blood, some may appear different because of morphologic changes induced by their presence in a focus of inflammation or simply because they are not well spread out. It is important to remember that it may not be possible to identify every cell present on a slide, or even on any given field, and that the interpretation is based on the entire cell population present.

If a lesion is found to be composed entirely of inflammatory cells, the relative percentages of the various types of inflammatory cells should be noted because this may provide clues as to the etiology of the inflammation. Finally, a search for infectious agents should be conducted. A discussion of the various inflammatory patterns and morphology of common infectious agents will be covered more completely in Chapters 3 and 5 of this text.

Neutrophils

Neutrophils are commonly found in cytologic specimens. Their morphology is often similar to that observed in peripheral blood smears (Figure 2-6). Normal neutrophil nuclei stain dark purple and contain one to multiple distinct segments or lobes. The neutrophil cytoplasm is typically clear. Neutrophils are phagocytic cells and typically are the cells that phagocytize pathogenic bacteria, if present (see Figure 2-6). Although neutrophils contain intracytoplasmic granules, in most domestic animals, these generally do not stain prominently with cytologic stains. Sometimes, however, these granules will be discernible as elongated, faintly eosinophilic structures, and they must not be confused with bacteria or lightly staining eosinophil granules.

Figure 2-6 Septic neutrophilic inflammation.
Many neutrophils are present, some of which contain phagocytized bacterial rods (arrows).

In thick preparations or in viscous fluids such as synovial fluid, neutrophils may not spread out well, and the segmented nature of their nucleus may be less evident. Sometimes, the nucleus will be essentially round mimicking a lymphocyte, but the cell can still be identified as a neutrophil by the lobulated outline of the nucleus. More normal neutrophil morphology can be observed in the thinner areas (often along the edges) of the smear.

Neutrophils may undergo several morphologic changes in tissues. Aging change is a commonly encountered phenomenon. The initial change seen is hypersegmentation of the nucleus (Figure 2-7). Sometimes, an elongated thin strand of nuclear material (Figure 2-8) connects the nuclear lobes of aged neutrophils. This is commonly seen in cytocentrifuged preparations from fluids. The end result of aging change is pyknosis of the nucleus. Pyknosis is condensation of the nuclear chromatin into one or more small discrete, densely staining spheres lacking any nuclear chromatin pattern (see Figure 2-7). Aging artifact simply represents neutrophils dying of “old age” and must not be confused with degenerative change.

Figure 2-7 Sample from a nonseptic inflammatory lesion.
Many of the neutrophils show nuclear hypersegmentation (arrows), an aging artifact. This will ultimately lead to pyknotic change (arrowheads) in which the nuclear chromatin condenses and fragments into several discrete, dense spheres.

Figure 2-8 Hypersegmentation of neutrophils in which elongated thin filaments connect the nuclear lobes (arrowheads).
This is a common manifestation of aged neutrophils in fluid samples prepared by cytocentrifugation.

Degenerative change occurs when neutrophils are present in an environment that is damaging to the cell. It is commonly seen in neutrophils from lesions in which endotoxin-producing bacteria are present. The presence of many neutrophils with marked degenerative change should prompt a diligent search for bacteria. Degenerative change is an acquired change and is distinct from the toxic changes noted in peripheral blood neutrophils. Degenerative change occurs when the neutrophil is unable to control water homeostasis and undergoes hydropic degeneration. The hallmark of degenerative change is nuclear swelling (Figure 2-9). The nucleus of the cell swells and appears thicker, stains a lighter eosinophilic color, and loses nuclear lobation. Degenerative neutrophils often resemble large band cells.

Figure 2-9 Neutrophils showing degenerative change.
Degenerative change is evidenced by nuclear swelling (black arrows), which leads to loss of distinct segmentation. As the nucleus swells the neutrophils may appear band shaped, or in more severe change, the nucleus may appear round or overtly lytic. Degenerative change is commonly associated with the presence of endotoxin-producing-bacteria. Many bacterial rods (red arrows) were present on this slide.

Macrophages

Macrophages in inflammatory lesions are derived from peripheral blood monocytes. Many tissues have low numbers of fixed tissue macrophages as normal resident cells (e.g., Kupffer cells in the liver). Macrophages may display extremely variable morphology in tissues, which can be somewhat confusing. Initially, they may resemble peripheral blood monocytes (Figure 2-10). With time, the nucleus becomes round and the cell enlarges as the cytoplasm becomes greatly expanded and, sometimes, extremely vacuolated. Macrophages are also phagocytic cells, typically phagocytizing larger structures such as fungal organisms and other cells. Many times, the cytoplasm of macrophages will contain partially phagocytized debris that cannot be identified but must not be misinterpreted as an infectious agent.

Figure 2-10 Pyogranulomatous inflammatory response demonstrating many macrophages.
Some macrophages resemble peripheral blood monocytes (arrowheads). Other macrophages are variably increased in size resulting from increased amounts of cytoplasm that is sometimes vacuolated (arrows).

Binucleate or multinucleate macrophages are commonly encountered in longstanding inflammatory lesions (Figure 2-11). Multinucleated macrophages can get very large and are referred to as inflammatory giant cells. In some chronic inflammatory lesions, epithelioid macrophages may be encountered (Figure 2-12). This term is applied to macrophages that are enlarged with expansive cytoplasm that stains uniformly basophilic, giving the cell the look of an epithelial cell. Because these macrophages can also show variation in size and multinucleation, they could potentially be misinterpreted as neoplastic epithelial cells. Extreme caution should be exercised when diagnosing malignancy in the face of inflammation because of the potential for macrophages to display atypical criteria.

Figure 2-11 Smear from a pyogranulomatous inflammatory reaction (Actinomyces infection).
An extremely large, multinucleated macrophage is present. The macrophage has phagocytized several neutrophils.

Figure 2-12 Impression smear of a nodule on pleural surface of a dog with pyothorax caused by Actinomyces spp. infection.
Numerous large epithelioid macrophages are present. These cells have abundant basophilic cytoplasm that may be nonvacuolated to minimally vacuolated. They may also occur in large aggregates resembling epithelial cell clusters.

Lymphocytes (Small, Medium, and Large)

Small lymphocytes are smaller than neutrophils with rounded nuclei and scant basophilic cytoplasm (Figure 2-13). The nucleus is generally not perfectly round but will have a flattened or indented area on one side. The nuclear chromatin has a smudged appearance. Nucleoli are not visible; however, darker areas (heterochromatin) and lighter areas (euchromatin) are often visible. Generally, the cytoplasm is not visible completely around the circumference of the nucleus but is visible only on one side. Medium-sized lymphocytes may be present and are similar to small lymphocytes, but they have moderately increased amounts of cytoplasm and may have nucleoli visible. Large, blastic lymphocytes (lymphoblasts) (Figure 2-14), commonly encountered in aspirates of lymphoid tissue and lymphoid neoplasms, may be present in low numbers in inflammatory lesions. Lymphoblasts are large cells with enlarged nuclei and dispersed chromatin which stains a lighter pink-purple than that of mature cells (see Figure 2-14). Cytoplasm is more abundant, often visible around the complete circumference of the nucleus, and is typically deeply basophilic. Distinct nucleoli are often visible, and multiple nucleoli may be observed.

Figure 2-13 Smear made from an aspirate of a reactive lymph node.
Many small lymphocytes are present (arrowheads). These cells have scant amounts of cytoplasm that do not appear to encircle the nucleus. Two plasma cells (arrows) are also present. One large, immature lymphoid cell (lymphoblast) is present (red arrow). Note that the lymphoblast and plasma cells are similar in size. However, the lymphoblast has a larger nucleus, whereas the nucleus of the plasma cell is similar in size to a small lymphocyte.

Figure 2-14 Image of a fine-needle aspirate smear of a lymph node from a dog with lymphoma containing numerous lymphoblasts (arrowheads) and lymphocytes (arrows).
The lymphoblasts are larger and have more abundant cytoplasm. Nuclei are large with light staining, dispersed chromatin and often show prominent nucleoli (red arrows).

Reactive lymphocytes are those responding to antigenic stimulation. They have moderately increased amounts of basophilic cytoplasm. Plasma cells are differentiated B-lymphocytes stimulated to produce antibodies. Plasma cells have a round, eccentrically placed nucleus, moderate amounts of deeply basophilic cytoplasm, and usually a distinct clear area located next to the nucleus (see Figure 2-13). This clear area represents the Golgi apparatus and is often located between the nucleus and the greatest volume of cytoplasm. Plasma cells and lymphoblasts are both larger than small lymphocytes, but in plasma cells, most of the increase in size is caused by more abundant cytoplasm, whereas in the lymphoblast, the nucleus has enlarged (see Figure 2-13). Some plasma cells (termed Mott cells) have numerous large clear to basophilic vacuoles (termed Russell bodies) filling their cytoplasm (Figure 2-15). These vacuoles represent retained immunoglobulin.

Figure 2-15 Image from a lymph node aspirate demonstrates small lymphocytes, plasma cells, and one Mott cell with numerous Russell bodies (arrow).
The Russell bodies may range from appearing as clear vacuoles or may be somewhat basophilic as in this image. (Courtesy Dr. Robin Allison, Oklahoma State University.)

Eosinophils

Eosinophils are slightly larger than neutrophils. Their nuclei are segmented but are commonly less lobated than those of neutrophils and often divided into only two distinct lobes (Figure 2-16). Rarely, eosinophils with perfectly round nuclei will be identified in cytologic specimens. The cytoplasm of eosinophils contains prominent orange-to-pink granules. In dogs, eosinophil granules are round and vary widely in size and number (Figure 2-17). Eosinophil granules are numerous, small, and rod shaped in cats (Figure 2-18). The delicate, densely packed granules of feline eosinophils are often less obvious than those of the dog, particularly in thick specimens such as transtracheal washes that may not stain well. Also, neutrophils in exudates will occasionally have mild eosinophilic stippling. Care must be taken not to confuse neutrophils and poorly stained eosinophils when trying to differentiate eosinophilic from neutrophilic inflammatory reactions in cats. The slightly larger and minimal nuclear lobation can help make the distinction. Often, it is easier to identify feline eosinophils that have been traumatized during slide preparation as their granules spread out and become more obvious. If many eosinophils have been ruptured during sample collection (such as with scraping of feline eosinophilic granuloma complex lesions), high numbers of eosinophil granules will be present throughout the background of the smear and may be identified before intact cells are seen.

Figure 2-16 Image from an inflammatory reaction in the intestines of a dog containing a mixture of neutrophils, eosinophils, and macrophages. The nuclei of the eosinophils are typically less lobulated (arrows) than those of the neutrophils (arrowheads).

Figure 2-17 Image from the same slide as Figure 2-16.
In the dog, eosinophils have numerous small granules (arrow) or just a few large granules (arrowhead).

Figure 2-18 Scraping from an eosinophilic granuloma complex lesion in a cat.
Cat eosinophils (arrows) have densely packed granules, often making it difficult to see the individual granules in intact cells. Numerous free granules (black arrowheads) released from ruptured cells are present and demonstrate the slender rod shape typical of feline eosinophil granules. Numerous bacteria (red arrowheads) are also present free in the background.

Occasionally, eosinophil granules will not stain well with Diff-Quik stain (similar to what sometimes occurs with mast cells) yet stain prominently with Wright stain or Wright-Giemsa stain.

If a Smear is Composed of Tissue Cells Rather than Inflammatory Cells, What Type of Cells are Present?

A wide variety of specific cells may be encountered from the various normal tissues and tumors sampled cytologically. With experience, most of these cells can be easily recognized, particularly with the knowledge of what structure is being sampled. However, even if the cells are not immediately recognizable, they can generally be classified into one of three major categories on the basis of certain common cytologic features (Table 2-1):

TABLE 2-1

General Cytologic Characteristics of Different Tissue Cell Types

1. Discrete cells (or round cells)

2. Epithelial cells

3. Mesenchymal cells

Categorization of cells according to the major group they belong to helps the evaluator identify the specific cell type present. Even if precise identification cannot be made, relevant information such as the presence of a cell type abnormal for the tissue sampled (e.g., epithelial cells in a lymph node aspirate) may be gained.

Discrete Cells (Round Cells)

Discrete cells are a group of cells that share certain cytologic features because they are present individually in tissues, not adhered to other cells or a connective tissue matrix. The majority of these cells are of hematogenous origin. Aspirates of normal lymphoid tissue such as the spleen and lymph nodes yield cell populations that have a discrete cell pattern. Other than normal lymphoid tissue, a discrete cell pattern usually indicates the presence of one of a group of tumors termed discrete cell tumors (or round cell tumors). Recognition of discrete cell tumors is important because these are some of the more common neoplasms encountered in small animal practice. Also, cells of most discrete cell tumors have cytologic characteristics that are sufficiently distinct to allow for a specific diagnosis.

General Cytologic Characteristics of Discrete Cell Populations

Because discrete cells are not adhered to other structures within the tissues, they generally exfoliate very readily during fine-needle biopsy (FNB). Hence, the cellularity of the resulting smears is usually very high. In addition, the individual cells are usually evenly spread throughout the smear (Figure 2-19). Cell clusters or aggregates are not present; however, the extremely high cellularity of the smears may result in cells being piled on top of each other in thicker areas of the smears and this may be misinterpreted as cell adhesion (or cell clustering). In the thinner areas of the smears, the cells can be seen to be individually oriented.

Figure 2-19 This slide, made from an aspirate of a transmissible venereal tumor, shows a typical discrete cell pattern. The slide is highly cellular and the cells are evenly spread out throughout the smear. This pattern can usually be recognized from low-power magnification.

The individual cells tend to be of small to medium size and round. If the cells have not been traumatized during sample preparation, they typically have distinct cytoplasmic borders (i.e., the boundary of the cell is well defined).

Specific Discrete Cell Tumors

The discrete cell tumors are mast cell tumor, lymphoma (lymphosarcoma), canine cutaneous histiocytoma, histiocytocytic sarcoma, plasmacytoma, and transmissible venereal tumor. In addition, melanoma is the great imitator, yielding cell populations that may appear discrete, epithelial, or mesenchymal.

Mast Cell Tumor

Mast cell tumors are the only lesions that will yield highly cellular smears consisting entirely or predominantly of mast cells. Mast cells are recognized by their distinctive small, red-purple intracytoplasmic granules (Figure 2-20). The number of granules in mast cells varies tremendously, even within cells from the same tumor (Figure 2-21). Most mast cell tumors yield cells that contain a sufficient number of granules to be easily recognized as mast cells. Sometimes, the cells are so densely packed with granules that the cytoplasm will appear diffusely dark purple and the individual granules difficult or impossible to discern. In this situation, the granules will be evident in cells that have been ruptured. Since mast cell granules have such a high affinity for most cytologic stains, the nucleus of a heavily granulated mast cell may appear pale or even totally unstained, giving the cell the look of a photographic negative with dark cytoplasm and a pale nucleus (Figure 2-22). Some of the components of mast cell granules are chemotactic for eosinophils. The number of eosinophils present in smears from a mast cell tumor varies from very few to many. Occasionally, an aspirate from a mast cell tumor will yield predominantly eosinophils with fewer mast cells (Figure 2-23). In this case, it can be difficult to differentiate a mast cell tumor from a hypersensitivity response. Generally, if there are areas on the slides containing large “sheets” where mast cells are present to the exclusion of other cells, mast cell tumor is most likely.