Cytology and Biopsies for the Practitioner: Part 1
WSAVA 2002 Congress
C. Guillermo Couto, DVM, ACVIM
College of Veterinary Medicine, The Ohio State University
Columbus, Ohio, USA

General Principles

Cytologic specimens are easily obtainable. Most collection techniques described below are relatively noninvasive, and the degree of morbidity or long-term complications associated with their use is low.

A multiplicity of tissues, organs and fluids can thus be sampled, including superficial cutaneous lesions, superficial and deep lymph nodes, parenchymal organs, metastatic and granulomatous pulmonary lesions, and fluid accumulations in body cavities (e.g.; pleural, pericardial, peritoneal cavities; CSF; joint fluid).

When obtaining cytologic specimens by FNA, it should be kept in mind that small gauge needles (i.e.; 23 to 25G) are preferable, since large gauge needles tend to yield cores of tissue, rather than single cell suspensions, making interpretation of the specimens extremely difficult due to "squashing" artifact.

Sample Collection

Cytologic specimens can be obtained by several means. Once smears are made, all the specimens are handled in the same fashion, regardless of the source or technique utilized.

A. Fine Needle Aspiration and Tissue Scrapings

With this technique, a single cell suspension is obtained by using a small gauge needle (i.e.; 23 to 25G) of the appropriate length for the desired specimen, coupled to a 12 or 20 ml sterile, dry, plastic syringe. Tissues easy to aspirate include the skin and subcutis, deep and superficial lymph nodes, spleen, liver, kidneys, lungs, thyroid, prostate, and intracavitary masses of unknown origin (e.g.; mediastinal mass).

When aspirating superficial masses, sterile preparation of the site is not necessary. However, clipping and sterile preparation should always be used when aspirating organs or masses within body cavities. Once the mass or organ is identified by palpation, radiography, or ultrasonography it should be manually isolated. A needle coupled to a syringe is then introduced in the mass or organ and suction is applied to the syringe 3 or 4 times. It is usually necessary to apply at least 6 to 8 ml of suction in order to obtain an adequate sample. Before withdrawing the needle and syringe, the suction is released in order to avoid aspirating blood that will contaminate the sample or air that will make the sample irretrievable from the barrel of the syringe. The needle is then detached, air is aspirated into the syringe, the needle is re-coupled and the sample is expelled on a glass slide or coverslip. In the majority of the cases, no material will be seen in the syringe, since the amount of cells present within the hub of the needle is usually adequate to obtain 4 to 8 good quality smears. "Pull" smears made with two 22 x 50 mm coverslips are usually of excellent quality. Once the smears are made, they are air-dried and stained with any of the techniques described below.

Superficial ulcerated masses can easily be sampled by scraping their surface with a sterile scalpel blade. Smears are then made as described below.

B. Cavity Centesis

Fluid from body cavities can be retrieved by centesis. Once a fluid sample is obtained, aliquots should be placed in both EDTA (purple top) and clot (red top) tubes, since effusions will occasionally clot. Analysis should be done to determine if the fluid is a transudate or an exudate (i.e.; cell count, protein content). A centrifuged (or cyto-centrifuged) sample should be used to make smears for cytologic evaluation as described above. Better results are obtained when centrifuging samples at low sped (under 1,500 rpm).

Staining Techniques

Several staining techniques are practical for in-office use, including rapid Romanowsky (e.g.; Diff-Quick®) and new methylene blue (NMB) stains. Most commercial laboratories use Romanowsky stains, such as Wright's or Giemsa's.

Interpretation of Cytologic Specimens

A. Hyperplastic Processes

Hyperplasia of different tissues commonly results in organ enlargement. Cytologically, hyperplastic changes may be difficult to recognize, since they may mimic either normal or neoplastic tissues. Care should be exerted when evaluating specimens from organs such as enlarged prostates, since the high degree of hyperplasia may be suggestive of malignancy.

B. Inflammatory Processes

Most inflammatory reactions are cytologically characterized by the presence of inflammatory cells and debris in the smear. The type of cell present will depend upon the etiologic agent (e.g.; neutrophils in pyogenic infections; eosinophils in parasitic or allergic reactions) and the course of the inflammatory process (i.e.; acute processes are usually characterized by predominance of granulocytes, while macrophages and lymphocytes predominate in chronic processes). Etiologic agents can occasionally be identified.

C. Etiologic Agents

The following etiologic agents are frequently identified in cytologic specimens: Histoplasma, blastomyces, cryptococcus, coccidioides, aspergillus/penicillium, ehrlichia, babesia, bacteria, demodex, toxoplasma

D. Malignant Cells

Most normal cells, with the exception of bone marrow precursors, are well-differentiated. That is, most of the cells are similar in size and shape, they have a normal nuclear:cytoplasmic ratio, the nuclei usually have condensed chromatin and absence of nucleoli, and the cytoplasms may exhibit features of differentiation (e.g.; keratin formation in squamous epithelium).

Malignant cells exhibit one or more of the following features: increased nuclear:cytoplasmic ratio; delicate chromatin pattern; presence of nucleoli (usually multiple); morphologic homogeneity (i.e.; all cells look alike); pleomorphism (i.e.; presence of cells in different stages of development); vacuolization (primarily in adenocarcinoma cells); anisocytosis (presence of cells of different sizes); and anisokaryosis (presence of cells with nuclei of different sizes). In addition, malignant cells are usually morphologically different from the progenitor cell population.

E. Carcinomas Vs. Sarcomas Vs. Round Cell Tumors

1. Carcinomas

Most carcinomas are composed of round or polygonal cells that tend to cling together forming clusters. Their cytoplasms are usually deep blue, and vacuolization is evident in most adenocarcinomas (aCA). In squamous cell carcinomas (SCC) cells are usually individualized, have deep blue cytoplasms, and lack vacuoles. Nuclei in both aCA and SCC are large, with fine chromatin and evident nucleoli.

2. Sarcomas

The cytologic features of sarcomas vary according to their histologic types. However, most mesenchymal tumors have spindle-shaped to oval cells, with reddish-blue cytoplasms, and irregularly shaped nuclei. Most cells are recognized as individuals, although clumping may occur. In most sarcomas, the cells have a tendency to form "tails," and the nuclei protrude from the cytoplasm. Intercellular matrix (e.g.; osteoid, chondroid) can be found occasionally .

As a general rule, since sarcomas do not exfoliate cells easily, aspirates of these masses may not yield any cells (remember this!!).

3. Round Cell Tumors

Tumors composed of a homogeneous population of round (or discrete) cells are referred to as round (or discrete) cell tumors. These tumors are common in the dog and the cat and include: lymphomas (or lymphosarcomas) [LSA], histiocytomas (HCT), mast cell tumors (MCT), transmissible venereal tumors (TVTs), and malignant melanomas (MM). Round cell tumors are easily diagnosed on cytology.

As a general rule, LSAs are characterized by a monomorphic population of individual undifferentiated round cells with large nuclei, coarse chromatin pattern, and 1 or 2 nucleoli; occasional cells can be vacuolated. Cells in HCTs are similar to LSA, except that the chromatin pattern is fine (not coarse), and most cytoplasms show vacuolization. Since inflammation is an important component of HCTs, inflammatory cells (i.e.; neutrophils, lymphocytes) are commonly found in these tumors. Mast cell tumors are characteristic in that the cytoplasm of these cells contains purple (metachromatic) granules; these granules can be so numerous as to obscure the nuclear features.

G. Fluids

The same principles described above apply to interpretation of cytologic features in fluid specimens.

H. Lymph Nodes

Cytologic evaluation of lymph node aspirates is commonly done in practice. When interpreting a lymph node aspirate, the clinician should keep in mind that these organs react to a variety of stimuli following distinct patterns. In general, 4 cytologic patterns are recognized:

1. Normal lymph node

Cytologic specimens from normal nodes are composed predominantly (75-90%) of small lymphocytes. These cells are approximately 7 to 10 um in diameter (1 to 1.5 times the diameter of an RBC), have dense chromatin pattern and absence of nucleoli.

2. Reactive lymphadenopathy

Lymphoid tissues reacting to different antigenic stimuli (e.g.; bacterial, immunologic, neoplastic, fungal) are cytologically similar in that they are composed of an admixture of small lymphocytes, prolymphocytes, lymphoblasts, plasma cells, and macrophages. In addition, depending upon the specific agent, other cell types may be present (e.g.; eosinophils in parasitic or allergic reactions). The presence of cells in different stages of development indicates that the lymphoid tissue is undergoing polyclonal expansion (i.e.; response to multiple antigens).

3. Lymphadenitis

Inflammatory processes affecting the lymph nodes result in cytologic changes similar to the ones described under reactive lymphadenopathy, although there is a profusion of inflammatory cells (e.g.; neutrophils in suppurative infections) and degenerative changes in most cell lines (e.g.; pyknosis, karyorrhexis); the etiologic agents may be visualized.

4. Neoplastic lymph node

Neoplastic cells can appear in a lymph node either due to lymphatic or vascular dissemination (i.e.; metastasis from a primary tumor distal to the node), or as primary processes affecting these tissues (i.e.; lymphomas). Cytologic features of metastatic lymph node lesions are characterized by a reactive pattern and presence of neoplastic cells; the morphology of the metastatic cells depends upon the primary tumor. Lymphomas are characterized by a predominance of a monomorphous population of large, immature lymphoid cells; these cells are usually large, have an abnormally high nuclear: cytoplasmic ratio, coarse chromatin, and evident nucleoli.

Speaker Information
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C. Guillermo Couto, DVM, ACVIM
College of Veterinary Medicine
The Ohio State University
Columbus, Ohio, USA

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