Cytology is a diagnostic tool used to examine exfoliated cells obtained by aspiration, impression, scraping, or natural release from any tissue. The basic indications for diagnostic cytology are to recognize the presence of inflammation, neoplasia, or effects of tissue injury, and then determine if possible an etiologic agent. If a specific disease condition is not possible to diagnose from cytologic evaluation, the procedure often rules out other disorders and may suggest other conditions to be pursued.

Specimen Collection and Preparation

Materials used for cytology include solid tissues, fluid pockets, tissue secretions or discharges, body cavity effusions, urine, feces, and tissue washings. The external site is either wiped with an alcohol soaked gauze pad or, preferably, scrubbed with warm water and antibacterial soap. Aspiration may be either with suction or without. For suction, a 6 or 12 ml syringe is attached to a 22-gauge needle for soft fluctuant masses. A 20-gauge needle is preferential for amplifying suction in firm or hard masses composed of dense connective tissue. The mass is isolated and immobilized between the thumb and forefinger. Place the needle into the center of the mass if it is small (< 2cm). For large masses, the center of the lesion should be avoided to prevent aspirating only necrotic material. Suction is applied (3 to 5 ml) once the needle is within the lesion, advancing the needle two or three times in different directions. Following aspiration, gently release the plunger to avoid splattering the aspirated material into the syringe barrel.

Aspiration may not even be necessary for obtaining a diagnostic cytologic specimen from certain tissues, such as lymph nodes and suspected mast cell tumors. Based on the principle of capillarity, a technique referred to as "fine needle capillary sampling" requires placement of a needle into the lesion without the syringe attached. One advantage is minimization of blood contamination and another is reduced risk of cell rupture, both common with the aspiration technique. The contents of the needle may not be visible in the hub of the needle. They are removed by filling a syringe with air and gently squirting a drop of the material onto a glass slide. A squash preparation is made by lightly pressing the material between two slides while sliding them apart horizontally.

Intact tissue or excised tissue must first be blotted onto absorbent paper to remove excess blood and tissue liquids. Gently touch the slide to create a "dry" imprint. In the case of skin, the surface is frequently contaminated with debris, bacteria, and a neutrophilic response, which might obscure the true pathogenesis.

Make thin smears by gently spreading materials onto glass slides or coverslips. In a humid environment, slides may be dried quickly with a hair dryer. Do not "heat fix" the slides for cytology as this procedure may rupture cells from the heat. Following preparation of the aspirate or imprint, air-dry the glass slides before staining.

Formalin fumes can permeate through plastic materials. It is necessary to send cytologic and histologic samples to referral labs in separate containers and avoid close proximity of cytologic samples with formalin fumes in the practice setting. Glass slides or coverslips should be placed in sturdy cardboard or plastic containers and then placed inside a thickly padded envelope or box for mailing to a referral diagnostic laboratory.

Staining Considerations

Slides should be stained as soon as possible to avoid poor stain results due to pH changes. If staining is to be performed at a later time, slides should be placed in a closed container to protect them from insects and light.

Once slides are air-dried, they may be stained with routine Romanowsky stains. Quick stains that take less than 5 minutes to perform are the most commonly used in clinical practices to evaluate cytologic specimens. These may be either alcohol-based Wright stains such as Hema-Quik II® or aqueous-based Wright stains, the latter stains known by a variety of trade names such as Diff-Quik® or Quik-Dip®. In general, there are few differences between the stain reactions of the alcohol-based stains and aqueous-based Wright stains. Compared with standard alcohol-based Wright or Wright-Giemsa stains, the aqueous-based Wright stains give similar cytomorphologic information but may be more expensive since they are prediluted. However some differences exist that the one should know. For example, mast cell tumors, especially poorly-differentiated ones, may not stain well relative to their cytoplasmic granules using an aqueous-based Wright stain because granule contents are washed away. Confirmation of a mast cell origin may require an alcohol-based Giemsa staining technique which does not affect granule contents.

New methylene blue (NMB) is another common stain in addition to the Romanowsky-type stains discussed above that can be used for cytology. It is used also for urine sediment examination, and for hematology with reticulocyte or Heinz body identification. Since it is a water soluble stain, it does not dissolve lipids and can be easily used to identify lipomas and cholesterol crystals. Fungi, bacteria, and mast cell granules are also easily visible with this stain. In cases of heavy blood contamination, nucleated cells are best identified using NMB since red cells do not stain well. A coverslip must be placed over the stain drop and the slide should be examined when the stain is applied as it will evaporate within hours.

Oil-red-O is a helpful stain in cytology to determine the presence of lipid materials, such as those found in vacuolated hepatocytes from a cat with hepatic lipidosis. A drop may be applied along with one of NMB to an air-dried slide to see both nuclear and cytoplasmic features.

Microscopic Examination

Artifacts are frequently encountered during cytologic examination. Fungal hyphae are often confused with the linear shapes formed from fractured cells forming nuclear strands or streaming and from strands of fibrin with entrapped platelets. Color, shape, and uniform of width are helpful in making this distinction. Basket cells are also formed from ruptured cells which resemble large intact cells. Bacteria, yeast, or rickettsial organisms are often mistakenly diagnosed as a result of stain precipitate following inadequate rinsing or with old stain material that has become contaminated. Plant spores or fragments may simulate pathogenic fungi. Poor cellular detail and staining often results from direct and indirect contact of the cytologic samples with formalin, for example, by making the cytologic prep in the same room as an open formalin container, cells appear blue-green.

Cytologic interpretations are generally classified into one of five cytodiagnostic groups. An exception is made for body cavity effusions which have their own classification categories. Interpretations of cytologic material may include more than one category, such as inflammation along with a response to tissue injury or neoplasia along with inflammation.

Cytodiagnostic Groups

 Normal/hyperplastic tissue

 Cystic mass

 Response to tissue injury

 Inflammation

 Neoplasia

Normal/Hyperplastic Tissue

Normal and hyperplastic tissues are composed primarily of mature cell types. Look for uniformity in cell, nuclear, and nucleolar size and shape. Cytoplasmic volume is usually high relative to the nucleus. Hyperplasia is a nonneoplastic enlargement of tissue that can occur relative to hormonal disturbances or tissue injury. Hyperplastic tissue has a tendency to enlarge symmetrically in size in comparison to neoplasia. Cytologically, hyperplastic cells have a higher nuclear to cytoplasmic ratio (N:C) than normal cells. For example, normal salivary gland epithelium appear as large foamy cells with condensed nuclei and a low N:C. Prostatic hyperplasia seen in intact male dogs with a symmetrically enlarged prostate have cuboidal epithelium of uniform size and differentiation commonly with a high N:C.

Cystic Mass

Cystic lesions contain liquid or semisolid material. The low protein liquid usually contains a small number of cells. These benign lesions may result from proliferation of lining cells or tissue injury. For example, seroma is a light yellow clear fluid resembling serum which may contain rare large mononuclear cells having cytoplasmic granularity. A follicular cyst is a common skin mass in many species composed entirely of keratinized anucleated squamous epithelium often with rectangular cholesterol crystals that are seen best against a light proteinaceous background or when NMB stain is used.

Response to Tissue Injury

Common changes include hemorrhage, proteinaceous debris, cholesterol crystals, necrosis, or fibrosis. Hemorrhage with the presence of erythrocytes is pathologic and should be distinguished from blood contamination encountered during the cytologic collection. Blood contamination contains numerous platelets in addition to unaffected erythrocytes. Acute hemorrhage is associated with the engulfment of erythrocytes by macrophages called erythrophages. Chronic hemorrhage is associated with active macrophages containing degraded blood pigment within their cytoplasm, for example, blue-green to black hemosiderin granules or yellow rhomboid hematoidin crystals. Hemosiderin represents an excess aggregation of ferritin molecules. This form of iron stores stains positive with the Prussian blue reaction. Hematoidin crystals may be formed during anaerobic breakdown of hemoglobin as occurs within tissues or cavities and do not contain iron. Hematomas often contain phagocytized erythrocytes if the lesion is acute or hemosiderin-laden macrophages if the lesion is chronic.

Proteinaceous debris may appear in the background of the preparation. This may include mucus that stains lightly as basophilic to purple amorphous strands. Lymphoglandular bodies are basophilic cytoplasmic fragments from fragile cells. Nuclear streaming is a strand of remnant nuclear material that stains pink to purple. Fibrovascular stroma appear as clear to light pink strands that often represent collagen and may be mixed with spindle cells and endothelium. Amyloid is an uncommon protein that is amorphous, eosinophilic, and hyalinized and associated with chronic inflammation.

Necrosis and fibrosis may occur together in some cytologic preparations. The death of cells is represented by fuzzy, indistinct cell outlines and poor definition of cell type. Accompanying tissue injury is the reparative response involving increased fibroblastic activity. It is common to see very reactive fibrocytes along with severe inflammation and this appearance often mimics a neoplastic condition.

Inflammation

Inflammatory conditions are classified cytologically by the predominance or mixture of the cell type involved such as neutrophils, macrophages, and eosinophils.

Neoplasia

Recognition of benign and malignant conditions may be challenging.

Cytomorphologic categories of neoplasia.

References

1.  Baker R, Lumsden JH, Colour Atlas of Cytology of the Dog and Cat, Mosby, St. Louis; 2000.

2.  Cowell RL, Tyler RD, Meinkoth JH, Diagnostic Cytology and Hematology of the Dog and Cat, Mosby, St. Louis; 2nd Ed. 1999.

3.  Radin MJ, Wellman ML, Interpretation of Canine and Feline Cytology, Ralston Purina Company Clinical Handbook Series. The Gloyd Group, Inc, Wilmington, DE; 2001.

4.  Raskin RE, Meyer DJ (eds), Atlas of Canine and Feline Cytology, WB Saunders Co, Philadelphia; 2001.