Diagnostic cytology--the basics (Proceedings)

Cells from ‘normal' tissues live harmoniously, maintaining an appropriate distance from each other without ‘piling up'.  They divide only at the rate necessary for replacement of tissue which has died from aging or injury.  Normal cells from the same organ are also homogeneous and predictable--each cell looks much like the next cell.   The nuclear/cytoplasmic (N/C) ratio should range from 1/3 to 1/10, depending on the tissue type. 

n exception to this is the lymphocyte, which normally has an N/C ratio of 1/1.  Nuclei and nucleoli should generally be uniform in number per cell, size, and shape--if they are even visible.  Mitotic figures are encountered only rarely or not at all in most tissues.  In fact, totally normal cells from the same organ are striking in their ‘sameness'. 

Normal cells react to their environment and to many pathologic processes by degenerating, repairing, and by reacting to chronic irritants.  These processes lead to dysplasia, in which certain nuclear and cytoplasmic characteristics can resemble neoplasia.  It is usually possible to differentiate neoplastic from dysplastic processes in cells exfoliated from a solid tissue, since one has a clinical history, radiographs, and the presence of a mass to suggest that a neoplasm is present.  In dysplasia, there is often a history of a chronic irritant or infection, and the presence of inflammatory cells along with the suspicious cell population makes one unwilling to diagnose a tumor without a histopathologic biopsy. 

Although tumors with marked cellular atypia are not uncommon, a large proportion of neoplasms are of low?grade cytologic malignancy.  The cytologist may be hard?pressed to make a definitive diagnosis??benign versus malignant??in these well?differentiated tumors.  It is a working principle of cytologic diagnosis that a tumor with many characteristics of malignancy may be interpreted as ‘malignant'. 

On the other hand, aspirates which have few malignant characteristics may not be interpreted as ‘benign'.  A carcinoma of low?grade malignancy may have cells which appear cytologically normal, but the cellular architecture of the tissue may be disarranged.  On histopathologic examination, the tumor may show invasion into the subcutaneous tissue, ‘piling up', and other structural abnormalities not noted on a cytologic preparation

The morphology of the nucleus is of the utmost importance in cytologic evaluation, since it reflects the biologic activity of the cell.  Enlargement of the nucleus is often found in cancer, and cells with nuclei larger than 10 microns in diameter are usually malignant.  With variation in nuclear size comes an increase in the N/C ratio, sometimes to 1:1.  In general, the degree of cellular maturity is inversely proportional to the N/C ratio, i.e., the more immature the cell the higher the N/C ratio.  To properly assess N/C ratio, however, the cytologist must be familiar with the normal range of N/C ratios for that cell type. 

Irregularity of the nuclear membrane is an abnormality which is generally not seen in benign or dysplastic cells, and as such is a strong indicator of malignancy.  These irregularities may appear as pointed spicules, sharp infoldings or outward projections, or nuclear ‘buds'.  Multinucleation as the result of abnormal mitosis is sometimes seen in neoplasia.  It must be remembered, however, that multinucleation occurs also in benign cells.  In general, nuclei of benign multinucleated cells are isokaryotic; multinucleated tumor cells often show mild (and sometimes marked) anisokaryosis.  However, abnormal mitotic figures or ‘stray' or ‘orphan' chromosomes left over after mitosis has been completed strongly suggest malignancy.

An increase in size and number of nucleoli may be seen in cells that are actively synthesizing protein.  Therefore, prominent or numerous nucleoli only reflect a heightened level of cell protein synthetic activity, which may be seen in dysplasia or neoplasia.  However, disparity in nucleolar size and shape within the same cell, or variation in the number of nucleoli in cells within the same tissue fragment strongly suggests malignancy.

Classifying tumors---epithelial, mesenchymal, or round-cell?

In order to differentiate epithelial, mesenchymal, and round cell tumors, the cytologist must evaluate individual cell morphology and patterns of cell arrangement.  As a group, the epithelial tumors exfoliate well, and the cells are often found in grape?like clusters.  Epithelial cells tend to be cohesive because of the presence of desmosomes (‘intercellular bridges'), and lines of cell?to?cell adherence may be found within the clusters.  Individually, the tumor cells are typically round with well?defined cell membranes.

It is typical of cells from tumors of mesenchymal origin to exfoliate poorly as compared to epithelial tumors and round cell tumors.  Rather than exfoliating in clusters, cells from mesenchymal tumors are found lying separately, in individual units.  However, examination of an area in which the cells are spread thinly will reveal that the cells show no evidence of cell?to?cell attachment, unlike the epithelial tumors. 

Typically, the cells of benign and malignant mesenchymal tumors have poorly?defined cell membranes as compared to epithelial tumors.  The cells are not generally round; they may be spindle?shaped, polygonal, or dendritic, but uniform ‘roundness' is a characteristic of epithelial cells or of cells from a round cell tumor.

The round-cell tumors are separated as a class from the epithelial and mesenchymal tumors because of cytologic characteristics which set them apart.  Typically, the round-cell tumors exfoliate well, and cytologic preparations may easily be too thick to interpret except at the edges of the aspirate.  The cells are round??like epithelial cells??and have distinct cell membranes.  Like mesenchymal tumors, however, the cells from a round-cell tumor exfoliate individually rather than in clusters. As a rule, the cytologic characteristics used to judge malignancy in epithelial and mesenchymal tumors are not applicable for the round-cell tumors, since the cells may be totally uniform and yet behave malignantly. 

Cytology of the Epithelial Tumors   

Keratin-Containing Tumors And Cysts

Several skin nodules are a frequent finding in the dog but occur only rarely in the cat.  Of these nodules, the epidermal inclusion cyst is the most common.  Epidermal inclusion cysts may be congenital or acquired, and arise within the epidermis itself, from a plugged hair follicle, or from an epidermal fragment buried in the dermis.  Grossly, the aspirated material is usually semisolid, yellow or tan, and granular.  Aspirations of these nodules will reveal masses of keratin which stain blue with Romanowsky stains; some areas of debris may be so thick that the material does not stain. 

Occasionally, well?differentiated epithelial cells from the cyst lining will be aspirated along with keratin.  Cholesterol crystals may also be present in the background of some epidermal inclusion cysts.  Since keratin acts as a foreign body, rupture of the cyst with release of keratin into the dermis will result in a sterile inflammatory reaction.  Generally, the inflammation is pyogranulomatous, with large numbers of macrophages and neutrophils present in the aspirate.Sebaceous Gland  Hyperplasia  And  Neoplasia

Adnexal structures are common sites of neoplasia in the canine species.  Hyperplasia and neoplasia of sweat glands also occur in canine skin, but with less frequency.  Ceruminous gland dysplasia, hyperplasia, and neoplasia are common pathologic findings in the ear canal of both the cat and the dog. 

In adenomatous hyperplasia of sebaceous glands, most of the cells are mature secretory cells.  The centrally?located nuclei are usually completely obscured by droplets of secretory material.  In adenomas, however, basilar ‘reserve' cells are commonly found; these are immature cells containing little or no secretory material.  Since the cytoplasm of reserve cells is basophilic and the N:C ratio is approximately 1:2, the cytologist may incorrectly diagnose these basilar cells as malignant.  Benign sweat gland and ceruminous gland tumors, on the other hand, are composed of cuboidal cells which sometimes contain a large droplet of secretory material.

Aspirates from sebaceous or sweat gland adenocarcinomas consist of clusters of highly basophilic reserve cells with malignant characteristics; only a few cells will contain any secretory material.  An occasional cell in an adenocarcinoma will produce a large amount of secretory material; in these cells a ‘signet?ring' appearance may be seen, with the nucleus pressed to the margin of the cell membrane. 

Perianal Gland Hyperplasia And Neoplasia

Perianal glands encircle the anus of the dog; small numbers of cells are also found in some dogs in the skin of the tail, prepuce, thigh, and over the dorsum of the back. Cytologically and histopathologically, it is difficult to differentiate hyperplasia of the perianal glands from adenoma.  The cells exfoliate individually and in clusters, have uniform nuclei and nucleoli, and contain a large amount of pink?staining cytoplasm which appears foamy at high magnifications.  The cells so closely resemble liver cells that European literature has termed perianal gland neoplasms ‘hepatoid gland tumors'. 

In some aspirates, a row of flattened ‘reserve' cells may be seen surrounding the larger cells.  The cytoplasm of these small cells is more basophilic and the N:C ratio ranges from 1:1 to 1:2.  These cells differentiate into the large hepatoid cells.   Perianal adenocarcinomas vary in the degree of cellular atypia present.  Both cytologically and histologically, it may be difficult to differentiate adenomas from well?differentiated adenocarcinomas.  Even one cytologic characteristic of malignancy may mean that the tumor is capable of metastasizing.  Variation in nuclear and nucleolar size and variation in nucleolar number per cell are the most common malignant characteristics found in perianal adenocarcinomas.  Basal  Cell  And  Adnexal 

Tumors

Basal cell tumors (aka trichoblastomas) are common in the skin of the cat and dog.  In the cat, they are likely to arise from sweat glands instead of hair follicles.  The tumors may be pigmented, especially in the cat, and often contain cystic spaces.  In many basal cell tumors, cords, or ribbons of palisaded epithelial cells are found embedded in a fibrous stroma.  Cytologically, a few fragments of these cords are usually found on a cytologic aspirate.  Individual cells within the cords are uniformly small and dark?staining.  Since the N:C ratio of the cells is 1:1, no cytoplasm can be visualized in most of the cells.  Since there is little to no visualized cytoplasm, the packets of basophilic-staining nuclei truly resemble ‘grapelike clusters'.

Squamous  Cell  Carcinoma

These cells have basophilic cytoplasm, are likely to occur in clusters, and have a N/C ratio of 1/2 or 1/3.  Dysplastic cutaneous lesions are very difficult to distinguish from neoplasia cytologically; if cellular evidence of obvious inflammation is present, one should be cautious in making a diagnosis of tumor.Normal keratinocytes have a low N:C ratio ranging from 1:8 to 1:10.  As a squamous cell keratinizes, the nucleus dies, undergoing pyknosis, karyorrhexis, and finally disappearing altogether, leaving an anuclear squame.  Well?differentiated squamous cell carcinoma with little cellular variation from normal may be difficult to distinguish from dysplastic conditions of the skin resulting from chronic irritation or infection. 

Tumor cells from these well?differentiated carcinomas show so little atypia that they might even be considered normal in another context.  Isolated keratinizing squamous cells exfoliate easily from the tumor, and are large with basophilic cytoplasm.  In the same smear, however, there are usually cells which are multinucleated or have large nuclei undergoing pyknosis.  These abnormal cells may have N:C ratios from 1:3 to 1:5, which would not be considered abnormal for epithelial cells from other organs.   Degree of differentiation of a squamous cell carcinoma is assessed by the degree of cellular anaplasia as well as the degree of keratinization

Thyroid  Carcinoma

Imprints or scrapings from the thyroid will reveal clusters and sheets of cuboidal or low columnar epithelial cells.  The nuclei are very uniformly round, and the cytoplasm is colorless to pale blue.  Although the N:C ratio of normal thyroid epithelial cells is about 1:2, this is difficult to discern on cytologic material, since the cell margins are indistinct.  Bare nuclei are common, and these must not be confused with lymphocytes.  Cells from an active thyroid gland may contain greenish-blue cytoplasmic granular material, which is probably iron.  Aspirates of thyroid nodules (follicular hyperplasia) in hyperthyroid cats contain cells with prominent granules identical to those from a normal active thyroid gland.Mammary  Neoplasia

Adenocarcinomas vary greatly in their cytologic malignancy; in some cases, there are very few cellular characteristics of malignancy, and it is only possible to determine whether the nodule is benign or malignant by examining a histopathologic section.  In other cases, many malignant characteristics are present, especially N:C ratio alteration, variation in nuclear size, and cytoplasmic hyperchromatism.  These cytologically malignant mammary tumors are often particularly ‘hot' in their biologic behavior, and metastasize rapidly both hematogenously and by lymphatics.  Typically, cells from malignant mammary neoplasms of epithelial origin exfoliate in grape-like clusters because of the presence of ‘intercellular bridges', and lines of cell-to-cell adherence may be observed.  The individual tumor cells are typically round with well-defined cell membranes.

Benign and malignant mixed mammary tumors contain a wide variety of tissues within one tumor mass, including epithelium, fibrous connective tissue, cartilage, bone, and myoepithelial cells.  These varied tissue types arise from proliferation of neoplastic epithelium, reactive fibrous stroma, metaplasia of myoepithelial cells into cartilage, and ossification of that cartilage into bone.  Any of these tissues may become malignant, so that it would not be unusual to obtain a diagnosis of ‘mammary osteosarcoma' or ‘mammary chondrosarcoma'.  Because these tumors usually contain more than one type of tissue, however, the pathologist will usually diagnose them as ‘malignant mixed mammary tumors'. 

Cytology of Mesenchymal Tumors

It is typical of cells from tumors of mesenchymal origin to exfoliate poorly as compared to epithelial tumors and round cell tumors.  Despite vigorous aspiration, the cytologist may obtain only a drop or two of blood and a few scattered cells; imprints of a biopsy sample may be equally unrewarding.  Generally, a scraping from a biopsy sample will provide the most cytologic material for interpretation.  Rather than exfoliating in clusters, cells from mesenchymal tumors are found lying separately, in individual units.  A vigorous scraping may result in thick sheets of cells on the slide, which can confuse the inexperienced cytologist into believing that the cells are ‘clustering'.  However, examination of an area in which the cells are spread thinly will reveal that the cells show no evidence of cell?to?cell attachment, unlike the epithelial tumors. 

Typically, the cells of benign and malignant mesenchymal tumors have poorly?defined cell membranes as compared to epithelial tumors.  The cells are not generally round; they may be spindle?shaped, polygonal, or dendritic, but uniform ‘roundness' is a characteristic of epithelial cells or of cells from a round cell tumor.

Spindle-Cell Tumors

The benign and malignant spindle?cell tumors comprise a group of mesenchymal tumors which are indistinguishable cytologically.  Exfoliation of cells from the spindle?cell tumors is typically extremely poor, but a scraping will provide adequate cells for examination.  Cytologically, cells from benign or well?differentiated malignant spindle?cell tumors will have fusiform or ovoid nuclei with little or no cellular atypia.

Osteosarcoma

Histologically, osteogenic sarcomas may contain osteoid, mature (usually reactive) bone, fibrous connective tissue, and/or cartilage. The normal osteoblast is a cell with an eccentric nucleus, well-delineated cell borders, and a ‘flag' of blue cytoplasm.  Most osteosarcomas are composed principally of osteoblasts with varying degrees of anaplasia.  Generally, characteristics of malignancy are prominent, with extreme variation in nuclear and nucleolar size and number and frequent mitotic figures.  Some osteosarcomas contain cells which are so poorly differentiated that they no longer resemble osteoblasts; a cytologic diagnosis of undifferentiated sarcoma must be made from such a specimen.  Osteoclasts, which resemble multinucleated giant cells, are prominent in some tumors.  These cells contain a few to a moderate number of nuclei of uniform size.  They may be round to ovoid or may have several long, sometimes tentacle-like cytoplasmic extensions.

Chondrosarcoma

Cells aspirated or scraped from a chondrosarcoma are generally spindle-shaped with plump, oval nuclei and a moderate amount of cytoplasm.  Although most of the cells are uniform in appearance, occasional cells can be found showing slight variation in nuclear size, enlarged nucleoli, or cytoplasmic hyperchromatism.  Mitotic figures are usually rare.  Some chondrosarcomas contain giant cells with multiple nuclei, which may make differentiation from giant cell tumor or osteosarcoma difficult.

Typically, chondroid is found in the background, both as solid fragments and as an amorphous background substance in which the cells are embedded.  It is bright pink to deep magenta in color, depending on its density, and may have a faintly fibrillar structure.

Lipoma/Liposarcoma

Older dogs commonly develop soft encapsulated or poorly?demarcated fatty tumors called lipomas.  The tumors are located in the subcutaneous tissue, vary enormously in size, and are composed of mature adipose tissue cells with no evidence of cytologic atypia.  These cells are exceptionally large and distended with fat, and usually rupture during the aspiration procedure.  The unstained smear will show droplets of glistening lipid which does not dry.  Staining of the aspirate in an alcohol?based stain will dissolve the fat, leaving a cell?free slide.  Occasionally, a group of lipocytes with small regular nuclei will remain unruptured. 

Although cytologic specimens from a lipoma are usually cell?free, aspirates from a liposarcoma contain many cells.  Histopathologic examination reveals that the cells contain abundant cytoplasm.  This feature is not noted in most cytologic preparations, however, since the cell membrane??and therefore the cell shape and size??is poorly visualized.  Most liposarcoma cells contain vacuoles of varying size and number.  Although the cell membrane cannot be seen, some of the vacuoles are obviously contained within the cells, since the nuclei are indented by the larger lipid droplets.  Other fat globules are scattered over the background, probably released from ruptured or dead cells.  The tumor cell nuclei are typically round to ovoid, and variation in nuclear size and multinucleation are usually present. 

Melanoma/Melanosarcoma

While aspirates of many other non?epithelial tumors are cell?poor, a moderate to large number of cells is generally obtained from dermal melanin?producing tumors.  Occasionally, only scattered cells mixed with blood are aspirated, but melanin pigment will almost always be found in the background.  In extremely melanotic tumors, the aspirated material and the unstained smear may contain so much pigment that they are colored brown?black. 

The presence of melanin pigment staining black or brown with Romanowsky stains is suggestive of the diagnosis, since the melanoma cells often rupture during the aspiration procedure.  It must be remembered, however, that pigment per se is not diagnostic of a melanin?producing tumor, since dark pigment is present in other benign cell types.  Pigment in the melanin?producing tumor cells may be absent (amelanotic melanosarcoma), appear as a salt?and?pepper sprinkling in occasional cells, or may fill the cell with black masses, sometimes nearly obscuring the nucleus.

Cytology of the ‘Round-Cell' Tumors

The round-cell tumors as classically defined consist of three sarcomas??the transmissible venereal tumor, the mast cell tumor, and the lymphoma??and the histiocytoma, a benign tumor arising from histiocytes.  However, other tumors may also fit morphologically into this ‘round-cell' classification--the plasmacytoma, histiocytic sarcomas, certain melanosarcomas, osteosarcomas, seminomas, and anaplastic carcinomas (which have lost their characteristic clustering).  The round-cell tumors are separated as a class from the epithelial and mesenchymal tumors because of cytologic characteristics which set them apart. 

Typically, the round-cell tumors exfoliate well, and cytologic preparations may easily be too thick to interpret except at the edges of the aspirate.  The cells are round??like epithelial cells??and have distinct cell membranes.  Like mesenchymal tumors, however, the cells from a round-cell tumor exfoliate individually rather than in clusters.  One has the overall impression of many homogeneous round cells with no cell?to?cell attachment.  As a rule, the cytologic characteristics used to judge malignancy in epithelial and mesenchymal tumors are not applicable for the round-cell tumors, since the cells may be totally uniform and yet behave malignantly. 

Mast Cell Tumor

Canine mast cell tumors are graded according to degree of granulation and cellular anaplasia, with well-granulated tumors being the most differentiated.Well-differentiated mast cell tumors are characterized by a uniform population of round cells containing distinct reddish?purple granules.  The cell nucleus may be so covered by granules that it is indistinguishable.  In other tumors, the granules will stain deeply but the nuclei will remain unstained. 

Mast cell tumors of intermediate differentiation show fewer granules and less homogeneity.  Nuclear and nucleolar variation are common, there may be a few mitotic figures, and some cells have no visible granules at all.  Anaplastic or poorly-granulated mast cell tumors may not be identifiable as mast cell tumors at all--the cytoplasmic granules are dust-like, and there is usually a great degree of anisokaryosis, some multinucleation, mitotic figures, and a variable amount of anisokaryosis. 

Transmissible  Venereal  Tumor

Typically, the TVT exfoliates extremely well, with many cells obtained by aspirate, impression smear, or scraping.  Although most of the cells are uniformly round, occasional large or multinucleated cells may be observed.  The TVT cell has a moderate amount of cytoplasm, which generally contains a few small, distinct vacuoles.  In some tumors, many mitotic figures are found; other tumors are heavily infiltrated by plasma cells and lymphocytes.

Histiocytoma, Cutaneous Histiocytosis, Histiocytic Sarcoma

Histiocytoma, cutaneous histiocytosis, and histiocytic sarcoma are neoplasms that all arise from the tissue histiocyte.  Differentiation of these is made by the biologic behavior of the particular tumor--its location, evidence of involvement of other organs, whether the tumor is single or multiple, etc.  All characteristically have round cells with a moderate amount of cytoplasm, a variable number of mitotic figures, and a few multinucleated cells.  Occasional nuclei are pleomorphic or indented like monocytes.  Histiocytomas have a a uniform population of individually exfoliating round cells, usually in a background of basophilic proteinaceous material (tissue fluid). 

The tumor cells have lacy chromatin, round to ovoid nuclei, relatively uniform nuclear size, and a moderate amount of lightly basophilic cytoplasm.  Occasional tumor cells show reniform or cleaved nuclear shapes.  No significant inflammation is noted, although a moderate number of small lymphocytes may be seen scattered among the histiocytic cells.  These tumors are common on the skin of young dogs, and generally regress spontaneously within a few months after they appear.  An infiltrate of small lymphocytes is commonly seen in histiocytomas as they begin to regress; these are presumed to be cytotoxic T cells and correspond with the onset of widespread single cell tumor necrosis and gradual regression of the mass. 

Generally, regression occurs within 2-3 months after the tumors appear.  In histiocytic sarcoma, the cells are larger and usually much more bizarre, there are many cells with marked nuclear pleomorphism, mitotic figures are very common, and phagocytic activity may be noted.

Large-Cell Lymphoma

Since erythrocytes are usually present on most cytologic aspirates, they are used for size comparison with other cells.  The small lymphocyte is somewhat larger than an erythrocyte but smaller than a neutrophil, while the large lymphocyte is two to three times the size of an erythrocyte, often contains an obvious nucleolus, and has a small amount of cytoplasm.  The tumor cells are often several times the size of an erythrocyte and have a small amount of basophilic cytoplasm.  Many of the cells contain one or more nucleoli, and mitotic figures are frequent.  Since these immature cells are fragile and cannot resist much shear force, it is common for the cells to rupture during preparation of the smear.  Naked, smeared nuclei and cytoplasmic fragments  (‘lymphoglandular bodies') will be found scattered among the intact cells.

Plasmacytoma

Extramedullary plasmacytomas have been diagnosed commonly in the skin and oral cavity of dogs; in some cases, especially in the oral cavity, these solitary tumors have progressed into disseminated myeloma, although this is apparently rare.  Typically, the tumors exfoliate well on aspiration. The cells are usually round to oval, with round, eccentrically-located nuclei with coarsely-granular chromatin and a moderate amount of basophilic cytoplasm.  The resemblance of the tumor cells to normal plasma cells is usually striking.  Although there are sometimes few characteristics of malignancy present in these tumors, bi- or multinucleate cells are often found, and there is often moderate variation in nuclear size.  A few mitotic figures may be found.  These tumors are commonly found in older dogs.