Diagnostic cytology involves the examination of cells which have either naturally exfoliated or been artificially removed from a body cavity or a tissue mass. The cytological interpretation is often valuable in establishing a diagnosis, identifying the disease process, directing therapy, forming a prognosis, and/or determining what diagnostic procedure should next be performed.
Some of the major advantages of cytological procedures are that they require minimal equipment, have rapid turn around time and most are minimally invasive and minimally stressful for the patient. However, since cytologic interpretation is based upon the distinct characteristics of individual cells on a smear that lacks the source tissue's architectural patterns, it should be mainly utilized as a rapid screening procedure. Histopathology is usually more diagnostic and definitive in that it allows study of both structure and form of a tissue or organ. Cytology should be considered a tentative diagnosis requiring a histologic confirmation, particularly in lesions with suspected neoplastic characteristics.
The particular method used for obtaining cytological specimens is determined by the characteristics of the lesion to be sampled. The major goal is to obtain a significant number of well-stained intact cells that reflect the composition of the lesion.
1. Touch impressions or imprints
Imprints can be prepared from excised external lesions on the living animal or from tissues removed during surgery or necropsy. The imprints are easy to obtain but they collect fewer cells than scrapings and contain greater contamination (bacterial and cellular) than aspirates. Therefore, imprints from superficial lesions often only reflect a secondary bacterial infection and/or inflammation-induced tissue dysplasia. This markedly hinders their use in diagnosis of neoplasia. To reduce contamination and to obtain a more representative sample, touch imprints are made on freshly excised surfaces. If the excised surface is very bloody or moist, it should be blotted with absorbent paper. Touch the slide gently to the specimen, remove and quickly air dry. Dragging of the slide across the specimen will distort the cells. Tissue rich in connective tissue imprints poorly.
Scraping has the advantage of collecting many cells from the tissue, and therefore, is advantageous when the lesion is firm and yields few cells. Disadvantages are similar to those described for tissue imprints. The surface of the lesion is freshly excised and scraped with a scalpel blade until material appears on the blade's surface. This material is then smeared thinly across a slide.
Swab smears are collected only when imprints, scrapings and aspirates cannot be made; e.g., fistulous tracts, nasal and vaginal collections. The lesion or tissue is swabbed with a moist, sterile cotton swab. Sterile isotonic fluid, such as 0.9% NaCl, should be used to moisten the swab. This helps minimize cell damage during sample collection and smear preparation. After sample collection, the swab is gently rolled, not rubbed, along the flat surface of a clean glass microscope slide.
4. Fine Needle Aspirations
Fine needle aspiration biopsies can be collected from masses such as lymph nodes, nodular lesions and internal organs. Aspiration of cutaneous masses avoids the superficial contamination common with imprints or scrapings, but collects fewer cells than scrapings. The tissue to be aspirated is immobilized as close to the skin surface as possible. A 21-25 gauge needle attached to a 3-20 ml syringe is used (a 12-ml syringe is a good all-around size). The softer the tissue being aspirated, the smaller the needle and syringe used. When needles larger than 21 gauge are used, tissue cores tend to be aspirated, resulting in a poor yield of free cells suitable for cytological preparation. Also, larger needles tend to increase the incidence of blood contamination.
The needle with syringe attached is introduced into the center of the mass and strong negative pressure is applied The cell population retrieved from a mass is highly dependent upon the site aspirated. In order to obtain a representative sample, the needle is redirected and moved to several areas in the mass, taking precautions to prevent the needle from leaving the mass. Negative pressure is maintained during the redirection. However, when the mass is not large enough for the needle to be redirected and moved without danger of the needle's leaving the mass, negative pressure is relieved during redirection and movement of the needle. In this situation, negative pressure is applied only when the needle is static. In both cases, aspirate may or may not appear in the syringe, but the needle will contain sufficient tissue for smears. Before removing the needle from the mass, the negative pressure is relieved. The needle is removed from the mass and skin and the needle contents are expressed onto a slide and a smear prepared.
Centesis is the process of perforation or tapping as with an aspirator, trocar, or needle, to obtain abdominal, thoracic, synovial or cerebrospinal fluid. The size of the trocar or needle used is dependent upon the species and centesis site. Whenever possible, at least 3 ml of fluid should be aseptically obtained to provide an amount sufficient for determination of specific and concentration for microscopic examination. Since exudates coagulate and modified transudates may coagulate, a portion of the specimen should be collected in an EDTA tube. A sterile tube should be used for one portion to assure a satisfactory specimen in the event that culture is indicated. Direct and sediment smears should be made for cytological examination. Sediment smears are prepared by centrifu-gation of the fluid for 5 minutes at 165-360 G to concentrate the cells. In addition to cytological examination, other properties (e.g., specific gravity, total protein, glucose) should be determined in order to pin-point the pathogenesis of the occurrence.
Catheterization procedures are used for transtracheal/ bronchial and prostatic washes. Once obtained, the fluid specimens are handled in a manner similar to centesis fluids.
Several methods can be used to prepare smears for cytologic evaluation of solid masses, including lymph nodes. The experience of the person preparing the smears and characteristics of the sample influence the choice of smear preparation technique. No matter what the technique used, the resultant smear must have a thin area where cells may settle flatly and expose a large surface area to view. If the smear is thick, the cells are supported more upright on the slide and have a smaller diameter. Cellsin thick areas are taller and thus stain darker. In addition, thick layers of proteinaceous and necrotic debris in the dried fluid surrounding cells interferes with staining. Some common cytological preparation techniques include:
1. Combination Technique
This method makes a squash prep of 1/3 of the aspirate, leaves the middle 1/3 untouched and thus concentrated, and gently spreads the remaining 1/3 of the aspirate. A portion of the aspirate is expelled onto a glass microscope slide (prep slide). Another glass microscope slide is placed over about one-third of the preparation. If additional spreading of the aspirate is needed, gentle digital pressure can be used. Excessive pressure should be avoided. The spreader slide is smoothly slid forward. This makes a squash prep of about one-third of the aspirate. The spreader slide also contains a squash prep. Next, the edge of a tilted glass microscope slide (second spreader slide) is slid backward from the end opposite the squash prep until it contacts about one-third of the expelled aspirate. Then, the second spreader slide is slid rapidly and smoothly forward. This produces an area that is spread with mechanical forces like those of a blood smear preparation. The middle area is left untouched and contains a high concentration of cells.
2. Squash Preparation
This method is used for spreading viscous samples and samples with flecks of particulate material. A portion of the aspirate is expelled onto a glass microscope slide and another slide is placed over the sample to spread it. If the sample does not spread well, gentle digital pressure can be applied to the top slide; however, care must be taken not to place excessive pressure on the slide causing the cells to rupture. The slides are smoothly slid apart. This usually produces well-spread smears but, even when care is take, may result in excessive cell rupture.
3. Modified Squash Preparation
This method is also used for spreading viscous samples and has less tendency to rupture the cells. A portion of the aspirate is expelled onto a glass microscope slide and another slide is placed over the sample. This causes the sample to spread. If necessary, gentle digital pressure can be applied to the top slide to spread the sample more. Care must be taken not to use excessive pressure and cause cell rupture. The top slide is rotated about 45 degrees and lifted directly upward, producing a spread preparation with subtle ridges and valley of cells.
4. Needle Spread or "Starfish" Preparation
This technique tends not to damage fragile cells, but allows a thick layer of tissue fluid to remain around the cells which may interfere with staining. A portion of the aspirate is expelled onto a glass microscope slide. The tip of a needle is placed in the aspirate and moved peripherally, pulling a trail of the sample with it. This procedure is repeated in several directions, resulting in a preparation with multiple projections.
Smears should be made immediately after fluid collection. Smears can be made directly from fresh, well-mixed fluid or from the resuspended sediment of a centrifuged sample. The cellularity, viscosity and homogeneity of the fluid determine the selection of smear technique used. The most common techniques include:
1. Blood Smear Technique
This procedure is identical to that used for preparing smears of blood and will result in a smear with a feathered edge. This method is generally not appropriate for viscous samples. A drop of fluid sample is placed on a glass microscope slide close to one end, then another slide is slid backward to contact the front of the drop. When the drop is contacted, it rapidly spreads along the juncture between the 2 slides. The spreader slide is then smoothly and rapidly slid forward the length of the slide, producing a smear with a feathered edge.
2. Line Smear Concentration Technique
When the fluid cannot be concentrated by centrifugation or the centrifuged sample is of low cellularity, the line smear technique can be used to concentrate cells in the smear. Unfortunately, an excessive amount of fluid may also remain in the "line" and prevent the cells from spreading well.
A drop of fluid sample is placed onto a glass microscope slide close to one end, and another slide is slid backward to contact the front of the drop. When the drop is contacted, it rapidly spreads along the juncture between the 2 slides. The spreader slide is then smoothly and rapidly slid forward. After the spreader slide has been advanced about two-thirds to three-fourths the distance required to make a smear with a feathered edge, the spreader slide is raised directly upward. This produces a smear with a line of concentrated cells at its end, instead of a feathered edge.
3. Squash Preparation (Same as described above)
Smears of viscous fluids such as synovial fluid can be prepared using this technique.
The two general types of stains most commonly used are the Romanowsky- type stains (Wright's stain, Giemsa stain, Diff-Quik) and Papanicolaou stain.
10.3.1 Romanowsky-type Stains
These are permanent stains which stain organisms and the cytoplasm of cells excellently. The stains tend to have a "smudging" effect on the nucleus, thus, nuclear and nucleolar detail cannot be perceived as well as with the Papanicolaou-type stains. However, the detail is usually sufficient for differentiating neoplasia and inflammation and for evaluating neoplastic cells for cytologic evidence of malignant potential. Smears to be stained with Romanowsky-type stains must first be air dried to fix the cells and prevent them from falling off the slide during the staining procedure. These stains tend to dissolve lipids from cells leaving vacuoles. RNA stains blue and DNA stains purple with variations to red or pink. Mast cell granules may not stain with Diff-Quik.
New Methylene Blue Stain (NMB) is a useful adjunct to Romanowsky-type stains. It stains cytoplasm weakly, if at all, but gives excellent nuclear and nucleolar detail. NMB does not stain RBC hemoglobin and is thus useful in interpretation of blood contaminated samples. Fungi are stained and lipid droplets are outlined. This stain is a wet mount of a dried smear and is not permanent; however, the smear can be counter stained with Wright's stain to provide a permanent preparation. A combination of a drop of NMB and Sudan's or other oil stain will selectively stain lipid and give adequate cell detail. This stain combination is useful for fatty livers, chylothorax, aspiration pneumonia or lipid granulomas.
10.3.2 Papanicolaou Stains
These stains use wet fixed smears (i.e., the smear must be fixed before the cells have dried) and clearly illustrate cell structure and nuclear characteristics. They do not demonstrate bacteria and other organisms as well as Romanowsky stains. Papanicolaou-type staining requires multiple steps and considerable time.
Once the smear has been prepared, stained and dried, it is scanned at low magnification (4-10X objective) to determine if all areas of the smear are stained properly and if there is adequate cellularity for evaluation. When proper staining is assured and all areas of increased and/or unique cellularity are recognized, magnification is increased to the 10X or 20X objective. An impression of the cellularity and cellular composition of the smear and of cell size are ascertained. Using the 40X objective, nucleoli and chromatin pattern are discerned. Cell morphology is evaluated in detail with the 100X (oil-immersion) objective.
An algorithm to aid in the general evaluation of all cytologic preparations is presented in Figure X.1. While a definitive diagnosis is not always achieved, the general process usually is recognized. Probably the most important judgment to be made when interpreting a cytologic sample is whether the lesion is inflammatory or neoplastic in nature. Samples containing only inflammatory cells or inflammatory cells and a few nondysplastic tissue cells indicate and inflammatory lesion, whereas samples containing only tissue cells indicate a neoplastic or hyperplastic process. An admixture of inflammatory cells and tissue cells suggests neoplasia with secondary inflammation, or inflammation with secondary tissue cell dysplasia. Lesions determined to be inflammatory in nature can be classified into different categories of inflammation.
The inflammatory process can be classified as acute when >70% of the inflammatory cells are neutrophils, as subacute or chronic-active when 50-70% of the inflammatory cells are neutrophils and 30-50% are macrophages, and chronic when >50% of the inflammatory cells are macrophages. If >85% of the inflammatory cells are neutrophils, the process is classified as purulent or suppurative, granulomatous if inflammatory giant cells and/or numerious epitheli.
macrophages are present, and eosinophilic or allergic if eosinophils are numerous.
Often neoplastic lesions can be recognized as epithelial, mesenchymal (spindle-cell) or discrete round-cell tumors. Important cytological characteristics of these tumor types are illustrated in Figure X.2. Evaluation for malignant potential of tumors is estimated by evaluating tumor cells for indications of anaplasia and asynchronous development. The cytomorphologic criteria for this assessment are illustrated in Figure X.3. Descriptions for the various terms used to assess nuclear chromatin patterns are listed in Figure X.4.
Nuclear criteria of malignancy are more reliable than cytoplasmic criteria for estimating malignant potential. Recognition of more than 3 nuclear criteria of malignancy in a high percentage of tumor cells is strong evidence that the tumor is malignant. If malignant criteria are not recognized, the tumor is most likely benign. However, some tumors, such as canine thyroid tumors, may be malignant but show few, if any, criteria of malignancy. Therefore, if the cell type of a tumor cannot be recognized, caution must be exercised in classifying the tumor as benign.
Abnormal accumulations of fluid in body cavities and joints are relatively common occurrences in animals. Cytological examination of these fluids is utilized to reveal the cellular characteristics of the fluid so that the presence or absence of inflammation or neoplasia can be detected. Fluids to be discussed will include body cavity effusions (abdominal, thoracic and pericardial), synovial fluid, cerebrospinal fluid prostatic fluid, and trans-tracheal bronchoalveolar aspirations.
The body cavities of animals normally contain small quantities of fluid which is essentially an ultrafiltrate of blood. This fluid has few cells, chiefly of the mononuclear type. a process that impairs absorption, alters vascular pressure, or alters the concentration of albumin in the blood can result in accumulation of fluid in a body cavity, which is termed an effusion. Four basic mechanisms by which fluid may accumulate in abnormal quantities includes:
Removal and examination of fluids from body cavities is indicated when there is excessive fluid accumulation or if the clinician suspects the presence of a lesion characterized by cellular exfoliation. An increased amount of fluid in a cavity is not a disease in itself but rather an indication of a pathologic process in the fluid production and/or removal system, or an accumulation from an ectopic source.
1. Fluid Collection
Most fluid transudates can be collected through a 16- to 20-gauge needle, whereas a 14- to 16-gauge needle may be required for exudates. A sample of the effusion should be collected in an EDTA tube to be used for a total nucleated cell count, total protein determination, and cytologic examination. Another sample should be collected in a serum tube if any biochemical analyses are to be performed.
Pleural effusions are typically abundant and bilateral but may be mild, unilateral and/or compartmentalized. Radiography is helpful in determining the extent and location of the effusion. Under most circumstances a 1- to 1 1/2-inch needle will suffice, except in some large domestic animals in which a 2- to 2 1/2- inch needle may be required. The site for thoracentesis varies, but the seventh or eighth intercostal space is chosen in most animals. The puncture should be made in the middle of the intercostal space to avoid damaging intercostal vessels found just caudal to the ribs. Thoracentesis is preferably performed with the animal in a standing position. In cases of accidental blood contamination, blood will be apparent either during the first or very last portion of the aspiration. Blood will be present throughout the aspiration if hemothorax is present
In small animals, the ventral midline of the abdomen, 1-2 cm caudal to the umbilicus, is the usual site of needle insertion. In the horse, the preferred site is at the lowest portion of the abdomen between the xiphisternum and umbilicus. A 1-inch needle is generally sufficient for small animals and a 2-inch needle or teat cannula for horses. Open-needle abdominocentesis is reportedly more sensitive than aspiration via syringe; therefore, a syringe is usually not attached. If a syringe is attached, only mild negative pressure should be applied because it is easy to aspirate omentum or other abdominal contents against the needle opening and inhibit fluid collection.
Fluid is less frequently removed from the pericardium, but if there is an abnormal accumulation or pericardial fluid, percardicentesis may be of diagnostic and therapeutic value. An 18- to 20-gauge needle of sufficient length to reach the pericardium is required. The site of for puncture is at the cardiac apex. After the cardiac apex beat is located, the needle is carefully advanced toward the heart. A constant negative pressure is maintained in the syringe. When the needle enters the pericardial sac, fluid will appear in the syringe. If the needle contacts the cardiac wall, pulsation occurs and the needle will move. Should this occur, the needle location is slightly altered in order to prevent damage to the heart or its blood vessels. A small quantity (0.5 to 1.0 ml) of fluid is adequate for cytologic evaluation. If the fluid collected is bloody and clotted, this usually indicates that a blood vessel has been entered during collection. If a bloody fluid does not clot, this is an indication that defibrinated blood is present in the pericardial sac.
2. Physical Classification of Effusions
On the basis of nucleated cell counts and total protein concentrations, effusions may be classified as either transudates, modified transudates or exudates. A transudate is an excess accumulation of normal fluid (filtrate of plasma) in any body cavity or space. Transudate accumulations result primarily from obstructive phenomena and usually contain few cells and have low specific gravity and protein content. However, any transudate that remains in a body cavity for a period of time causes irritation to the lining of that cavity. This irritation generally results in increased cellularity, primarily due to proliferated mesothelial cells, and an increase in protein content that is generally due to fluid leakage from lymphatics. Fluids of this type are termed modified transudates. An exudate is an excess accumulation of abnormal fluid in any body cavity or space. It is usually the result of both obstructive and productive phenomena. The cellular and protein content are generally much higher than that of transudates. Since most exudates are inflammatory, this increased cellularity is usually due to accumulations of white blood cells.
The general parameters evaluated in effusion cytology include:
a. Physical and Chemical Characteristics
- 1) Color
- a) serum or plasma - light or dark yellow
- b) Erythrocytes - red, dark red, or reddish brown
- c.) Leukocytes - white, cream, yellow, gray or green
- d) Chyle - milky white (after centrifugation), peach or pink
- e) Bilirubin - yellow, orange, red or brown
- 2) Transparency or Turbidity
- a) Erythrocytes - grayish-red turbidity
- b) Leukocytes - from light turbidity to thick, creamy pus
- c) Fat droplets - milky turbidity
- 3) Coagulation - depends upon the amount of fibrin present.
- a) Transudates - rarely show spontaneous complete or partial coagulation
- b) Exudates - usually coagulate very quickly after removal from the serous cavity.
- 4) Protein Concentration - Refractometer
- a) Transudates less than 3 g/dl
- b) Exudates more than 3 g/dl
- 5) Specific Gravity - Refractometer
- a) Transudates under 1.017
- b) Exudates over 1.017
- 6) Bilirubin, BUN, Creatinine, globulin, amylase, cholesterol, triglycerides and other determinations may facilitate interpretation.
b. Cellular Elements
The approach to cytologic evaluation of fluids is essentially the same as the approach to the cytologic evaluation of solid masses. The populations of cells present is determined and compared to those found in the normal fluid of the given cavity. It must then be decided if the cells in the fluid accumulation are normal, the result of inflammation, or the result of neoplasia.
Normally, fluid content of the body cavities is low and contains few cells. Occasional white blood cells with the same features as those in the peripheral blood will be seen. Additionally, mesothelial lining cells are seen. The following is a list and description of those cells which may be present in variable numbers in effusions:
1) Mesothelial Cell
These cells line the serous surfaces of the pleural, pericardial and peritoneal cavities. With Romanowsky stain, the normal exfoliated cell is a large cell (12-30 microns), with extreme cytoplasmic basophilia which may obscure the nucleus. The cytoplasmic margin may or may not present reddish hairlike processes (eosinophilic brush border). Nuclei are of uniform size and are hyperchromic. Binucleation may be observed.
Pale-staining mesothelial cells may also be observed and represent in situ cells removed during the centesis procedure. The cells are usually in clusters. The nuclei are of uniform shape and size. The density of RNA and DNA is low, giving a pale-staining overall appearance to the cells.
The accumulation of large quantities of transudative fluid causes mesothelial cells to exfoliate in relatively large numbers. The transudate fluid is biologically a perfect cell-culture medium in which the mesothelial cells may replicate and transform into so-called blast-transformed, reactive or activated mesothelial cells. These are highly variable in cytologic appearance. The nuclei are generally less hyperchromic than those of non-transformed mesothelial cells. The chromatin generally has a finely reticular pattern and nucleoli may be present. Mitotic figures and multinucleate forms are common. The cytoplasm is slightly basophilic and may contain phagocytic debris, since activated mesothelial cells may become phagocytic. For this reason, they cannot be differentiated from the monocyte-derived macrophage.
Blast-transformed mesothelial cells often imbibe water and swell to unusually large size. The swollen cells have a clear cytoplasmic area with the nucleus displaced, giving a signet-ring appearance to the cell. The transformed cells may occur singly but more often , the cells suspended in the fluid do not divide completely and result in clusters of cells.
Since many of the morphologic criteria of malignancy are observed in blast-transformed mesothelial cells, it is important that the nuclear criteria be stringently applied when differentiating reactive mesothelial cells from neoplasia.
Mesothelial-cell reactions are accompanied by variable numbers of macrophages, depending on the amount of particulate detritus. Macrophages tend to be sticky and form cell clusters of a pale or light appearance. The cells are large (15-50 microns) and they generally have a single oval to bean-shaped nucleus; however, multinucleation and variable nuclear shapes may be present. Their nuclear chromatin is lacy and their cytoplasm is frequently vacuolated and may contain phagocytic debris.
Neutrophils are present to some degree in most effusions and tend to predominate in effusions associated with inflammation. The two general cytological classifications, non-degenerate and degenerate, and their significance have been discussed in a previous section of this syllabus.
4) Lymphocytes and Plasma Cells
Lymphocytes are present in small numbers in many effusions and may be predominant in chylous, pseudochylous and lymphosarcomatous effusions. Care must be taken not to confuse reactive lymphocytes in inflammatory conditions with neoplastic lymphocytes. Neoplastic lymphocytes are usually lymphoblasts and are characterized by a moderate amount of clear to blue cytoplasm, variably shaped nuclei, finely stippled nuclear chromatin and nucleoli of variable shape and size. These cells are larger than neutrophils.
Moderate to large numbers of eosinophils may be seen in effusions secondary to mast-cell tumors, heartworms, allergic reactions and hypersensitivity. They are commonly seen in bovine aspirates, perhaps due to the high incidence of Setaria nematodes within the peritoneal cavity of cattle.
6) Mast Cells
Mast-cell tumors within body cavities may be associated with effusions and frequently exfoliate large numbers of mast cells into the effusion. However, mast cells are observed in small numbers in effusions from dogs and cats with many different inflammatory disorders.
Erythrocytes may be seen cytologically within effusions secondary to hemorrhage or contamination with peripheral blood. Erythrocytes are useful as a marker in measuring size of other cells. It is important to differentiate bloody taps from true intracavity hemorrhage. This differentiation is based upon clinical signs and the presence or absence of intact platelets and erythrophagocytosis. Macrophages containing hemoglobin pigment are indications of chronic hemorrhage. A hemorrhagic effusion has a PCV and WBC counts similar to pripheral blood. Modified transudates and exudates containing blood usually have a PCV of 5% or less.
Neoplastic cells may be observed in effusions with many different types of neoplasia. Distinction of transformed mesothelial cells from malignant cells in effusions may be difficult by ctyologic study. Identification of the neoplastic cells depends upon recognition of the cell type and malignancy criteria. Various carcinomas, adenocarcinomas, lympho-sarcomas, mast-cell tumors, hemangiosarcomas and mesotheliomas may exfoliate neoplastic cells.
c.Type of Effusions and Their Causes
Attempts are made to classify effusions as transudates, modified transudates and exudates. This is often difficult because of the overlapping of many of the parameters. In attempts to simplify this procedure, the following discussion on classification is based solely on the total nucleated cell count (TNCC) and total protein (TP) concentration of the fluids.
- 1)Pure Transudate
Transudates are usually due to decreased plasma oncotic pressure,principally from hypoalbuminemia. Characteristics of a transudate are:
Colorless and clear fluid
- Less than 3.0 g/dl protein in canine and feline thoracic and abdominal fluid.
- Less than 2.0 g/dl for thoracic fluid in large animals
- Less than 1.5 g/dl for horses and less than 3.5 g/dl for ruminant abdominal fluids
- Less than 1000 nucleated cells/ul in canine and feline fluids
- Less than 5000 nucleated cells/ul in large animal fluids
Cell population consists of nondegenerate neutrophils, macrophages and reactive or trans-formed mesothelial cells in low numbers
- 2)Modified Transudate
Most modified transudates occur as a result of fluid leakage from lymphatics or blood vessels. Such leakage is caused by increases in hydrostatic pressure or permeability. Both of these conditions allow high-protein ultrafiltrate fluid to pass into the cavity. Neither of these conditions results in chemotactants in the cavity; therefore, large numbers of inflammatory cells do not migrate into the fluid. This produces a high protein fluid with low to moderate nucleated cell counts.
Modified transudates are the least specific classification of effusions. Effusions in this classification can develop secondary to many nonspecific disorders. Most modified transudates are transitory and may progress to nonseptic exudates. The effusion itself may initiate an inflammatory response. Some of the general characteristics of modified transudates are as follows:
- Low to moderate cellularity with TNCC or between 1000 - 7000 cells/ul
- Total protein concentrations of between 2.5-7.5 g/dl
- Color variation from amber to white to red and frequently slightly turbid to turbid
Nondegenerate neutrophils, meosthelial/macro-phage cell types, small lymphocytes or neoplastic cells may predominate, depending on the cause of the effusion.
Exudates occur most commonly due to chemotactants in the cavity as a consequence of an inflammatory process. Occasionally, an exudate may develop due to abundant exfoliation of cells from a tumor or secondary to a chylous effusion. Any modified transudate may develop into an exudate. The general characteristics of exudates are:
- High protein concentrations of greater than 3.0 g/dl
- High TNCC or >7000 cells/ul
- Color variation from amber to white to red
- Turbid to cloudy
The predominant cell type is dependent upon the cause of the exudate. In inflammatory exudates, the neutrophil generally predominates with variable numbers of macrophages and lymphocytes.
Inflammatory exudates are generally divided into two main categories, non-septic and septic.
- This type of effusion is the product of inflammation caused by irritants that are not toxic to neutrophils.
- Causes include gallbladder or urinary bladder rupture and sterile foreign bodies.
- It may develop from any of the causes of modified transudates.
- (1)White or pink and cloudy fluid
- (2)3.0-5.0 g/dl of protein
- (3)3000-50,000 or more cells/ul
- (4)Nondegenerate neutrophils and variable numbers of macrophages, erythrocytes and lymphocytes. Small numbers of cells may have pyknotic and karyorrhectic nuclei.
- They are caused by a wide variety of microorganisms that are toxic to neutrophils
- Septic exudates are characterized by:
- (1)White, red, or yellowish, cloudy fluid
- (2)3.0-5.5 g/dl of protein
- (3)300-100,000 or more cells/ul
- (4)Degenerate neutrophils and variable numbers of macrophages
- (5)Intracellular and/or extracellular bacteria
- Exudates caused by Nocardia sp. or Actinomyces sp. in the dog have special features:
- (1)Microcolonies composed of pleomorphic, filamentous rods are observed. Some organisms occur in branching chains with a beaded appearance.
- (2)Neutrophils around the microcolony are degenerate, whereas neutrophils distant from the microcolony or in aspirates not containing microcolonies are nondegen-erate.
d.Effusions in Selected Disorders
1)Infectious Peritonitis and Pleuritis
Peritonitis and pleuritis are associated with release of chemotactants and vasoactive substances within the respective cavities. This results in an exudative effusion due to increased capillary permeability with a massive outpouring of peripheral blood neutrophils and high-protein plasma filtrate. Degenerate neutrophils generally predominate in bacterial infections and intracellular and/or extracellular organisms may be seen. Spirochetes occasionally are seen in association with bacterial peritonitis and pleuritis, especially secondary to bite wounds. Mycotic, protozoal and rickettsial organisms may also be etiological agents in infectious peritonitis/pleuritis.
Intracavity organ inflammation (e.g., heart, liver, spleen, lymph nodes, lungs, etc.) or a walled-off abscess may result in an associated body cavity effusion. The mechanisms are similar to those described for infectious peritonitis/pleuritis in that inflammation causes the release of chemotactic and vasoactive substances. These in turn result in the influx of inflammatory cells and high protein fluid into the involved area. When this process extends into the associated cavity, effusive inflammation results. TNCC and TP levels will determine if this effusion is classified as a modified transudate or exudate. In this type of effusion, nondegenerate neutrophils generally predominate, but macrophages, mesothelial cells and lymphocytes are also present. The mononuclear cell component will increase with chronicity.
Cytological evaluation of these effusions is typically nondiagnostic as to etiology or to the site of original tissue involvement and must be correlated with physical findings, history and other laboratory test results to pinpoint the exact problem.
3)Feline Infectious Peritonitis (FIP)
An effusion associated with FIP is one cause of a nonseptic exudate (pleural and/or peritoneal) in cats. This effusion is an odorless, straw-colored to golden, tenacious fluid that may contain flecks or fibrin strands. The fluid is generally cloudy and has an extremely high protein concentration (3.5 to 8.0 g/dl) consisting predominantly of gamma globulin. This high protein concentration and fine particles of cell detritus give Wright's stained smears of the fluid a precipitous eosinophilic background. NMB-stained fluids may show a background of fibrin in needle-like meshwork form. There is a mild to marked increase in the TNCC (can vary from less than 1000 to 23,000 or more cells/ul) consisting primarily of nondegenerate neutrophils (60-80%) and lesser numbers of macrophages, lymphocytes and occasionally plasma cells. In chronic effusions, the percentage of neutrophils may decrease as the number of mononuclear cells increases. Blast-transformed mesothelial cells and associated cell clusters are seldom found in FIP fluids.
While the cytological findings are not diagnostic alone, when associated with history and clinical findings, a presumptive diagnosis of FIP can be made.
Rupture of the gall bladder or bile duct can produce a nonseptic exudative effusion. The effusion is discolored by bile pigments giving it a typical yellow-orange color. Bile is an irritant and results in increased cell counts, particularly neutrophils and macrophages. The fluid will give a positive icotest and is high in cholesterol. In Wright's stained smears, bile peritonitis is characterized by phagocytized yellow green to brown green pigment within macrophages.
Rupture of the kidney, urinary bladder, urinary tract or patent urachus that results in release of urine in the abdominal cavity can also produce a nonseptic exudative effusion. Urine is an irritant and results in increased in mononuclear cell count. Very acute rupture causes an elevated urea nitrogen in the abdominal fluid (compared to blood urea nitrogen levels); however, this is rapidly reabsorbed by the lining tissues and levels soon equilibrate with blood levels (generally within 24 hours). Creatinine is more poorly absorbed by the lining tissues and will remain elevated above blood levels for longer periods. Therefore, measurement of the creatinine level is more reliable than measurement of the urea nitrogen level.
Chylous effusions can occur secondary to many disease processes, including trauma, heart disease, inflammation and neoplasia. These effusions in dogs and cats seem to occur most frequently in the thorax and are generally bilateral. Chylous effusions occur as a result of lymphatic duct leakage or rupture. These effusions are odorless and vary in color from milky white to yellow or pink, depending on the animal's diet and the number of RBCs in the fluid. Overnight refrigeration of the chylous fluid will typically produce a "cream layer". Tests for protein levels give inaccurate values because of interference by lipid droplets. TNCC are usually less than 10,000/ul. While small lymphocytes are typically thought of as the predominant cell type, chylous effusions occur in which neutrophil and/or macrophages predominate. Lysed cells are common in effusions rich in lymphoctyes. Bacterial infection is uncommon due to the bacteriostatic effect of the fatty acids in chyle.
Most milky white effusions are chylous, but occasionally they are pseudochylous. These effusions most commonly occur with neoplastic of inflammatory disorders, but may be associated with any chronic effusion. Unlike chylous effusions, the white color of pseudochylous effusions is not due to fat (chyle) but to cellular debris, protein, lecithin globulin complexes and/or cholesterol granules.
Chylous and pseudochylous effusions have been differentiated by staining smears with fat stains such as Sudan III. A positive stain confirms the presence of a chylous effusion; however, since cytologically identifiable fat may be absent from some chylous effusions, a negative result does not confirm pseudochylous effusion. The use of ether to clear the chylous effusion is also not a reliable differentiation test.
The best available diagnostic test for differentiation between chylous and pseudochylous effusion is measurement of the triglyceride and cholesterol concentrations in both the effusion and peripheral blood. With chylous effusions, the triglyceride concentration is higher in the effusion than in the serum and the cholesterol concentration is higher in the serum than in the effusion. The reverse is true of pseudochylous effusions. Also, the cholesterol/ triglyceride ratio of the effusion fluid is <1.0 in chylous effusions.
7)Heart Disease Characterized by Heart Failure
A common sequelae to heart failure or liver congestion is the development of a modified transudate effusion termed ascites. This effusion develops secondary to increased intrahepatic pressure and leakage of high-protein hepatic lymph. Some specific characteristics of this fluid are:
- -pink to red and cloudy fluid
- -2.5-5.0 g/dl protein
- -300-5500 cells/ul
- -Variable numbers of erythrocytes, nondegenerate neutrophils, mesothelial cells, macrophages , and occasionally eosinophils and lymphocytes
There is no cytologic finding in these effusions that is pathognomonic for heart failure and other clinical examinations are necessary to establish the diagnosis.
Cats may develop a pleural effusion secondary to feline chronic congestive heart failure (cardio-myopathy). These effusions are yellow to milky white and typically consist of >50% small mature lymphocytes. While the color of the fluid may be similar to that of a chylous effusion, the triglyceride concentration may or may not be increased.
Hemorrhagic effusions are usually caused by trauma, surgery, infarction of the intestine, neoplasia and hemostatic defects. Hemorrhagic effusion must be differentiated from blood contamination due to bloody taps and organ taps (e.g., liver and spleen). Unfortunately, differentiating blood contaimination from acute hemorrhage can be difficult. However, if acute hemorrhage is severe, clinical signs of blood loss should be evident. When blood enters a body cavity, the platelets quickly aggregate, degranulate and will disappear within 45 minutes. In conjunction with these platelet changes, the blood will coagulate rapidly and then undergo mechanical defibrination. Therefore, hemorrhagic effusions do not clot. Also, within hours of release into the cavity, the RBCs are phagocytized and digested by macrophages. These and other factors can be used to confirm the existence of hemorrhage and to estimate its duration.
Recent hemorrhage (within several hours) is characterized by:
- Clear supernatant fluid and red sediment
- Protein and cell count values similar to those of peripheral blood.
- Intact erythrocytes
- Leukocytes of similar morphology and distribution as those in peripheral blood
- Platelet aggregates
More longstanding or resolving hemorrhagic effusions are characterized by:
- Erythrocytes and leukocytes that appear older and distorted.
- Supernatant fluid may be pink due to hemolysis
- Absence of platelets
- Macrophages with phagocytized erythrocytes and hemosiderin and/or hematoidin crystals.
In summary, the absence of erythrophagocytosis and presence of platelets suggest either peracute hemorrhage or a bloody tap. The presence of erythrophagocytosis and platelets suggests either chronic persistent hemorrhage or previous hemorrhage and a bloody tap. The presence of erythrophagocytosis and absence of platelets suggest chronic or previous hemorrhage.
The characteristics of an effusion associated with neoplasm will be variable depending upon the tumor involved. Neoplastic effusions may have characteris-tics of obstructive, inflammatory, or hemorrhagic effusions. Most effusions caused by tumors not exfoliating neoplastic cells are modified transudates. Effusions caused by tumors that are exfoliating cells into the cavity and/or are secondarily inflamed will be exudative in nature. In all cases, particularly in inflammatory exudates, care must be taken not to confuse dysplastic cells and/or normal or reactive mesothelial cells with neoplastic cells.
The most common cause of feline neoplastic pleural effusion is mediastinal Lymphosarcoma. In horses, lymphosarcoma is also the most commonly identified neoplasm in pleural and peritoneal effusions. These fluids are generally characterized by:
- Whitish to pink and cloudy fluid
- 3.0-6.0 g/dl of protein
- 4500-45,000 cells/ul
- Immature lymphocytes, usually lymphoblasts
Other neoplasms which may exfoliate into the body cavities include mast-cell tumors, mesothelioma and various carcinomas, adenocarcinomas and sarcomas. Malignant mesothelioma, a primary pleural or peritoneal tumor, is a rare cause of effusion in animals. They are extremely difficult to diagnose cytologically because of the variability of normal mesothelial cells. Diagnosis generally requires a biopsy and it is difficult even on histopathologic examination to make a definitive diagnosis.
Since many tumors do not exfoliate neoplastic cells, the absence of neoplastic cells within effusions does not rule out neoplasia. As in many instances, a positive finding is positive but a negative finding necessitates further diagnostic evaluation.
Evaluation of synovial fluid is a valuable adjunct to assessment of many lameness cases. In addition to cytologic evaluation, the fluid should be assessed for volume obtained, color, turbidity, viscosity, mucin (hyaluronic acid) quality/concentration, and protein concentration.
Based on laboratory tests and cytological findings, arthropathies can usually be categorized as hemorrhagic, degenerative (non-inflammatory, non-exudative) or inflammatory (exudative) in origin. Unfortunately, all synovial fluid specimens will not fall neatly into one of these categories. This is especially true of longer-standing problems which tend to develop characteristics of more than one category, thus making classification and interpretation more difficult.
Degenerative or non-inflammatory joint diseases usually result from trauma to a joint and/or degenerative changes of the articular surfaces. The latter may be caused by acute or chronic trauma as a result of accidental injury or congenital or acquired anatomic disorders that predispose articular surfaces to abnormal stresses. Additional causes include metabolic and nutritional abnormalities and neoplasia.
Inflammatory joint diseases include those caused by both infectious and noninfectious causes. Both are usually associated with an effusive exudate showing a moderate to marked increase in neutrophil numbers and variable increase in large mononuclear cell numbers. Etiological agents associated with septic arthritides in the various species include bacteria, fungus, Mycoplasma, Protozoa, Rickettsia and Virus. The noninfectious inflammatory joint diseases can be subgrouped into immune-mediated and nonimmune-mediated causes.
Normal synovial fluid is essentially a dialysate of plasma. Hence, concentrations of electrolytes and nonelectrolytes (such as glucose and urea) are similar to those in blood. It is essentially without fibrinogen and most other clotting factors and therefore does not clot, even in vitro. With blood contaimination, intraarticular hemorrhage or protein exudation in various inflammatory diseases, samples may clot unless processed immediately or added to an anticoagulant. EDTA is preferred for cytological examination. Heparin is recommended for the mucin clot test because EDTA interferes with the test by degrading hyaluronic acid. Both anticoagu-lants are suitable for other routine tests.
Cytololgical examination of synovial fluid requires properly made smears. Since normal synovial fluid is viscous, direct smears must be made by slowly advancing the spreader slide to create thin smears. The high viscosity of the fluid will result in cells evenly dispersed in the smear. Cells will not accumulate at the feathered edge unless the viscosity of the fluid has been altered by dilution with exudative effusion.
The background material is mucin and the density reflects the thickness of the smear. In Wright's stained specimens, the background is granular. Clefts in the background may result from fibrin formation and separation of the mucin.
Synovial fluid has a limited number of ways in which it can respond in disease. Therefore, the major questions in examination of synovial fluid are, Is there inflammation? and is there sepsis? Normal synovial fluid contains low numbers of mononuclear cells. Lymphocytes predominate and monocytes-macrophages are variable. Occasionally, synovial lining cells may be present. Neutrophils and RBCs are rare in normal synovial fluid, and when found, are usually the result of blood contamination from the sampling procedure. In small animals, inflammation is determined by finding more than 3000 WBCs/mm3 or greater than 12% of the cells are neutrophils. In large animals, nucleated cell counts greater than 1000 cells/ul (500 cells/ul in the horse) with similar increases in the percentage of neutrophils is indicative of inflammation. In all species, sepsis is determined by finding degenerated WBCs or bacterial (especially if engulfed by WBCs). For unknown reasons, it is often much more difficult to detect etiologic agents, particularly bacteria, in synovial fluid than in aspirates from other body sites. Culture of the fluid is generally required to detect or confirm infectious agents.
RBC counts increase with blood contamination, traumatic conditions and with certain inflammatory conditions. The highest elevation of WBC cell counts occur in septic and autoimmune arthritis. Mild increases occur in traumatic and degenerative disease. Lymphocytes and monocytes comprise 90% of the cells in normal fluid and predominate in tarsal hydrarthrosis and traumatic and degenerative joint diseases. Neutrophils predominate in bacterial and certain nonseptic inflammatory diseases; e.g., lupus erythematosus (SLE) and rheumatoid arthritis (RA). Occasionally, osteoclasts, multinucleated giant cells, may be found in smears and are indicative of cartilage erosion and exposed bone.
Removal and laboratory examination of CSF are indicated whenever there is clinical evidence suggesting CNS disease. Lesions involving the CNS do not consistently or uniformly cause changes in the CSF. The results of CSF analysis may be within normal limits in many instances of neurologic disease, and even when CSF cytologic abnormalities are present, they are often nonspecific. Alterations of CSF probably depend more on the location and extent of the CNS lesion than on cellular abnormalities. Thus, disease of the meninges produces greater alterations in CSF than do most diseases of CNS parenchymal tissue. Septic meningitis may cause suppuration within the CSF; inflammatory cells may be numerous and levels of exudative protein markedly increased. In contrast, viral disease affecting the CNS, such as canine distemper, usually cause only a mild increase in CSF nucleated cell numbers.
Collected CSF should be placed in EDTA anticoagulant at the same concentration as used for blood samples. Refrigeration at 4o C also aids cell preservation. Because cells in CSF sometimes degenerate rapidly, cell counts and cytologic examination should be performed within 30 minutes of collection.
Turbidity will be apparent when the CSF contains 300-500 cells/ul or more. Bacteria or mycotic agents can contribute to turbidity. Bacterial meningitis may be only slightly turbid to almost pure pus which may clot. Viral encephalitis, trauma, tumor, abscess may show turbidity due to large amounts of protein, fibrin and/or cells.
Normal CSF does not coagulate. Coagulation may occur if damage to the blood brain barrier permits fibrinogen to enter the CSF or if CSF collection results in hemorrhage.
a.Total Nucleated Counts
Total cell counts of CSF can be determined by using a hemocytometer with a Neubauer ruling. A capillary pipette is used to place CSF on one chamber of the hemocytometer. The cells in all 9 squares are counted and the total multiplied by 1.1 to determine the total cell count/ul. Differentiation of RBC and nucleated cells is done using the high-dry objective of the microscope. Nucleated cells are larger than RBC and have a granular appearance.
Normal CSF is free of RBCs and contains <8 nucleated cells/ul in dogs and cats and <5/ul in all large animal species. Total nucleated cells counts in the CSF are mildly elevated (pleocytosis) in a variety of disorders, and, though cytologic findings may suggest that inflammation is chronic (primarily macrophages) or chronic-active (nearly equal numbers of macrophages and neutrophils), no specific etiology may be apparent. Nucleated cell counts and cytologic findings of CSF examination can be difficult to fit into classifications conforming to specific disease. All classifications appear to overlap. Pleocytosis of CSF indicates abnormality, but CNS disease may exist in which there is no pleocytosis and the cells which are present are normal.
b.Differential Cell counts
The nucleated cell population of normal CSF is predominantly mononuclear consisting of small lymphocytes and a few macrophages, endothelial cells and histiocytes. Neutrophils are not normally found in CSF unless the tap is traumatic. They should not constitute >10% of the total cell count.
Neutrophilic pleocytosis usually indicates pyogenic or bacterial infection, an abscess, bacterial encephalitis or meningitis. Under these conditions, counts may vary from 50 to over a 1000 cells/ul. Elevated neutrophil numbers may also occur in noninfectious inflammatory conditions. Attempts should be made to identify bacteria and the fluid should be cultured. In ruminants, pigment granules are common and must be distinguished from bacteria.
The CSF of animals with mycotic and protozoal encephalitides will have varied pleocytosis, consisting of neutrophils, mononuclear cells or eosinophils. An increase in TNCC consisting primarily of lymphocytic cells may indicate viral infections, uremia, fungal infections, postvaccinal inflammation, chronic infection and toxic conditions. Monocytes/macrophages are often associated with lymphocytes and will increase in conjunction with lymphocytes. They may predominate in the CSF in FIP.
Pleocytosis is rarely observed in noninflammatory conditions involving the CNS. In animals with spinal trauma, a slight increase in cell numbers may occur. Animals with congenital malformations of the CNS generally have normal CSF. Animals with cerebral infarcts may have increased numbers of RBC in the CSF and erythrophagocytosis by macrophages is a diagnostic feature of this condition.
Neoplastic cells may be evident if the meninges are involved. Filtration techniques are usually needed to demonstrate these cells. In most CNS tumors, CSF samples will have a normal or only slightly increased nucleated cell count consisting predominantly of mononuclear cells.
Evaluation of an enlarged prostate may be greatly enhanced by cytological evaluation. Clinical signs suggestive of prostatic enlargement include difficult defecation or difficult micturition. Rectal palpation of the prostate may reveal symmetric enlargement, unilateral enlargement or focal irregularities.
1.Collection and Preparation of Specimens
There are several ways in which cytological specimens may be obtained from the prostate gland. Prostatic fluid may be obtained by digital massage while aspirating with a syringe through a urinary catheter passed to the level of the gland. In this procedure, the tip of the catheter can be palpated per rectum as it approaches the base of the prostate. Care should be taken to properly lubricate the catheter and cleanse the tip of the penis to assure clean technique and to avoid introducing infection. This technique may cause fluid to be drawn into the syringe or only a few cells may be drawn into the tip of the catheter. Following massage and aspiration, negative pressure is released and the catheter withdrawn. Cells collected at the tip of the catheter are placed on a slide, spread by the "squash-prep" technique and evaluated. Fluid should be kept sterile for bacterial culture if warranted.
More prostatic material can be obtained by prostatic washing. In this procedure, a small amount of saline is used to wash the urethral lumen in the area of the prostate. Gentle injection and aspiration is used while gently massaging the prostate. Fluid with low cellularity should be centrifuged to concentrate the cells for slide preparation.
In addition, direct fine-needle aspiration techniques can be used to obtain prostatic material for evaluation, especially if prostatic cysts are suspected.
Another less desirable method is to obtain material by inducing ejaculation. This procedure produces samples that contain contaminant substances from other parts of the reproductive tract.
The prostate consists of numerous crypts lined by cuboidal epithelium. In cytological preparations, sheets of cuboidal epithelium are commonly seen. In normal smears, these epithelial cells will be uniform with round to oval nuclei. The nuclei are central in location and have a homogeneous to fine reticular chromatin pattern. The cytoplasm may be granular and slightly acidophilic. Aspiration and wash specimens frequently contain cells of nonprostatic origin including spermatozoa, squamous cells from the distal urethra or external genitalia and urothelial cells.
A condition commonly seen in older dogs and believed to be caused by sex hormone imbalances is prostatic hyperplasia. Cytologically, the hyperplastic prostatic epithelial cells resemble normal cells but are usually greater in number. Large sheets and clusters may exfoliate. In these cell clusters, cytoplasmic boundaries are often indistinct. The cytoplasm is basophilic and slightly granular. Nuclei are round to oval and may be eccentric in location. Chromatin patterns are fine and nucleoli are generally not discernible. The nuclear/cytoplasmic ratio is increased as compared to normal prostatic epithelial cells. Inflammatory cells may occasionally be seen and suggest that the hyperplasia may be a result of inflammation.
Aspiration specimens of prostatic cysts are highly variable cytologically. Some cysts may contain poorly cellular fluid containing only a few epithelial cells and cellular debris. Sometimes moderate numbers of normal or slightly hyperplastic epithelial cells are found.
Squamous metaplasia of the prostate may occur under the influence of estrogen-like hormone activity, such as occurs in Sertoli-cell tumors, or as a sequel to chronic prostatic irritation or inflammation. Under these influences, the prostatic epithelium undergoes metaplasia to a squamous-like epithelium. Similar changes may also occur to some minor degree in normal older animals. Aspirates are moderately cellular with clusters of slightly basophilic to slightly acidophilic pale-staining cells. These cells are very large and have a flattened appearance. An occasional cell may contain a pyknotic or karyorrhectic nucleus. Occasional inflammatory cells and hyperplastic epithelial cells may be present.
Cytological specimens from an inflamed prostate will contain large numbers of inflammatory cells. Neutrophils are usually most numerous, but variable numbers of macrophages may also be present. Bacteria are frequently found and may represent an etiologic agent or a contaminant from some other region of the genital or urinary tract. Phagocytized bacteria are considered to be involved in the etiology of the prostatitis.
Specimens from prostatic abscesses will contain large numbers of degenerated neutrophils with a background of cellular debris. Single or small clusters of prostatic epithelial cells may be present and may exhibit hyperplastic to metaplastic changes.
Prostatic neoplasia generally results in prostatomegaly. The prostate may be very large, irregular and asymmetric. Aspirates of prostatic adenocarcinoma are moderately to markedly cellular. The neoplastic cells have the cytologic features common to adenocarcinoma of other organs. Anisokaryosis, nuclear enlargement, nuclear irregularity and an increased nuclear/cytoplasmic ratio are often evident. Nucleoli are present and are usually small, single and uniform, but large and sometimes irregular forms may occur. Cell membranes are usually distinct in well differentiated tumors but are indistinct in more poorly differentiated tumors. Cohesion of cells is often apparent and acinus formation may occur. Prostatic adenocarcinomas frequently metastasize to the iliac lymph nodes and cytological examination of these nodes should be performed in suspected adenocarcinoma cases. In addition to adenocarcinoma, transitional cell carcinomas may sometimes involve the prostate.
Diagnostic cytology of samples from the respiratory tract has been used extensively in dogs, cats and horses and to a lesser degree in ruminants. These procedures are used routinely in the evaluation of pulmonary disease. Examination of secretions of the tracheobronchial tree and associated structures can yield useful information on developing or established lower respiratory tract disease including hypersensitivity reactions, inflammation, infectious agents and neoplasia. Depending upon the method of collection used, the specimens obtained are of variable volume and concentration techniques may be used in conjunction with direct smears.
1.Methods of Collection
The most common procedures for obtaining specimens from the tracheal/bronchial tree include percutaneous or endoscopic tracheobronchial aspiration (TT/B) and bronchoalveolar lavage (BAL). The fluid used in these procedures tends to damage and/or distort cells and induces changes in inflammatory cells. Brush or punch biopsy of bronchial lesions is a more direct and satisfactory method for obtaining specimens when lesions are visible during bronchoscopy.
2.Cytological Evaluation of Tracheal/Bronchial Washes and Lavages
Oropharyngeal contamination is indicated by the presence of superficial squamous cells and Simonsiella bacteria. Superficial squamous cells are large epithelial cells with abundant, angular cytoplasm and a small round nucleus. Simonsiella bacteria divide lengthwise and line up in parallel rows. This gives the impression of a single large bacterium. These organisms are nonpathogenic and are often seen free in the smear or adhered to the surface of superficial squamous cells. Specimens from animals with aspiration pneumonia may also contain squamous epithelial cells; however, in these cases, there should also be an accompanying increase in degenerated neutrophils and a mixed intracellular bacterial population. Plant material and fungal hyphae or spores are occasionally observed in normal herbivores and do not necessarily indicate oral contamination in these species.
Mucus is seen in virtually all wash specimens, including those from normal animals. Mucus appears as blue to pink homogeneous strands. These may frequently be twisted or whorled. Mucus in inflammatory conditions stains eosinophilic due to incorporation of inflammatory proteins and material from lysed cells. Specimens from animals with chronic respiratory conditions causing excessive production of mucus, such as chronic obstructive pulmonary disease, may have mucus casts of small bronchioles. These are called Curschmann's spirals and appear as spirally twisted masses of mucus that may have perpendicular radiations that impart a test-tube-brush-like appearance.
Total cells counts on TT/B wash fluids are generally not performed because of the mucus content and the dilution of the specimen with saline. Total cell counts are usually performed on BAL specimens but care must be taken in their interpretation because of the dilution factor.
Erythrocytes may be seen in TT/B and BAL fluids with disorders causing vascular damage or RBC diapedesis into the lung. Intrapulmonary hemorrhage may be of pulmonary origin (e.g., trauma, infarct, foreign bodies, infectious diseases, neoplasia) or also may occur with disorders of non-pulmonary origin (e.g., heart failure, pulmonary embolism, hemostatic disorders). RBCs and/or RBC breakdown produces (hemosiderin, hematoidin) within alveolar macrophages is an indication of chronic hemorrhage. These cells are often referred to as heart failure cells; however, they may occur as a result of any disorder which causes intrapulmonary hemorrhage. Hemosiderin-laden macrophages are common in high-speed performance horses that have experienced pulmonary hemorrhage at least 72 hours previously. The presence of hemosiderin can be confirmed with special stains (e.g., Prussian blue) which stain iron-containing compounds, such as hemosiderin, blue.
The predominant cell types observed in TT/B wash specimens from normal animals include respiratory epithelial cells and alveolar macrophages with low numbers of nondegenerate neutrophils and small lymphocytes. In pathologic conditions, increased numbers of inflammatory cells (neutrophils, eosinophils, lymphocytes and macrophages), RBCs, mast cells, and/or dysplastic and neoplastic cells may be present.
Types of epithelial cells that may be observed in TT/B wash and BAL specimens include ciliated and nonciliated columnar and cuboidal cells and mucus-secretory cells.
Generally, the most abundant cell type is the ciliated columnar epithelial cell. These cells are elongated or cone shaped with apically located cilia and a basally located nucleus. The basal cytoplasm frequently terminates in a tail. The nucleus is round to oval with a finely granular chromatin pattern. The non-ciliated columnar cell is identical to these cells but does not have cilia. Ciliated and nonciliated cuboidal epithelial cells resemble their columnar counterparts except that they are as wide as they are tall. These latter cells are generally from the bronchial regions and are, therefore, more common in BAL specimens.
Goblet cells are not commonly seen in specimens from normal animals; however, any chronic pulmonary irritant may result in increases in their numbers. These cells appear as an elongated or columnar cell with a basally placed nucleus. They secrete mucus which appears as coarse metachromatic cytoplasmic granules which frequently distend the cytoplasm. Occasionally, the cytoplasm may be so distended as to give a rounded shape to the cell. Granules from ruptured goblet cells may be seen free in the smear.
Alveolar macrophages are readily observed in TT/B washings from normal animals and may be the predominant cell type. Depending upon the pathologic state, macrophages vary considerably in size and activity level. Unactivated alveolar macrophages are rounded mononuclear cells 15 to 20 microns in diameter with abundant blue-gray, granular cytoplasm. The nucleus is round to bean shaped and eccentrically positioned. Activated macrophages will have more abundant cytoplasm that will be vacuolated or foamy and may contain phagocytized material. These activated cells may form huge, multinucleated phagocytic cells that may exceed 80 microns in diameter. These multinucleated forms are common in chronic lung diseases such as chronic obstructive pulmonary disease.
Increased numbers of macrophages are seen in many subacute and chronic lung disorders such as congestive heart failure, granulomatous pneumonia, lipid pneumonia and various chronic persistent inflammatory processes. Various pigments from RBC breakdown (hematoidin, hemosiderin) or inhalation of polluted air (anthracotic pigment) may be found in the cytoplasm of phagocytic macrophages.
In specimens from normal animals, low numbers (generally <5%) of nondegenerate neutrophils may be present. These cells will resemble those of peripheral blood. Neutrophil numbers will be increased in nearly all conditions that cause inflammation, including both infectious and noninfectious conditions. Infectious disorders include bacterial, mycotic, viral and protozoal diseases. Noninfectious disorders include tissue irritation or necrosis secondary to inhalation of toxic substances and tissue necrosis resulting from neoplasia. In clinical cases of chronic obstructive pulmonary disease, tracheobronchial aspirates often show a rise in the numbers of nondegenerate neutrophils along with activated macrophages, particularly multinucleated giant cells.
As with other cytological specimens, assessment of neutrophil preservation is critical and the cells must be examined for degenerative changes indicative of infectious or toxic processes. Care must be taken in making this assessment in that smudging and/or rupture of neutrophils may result during collection and preparation of the specimen.
Eosinophils are present only in very small numbers (<5%) in TT/B and BAL fluids from normal animals. Eosinophils may be seen in increased numbers (>10%) with type I hypersensitivity reactions such as allergic processes (e.g., allergic bronchitis/ pneumonitis, feline asthma) and parasitic migration. Eosinophlic granuloma may demonstrate eosinophilic lung infiltrates in affected cats. Increased numbers are commonly seen in specimens from dogs infected with heartworm, dogs and cats infected with lungworm, foals infected with Parascaris equorum (during the pulmonary migration stage of the parasite), horses with Dictyocaulus arnfieldi infection and in cattle infected with Dictyocaulus viviparus. In addition to increased eosinophil numbers, parasite ova and/or larvae may occasionally be identified.
Rupture of eosinophils results in free granules present in smears and can be confused with bacteria. Eosinophil granules may coalesce into a large crystal known as a Charcot-Leyden crystal. These are elongated double pyramids and may occur in any condition that results in accumulation of large numbers of eosinophils. Increased numbers of neutrophils and macrophages may be seen along with increased numbers of eosinophils if tissue irritation is sufficient to induce an inflammatory response.
e.Miscellaneous Cell Types
Increased numbers of small lymphocytes in TT/B and BAL specimens generally denote nonspecific inflammation and are of limited diagnostic value. Mild increases in their numbers may occur with airway hyperreactivity, viral disease of the tracheobronchial tree and in chronic infections. Immune stimulated lymphocytes have abundant cytoplasm which stains deep-blue because of increased protein synthesis. Transformation into plasma cells with the characteristic nuclear halo or negative golgi apparatus may occur.
Increased numbers of mast cells may occur in specimens from animals with various inflammatory lung disorders. The presence is generally of little diagnostic significance.
Various atypical cells may be seen with pulmonary metaplasia, dysplasia or primary and metastatic neoplasia. Epithelial cells adapt to chronic irritation by undergoing metaplasia. The normal respiratory epithelium is replaced by stratified squamous epithelium. The metaplastic cells mimic maturing squamous epithelium and must not be confused with neoplastic cells. Epithelial cells will also react to irritation by becoming dysplastic. Dysplastic changes include variation in cell size and shape, increased nucleus/cytoplasm ratio, increased cell basophilia and increased numbers of immature cells. Care must be taken not to confuse inflammation induced dysplastic changes with neoplasia.
Unless the neoplasm has invaded the tracheobronchial tree and invaded bronchioles are not blocked by mucus plugs, neoplastic cells are rarely found in TT/B and BAL fluids. When neoplastic cells are observed, they are generally from adenocarcinomas. These large epithelial cells may be present as single cells or as clusters. They generally have basophilic, vacuolated cytoplasm and high nucleus/ cytoplasm ratios, coarse nuclear chromatin and prominent nucleoli.
Cytological procedures can be used to evaluate tissue masses and organ parenchyma for the purposes of detection and/or classification of pathological conditions. One of the most widely used applications of cytological evaluation is the identification and classification of tissue masses.
The usual composition of a mass is a proliferation of tissue cells, an accumulation of inflammatory cells, or both. The algorithm presented in Figure X.1 indicates the procedures for evaluating a cytological specimen from a mass. If it is determined that the cell population is primarily inflammatory, attempts should be made to classify the reaction and to identify an etiologic agent.
Tissue cells found on cytologic smears may arise from normal tissue, hyperplastic and/or dysplastic tissue, or neoplastic tissue. The observed cells indicate a proliferative tissue mass if they are noninflammatory tissue cells of one type; i.e., a uniform population. This conclusion is often made difficult by concurrent inflammation and necrosis in many neoplasms. The higher the percentage of inflammatory cells in the population, the lower one's confidence that the mass is not primarily inflammatory. The presence of inflammation can induce dysplastic changes in surrounding tissue cells which can be confused with neoplastic processes. Cells undergoing dysplasia may show mild to moderate variation in cell, nuclear and nucleolar size and shape, increased nucleus:cytoplasm ratio, and coarse chromatin. Dysplasia generally does not cause the bizarre nuclear and nucleolar morphology characteristic of neoplasia. In any case, care must be taken in the interpretation of these lesions. They should be biopsied or treated and re-evaluated cytologically when the inflammatory process has subsided.
The shape of the cells, their association with other cells, especially in tissue fragments, and cytoplasmic features are used to indicate the tissue of origin of a tumor (See Figure X.2). Once the cell type of a neoplasm is ascertained, the mass is evaluated for malignancy based upon the criteria listed in Figure X.3.
a.Basal Cell Tumor
Basal cell tumors are neoplasms of the basilar layer of the epidermis. They are common in cats and dogs. They can be pigmented, especially in cats, and often contain cystic spaces. The tumors are generally benign but can be locally invasive or malignant. Their malignancy potential is difficult to predict cytologically; however, it is reported that those exhibiting solid or basosquamous tendencies tend to behave malignantly.
The cell type found in these tumors is a primitive epithelial germ cell that does not exhibit differentiation toward squamous cells or adnexal structures. Histologically, these cells are arranged as palisaded cords or ribbons embedded in fibrous stroma. This pattern is caused by the tendency of basal cells to line up along basement membranes within the tumor.
The tumor is readily imprinted and aspirated and yields large numbers of cells arranged singly or in groups. Sometimes a row or ribbon of several cells may be found. Individual cells are uniformly small, approximately the size of a RBC, and dark staining. The N:C ratio is generally 1:1; therefore, only a scant amount of pale blue cytoplasm can be visualized in most of the cells. Their nuclei are variably-sized but regular in shape and have finely etched, lacy to finely stippled chromatin and contain multiple indistinct nucleoli. Mitotic figures may be common.
b.Squamous Cell Carcinoma
Squamous cell carcinoma originates in surface stratified squamous epithelium. It is among the most common of all dermal and oral neoplasms and is found in all domestic species. Due to its tendency to become ulcerated and inflamed, deep aspiration is the preferred method for obtaining cells for cytological examination. The cells generally exfoliate in clusters with variable numbers of individual cells.
Tumor cells from well-differentiated squamous cell carcinomas show slight cellular atypia and little evidence of neoplasia. These tumors are difficult to distinguish from dysplastic conditions of the skin resulting from chronic irritation or infection. The degree of differentiation of a squamous cell carcinoma is directly proportional to the degree of cellular anaplasia and indirectly proportional to the degree of keratinization. In poorly differentiated tumors, the cells exfoliate in clusters typical of other epithelial neoplasm; however, a few individual keratinizing cells usually are present which confirm an interpretation of squamous cell carcinoma.
In moderately to poorly differentiated tumors, marked variation in cell, nuclear and nucleolar size, nuclear and nucleolar number and shape, nucleus:cytoplasm ratio (1:3 to 1:5) and cytoplasmic basophilia occurs. Individual cellular morphology varies from normal large, mature squamous cells to small or medium-sized round cells, with a small amount of very basophilic cytoplasm and large, round nuclei that may have a very coarse, ropy chromatin pattern and contain multiple, prominent, irregularly shaped and sized nucleoli. Some cells may contain small clear vacuoles which occasionally aggregate around the nucleus and appear to coalesce, forming a clear ring around the nucleus (perinuclear halo).
c.Circumanal Gland Adenoma or Perianal Adenoma/ Adenocarcinoma
The perianal glands encircle the canine anus, and a few cells can be located in the skin of the tail, the prepuce, the thigh, and over the dorsum of the back. Perianal adenomas and adenocarcinomas can occur at any of these locations. These tumors are most commonly seen in the male. Perianal adenomas and adenocarcinomas often ulcerate because of pressure necrosis of the overlying epidermis and mechanical irritation of the tumor.
Cytologically and histopathologically, it is difficult to differentiate between perianal gland hyperplasia and benign neoplasia. Generally, large numbers of cells exfoliate and occur in clusters with a few scattered individual cells. The cells are uniform and medium in size and shape. They have a moderate amount of pink to tan-staining foamy cytoplasm. At high magnification, the cytoplasm may appear granular. The cells contain uniform rounded nuclei with 1-2 small round nucleoli. Because the cells resemble hepatocytes, the tumors are sometimes referred to as hepatoid gland tumors. Keratinization may be a feature in cells which have undergone squamous metaplasia. In some aspirates, a row of flattened reserve cells may be seen surrounding the clusters of larger cells. These cells are more basophilic and have N:C ratios of from 1:1 to 1:2.
Perianal gland adenocarcinomas may demonstrate numerous criteria of malignancy or they may be well-differentiated and difficult to distinguish from the benign tumor. The most common malignancy criteria demonstrated by these tumors are variation in nuclear and nucleolar size and variation in nucleolar number/cell.
d.Sebaceous Gland Adenomas/Adenocarcinomas
Tumors of the sebaceous glands are more common in older dogs and usually appear as wart-like growths. They generally exfoliate cells in groups and clusters and acinar patterns may be seen.
The predominant cell type in the sebaceous gland adenoma is the large mature secretory cell which has foamy cytoplasm and a small, central to slightly eccentric nucleus. The nucleus has dark staining slightly coarse chromatin and an indistinct or indiscernible nucleolus. The nucleus may be totally obscured by secretory material. In addition to these mature cells, adenomas may also contain basilar reserve cells. These cells are immature and contain little or no secretory material. Their cytoplasm is basophilic and the N:C ratio is approximately 1:2. Care must be taken not to interpret these cells as malignant.and contain little or no secretory material. Their cytoplasm is basophilic and the N:C ratio is approximately 1:2. Care must be taken not to interpret these cells as malignant.
Sebaceous gland adenocarcinomas are relatively uncommon. Cytologic preparations usually consist of groups of extremely basophilic reserve cells showing numerous criteria of malignancy. Only a few cells will contain secretory material. An occasional cell will produce a large amount of secretory material which presses the nucleus to the cell margin resulting in a signet-ring appearance.
e.Transitional Cell Carcinoma
Transitional cell carcinoma may have papillary, nonpapillary or invasive growth patterns. The most common site is the bladder trigone, but they may also occur in other areas of the bladder, the renal pelvis and the tubular system. Direct tumor aspiration and urine sediment evaluation reveal cells with similar cytologic features. Transitional cell carcinomas exfoliate isolated cells and cell clusters, frequently including very large clusters of cells with marked pleomorphism. The cytoplasm of these exfoliated cells is often deeply basophilic. In urine specimens, the cytoplasm may contain relatively light-staining vacuoles, indicating that the cells are undergoing hydropic degeneration. This degeneration may be a result of the toxic effects of urine upon the cells. Within the cellular clusters, cytoplasmic borders are usually indistinct and cell crowding is evidenced by cellular and nuclear molding. The nucleus: cytoplasm ratio is high in most cells; however, a few very large cells with abundant cytoplasm and a low nucleus:cytoplasm ratio are generally present. Nuclei are large, circular to ovoid, with finely to coarsely reticulated chromatin. Binucleate forms are common. Nucleoli may be indistinct to large and prominent.
Inflammation and other irritation can cause mesenchymal cells to become dysplastic and exhibit changes similar to neoplastic manifestations. In many cases, histopathologic examination is necessary to definitively diagnose these tumors. Since many of the spindle cell tumors are embedded in a dense ground substance, imprints generally contain few neoplastic cells and the ones obtained are discrete and individual. Scrapings generally results in a greater population of cells. As with imprints, cells obtained by scraping are generally not intimately associated.
Granulation tissue is composed of young proliferating fibroblasts and vascular tissue. Since these young fibroblasts are plump spindle cells with anaplastic characteristics, it is difficult cytologically to differentiate granulation tissue from fibrous tissue neoplasia. Like neoplastic fibroblasts, hyperplastic fibroblasts are active proliferating cells and exhibit large nuclei with multiple nucleoli and abundant cytoplasm with increased basophilia. However, unlike neoplasia, hyperplastic cells are proliferating under control mechanisms and therefore maintain a constant nuclear/cytoplasmic ratio. Additionally, nuclei of proliferating fibroblasts retain the normal finely granular chromatin pattern of the more mature normal fibrocyte. The differentiation between fibrous hyperplasia and fibrous neoplasia is particularly important in the equine where both sarcoids and excessive granulation tissue are common. Histopathological evaluation should be performed to make this definitive assessment.
Fibromas can occur in the dermis or subcutaneous tissue. Unlike fibrosarcomas, they seldom ulcerate. Scrapings yield a small number of cells arranged individually or in small groups. The cells are relatively uniform in size and shape and tend to have very elongated spindle shapes with a moderate amount of light blue cytoplasm. The nuclei are round to oval with a smooth to lacy chromatin pattern and may contain 1-2 small, round, indistinct nucleoli.
Fibrosarcomas can arise from cutaneous or subcutaneous tissues and may ulcerate and become secondarily infected. Fibrosarcomas of the dermis do not occur with great frequency in the dog but oral canine fibrosarcomas are relatively common. Sarcoids in horses resemble fibrosarcomas cytologically; however, they generally do not demonstrate malignant neoplastic behavior.
On cytological preparations, cells taken from fibrosarcomas are generally less spindle shaped than cells from fibromas. Many of the cells may be plump and/or oval shaped. Others may be stellate or have only a single indistinct tail of cytoplasm. The cytoplasm is often coarse and basophilic with Wright's stain or Diff Quik. Often small vacuoles are seen in the cytoplasm. Nucleoli may be singular and very large or multiple. With increased malignancy potential, large nucleoli are present and are often very irregular and angular. Chromatin is frequently clumped, particularly along the nuclear margin.
Mature lipocytes are large and distended with fat. They usually rupture during the aspiration procedure; therefore, aspirates of lipomas usually yield abundant free fat with a few lipocytes giving the smears an oily appearance which will not dry. Staining of the aspirate in an alcohol-based stain will dissolve the fat leaving clear areas and a few intact lipocytes on the smear. Lipocytes have pyknotic nuclei that are pressed against the side of the cell membrane by huge fat globules. Fat stains, such as Sudan IV and oil red O, may be used on fresh smears before alcohol fixation to verify the presence of these cells. New methylene blue stain may also be used. Lipomas are benign tumors but they can occasionally infiltrate between muscle masses. These infiltrative lipomas are difficult or impossible to remove and can cause death of the animal. In horses, pedunculated lipomas may be life threatening because of their potential for causing strangulation of the gut.
Unlike lipomas, cytologic preparations from liposarcomas may contain numerous cells. Aspirates, imprints and scrapings from liposarcomas may contain free fat, some mature lipocytes and lipoblasts, and appear greasy. Or they may contain very little free fat, few mature lipocytes, many lipoblasts and don't appear greasy. Histopathologic examination demonstrates that the cells contain abundant cytoplasm. This feature is not noted in most cytologic preparations because the cell membrane is visualized poorly. Cytologically, the cells have very light cytoplasm containing vacuoles of varying size and number. Some of the larger vacuoles may cause indentation of the cell nucleus. In general, the more immature and anaplastic cells have fewer and smaller fat globules. The lipoblast nucleus is typically round and variation in nuclear size and multinucleation is usually present in the preparation.
Dermal melanomas are common in dogs and horses but rare in cats. Although generally benign in dogs, cutaneous melanomas of the oral cavity, lips and digits have a high incidence of malignancy. Aspirates generally contain a large to moderate amount of cells but, occasionally, only scattered cells mixed with blood are aspirated. In all instances, melanin pigment almost always will be found in the background. The tumor aspirate contains predominantly individual cells, but sheets or rare clusters may be found. Cell morphology varies from round, oval, stellate or spindle shaped.
The cells from both benign and malignant tumors have a moderate to abundant amount of cytoplasm with a low N:C ratio. Melanomas are often extremely malignant and bizarre forms are common. Tumor giant cells and cells with giant nuclei are often seen. Melanin pigment appears as brown-black to green-black cytoplasmic granules of irregular size and shape. This pigment might appear as a salt-and-pepper sprinkling in occasional cells; or might be densely packed and obscure the nucleus. Even in tumors apparently devoid of pigment, a thorough examination of the smear will generally reveal a few cells containing a few small pigment granules interspersed throughout their cytoplasm. Anisokaryosis and numerous mitotic figures are usually additional features of these "amelanotic" neoplasms. In general, the less pigmented the melanoma, the more malignant the neoplasm.
Hemangiomas and hemangiosarcomas are tumors of blood vessel endothelium and are contiguous with the blood vascular system. The spindle cell tumor origin of hemangiomas and hemangiosarcomas may be difficult to recognize on cytologic preparations because of the massive blood contamination that may occur.
The cells of hemangiomas are difficult to differentiate from non-neoplastic endothelial cells. They tend to be oval, spindle or stellate in shape, have moderate to abundant amounts of light to medium blue cytoplasm, and contain a medium-sized round to slightly oval nucleus. The nucleus has a smooth to fine lacy chromatin pattern and may have 1-2 small, round, indistinct nucleoli.
Neoplastic endothelial cells aspirated from hemangio-sarcomas range in morphology from apparently normal endothelial cells to medium or large cells with marked cytologic criteria of malignancy (e.g., marked variation in cell, nuclear and nucleolar size; increased N:C ratios, nucleolar prominence and angularity, cytoplasmic basophilia.
Osteosarcoma is the most common tumor involving bone. A striking feature is that some of these malignant osteoblasts can be seen embedded in and apparently secreting an osteoid matrix which appears as fibrillar bright-pink material on Wright's stained slides. Osteoid is not found in most aspirates from osteosarcomas; however, when found, its presence provides strong evidence that a tumor is of bone origin.
The principal cell is the malignant osteoblast which may appear singly or in clusters. The cells are polygonal to fusiform with abundant foamy basophilic cytoplasm. Several clear cytoplasmic vacuoles and/or scattered pink cytoplasmic granules may be present. A Golgi area is often apparent. Nuclei of malignant osteoblasts are highly variable in size, exhibiting coarse chromatin patterns and multiple nucleoli. The nuclei may be eccentrically located and appear as if they are being "spit out" by the cell. Multinucleate forms may be present due to incomplete cellular division. These cells resemble osteoclasts; however, they are not osteoclasts but tumor giant cells and will exhibit nuclear criteria of malignancy.
Chondrosarcomas are the second most common tumor of bone and are difficult to differentiate from osteosarcomas cytologically. The ribs, turbinates and pelvis are the most common sites for chondrosarcomas of dogs, while the scapulae, vertebrae and ribs are most common sites in cats. One useful but inconsistent cytologic feature on low-power examination of aspirates is lakes of bright pink, smooth or slightly granular material in which cells may be embedded. This material is the intercellular matrix of cartilage sometimes called chrondroid.
Individual neoplastic chondroblasts have cytologic features similar to those of osteosarcoma cells. They vary from round to fusiform, with large nuclei and dark-blue cytoplasm. Anisokaryosis is prominent, and multinucleate tumor cells may be found. The cytoplasm Often contain several small clear vacuoles, and occasional cells may contain fine pink cytoplasmic granules. If the tumor is causing bone lysis, osteoclasts may also be found in cytologic specimens.
The round cell tumors consist of three sarcomas (transmissible venereal tumor, mast cell tumor and lymphosarcoma) and the histiocytoma, a benign tumor or questionable origin. The round cell tumors typically exfoliate well. Like epithelial cells, the cells are round and have distinct cell membranes. Like mesenchymal tumors, however, the cells from round cell tumors exfoliate individually rather than in clusters.
a.Mast Cell Tumor
Smears made from Mast cell tumors contain few to many round to oval cells containing a moderate amount of cytoplasm with variable numbers of small, red-purple granules (when stained with Wright's stain). The number of granules seen within a given neoplastic mast cell may vary from tumor to tumor and even within a tumor. Generally, mast cell neoplasms containing relatively few granules are considered to be formed by less differentiated, more primitive cells than are highly granulated neoplasms.
The nuclei of neoplastic mast cells are round to oval, variably sized and generally eccentric. They appear to stain palely because of the intense staining of the cytoplasmic granules. Nuclear criteria of malignancy are present although nuclei may be so obscured by granules that these features are indistinguishable. Mitotic figures may be present.
Mast cells contain heparin and its anticoagulant effect may result in local hemorrhage. Because of the local histamine release that occurs when mast cells degranulate, inflammatory cells are commonly noted in aspirates. Some tumors have a marked infiltrate of eosinophils which may be a distinct cytologic feature.
b.Transmissible Venereal Tumor (TVT)
The TVT is a discrete cell tumor of dogs most commonly found on mucous membrane surfaces, such as the penis, the vagina, and the oral or nasal cavities. The neoplasm is believed to have a viral etiology, currently unclassified. The TVT meets both cytologic and histologic criteria for malignancy but reports of metastasis are rare. In many cases the neoplasm will regress spontaneously.
Cells from TVTs exfoliate extremely well giving numerous cells in aspiration, impression or scraping preparations. Most of the cells are uniformly round; however, an occasional large or multinucleated cell may be observed. The cells have a moderate amount of light gray to blue cytoplasm with distinct boundaries. Numerous cytoplasmic vacuoles may be present. The nuclei are usually round and eccentrically placed and contain coarse, cord-like chromatin and 1-2 large prominent nucleoli. Variation in N:C ratio is a common feature. TVTs generally exhibit a high mitotic index. Tumor infiltration by plasma cells and and macrophages is common.
Histiocytomas are benign dermal neoplasms that occur most commonly as solitary growths on the head or the extremities of young dogs. The overlying epidermis is often ulcerated and accompanied by secondary inflammation of the neoplasm. Histiocytomas usually yield only a few cells on cytological preparations. The tumor cells are pleomorphic and resemble monocytes and epitheliod cells. They have a moderate amount of pale blue cytoplasm that often has ill-defined boundaries and may contain a few small vacuoles. Nuclei of histiocytoma cells are variable in both size and shape and a few multinucleated cells may be present. The nuclei may be indented like those of monocytes. Although the mitotic index may be quite high, nuclei of most cells appear benign and do not exhibit nuclear criteria of malignancy. The nuclei have finely etched, lacy to finely stippled chromatin patterns and contain multiple indistinct nucleoli. Lymphocytic infiltration of the tumor is a common feature.
Cutaneous lymphosarcoma can occur as a part of systemic lymphosarcoma or, rarely, as a disease localized to the skin only. It is usually manifested as multiple plaque-like lesions, but may occasionally occur as solitary nodules. Aspirates of these lesions usually yield highly cellular cytological preparations. Although numerous histo-pathologic classification schemes based on tumor architecture have been proposed for lymphosarcoma, the cytologist is limited to describing the tumor based on the predominant cell type. These cytologic classifications are lymphoblastic, lymphocytic and histiocytic lympho-sarcoma.
In canine multicentric lymphosarcoma, the lymphoblastic form of the disease is much more common than the lymphocytic or histiocytic forms. The neoplastic lymphoblasts are large, often several times the size of erythrocytes, and have a small to moderate amount of light to medium blue-staining cytoplasm. There is often displacement of this cytoplasm to one side of the nucleus. The nuclei are indented to irregular with smudged to stippled chromatin patterns and contain one or more nucleoli. Mitotic figures can be frequent. Because these immature cells are fragile, it is common for them to rupture during preparation of the smear. Naked, smeared nuclei and cytoplasmic fragments frequently are observed scattered among the intact cells.
Smears from cytologic preparations of lymphocytic lymphosarcoma consist of small lymphocytes that cannot be readily differentiated from normal lymphocytes. These tumors require histopathologic examination for definitive diagnosis.
1.Mammary Gland Abnormalities
Aspirates from normal mammary glands are generally acellular or contain only blood. If mammary tissue is present, it consists of acini of secretory cells. These cells have a moderate amount of basophilic cytoplasm and round, dark uniform nuclei. Active cells will appear foamy due to the accumulation of secretory material. Sheets or fragments of simple cuboidal duct epithelium may be present and appears as small cells with scanty cytoplasm and basally located ovoid nuclei. A third cellular component, the myoepithelial cell, may also be present and appears as a dark staining, naked, oval nucleus or as a spindle shaped cell.
Mammary cysts result when a dysplastic process causes dilatation of the duct system with the formation of large cavitations. They are common in middle-aged and older bitches, but may occasionally occur in younger dogs. Though considered benign, mammary cysts may be precancerous. Aspirated fluid is yellow, brown, green or blood-tinged and is of low cellularity consisting primarily of "foam cells" and pigment-laden macrophages. Dense clusters of cyst epithelial lining cells exhibiting minor degrees of nuclear pleomorphism may also be present.
Dysplastic and benign neoplastic lesions include lobular hyperplasia, adenosis, adenomas and papillomas. Aspirates from these all appear similar cytologically and contain numerous epithelial cells occurring singly or in sheets and clusters. The cells exhibit little or no pleomorphism. The nuclei have evenly dispersed chromatin patterns and contain single, small, round nucleoli. Pigment-laden macrophages may be present.
Complex adenomas, fibroadenomas and benign mixed tumors involve both epithelial and stromal elements. Smears of aspirates from these lesions contain spindle-shaped connective tissue cells and clusters of epithelial cells similar to those described in the preceding paragraph.
Numerous classifications of malignant neoplasms may involve the mammary gland, including various carcinomas, sarcomas and mixed origin tumors. Histologically, areas of cartilagenous/osseous metaplasia may be found in these various tumor types. Nests of these metaplastic connective tissue cells may also be seen in cytologic preparations.
Adenocarcinomas are the most common malignant tumor and have the classic cytological criteria previously described for this tumor type as do the less common mammary squamous cell carcinomas. Aspirates of anaplastic carcinomas contain very large, very pleomorphic epithelial cells occurring individually or in small clusters. These cells have a high N:C ratio, bizarre nuclear and nucleolar forms and exhibit a high mitotic index. Multinucleate forms are common.
2.Lymph Node Cytology
Three general processes can cause lymph node enlargement, hyperplasia, inflammation and neoplasia. Cytological examination can be used to differentiate between these conditions.
a. Normal Lymph Nodes
Cytologic examination of a normal size lymph node is warranted to ascertain the presence of certain microbial agents and to determine whether neoplastic metastasis has occurred.
Small (9 micron) mature lymphocytes are the predominant cell type in aspirates of normal lymph nodes and make up 85 to 90% of the cells observed. These cells have condensed nuclei, only slightly larger than a red blood cell, and scant cytoplasm consisting of a narrow rim around the nucleus.
The medium lymphocyte, or prolymphocyte, is somewhat larger than the small lymphocyte and contains more abundant pale cytoplasm. It has a nucleus with less densely packed nuclear chromatin and a nuclear diameter approximately equal to two red blood cells (i.e., 10-15 microns).
Large lymphocytes, or lymphoblasts, are the less common than either small or medium lymphocytes in normal nodes. They are large cells (15-25 microns) with basophilic cytoplasm which may appear as a broad or narrow rim around the nucleus. The nucleus is vesicular with a fine, diffuse chromatin pattern and may contain multiple distinct nucleoli.
Plasma cells and plasmablasts are derived from antigen stimulated B-lymphocytes and may be present in small numbers in normal lymph nodes. Plasma cells have small, round, eccentric nuclei with condensed chromatin. They have abundant deep blue cytoplasm with a prominent clear Golgi zone. Plasmablasts are more immature and are larger with less aggregated chromatin and a higher nucleus:cytoplasm ratio. Their very blue cytoplasm often contains vacuoles.
Cytoplasmic fragments called lymphoglandular bodies are a characteristic feature of lymphoid tissue. They are round, homogeneous, basophilic structures similar in size to platelets. They should not be confused with organisms.
b. Reactive Hyperplasia
Lymph nodes become reactive or hyperplastic when they are antigenically stimulated, such as occurs when high concentrations of antigens reach the draining nodes. A reactive hyperplastic lymph node resembles a normal lymph node cytologically; however, the size of the lymph node aids in differentiation since a normal node should not be enlarged. As with normal lymph nodes, specimens from reactive hyperplastic nodes contain a heterogeneous cell population. The small lymphocyte predominates, composing approximately 60-70% of the cells. The numbers of medium and large lymphocytes are increased and may make up to 15% of the total cell population. Mitotic figures may be prominent. Mature and immature plasma cells are generally increased in number and make up to 5-10% of the cell population in some areas of the smear. Plasma cells that contain many discrete vacuoles (Russell bodies) are called Mott cells or Russell body cells and are commonly seen in reactive lymph nodes. Macrophages may be increased in number, particularly when hyperplasia of sinus macrophages occurs. Increased numbers of lymphoglandular bodies are generally present.
The cytology of an inflamed lymph node will vary depending upon the etiology. Neutrophils (degenerate or nondegenerate), eosinophils, or macrophages can predominate. Inflammation is probably present when the total cell population of the lymph node is >5% neutrophils or >3% eosinophils. Macrophage numbers can increase in inflammation, hyperplasia and sometimes in neoplasia. Macrophages may also appear as epithelioid cells and multinucleated giant cells in granulomatous inflammation.
Lymphosarcoma is characterized by a uniform population of large, intensely basophilic immature cells (usually prolymphocytes or lymphoblasts) which give the smear a homogeneous cellular appearance. When these immature cells comprise >50% of the cell population, a diagnosis of lymphosarcoma can be reliably made, but smaller numbers may be present in early stages. Mitoses may be more numerous than in hyperplasia. Macrophages, called "tingible-body" macrophages, containing bluish cytoplasmic globules may be present and indicate intense lymphopoiesis and cell turnover. Lymphoglandular bodies are more numerous than in hyperplasia. Unlike lymphoid hyperplasia, plasma cells are usually few in number. Occasionally, lymphosarcoma is manifested as the small, well-differentiated lymphocyte type. This form is very difficult to distinguish from hyperplasia because the small lymphocyte predominates in each.
e. Metastatic Neoplasia
Malignant tumors frequently metastasize via lymphatics. This can result in the proliferation of neoplastic tissue in the draining lymph node. The presence of cells not normally found in lymph nodes or an increase in numbers of certain cell types normally present may suggest metastatic neoplasia.
Unilateral or bilateral enlargement is the major indication for fine-needle aspiration biopsy and cytologic evaluation of the testis. Fine needle aspiration may also be used to evaluate an enlarged epididymis. Enlargement of these organs can be due to inflammatory or neoplastic conditions.
The three major neoplasms of importance in the testis include seminomas, Sertoli-cell tumors and interstitial-cell tumors. These tumors are difficult to differentiate by cytologic examination alone and interpretation must be accompanied by a thorough history and clinical examination.
Aspirate from seminomas are usually highly to moderately cellular. The cells are of variable size with high nuclear:cytoplasm ratios. Distinct cells with intact cytoplasmic membranes may be sparse and ruptured cells are common. The nuclei are variable in size with homogeneous to finely reticular chromatin. Bi- and multiple nucleation is common. Nucleoli are prominent, relatively large and frequently multiple and irregular. Mitotic figures are common. Small lymphocytes are frequently scattered throughout the stroma.
b. Sertoli-Cell Tumor
Aspirates from Sertoli-cell tumors are highly cellular. The tumor cells usually have abundant light-staining cytoplasm which contains numerous distinct variable sized vacuoles. Nuclear chromatin patterns are coarsely reticular and small to large nucleoli may be present. Mitotic figures may be found. Rarely, spindle-shaped cells with abundant cytoplasm may be present.
The vaginal epithelium is a target tissue for ovarian hormones, changing from 2-4 layers thick to a multilayered epithelium under their influences. These changes result in exfoliation of large numbers of superficial epithelium cells. Examination of these cells from the vagina is a simple technique that can be used to accurately monitor the progression of proestrus and estrus in dogs and cats. Cytologic examination of vaginal smears is also useful in detecting inflammation and neoplasia in the female reproductive tract.
a. Classification of Vaginal Epithelium
Vaginal epithelial cells will vary in age and morphology during the various stages of the estrous cycle. The four main classifications include basal, parabasal, intermediate and superficial epithelial cells.
Basal cells are from the deepest layer of the epithelium lining, near the basement membrane. These cells give rise to the other epithelial cell types in the vaginal lining. Basal cells are small with a small amount of cytoplasm and a relatively high nuclear: cytoplasm ratio. They are rarely seen in vaginal smears.
Parabasal cells are located just above the basal cell layer of the epithelium. They are small round cells with round nuclei and a small amount of cytoplasm; however, they are considerably larger than basal cells. Large numbers of parabasal cells may exfoliate when the vagina of a prepubertal animal is swabbed. These cells are usually quite uniform in size and shape.
Intermediate cells vary in size depending on the amount of cytoplasm present but are generally twice the size of parabasal cells. Their nuclei are approximately the same size as those of parabasal cells, no matter how large the intermediate cell is. As intermediate cells increase in size, their cytoplasm becomes irregular, folded or angular, similar to the cytoplasm of more superficial epithelial cells.
Superficial cells are the largest epithelial cells seen in vaginal smears. As they age and degenerate, their nuclei become pyknotic and then faded, and occasionally they disappear. Their cytoplasm is abundant, angular and folded. As the cells degenerate, they undergo cornification.
b. Characteristics of the Stages of the Canine Estrous Cycle
Depending upon the reference used, the canine estrous cycle is divided into four or five stages. These stages are anestrus, proestrus, estrus and metestrus. The fifth stage, diestrus, is described as occurring after metestrus and before anestrus. In some references, diestrus and metestrus are used synoymously.
Smears obtained in early and mid-proestrus contain a mixture of epithelial cells, including parabasal, small and large intermediate and superficial cells. RBCs may be numerous and a few neutrophils may be present. As proestrus progresses, there is a steady increase in the number of cornified epithelial cells so that by late proestrus, large intermediate and superficial cells predominate. Parabasal and small intermediate cells are no longer seen on smears about 4 days before the LH peak of estrus. As estrus approaches, neutrophils totally disappear and RBC numbers decrease substantially.
Cytologically, early estrus is characterized by a marked decrease in red blood cells, an absence of neutrophils and a continuing increase in the number of cornified epithelial cells. The time of maximum cornification is variable and may range from as early as 6 days before to 3 days following the LH peak. Large numbers of bacteria are commonly observed on and around superficial epithelial cells. Neutrophils may reappear during tthe last day or two of estrus and indicate its end.
Cytologically, metestrus is characterized by an abrupt change in relative numbers of superficial epithelial cells with a marked increase in the proportion of parabasal and intermediate cells. Neutrophils appear in variable numbers and usually coincide with increased numbers of parabasal and intermediate cells. "Metestral cells", which are parabasal epithelial cells containing a neutrophil in the cytoplasm, are often present. Individual cytologic preparations made during the transition period from late estrus to early metestrus, without benefit of prior preparations, can appear very similar to smears made in early or mid-proestrus. At both times there can be a similar mixture of superficial and nonsuperficial cells.
4) Diestrus and Anestrus
Diestrus follows metestrus and is characterized by increasing numbers of parabasal and intermediate cells and a few of the more superficial cells. If present, neutrophils and bacteria are few in number. This stage will blend into the quiescent phase of the heat cycle, anestrus. during normal anestrus, the vaginal mucosa is dry and secretions are absent. Parabasal and intermediate cells predominate.
c. Vaginitis and Metritis
Vaginitis and metritis can be suspected in animals with a history of a lack of sequential estrus cycles and when mild vulvar swelling is observed with a white to pink vulvar discharge. The vaginal smear will demonstrate a large number of neutrophils, some in clumps. When these occur with noncornified epithelium, it may be difficult to differentiate inflammation from proestrus or metestrus. Neutrophils associated with cornified epithelium indicate inflammation because the two cell types do not normally occur together. Degenerate neutrophils indicate septic inflammation. Vaginal smears from animals with pyometra or metritis usually contain large numbers of very degenerate neutrophils. Clusters of foamy secretory endometrial lining cells may also be seen in endometritis. Endometrial hyperplasia is characterized by the presence of endometrial lining cells without associated inflammation.