UPDATE ON RESPIRATORY DISEASES

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CYTOLOGIC EVALUATION OF THE RESPIRATORY TRACT A. H. Rebar, DVM, PhD, Eleanor C. Hawkins; DVM, and Dennis B. DeNicola, DVM, PhD

Evaluation of respiratory tract disease has long been a diagnostic challenge for the veterinarian. There are currently no serum biochemical or hematologic tests that localize injury to the respiratory system. Imaging techniques are useful in identifying and localizing respiratory disease but generally do not lead to etiologic diagnosis. Obtaining excisional biopsies from respiratory lesions is often difficult, if not impossible. For these reasons, specific diagnosis of respiratory tract disease often resides in cytologic evaluation.20 This article reviews the various cytologic collection techniques that yield high-quality specimens from the components of the upper and lower respiratory tract. Additionally, the cytologic features of both the normal respiratory tract and common respiratory disorders are described and illustrated. COLLECTION TECHNIQUES Upper Respiratory Tract (Nose)

The collection of cytologic specimens representative of nasal disease is difficult. Most nasal diseases result in marked superficial inflammation and secondary bacterial infection, which can obscure the underlying disease. Also, the disease may be localized or multifocal rather than diffuse. From the Department of Veterinary Pathobiology, Purdue University School of Veterinary Medicine, West Lafayette, Indiana (AHR, DBD); and the Department of Companion Animal and Special Species Medicine, North Carolina State University College of Veterinary Medicine, Raleigh, North Carolina (ECH)

VETERINARY CLINICS OF NORTH AMERICA: SMALL ANIMAL PRACTICE VOLUME 22 • NUMBER 5 • SEPTEMBER 1992

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Nonsurgical techniques for the collection of nasal specimens include superficial and deep nasal swab, nasal flush, 23 traumatic nasal flush, 13• 35 pinch biopsy, 9• 13 and core biopsy. 22· 36 Superficial nasal swabs potentially can provide a rapid diagnosis of cryptococcosis25 and should be performed in all cats with chronic nasal discharge. Otherwise, swabs and nasal flush specimens often represent only the nonspecific, superficial processes. The latter three techniques are preferred because they result in deeper tissues, which can be examined both cytologically by impression smear and histologically. The potential risks of these tissue collection techniques include hemorrhage and inadvertent passage of the instrument through the cribriform plate. The patient should be evaluated for bleeding tendencies prior to the procedure. A cuffed endotracheal tube is placed and the caudal pharynx packed with gauze to prevent aspiration. Intravenous fluids are administered to replace blood volume. The vascular floor of the nasal cavity should be avoided. Hemorrhage, if encountered, generally resolves spontaneously. Excessive hemorrhage can be controlled through gentle infusions of cold saline with epinephrine or by packing umbilical tape through both the external and internal nares. Instruments should never be passed blindly through the external nares to a depth greater than the medial canthus of the eye in order to avoid penetrating the cribriform plate.22• 35 Traumatic nasal flushing 13• 35 provides very small, random tissue specimens but requires no special equipment. It can be performed using a stiff polypropylene urinary catheter, usually 8-10 French, which has been cut at an angle. Wings can be added to the catheter by making nicks in the wall with a blade and bending these 'ears' of plastic out. With the patient's nose tipped toward the floor, saline is instilled and aspirated through. the catheter as it is aggressively raked along the mucosa. Pieces of tissue may be recovered within the syringe or catheter or caught in a bowl with fluid draining from the nares. A nasal pinch biopsy is typically performed using alligator biopsy forceps. Fifteen-centimeter-long forceps with a biopsy cup of 2.7 mm (R. Wolf, Rosemont, IL) provide a good size specimen that contains tissue deep to the mucosal surface. 9• 13 The instrument can be guided rhinoscopically in many cases, or the location of radiographic lesions relative to the teeth can be used as a guide for depth of collection. Three to six biopsy specimens should be obtained. A large core of deep tissue can be obtained when a soft-tissue mass is present. The plastic sleeve of a Sovereign indwelling catheter (Sherwood Medical, St. Louis, M0) 36 or a large, polypropylene urinary catheter22 is used, with the end cut at an angle. The catheter is placed against the mass, then is twisted into it while suction is applied with a 12-cc syringe. Tracheobronchial Tree

Specimens can be obtained from the major airways by tracheal wash, bronchial brushing, or impression smears from bronchial biopsy.

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Tracheal wash is the most useful because it is minimally invasive and requires no special equipment. Bronchial brushings and biopsies allow for directed sampling from localized lesions but generally require bronchoscopy. 11 • 24 Tracheal wash can be performed either trans-tracheally6 or through an endotracheal tube. 26 Both methods avoid contamination of the specimen with organisms and cells from the oropharynx. The transtracheal wash is best performed in a well-restrained or lightly .sedated animal. Surgical preparation of the skin and local anesthesia is required. A catheter is placed using sterile technique through the cricothyroid ligament, into the tracheal lumen and is then passed down to the carina.* An 18 to 22 gauge through-the-needle vascular catheter is convenient to use; however, if the length of available catheters is inadequate to reach the carina, a 3.5-french polypropylene urinary catheter can be passed through a pre-placed 14-gauge over-the-needle catheter. Once the catheter is in place, 3 to 5 mL aliquots of sterile saline are instilled through the catheter and immediately withdrawn. Coughing is frequently induced during the procedure, and may facilitate retrieval of material .from the more distal airways. Diagnostic material must be aspirated the entire length of the catheter, so air must be discharged from the syringe and many more aspiration attempts made. Additional aliquots of saline are instilled and withdrawn until 0.5 mL or more of turbid fluid is recovered. The endotracheal technique is advantageous in animals undergoing anesthesia for another procedure and in cats and puppies, which are difficult to restrain. A potential disadvantage is the lack of a cough reflex. (Coughing helps to distribute and mix the saline within the lungs and to bring it back into the trachea, where aspiration attempts will allow it to be recovered.) This problem usually can be overcome with conscientious aspirations or by using a light plane of anesthesia and performing the wash as the gag reflex returns. Short-acting barbiturates can be used in dogs, and intravenous ketamine and acepromazine combinations can be used in cats for this procedure. Lidocaine should be sprayed on the larynx of cats to decrease the chance of laryngospasm. A sterile endotracheal tube is placed using a laryngoscope in order to minimize oral contamination. A sterile 3.5-french polypropylene catheter is passed through the tube and the wash procedure performed as described previously. Deep Lung

Nonsurgical methods for obtaining specimens from the deep lung include transthoracic aspiration or biopsy, 5• 33· 34 bronchoalveolar lavage *Editor's Note: Alternatively, the catheter may be threaded down the trachea after inserting the needle through the skin and tracheal wall in the midcervical region. Lateral restraint of the animal with the (radiographically) worst side ventral will facilitate the mixing of the injected saline with the more affected airways.

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(BAL), 16 and transbronchial biopsy. Transbronchial biopsy requires special instrumentation and results in extremely small specimens containing primarily airway epithelium. Transthoracic lung aspiration or fine-needle biopsy is the technique of choice for collection of specimens from pulmonary mass lesions located adjacent to the body wall. There is little risk in passing a needle directly into the mass, and the diagnostic yield is high. Careful localization of the mass in all three dimensions with two radiographic views of the chest is essential if ultrasonographic or fluoroscopy guidance is not available. In cases with diffuse interstitial disease, the diagnostic yield is less and the risk for complications greater.* Potential complications include pneumothorax and hemothorax, 34 and the animal should be observed carefully afterward. Bronchoalveolar lavage provides a large specimen from a relatively large area of the lung; however, general anesthesia is required. The technique generally is performed through a flexible bronchoscope. After routine bronchoscopic examination, the scope is lodged snugly in an airway. In dogs, several lobes are lavaged, including lobes suspected of having localized involvement. In cats, it may not be possible to isolate specific lobes owing to the relative size of the scope to the bronchial lumen size; therefore, one side of the lungs (i.e. a principal bronchus) is lavaged. Saline is instilled by syringe through the biopsy port of the scope, and suction is immediately applied using the same syringe. Without moving the scope, the procedure is repeated. The volume of fluid instilled should be standardized to improve consistency of results. The authors use the following volumes: in dogs, two aliquots of 25 mL each for each lobe lavaged; and in cats three aliquots of 5 mLI kg body weight. Following the procedure, several positive pressure ventilations are administered by hand to encourage expansion of collapsed alveoli. An unguided BAL can be performed in cats using a sterilized endotracheal tube. 15 The cat is given atropine, anesthetized, and placed in lateral recumbency. A sterile endotracheal tube is passed as described for tracheal wash and the cuff expanded. A syringe adapter is placed on the end of the tube. Saline is instilled directly through the tube and recovered by syringe using the fluid volumes described previously. Slightly elevating the rear of the cat during suction may facilitate fluid return. Other nonbronchoscopic methods for BAL have been used, generally by passing soft catheters or sterile tubing blindly into the lung until they have lodged in an airway. Primary disadvantages include inability to direct the catheter and lack of consistent results or reference values. Hypoxemia does occur following BAL. 15• 27 It is transitory in the majority of cases, but only animals able to withstand anesthesia and additional respiratory compromise are considered as candidates for the *Editor's Note: This is especially important in those cases that are noted to have an expiratory effort/heave/wheeze during quiet breathing .

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procedure. The hypoxemia generally responds well to increased concentrations of inhaled oxygen. Animals are allowed to breath 100% oxygen until extubation becomes necessary. If clinical signs are suggestive of poor oxygenation, oxygen supplementation is continued by face mask or oxygen cage. Such supplementation is rarely needed for longer than 15 minutes. 15 Bronchospasms may contribute to respiratory distress, particularly in cats. Animals that fail to respond to supplemental . oxygen therapy may be treated with a bronchodilator.

CYTOLOGY OF THE NOSE Normal Findings

The most anterior segments of the nose are lined by stratified squamous epithelium, whereas the turbinates are lined by pseudostratified columnar epithelium; most cytologic specimens contain some representatives of both populations. Columnar cells are elongate with basal oval nuclei. 1 At low magnification, they can be easily confused with mesenchymal elements. At high magnification, apical cilia may be seen; however, in many cells, cilia are not apparent (Figs . 1 and 2). Scattered among the more numerous ciliated columnar cells are the goblet cells. Goblet cells are not ciliated and are characterized as elongate cells with basal nuclei and moderate amounts of cytoplasm with distinct coarse metachromatic cytoplasmic granules or droplet(s) of weakly or unstained mucus. Immature goblet cells contain metachro-

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Figure 1. Normal columnar nasal epiihelial cells. Cilial remnants are seen on the top cell but not the others. Wright; original magnification, x 100.

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Figure 2. Normal columnar nasal epithelial cells. Cilia are found only on the central cell. Columnar respiratory epithelial cells from all segments of the respiratory tract are morphologically similar. Wright; original magnification, x 100.

matic cytoplasmic granules. Goblet cells are present in increased numbers whenever there is chronic irritation of the nasal lining (goblet cell hyperplasia). Basophilic basal epithelial cells are a component of pseudostratified epithelium and are present in low numbers. In samples from normal animals, they are visualized as small rafts of deeply stained round to polygonal cells. Like goblet cells, they are present in increased numbers whenever there is chronic nasal irritation; they represent benign epithelial hyperplasia. Inflammation of the Nose (Rhinitis)

Rhinitis is the most common nasal abnormality recognized cytologically. Inflammatory lesions are classified as neutrophilic, mixed (neutrophils and mononuclear cells), or macrophagic depending on which inflammatory cells are the most prevalent. In general, neutrophilic inflammation (Fig. 3) suggests the possibility of bacterial infection, especially if the neutrophils have features of degeneration (pyknotic, karyorrhectic, or karyolytic nuclei). Often, bacteria may be found in the cytoplasm of neutrophils (Fig. 4). Even when organisms are not seen, bacterial rhinitis is still a consideration. Mycotic agents, especially Aspergillus fumigatus, the most common cause of mycotic rhinitis, can be associated with neutrophilic inflammation; however, they more typically cause mixed or predominantly

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Figure 3. Neutrophilic rhinitis. Neutrophils are well preserved and no organisms are seen. Wright; original magnification, x 100.

Figure 4. Neutrophilic rhinitis. Neutrophils are degenerate and bacteria are seen within neutrophil cytoplasm. Wright; original magnification, x 100.

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macrophagic1 reactions. Cytologic support for the diagnosis of mycotic rhinitis depends on finding either fungal hyphae or microconidia (Figs. 5 and 6). Etiologic diagnosis should be established through fungal culture. 30 A diagnosis of mycotic rhinitis is confirmed by identifying other abnormalities that differentiate fungal infection from normal nasal flora. Such abnormalities include radiographic signs of mycotic infection, visible fungal plaques through rhinoscopy, invasion of organisms into the mucosa on nasal biopsy, and positive serum antibody titers. A diagnosis of either bacterial or mycotic rhinitis does not preclude the possibility of other nasal disorders. Infectious rhinitis may occur secondarily to underlying nasal neoplasia or foreign bodies. 31 Cases that do not respond quickly to aggressive antimicrobial therapy should be further evaluated with these differentials in mind. Eosinophilic rhinitis is a unique form of inflammation occasionally encountered in both dogs and cats. Cytologic findings include large numbers of eosinophils, perhaps admixed with neutrophils, as well as mast cells and occasional plasma cells. Goblet cell hyperplasia as well as an increased prominence of rafts of basal epithelial cells are typically noted. Such findings generally are regarded as evidence of a local hypersensitivity response. Allergic rhinitis may be seen in conjunction with similar changes in the tracheobronchial tree and/or deep lung or may occur without any other evidence of respiratory involvement. L31 Another inflammatory lesion encountered with some frequency is the inflammatory polyp. Radiographically, this lesion may resemble nasal neoplasia, even to the extent of having bony lysis. Cytologically, the reaction is largely comprised of lymphocytes and plasma cells with lesser numbers of macrophages and neutrophils. Epithelial cell rafts with evidence of squamous metaplasia are common and must be closely evaluated for criteria of malignancy to rule out the possibility of true nasal carcinoma. Histologic assessment may be required to definitively rule out well-differentiated nasal carcinoma.

Neoplasms of the Nose*

Nasal neoplasms can be placed into either primary epithelial and mesenchymal or metastatic disease categories. Primary epithelial neoplasms are most common. 22 • 28 The morphologic presentation is quite variable; however, in many cases the cytologic specimen contains variably sized cohesive rafts of round to polygonal cells that fulfill various nuclear criteria of malignancy, including anisokaryosis, variable nuclear/cytoplasmic ratios, abnormally coarse chromatin, and multiple large and/or irregularly-shaped nucleoli (Figs. 7 and 8). 32 Specific variants of primary nasal epithelial neoplasms are recognized. Adenocarcinomas may be identified by the presence of secretory *Editor's Note: Additional information on nasal tumors may be found elsewhere in this issue in the article entitled " Canine and Feline Nasal and Paranasal Sinus Tumors."

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Figure 5. Mycotic rhinitis caused by Aspergillus fumigatus. Microconidia and lysed neutrophils are present. Wright; original magnification, x 100.

Figure 6. Mycotic rhinitis caused by Aspergillus fumigatus. A mat of branching septate hyphae are enmeshed in cellular debris. Wright; original magnification, x 100. (From Rebar AH: Handbook of Veterinary Cytology. St. Louis, Ralston Purina Co, 1978; with permission.)

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Figure 7. Low magnification of a nasal swab in a case of nasal carcinoma. Rafts of cells exhibiting anisocytosis and anisokaryosis are readily apparent. Diff Quik; original magnifi· cation, x 16.

Figure 8. Oil immersion of nasal carcinoma. Diff Quik; original magnification, x 100. (From Rebar AH: Handbook of Veterinary Cytology. St. Louis, Ralston Purina Co, 1978; with permission.)

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cells, which often contain single, large, unstained vacuoles within their cytoplasm (signet ring cells). Squamous cell carcinomas are identified by finding neoplastic cells with relatively abundant glassy or hyalinized cytoplasm, angulated cell margins, and central nuclei with potentially bizarre chromatin patterns. 1• 32 Finding squamous epithelial cells alone does not establish the diagnosis of squamous cell carcinoma, especially when inflammation is present, because squamous metaplasia is possible with chronic irritation. Primary connective tissue neoplasms can occur in the nOSf but are less frequent than the epithelial neoplasms. Possible specific diagnoses include osteosarcoma, chondrosarcoma, and fibrosarcoma. 28 These neoplasms present with individual or cohesive spindle to flame-shaped cells. Because all three tissue types (bone, cartilage, and fibrous tissue) may be present, definitive diagnosis may require histologic evaluation. The most common metastatic neoplasm in the nose is lymphosarcoma. Nasal lymphosarcoma has similar features to lymphosarcoma elsewhere in the body, being composed of a uniform population of large, discrete, round cells with very high nuclear/cytoplasmic ratios. 32 Nasal lymphosarcoma must be distinguished cytologically from an inflammatory polyp, which contains a mixture of lymphocytes and plasma cells. 1 In many cases, there is a prominent inflammatory process associated with these locally aggressive neoplasms. Secondary sepsis is common. When the cytologic criteria of malignancy are prominent, identification of the primary carcinoma is relatively easy; however, separating well-differentiated nasal carcinomas from benign hyperplasia and dysplastic changes associated with active rhinitis, can be extremely difficult. 1 In these situations, biopsy and histologic confirmation are indicated.

CYTOLOGY OF THE TRACHEOBRONCHIAL TREE Normal Findings

Normal findings depend upon the type of sample collected. The predominant cells in mucosal brushings and imprints from pinch biopsies are ciliated epithelial cells, which often are found forming distinctive rows. They are elongate with basally oriented oval nuclei. The cilia are hair-like projections arising from the apical cytoplasm. 32 Goblet cells are present, but in far lesser numbers than the ciliated cells. They are morphologically similar to those of the nose, Goblet cells may be found as individuals or interspersed among the ciliated cells. Occasional rafts of basal epithelial cells also are present. These are comprised of cohesive aggregates of uniform polygonal cells with central round nuclei and basophilic cytoplasm. Samples collected by tracheal wash usually are very low in cellularity. In contrast to brushings or imprints, the predominant cell

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normally is the alveolar macrophage. The presence of macrophages indicates that the deep lung as well as the tracheobronchial tree is sampled. Epithelial cells are present in extremely low numbers. 4• 32

Tracheobronchial Cytology in Disease

In our experience, tracheal brushings and imprints of pinch biopsies are of limited diagnostic usefulness, because they only yield information regarding changes that directly involve the tracheobronchial mucosa. They provide little or no information about diseases localized to the lung. In humans, however, brushings of early mucosal neoplastic lesions have been found to be very diagnostic where brushings of larger advanced neoplastic processes tend to yield poor diagnostic specimens owing to variable amounts of secondary inflammation. 3 Probably the most commonly recognized abnormality in veterinary specimens is allergic tracheobronchitis, which is characterized by large numbers of eosinophils and mast cells. Chronic tracheobronchial irritation, characterized by an obvious increase in goblet cell numbers and squamous metaplasia of ciliated epithelium, also is observed frequently.19· 31 Tracheal wash cytology is of somewhat greater utility because the deep lung often is sampled. Lesions that can be identified via tracheal wash include allergic tracheobronchitis, chronic obstructive disease of small airways, parasitic diseases of the tracheobronchial tree, and selected diseases of the deep lung such as bacterial pneumonia, mycotic pneumonia, viral pneumonia, and primary and metastatic lung neoplasia. Diseases of the deep lung are better demonstrated via bronchoalveolar lavage and are described later. The principal cytologic feature of allergic tracheobronchitis is the presence of large numbers of eosinphils. 10· 19· 31 Other inflammatory cells, such as neutrophils, differentiated alveolar macrophages, and lymphocytes also may be present in significant numbers (Fig. 9). Mast cells may be observed. If the condition is chronic, rafts of basophilic hyperplastic bronchial epithelial cells also may be present (Fig. 10). 19 Obstructive small airway disease can occur as an accompaniment of a variety of chronic pulmonary disorders including allergic tracheobronchitis, emphysema, and chronic bacterial pneumonia. Cytologically, the presence of tight coils of inspissated mucus (presumably washed out of plugged airways) is associated with small airway disease. These coils are known as Curschman's spirals (Fig. 11).4 Parasitic diseases are sometimes diagnosed by tracheal washes. Occasionally, Filaroides sp. larvae are washed out of lesions at the tracheal bifurcation (Filaroides osleri) or from the alveoli of the deep lung (Filaroides hirthi). 11 These larvae are typical nematode larvae with a welldefined oral cavity and gut but lack reproductive organs. They u sually are tightly coiled in cytologic preparations. Microfilariae of Dirofilaria immitis also can be seen in tracheal washes, particularly if the collection

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Figure 9. Allergic tracheobronchitis in a dog. Numerous eosinophils are present. Wright; original magnification, x 100.

Figure 10. A raft of hyperplastic epithelial cells in a case of allergic tracheobronchitis. Wright; original magnification, x 100.

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Figure 11 . Tightly coiled spirals of inspissated mucus (Curschman's spirals) are thought to indicate obstructive small airway disease. Wright; original magnification, x 100.

technique has been slightly traumatic and there is evidence of hemorrhage in the specimen. Eggs of Paragonimus kellicotti may be seen occasionally in tracheal samples from infected animals. These eggs are large (30-40!-l- by 80-lOO!J.), brown, have a single operculum, 4• 10 and are readily visualized under low-power magnification. A wide variety of inflammatory responses are possible with these various parasitic diseases. Mixed inflammatory responses are common. When a significant eosinophilic component is identified and the clinical or radiographic findings are supportive, thorough examination of the specimen for larvae or ova is recommended.

CYTOLOGY OF THE DEEP LUNG Normal Findings

The predominant cell in BAL fluid from normal dogs and cats, as with humans and many other mammals, is the undifferentiated alveolar macrophage. 4 • 7• 8• 12• 19• 20• 30 These cells comprise approximately 80 to 90% of the total cells present. The remainder are composed of an admixture of differentiated macrophages, lymphpcytes, ciliated and nonciliated epithelium, eosinophils, neutrophils, and perhaps a few mast cells. 2• 4• 21 • 30 A significant eosinophilia has been reported in the BAL cytology taken from normal cats.29 Undifferentiated alveolar macrophages are moderately large, discrete round cells (15- 301-1-) with fairly uniform basophilic granular

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Figure 12. Normal undifferentiated ·alveolar macrophages. Wright; original magnification, X 16.

cytoplasm and eccentric round finely granular nuclei (Figs. 12 and 13). Differentiated macrophages are larger (30-801-1-) with vacuolated paler basophilic cytoplasm that often contains phagocytized debris. Differentiated macrophages may be binucleate or multinuclea.te. If found in

Figure 13. High magnification of normal undifferentiated alveolar macrophages. Wright; original magnification, x 100.

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a clinically normal animal, the activated macrophages tend to be on the smaller end of the described size range. 7• 30• 31

Inflammation of the Lung

Inflammatory reactions in the lung may be neutrophilic, eosinophilic, mixed, or predominantly macrophagic. The type of inflammation often suggests possible etiology. 16 Septic pneumonias generally are neutrophilic, although the more chronic or resolving forms may be characterized by mixed inflammation. Degenerate neutrophils with pyknotic, karyorrhectic, or karyolytic nuclei are less frequently observed than with other body fluid exudates (Figs. 14 and 15). Bacteria may be present within neutrophils; however, even when they are not specifically observed, samples for microbiologic evaluation (culture and sensitivity) should be collected. 31 Eosinophilic exudates are most commonly associated with steroidresponsive allergic reactions. Infectious agents, particularly parasites, also can elicit eosinophilic inflammation and must be ruled out. Radiography can be particularly useful in this regard; allergic pneumonitis has a predominantly reticular interstitial pattern, whereas infectious causes of eosinophilic inflammation may produce multifocal and nodular lesions. 4• 16• 31 Systemic mycotic and protozoal agents generally cause mixed or histiocytic inflammatory reactions (Fig. 16); a significant eosinophilic component is possible. When such reactions are present in BAL specimens, a systematic search for causative organisms should be made. Specific diagnoses can be made on the basis of the characteristic morphology of the various causative agents. 14· 16 Viral pneumonias cause varying degrees and types of inflammation.4 Diagnosis therefore depends upon finding direct evidence of viral infection, that is, viral inclusion bodies. Inclusion bodies can be found in active cases of canine distemper and adenoviral pneumonia. DiffQuik stain is the stain of choice for maximizing the identification of viral inclusion bodies. The inclusion bodies of canine distemper are eosinophilic, of variable size and shape, and intracytoplasmic or intranuclear. They may be found in various cell types, including macrophages, lymphocytes, and epithelial cells (Fig. 17). Adenoviral inclusions are large, round, and intranuclear. They either fill the entire nucleus or are located centrally with clearing of the surrounding nucleoplasm and margination of chromatin.

Cytology of Pulmonary Neoplasms

Neoplastic involvement of the lung may be either primary or metastatic. 17 The vast majority of th~ primary neoplasms are carcinomas,

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Figure 14. Neutrophilic pneumonia. Intact neutrophils predominate; occasional degenerating epithelial cells and macrophages are also present. The pink granular background reflects the presence of lysed cells. Wright; original magnification, x 100.

Figure 15. Neutrophilic pneumonia, high magnification. The process is bacterial, but the neutrophils are nondegenerate. No organisms are seen, but bacterial culture was positive. Wright; original magnification, x 100.

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Figure 16. Bronchoalveolar lavage of a mixed inflammatory pneumonia. The large central histiocyte contains large numbers of histoplasma organisms. Wright; original magnification, x 100.

Figure 17. Canine distemper. Intracytoplasmic eosinophilic inclusion bodies are seen adjacent to the nuclei in the two degenerating columnar cells. Wright; original magnification, x 100. (Courtesy of Dr. Donald Meuten, North Carolina State University.)

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Figure 18. A raft of neoplastic epit~elial cells in a bronchoalveolar lavage from a case of primary pulmonary carcinoma. Wright; original magnification, x 100.

as are the vast majority of metastases. Both primary and metastatic carcinomas exfoliate into BAL samples. 3• 18 Carcinoma cells in BAL samples are round to polygonal, deeply basophilic cells found as individuals or as small rafts and sheets (Fig. 18). The nuclei may be difficult to visualize in th~ larger clumps of cells because of the intense cytoplasmic staining. Where nuclear morphology can be evaluated, criteria of malignancy (e.g., anisokaryosis, variable nuclear/cytoplasmic ratio, abnormal chromatin clumping, multiple nucleoli, and irregularly shaped nucleoli) are readily apparent. 3 In general, primary pulmonary carcinomas cannot be readily distinguished from metastatic ones. Evaluation of clinical and radiographic information may aid in this differentiation. The only other neoplasms identified with some frequency in BAL samples are metastatic discrete cell neoplasms, most notably lymphosarcoma. Lymphosarcoma of the deep lung has similar cytologic features to lymphosarcoma from any other site (see the article on neoplasms of the nose elsewhere in this issue).

References I. Andreasen CB, Rakich PM, Latimer KS: Nasal exudates and masses. In Cowell RL,

Tyler RD (eds): Diagnostic Cytology of the Dog and Cat. Goleta, CA, American Veterinary Publications, 1989, pp 47- 53 2 Brownstein DG, Rebar AH, Bice DE, et al: Immunology of the lower respiratory tract. Am J Pathol 98:499-514, 1980

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3. Chalon J, Tang CK, Klein GS, et al: Routine cytodiagnosis of pulmonary malignancies. Arch Pathol Lab Med 105:11-14, 1981 4. Cowell RL, Tyler RD, Baldwin CJ: Transtracheal and bronchial washes. In Cowell RL, Tyler RD (eds): Diagnostic Cytology of the Dog and Cat. Goleta, CA, American Veterinary Publications, 1989, pp 167-177 5. Cowell RL, Tyler RD, Baldwin CJ: The lung parenchyma. In Cowell RL, Tyler RD (eds): Diagnostic Cytology of the Dog and Cat. Goleta, CA, American Veterinary Publications, 1989, pp 179-187 6. Creighton SR, Wilkins RJ: Transtracheal aspiration biopsy: Technique and cytologic evaluation. JAm Anim Hosp Assoc 10:219, 1974 7. DeNicola DB, Rebar AH, Henderson RF: Early damage indicators in the lung: V. Biochemical and cytologic response to NO, inhalation. Toxicol Appl Pharmacal 60:301-312, 1981 8. Fahey PJ, Utell MJ, Mayewski RJ, et a!: Early diagnosis of bleomycin pulmonary toxicity using bronchoalveolar lavage in dogs. Am Rev Respir Dis 126:126- 130, 1982 9. Forbes S, Hawkins EC: Gross rhinoscopy and rhinoscopy assisted pinch biopsy in 119 dogs (abstr). In Proceedings of the 9th Annual Veterinary Medical Forum, American College of Veterinary Internal Medicine, New Orleans, 1991 10. Ford RB: The respiratory system. In Prat PW (ed): Feline Medicine. Santa Barbara, CA, American Veterinary Publications, pp 299-320, 1983 11. Ford RB: Endoscopy of the lower respiratory tract of the dog and cat. In Tams TR (ed): Small Animal Endoscopy. St. Louis, CV Mosby, 1990, pp 309-326 12. Gee JBL, Fick RB Jr: Bronochoalveolar lavage. Thorax 35:108, 1980 13. Hawkins EC: Chronic viral upper respiratory disease in cats: Differential diagnosis and management. Compend Conlin Educ Pract Vet 10:1003, 1988 14. Hawkins EC, DeNicola DB: Cytologic analysis of tracheal wash specimens and bronchoalveolar lavage fluid in the diagnosis of mycotic infections in dogs. JAm Vet Med Assoc, 197:79-83, 1990 15. Hawkins EC, DeNicola DB: Collection of bronchoalveolar lavage fluid in cat, using an endotracheal tube. Am J Vet Res 50:855, 1989 16. Hawkins EC, DeNicola DB, Kuehn NF: Bronchoalveolar lavage in the evaluation of pulmonary disease in the dog and cat: State of the art. J Vet Intern Med 4:267, 1990 17. Hess FG, McDowell EM, Trump BF: Review article. Pulmonary cytology: Current status of cytologic typing of respiratory tract tumors. Am J Pathol 103:323-333, 1981 18. Jay SJ, Wehr K, Nicholson DP, et al: Diagnostic sensitivity and specificity of pulmonary cytology. Comparison of techniques used in conjunction with flexible fiber optic bronchoscopy. Acta Cytol 24:304-312, 1980 19. Johnston WW, Frable WJ: The cytopathology of the respiratory tract. A review. Am J Pathol 84:372- 424, 1976 20. Johnston WW, Frable WJ: Diagnostic Respiratory Cytopathology. New York, Masson, 1979 21. King RR, Zeng QY, Brown D), et al: Bronchoalveolar lavage cell populations in dogs and cats with eosinophilic pneumonitis (abst) . In Proceedings of the Seventh Veterinary Respiratory Symposium. Chicago, Comparative Respiratory Society, 1988 22. Legendre AM, Spaulding K, Krahwinkel DJ, )r: Canine nasal and paranasal sinus tumors. JAm Anim Hosp Assoc 19:115, 1983 23. MacEwen EG, Withrow SJ, Patnaik AK: Nasal tumors in the dog: Retrospective evaluation of diagnosis, prognosis, and treatment. JAm Vet Med Assoc 170:45, 1977 24. McKiernan BC: Bronchoscopy in the small animal patient. In Kirk RW (ed): Current Veterinary Therapy X. Philadelphia, WB Saunders, 1989, p 219 25. Medleau L, Barsanti JA: Cryptococcosis. In Greene CE (ed): Infectious Diseases of the Dog and Cat. Philadelphia, WB Saunders, 1990, p 687 26. Moise NS, Blue): Bronchial washings in the cat: Procedure and cytologic evaluation. Compend Conlin Educ Pract Vet 5:621, 1983 27. Muggenburg BA, Mauderly JL, Pickrell JA, et al: Pathophysiologic sequelae of bronchopulmonary lavage in the d og. Am Rev Respir Dis 106:219, 1972 28. Norris AM: Intranasal neoplasms in the dog. J Am Anim Hosp Assoc 15:231-237, 1976 29. Padrid PA, Feldman BF, Funk K, et a!: Cytologic, microbiologic, and biochemical

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analyses of bronchoalveolar lavage fluid obtained from 24 healthy cats. Am J Vet Res 52:1300-1307, 1991 Rebar AH, DeNicola DB, Muggenburg BA: Bronchopulmonary lavage cytology in the dog: Normal findings. Vet Pathol 17:294-304, 1980 Rebar AH, DeNicola DB: The cytologic examination of the respiratory tract. Semin Vet Med Surg (Small Anim) 3:109-121, 1988 Rebar AH: Handbook of Veterinary Cytology. St. Louis, Ralston Purina, 1978 Roudebush P, Green RA, Digilio KM: Percutaneous fine-needle aspiration biopsy of the lung in disseminated pulmonary disease. JAm Anim Hosp Assoc 17:109, 1981 Teske E, Stokhof AA, van den Ingh TSGAM, et a!: Transthoracic needle '!spiration biopsy of the lung in dogs with pulmonic diseases. JAm Anim Hosp Assoc 27:289, 1991 Withrow SJ: Diagnostic and therapeutic nasal flush in small animals. J Am Anim Hosp Assoc 13:704, 1977 Withrow SJ, Susaneck SJ, Macy OW, et a!: Aspiration and punch biopsy techniques for nasal tumors. JAm Anim Hosp Assoc 21:551, 1985

Address reprint requests to A. H. Rebar, DVM, PhD Purdue University School of Veterinary Medicine Lynn Hall, Room 113 West Lafayette, IN 47907