Department of Companion Animal Clinical Studies, Section Clinical Pathology, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, Pretoria, South Africa
Cytological evaluation of a high-quality sample from the respiratory tract can, together with history, clinical data and imaging, provide invaluable diagnostic information towards patient management. One of the major factors determining the diagnostic value of cytology specimens is the quality of the sample. Samples obtained through transtracheal aspiration/wash (TTA) or bronchoalveolar lavage (BAL) should be divided into two portions: one portion should be placed into an EDTA tube to preserve cellular morphology, and the other portion into a sterile container for possible microbial culture. Macrophages and neutrophils may phagocytise red blood cells, bacteria, and other debris if the sample is not processed within a short period of time, ideally within one hour. The cellular component of the fluid should be concentrated either through standard- or cytocentrifugation. Squash preparations should be made of large mucus plugs observed in the sample, as cells and organisms are frequently embedded in the mucus. If a TTA/BAL sample is deemed unacceptable for various reasons, it should be repeated either immediately or only after 48 hours. Even though a sterile solution is used during the procedure, it induces a neutrophilic response that peaks 24 hours after the procedure was performed.
Nasal Washes and Smears
In healthy animals, few cells are normally present with nasal washes or smears, combined with small amounts of mucus and low numbers of a mixed population of extracellular bacteria (normal bacterial flora) colonising the surface of epithelial cells. The predominant cell type is typically ciliated respiratory epithelial cells, with lesser numbers of squamous epithelial cells that originate from the anterior nasal cavity.
Inflammation: Neutrophils predominate in nasal exudates associated with bacterial, viral, and some fungal infections, and foreign material. Various numbers of macrophages, lymphocytes, and plasma cells may also be present. Phagocytised bacteria consisting of a monomorphic population suggest infection.
Hyperplasia/dysplasia: To survive amid a pathologic stimulus such as chronic inflammation, cells within the nasal cavity may undergo adaptive changes. Hyperplasia (increased number of cells) is often accompanied by dysplasia (loss of architectural organisation) but difficult to identify cytologically. Chronic ongoing inflammation may result in squamous metaplasia, where the normal cell type is transformed into one that is better able to endure the environmental stress while losing specialised function (squamous cells). Both hyperplasia and metaplasia may result in neoplasia.
Neoplasia: The most common nasal tumours are of epithelial cell origin (e.g., adenocarcinoma, squamous cell carcinoma) and are malignant in 80–90% of cases; however, metastasis is uncommon.
Transtracheal and Bronchoalveolar Washes
1. Cytological Evaluation
Normal findings include a small amount of mucus; low numbers of columnar or cuboidal epithelial cells, goblet cells, alveolar macrophages, and neutrophils (< 5%); occasional lymphocytes; and superficial squamous cells (less with BAL).
A small amount is usually present in clinically normal dogs and cats. Mucus appears as amorphous sheets ranging from blue to pink or as homogenous strands that are frequently twisted or whorled. An increased amount can be seen with inflammation, irritation, or upper airway damage (chronic respiratory disease), due to increased numbers of goblet cells.
Mucus casts of small bronchioles that appear as spiral, twisted masses of mucus that may have perpendicular radiations (bottle-brush-like appearance). Seen with disorders that result in chronic, excessive mucus production and are usually an indication of bronchiolar obstruction.
Respiratory Epithelial Cells
Ciliated and non-ciliated epithelial cells can be seen in washings from normal dogs and cats. Ciliated columnar cells are elongated or cone shaped with cilia on their flattened apical ends. The nucleus is present in the basal end of the cell. Ciliated cuboidal cells look similar except that these cells are as wide as they are tall. Non-ciliated cells look exactly the same except for the absence of cilia. If these cells are predominant in a sample, the procedure probably sampled mainly bronchi and bronchioles (as opposed to alveolar space). These cells may be present individually or in clusters and are usually of no clinical significance.
Mucus-producing bronchial cells that are generally columnar with round granules of mucin that stain from red to blue to clear with Diff-Quik stains. Free granules from ruptured cells may be seen on smears. Goblet cells can be differentiated from mast cells in that they have larger granules. They are not commonly seen; however, any chronic pulmonary irritant may result in an increased number.
Alveolar macrophages are commonly seen in the washes of normal dogs and cats and are useful indicators of washes that have adequately washed the alveolar spaces. They have abundant blue-grey cytoplasm and an eccentrically positioned, round to bean-shaped nucleus. When activated, their cytoplasm becomes more abundant and vacuolated and may contain phagocytised material.
Neutrophils and eosinophils look similar to those seen in the blood. Increased neutrophils indicate inflammation and increased eosinophils indicate a hypersensitivity reaction that is either allergic or parasitic. A small percentage of lymphocytes may be present in the washes of normal dogs and cats and resemble those seen in the blood. Increased numbers of lymphocytes may generally indicate nonspecific inflammation and are of limited diagnostic value. The presence of high numbers of lymphoblasts may indicate pulmonary lymphoma.
Low numbers are occasionally observed in samples of dogs and cats with many different inflammatory lung disorders, but are of little diagnostic significance.
2. Cytological Interpretation
Samples of TTAs and BALs are usually interpreted according to type, quantity, and proportion of cells recovered.
Contamination is much more likely when TTAs or BALs are performed through an endotracheal (ET) tube. Superficial squamous cells and a mixed population of bacteria (specifically Simonsiella spp.) are the hallmark for oropharyngeal contamination. Many bacteria may be seen adhered to the surface of squamous epithelial cells and generally without the presence of neutrophils, although neutrophils may be present in a contaminated sample if the patient has a purulent or ulcerative oropharyngeal lesion or suffers from dental disease.
Normal animals have a very low percentage (< 5%) of eosinophils in TTA or BAL samples, but clinically healthy/asymptomatic cats may have significantly higher numbers (up to 25%). In dogs, samples with > 10% eosinophils are indicative of a significant hypersensitivity disease process. Increased numbers of neutrophils, macrophages, mast cells, lymphocytes, and plasma cells may also be seen if tissue irritation is sufficient to induce an inflammatory response. Eosinophils trapped in strands of mucus may predominate in certain areas of the slide and sometimes do not stain completely. Disorders include allergic bronchitis/pneumonitis, feline asthma, lung worms, eosinophilic bronchopneumopathy, and heartworms.
Normally, neutrophils are present in very low numbers (< 5%) in TTAs and BALs of dogs and cats. Neutrophil numbers are increased in nearly all conditions (infectious and noninfectious) that cause inflammation. Neutrophils may show degenerative changes because of bacterial toxins or may be smudged (ruptured) secondary to trauma from collection and preparation. Infectious disorders include bacterial, mycotic, viral, or protozoal diseases. Noninfectious disorders include tissue irritation or necrosis secondary to inhalation of a toxic substance, as well as neoplasia. Increased numbers of macrophages are seen with subacute and chronic pulmonary disorders. Alveolar macrophage numbers frequently increase with chronic persistent inflammation.
Anthracotic pigment (dark or black granules) may be present within macrophages from clinically normal animals living in large cities or areas with polluted air.
Erythrophagocytosis or erythrocyte-breakdown products (i.e., haematoidin or hemosiderin) may be seen within macrophages in conditions that cause pulmonary haemorrhage. To differentiate between iatrogenic haemorrhage and haemorrhage caused by pulmonary pathology, erythrophagocytosis, haemosiderophages, or haem pigment should be identified on the smear.
Lung Parenchyma (Fine-Needle Aspiration)
Lung parenchyma is composed of alveoli that are lined by type I and type II alveolar epithelial cells. Aggregates of lymphoid tissue are also present in the lung and may be sampled during evaluation of the lung. Samples from healthy lung tissue are sparsely cellular and contain primarily respiratory epithelial cells. A small number of alveolar macrophages, erythrocytes, and white blood cells may also be seen.
Refer to previous section.
Cytological evidence of allergic bronchitis include increased mucus, Curschmann's spirals, and increased numbers of eosinophils, as well as variable numbers of macrophages, neutrophils, and mast cells. Eosinophilia in the lung may be seen with or without blood eosinophilia.
Refer to previous section.
Adenocarcinoma of bronchogenic or bronchiolar-alveolar origin is the most common neoplasm of the lung; however, cytological differentiation is not possible. Aspirates of lung carcinomas typically contain moderate numbers of epithelial cells in sheets, aggregates, and clusters with lesser numbers of individualised cells. Moderate-to-marked pleomorphism between clumps of cells, as well as within cells of the same cluster is common in pulmonary carcinoma. Various cellular criteria of malignancy can be seen, which include an eccentrically placed nucleus with coarsely clumped chromatin and prominent, single to multiple nucleoli. Anisokaryosis is common with deeply basophilic cytoplasm. Cytoplasmic vacuolation, particularly around the perinuclear region, is frequently prominent.
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