Cytology of Inflammatory Cutaneous lesions in the Dog and Cat

Transcription

1 Cytology of Inflammatory Cutaneous lesions in the Dog and Cat Rick L. Cowell, DVM, MS, MRCVS, Diplomate ACVP Clinical Pathologist IDEXX Laboratories Inc A. Cytologic Patterns of Inflammation: 1. Neutrophilic (Acute) Inflammation: Preparations in which greater than 70% of the cells are neutrophils. Suppurative or Purulent Inflammation are other terms used when there is a marked predominance of neutrophils (greater than 85%). The most common cause is a bacterial infection but other organisms (e.g., Sporothricosis) and many noninfectious disorders (e.g., necrotic areas in tumors, immune mediated disorders, etc) can cause neutrophilic inflammation. 2. Pyogranulomatous (Chronic Active) Inflammation: Preparations with an inflammatory population that contains both neutrophils and a prominent fraction of macrophages (15 to 50% macrophages). Multinucleated giant cells, reactive fibroblasts, and lymphocytes may be present also. Pyogranulomatous inflammation suggests a cause other than routine bacterial infection. Fungal infections (e.g., Blastomycosis), higher bacteria (e.g., Actinomyces), mycobacterium, protozoa, and noninfectious disorders (e.g., foreign bodies, necrosis) are common causes of pyogranulomatous inflammation. 3. Granulomatous (Chronic) Inflammation: Preparations in which greater than 50% of the cells are macrophages. Multinucleated inflammatory giant cells, reactive fibroblasts, and lymphocytes may be present also. Causes of granulomatous inflammation are similar to those that cause pyogranulomatous inflammation (e.g., fungal, mycobacterium, protozoa, foreign bodies, and necrosis). 4. Eosinophilic Inflammation: Preparations in which large proportions of the cells are eosinophils (greater than 10 to 20% eosinophils). The other cell types are often an admixture of neutrophils, macrophages, mast cells, and lymphocytes. High numbers of eosinophils occur with immune/allergic reactions, parasitic disorders (e.g., lungworms), certain fungal infections (e.g., phycomycosis), and neoplasia (e.g., mast cell tumors). Occasionally, eosinophil granules may stain a tan or muddy-brown in tissue preparations making them somewhat more difficult to recognize. However, their distinct granules allow for there identification. Also, neutrophils occasionally have a fine eosinophilic stippling in thick exudates and should not be confused with eosinophils. 5. Lymphocytic or Lymphocytic/Plasmacytic Inflammation: Preparations from nonlymphoid tissue that contain a large proportion of mature lymphocytes (small lymphocytes and plasma cells). This is differentiated from cutaneous lymphoma in 1

2 that lymphoma consists almost totally of large lymphoblasts. Lymphocytic/plasmacytic inflammation occurs with some injection site reactions, feline stomatitis/gingivitis, and lymphocytic/plasmacytic gastroenteritis. B. Neutrophils: Degenerative Vs Nondegenerative Neutrophils: 1. Degenerative Neutrophils: These are neutrophils that have lost their ability to control water homeostasis and are undergoing hydropic degeneration. As water diffuses into the cell and into the nucleus via nuclear pores, it causes the nucleus to swell, fill more of the cytoplasm, and stain homogeneously eosinophilic. The presence of degenerative neutrophils suggests a bacterial cause because these changes are due primarily to toxins produced by bacteria (e.g., endotoxin). However, the absence of degenerative neutrophils does not rule out a bacterial cause because bacteria may be present in low numbers and some bacteria produce low levels of toxin. Therefore, when degenerative neutrophils are present your suspicion of a bacterial infection is increased. Although all cell types are subjected to the same toxin, degenerative change is evaluated in neutrophils only. Also, degenerative changes can be induced by autolysis (rot), therefore, samples made from dead animals or samples collected and held for a long time before smears are made may show degenerative as well as pyknotic (see below) changes. 2. Nondegenerative Neutrophils: These are neutrophils with tightly clumped, basophilic nuclear chromatin. A few to many of the neutrophils may be hypersegmented or pyknotic (nuclear chromatin broken into tight, round spheres). These are aging changes that may occur in vivo or in vitro. C. Pathogenic Bacteria: 1. General comments: a. With the routine blood stains (Wrights, Diff-Quik, Dip-Stat) all bacteria (Grampositive and Gram-negative) stain blue to purple (basophilic) with a few exceptions like Mycobacterium that do not stain. Mycobacterium spp. have a lipid cell wall and therefore do not stain but appear as nonstaining small rods. Intracellular bacteria indicate a bacterial infection (primary or secondary) while extracellular bacteria may represent a bacterial infection or contamination. Bacterial infections often have bacteria intracellularly and extracellularly while contamination has only extracellular bacteria. Also, evaluate the bacteria to determine if only one population exists (e.g., monomorphic population of cocci or rods) or if a mixture of rods and cocci or variably sized rods exists (e.g., pleomorphic population). Pleomorphic bacteria are common with infections from GI origin, bite wounds, etc. b. Gram Stains can be used but it is very difficult to get reproducible, accurate staining of bacteria when they are in exudative effusions. Cells and exudative 2

3 proteins stain red with Gram Stain and bacteria, whether Gram-positive or Gram-negative, tend to stain red in exudative effusions. This makes Gram stain a poor choice when trying to confirm the presence of bacteria since it is difficult to see red bacteria in a red-staining background. 2. Coccoid bacteria: Pathogenic bacterial cocci in veterinary medicine are typically Gram-positive (Staphylococcus spp. and Streptococcus spp.). Dermatophilus congolensis replicates by transverse and longitudinal division, producing long chains of coccoid bacterial doublets that resemble small, blue, railroad tracts. 3. Bacterial rods: Small bacterial rods are typically gram-negative, and all pathogenic small, bipolar rods are Gram-negative. Common small bacterial rods include E. coli and Pasteurella spp. Large spore forming bacterial rods usually indicate Clostridium spp. Filamentous rods are usually Actinomyces or Nocardia spp. They are characterized by long, slender (filamentous) strands that stain pale blue, and have intermittent, small, pink or blue areas. D. Nonpathogenic Bacteria: 1. Contaminants from the oral cavity like Simonsiella spp. are considered nonpathogenic. Simonsiella organisms are found extracellularly and often adhered to superficial squamous epithelial cells. They are bacteria that divide lengthwise and line up in parallel rows giving the appearance of a single, large bacterium. Their presence indicates oropharyngeal contamination. E. Common Pathogenic Fungi: 1. Small Yeast s: a. Sporothrix schenckii organisms are round to oval to fusiform (cigar-shaped) and are about 3-9 u long and 1-3 u wide. They stain pale to medium blue with a slightly eccentric pink or purple nucleus. b. Histoplasma capsulatum organisms are round to slightly oval, but are not fusiform. They are about 2-4 u in diameter, stain pale to medium blue, and contain an eccentric pink to purple staining nucleus that is often crescent shaped. There is usually a thin clear halo around the yeast. 2. Medium sized yeast s: a. Blastomyces dermatitidis organisms are blue, spherical, 8-20 u in diameter, and thick walled. Occasional organisms showing broad-based budding may be found. 3

4 b. Cryptococcus neoformans organisms are extremely variable in size but are generally 4-15 u in diameter without their capsule and 8-40 um in diameter with their capsule (thick mucoid). The organism stains pink to blue-purple and may be slightly granular. The capsule is usually clear and homogenous. Also, nonencapsulated forms of Cryptococcus may be found. These appear similar but have a thin clear capsule. Occasional organisms showing narrow-based budding may be found. 3. Large yeast s: a. Coccidioides immitis organisms range from 10 to greater than 100 u in diameter. They appear as blue or clear, double contoured spheres with finely granular protoplasm and will often appear folded or crumpled. Round endospores, 2-5 u in diameter may be seen in some of the larger organisms. 4. Fungi that primarily form hyphae: a. Fungi that form hyphae require culture for specific identification. Some hyphating fungi do not stain well with the hematologic-type stains (Phycomycosis). 5. Dermatophytes: a. Microsporum and Trichophyton spp. are the dermatophytes that commonly cause ringworm. Scrapings from the edge of the lesion, stained with Wright s stain, readily show small spores and mycelia on and within hair shafts. F. Other organisms: 1. Protozoal organisms: 2. Algae: a. Leishmania donovani: Tend to be associated with a granulomatous or pyogranulomatous reaction. Leishmania organisms are oval, 2-4 microns in diameter, have a blue to light purple nucleus, and a small dark blue to purple, rod-shaped kinetoplast. Though variable, the kinetoplast tends to be located between the nucleus and greatest volume of cytoplasm. a. Protothecal infections tend to be associated with a granulomatous or pyogranulomatous reaction. Prototheca spp. are round to oval organisms that are 1 to 14u wide and 1 to 16u long, have a granular basophilic cytoplasm, and a thin clear cell wall. They are associated with cutaneous infections in cats and disseminated disease in dogs (often with GI signs). G. Noninfectious inflammatory lesions: 4

5 1. Many noninfectious inflammatory lesions are encountered in Veterinary Medicine. Some examples are: a. Cysts: Fluid from a variety of cystic lesions (salivary mucocele, seroma, hygroma) is commonly evaluated cytologically. Most cystic fluids contain moderate to high numbers of macrophages, although some cysts are of low cellularity. The macrophages may be relatively small, unstimulated macrophages resembling blood monocytes or large, actively phagocytic macrophages with abundant vacuolated cytoplasm. Neutrophils may be present in low, moderate or high numbers resulting from inflammation induced by the pressure of the cyst (remember, neutrophils mean inflammation not infection). Intracystic hemorrhage occasionally occurs and is recognized by the presence of RBC breakdown products (hemosiderin, hematoidin). Fluid from a salivary mucocele can often be recognized by the presence of amorphous blue mucus tufts or clouds or pillows scattered amongst the cells. Follicular or epidermal cysts typically contain only high numbers of mature (often nonnucleated) superficial squamous epithelial cells and some epithelial debris. If cyst contents rupture into the tissue they invoke a foreign body inflammatory response resulting in variable numbers of inflammatory cells (typically neutrophils and macrophages). b. Neoplasia: Tumors often out grow their blood supply and create necrotic areas that contain many inflammatory cells (typically neutrophilic or pyogranulomatous inflammation). c. Injection site reactions: These may be neutrophilic, pyogranulomatous, granulomatous, or lymphocytic. Occasionally amorphous, homogeneous, brightly eosinophilic material may be seen extracellularly and within macrophages with vaccine reactions. d. Eosinophilic granuloma: Scrapings or aspirates usually contain high numbers of eosinophils. Often, especially in scrapings, numerous free eosinophil granules are present in the background of the smear. Fibroblasts are present in variable numbers. e. Steatitis (fat necrosis, panniculitis): Aspirates from areas of inflamed fat typically consist of inflammatory cells and high numbers of variably sized lipid vacuoles. Granulomatous (histiocytic) inflammation is most common, although pyogranulomatous and purulent inflammation may be seen. The macrophages often contain numerous small, clear, lipid vacuoles within their cytoplasm. Large, multinucleated macrophages are commonly seen and must not be confused with neoplastic cells. f. Immune-mediated skin lesions: Many nondegenerative neutrophils and necrotic debris are seen on aspirates from pustules associated with pemphigus. 5

6 Occasionally, acantholytic cells (rounded squamous epithelial cells) are observed. g. Insect bites: May range from neutrophilic to eosinophilic to mixed to all basophils (Jones-Mote reaction). 6