CHAPTER 2 Cutaneous and Subcutaneous Lesions
Masses, Cysts, and Fistulous Tracts
Cytologic examination can be very useful when a cutaneous or subcutaneous lesion is not easily diagnosed by simple clinical evaluation, especially when such lesions are not responsive to therapy. Cutaneous and subcutaneous lesions are easily accessible, and there are no significant contraindications to collecting samples from them. Tranquilization and/or anesthesia is seldom needed for sample collection. Often the cytologic preparation can be collected, prepared, stained, and microscopically evaluated in minutes, providing a diagnosis, prognosis, indication of appropriate therapy, and/or indication of the next diagnostic procedure.
Lesions may be swabbed, imprinted, scraped, and/or aspirated, depending on the character of the lesion and the tractability of the patient. Ulcerated lesions should be imprinted, then cleaned, dried, and reimprinted. After imprints are made, scrapings should be obtained. Aspirates are then collected from deep within the lesion or mass. Obviously, lesions without an eroded or ulcerated surface must be aspirated.
Generally, cytologic swab smears are collected only when imprints, scrapings, and aspirates cannot be made. Sterile cotton swabs moistened with sterile isotonic fluid, such as 0.9% NaCl, are used. Moistening the swab helps minimize cell damage during sample collection and smear preparation but is unnecessary if the lesion itself is very moist. After sample collection, gently roll the swab along the flat surface of a clean glass slide. Do not rub the swab across the slide surface, since this causes excessive cell damage. If a Romanowsky-type stain is used, air dry the smears before staining (see Chapter 1).
Imprints are made by removing any scab covering the lesion and then touching the surface of a clean glass slide to the surface of the lesion. If Dermatophilus congolensis infection is suspected, the underside of the scab is imprinted also. The lesion is then cleaned with a nonirritating antiseptic, wiped dry with a sterile gauze sponge or other clean absorbent material, and reimprinted. High-quality cytologic smears can also be made by imprinting biopsy specimens (see Chapter 1).
Scrapings of cutaneous lesions are made by rubbing the edge of a blunt instrument, such as a glass slide or the back of a scalpel blade, across the lesion. This results in accumulation of cells along the edge of the blunt instrument. These cells are then spread onto a clean, dry glass slide by one of the techniques described in Chapter 1.
Aspiration Technique: Aspirates are obtained by using a 20- to 25-gauge needle attached to a 3- to 20-ml syringe. If microbiologic evaluation is to be performed on a portion of the sample, the area of aspiration should be surgically prepared. Otherwise, skin preparation is essentially that required for vaccination or venipuncture. An alcohol swab can be used to clean the area.
Hold the mass to be aspirated firmly to aid penetration of the skin and mass and control the direction of the needle. Introduce the needle, attached to a syringe, into the center of the mass and apply strong negative pressure by withdrawing the plunger about one-half to three-fourths the volume of the syringe (see Fig. 1-1). Sample several areas of the mass, but avoid aspiration of the sample into the barrel of the syringe and contamination of the sample by aspiration of tissue surrounding the mass. To accomplish this, when the mass is large enough to allow the needle to be redirected and moved to several areas in the mass without danger of the needle’s leaving the mass, maintain negative pressure during redirection and movement of the needle. However, when the mass is not large enough for the needle to be redirected and moved without danger of the needle leaving the mass, relieve the negative pressure during redirection and movement of the needle. In this situation, apply negative pressure only when the needle is static.
When aspiration is complete (often, high-quality collections do not have sample showing in the syringe and sometimes not even in the hub of the needle), release negative pressure from the syringe and remove the needle from the mass. If negative pressure is not released before the needle is removed from the mass, cells and blood from the subcutaneous tissues and skin may be aspirated into the sample and interfere with interpretation of the aspirate. If negative pressure remains on the syringe when the needle exits the skin, the portion of the sample in the barrel and hub of the needle will be aspirated into the syringe. Frequently, when only a small amount of sample has been collected and it is aspirated into the syringe, the sample cannot be recovered from the syringe and another sample must be collected. Once the negative pressure has been fully released, remove the needle from the mass and skin.
Then remove the needle from the syringe and aspirate air into the syringe. Next, replace the needle onto the syringe and expel some of the tissue in the barrel and hub of the needle onto the middle of a glass slide by rapidly depressing the plunger.
Nonaspiration Technique: Alternatively, a nonaspiration technique may be used. This has proved useful in some instances where adequate numbers of cells are difficult to retrieve without contaminating the sample with peripheral blood. With an aspiration technique, the tissue obtained will be the tissue of least resistance. Once small blood vessels are ruptured, peripheral blood will be the tissue of least resistance. The nonaspiration technique collects cells within the needle by capillary action after shearing cells loose from surrounding tissue with the point of the needle.
Use a needle and syringe of similar size as with the aspiration technique; however, fill the syringe with air before the aspiration procedure. Since aspiration is not required, hold either the distal end of the barrel of the syringe or the hub of the needle, gripping it like a pencil. This allows much greater control over directing the tip of the needle to the proper area. Firmly hold the mass to be aspirated with one hand, while using the other hand to guide the tip of the needle into the mass. Instead of aspirating, rapidly and repeatedly advance the needle through most of the thickness of the mass, using an action like a sewing machine. The repeated needle puncture of the tissue will dislodge some cells from the mass, creating a slurry of cells and tissue fluid. Some of this material will enter the needle. After 8 to 10 passages, withdraw the needle from the mass and expel the material onto a glass slide as usual. There is often enough material for just one slide in the needle. Repeated collections at various sites within the mass is recommended to get a representative sampling of the lesion.
A combination of slide preparation techniques can be used to spread aspirates of solid masses (see Figs. 1-2 to 1-5). One combination procedure (see Fig. 1-2) is to spray the aspirate onto the middle of a slide (prep slide). Keeping the prep slide on a flat, solid, horizontal surface, pull another slide (spreader slide) backward at a 45-degree angle to the first slide until it contacts about one third of the aspirate. Then slide the spreader slide forward smoothly and rapidly as if making a blood smear. Next, place the spreader slide horizontally over the back third of the aspirate at a right angle to the prep slide. Use the weight of the top slide to spread the material, resisting the temptation to compress the slides manually. Keeping the top slide flat and horizontal, slide it quickly and smoothly across the prep slide. This makes a squash prep of the back third of the aspirate. The middle third of the aspirate is left untouched.
This procedure leaves the front third of the aspirate gently spread. If the aspirate is of fragile tissue, this area should contain sufficient unruptured cells to interpret. The back third of the aspirate has been spread with the shear forces of a squash prep. If the aspirate contains clumps of cells that are difficult to spread, there should be some clumps sufficiently spread in the back third of the preparation. If the aspirate is of very low cellularity, the middle third will be more concentrated and the most efficient area to study.
The squash prep (see Fig. 1-3) is commonly used to spread samples. In expert hands this procedure can yield excellent cytologic smears; however, in less experienced hands it often yields smears that cannot be evaluated because too many cells are ruptured or the sample is not sufficiently spread. Make a squash prep by expelling the aspirate onto the middle of a microscope slide (prep slide). Then place a second slide (spreader slide) over the aspirate at right angles to the first slide. Keeping the spreader slide horizontal with the prep slide, slide the spreader slide rapidly and smoothly across the prep slide. A modification of the squash preparation that has less tendency to rupture cells can be performed by placing the second slide (spreader slide) over the aspirate at right angles to the first slide (prep slide), then rotating the second slide 45 degrees and then lifting it upward (see Fig. 1-4).
Another technique for spreading aspirates (see Fig. 1-5) is to drag the aspirate peripherally in several directions with the point of a needle, producing a starfish-shaped preparation. This technique tends not to damage fragile cells but leaves a thick layer of tissue fluid around the cells. Sometimes the thick layer of fluid prevents individual cells from spreading well, causing them to appear contracted and interfering with evaluation of cell detail. Usually, however, some acceptable areas are present.
Aspirates can be collected from fluid-filled masses and cysts with a 20- to 25-gauge needle attached to a 3-ml syringe. When possible, aspirate enough fluid to prepare several cytologic smears, perform a nucleated cell count and total protein analysis, and obtain a sample for culture. A sample of 1 to 3 ml usually is sufficient.
The lesion is prepared as described for solid masses. Smears can be prepared directly from the aspirated fluid or from the sediment of centrifuged fluid using the blood smear, line smear, and/or squash prep techniques as described in Chapter 1.
Fistulous tracts are usually caused by foreign bodies, bone sequestra, or infectious agents. They should be probed for foreign bodies and, if possible, radiographed for sequestra or osteomyelitis. Culture and cytologic swab samples should be collected from deep within the tracts. Cytologic preparations should be carefully perused for filamentous rods staining light blue with intermittent pink to purple dots with Romanowsky-type stains (Plate 3F). This morphology is characteristic of Nocardia spp. and Actinomyces spp., which can cause fistulous tracts, but occasionally occurs with some anaerobic bacteria also, such as Fusobacterium spp.
Ulcerated lesions may be areas of skin that have been injured and/or infected and become ulcerated and indurated due to the subsequent inflammatory reaction or may be areas of ulcerated skin overlying a cutaneous or subcutaneous mass. Generally, physical examination of the lesion indicates whether there is an underlying mass. Ulcerated lesions can result from infectious, foreign body, allergic, parasitic, or neoplastic causes.
Nonulcerated masses may be solid or fluid filled. Slowly developing, nonulcerated solid masses usually are neoplastic in origin. However, inflammatory conditions can produce rapidly developing, nonulcerated, solid masses. Fluid-filled nonulcerated masses usually are nonneoplastic in origin, but occasionally they represent cystic neoplasia.
The first step in cytologic evaluation of a smear is to determine whether sufficient numbers of intact cells are present and whether the sample is spread and stained adequately to allow evaluation of cell morphology. If repeated collection attempts fail to yield sufficient numbers of cells for cytologic evaluation, an alternative procedure, such as biopsy or culture (depending on the character of the lesion), may be necessary.
Once a suitable cytologic preparation has been produced, the smears are evaluated for evidence of inflammation and/or neoplasia (Fig. 2-1). If all the cells from a solid mass are tissue cells (ie, no inflammatory cells are present), either the lesion is caused by neoplasia or hyperplasia or the lesion was not sampled and surrounding tissues were sampled. If all the cells are inflammatory cells, an inflammatory process is most likely the primary cause of the lesion, but an inflamed neoplasm cannot be ruled out. An admixture of inflammatory cells and dysplastic tissue cells can be caused by inflammation with secondary tissue-cell dysplasia or neoplasia with secondary inflammation. Therefore, caution must be used in diagnosing neoplasia if evidence of inflammation is detected.
Fig. 2-2 provides an algorithm to aid evaluation of the inflammatory-cell component of cutaneous and subcutaneous lesions. Table 2-1 gives some general considerations for some inflammatory responses. If most of the inflammatory cells are neutrophils (Plates 1A–D, 3A) but no bacteria are found, a covert infection may be present or the neutrophilic inflammatory response may be due to one of the conditions listed under Marked predominance of neutrophils in Table 2-1. The lesion can be cultured to identify a covert infection. If culture results reveal an infectious agent, appropriate therapy can be instituted. If culture results do not reveal an infectious agent or if therapy for the infectious agent identified by culture is not effective, cytologic evaluation can be repeated or a biopsy can be submitted for histopathologic evaluation.
|Inflammatory Cell Population||First Considerations||Second Considerations|
|Marked Predominance (85%) of Neutrophils|
|Many neutrophils degenerate||Gram-negative bacteria Gram-positive bacteria||Abscess secondary to neoplasia, foreign bodies, etc|
|Few degenerate neutrophils|
|No degenerate neutrophils|
|Admixture of Inflammatory Cells|
|Giant inflammatory cells present|
When >15% of the inflammatory cells are macrophages (Plates 2A–D, 3B) and/or giant inflammatory cells are present (Plate 3B), fungal infection or foreign body granuloma should be considered. The slide should be carefully perused for organisms or signs of foreign material, such as refractile debris (Fig. 2-3, A) or eosinophilic material typical of adjuvant (Fig. 2-3, B). Also, historical information concerning possible introduction of foreign material should be sought. If no organisms or foreign materials are found and there is no historical information indicating introduction of a foreign substance into the area, the tissue can be cultured or a biopsy can be submitted for histopathologic examination.
Fig. 2-3 Fine-needle aspirate.
A, Fine-needle aspirate from foreign body reaction. Refractile foreign material is scattered throughout micrograph. Large clump of cell debris and refractile foreign body is in center. (Wright’s stain; original magnification 100X) B, Aspirate from injection site reaction in gelding. Large macrophage contains brightly eosinophilic noncellular material typical of adjuvant. Scattered neutrophils are also present. (Wright’s stain; original magnification 125X)
If the proportion of eosinophils exceeds 10% (Plate 3C), an allergic, parasitic, or foreign body reaction and certain hyphating fungi (eg, phycomycosis) should be considered. Again, the slide should be carefully searched for organisms or signs of foreign material. If none is found, the lesion can be cultured (including fungal cultures) or a biopsy can be submitted for histopathologic evaluation.
When tissue cells showing criteria of malignancy are accompanied by inflammatory cells (Fig. 2-4), the sample should be interpreted cautiously. Dysplasia in tissue cells adjacent to inflammatory reactions can alter tissue cell morphology. As a result, cells undergoing dysplasia in response to a local inflammatory process can be erroneously classified as neoplastic cells. As the intensity of the inflammatory reaction increases, the assurance with which neoplasia can be diagnosed decreases.
Fig. 2-4 Aspirate from nasal polyp caused by Rhinosporidium seeberi.
Note dysplastic epithelial cells (mild anisocytosis, anisokaryosis, prominent nucleoli, coarse chromatin, cytoplasmic basophilia) and numerous neutrophils. Rhinosporidium organisms were found in other areas of smear. (Wright’s stain; original magnification 100X)
Infectious agents invariably cause lesions containing inflammatory cells. Bacteria usually produce lesions characterized by >85% neutrophils (Plates 1D, 3A) (many of which may be degenerate), a few macrophages, and a few lymphocytes and plasma cells. On the other hand, fungi tend to produce lesions containing more macrophages than in bacterial lesions, but neutrophils often predominate and occasionally eosinophils are plentiful with certain hyphating fungi. Mycotic lesions also often contain lymphocytes, plasma cells, and fibroblasts. The infectious agent, location of the lesion, chronicity of the lesion, and immune status of the animal influence the character of the lesion.
Most pathogenic bacterial cocci are gram positive and of the genus Staphylococcus or Streptococcus (Plate 3E). Staphylococci usually occur in clusters of 4 to 12 bacteria, while streptococci tend to occur in short or long chains of organisms. When cocci are identified in cytologic preparations and are considered as causing or contributing to the lesion, aerobic and anaerobic cultures and sensitivity tests should be performed to identify the organism and appropriate antibacterial therapy. Because most pathogenic cocci are gram positive, antibacterial therapy effective against gram-positive organisms should be used when it is necessary to initiate therapy before culture and sensitivity results are received.
Dermatophilus congolensis: Dermatophilus congolensis is an aerobic to facultatively anaerobic actinomycete that infects the superficial epidermis, causing exudative, crusty lesions. Removal of these crusty lesions reveals eroded to ulcerated skin lesions. Cytologic preparations from the undersurface of scabs from these crusty lesions are most rewarding in demonstrating organisms. These preparations usually contain mature epithelial cells, keratin bars, debris, and organisms. A few neutrophils may also be found. If the undersurface of scabs is dry and does not yield adequate cytologic preparations, crusts and scabs may be minced in saline and smears made for cytologic evaluation. Dermatophilus congolensis replicates by transverse and longitudinal division, producing chains of coccoid cells arranged in two to eight parallel rows. These chains resemble small, blue railroad tracks (Fig. 2-5). Also, many individual coccoid cells may be seen cytologically.
Fig. 2-5 Imprint from underside of scab caused by Dermatophilus congolensis.
There is a background of squamous debris and numerous chains of bacterial doublets. Scattered individual bacteria are also present. (Wright’s stain; original magnification 250X)
Small Bacterial Rods: Most small bacterial rods are gram negative; however, some, such as Corynebacterium spp, are gram positive. Some gram-negative rods can be recognized cytologically as bipolar (Plate 3D). All pathogenic bipolar bacterial rods are gram negative. Rod bacterial infections are usually associated with a marked neutrophilic inflammatory response. When small bacterial rods are recognized in cytologic preparations, the lesion should be cultured to identify the organism and sensitivity tests performed to determine appropriate antibacterial therapy. If it is necessary to institute antibacterial therapy before culture and sensitivity results are received, therapy employed should be effective against gram-negative organisms, since most pathogenic small rods are gram negative.
Filamentous Rods: Filamentous rods that can cause cutaneous infections include Nocardia spp., Actinomyces spp., certain anaerobes (eg, Fusobacterium spp.), and Mycobacterium spp. Because these organisms are often refractory to common antibacterial therapy and reliable culture of these organisms has special requirements, cytologic evaluation is very useful in indicating that special cultures are needed.
Rarely, the pathogenic filamentous rods of Nocardia spp or Actinomyces spp. (Plate 3F) may cause cutaneous or subcutaneous lesions (abscesses, ulcers, draining tracts, lumps) in horses. These lesions are sometimes referred to as actinomycotic mycetomas. Infection with these agents is uncommon and usually occurs secondary to contamination of existing wounds.1 Also, Mycobacterium spp. and some anaerobes, such as Fusobacterium, rarely may be filamentous. Nocardia and Actinomyces generally have a distinctive morphology in cytologic preparations stained with Romanowsky-type stains. They are characterized by long, slender (filamentous) strands that stain pale blue and have intermittent, small, pink to purple areas (dots). This morphology is characteristic of both Nocardia and Actinomyces spp. and the filamentous form of Fusobacterium spp. When these features are recognized cytologically, cultures should be performed specifically for Nocardia, Actinomyces, and anaerobes.
Mycobacterium spp. (atypical mycobacterial infections and cutaneous tuberculosis), on the other hand, often do not stain with Romanowsky-type stains. As a result, negative images (Plate 4A–B) may be observed in the cytoplasm of macrophages and/or inflammatory giant cells. When epithelioid macrophages and/or inflammatory giant cells are encountered in cytologic preparations not containing any obvious organisms, a careful search for negative images of Mycobacterium spp. should be made. Mycobacterium spp. stain with acid-fast stains. Therefore, when negative images are encountered or when the character of the lesion suggests Mycobacterium spp., an acid-fast stain can be performed to demonstrate the organism and/or cultures for Mycobacterium spp. can be performed to identify the organism.
Large Bacterial Rods: Large bacterial rods found in cytologic preparations may be pathogenic or nonpathogenic. Those that are pathogenic and sometimes infect cutaneous and subcutaneous tissues include Clostridium spp. and, infrequently, Bacillus spp. When large bacterial rods are thought to be pathogenic, both aerobic and anaerobic cultures should be performed. Also, the smears should be inspected for large bacterial rods that contain spores.
Sporothrix schenckii: Sporothrix schenckii infection (sporotrichosis) (Plate 4D) most commonly occurs in a cutaneolymphatic form; however, a primary cutaneous form with no lymphatic involvement is seen occasionally. In the cutaneolymphatic form, hard subcutaneous nodules develop along lymphatics and the lymphatics may become corded. The nodules may ulcerate. In horses, the organisms are scarce and cytologic preparations must be perused carefully. If organisms are not found, the lesion should be cultured and a biopsy of the lesion should be submitted for histopathologic evaluation.
In cytologic preparations stained with Romanowsky stains, Sporothrix schenckii organisms are round to oval to fusiform (cigar shaped). They are 3 to 9 μ long and 1 to 3 μ wide and stain pale to medium blue with a slightly eccentric pink to purple nucleus (Plate 4D). They may be confused with Histoplasma capsulatum if only a few organisms are found and the classic fusiform (cigar shape) is not seen.
Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans, and Coccidioides immitis: Cutaneous lesions secondary to infection with Blastomyces, Cryptococcus, Coccidioides, or Histoplasma organisms are rare. These organisms may on rare occasion produce a primary cutaneous lesion or disseminate from other sites and secondarily infect the skin.2–4 Characteristics of these organisms in cytologic preparations stained with Romanowsky-type stains are as follows:
Histoplasma organisms (Plate 4C) are round to slightly oval but are not fusiform or cigar shaped. They are 2 to 4 μ in diameter (about half the size of a RBC), stain pale to medium blue, and contain an eccentric pink to purple nucleus. There is usually a thin, clear halo around the yeast.
Blastomyces dermatitidis organisms are blue, spherical, 8 to 20 μ in diameter, and thick walled (Plates 4E–G). Most organisms are single, but occasionally those showing broad-based budding are found. Imprints of these lesions usually have a cell composition characteristic of pyogranulomatous inflammation (Plate 3B) and few to many organisms.
Cryptococcus neoformans organisms are spherical and usually have a thick mucoid capsule; occasionally, nonencapsulated (rough) forms are found. The organism is 4 to 8 μ in diameter excluding the capsule and 8 to 40 μ in diameter including the capsule. The organism stains light pink to blue-purple and may be slightly granular (Plate 4H). The capsule usually is clear and homogeneous, but it may stain light to medium pink. Cryptococcosis usually evokes a minor granulomatous response of epithelioid macrophages and/or inflammatory giant cells. In some cytologic preparations, Cryptococcus organisms may outnumber inflammatory and tissue cells. Weakly encapsulated (rough) forms tend to elicit a greater inflammatory response than heavily encapsulated forms.
Coccidioides immitis organisms are large (10 to 100 μ in diameter), double-contoured, blue to blue-green spheres with finely granular protoplasm (Plate 5A). Round endospores 2 to 5 μ in diameter may be seen in larger organisms. Cytologic preparations usually have a cell composition characteristic of pyogranulomatous or granulomatous inflammation (Plate 3B). Coccidioides organisms usually are scarce. The tremendous variation in size, presence of endospores, and green tint to the organism differentiate Coccidioides immitis from nonbudding Blastomyces dermatitidis.
Dermatophytes: The dermatophytes Trichophyton spp. and Microsporum spp. cause cutaneous lesions that may have the typical ringworm-like appearance or appear as gray to yellow-brown crusty lesions or as follicular papules.2 Scrapings from the edge of the lesion are most rewarding when searching for dermatophytes. They can be identified in cytologic preparations using the standard 20% potassium hydroxide, in wet mount preparations stained with new methylene blue, or in air-dried preparations stained with Romanowsky-type stains. Cytologically, conidia are found free within the smears as well as within hair shafts (endothrix invasion) or on the hair shaft surface (ectothrix invasion). With Romanowsky-type stains, conidia stain medium to dark blue with a thin clear halo (Fig. 2-6). An inflammatory reaction composed of an admixture of neutrophils, macrophages, lymphocytes, eosinophils, and plasma cells may be seen in cytologic preparations from skin scrapings.
Fig. 2-6 Scraping from animal with ringworm.
Several degenerating neutrophils are present, along with RBCs and a row of dermatophyte organisms attached to a hair shaft. (Wright’s stain; original magnification 330X)