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Practical Cytology For Inflammatory Skin Diseases
Practical Cytology For Inflammatory Skin Diseases Colleen Mendelsohn, DVM, DACVD, Wayne Rosenkrantz, DVM, DACVD, and Craig E. Griffin, DVM, DAVCD Cytology is the most common and immediately informative diagnostic tool in the practice of dermatology. It is simple to perform and requires equipment usually already present in most practices such as a microscope and slides, cotton-tipped applicators and stains. Cytology interpretation is also easily self taught. With little practice the practitioner can easily become familiar with sample interpretation from inflammatory lesions; including recognition of bacteria, yeast, dermatophyte spores and hyphae, deeper fungal elements and parasites. Additionally, the practitioner should become familiar with the variety of inflammatory cells seen with these conditions, as well as keratinocyte morphology. This article provides an overview and the initial steps on how to become a more proficient cytologist in practice. Clin Tech Small Anim Pract 21:117-127 © 2006 Elsevier Inc. All rights reserved. KEYWORDS cytology, cytological techniques, microscope, bacteria, yeast
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utaneous cytology is one of the most valuable tools in the practice of veterinary dermatology. It gives the practitioner immediate information regarding presence of infectious organisms, inflammatory cells and other processes such as allergic inflammation, acantholysis, and others that occur in the skin. It is inexpensive to perform and will often allow the practitioner to make immediate decisions on the direction of further diagnostics. Cytological evaluation skills are easily honed in practice. In the event a practitioner is not comfortable making diagnostic decisions based on their cytological evaluation, duplicate samples can be obtained and an unstained sample can be submitted to a clinical pathologist for corroboration. In addition, it is important to get samples from nondiseased skin and ears to become comfortable with normal findings such as melanin granules and keratinocytes. The practitioner also needs to be able to identify artifacts such as stain precipitates, which can be confused with infectious microorganisms. Cytology can aid in the diagnosis of numerous types of cutaneous conditions. The interpretation of samples taken from neoplastic lesions requires additional expertise and specialized training and is beyond the scope of this paper. However, in veterinary dermatology, the vast majority of cases in which cytological evaluation is utilized are inflammatory and/or infectious in nature. This paper will focus on sample collection techniques and the identification of infectious organisms, inflammatory cells, changes in keratinocyte morphology and artifacts that may be encountered.
Animal Dermatology Clinic, Tustin, CA. Address reprint requests to Colleen Mendelsohn, DVM, DACVD, Animal Dermatology Clinic, 2965 Edinger Avenue, Tustin, CA 91780. E-mail: [email protected]
1096-2867/06/$-see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1053/j.ctsap.2006.05.004
Equipment and Techniques Equipment The equipment that is required for collection and evaluation of cytological samples include: Microscope, microscope slides and cover slips, syringes and needles, cytologic stain, cotton-tipped applicators, and a heat source. Other optional equipment includes: transparent acetate tape and mounting media (Fig. 1).1-4
Microscope A good quality, well maintained, microscope with multiple objectives is imperative in cytologic evaluation (4⫻, 10⫻, 40⫻, and oil immersion 100⫻ lenses). Binocular scopes are vastly superior to monocular lens scopes. Halogen bulbs are also preferred over incandescent light sources. There are three main types of immersion oil: Types A, B, and NVH. These represent different viscosities with Type A being the least viscous and Type NVH being the most viscous. Overall, for in-house use type B is probably best, but Type A is often preferred because it is less messy, easier to clean up and less expensive.5 When examining a sample, it is best to scan slides under low power first to identify location and types of cells before choosing an area to examine under oil immersion. When an area is examined under low power it allows the practitioner to get a general idea of the amount and types of cells present. Often, the entire sample will be covered with keratin debris or corneocytes and the inflammatory cells will only be found in a few areas. Examining the area that contains inflammatory cells under higher magnification can aid in identifying more detailed cellular morphology and the presence of microbes. 117
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stained and viewed normally. In the authors’ experience, these slides are unnecessary and if not handled just right, will pick up environmental debris.
Syringes and Needles These are used for aspiration of samples from nodules, tumors, plaques, abscesses, or other lesions where a deep (nonsurface) sample is required. Ideally, a 20 or 22 gauge needle is attached to a 6 or 12 mL syringe. It is important that the needle diameter be large enough destruction of cells as they pass through the lumen of the needle and allow passage of material and; 6 to 12 mL syringes are necessary to create enough suction to collect an adequate sample. Figure 1 Staining station. The picture shows a typical staining station with Wright’s stain, wash tub and bottle of water for rinsing the slides. Matches for heat fixation and clothes pins to grasp the slides during the heating process. Also photographed is bibulous paper for drying the slides. (Color version of figure is available online.)
The presence of intracellular organisms indicates that the microbes are pathogens and associated with a true infection. Finding microbes without the presence of inflammation is more indicative of either nonpathogenic bacteria, bacterial colonization or failure to select the infected area for sampling. When examining oil immersion fields, it is important to constantly manipulate the fine focus. This is done so that organisms at different levels can be found and their refractory characteristics can be evaluated. This can help differentiate melanin granules and stain precipitates from bacteria and helps identify the clear capsules often found around fungal elements.1-4,6 Generally, it is important to keep the light condenser up when examining a cytology sample (Köhler illumination). This is in contrast to examining skin scrapes or unstained tape strip samples where the shadows and contrasts created by keeping the condenser down can aid in the identification of fungal spores, hyphae, parasites, and hair shaft abnormalities. If the microscope is used more than once or twice daily in practice, it is strongly recommended that the light source be dimmed, but kept switched on. Repeatedly turning the microscope on and off leads to the bulb burning out more rapidly than when the light source is kept on.
Microscope Slides and Cover Slips Slides with frosted ends are easier to label for identification. Most commercial laboratories will provide microscope slides at little or no cost as part of their services. Cover slips must be used with wet stains such as New Methylene Blue, as these cannot be examined under high power without a cover slip. Cover slips are also used for permanent mounting (see below). Unless the slides are going to be kept permanently, cover slips are usually not required with routine Wright’s stains. In addition to traditional glass microscope slides, there are “Sticky Slides” (Sticky Slides Nasco, Fort Atkinson, WI). To use these slides, the protective covering is removed and then the slide is applied directly to a lesion. The slide is then
Stains The easiest stain to use in-house is a commercial modified Wright’s stain like Diff Quik (Diff Quik Baxter Diagnostics, McGaw Park, IL). This is a Romanovsky-type stain that gives less nuclear detail than a vital stain such as New Methylene blue. It is important to keep the stain clean. Staining tubs should be changed weekly and separate tubs should be used for “dirty” samples such as anal gland cytology and fecal cytology, because the potential for bacterial contamination is high. Other stains used in cytology include New Methylene Blue (NMB), acid fast, Gamori methanine silver (GMS), Giemsa, and others. NMB is a vital stain and gives better nuclear detail than Wright’s stains. Gram Stains are rarely performed in practice because of the time commitments and minimal benefit of differentiating Gram-negative versus. Gram-positive organisms, as this is doesn’t change immediate clinical decisions. Other stains that are utilized in cytology require professional laboratory techniques. Ordering acid fast staining is the best diagnostic test of identifying Mycobacterium spp infections. Occasionally, other stains can be applied to cytology samples.1
Cotton-Tipped Applicators (CTA) These are used to obtain samples from exudative or purulent areas such as ears, skin folds and interdigital spaces that do not lend themselves to direct contact with a microscope slide.
Heat The importance of heat fixation on waxy, greasy, or exudative samples is open to debate, however, in the authors’ experience; heat fixation helps to fix samples to the glass slide and prevents sample loss during the staining process. Heat fixation of a slide can be done with a variety of methods. One method is to use a blow dryer directly on the slide from a distance of about 10 inches. A Bunsen burner, a cigarette lighter or even a match can also be used. Hold the slide over the flame for 5 seconds without burning the slide or damaging the sample by overheating. If the flame is not burning “clean,” (as with a Bunsen burner) soot will accumulate on the bottom of the slide. This is easily wiped off either before or after the slide is stained (Fig. 2).
Transparent Acetate Tape Transparent acetate tape can be used to pick up epidermal debris, obtain samples from dry lesions that will not stick to
Practical cytology for inflammatory skin diseases
119 check that the cotton tip is attached to the stick of the CTA well and will not become dislodged. If the patient allows, the ear flap should be lifted up at about a 30 degrees from dorsal, and the CTA is applied to the vertical ear canal as deep as possible, being careful not to damage the tympanic membrane (Fig. 3). The exudate is then rolled on a glass slide, and a notation is made on the slide to differentiate right from left ears. Using a CTA within exudative skin folds, such as brachycephalic facial folds and tail folds or vulvar folds is also an excellent way of collecting intertriginous samples.1
Moist or Exudative Skin Lesions
Figure 2 Heat fixation with a match. The slide is held with a clothes pin just above the flame of a match for 5 to 10 seconds. Care must be taken not to overheat and ruin the sample. Soot can be easily wiped away both before and after staining. (Color version of figure is available online.)
a glass slide easily or to obtain samples from areas that are difficult to get to with a glass slide. To examine a sample obtained with acetate tape, you can either stain the tape directly [skipping the fixation (clear) solution as this will prevent the tape from laying flat on the glass slide] or place a drop of the third solution (the purple stain) or NMB onto a glass slide before placing the unstained tape directly over it. These samples can be examined with all of the objectives, including oil.
Cytology of crusts, exudation or purulent debris, pustules or papules, or other lesions in which the surface of the skin is sampled, are usually best obtained by directly impressing or firmly rubbing the glass slide firmly across the surface of the lesion in a single motion. Rubbing back and forth should be avoided. When there are crusts present it is important to obtain the sample from the skin beneath the crust. If a pustule or papule is sampled it is recommended to tease the lesion open first with a 25 gauge needle (Fig. 4).
Dry or Waxy Skin Lesions The acetate tape technique is used on dry, scaly lesions, or lesions that are anatomically difficult to reach, such as interdigital spaces or tragal folds. To obtain an acetate tape sample, the sticky side of the tape is pressed firmly against the area several times, then stained and prepped as described above. Direct impression with a glass slide can also be used and requires firm impression techniques and then heat fixation is critical. In the authors’ experience direct impression is just as effective in obtaining informative samples as using the acetate tape technique.
Mounting Media: Permount Mounting Medium (optional) This is used if a slide is of particular interest or if the practitioner is creating a permanent library of cytology samples and slide is to be kept permanently. To “Permount” a slide, a drop of Permount solution (Permount ProSciTech, Kirwin, Australia) is applied to the top of a clean sample (any oil should be removed) and a cover slip is placed on top, avoiding air bubbles. Once the slide is dry, it should be stored away from light.
Sample Collection Depending on location or the type of lesion, there are several ways to obtain samples for evaluation.
Ears Ear cytology is the most common sample to collect and is often the easiest to interpret, because the material is being evaluated for infectious organisms and the interpretation of cell morphology or type is not as relevant to the diagnosis. The most straight forward way to obtain a sample from the ear is with a CTA. Before obtaining a sample it is important to
Figure 3 Obtaining an ear sample with a CTA. The pinna is held about 30 degrees from vertical to straighten the canal and the CTA is applied as deep as possible, being careful not to damage the tympanic membrane. (Color version of figure is available online.)
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C. Mendelsohn, W. Rosenkrantz, and C.E. Griffin
Figure 4 Obtaining a direct impression sample from the periocular skin. (Color version of figure is available online.)
Figure 6 Yeast organisms (100⫻). The footprint-shaped organisms are found in abundance adherent to keratin and keratinocytes. (Color version of figure is available online.)
Claw Folds
Nodules/Plaques/Abscesses
Claw folds often contain infectious microorganisms that can contribute to pedal pruritus or discomfort. Occasionally, the infection within claw folds is different from what is found on the surface of the paws. Obtaining a sample from this area can be challenging. The most valuable technique is to break a wooden CTA so that a tapered firm edge is created. The tapered tip can then be inserted and rubbed gently within the claw folds and then rolled on a glass slide (Fig. 5).7
These lesions are best sampled by aspiration. For aspirate samples, ensure the needle is firmly attached to the syringe and insert it into to the center of the lesion. The needle should be held stable and the plunger pulled back fully several times. The plunger should be neutral with no negative pressure when the needle and syringe are withdrawn. Unless the sample is fluid, the needle will contain most of the desired sample. Remove the needle from the syringe, pull the plunger back and replace the needle. The sample can then be expelled onto the glass slide. The sample on the slide can be smeared evenly by using an additional slide before fixation and staining.
Sample Interpretation Infectious Organisms
Figure 5 Collecting sample from claw folds. Clean tapered edge from a broken CTA is gently inserted within the claw fold to collect sample. (Color version of figure is available online.)
The most common types of organisms encountered in cutaneous cytology are yeast and bacteria. The most common yeast organism found on samples from dogs and cats is Malassezia pachydermatis, although Candida spp can also be found, on the skin, in the ears and in the oral cavity. This organism usually has the classic “foot print” shape of a budding yeast organism. M. pachydermatis is usually 3 to 5 m in diameter (red blood cells are 6-8 m in diameter). Yeast organisms are usually found adherent to corneocytes (Fig. 6). In one study, average yeast counts greater than 5 organisms per 40⫻ high powered field (HPF) were considered significant.8 However, in the authors’ experience, average yeast counts as low as 0.5 to 1 organism per oil immersion field (OIF) are often significant when the sample is from an inflamed ear and even lower than that for samples obtained from the skin.1,3,8,9 The variety of bacteria that can be found on cutaneous cytology is vast. However, Staphylococcus intermedius infec-
Practical cytology for inflammatory skin diseases
Figure 7 Mixed infection of bacteria and yeast (100⫻). This sample contains rod-shaped Pseudomonas spp. (as verified based on a culture), coccoid Staphylococcus intermedius as well as Malassezia pachydermatis organisms. (Color version of figure is available online.)
tion is the most common bacteria isolated from infections of the ears and skin of dogs and cats. S. intermedius is a coccoid bacterium, 0.5 to 1.5 m in diameter and is often found in pairs. S. intermedius and other coccoid bacteria can be normal inhabitants of the ear and skin. Previous studies have determined that average otic bacterial counts greater than 25 organisms/HPF are significant.8 In another study cocci counts greater than 5/HPF or rod counts greater than 1/HPF should be considered pathogenic.1,8,10 Another commonly encountered type of bacteria, especially from ear samples, is Pseudomonas aeruginosa. These are rod-shaped organisms, approximately 0.3 to 0.8 m wide by 1.0 to 1.2 m long and are often found in short chains. Because Pseudomonas spp and other rod-shaped organisms often have high incidence of antibiotic resistance, seeing these bacteria on cytology indicates the need for a bacterial culture and sensitivity (Fig. 7).1,3 Mycobacteria can also be seen on cytology with the use of acid-fast stains. The authors prefer Nocardia acid fast stains for suspect mycobacterium infections, as it tends to yield better staining for the common species seen in dogs and cats. Aspiration is the best reported method for identification. Aspirate cytology should be submitted with biopsies for acid fast staining if these organisms are suspected.1 Another bacterium occasionally encountered is Simonsiella spp. These are harmless very large filamentous Gram-negative saprophytes that inhabit the oral cavity of a variety of warmblooded vertebrates. They are 6 to 8 m long and 2 to 3 m wide. There are usually found in small numbers and are not intracellular (although they can be adherent to keratinocytes). These organisms are not known to be pathogenic; however, when found on areas not associated with the oral cavity, such as the lip folds or nasal planum, their presence is likely associated with licking. If the practitioner is not famil-
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Figure 8 Simonsiella organisms (100⫻). They are much larger than the rod-shaped bacteria also seen in the picture. (Color version of figure is available online.)
iar with this organism it can create some concern because of its unique morphology (Fig. 8).1,11 Numerous dermatophyte and other fungal organisms can be demonstrated with either exfoliative or aspiration cytology. Dermatophyte spores and hyphae are visible, even with Wright’s stain. These organisms are usually more easily demonstrated in samples from cats than from dogs. Dermatophyte spores often appear as round spheres, usually about twice the size of coccoid bacteria. The spores are
Figure 9 Dermatophyte spores (100⫻). These are about twice the size of cocci with a clear “ghost” halo outlining them on the background of the keratinocyte. Courtesy Michael Canfield, DVM, Animal Dermatology Clinic, Tustin, CA.
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Figure 10 Dermatophyte hyphae (100⫻) seen in the midst of inflammatory cells. This sample was taken from a fungal furunculosis secondary to a Trichophyton spp. infection in a dog. (Color version of figure is available online.)
surrounded by a capsule that limits staining and gives the appearance of a clear halo (Fig. 9). Hyphae recognition is a bit more challenging as they can be easily confused for debris and hair keratin in the field. Dermatophyte hyphae are uniform 2 to 3 m wide filamentous structures often with poor staining characteristics and may appear as “ghost-like” shadows found free in the specimen (Fig. 10).1 Wet mount cytology is another technique used in the identification of dermatophytes. Here a solution is used to dissolve keratins and other debris and the sample is examined “wet” and without staining. This method has very low sensitivity and for that reason is not commonly employed by the authors. When clinical suspicion exists for a dermatophyte, negative findings here do not negate the necessity for further diagnostics. Also, if KOH tests are positive, the authors and others have found that the spores and hyphae can be also found on routine cytology. To utilize this method, KOH is the most commonly used solution; however the use of chlorphenolac has also been recommended. The solution is applied to a slide and hairs and keratin debris are added to it. With KOH, the specimen is left to dry for 30 minutes (or gently heated by leaving on the microscope stage with the light on) and examined. With chlorphenolac, the slide can be examined immediately. These solutions allow the identification of yeasts, conidia, hyphae, or pseudohyphae. For best results, fragmented or distorted hairs, usually near the hair bulbs, should be examined.1 Other subcutaneous (SQ) or deep fungal organisms can also be identified with exfoliative or aspiration cytology, including but not limited to; Cryptococcus spp. Coccidioides spp, organisms of phaeohyphomycosis, Pythiosis, organisms of zygomycosis, Sporotrichosis, Blastomycosis, and Histoplasmosis (Figs. 11-13).1 Ectoparasites are not commonly diagnosed with exfoliative
C. Mendelsohn, W. Rosenkrantz, and C.E. Griffin
Figure 11 Cryptococcus organisms (100⫻). Smear obtained from a cat with generalized cutaneous lesions. (Color version of figure is available online.)
cytology. However, in exudative processes with severe, deep secondary pyodermas, organisms such as Demodex spp. and Sarcoptes spp. can be identified when the slide is scanned under low power (Figs. 14 and 15).
Inflammatory Cells Identifying inflammatory cells on cytology gives the practitioner a clue as to the pathogenesis of the lesion. Presence of degenerate neutrophils, with (Fig. 16) intracellular bacteria, is most often indicative of a pyoderma. If well-preserved neutrophils are seen, then sterile inflammatory conditions such
Figure 12 Coccidiomycosis organism (100⫻) as seen from a cutaneous lesion on a cat. (Color version of figure is available online.)
Practical cytology for inflammatory skin diseases
Figure 13 Sporotrichosis fungal elements (100⫻). Classic cigar shaped yeast organisms with a clear halo around them.
as pemphigus foliaceus, primary allergic inflammation, irritant reaction, and others must be considered. At times, the force that is required to obtain an exfoliative sample ruptures any present inflammatory cells, especially neutrophils. When this occurs, the nuclear material from these cells often streaks the slide and can be identified on low power (Fig. 17). The presence of eosinophils indicates inflammation secondary to parasites, hypersensitivity reactions such as food allergies or atopy, pemphigus foliaceus, foreign body reaction (such as free keratin within the dermis), eosinophilic granuloma, mast cell tumors, or other sterile eosinophilic
Figure 14 Demodex canis (10⫻). These poorly staining organisms are seen in a background of inflammatory cells. (Color version of figure is available online.)
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Figure 15 Sarcoptes (10⫻) Poorly staining organism that could only be found if scanning on low magnification lens. It is better visualized by dropping the condenser light as seen here.
diseases (Fig. 18). Eosinophils can also be seen in ear cytology with topical reactions. Intracellular bacteria can also be seen within eosinophils.1 Other common inflammatory cells recognized are mononuclear macrophages and lymphocytes. These are usually seen from more chronic or deeper lesions. Macrophages are efficient phagocytic cells and often contain effete neutrophils, red blood cells, bacteria, fungal elements and even intracellular parasites such as Leishmania spp organisms and others.
Figure 16 Intracellular rods (100⫻) Phagocytosed rods within a neutrophil as seen from a Pseudomonas aeruginosa infection on the skin. Courtesy Michael Canfield, DVM, Animal Dermatology Clinic, Tustin, CA. (Color version of figure is available online.)
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Figure 17 Ruptured cells with streaks of nuclear material (100⫻). Numerous coccoid bacteria seen streaked within the nuclear material; indicating likely intracellular organisms. There is an intact degenerated eosinophil at the bottom left of the picture as well. (Color version of figure is available online.)
C. Mendelsohn, W. Rosenkrantz, and C.E. Griffin
Figure 19 Mycobacteria (100⫻) Acid fast staining reveals the rodshaped organisms within macrophages. Aspirate sample taken from atypical mycobacterial lesions on the pinnae of a dog.
Depending on the severity of the wound or lesion contamination, macrophages will often be markedly vacuolated and degenerate. Mycobacteria and fungal elements are often found within these vacuoles when stained with acid-fast or GMS stains, respectively (Fig. 19). When samples from deep infections are examined, the offending organism is often not readily identified; however, the presence of degenerated neutrophils combined with degenerated and vacuolated macrophages or lymphocytes indicates infection and the possible
need for a tissue culture. Xanthoma cells and multinucleated giant cells are unique presentations of macrophages. Multinucleate giant cells represent numerous macrophages that have fused together in an attempt to phagocytose large or toxic foreign material (Fig. 20). These are more often noted in histological lesions, but can be seen from deep pyoderma secondary to Demodicosis, ruptured hair follicles, foreign bodies and others. Xanthoma cells are macrophages from xanthoma lesions in cats that have phagocytosed lipid material and are often multinucleated giant cells that have markedly foamy and granular cytoplasm.1,3
Figure 18 Eosinophil (100⫻) from canine allergic patient with intracellular cocci.
Figure 20 Multinucleated giant cell (100⫻). Large cell with two obvious nuclei.
Practical cytology for inflammatory skin diseases
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Figure 21 Feline mast cell (100⫻) taken from area of a deep pyoderma. (Color version of figure is available online.)
Mast cells are often sparsely present in cytology from parasitic or allergic lesions (especially from cats) (Fig. 21). However, if full fields of mast cells are present that is usually indicative of a mast cell tumor. Plasma cells are often seen from deep chronic lesions, but when full fields are noted, plasmacytomas or plasma cell pododermatitis become the primary differentials. Chronic healing wounds, such as acral lick lesions often have spindle-shaped fibroblasts that are not necessarily neoplastic (Fig. 22). However, like with the presence of full fields of mast cells or plasma cells, the presence of numerous spindle cells indicates the need for a biopsy to rule out neoplasia.1-3
Figure 23 Keratinocytes containing multiple keratohyaline granules from a cat (A) and a dog (B). (Color version of figure is available online.)
Epithelial Cells
Figure 22 Fibroblast (100⫻).
Epithelial cells encountered normally on exfoliative cytology are corneocytes (nonnucleated keratinocytes). Keratin and keratinocytes are a normal finding; however, in diseased skin, keratinocyte morphology often has diagnostic value. Nucleated keratinocytes are normally found in samples from mucous membranes, when the surface of the skin was scraped before cytology or occasionally from ear samples. When found from surface skin samples it is associated with parakeratosis and suggestive of abnormal turnover of the epithelial layers or erosion of the cornified layers. These keratinocytes often contain keratohyaline granules and should not be confused with bacteria (Fig. 23). Instead of appearing as flat sharply angulated large cells, some keratinocytes or
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Figure 24 Raft of acantholytic cells (40⫻). These large round cells have a dark-staining periphery. They are found among numerous well preserved neutrophils and eosinophils.
corneocytes can appear rolled or more spindle shaped on routine cytology because of folding and rolling during the sampling and fixation process. Acantholytic cells are lower layer keratinocytes that have lost their adhesion to neighboring keratinocytes. They are most commonly found in pemphigus foliaceus lesions, but can also be seen with deep furunculosis secondary to conditions such as Demodicosis and dermatophytosis. Acantholytic cells are large, round cells with a central nucleus. They often stain with a dark blue periphery that may indicate antibody coating. In cases of pemphigus, they are often found in rafts and among well preserved neutrophils and/or eosinophils. In cases of Demodicosis or dermatophytosis, acantholytic cells are usually sparse and found among degenerate inflammatory cells (Fig. 24).1-3
C. Mendelsohn, W. Rosenkrantz, and C.E. Griffin
Figure 25 Melanin granules (100⫻). These rod-shaped brown-black organelles are the same size and shape as most rod-shaped bacteria. Their color, distribution and refractory nature differentiates them from pathogenic organisms.
tate is very dark purple staining and has an irregular appearance and distribution.1-3 Pollen can also be confusing. It comes in multiple shapes and sizes and is often confused for fungal elements. Pollen tends to be dramatically geometric and is usually not present in large numbers. The presence of pollen is not pathogenic, however, when seen on a sample from a dog with pododermatitis and clinical symptoms consistent with atopic derma-
Artifacts and Other Findings Artifacts are often encountered on cytology slides. Practitioners need to be familiar with these, as they can often be confused for significant findings. Many exudative samples have a markedly proteinaceous background, which often looks light purple or pink with fine darker staining granules that can be mistaken for small bacteria. However, they are generally smaller, more evenly sized and uniform in appearance than bacteria. Melanin granules can also be mistaken for rod-shaped bacteria such as Pseudomonas spp. However, melanin granules are most often found in and around keratinocytes, stain a refractory brown to black color and are very even in size. Their refractory nature is more easily appreciated when the fine focus is manipulated (Fig. 25). Medication debris often appears as crystalloid poorly staining debris in a variety of sizes. It is most often seen in samples taken from the ear where large amount of medication has accumulated within the debris. Stain precipitate can also be mistaken for bacteria. It is a problem mostly with the NMB stain. Precipi-
Figure 26 Pollen grain (100⫻) found on sample from ventral interdigital webbing on a dog with pododermatitis secondary to atopy. (Color version of figure is available online.)
Practical cytology for inflammatory skin diseases titis, it suggests possible exposure to environmental pollens and may play a role in the patient’s clinical disease (Fig. 26).1
Summary Overall, when evaluating inflammatory skin conditions, cytology is the single most commonly used diagnostic test performed in the practice of dermatology. In a given month at a dermatology referral practice, cytological samples were taken from approximately 60% of patients presented, comprising about 8 to 9% of the clinic’s total income! It is not only used when cases initially present, but used to monitor therapy and indicate when an infectious or inflammatory condition may have changed. For example, if a dog with pemphigus foliaceus that was previously controlled seems to flare, cytology should be one of the first tests performed to evaluate whether the flare is because of the primary disease, or a pyoderma secondary to immune suppression. The finding of numerous intracellular cocci and no acantholytic cells would support a pyoderma that maybe secondary to therapy and further immune suppression would be contraindicated. Cutaneous cytology is inexpensive, highly informative and easily self-taught. By making cytology part of everyday practice, diagnostics and treatments become more proficient and case management is immediately more rewarding. The ultimate result is significantly happier patients and clients.
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References 1. Scott DW, Miller WHJ, Griffin CE: Muller & Kirk’s small animal dermatology (ed 6). Philadelphia, W.B. Saunders Company, 2001 2. Baker R, Lumsden JH: Color atlas of cytology of the dog and cat. St. Louis, MO, Mosby, 2000 3. Medleau L, Hnilica KA: Small animal dermatology: A color atlas and therapeutic guide (ed 2). St. Louis, MO, Saunders, 2006 4. Nesbitt GH, Ackerman LJ: Canine and feline dermatolog. (ed 2). Trenton, NJ, Veterinary Learning Systems, 1998 5. Cargille JJ: Immersion oil and the microscope (ed 2). New York Microscopial Society Yearbook, Cargille-Sacher Laboratories, Inc., 1985 6. Pappalardo E, Martino PA, Noli C: Macroscopic, cytological and bacteriological evaluation of anal sac content in normal dogs and in dogs with selected dermatological diseases. Vet Dermatol 13:315322, 2002 7. Mueller RS: Diagnosis and management of canine claw diseases. Vet Clin North Am Sm Anim Practice 29:1257-1371, 1999 8. Ginel PJ, Lucena R, Rodriguez JC, et al: A semiquantitative cytological evaluation of normal and pathological samples from the external ear canal of dogs and cats. Vet Dermatol 13:151-156, 2002 9. Bensignor E, Jankowski F, Seewald W, et al: Comparison of two sampling techniques to assess quantity and distribution of Malassezia yeasts on the skin of Basset Hounds. Vet Dermatol 13:237-241, 2002 10. Columbo S: Quantitative evaluation of cutaneous bacteria in normal dogs and dogs with pyoderma by cytological evaluation. Faculty of Veterinary Medicine. Doctoral Thesis Milan: University of Milan, 1997 11. Carandina G, Bacchelli M, Virgili A, et al: Simonsiella filaments isolated from erosive lesions of the human oral cavity. J Clin Microbiol 19:931933, 1984
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utaneous cytology is one of the most valuable tools in the practice of veterinary dermatology. It gives the practitioner immediate information regarding presence of infectious organisms, inflammatory cells and other processes such as allergic inflammation, acantholysis, and others that occur in the skin. It is inexpensive to perform and will often allow the practitioner to make immediate decisions on the direction of further diagnostics. Cytological evaluation skills are easily honed in practice. In the event a practitioner is not comfortable making diagnostic decisions based on their cytological evaluation, duplicate samples can be obtained and an unstained sample can be submitted to a clinical pathologist for corroboration. In addition, it is important to get samples from nondiseased skin and ears to become comfortable with normal findings such as melanin granules and keratinocytes. The practitioner also needs to be able to identify artifacts such as stain precipitates, which can be confused with infectious microorganisms. Cytology can aid in the diagnosis of numerous types of cutaneous conditions. The interpretation of samples taken from neoplastic lesions requires additional expertise and specialized training and is beyond the scope of this paper. However, in veterinary dermatology, the vast majority of cases in which cytological evaluation is utilized are inflammatory and/or infectious in nature. This paper will focus on sample collection techniques and the identification of infectious organisms, inflammatory cells, changes in keratinocyte morphology and artifacts that may be encountered.
Animal Dermatology Clinic, Tustin, CA. Address reprint requests to Colleen Mendelsohn, DVM, DACVD, Animal Dermatology Clinic, 2965 Edinger Avenue, Tustin, CA 91780. E-mail: [email protected]
1096-2867/06/$-see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1053/j.ctsap.2006.05.004
Equipment and Techniques Equipment The equipment that is required for collection and evaluation of cytological samples include: Microscope, microscope slides and cover slips, syringes and needles, cytologic stain, cotton-tipped applicators, and a heat source. Other optional equipment includes: transparent acetate tape and mounting media (Fig. 1).1-4
Microscope A good quality, well maintained, microscope with multiple objectives is imperative in cytologic evaluation (4⫻, 10⫻, 40⫻, and oil immersion 100⫻ lenses). Binocular scopes are vastly superior to monocular lens scopes. Halogen bulbs are also preferred over incandescent light sources. There are three main types of immersion oil: Types A, B, and NVH. These represent different viscosities with Type A being the least viscous and Type NVH being the most viscous. Overall, for in-house use type B is probably best, but Type A is often preferred because it is less messy, easier to clean up and less expensive.5 When examining a sample, it is best to scan slides under low power first to identify location and types of cells before choosing an area to examine under oil immersion. When an area is examined under low power it allows the practitioner to get a general idea of the amount and types of cells present. Often, the entire sample will be covered with keratin debris or corneocytes and the inflammatory cells will only be found in a few areas. Examining the area that contains inflammatory cells under higher magnification can aid in identifying more detailed cellular morphology and the presence of microbes. 117
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stained and viewed normally. In the authors’ experience, these slides are unnecessary and if not handled just right, will pick up environmental debris.
Syringes and Needles These are used for aspiration of samples from nodules, tumors, plaques, abscesses, or other lesions where a deep (nonsurface) sample is required. Ideally, a 20 or 22 gauge needle is attached to a 6 or 12 mL syringe. It is important that the needle diameter be large enough destruction of cells as they pass through the lumen of the needle and allow passage of material and; 6 to 12 mL syringes are necessary to create enough suction to collect an adequate sample. Figure 1 Staining station. The picture shows a typical staining station with Wright’s stain, wash tub and bottle of water for rinsing the slides. Matches for heat fixation and clothes pins to grasp the slides during the heating process. Also photographed is bibulous paper for drying the slides. (Color version of figure is available online.)
The presence of intracellular organisms indicates that the microbes are pathogens and associated with a true infection. Finding microbes without the presence of inflammation is more indicative of either nonpathogenic bacteria, bacterial colonization or failure to select the infected area for sampling. When examining oil immersion fields, it is important to constantly manipulate the fine focus. This is done so that organisms at different levels can be found and their refractory characteristics can be evaluated. This can help differentiate melanin granules and stain precipitates from bacteria and helps identify the clear capsules often found around fungal elements.1-4,6 Generally, it is important to keep the light condenser up when examining a cytology sample (Köhler illumination). This is in contrast to examining skin scrapes or unstained tape strip samples where the shadows and contrasts created by keeping the condenser down can aid in the identification of fungal spores, hyphae, parasites, and hair shaft abnormalities. If the microscope is used more than once or twice daily in practice, it is strongly recommended that the light source be dimmed, but kept switched on. Repeatedly turning the microscope on and off leads to the bulb burning out more rapidly than when the light source is kept on.
Microscope Slides and Cover Slips Slides with frosted ends are easier to label for identification. Most commercial laboratories will provide microscope slides at little or no cost as part of their services. Cover slips must be used with wet stains such as New Methylene Blue, as these cannot be examined under high power without a cover slip. Cover slips are also used for permanent mounting (see below). Unless the slides are going to be kept permanently, cover slips are usually not required with routine Wright’s stains. In addition to traditional glass microscope slides, there are “Sticky Slides” (Sticky Slides Nasco, Fort Atkinson, WI). To use these slides, the protective covering is removed and then the slide is applied directly to a lesion. The slide is then
Stains The easiest stain to use in-house is a commercial modified Wright’s stain like Diff Quik (Diff Quik Baxter Diagnostics, McGaw Park, IL). This is a Romanovsky-type stain that gives less nuclear detail than a vital stain such as New Methylene blue. It is important to keep the stain clean. Staining tubs should be changed weekly and separate tubs should be used for “dirty” samples such as anal gland cytology and fecal cytology, because the potential for bacterial contamination is high. Other stains used in cytology include New Methylene Blue (NMB), acid fast, Gamori methanine silver (GMS), Giemsa, and others. NMB is a vital stain and gives better nuclear detail than Wright’s stains. Gram Stains are rarely performed in practice because of the time commitments and minimal benefit of differentiating Gram-negative versus. Gram-positive organisms, as this is doesn’t change immediate clinical decisions. Other stains that are utilized in cytology require professional laboratory techniques. Ordering acid fast staining is the best diagnostic test of identifying Mycobacterium spp infections. Occasionally, other stains can be applied to cytology samples.1
Cotton-Tipped Applicators (CTA) These are used to obtain samples from exudative or purulent areas such as ears, skin folds and interdigital spaces that do not lend themselves to direct contact with a microscope slide.
Heat The importance of heat fixation on waxy, greasy, or exudative samples is open to debate, however, in the authors’ experience; heat fixation helps to fix samples to the glass slide and prevents sample loss during the staining process. Heat fixation of a slide can be done with a variety of methods. One method is to use a blow dryer directly on the slide from a distance of about 10 inches. A Bunsen burner, a cigarette lighter or even a match can also be used. Hold the slide over the flame for 5 seconds without burning the slide or damaging the sample by overheating. If the flame is not burning “clean,” (as with a Bunsen burner) soot will accumulate on the bottom of the slide. This is easily wiped off either before or after the slide is stained (Fig. 2).
Transparent Acetate Tape Transparent acetate tape can be used to pick up epidermal debris, obtain samples from dry lesions that will not stick to
Practical cytology for inflammatory skin diseases
119 check that the cotton tip is attached to the stick of the CTA well and will not become dislodged. If the patient allows, the ear flap should be lifted up at about a 30 degrees from dorsal, and the CTA is applied to the vertical ear canal as deep as possible, being careful not to damage the tympanic membrane (Fig. 3). The exudate is then rolled on a glass slide, and a notation is made on the slide to differentiate right from left ears. Using a CTA within exudative skin folds, such as brachycephalic facial folds and tail folds or vulvar folds is also an excellent way of collecting intertriginous samples.1
Moist or Exudative Skin Lesions
Figure 2 Heat fixation with a match. The slide is held with a clothes pin just above the flame of a match for 5 to 10 seconds. Care must be taken not to overheat and ruin the sample. Soot can be easily wiped away both before and after staining. (Color version of figure is available online.)
a glass slide easily or to obtain samples from areas that are difficult to get to with a glass slide. To examine a sample obtained with acetate tape, you can either stain the tape directly [skipping the fixation (clear) solution as this will prevent the tape from laying flat on the glass slide] or place a drop of the third solution (the purple stain) or NMB onto a glass slide before placing the unstained tape directly over it. These samples can be examined with all of the objectives, including oil.
Cytology of crusts, exudation or purulent debris, pustules or papules, or other lesions in which the surface of the skin is sampled, are usually best obtained by directly impressing or firmly rubbing the glass slide firmly across the surface of the lesion in a single motion. Rubbing back and forth should be avoided. When there are crusts present it is important to obtain the sample from the skin beneath the crust. If a pustule or papule is sampled it is recommended to tease the lesion open first with a 25 gauge needle (Fig. 4).
Dry or Waxy Skin Lesions The acetate tape technique is used on dry, scaly lesions, or lesions that are anatomically difficult to reach, such as interdigital spaces or tragal folds. To obtain an acetate tape sample, the sticky side of the tape is pressed firmly against the area several times, then stained and prepped as described above. Direct impression with a glass slide can also be used and requires firm impression techniques and then heat fixation is critical. In the authors’ experience direct impression is just as effective in obtaining informative samples as using the acetate tape technique.
Mounting Media: Permount Mounting Medium (optional) This is used if a slide is of particular interest or if the practitioner is creating a permanent library of cytology samples and slide is to be kept permanently. To “Permount” a slide, a drop of Permount solution (Permount ProSciTech, Kirwin, Australia) is applied to the top of a clean sample (any oil should be removed) and a cover slip is placed on top, avoiding air bubbles. Once the slide is dry, it should be stored away from light.
Sample Collection Depending on location or the type of lesion, there are several ways to obtain samples for evaluation.
Ears Ear cytology is the most common sample to collect and is often the easiest to interpret, because the material is being evaluated for infectious organisms and the interpretation of cell morphology or type is not as relevant to the diagnosis. The most straight forward way to obtain a sample from the ear is with a CTA. Before obtaining a sample it is important to
Figure 3 Obtaining an ear sample with a CTA. The pinna is held about 30 degrees from vertical to straighten the canal and the CTA is applied as deep as possible, being careful not to damage the tympanic membrane. (Color version of figure is available online.)
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Figure 4 Obtaining a direct impression sample from the periocular skin. (Color version of figure is available online.)
Figure 6 Yeast organisms (100⫻). The footprint-shaped organisms are found in abundance adherent to keratin and keratinocytes. (Color version of figure is available online.)
Claw Folds
Nodules/Plaques/Abscesses
Claw folds often contain infectious microorganisms that can contribute to pedal pruritus or discomfort. Occasionally, the infection within claw folds is different from what is found on the surface of the paws. Obtaining a sample from this area can be challenging. The most valuable technique is to break a wooden CTA so that a tapered firm edge is created. The tapered tip can then be inserted and rubbed gently within the claw folds and then rolled on a glass slide (Fig. 5).7
These lesions are best sampled by aspiration. For aspirate samples, ensure the needle is firmly attached to the syringe and insert it into to the center of the lesion. The needle should be held stable and the plunger pulled back fully several times. The plunger should be neutral with no negative pressure when the needle and syringe are withdrawn. Unless the sample is fluid, the needle will contain most of the desired sample. Remove the needle from the syringe, pull the plunger back and replace the needle. The sample can then be expelled onto the glass slide. The sample on the slide can be smeared evenly by using an additional slide before fixation and staining.
Sample Interpretation Infectious Organisms
Figure 5 Collecting sample from claw folds. Clean tapered edge from a broken CTA is gently inserted within the claw fold to collect sample. (Color version of figure is available online.)
The most common types of organisms encountered in cutaneous cytology are yeast and bacteria. The most common yeast organism found on samples from dogs and cats is Malassezia pachydermatis, although Candida spp can also be found, on the skin, in the ears and in the oral cavity. This organism usually has the classic “foot print” shape of a budding yeast organism. M. pachydermatis is usually 3 to 5 m in diameter (red blood cells are 6-8 m in diameter). Yeast organisms are usually found adherent to corneocytes (Fig. 6). In one study, average yeast counts greater than 5 organisms per 40⫻ high powered field (HPF) were considered significant.8 However, in the authors’ experience, average yeast counts as low as 0.5 to 1 organism per oil immersion field (OIF) are often significant when the sample is from an inflamed ear and even lower than that for samples obtained from the skin.1,3,8,9 The variety of bacteria that can be found on cutaneous cytology is vast. However, Staphylococcus intermedius infec-
Practical cytology for inflammatory skin diseases
Figure 7 Mixed infection of bacteria and yeast (100⫻). This sample contains rod-shaped Pseudomonas spp. (as verified based on a culture), coccoid Staphylococcus intermedius as well as Malassezia pachydermatis organisms. (Color version of figure is available online.)
tion is the most common bacteria isolated from infections of the ears and skin of dogs and cats. S. intermedius is a coccoid bacterium, 0.5 to 1.5 m in diameter and is often found in pairs. S. intermedius and other coccoid bacteria can be normal inhabitants of the ear and skin. Previous studies have determined that average otic bacterial counts greater than 25 organisms/HPF are significant.8 In another study cocci counts greater than 5/HPF or rod counts greater than 1/HPF should be considered pathogenic.1,8,10 Another commonly encountered type of bacteria, especially from ear samples, is Pseudomonas aeruginosa. These are rod-shaped organisms, approximately 0.3 to 0.8 m wide by 1.0 to 1.2 m long and are often found in short chains. Because Pseudomonas spp and other rod-shaped organisms often have high incidence of antibiotic resistance, seeing these bacteria on cytology indicates the need for a bacterial culture and sensitivity (Fig. 7).1,3 Mycobacteria can also be seen on cytology with the use of acid-fast stains. The authors prefer Nocardia acid fast stains for suspect mycobacterium infections, as it tends to yield better staining for the common species seen in dogs and cats. Aspiration is the best reported method for identification. Aspirate cytology should be submitted with biopsies for acid fast staining if these organisms are suspected.1 Another bacterium occasionally encountered is Simonsiella spp. These are harmless very large filamentous Gram-negative saprophytes that inhabit the oral cavity of a variety of warmblooded vertebrates. They are 6 to 8 m long and 2 to 3 m wide. There are usually found in small numbers and are not intracellular (although they can be adherent to keratinocytes). These organisms are not known to be pathogenic; however, when found on areas not associated with the oral cavity, such as the lip folds or nasal planum, their presence is likely associated with licking. If the practitioner is not famil-
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Figure 8 Simonsiella organisms (100⫻). They are much larger than the rod-shaped bacteria also seen in the picture. (Color version of figure is available online.)
iar with this organism it can create some concern because of its unique morphology (Fig. 8).1,11 Numerous dermatophyte and other fungal organisms can be demonstrated with either exfoliative or aspiration cytology. Dermatophyte spores and hyphae are visible, even with Wright’s stain. These organisms are usually more easily demonstrated in samples from cats than from dogs. Dermatophyte spores often appear as round spheres, usually about twice the size of coccoid bacteria. The spores are
Figure 9 Dermatophyte spores (100⫻). These are about twice the size of cocci with a clear “ghost” halo outlining them on the background of the keratinocyte. Courtesy Michael Canfield, DVM, Animal Dermatology Clinic, Tustin, CA.
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Figure 10 Dermatophyte hyphae (100⫻) seen in the midst of inflammatory cells. This sample was taken from a fungal furunculosis secondary to a Trichophyton spp. infection in a dog. (Color version of figure is available online.)
surrounded by a capsule that limits staining and gives the appearance of a clear halo (Fig. 9). Hyphae recognition is a bit more challenging as they can be easily confused for debris and hair keratin in the field. Dermatophyte hyphae are uniform 2 to 3 m wide filamentous structures often with poor staining characteristics and may appear as “ghost-like” shadows found free in the specimen (Fig. 10).1 Wet mount cytology is another technique used in the identification of dermatophytes. Here a solution is used to dissolve keratins and other debris and the sample is examined “wet” and without staining. This method has very low sensitivity and for that reason is not commonly employed by the authors. When clinical suspicion exists for a dermatophyte, negative findings here do not negate the necessity for further diagnostics. Also, if KOH tests are positive, the authors and others have found that the spores and hyphae can be also found on routine cytology. To utilize this method, KOH is the most commonly used solution; however the use of chlorphenolac has also been recommended. The solution is applied to a slide and hairs and keratin debris are added to it. With KOH, the specimen is left to dry for 30 minutes (or gently heated by leaving on the microscope stage with the light on) and examined. With chlorphenolac, the slide can be examined immediately. These solutions allow the identification of yeasts, conidia, hyphae, or pseudohyphae. For best results, fragmented or distorted hairs, usually near the hair bulbs, should be examined.1 Other subcutaneous (SQ) or deep fungal organisms can also be identified with exfoliative or aspiration cytology, including but not limited to; Cryptococcus spp. Coccidioides spp, organisms of phaeohyphomycosis, Pythiosis, organisms of zygomycosis, Sporotrichosis, Blastomycosis, and Histoplasmosis (Figs. 11-13).1 Ectoparasites are not commonly diagnosed with exfoliative
C. Mendelsohn, W. Rosenkrantz, and C.E. Griffin
Figure 11 Cryptococcus organisms (100⫻). Smear obtained from a cat with generalized cutaneous lesions. (Color version of figure is available online.)
cytology. However, in exudative processes with severe, deep secondary pyodermas, organisms such as Demodex spp. and Sarcoptes spp. can be identified when the slide is scanned under low power (Figs. 14 and 15).
Inflammatory Cells Identifying inflammatory cells on cytology gives the practitioner a clue as to the pathogenesis of the lesion. Presence of degenerate neutrophils, with (Fig. 16) intracellular bacteria, is most often indicative of a pyoderma. If well-preserved neutrophils are seen, then sterile inflammatory conditions such
Figure 12 Coccidiomycosis organism (100⫻) as seen from a cutaneous lesion on a cat. (Color version of figure is available online.)
Practical cytology for inflammatory skin diseases
Figure 13 Sporotrichosis fungal elements (100⫻). Classic cigar shaped yeast organisms with a clear halo around them.
as pemphigus foliaceus, primary allergic inflammation, irritant reaction, and others must be considered. At times, the force that is required to obtain an exfoliative sample ruptures any present inflammatory cells, especially neutrophils. When this occurs, the nuclear material from these cells often streaks the slide and can be identified on low power (Fig. 17). The presence of eosinophils indicates inflammation secondary to parasites, hypersensitivity reactions such as food allergies or atopy, pemphigus foliaceus, foreign body reaction (such as free keratin within the dermis), eosinophilic granuloma, mast cell tumors, or other sterile eosinophilic
Figure 14 Demodex canis (10⫻). These poorly staining organisms are seen in a background of inflammatory cells. (Color version of figure is available online.)
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Figure 15 Sarcoptes (10⫻) Poorly staining organism that could only be found if scanning on low magnification lens. It is better visualized by dropping the condenser light as seen here.
diseases (Fig. 18). Eosinophils can also be seen in ear cytology with topical reactions. Intracellular bacteria can also be seen within eosinophils.1 Other common inflammatory cells recognized are mononuclear macrophages and lymphocytes. These are usually seen from more chronic or deeper lesions. Macrophages are efficient phagocytic cells and often contain effete neutrophils, red blood cells, bacteria, fungal elements and even intracellular parasites such as Leishmania spp organisms and others.
Figure 16 Intracellular rods (100⫻) Phagocytosed rods within a neutrophil as seen from a Pseudomonas aeruginosa infection on the skin. Courtesy Michael Canfield, DVM, Animal Dermatology Clinic, Tustin, CA. (Color version of figure is available online.)
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Figure 17 Ruptured cells with streaks of nuclear material (100⫻). Numerous coccoid bacteria seen streaked within the nuclear material; indicating likely intracellular organisms. There is an intact degenerated eosinophil at the bottom left of the picture as well. (Color version of figure is available online.)
C. Mendelsohn, W. Rosenkrantz, and C.E. Griffin
Figure 19 Mycobacteria (100⫻) Acid fast staining reveals the rodshaped organisms within macrophages. Aspirate sample taken from atypical mycobacterial lesions on the pinnae of a dog.
Depending on the severity of the wound or lesion contamination, macrophages will often be markedly vacuolated and degenerate. Mycobacteria and fungal elements are often found within these vacuoles when stained with acid-fast or GMS stains, respectively (Fig. 19). When samples from deep infections are examined, the offending organism is often not readily identified; however, the presence of degenerated neutrophils combined with degenerated and vacuolated macrophages or lymphocytes indicates infection and the possible
need for a tissue culture. Xanthoma cells and multinucleated giant cells are unique presentations of macrophages. Multinucleate giant cells represent numerous macrophages that have fused together in an attempt to phagocytose large or toxic foreign material (Fig. 20). These are more often noted in histological lesions, but can be seen from deep pyoderma secondary to Demodicosis, ruptured hair follicles, foreign bodies and others. Xanthoma cells are macrophages from xanthoma lesions in cats that have phagocytosed lipid material and are often multinucleated giant cells that have markedly foamy and granular cytoplasm.1,3
Figure 18 Eosinophil (100⫻) from canine allergic patient with intracellular cocci.
Figure 20 Multinucleated giant cell (100⫻). Large cell with two obvious nuclei.
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Figure 21 Feline mast cell (100⫻) taken from area of a deep pyoderma. (Color version of figure is available online.)
Mast cells are often sparsely present in cytology from parasitic or allergic lesions (especially from cats) (Fig. 21). However, if full fields of mast cells are present that is usually indicative of a mast cell tumor. Plasma cells are often seen from deep chronic lesions, but when full fields are noted, plasmacytomas or plasma cell pododermatitis become the primary differentials. Chronic healing wounds, such as acral lick lesions often have spindle-shaped fibroblasts that are not necessarily neoplastic (Fig. 22). However, like with the presence of full fields of mast cells or plasma cells, the presence of numerous spindle cells indicates the need for a biopsy to rule out neoplasia.1-3
Figure 23 Keratinocytes containing multiple keratohyaline granules from a cat (A) and a dog (B). (Color version of figure is available online.)
Epithelial Cells
Figure 22 Fibroblast (100⫻).
Epithelial cells encountered normally on exfoliative cytology are corneocytes (nonnucleated keratinocytes). Keratin and keratinocytes are a normal finding; however, in diseased skin, keratinocyte morphology often has diagnostic value. Nucleated keratinocytes are normally found in samples from mucous membranes, when the surface of the skin was scraped before cytology or occasionally from ear samples. When found from surface skin samples it is associated with parakeratosis and suggestive of abnormal turnover of the epithelial layers or erosion of the cornified layers. These keratinocytes often contain keratohyaline granules and should not be confused with bacteria (Fig. 23). Instead of appearing as flat sharply angulated large cells, some keratinocytes or
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Figure 24 Raft of acantholytic cells (40⫻). These large round cells have a dark-staining periphery. They are found among numerous well preserved neutrophils and eosinophils.
corneocytes can appear rolled or more spindle shaped on routine cytology because of folding and rolling during the sampling and fixation process. Acantholytic cells are lower layer keratinocytes that have lost their adhesion to neighboring keratinocytes. They are most commonly found in pemphigus foliaceus lesions, but can also be seen with deep furunculosis secondary to conditions such as Demodicosis and dermatophytosis. Acantholytic cells are large, round cells with a central nucleus. They often stain with a dark blue periphery that may indicate antibody coating. In cases of pemphigus, they are often found in rafts and among well preserved neutrophils and/or eosinophils. In cases of Demodicosis or dermatophytosis, acantholytic cells are usually sparse and found among degenerate inflammatory cells (Fig. 24).1-3
C. Mendelsohn, W. Rosenkrantz, and C.E. Griffin
Figure 25 Melanin granules (100⫻). These rod-shaped brown-black organelles are the same size and shape as most rod-shaped bacteria. Their color, distribution and refractory nature differentiates them from pathogenic organisms.
tate is very dark purple staining and has an irregular appearance and distribution.1-3 Pollen can also be confusing. It comes in multiple shapes and sizes and is often confused for fungal elements. Pollen tends to be dramatically geometric and is usually not present in large numbers. The presence of pollen is not pathogenic, however, when seen on a sample from a dog with pododermatitis and clinical symptoms consistent with atopic derma-
Artifacts and Other Findings Artifacts are often encountered on cytology slides. Practitioners need to be familiar with these, as they can often be confused for significant findings. Many exudative samples have a markedly proteinaceous background, which often looks light purple or pink with fine darker staining granules that can be mistaken for small bacteria. However, they are generally smaller, more evenly sized and uniform in appearance than bacteria. Melanin granules can also be mistaken for rod-shaped bacteria such as Pseudomonas spp. However, melanin granules are most often found in and around keratinocytes, stain a refractory brown to black color and are very even in size. Their refractory nature is more easily appreciated when the fine focus is manipulated (Fig. 25). Medication debris often appears as crystalloid poorly staining debris in a variety of sizes. It is most often seen in samples taken from the ear where large amount of medication has accumulated within the debris. Stain precipitate can also be mistaken for bacteria. It is a problem mostly with the NMB stain. Precipi-
Figure 26 Pollen grain (100⫻) found on sample from ventral interdigital webbing on a dog with pododermatitis secondary to atopy. (Color version of figure is available online.)
Practical cytology for inflammatory skin diseases titis, it suggests possible exposure to environmental pollens and may play a role in the patient’s clinical disease (Fig. 26).1
Summary Overall, when evaluating inflammatory skin conditions, cytology is the single most commonly used diagnostic test performed in the practice of dermatology. In a given month at a dermatology referral practice, cytological samples were taken from approximately 60% of patients presented, comprising about 8 to 9% of the clinic’s total income! It is not only used when cases initially present, but used to monitor therapy and indicate when an infectious or inflammatory condition may have changed. For example, if a dog with pemphigus foliaceus that was previously controlled seems to flare, cytology should be one of the first tests performed to evaluate whether the flare is because of the primary disease, or a pyoderma secondary to immune suppression. The finding of numerous intracellular cocci and no acantholytic cells would support a pyoderma that maybe secondary to therapy and further immune suppression would be contraindicated. Cutaneous cytology is inexpensive, highly informative and easily self-taught. By making cytology part of everyday practice, diagnostics and treatments become more proficient and case management is immediately more rewarding. The ultimate result is significantly happier patients and clients.
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References 1. Scott DW, Miller WHJ, Griffin CE: Muller & Kirk’s small animal dermatology (ed 6). Philadelphia, W.B. Saunders Company, 2001 2. Baker R, Lumsden JH: Color atlas of cytology of the dog and cat. St. Louis, MO, Mosby, 2000 3. Medleau L, Hnilica KA: Small animal dermatology: A color atlas and therapeutic guide (ed 2). St. Louis, MO, Saunders, 2006 4. Nesbitt GH, Ackerman LJ: Canine and feline dermatolog. (ed 2). Trenton, NJ, Veterinary Learning Systems, 1998 5. Cargille JJ: Immersion oil and the microscope (ed 2). New York Microscopial Society Yearbook, Cargille-Sacher Laboratories, Inc., 1985 6. Pappalardo E, Martino PA, Noli C: Macroscopic, cytological and bacteriological evaluation of anal sac content in normal dogs and in dogs with selected dermatological diseases. Vet Dermatol 13:315322, 2002 7. Mueller RS: Diagnosis and management of canine claw diseases. Vet Clin North Am Sm Anim Practice 29:1257-1371, 1999 8. Ginel PJ, Lucena R, Rodriguez JC, et al: A semiquantitative cytological evaluation of normal and pathological samples from the external ear canal of dogs and cats. Vet Dermatol 13:151-156, 2002 9. Bensignor E, Jankowski F, Seewald W, et al: Comparison of two sampling techniques to assess quantity and distribution of Malassezia yeasts on the skin of Basset Hounds. Vet Dermatol 13:237-241, 2002 10. Columbo S: Quantitative evaluation of cutaneous bacteria in normal dogs and dogs with pyoderma by cytological evaluation. Faculty of Veterinary Medicine. Doctoral Thesis Milan: University of Milan, 1997 11. Carandina G, Bacchelli M, Virgili A, et al: Simonsiella filaments isolated from erosive lesions of the human oral cavity. J Clin Microbiol 19:931933, 1984