Diagnostic Testing

The dermatologic diagnostic minimum database includes skin scrapes, otic swabs, and cutaneous cytology. The goal should be to identify all secondary infections (e.g., pyoderma, demodicosis, dermatophytosis, otitis, Malassezia dermatitis, infectious pododermatitis), then formulate a diagnostic plan for identifying and controlling the underlying/primary disease (i.e., allergies, endocrinopathies, keratinization defects, and autoimmune skin diseases) (Box 2-1).

BOX 2-1

What Are the Infections?

For every dermatitis case, every time you evaluate the patient, ask yourself, “What are the infections?”

Using diarrhea and the microscopic fecal exam as a comparison works well because both skin cytology and fecal exams involve the use of a microscope, can easily identify the type of infection, and can be performed by trained technical staff. So why does your clinic perform fecal exams? When is a fecal exam performed (before the doctor’s examination)? Who performed the fecal? Does the clinic charge for the fecal exam? The answers to these questions should be the same for skin cytology (skin scrapings, impression smears, tape preps, and otic swabs).

FIGURE 2-1 The 3-Slide Technique.

Skin scrapes, cutaneous cytology, and otic swabs.

The practical solution for determining the best method by which to answer the question, “What are the infections?” is to implement a minimum database “infection screening” procedure to be performed by the technician before the veterinarian examines the patient. Every dermatology patient should undergo otic cytology, skin cytology (an impression smear or a tape prep), and a skin scrape at every examination (initially and at every recheck visit). This 3-Slide Technique can be performed easily and interpreted by a technician before the doctor completes an evaluation—which is exactly how diarrhea cases and fecal exams are handled in most clinics.

FIGURE 2-2 Diagnostic Techniques.

A, This microscopic image (10× objective) demonstrates the typical angular shape of a normal, mature keratinocyte. Note the absence of a nucleus and and the small melanin granules (often mistaken for bacteria but pigmented brown). B, This microscopic image (10× objective) reveals the brown pigmented melanin granules within the anuclear keratinocyte with typical angular cell walls. Note the dark blue structure, which is likely a rolled-up keratinocyte. C, This microscopic image demonstrates the typical appearance of a neutrophil at 10× magnification. D, To collect a sample ideal for cytologic evaluation, the superficial crust should be gently scraped away and the glass slide applied firmly to the skin. E, To collect a sample ideal for cytologic evaluation, the superficial crust should be gently scraped away and the glass slide applied firmly to the skin. Note: It may be easier to collect the sample if the skin is slightly elevated by pinching or rolling the skin into a mound.

Skin Scrapes

Skin scrapes are the most common dermatologic diagnostic tests (slide #1 in the 3-slide technique). These relatively simple and quick tests can be used to identify many types of parasitic infections (Table 2-1). Although they are not always diagnostic, their relative ease and low cost make them essential tests in a dermatologic diagnostic minimum database.

TABLE 2-1 Diagnosing Common Cutaneous Parasites

Many practitioners reuse scalpel blades when performing skin scrapes; however, this practice should be stopped because of increased awareness of transmittable diseases (e.g., Bartonella, Rickettsia, feline leukemia virus [FeLV], feline immunodeficiency virus [FIV], herpes, papillomavirus).

Procedure

Superficial Skin Scrapes (for Sarcoptes, Notoedres, Demodex gatoi, Cheyletiella, Otodectes, chiggers)

A dulled scalpel blade is held perpendicular to the skin and is used with moderate pressure to scrape in the direction of hair growth. If the area is covered with hair, it may be necessary to clip a small window to access the skin. In an attempt to find the relatively few sarcoptic mites that may be present on a dog, large areas (1–2 inches) are scraped. Applying mineral oil directly to the skin to be scraped helps dislodge debris and makes it easier to collect the scraped material. Because these mites do not live deep within the skin, it is not necessary to visualize capillary oozing or blood. The most productive sites for sarcoptic mites include the ear margin and the lateral elbows. Anecdotal reports suggest that Demodex gatoi in cats may be found more easily on the lateral shoulder. Usually, several slides are needed to spread the collected material thinly enough for microscopic examination.

Deep Skin Scrapes (for Demodex spp. except D. gatoi)

A dulled scalpel blade is held perpendicular to the skin and is used with moderate pressure to scrape in the direction of hair growth. If the area is covered with hair (usually, alopecic areas caused by folliculitis are selected), it may be necessary to clip a small window to access the skin. After several scrapes, the skin should appear pink, with the capillaries becoming visible and oozing blood. This ensures that the material collected comes from deep enough within the skin to allow the collection of follicular Demodex mites. Most people also squeeze (pinch) the skin to express mites from deep within the follicles into a more superficial area, so that they may be collected more easily. If scraping fails to provide a small amount of blood, then the mites may have been left in the follicle, resulting in a false-negative finding. In some situations (with Shar peis or deep inflammation with scarring), it may be impossible to scrape deeply enough to harvest Demodex mites. These cases are few in number but require biopsy for identification of mites within the hair follicles. Hair-plucks from an area of lesional skin may be used to help find mites, but the accuracy of this technique compared with skin scrapes is unknown.

Regardless of the collection technique used, the entire slide should be searched for mites with the use of low power (usually a 10× objective). A search of the entire slide ensures that if only one or two mites are present (as is typical of scabies infection), the user will likely find them. It may be helpful to lower the microscope condenser; this provides greater contrast to the mites, thereby enhancing their visibility. (One must be sure to raise the condenser before looking for cells or bacteria on stained slides.)

Author’s Note

There is no excuse for mistreating a patient who has demodicosis. Lesions caused by demodicosis can look identical to folliculitis lesions caused by bacterial pyoderma and dermatophytosis. Clinical appearance is not an acceptable criterion for ruling in or ruling out demodicosis. When the technician performs a skin scrape as part of the infection screen called the 3-Slide Technique, demodicosis can be identified and treated easily and accurately.

FIGURE 2-3 Skin Scrape.

A, A new, dulled scalpel blade is used to scrape in the direction of hair growth. B, For deep skin scrapes, once capillary oozing is initiated, the skin is usually squeezed before a final scrape is performed to collect the material. C, Capillary oozing is apparent as the sample material is collected. D, The collected sample is evenly distributed in mineral oil on a glass slide. E, Microscopic image of the Demodex mite as seen with a 10× objective.

Cutaneous Cytology

Cutaneous cytology is the second most frequently employed dermatologic diagnostic technique (slide #2 in the 3-slide technique). Its purpose is to help the practitioner to identify bacterial or fungal organisms (yeast) and assess the infiltrating cell types, neoplastic cells, or acantholytic cells (typical of pemphigus complex).

Procedure

Direct Impression Smear

Moist exudate is collected from pustules, erosions, ulcers, or draining lesions. Alternatively, crusts can be lifted, revealing a moist undersurface. Papular lesions may be traumatized by the corner of a glass slide or a needle, then squeezed to express fluid. Yeast dermatitis can be sampled by repeated sticking of the slide onto lichenified lesions, or through the use of a dry scalpel blade to collect material that is then smeared onto a dry slide. Regardless of which technique is used, the moist exudate collected on the slide is allowed to dry. The slide is then stained with a commercially available cytology stain (e.g., modified Wright’s stain [Diff-Quik is the most common]), and it is gently rinsed. A low-power objective is used to scan the slide to allow selection of ideal areas for closer examination. A high-power (40× or, preferably, 100× oil) objective is used to identify individual cell types, as well as bacterial or fungal organisms.

Fine Needle Aspirate Method

A needle (22–25 gauge) and a 6-mL syringe should be used to aspirate the mass. The area should be cleaned if necessary with alcohol or chlorhexidine. The lesion is then immobilized. The practitioner should insert the needle into the nodule while aiming for the center of the lesion, pull back on the plunger to apply suction, release and redirect, pull back on the plunger again, and stop if any blood is visible in the hub of the needle, as this will dilute the cellular sample. Negative pressure should be released before the needle is removed from the lesion. An alternative technique involves repeated insertion of the needle without the syringe into the lesion, while redirecting several times. This latter technique (without negative pressure), which decreases the frequency of inadvertent dilution of the sample with blood, works best for soft masses. Once the sample has been collected, the material is expressed onto a microscope slide by blowing a syringe-full of air through the needle to spray the cells onto the slide. The material is smeared gently to thin the clumps of cells stained with cytology stain. The slide should be scanned with low power (4×–10×) to reveal a suitable area for closer examination. A high-power (40×) objective may be used to reveal the infiltrating cell type and the cellular atypia.

Author’s Note

The infections are always secondary to a primary disease; however, all too often, the patient is not evaluated or treated for the primary disease for three major reasons:

1 Only the secondary infections are treated over and over again.

2 The nature of the allergy is confusing.

3 Cheap steroids that have delayed repercussions are available.

FIGURE 2-4 Cytology.

A, A glass slide is pressed onto a cutaneous lesion to collect the moist exudates for cytologic evaluation (impression smear). B, A needle is inserted into a nodular lesion to collect cells for cytologic evaluation (fine needle aspirate). C, Once dry, the cytology slide is processed with the use of a modified Wright’s stain (Diff-Quik). D, Microscopic image of neutrophils and Staphylococcus organisms, as viewed with a 100× (oil) objective. E, Microscopic image of Malassezia yeast, as viewed with a 100× (oil) objective. F, Microscopic image of a keratinocyte, melanin granules, and Simonsiella organisms, as viewed with a 100× (oil) objective. Simonsiella is a common oral bacterium; its presence suggests that the patient has been licking (pruritus). G, Microscopic image of neutrophils and acantholytic cells, as viewed with a 100× (oil) objective. Acantholytic cells are suggestive of pemphigus.

Acetate Tape Preparations

Tape preps are used to evaluate a variety of different conditions. The basic technique involves the use of crystal clear tape (single- or double-sided) to collect a sample of hair or superficial skin debris.

Tape Preps for Yeast

Tape preps for yeast dermatitis are some of the most efficient and effective methods of identifying Malassezia skin infections. Although they are not as reliable and quantitative as impression cultures that use Sabouraud’s media, the speed and ease of tape preps for yeast make them the techniques most commonly employed for identifying Malassezia. The lichenified lesion (elephant skin on the ventral neck or ventrum) is sampled by repeated application of the sticky side of the tape onto the lesion. The tape is then adhered to a glass slide and is stained with a cytology stain (while the first alcohol stain solution is admitted). The tape serves as a coverslip and can be examined under high power (100× oil immersion) for visualization of Malassezia organisms. This technique is useful, but false-negative results are common with all yeast collection techniques.

FIGURE 2-5 Tape Preps.

A, Clear acetate tape is pressured repeatedly into the interdigital space for collection of a superficial sample. B, The tape is processed with a modified Wright’s stain (Diff-Quik) with omission of the first light blue alcohol solution, which dissolves the tape adhesive. C, After processing has been completed, the sample material is easily visible under the tape. D, Microscopic image of Malassezia organisms and keratinocytes, as viewed with a 100× (oil) objective.

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