Dermatology
BACK TO THE BASICS: INTRODUCTION TO DERMATOLOGY SAMPLING AND TECHNIQUES
Kinga Gortel DVM, MS, Diplomate ACVD and Candace Dietzen RAHT
The diagnostic tests used most often in veterinary dermatology are skin cytology and skin scrapings. Becoming pro-ficient in these two techniques, and more importantly, taking the time to do them, will take you a long way in diag-nosing patients with skin disease. Other tests may be used less frequently, but are still very useful in many situations.
Skin Surface Cytology
Why do skin surface cytology?
Skin cytology is a very high-yield, inexpensive procedure for patients with skin disease. It tells us more about what is going on with the skin, more quickly, than any other diagnostic test. Nearly all patients with skin disease, and par-ticularly those with skin lesions, should have this test performed. It is also very useful for monitoring therapy. The primary purpose of skin cytology is to assess the presence of bacterial or yeast overgrowth or infection. Another important purpose is to assess what the skin is “doing” – particularly to detect and characterize the degree and type of inflammation.
How is cytology collected?
Cytology can be collected by direct or tape techniques. Within these two techniques, there are many variations in methodology.
For the direct techniques, material can be collected from the surface of the skin as follows:
- impressions: directly pressing the slide on the skin several times. Although a regular glass slide suffices, adhesive-coated slides (Duro-Tak™) are preferred by some.
- swabbing the affected area vigorously, then rolling swab onto a slide (like making an ear cytology preparation)
- scraping the surface debris from the skin using a scalpel blade (or a broken cotton-tipped applicator, e.g. in the nail fold) and smearing the material onto a slide like buttering bread – these scrapings are superficial (do not draw blood) and no oil is used in their collection
Heat-fixing is generally not necessary, but may help with preparations that are dry or contain little material. All di-rect techniques are stained with Diff Quik (fixative + 2 stains) and examined microscopically. Although the slide can be stained at lower magnification, high power (1000x) magnification and oil immersion should be used as the final step.
For the tape wet mount technique, samples are collected as follows:
- A 3-4 cm piece of tape is pressed onto the skin several (3-4) times, sticky side down. Clear tape or Scotch® tape can be used.
- The piece of tape is placed sticky side down on the microscope slide. It is only attached to the slide by one end in order to hold it in place. Most of the tape is resting on, but not adhering to, the glass slide.
- A drop of blue (last) stain of Diff Quik or new methylene blue is placed under the loose part of the tape to make a wet mount. Alternatively, the slide is immersed in the purple (last) stain of Diff Quik for a few seconds to allow the stain to enter the space between the tape and the slide. The latter is simpler but contaminate the stain.
- The back of the slide is rinsed gently.
- The slide is blotted in a paper towel or bibulous paper.
- The tape is examined under high power (1000x) oil immersion. Note that if Scotch® tape is used, the image will be cloudy on lower powers due to its opacity - only under oil immersion does the tape become clear.
* In alternative tape method - a dry preparation - the tape is stained with the blue Diff Quik stain, rinsed, dried with a blow dryer, placed sticky-side down on the glass slide and examined as above.
Why the different direct techniques?
Some of it depends on personal preference - many practitioners always prefer to use one technique. However, each one is best for certain situations and has advantages over the other techniques:
Direct techniques:
Impression
Works well for: moist, exudative lesions, pustules (open with needle), crusts (peel crust, touch skin), very greasy skin, draining lesions
Not as well for: dry or minimally exudative lesions, small areas (e.g.. nail folds), awkward areas (e.g.. interdigital)
Swab
Works well for: moist, exudative lesions, small areas (e.g.. face & lip folds, nail beds), ears
Not as well for: dry or minimally exudative lesions
Scraping
Works well for: large greasy lesions
Not as well for: sensitive areas, rambunctious patients, near eyes
Tape wet mount technique:
Works well for: greasy or dry skin, minimally abnormal skin, awkward areas, small areas, sensitive areas
Not as well for: purulent lesions, pustules, wet skin
Direct technique or tape technique?
Each technique provides certain advantages and none are perfect. In fact, they are complementary and it is ideal to try both the tape technique and one of the direct techniques on each patient. Here are some tips about their relative merits:
Direct techniques
Good:
1) The direct techniques make a smear that is somewhat easier to read.
2) It is a thinner preparation so you don’t need to focus up and down as much. Everything adhering to the slide, there is no movement as can be seen in a wet mount.
3) Most organisms stain deeply.
4) It is easier to identify and quantitate bacteria on direct techniques - rods vs. cocci can be differentiated more easily than on a wet mount.
5) Inflammatory cells and other cells such as acantholytic cells can be identified more easily, particularly on the direct impression smears, in which they are not usually damaged.
6) These techniques make nice preparations with moist, exudative lesions.
Bad:
1) Picks up less material than a tape smear from many skin lesions.
2) May not obtain adequate samples from dry or minimally greasy skin.
3) Staining takes longer than with the tape preparations, as all 3 Diff Quik steps are used, and the slide must dry.
Tape wet mount technique
Good:
1) Better at picking up material from minimally exudative or dry skin.
2) Faster staining and no air-drying.
3) Well tolerated by pets in areas such as interdigital spaces.
4) An easier way to find Malassezia when it is present in low numbers - this is the major advantage.
Bad:
1) The tape technique makes a very “busy” slide with lots of material. It can be a bit overwhelming when you first look at these types of preparations.
2) Bacteria may not be as easy to see and quantitate with wet-mount preparations.
3) Not as good for identifying cellular inflammation, acantholytic cells, etc.
Interpretation
First, assess the presence and numbers of microorganisms. Remember that recent bathing interferes significantly with cytologic assessment. Examine approximately 10 representative high power oil immersion fields (hpOIF). Record the approximate numbers of yeast per field. For bacteria, it may be easier to score them as 0 to 4+ rather than counting. Record whether they consist of rods, cocci, or a mixed population.
Yeast organisms are most often Malassezia pachydermatis. Cocci are most often Staphylococcus pseudintermedius. Rod-shaped organisms are less commonly found on skin and ear preparations and should be considered significant pathogens in most sites.
What numbers are significant? It’s difficult to establish a “cut-off”. Although Malassezia and cocci inhabit some areas of normal skin in low numbers, we don’t find many using cytologic techniques. On the skin, I consider one or more Malassezia per hpOIF significant (3 or more in the canine ear canal). However, since Malassezia hypersensi-tivity can be present with even lower numbers, finding even one yeast per 5 hpOIF from inflamed skin (or 1 per hpOIF in an inflamed ear canal) may warrant antifungal therapy (see Morris DO, 2008). Bacterial numbers are harder to quantitate. Finding them within inflammatory cells is significant in most cases.
Cytology is very useful for monitoring response to therapy for infections in the skin or ears. In some pets, recheck-ing cytology at every is warranted because the nature of infection can be dynamic and changes in therapy can be made quickly.
The next step after assessing microorganisms is to assess the cellular response of the skin. This may include the presence of neutrophils, eosinophils, macrophages, acantholytic cells, or neoplastic cells.
The key is to get familiar with cytologic techniques and to use them often; you will become more familiar with col-lection and interpretation very quickly.
One last note: Learn the difference between bacteria (which stain blue/purple) and melanin (similar in size, oval, but always brown/black and NOT blue/purple).
Skin scraping
Why do skin scrapings?
Skin scrapings are primarily indicated for the diagnosis of mite and louse infestations. Before performing skin scrap-ings, consider which parasite you are looking for, as the technique will vary.
Technique
Skin scrapings are readily performed in most sites; however, skin scrapings of sensitive skin around the face and feet may require sedation.
Deep skin scrapings are necessary to find the deep follicular dwellers Demodex canis and D. cati mites. The skin should be squeezed before and/or during the scraping. A new (or clean dulled) #10 scalpel blade dipped in mineral oil is scraped in the direction of hair growth until slight dermal bleeding is observed. At least 3 sites should be sam-pled if multiple areas are affected, and each one placed on a separate site on the slide (e.g. 2 scrapings per slide) so the site of origin can be recorded. Droplets of blood should be seen microscopically if the scrapings are of adequate depth. An additional way to look for Demodex is with a trichogram (see below).
More superficial skin scrapings are useful for Sarcoptes, Notoedres, surface-dwelling Demodex gatoi, and Cheyle-tiella. In superficial scrapings, squeezing the skin and drawing blood is not necessary. The aim is to collect surface scale, crust, and epidermis. Since finding these mites is always significant, the site of origin is not important and you can pool the material from multiple scrapings on 1 or 2 slides. For Sarcoptes, scrape multiple (5-10) areas, concen-trating on ear margins, elbows, hocks, and any areas with excessive crust and scale. For Cheyletiella and Demodex gatoi, scrapings should be broad and superficial, and you may first apply mineral oil to the skin to aid in retrieval. If in doubt, perform both deep and superficial scrapings.
For any of these scrapings, the material is collected on a slide and covered with a coverslip. Examine the material under 40x (low power) and then 100x (medium power). Lowering the condenser increases contrast and makes scarce parasites easier to find. Scan the slide completely.
An additional test similar to skin scrapings involves looking for Otodectes mites in ear canals. Exudate is collected with a cotton swab and applied to a glass slide with mineral oil.
Interpretation
Although Demodex canis mites are normal inhabitants of the skin, in reality they are rarely found on skin scrapings from normal skin. The finding of any Demodex canis mites should be considered “suspicious”, but a diagnosis of demodicosis is made by finding multiple mites. Note the relative proportion of adult vs. immature mites and eggs. Record the approximate proportion of live mites and track these parameters at recheck examinations. The sites of the scrapings should be recorded so on subsequent visits, the same sites can be scraped.
Finding of one mite or egg of Sarcoptes, Notoedres, Cheyletiella, Demodex gatoi, or Otodectes suffices for the diag-nosis. The same is true for lice. Fecal pellets, which are easily mistaken for skin debris, should raise suspicion for skin mites. It is very important to realize that negative scrapings do not rule out these parasites.
Pitfalls
Demodex canis should be easily retrieved on skin scrapings. However, it may be harder to find in scarred skin, and from sites such as the feet. It may be more difficult to find mites on certain breeds as well; the Shar pei is commonly mentioned. It is often useful to supplement scrapings with hair plucks, which can retrieve Demodex in areas that are difficult to scrape.
Sarcoptes is notoriously elusive and skin scrapings only find it in about 20-30% of cases. A treatment trial is rec-ommended if these mites are suspected.
Cats often remove surface dwelling parasites by fastidious grooming. Fecal floatations are sometimes useful for finding Cheyletiella and other parasites in this species.
Clear tape preparation
Clear tape preparations are used to find some of the same surface dwellers that superficial skin scrapings target, but can sample larger areas. They are useful for Cheyletiella, lice, poultry mites (Dermanyssus), and fur mites (Lynxaca-rus). The tape is pressed on the affected skin and fur multiple times, then placed on a slide sticky-side down with a drop of mineral oil underneath it for easier examination. It is examined like a skin scraping, with the tape acting as a coverslip.
Flea examination
Fleas are visible to the naked eye, but are quick and elusive. Parting of the hair on the caudal dorsum and turning the pet over can help reveal them. Flea combs can also help. Often, only flea feces or “dirt” is seen. To differentiate flea feces from soil or other debris, rub the material on a moistened white tissue; flea dirt will dissolve into a brownish red smear. Note that material collected by flea combings can also be collected and placed in fecal floatation solution to find some of the more elusive parasites such as Cheyletiella.
Flea dirt can harbor Bartonella henselae, a causative agent of cat scratch fever - avoid contact with broken skin.
Trichogram (hair pluck)
A forceps is used to forcefully pluck hairs from affected skin. The hairs are placed onto a drop of mineral oil under a coverslip and examined under a low power objective. Although there are many reasons to collect a trichogram, my (biased) order of its usefulness is:
To find Demodex mites in areas that are difficult to scrape.
To find louse ova (nits) or occasionally Cheyletiella ova.
To diagnose color dilution alopecia or other structural abnormalities of hair.
To look for evidence of self-inflicted alopecia, in which the ends of the hairs appear broken. This is particularly use-ful in alopecic cats, which can be “closet” over-groomers.
To look for dermatophyte-infected hairs, which can look fuzzy, swollen, and distorted. Note that this is neither a sensitive nor specific way to diagnose a dermatophyte infection, but can provide support for your tentative diagnosis while you await a fungal culture.
To determine the approximate ratio of hairs in telogen (resting) vs. anagen (growth) phase. Telogen hair bulbs are spear-shaped while anagen bulbs are rounded, soft, and may be bent. This takes practice and a truly forceful pluck, and “normal” ratios have not been established for pets. There is great variation between breeds of dogs, so the re-sults are difficult to interpret.
Wood’s Lamp examination
In approximately half of Microsporum canis dermatophyte infections, apple-green fluorescence of tryptophan metabolites is seen under ultraviolet light (Wood’s lamp). No other dermatophytes of veterinary importance fluoresce. The fluorescence must be seen along the hairs, rather than on the skin. Some drugs, soaps, and bacteria may also show fluorescence but are not associated with hair shafts; they are nonetheless the source of many false positives. The lamp should be warmed up for 5 minutes before use.
Dermatophyte fungal culture
A fungal culture is indicated in any pet with possible dermatophytosis as it confirms the diagnosis and allows identi-fication of the causative fungus. Hairs and scale from the edge of a lesion (or those fluorescing under a Wood’s lamp) are collected using forceps. Broken hairs or those which epilate easily are best. Swabbing the lesion with al-cohol has been recommended to reduce contaminant molds, but is not necessary. If lesions are generalized or if asymptomatic carriers are suspected, the Mackenzie toothbrush technique is best. The hair is brushed with a new toothbrush for 1 minute, and the loose hair and scale are gently pressed onto the dermatophyte culture medium.
Fungal cultures can be submitted to a laboratory or kept in-house for at least 3 weeks. If they are kept in house, it is imperative to check them and record findings daily. DTM (dermatophyte test medium) contains a color indicator that turns from yellow to red when proteins in the medium are used. Since dermatophytes preferentially use proteins over carbohydrates, colony growth is preceded by focal color change of the medium. Contaminant molds may also eventually use proteins after their carbohydrate sources run out, so they may turn the medium red once they are well established. Thus, it is the timing of the red color change that is most useful and recording any color change daily is very important. Dermatophytes are typically incubated in a dark area at room temperature, though 25 to 30° C results in faster growth. There are various dermatophyte media available, e.g. Derma-SAB™ plates (Impact Diagnostic International, www.impactdiagnostic.com) as they are easy to inoculate and follow, and contain both DTM medium and Sabourad’s medium. In a dry climate they need protection from desiccation.
All positive cultures should be examined for colony characteristics and microscopic appearance. Dermatophyte colonies are always white, cream, or tan while gray or green colonies are contaminants. Microscopic examination should yield the typical macroconidia (Microsporum species) or microconidia (Trichophyton species); if these are not found, incubate the culture for a few more days. Microscopic examination consists of a wet mount: material is picked up from the surface of the colonies using clear tape, and placed on a drop of lactophenol cotton blue stain. Overall, fungal cultures can be difficult to carefully monitor and interpret in-house, and submission to a reference laboratory is recommended unless the above conditions can be met.
Skin biopsy
Why do skin biopsies?
Skin biopsies are very useful tests in veterinary dermatology. They are indicated when dealing with a serious, un-usual, or poorly responsive skin disease. They are recommended when neoplasia, immune mediated disease, or con-ditions only found by histopathology are suspected. Biopsies cannot replace “derm due diligence”, most importantly skin scrapings and cytology, but they can often narrow down the list of differential diagnoses.
Techniques
If the lesions are secondarily infected, treatment with antibiotics for 1-2 weeks before biopsy is ideal. Select a varie-ty of the most representative samples, including both primary and secondary lesions. Multiple samples (3 or more) should be submitted, as well as a normal sample of skin in most cases. In general, ulcers and erosions should be avoided; the affected but still intact periphery of these lesions is more useful. Alopecic skin should be biopsied in the center of the most hairless areas as well as in the junctional and normal areas. Depigmenting lesions should be biopsied in an area of active depigmentation (gray color) rather than the final stage (white/pink). Pustules, papules, and vesicles are all very useful primary lesions. If taking biopsies of crusted areas, make sure to include the entire crust (it can sometimes fall off during collection).
Local anesthesia often suffices for biopsy collection. If biopsies involve the face, feet, ears, or other sensitive areas, if the patient is intractable very small, or if a larger wedge biopsy is needed, sedation (in conjunction with local anesthesia) or general anesthesia may be used.
The sites to be biopsied are clipped very gently using scissors or a clipper blade held several millimeters from the skin. The lesions are marked. There should be no surgical preparation of the skin, with the exception of biopsies collected solely for cultures. This is not a sterile procedure and infection of biopsy sites is very uncommon. Approx-imately 0.5 to 1.0 ml of local anesthetic (usually 2% lidocaine, without epinephrine) is injected subcutaneously be-neath the lesion in a fanning motion using a single needle stick. Lidocaine stings. Lidocaine injections can be ren-dered less painful (and perhaps more effective) by mixing a small amount of sodium bicarbonate (approximately 1 part to 10 parts lidocaine) into the syringe prior to injection. This combination is not stable. Injecting lidocaine slow-ly seems to be better tolerated. Allow 3-5 minutes for the skin to become numb. Keep in mind that for small pets, multiple lidocaine injections can exceed the toxic dose. Avoid exceeding 3 mg/kg in cats and 6 mg/kg in dogs. For example, a cat weighing 4 kg should get no more than 12 mg or 0.6 ml of 2% (20 mg/ml) lidocaine. In smaller pets, you may dilute the lidocaine 50% with saline to avoid causing toxicity.
Punch biopsies are most commonly used. A biopsy punch of appropriate size is rotated with moderate pressure in a position perpendicular to the skin, in one direction until a reduction in resistance is felt. A 6 mm punch is appropriate for most sites; a 4 mm punch may be preferred on eyelids, the nasal planum, and small footpads. A small thumb forceps is used to remove the biopsy “plug”, which is usually attached by the subcutaneous tissue, and the attach-ment is cut using iris scissors. The sample should be handled gently, grasping the edge of the subcutaneous tissue rather than by crushing the entire plug. Elliptical biopsies are sometimes used to remove nodules, fragile lesions such as bullae, or deep lesions involving the subcutaneous tissue. Finally, biopsy sites are sutured: a cruciate pattern using 4-0 or 3-0 nylon suffices for most 6 mm biopsies. Pets generally do not bother their biopsy sites, but consider a physical barrier such as an e-collar when collecting biopsies from very pruritic animals or from the feet. Biopsies are submitted in formalin with a thorough history.
Interpretation
Send skin biopsies to a pathologist with a special interest in dermatology. Remember that a skin biopsy can some-times “miss” a diagnosis that is made more easily another way. Examples include most ectoparasite infestations, dermatophytosis, and Malassezia dermatitis. Skin biopsies are not always helpful in animals suspected of being allergic, except to rule out similar-appearing “zebras”. One of the most important factors aiding the usefulness of a skin biopsy is the submission of a good history, examination findings, and differential diagnoses. In addition to his-topathology, biopsies can be used for immunohistochemical staining to look for various organisms or for antibody deposition in immune-mediated diseases.
Serum testing for Allergen-specific IgE
Serum allergy tests use ELISA methodologies to quantitate circulating allergen-specific IgE levels. Like the intra-dermal allergy test, these tests are never used to diagnose atopic dermatitis, but to identify offending environmental allergens. Again, this is mostly useful if immunotherapy is to be pursued. In general, serum allergy tests do not cor-relate closely with intradermal allergy tests. There has been no conclusive evidence for the superiority of one test over the other, as no “gold standard” exists. However, it is known that when technical performance is used as a criterion, there are substantial variations between certain laboratories. Finally, although food-specific IgE testing is offered by many of these laboratories, these tests should not be used for the investigation of food allergy as the results can be highly misleading.
Elimination diet trial
An elimination diet trial is the only way to evaluate food adverse reactions in dogs and cats. Adverse food reactions can occur at any age and are rarely associated with a recent food change. Dogs and cats with nonseasonal pruritus, recurrent pyoderma, or cats with miliary dermatitis, self-induced alopecia, eosinophilic granuloma complex, and head/neck pruritus should be investigated with a diet trial. The diet trial is an 8-10 week period during which only one protein and one carbohydrate source are fed to the dog. Either a home cooked diet or a prescription diet may be used. Of the prescription diets, novel proteins (fish, kangaroo, venison, etc) and hydrolyzed proteins (soy or chicken protein) are available. Maintaining exclusivity is critical, and it is important to educate owners to the fact that:
- It may take 8 weeks to see a response.
- May find some patients still react to a novel or hydrolyzed protein within the same group as the initial food allergy, ie. if they have a chicken allergy, they may react to Duck.
- Any divergence from the trial can result in a failure to make the diagnosis. This includes the other pets’ food, fec-es, chewable supplements, rawhides, plates, handouts from well-meaning neighbors or children, and many others.
If the pet improves with the elimination diet, it is important to confirm the diagnosis of an adverse food reaction by resuming feeding of the suspected offending food. A relapse of symptoms should be seen within 5 days in most cases (sometimes immediately), but within 14 days in all cases. Some owners agree to investigate further to deter-mine the specific allergenic ingredients. While feeding the well-tolerated diet, one ingredient from the offending diet is added for 7 to 14 days at a time to try to elicit the symptoms.
Sources:
Mueller RS. Dermatology for the Small Animal Practitioner, 2000: available online at www.vin.com
White SD et al. Evaluation of aerobic bacteriologic culture of epidermal collarette specimens in dogs with superficial pyoderma. J Am Vet Med Assoc. 2005; 226(6): 904-8.
Morris DO. Therapy of Malassezia Infections and Malassezia Hypersensitivity. In Bonagura JD, Twedt DC, Eds. Kirk’s Current Veterinary Therapy XIV, 2008.
Saridomichelakis MN et al. Relative sensitivity of hair pluckings and exudate microscopy for the diagnosis of canine demodicosis. Vet Dermatol. 2007; 18(2): 138-41.
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