fbpx

BASIC DIAGNOSTIC TECHNIQUES IN DERMATOLOGY – USING YOUR MICROSCOPE

BASIC DIAGNOSTIC TECHNIQUES IN DERMATOLOGY – USING YOUR MICROSCOPE

I believe that no dermatological examination should pass without doing basic in-house cutaneous cytology. Your microscope is a relatively simple tool, that with some diagnostic tests helps to identify infections and formulate a diagnostic plan to search for possible underlying disease.

Skin scrapes

Skin scrape is the most common and simple diagnostic test for identifying ectoparasitic infections.

  • Superficial skin scrapes are used for detecting Sarcoptes, Notoedres, Cheyletiella and Demodex gatoi. Select the area of the skin with the lesion, clip the hair if necessary and apply a small amount of mineral oil on it (that will ease the collection of scraped material). Scrape it with a scalpel blade or dermal spatula in the direction of hair growth and transfer accumulated material to the glass slide. Sample several sites or, in case of suspected Sarcoptes mites, large surface area. Best sites for detecting sarcoptic mites are ear margins and lateral elbows. D. gatoi is anecdotally more easily found on the lateral shoulders. Scan the entire slide using the low power and adjust the microscope diaphragm and/or condenser to provide greater contrast and enhance the visibility of the mites.
  • Deep skin scrapes are used to diagnose infestation with Demodex (except D. gatoi). The technique is similar to superficial scrape, but the scraping should be strong enough to induce capillary oozing. This ensures that the collected material comes from deep enough to collect follicular Demodex mites. Sampling site can be previously squeezed between fingers to force the mites toward the surface of the hair follicles.

Hint: the area with comedones is a good place to scrap for Demodex mites.

Trichoscopy (hair plucks)

Trichoscopy is used to visualize the hair for evidence of pruritus, fungal infection and pigmentation defects and to assess the growth phase. Pluck a small sample of hairs with hemostat from lesional and/or perilesional skin and place it onto a glass slide. Hairs should be placed on the slide in a unidirectional pattern, so that hair bulbs line up, to make the evaluation of the slide easier. Tape or mineral oil is used to secure the hair in position. Scan the slide under low power and adjust the diaphragm and/or condenser to provide greater contrast.

  • Broken hair tips instead of the tapering tips of normal hair are indicative of pruritus (chewing or excessive grooming).
  • Observe the hair roots for identification of anagen (growing) and telogen (resting) hairs. Anagen hair bulbs have rounded ends and may curl or bend, telogen hairs have a pointed or tapered end. In most breeds, hair plucks are a mixture of anagen and telogen hairs, with telogen hair predominating. In breeds with prolonged growth periods (poodles), most of the hairs would be in anagen and relatively few in the telogen state. Many Nordic breeds have long resting phases with telogen-dominated hairs. An excess of telogen hairs or completely telogenised coat may indicate an endocrinopathy or metabolic disorder. Predominantly telogen hair may also be seen after severe illness, pyrexia, pregnancy and lactation, malnutrition etc., and with dermatoses characterised by follicular arrest (cyclical flank alopecia, alopecia X).

“Fun” fact: breeds with predominantly anagen coats suffer anagen defluxion and hair loss during chemotherapy and other treatments with cytotoxic drugs (like humans). This is otherwise uncommon in animals.

  • Clumping of melanin within the hair shafts may indicate a colour dilution alopecia or follicular dysplasia. Pale, irregular appearance of the hair shaft may be caused by the invasion of the keratin by arthroconidia and hyphae in case of dermatophyte infection. Spores may be seen inside (endothrix) or around hair shafts (ectothrix). Sometimes, ectoparasite eggs attached to the hair shaft may be visible with pediculosis and cheyletiellosis.

Demodex mites may be seen clustered around hair bulbs. Hair pluck can be used for detecting Demodex in areas or situations, when a skin scrape would be difficult – around the eyes, in pododermatitis or with excited puppies and other fractious animals.

Acetate tape preparation

A tape can be used for identification of mites (Cheyletiella, lice) and yeast. For detecting mites, press the tape to multiple sites and collect as much scale as possible, then stick the tape onto a glass slide and examine it under low power. Tape preparations are also a very efficient method for identifying Malassezia skin infections. After pressing the tape on the lichenified lesion or moist pedal erythematous skin multiple times, stain it with the last dark blue stain of a cytology stain (you can put a drop on the slide under the tape) or use eosinophilic and basophilic stain, and examine it under high power. Do not put the tape into the fixative – it will dissolve the adhesive.

Direct impression smear (touch imprint)

Touch imprints can be used to diagnose Malassezia overgrowth or to evaluate moist lesions like ulcers, erosions and plaques. Press a glass slide several times directly on the surface of the skin or a plaque/ulcer. Press stronger for greater adherence. Papular lesions may be traumatized by the corner of a glass slide or a needle and squeezed to express fluid. Sample crusted lesions by removing the surface or margins with a sterile needle and imprinting the subjacent surface on a slide. For yeast identification, you can also collect samples by cotton swab, which is then rolled onto the slide (used for sensitive areas such as the perivulvar or perineal region), or by superficial skin scraping. Allow moist samples to dry or heat-fix them before staining. Scan the slide with low power to find areas rich in basophilia for closer examination. High power is then used to identify cell types and microorganisms.

Hint: When staining, avoid contamination of the stains – yeast organisms, in particular, seem to be able to persist in the stain and appear artefactually on subsequent samples. This can be avoided by adequate heat fixing of exudate samples.

Fine-needle aspiration

Fine needle aspiration is used to examine pustules, nodules and tumours. A 22-25 gauge needle with an attached syringe (6 ml) is used. Clean (clip) and disinfect the surface of the lesion and immobilize it. Insert the needle into the centre of the lesion, apply negative pressure by pulling back the plunger, then release the negative pressure and repeat it several times while redirecting the needle. Don’t forget to release negative pressure before removing the needle from the lesion in order to keep the sample in the needle. Stop if blood is visible in the needle hub because this will dilute the cellular sample. Another method, which works best for soft masses, is to draw a small amount of air into the syringe, or use the needle without the syringe, and insert the needle into several areas of the lesion using a stabbing motion. After sampling, remove the needle from the syringe, fill the syringe with air, place the needle back and expel the sample on the glass slide. Distribute the sample in a thin layer using a second slide. Stain and examine the slide as with touch imprint.

Otic swabs

Otic cytology should be performed in every case of otitis. It is used mostly to diagnose bacterial and yeast overgrowth but is also useful to confirm other differentials – neoplasia, keratinization disorders, mites and fungal infections. Swab the ear canal with a cotton swab and roll it on the glass slide. Hint: smear a left ear sample on the left side of a glass slide and right ear sample on the right side. In case of a black waxy exudate, place some mineral oil on it and examine the slide under low power for mites. If the sample is light brown or purulent, stain the slide and examine it under greater magnification to identify organisms and/or cellular population. If the material is very waxy, the end of the slide could be heated to melt the wax and allow the stain to penetrate the sample. Normal cerumen does not take up the stain. Stain and examine the slide as with touch imprint.

Note: Keratinocytes may include melanin granules, which should not be mistaken for bacteria. Melanin granules are typically round to oblong and have green/brown colouration.

LITERATURE:

Rhodes K. H., Werner A. H.: Blackwell’s Five-Minute Veterinary Consult. Clinical Companion. Small Animal Dermatology, 3rd Ed. Wiley Blackwell, 2018

Hnilica K. A., Patterson A. P.: Small Animal Dermatology. A Color Atlas and Therapeutic Guide, 4th Ed. Elsevier, 2017

Jackson H. A., Marsella R.: BSAVA Manual of Canine and Feline Dermatology, 3rd Ed. BSAVA, 2012

Neuber A., Nuttall T.: Diagnostic Techniques in Veterinary Dermatology. Wiley Blackwell, 2017