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CAL 27/09/2011

Dermatology Diagnostic Procedures

Coat Brushings
Confirms presence of surface dwelling ectoparasites Parasites that can be identified in this way are: - Fleas and flea faeces - Cheyletiella (mites) - Lice Technique: 1. Place px on floor standing on white or brown piece of packing paper. 2. Vigorously rub skin and haircoat to help dislodge material that may be adherent. 3. Follow by combing vigorously with a flea comb including the entire body surface. 4. Move px away from paper and fold paper so all material in middle. 5. Remove the hair- parasites likely to be amongst the smaller debris. 6. Use strips of clear adhesive tape to remove remaining material from central part of paper. 7. Place on microscope slide + examine.

Cytology
Rapid way of gaining important info about a skin disorder. Indicated in most cases of skin dz and in ALL cases of otitis externa. Stains used: Diff Quik = modified Wrights stain (Romanowski). Methylene blue may also be used. Diff Quik = Alcohol fixative (clear) Eosinophillic xanthene dye (red) Basophilic thiazine dye (blue) Distilled water. Permits visualisation of bacteria, although sometimes a gram stain may be helpful as well. If mycobacteria are suspected an acid fast stain should be employed. Technique: Impression Smear 1. Press a microscope slide onto skin (clip hair away) or lesions/pustules if the epidermis is eroded. This can also be used for sampling ruptured pustules and the cut surface of tumours biopsied or removed, (squeeze +express the contents onto the slide). 2. This is useful for assessing levels of Malassezia (yeast organisms naturally present on skin surface but if overgrowth occurs= dermatitis +intense pruritis) or bacteria on the skin surface. 3. Also useful for open, exudative, or draining lesions. 4. Be sure that the material is not too thick on the slide-smear it with the aid of another slide if necessary. 5. Demodex can often be found in this way from lesions of associated deep pyoderma.

6. Before staining the slide should be air-dried and then alcohol fixed. When evaluating samples for Malassezia or ear samples heat is used. 7. In assessing levels of Malassezia it may be preferable to omit the alcohol fixative stage of Diff Quik stain, as this may remove some of the lipid in which the yeast is generally situated and with it some of the organisms. If examining material from suspected pyodermas, look for material from intracellular cocci. Fig 1. Malassezia

Tape Strips for Cytology Clear cellulose tape is used (sellotape). Press firmly on area of skin or lesion to be sampled. For Cheyletiella place straight onto slide +examine 1st under low power. For Malassezia of bacteria each end of tape is then attached to the end of a microscope slide to facilitate handling for staining (allows dunking) Stain with Diff Quik omitting the fixative stage which would destroy the tape. One end of the tape is then freed and the tape pressed down onto the surface of the microscope slide. A drop of immersion oil is placed upon the tape, a coverslip applied, and the sample the examined under a high power and oil. Samples Procured with a Needle Using needle to rupture the lesion: A suitable lesion is gently ruptured using a 23-26g hypodermic needle Material from the needle tip is then placed on a microscope slide and smeared using the needle or, if there is abundant material, using another microscope slide. Additional material is then obtained by an impression smear placing a microscope slide onto the ruptured lesion. The material is then air-dried and stained using the usual protocol.

FNA: For obtaining material from cysts or deep inflammatory lesions; or a cutaneous mass. Diagnostic cytology may be obtained for skin tumours but in many cases biopsies for histopathology are required to confirm the dx. Use 20g needle or 18g if sampling more dense lesions. Attach needle to 3ml syringe or 10ml if greater suction is required. Be careful when sampling possible mast cell tumours as their manipulation can cause adverse reactions due to HISTAMINE release. Pre-tx with antihistamines is a wise precaution.

2 main approaches for FNA: 1) Using a needle: a. After application of a suitable antiseptic to sterilise the surface the needle is introduced into the lesion at a 30 angle to the skin surface. b. Partly withdraw and redirect 4-5 times to obtain additional material. c. Withdraw the needle, the plunger of the syringe is withdrawn half way or more, the needle is attached and the contents of the needle expressed onto the surface of a microscope slide. d. Material is gently smeared to avoid too thick a preparation using either the needle or another slide. e. Air dry and stain. f. If the mass was a lipoma, lipid material only will appear on the slide. g. If the mass were an epidermal cyst, granular cheesy material that is amorphous will be obtained and is also readily expressed from the puncture wound. However, routine expression of epidermal cysts in this way is not recommended. 2) Using a syringe and needle: a. A 20g needle is attached to a 3 or 5 ml syringe with the barrel pressed down. b. Sterilise the surface of the mass with a suitable antiseptic. c. The needle is introduced into the mass at 30 and suction is applied. Dont let the needle penetrate the deep surface of a potentially malignant neoplasm as it could aid in its spread. d. Withdraw the needle, remove the syringe and withdraw the plunger. e. Reattach the needle and express the contents onto a slide. f. Smear, dry, stain. Samples Procured with a Cotton Swab Indicated for sampling fistulous tracts and deep draining lesions. It is also used for obtaining samples from ears. A cotton tipped applicator is introduced into the lesion After withdrawing material is transferred to a microscope slide by gently rolling the tip on the slide rather than smearing it. Air-dry, heat or alcohol fixation is undertaken prior to staining. The latter is used where preservation of cell morphology is important. Cytological Examination of Exudate from the Ear Canal A cotton tipped applicator is introduced gently to the level of the horizontal canal. If ear canal is dry or Otodectes suspected, moisten tip w. liquid paraffin prior to introducing. Gently rotate the tip in a circular motion to obtain the exudate then smear onto microscope slide.

In looking for parasites additional oil is placed onto the slide which is then cover-slipped prior to microscope examination. For examination of bacteria and yeasts, moisten the ear canal prior to sampling. Fix slide by heat rather than by alcohol then stain.

Hair Plucking
Useful where there is hair loss or abnormal appearance of the hairs. Technique: Sample of hairs grasped either with eyebrow forceps or haemostat. Transferred to slide, placed on oil, and examined microscopically first under low power. If a dermatophyte is suspected a sample is also placed either in 10% potassium hydroxide and allowed to clear for 30 mins prior to examination OR in chlorphenolac which is - 50g chloral hydrate - 25ml liquid phenol - 25ml lactic acid Both KOH and Chlorphenolac are CAUSTIC- caution! Findings: The nature of the hair bulb - Look for anagen and telogen hairs - Anagen hair bulbs have rounded and smooth ends that are usually bent - Telogen hairs have spear-shaped heads

Telogen -

Anagen

Assessment of Anagen/Telogen ratios is not generally helpful as normal animals have widely ranging ratios, dependant upon the stage of the hair cycle.

Evidence of parasites - Look for evidence of eggs of Cheyletiella. (A1) - Look for the nits of lice, which may be attached to the hair shaft. (A2) - Look for evidence of Demodex mites which may accompany epilation of the hair from the hair follicle. (A3)

A1

A2

A3

The nature of the hair shaft - Examine the tips and ascertain whether they have pointed ends which is normal (Pic 1) - Assess whether the ends appear to have been sheared-off, e.g. by excessive licking (Pic 2) - Look to see if the hair shaft appears to have normal texture or of it appears more like a ghost-shaft due to digestion by fungal keratinases. (Pic 3) - Look to see whether the melanin granules appear normal or if there are aggregates of melanin- macromelanosomes (Pic 4). The latter are seen in COLOUR DILUTION ALOPECIAS e.g. blue Dobermans (Pic 5), blue Yorkshire Terriers (Pic 6), and fawn Irish Setter and in black hair follicle dysplasia (Pic 7). - Look to see if there is evidence of fracture of the hair shaft, which can suggest colour dilution conditions and excessive grooming. - Look for evidence suggestive of trichorrhexia nodosa (Pic 8) which is a dystrophic condition of the hair or any other evidence of hair shaft dystrophy.

Pic 2 Pic 1

Pic 4 Pic 3

Pic 5

Pic 6 Pic 7

Pic 8

Pic 9

Evidence of dermatophytes (Ringworm!) If you suspect dermatophycosis, first examine the haircoat with a Woods lamp. Some 50% of cases of Microsporum canis will fluoresce green (A). Choose hair shafts that appear as ghosts, suggesting digestion by fungal keratinases. Carefully examine the borders under high power with a partially closed condenser (to find suspicious areas) and then oil immersion for confirmation. Dermatophyte arthrospores have a slightly greenish tinge(B) and a solid appearance that is appreciable upon focussing the microscope up and down. Those of M. canis are smaller (1-2) than are those of Microsporum gypseum. Fungal hyphae(C) may also be seen, either in the hair shaft or in scale.

A C B

Intradermal Test
Points to consider before deciding to set up allergy testing: How many pxs will you potentially skin test in a year? The outlay is expensive and the procedure is unlikely to be economically viable if used less than once per week. Whose allergen extracts should you use? There are a number of reliable allergy houses that specialise and supply aqueous extracts. Human allergists usually use glycerinated extracts that are unsuitable for veterinary use. How many allergens should you use? There is no answer to this! Most use around 50 but it is acceptable to limit the screen to ~ 20 of the most common. Which ones should I include? There is now good evidence on the incidence of positive skin tests in countries. Consult a veterinary dermatologist. House dust mitesDermatophagoides farina- should always be included.

Should I purchase diluted allergens ready for use or allergens in concentrated form? Diluted allergens are only stable for around 6 months so if you undertake a reasonable amount of skin tests it could be wiser to purchase small volumes of conc extracts which are stable for 2 years and dilute them for use every 3-6 m. One advantage is that you can then use the same preparations to make up the vaccine for immunotherapy. Getting started: The fundamentals of why we skin test Intradermal tests may be used to 1) Confirm a clinical dx of atopic dermatitis 2) Enable selection of allergens for immunotherapy Equipment required= 1ml syringes with 25-26g needles, clippers, marker pen, good light for reading results. Storage of allergens at +4C, NEVER FROZEN. Keep best in glass vials. Which px should I select for skin testing? - 1st rule out other diseases which could account for the clinical signs. e.g. parasitic and food allergy. - If flea allergy is suspected testing with that allergen alone is undertaken. - If atopic dermatitis is still suspected and the problem is seasonal, skin testing is best done after the major allergy season. If it is perennial it does not matter. Preparation of the patient for skin testing: Must have been off antihistamines for 10d. Corticosteroids also interfere with the IDT. Generally the px should have been off for at least one week for every month of continual therapy- where more long-acting injectables have been used and possibly less where low dose alternate day therapy has been given. If in doubt give an injection of the positive control (histamine phosphate 1/100,000 w/v histamine phosphate. If this does not induce a good weal, do not proceed). Sedate the px with a product that has no antihistaminic activity (e.g. xylazine). Clip the hair from the lateral chest. This is the preferred area as it is the most sensitive. Mark the sites to be injected using a suitable marking pen.

Giving the injections: Hold the syringe so that you can inject immediately once the needle enters the skin, without having to shift your hold on the syringe. This is the key to successful technique. Introduce the needle into the skin, bevel upwards, and inject around 0.05 ml. This should cause a readily visible bleb. Two common errors: If you are subcutaneous you will not appreciate resistance and there will be no discernible bleb. If you inject air, you will see a rapid blanching of the site.

In either case rpt the inj at an alt site. Continue with each allergen and with the positive and negative controls. The most important thing is to ensure that all blebs are the same size.

Reading the skin test: The time -

Some skin tests are read at 15-20mins after injection Sometimes the immediate reactions are negative, with reactions appearing at 4-8 hours. The significance of these is unclear and many dermatologists do not routinely record these. In the case of the flea allergen, reactions can occur either immediately or at 24-48 hours. Thus if the immediate reaction is negative, the patient should be reassessed at the later time. The delayed reaction is often very subtle and not obviously urticarial in nature.

Two approaches to grading Assuming that the size of the histamine weal is +4 and the diluent control is negative, each reaction is graded on a +1-+4 scale taking into account both the elevation above the skin, the degree of erythema and the weal diameter. Alternatively: The diameter of each reaction is measured. A positive reaction is any that exceeds 50% of the mean of the weal diameter of the histamine and diluent controls.

Interpreting the test Remember, again, that a positive skin test merely indicates that the patient has allergen specific IgE antibodies. It does not imply that these are the cause of the disease. The results are interpreted having regard to: o The presence of the allergens in the env and o The clinical signs as related to the above.

Skin Biopsy
Indications for performing a skin biopsy can be summarised as: Any primary eruption of whose nature you are unaware should be sampled. Any dermatosis whose aetiology is still obscure after clinical and other examinations.

Site selection: Choose primary lesions wherever possible In the case of puch biopsies, the punch should be placed in the centre of the lesion, with normal skin excluded (unless the lesion is very small). If excisional biopsies are undertaken, e.g. in the case of a suspected tumour normal skin should be included in the margins. This is also the case where wedge shaped or elliptical biopsies are concerned. Sample a number of lesions in varying stages.

Technique: Disinfection of skin is not essential If you chose to do it the skin should be dabbed lightly only with 70% ethanol or with isopropyl alcohol. No other disinfectants. Do not rub! Inject 1-3ml of 2% lidocaine or other suitable LA S/C well clear of the biopsy site. 6-8mm disposable punch biopsies are ideal. Gently rotate biopsy punch, moving always the same way until through skin. Withdraw punch Pick up biopsy using forceps and handling only the undersurface Excisional, wedge shaped or elliptical biopsies are taken using a scalpel. Handle tissue gently. To prevent distortion (occurs readily with larger biopsies) place biopsy with cut surface down on a piece of cardboard or wood. After adherence is assured, it is then transferred to a vial of 10% neutral buffered formalin.

Working with the pathologist: Form a team and co-operate Choose pathologist with interest in skin dz and who attends derm meetings Give complete hx, lab findings, response to therapy, your list of ddxs in rank order. Give pathologist feedback- learn from each other Dermopathology is v limited as many dzs look the same. Dx may not be definitive.

Skin Scrapings
Indications For dx of parasitic dz Cheap and simple, indicated in majority of cases if not all!!!!!

Selection of area to be scraped Choose abnorm skin Select areas where there is excessive scale, loss of hair, papules, comedones. Ear margins are favourites for scabies.

Location of parasites Harvest mites (chiggers or berry bugs) are on the SURFACE and just visible as orange-red dots. (A) - Cheyletiella on SURFACE and just visible to the naked eye as white dots WALKING DANDRUFF (B) - Male scabies mites are on the SURFACE. Females burrow into the EPIDERMIS, where the eggs are laid. Same is true of Notoedres. - Demodex mites are in the hair follicle. (C) - Otodectes usually found in ear but may be on surrounding skin. SURFACE dwellers just visible to the naked eye as grey dots. (D) Superficial and deep scrapings should be taken to cover entire spectrum of parasites. Do these separately i.e. superficial first, place on slide, then return to do deep. -

Technique: Clip hair from area to be scraped If Cheyletiella suspected clip with scissors as otherwise mites will be removed by close use of clippers. If Demodex suspected squeeze skin gently btwn finger and thumb to force any more mites more superficially in the hair follicle Moisten skin with oil (mineral oil, liquid paraffin) Take a number 10 scalpel blade Blunt the cutting edge on a hard surface Moisten the blade and skin surface with oil and place a drop of oil on the microscope slide. Gently but firmly scrape in the direction of the hair growth at an angle of about 45-70 to the skin surface. Transfer to slide. Return for deeper scrapings and continue until there is obvious capillary bleeding.

Sarcoptes Notoedres

Therapeutic trial Specific therapy = dx Combination tx eg abs, corticosteroids, and anti-parasitic tx should NOT be used. If there is a response it is impossible to ascribe the improvement to any one of the drugs administered. Conditions amenable to this approach:

Sarcoptic mange mites often dif to find. Choose approved prod. Ivermectin 100% effective but NOT approved for this purpose. Also sp cautions relating to sheepdogs and other herding breeds. Pyodermas: If a bacteria has been isolated from suspected pyoD, tx with abx to which sensitive (shown in vitro) is best.

Key concepts Conditions amenable to this approach included: Flea allergy: sometimes evidence of parasite hard to find. In such cases use a combo of suitable parasiticide on pet AND environmental control. Cheyletiella and harvest mites sometimes hard to confirm. Response to tx may be confirmatory. Vit A responsive dermatosis: compare before and after tx Zinc responsive dermatosis: compare before and after zinc sup. Conditions NOT amenable to this approach: Endocrine dz hypoT. Response may be seen in normal animals resulting from supraphysiological doses. Cushings a ddx must be made and if borderline results are obtained rpt testing should be done in 3 months rather than trial tx which could be dangerous.

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