Superficial skin cytology
This article was kindly provided by Royal Canin:
1/ 5-year-old German Shepherd cross, neutered bitch
At presentation the dog had intense pruritus in its face, paws and groin that was not responding to prednisone (0.5 mg/kg once daily).
Apart from the dermatology problem, the dog was apparently healthy, fully vaccinated and was given spot-on flea prophylaxis irregularly (every two months and only in summer).
The general physical examination did not show any abnormal changes, while the dermatological examination showed alopecia with erythema and crusts in the skin of the lower lips (Figure 1), erythema in the ventral interdigital spaces of the forelegs and perianal area (Figures 2 and 3) and erythema with hyperpigmentation and mild lichenification on the abdomen and groins (Figure 4).
What diagnostic and/or therapeutic steps do you suggest?
1. Commence allergen-specific immunotherapy immediately.
2. Increase the prednisone dose to 0.5 mg/kg twice daily.
3. Administer cyclosporine A orally at a dose of 5 mg/kg once daily.
4. Perform a cytology examination of the surface of the affected areas and also multiple microscopic examinations of hair and superficial skin scrapes.
Based on the clinical symptoms observed (pruritic erythematous dermatitis of the face, paws and abdomen) and the dog's clinical history (seasonal atopic dermatitis), the most likely differential diagnoses are, in this order:
1. Atopic dermatitis (seasonal or otherwise) with or without:
a. Superficial pyoderma and/or Malassezia-induced dermatitis,
b. Flea infestation,
c. Flea bite-induced allergic dermatitis (this is unlikely due to the absence of dorsal-lumbar pruritus and specific IgE against flea-saliva allergen in the serology test).
2. Sarcoptic mange (unlikely because the distribution of the pruritus is not consistent with this).
3. Demodicosis (unlikely due to the presence of pruritus and only moderate alopecia).
The clinical manifestations of patients with atopic dermatitis are often exacerbated by the presence of factors that are considered to aggravate or trigger allergic inflammation. In dogs, these factors are typically skin infections caused by Staphylococcus and Malassezia, fleas and food allergens.
Therefore, in dogs with atopic dermatitis, it is necessary to identify and control these factors before deciding on a specific treatment option for the allergic reaction (anti-inflammatory treatment or prophylaxis with specific allergen immunotherapy).
Microscopic examinations on plucked hair and superficial skin scrapes did not reveal the presence of parasites. Brushing to look for fleas did not show the presence of adult fleas or faeces. In addition, direct impression samples were taken of the abdominal skin and adhesive tape impressions of the skin on the lip and interdigital spaces for cytology examination, showing the presence of abundant neutrophils with some eosinophils and intracellular cocci in all samples (Figure 5).
Final diagnosis ---> Superficial pyoderma
Empirical antibiotic treatment was commenced with oral cephalexin (22 mg/kg twice daily) along with baths using a 3% chlorhexidine-based shampoo every 2-3 days. A parasiticidal treatment was also commenced with a selamectin-based spot-on application every 21 days. At 21 days, the clinical response was excellent with almost complete absence of lesions (Figures 6 and 7) and highly notable reduction in pruritus, suggesting a final diagnosis of superficial pyoderma secondary to seasonal atopic dermatitis. It was not necessary to prescribe any anti-inflammatory treatment to control the clinical symptoms and no recurrence of pruritus was observed until the following year, and so it was not necessary either to commence specific allergen immunotherapy.
2/ Superficial skin cytology examination (non-neoplastic skin cytology)
Superficial skin cytology examination is a very valuable test in veterinary dermatology practice. It provides very useful information in a short space of time so that an action plan can be made quickly (e.g. deciding between empirical antibiotic therapy or performing a culture and antibiogram).
It permits inflammatory and neoplastic cell types to be defined and microorganisms to be identified.
A superficial skin cytology examination is indicated in all conditions that present:
• Papules, pustules and/or epidermal collarettes.
• Erosions, ulcers and/or crusts.
• Dry or oily scale.
• Increased secretions or exudate present in the ear canals.
In addition, in dogs with facial pruritus, cytology examination of conjunctival mucosa may be indicated if there is:
• Presence of mucopurulent discharge.
B) Material required
• Curved scissors,
• Sterile 25-gauge needles,
• Transparent adhesive tape, ideally Scotch® Crystal Clear Tape, 3M,
• Scalpel blades, no 10 or 20,
• Cotton-tipped swabs,
• Ground-edge slides,
• Pencil or permanent pen to mark the slide with the case name and the body area of sample,
• Rapid staining,
• Cover glasses (slides),
• Immersion oil.
C) Sample collection techniques
Different techniques exist and the choice depends on the type and location of the lesion. For example, samples to be taken in areas that are hard to access (skin surface of the ear canal, skin folds, interdigital spaces, perianal area) can be obtained more easily with the adhesive tape technique or a swab.
The different techniques are described below, specifying the lesions for which they are indicated.
1) Direct impression smear
This technique is used for flat, accessible body areas in the presence of:
• Pustules (Figure 8),
• Epidermal collarettes (not very chronic, when exudate is still present) (Figure 9),
• Erosions, ulcers or surfaces underneath a haemorrhagic crust (Figure 10).
This technique involves directly pressing the slide gently on the affected skin surface. Before collecting the sample it is best to cut the hair surrounding the lesion with scissors. If pustules are intact they should be gently broken with the 25-gauge needle. The purulent material found is then collected by gently pressing the slide on the surface. This procedure may be repeated several times in order to collect a lot of samples from the same pustule.
Crusts, if present, must be lifted with a fine-gauge needle to expose the surface exudate. In the presence of erosive-ulcerative lesions, it is recommended to dry the exudative surface by rubbing it and then collect fresh exudate.
2) Collection with a cotton-tipped swab
This technique is used to collect the following samples:
• Secretions inside the ear canals,
• Exudate from fistula tracts (Figure 11),
• Exudate present in the interdigital spaces or between skin folds (Figure 12),
• Conjunctiva (muco-purulent material present in the conjunctival sac in dogs with suspected allergic conjunctivitis).
It consists of collecting the exudate by exerting moderate pressure. The swab is then rolled gently on the slide.
3) Collection using adhesive tape
This technique is used to sample skin areas that would be hard to reach using a direct impression smear technique (interdigital spaces, eyelids, lips or perianal area). It is ideally used in the presence of oily scale in order to identify the presence of Malassezia on the skin surface (Figure 13).
The technique consists of cutting a 3-5 cm strip of adhesive tape and pressing one end moderately but repeatedly on the surface to be sampled. After collecting the sample, the other end of the adhesive tape is stuck to the slide, leaving the end with the sample free for staining. These samples are stained directly, without using the alcohol fixative provided in the staining kit because the alcohol might remove the adhesive along with the sample from the tape. After staining, the tape is stuck to the slide with the sticky side down, avoiding making creases. The sample is examined under the microscope through the tape without needing a cover glass(slide).
4) Collection by means of superficial scrape
This technique is indicated for:
• Oily scale,
• Lichenification (Figure 14),
• Ulcers with thickened margins.
It consists of scraping the skin surface with a number 10 or 20 scalpel blade. In lichenified areas it is advisable to spread out the skin by stretching it between your first finger and thumb to expose the skin surface that is hidden in the folds. Once obtained, the material is spread on the slide like "butter on bread".
D) Fixing and staining of samples
In all cases, the slide should be marked with the case name and the site or lesion where the sample was taken from.
Most samples should be air dried, except for samples collected using adhesive tape, which can be stained directly. However, for very oily samples (ear wax secretions, oily scale) it is recommended to heat fix them before immersion into alcohol. To do this, simply pass a flame a couple of times some two centimetres' distance under the slide (on the opposite side from the sample).
The most commonly used stain in clinical practice is the Diff Quick stain. It is a Romanovsky-type panoptic stain that makes it possible to differentiate basophilic and acidophilic areas in a preparation, for the purpose of cytology study. Its main advantage over other similar types of stains is that it is simple and quick to perform (15-30 seconds). However, the quality is slightly lower than stains such as the May-Grundwald-Giemsa stain or Wright's stain that are used in diagnostic laboratories, especially for cytological preparations of neoplastic lesions.
The sample is immersed for 5-10 seconds in each step (1- fixation, 2-red dye, 3-blue dye). Between each step, the excess liquid should be allowed to drain onto a paper towel. After staining, the samples are rinsed rapidly under running water and left to air dry in a vertical position for later examination.
Tips for maintaining the staining kit in good condition:
• Number the bottle caps (1, 2 and 3): this should avoid mixing the caps up and staining the fixative with remnants of dye that may be left in the cap.
• Close the bottles immediately after use: this should prevent the volatile products from evaporating, with unnecessary product loss.
• Use two different fixative and dye kits: one for "clean" samples (e.g. nodule cytology, contents of intact pustules) and one for "dirty" samples (ear secretions, adhesive tapes, direct impressions, etc.).
• Filter the liquids regularly: this removes precipitate and sample remnants (wax, hairs).
• Change the kit regularly, depending on use, to prevent the formation of precipitate in the samples and to preserve the staining quality.
E) Examination of the sample
The samples are initially examined at low magnification (40x) to determine the sample area that contains the best characteristics to study (staining quality, high cellularity, single sample layer). Samples obtained by direct impression have the advantage of not misshaping the cells, although sometimes the material is too dense. In these cases it is recommended to identify outlying areas in the sample where it is possible to examine a single cell layer. Examination with low-power magnification permits larger structures to be observed, such as Demodex (Figure 15) or fungal spores (Figure 16), which could be overlooked if the examination were started at a higher magnification. However, by moving up to a higher magnification (100x), the entire cell population can be seen, evaluating signs of inflammation and selecting areas on the slide that seem to be the best for studying at a higher magnification (400x). Under high magnification, cell components can be clearly defined, and some micro-organisms (yeasts and bacteria) can be identified. Finally, the sample is examined using maximum magnification (1000x, in oil immersion), which is indicated for identifying infectious agents accurately (e.g. Leishmania and small bacilli) and cell details.
To conserve samples for a long period, they should be covered with a cover glass mounted with special glue (Eukitt), and they should be protected from the light. In daily practice, and in the case of samples that do not have to be kept, it is still recommended to cover the sample with a cover glass, placing a drop of liquid paraffin between the sample and the cover glass. This improves the quality of the image when examined at low (40x), medium (100x) or high (400x) power.
1) Normal findings on the skin surface
• Keratinocytes are mostly enucleated (corneocytes) and a smaller proportion is nucleated. Frequently melanin granules are seen on the surface of epiteliated cells (Figure 17).
• A small number of cocci and Malassezia may be present.
Numbers vary depending on the breed, sample site, and the technique used. For example, a Basset Hound has numerous Malassezia without any evident clinical signs. Although there is no broadly-defined number of micro-organisms that is considered normal in canine healthy skin, it is commonly agreed that the maximum number of observable cocci is <2 per immersion field (1000x), and <1-2 per dry field (400x) for Malassezia.
• Absence of bacilli.
• Absence of inflammatory cells.
2) Normal findings on the skin surface of ear canals
• Enucleated and nucleated keratinocytes,
• <5 cocci per immersion field (1000x),
• <4 Malassezia per immersion field (1000x),
• Absence of bacilli,
• Absence of inflammatory cells.
3) Normal findings of the conjunctiva
• Keratinised and non-keratinised epithelial cells,
• A few cocci and bacilli.
4) Abnormal findings
• The presence of degenerate neutrophils with intracellular cocci is diagnostic of bacterial infection (Figure 18).
• The presence of an elevated numbers of cocci or the presence of bacilli in the absence of inflammatory cells is diagnostic of bacterial overgrowth (Figure 19). This cytological diagnosis should be reached after an exhaustive examination of the sample to ensure absence of neutrophil and/or eosinophil granulocytes and of nuclear remnants that could suggest its presence. Cytological examination of pruritic areas where the dog licks may revealed bacteria from the oral cavity, without a pathological significance (Figure 20).
• The presence of an elevated number of Malassezia is diagnostic of overgrowth due to Malassezia (Figure 21).
• In general, when interpreting a sample and when significance should be given to the number of micro-organisms in the absence of inflammatory cells (i.e. in microbial overgrowth), the clinical presentation should be taken into account. Atopic patients may develop a hyper-sensitivity reaction to components of bacteria or Malassezia. In such cases, even a very few organisms may be clinically relevant and cause inflammation and/or pruritus, and therefore anti-microbial therapy would be indicated.
• The presence of non-degenerate neutrophils and numerous acantholytic keratinocytes (keratinocytes with a large nucleus and rounded hyperbasophilic cytoplasm) in a sample collected from the contents of a pustule suggests the diagnosis of pemphigus foliaceus, which justifies carrying out a bacterial culture and biopsy to confirm that the lesion is sterile and to make the final dermatopathological diagnosis of pemphigus).
• The presence of eosinophils in a cytology examination of superficial skin samples in dogs is uncommon and initially suggests allergic dermatitis and ectoparasitosis. Eosinophils may occasionally be seen inside pustules, suggesting an immune-mediated origin of the pustule, such as pemphigus foliaceus, pemphigus erythematosus or sterile eosinophilic pustulosis.
• The presence of macrophages, lymphocytes or plasma cells together with degenerate neutrophils in a deep lesion sample (ulcers) suggests a deep pyoderma (furunculosis). In deep pyoderma there are very few bacteria and they are very difficult to identify (Figure 22).
• The presence of eosinophils and Malassezia in specimens collected in dogs with conjunctivitis is diagnostic of allergic conjunctivitis. In dogs with pruritus it is suggestive of atopic dermatitis.
This article was kindly provided by Royal Canin. If you would like printed copies of this material or other Focus publications please contact your Veterinary Business Manager:
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