Overview of the Issue

Cutaneous cytology is an important diagnostic tool to definitively diagnose secondary infections of the skin and ears due to bacteria (cocci and rods) and yeast (Malassezia). Cytology is an inexpensive tool with a short turnaround time as it can be performed in clinic.

Cytology can provide valuable information about skin/ ear infections that need to be treated in our dermatologic patients. Cytology is also a valuable tool for determining response to treatment. Cytology can help characterize the microbial population on the surface of the skin when lesions are present.

Objectives of the Presentation

1.  To review cytology techniques

2.  How to stain and interpret cytology

When taking cytology, it is important to determine what micro-organism you are most suspicious of when looking at your samples. Bacteria and Malassezia can be found in higher numbers using certain cytology techniques over another. Certain cytology techniques will be more favourable based on lesion appearance. For a moist dermatitis, an impression smear might be the technique of choice. If samples are to be taken from a crusting dermatitis, the edge of a glass slide can be used to gently lift the corner of the crust and an impression smear obtained from under the crust. If pustules are present, a sterile 25-G needle can be easily used to lift the top of the pustule and then an impression smear obtained from the open pustule. If epidermal collarettes are present, a cotton tipped applicator can be rubbed under the scaly ring for the best sample. Cotton tipped applicators can be used to collect other cytology samples. When using a cotton tipped applicator, the skin should be rubbed vigourously until there is a colour change on the applicator. The sample should be rolled onto a slide. Multiple samples can be rolled onto one labelled slide to allow for more efficient reading of the cytology samples.

I personally find tape preparations the best way to find Malassezia. Acetate tape preparations can also be used for dry lesions. Clear acetate tape should be used for these samples. A small piece of tape is taken and then “stuck” onto the lesion multiple times¹. Once the tape is no longer “sticky” it can be stained. Tape preparations are also a great way to collect samples from areas such as the lip margin, interdigital regions and nail beds.

One study by Lo et al., showed that by inserting a toothpick into the claw folds on dogs with suspected claw fold dermatitis, a higher yield of yeast and cocci was noted when compared to using acetate tape or impression smears².

Ear Cytology

When doing ear cytology, it can be helpful, when taking the sample, to rub the opposite ear to prevent the pet pulling away from you or jumping as you are taking the sample. Ear cytology and skin cytology can be performed with a cotton tipped applicator. The cotton tipped applicator should be rubbed vigorously onto the skin until there is a colour change on the applicator. If ear cytology is being taken, the cotton tipped applicator should be inserted into the ear canal to the junction of the vertical and horizontal canals. The cotton tipped applicator can then be gently rotated a few times and removed from the ear.

Cytology samples should be heat fixed and then stained using Diff-Quik® and viewed under the microscope. I use the 10-10-20 rule for dipping into the stain (more dips into the blue stain).

Staining Cytology

If an impression smear or sample from a cotton tipped applicator are obtained, these slides should be heat fixed and then stained. A modified Wright stain, or Diff-Quik®, is the most common stain used for cytology preparations^3,4. Once stained the slide is rinsed with water and then dried before being examined under the microscope. If an acetate tape preparation is obtained, this does not need to be heat fixed but does need to be stained. Do not put the tape into the fixative as this will cause the adhesive in the tape to break down and your sample will be left at the bottom of the fixative jar! I like to stick one end of my tape onto a glass slide to hold the tape. I then use the slide to dip the tape into the red and blue stains (10 dips in the red and 20 in the blue).

The “sticky” side of the tape is then laid onto the glass slide. With any cytology samples, a cover slip can be placed onto the slide to allow for better viewing under the microscope.

Viewing Cytology

Slides should first be scanned at a low power for aggregates of inflammatory cells, nuclear streaming or acantholytic keratinocytes^4,5. Any identified areas are then examined under higher power. Multiple regions on the slide should be viewed as findings can differ between different areas of the slide.

Interpreting Cytology

Classification of a semi-quantitative method for assessing cytology has shown good intra-observer and inter-observer reproducibility. The Canadian Academy of Veterinary Dermatology has one of these types of scales available on their website. You can also grade cytology using number estimates of how many organisms are noted e.g., 0-2 yeast/OIF.

In my opinion, I do not have a rule as to “how many organisms are too many and require treatment”. I always pair my cytology findings with what my patient is showing me and their clinical signs. However, documentation of an inflammatory reaction and intracellular bacteria provides confirmation of pyoderma³. Minimal data exists documenting normal yeast numbers on feline skin so the presence of any yeast on cutaneous cytology taken from a cat should be considered abnormal. When viewing otic cytology, rods are not commensals of the ear canal in dogs or cats, so the presence of any rods should be considered abnormal.

Normal Findings on Cytology

It is important to be familiar with what constitutes normal findings on cytology. Keratinocytes and melanin granules are considered normal findings. Simonsiella organisms can also be found on cutaneous cytology. This is a bacterial species that lives commonly within the oral cavity. It is not a pathogenic organism but does indicate that the patient has been licking or chewing at the region the cytology was obtained from.

Summary Including 5 Key “Take Home” Points:

1.  Cytology is a simple and inexpensive way to determine whether a cutaneous infection is present and contributing to your patient’s pruritus or skin disease.

2.  Knowing what is “normal” on cytology is important to allow interpretation of your samples.

3.  There are multiple ways to obtain cytology (cotton tipped applicators, tape preparations, etc.), some of which are better for specific lesions or micro-organisms.

4.  Diff-Quik® can be used to allow better identification of micro-organisms on your samples.

5.  Always pair cytology findings with the clinical signs your patient is exhibiting.

References

1.  Besignor E, Jankowski F, Seewald W, Touati F, Deville M, Guillot J. Comparison of two sampling techniques to assess quantity and distribution of Malassezia yeasts on the skin of Basset hounds. Vet Dermatol. 2002;13:237–241.

2.  Lo, KL, Rosenkrantz WS. Evaluation of cytology collection techniques and prevalence of Malassezia yeast and bacteria in claw folds of normal and allergic dogs. Vet Dermatol. 2016;27(4):279–e67.

3.  Miller WH, Griffin CE, Campbell KL. Muller & Kirk’s Small Animal Dermatology. 7th ed. St. Louis, Missouri: Elsevier; 2013. pp. 81–92.

4.  Hnilca KA. Small Animal Dermatology: A Color Atlas and Therapeutic Guide. 3rd ed. Elsevier Saunders; 2011. pp. 22–28.

5.  Udenberg TJ, Griffin CE, Rosenkrantz WS, et al. Reproducibility of a quantitative cutaneous cytological technique. Vet Dermatol. 2014;25:435–440.