Cytology should be the most frequently utilized diagnostic test in veterinary dermatology. It is the minimum database for cases whether identifying the etiology of new lesions or rechecking a resolving infection. Dermatologists use cytology to determine response to therapy and, subsequently, guide therapeutic treatment changes. The results of a quick cytology may also indicate the need for additional testing such as skin biopsy or culture and sensitivity (C/S).

Why Should I Do It?

There are many different pathologies the skin can undergo when damaged. It can become erythematous, alopecic, exfoliative, hyperpigmented, eroded, lichenified, etc. These presentations can be due to various primary, secondary, and/or perpetuating causes. Cytology helps you identify these causes and put together a diagnostic and treatment plan.

Dermatologic conditions are often chronic or recurrent. The skin is ever changing, so repeat cytology is critical for successful management. Cytology allows veterinarians to determine response to treatments on recheck. Not just infection, but also the inflammatory response.

What Are You Looking For?

Cytology can tell you so much information! Most commonly, infectious organisms, such as bacteria and yeast, can be readily identified as a secondary issue, but other infectious organisms, such as dermatophyte spores or hyphae, can also be seen.

Inflammatory cells can be viewed under the microscope, especially in allergic patients with infection. Neutrophils and macrophages are often suggestive of infection or inflammation, while eosinophils may indicate a hypersensitivity or parasitic issue. An important thing to recognize is whether inflammation is accompanied with infection or if it is a sterile process.

Other cells that can be seen include acantholytic keratinocytes (often suggestive of pemphigus foliaceus) or neoplastic cells.

What Do I Need?

A quality microscope and lots of microscope slides! The more you do cytology, the better you become at it. I suggest having the frosted slides to help identify which side of the slide your sample is on. After staining the slide, it can be hard to tell. If you can visualize the sample on lower power (4x or 10x), but struggle to focus on 100x your sample may be on the opposite side of the slide that you are viewing.

In order to interpret cytology appropriately you need a functioning microscope. That means it has to be maintained well, serviced regularly, and clean with working objectives and stage.

A modified Romanowski stain (Diff-Quik™) is most commonly used in veterinary clinics. Compared to other rapid staining techniques, this process provides the best quality of detail.¹

Acetate tape can be used to sample dry, scaly material from skin lesions (often used interdigitally). A cotton-tipped applicator is mostly commonly used to collect samples from the ear canals. However, it can be used to sample draining tracts and difficult anatomical regions as well. Toothpicks are extremely helpful when sampling claw folds. Acetate tape, swab, and toothpick collection techniques are explained in more detail below.

How Do I Do It?

There are several different techniques for cytology collection. Depending on the location, patient temperament, and lesion, you may adjust which method is the best for that particular case. Also, different practitioners have different preferences. Find the techniques that work the best for you and become an expert.

Direct Impression

This allows you to obtain samples by directly using the slide to collect exudate and debris. Care should be taken not to rub back and forth because this can rupture cells. Also, don’t press too firmly since this can break the slide. Crusted lesions can be teased open with the slide edge. After lifting the crust, the slide can be moved underneath and gently pressed on the underlying tissue.

A reproducible, quantitative cytological technique with direct impression has been identified with polymorphonuclear cells (PMNs), nuclear streaming, extracellular cocci, and intracellular cocci.²

Swab

This technique utilizes a cotton-tipped applicator (CTA) to collect samples from tight spaces including ear canals or delicate anatomic regions such as the periocular tissue. Rub or roll a CTA onto the affected skin surface and roll the sample onto the slide. When sampling the ear canal, insert the CTA into the ear canal to the junction of the vertical and horizontal canal, gently rotate within the canal to collect exudate, and roll sample onto the slide. Samples can be heat-fixed if exudative, although most don’t have to be if they are allowed to sit in the fixative.

Tape Prep

Samples can be obtained by firmly pressing the sticky side of the tape to the skin surface repeatedly. The tape can be stained two different ways. You can place the tape adhesive side down onto the slide, lift edge of tape and apply a drop of purple stain from the Diff-Quik™ to the slide. You can also stain the tape itself in the red and purple stain and then place on a microscope slide for evaluation. You don’t need to use the fixative (first Diff-Quik™ stain) since it will remove the sticky portion of tape and your sample.

Tape prep can be useful for dry, scaly lesions or tricky anatomical spaces such as lip margins or between the digits.

Toothpick Method for Claw Folds

You will be amazed what a little toothpick can collect! Gently insert toothpick into the claw fold, scrape material from proximal claw, and roll toothpick onto slide.

It is a great technique to use when debris is noticed by the claw fold or any pet chewing their paws. One study compared toothpick, direct impression smear, and tape prep methods for sampling allergic dogs with clinical paronychia. The toothpick method was found to be the optimal collection technique when identifying populations of Malassezia sp. overgrowth.³

Scraping

This is different from a skin scrape looking for mites. Use a scalpel blade to sample dry debris by scraping superficially in one direction. Then, gently smear collected material on a slide. Scrapings can be useful for scaly, seborrheic samples.

Staining Slides

Collected material should be allowed to dry after you collect it. Length of time to dry depends on the thickness and consistency of the sample. It can be allowed to air dry or heat fix with a hair dryer on low heat or lighter on side of slide lacking sample.

As mentioned above, Diff-Quik™ stain is the most commonly used. It contains three different stains: 1) fixative (methanol), solution I (cytoplasmic, eosinophilic), and solution II (nuclear, basophilic).

Dip each slide in each solution 5–8 times (many sources vary on time in each fix). Allow excess solution to drain into jar or touch end of slide on paper towel to take away excess. This prevents dilution of the subsequent solution. After solution II, dip in distilled water or rinse under tap water (side lacking sample exposed to stream). Then, allow to dry through air drying, hair dryer on low heat, or blotting in bibulous paper.

Interpreting Cytology

Make sure you have the light adjusted accordingly. For viewing bacteria and yeast at 100x (viewed with immersion oil), you will need to have the condenser open and the light up, in contrast to other samples, such as skin scrapes or fecal floats, which are easier to read with the condenser mostly closed to enhance contrast.

Always start at low power. Scanning slides at 4x allows you to identify specific areas of the slide that would be more beneficial for 100x magnification and diagnostic evaluation. Avoid areas where the sample is too dense or too scarce, since this may not provide a good representation for diagnosis.

Neutrophils

These are acute inflammatory cells that destroy infectious organisms such as bacteria and fungus. A true pyoderma includes neutrophils with intracellular bacteria. However, bacterial overgrowth often reveals as neutrophils with several extracellular bacteria.

Eosinophils

These white blood cells have pink/red granules. Eosinophils phagocytize infectious organisms. They are most known for presenting due to hypersensitivity disorders. In dogs, parasites and food allergies are the most common reasons for eosinophils to present on cytology samples. However, cats commonly have eosinophils on cytology with any of the hypersensitivity disorders including atopic dermatitis.

Macrophages

A large, vacuolated white blood cell that engulfs and digests cellular debris, infectious organisms, foreign substances, etc. Seen more in the chronic stages of inflammation, but may be seen concurrently with neutrophils with pyogranulomatous inflammation.

Nuclear Streaming

Commonly seen on skin cytology and secondary to cells rupturing during sampling. This is composed of DNA smearing across the slide in fine threads.

Yeast

Malassezia spp. are considered normal inhabitants on canine and feline skin.³ Malassezia pachydermatis is the most common species found associated with clinical disease in the dog.⁴

These organisms usually present bilobed and are described as peanut- or snowman-shaped. However, you can get a non-budding yeast that appears as a large round organism. Malassezia are typically dark purple with Diff-Quik™ stain with a distinct capsule. The fine focus knob may need to be adjusted up and down to look for Malassezia since organisms can sit in different planes on keratinocytes.

Cocci Bacteria

Staphylococcus pseudintermedius is the most commonly isolated strain of cutaneous bacteria from dogs. In low numbers, it is considered a commensal organism. For this reason, it is important to quantify the number of bacteria present when reading cytology. On cytology, numerous sizes of round, basophilic cocci can be appreciated. Some strains of coccoid bacteria tend to form chains (Streptococcus sp.), while others group in clusters or can be found in pairs (Staphylococcus sp.).

Rod-Shaped Bacteria

Pseudomonas aeruginosa are rod-shaped bacteria that will appear basophilic with Diff-Quik™ stain. However, other rod-shaped bacteria, such as Escherichia coli and Klebsiella sp., can also occur. A bacterial C/S is necessary to truly determine the species. Gram-negative rods can become resistant quickly, so C/S should be considered if overgrowth is present.

Dermatophytes

Macroconidia are not found on cytology. This morphologic structure occurs only on fungal culture medium. On cytology, you may find fungal hyphae or spores amongst inflammation or debris. Dermatophyte spores and hyphae can be difficult to locate on cytology. Not finding dermatophyte organisms on cytology does not rule out dermatophytosis.

Acantholytic Keratinocytes

These are keratinocytes that have lost their intercellular connections deeper in the epidermis. They have a basophilic, centrally located nucleus. Often described as “fried eggs.” Most commonly noted with pemphigus foliaceus, especially if large numbers or “rafts” of acantholytic keratinocytes are present. However, low numbers of acantholytic keratinocytes have also been reported in cases of Trichophyton mentagrophytes and Staphylococcus pseudintermedius.

Melanin Granules

These are commonly mistaken for bacteria. Melanin granules can be plentiful on pigmented areas of the skin. Granules will be tan or brown and appear as small rods. You can appreciate refractile properties of melanin granules by adjusting the fine focus.

Conclusion

Do not underestimate the value of cytology! It is the most valuable tool in veterinary dermatology. Once you feel comfortable collecting and reading samples, you will be able to explain the value to clients and appropriately manage your dermatologic cases. Cytology everything!

References

1.  Bouassiba C, Osthold W, Mueller RS. Comparison of four different staining methods for ear cytology of dogs with otitis externa. Tierarztl Prax Ausg K Kleintiere Heimtiere. 2013;41(1):7–15.

2.  Udenberg T, Griffin C, Rosenkrantz W, et al. Reproducibility of quantitative cutaneous cytological technique. Vet Dermatol. 2014;25(5):435–367.

3.  Lo K, Rosenkrantz W. 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.

4.  Cafarchia C, et al. Occurrence and population size of Malassezia spp. in the external ear canal of dogs and cats both healthy and with otitis. Mycopathologia. 2005;160(2):143–149.

5.  Cafarchia C, et al. Frequency, body distribution, and population size of Malassezia species in healthy dogs and in dogs with localized cutaneous lesions. J Vet Diagn Invest. 2005;17(4):316–322.