A Step-by-Step Guide to In-House Cytology in Veterinary Dermatology

Appropriate sampling techniques are key to obtaining diagnostic cytology.¹ The appropriate sampling method depends on the clinical concern (e.g., pruritus, inflammation, open lesions, alopecia, crusts, pustules, dry or moist areas) and the location and accessibility of the area, which will determine whether to collect the sample by swab, impression smear, tape preparation, skin scraping, or fine-needle aspirate (Table 1). If clipping or scissor-trimming the fur is necessary, avoid rupturing fragile pustules, which can compromise sample integrity and lose valuable diagnostic material (Box 1).

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Step 1: Gather Materials

Before beginning cytologic sampling, have the necessary supplies, depending on the type of sampling technique planned, readily accessible and organized. Having a dedicated drawer or station with all potentially needed supplies can be helpful.

• Scissors or clippers
• Frosted slides (labeled with patient information and sample site), glass coverslips, and mineral oil
• Cotton swabs, clear tape, scalpel blades, scraping spatula
• Needles (22 to 25 gauge), syringes (3 to 6 mL)
• Heat source (e.g., lighter)
• Small air dryer or fan
• Stain (e.g., Diff-Quik, new methylene blue)
• Microscope

Step 2: Base Appropriate Sample Collection Technique on Lesion Type and Location

Swab Smear

The swab technique is best used for ears, skin folds, and areas with discharge.

Ears: Obtain swabs from both ears, even if only 1 ear seems to be symptomatic.²

• Pull up the pinna, gently insert the cotton swab into the ear where the horizontal and vertical canals intersect, gently rotate the swab to lift out the debris, and roll the swab onto the slide (FIGURE 1A).³ Be consistent with how you apply the samples from each ear on the left or right side of the slide so there is no confusion as to which ear you are evaluating (FIGURE 1B).
• To look for mites, apply a small amount of mineral oil to the slide and the cotton swab, insert the swab into the ear canal, and lift out any exudate. Roll the swab into the mineral oil on the slide, distributing the exudate so it is not too thick. Apply a coverslip.

Figure 1A. Collecting a swab sample from a cat’s ear.

Figure 1B. Transfer the debris from the swab to the slide.

Skin lesions: A swab can be used to obtain the sample and rolled onto the slide.

• In collecting the sample, the swab can sometimes be moistened with sterile saline.¹
• Swabs can also be used to access samples from hard-to-reach areas (e.g., crevices, claws, lips, facial or tail folds, interdigital areas).

Direct Impression Smear

Direct impression smears are best used for moist, exudative lesions such as ulcers, pustules, blisters, or vesicles (FIGURE 2). Methods for obtaining direct smear samples depend on the type of lesion.

Open lesions: Press a slide firmly to the affected area.

Lesions with crust: Lift the crust and gently press the slide underneath.

Pustules or vesicles: Carefully puncture the lesion with a 25-gauge needle, aspirate the material, and spray it onto a slide or directly press the slide to the exudate. To distribute the cells more evenly, a second slide can be gently applied perpendicular to the first slide and slid along the length of it.¹

Figure 2. Collecting an impression smear from a chinchilla.

Clear Tape Preparation

This method is best used for dry, scaly, or alopecic areas and hard-to-reach areas (e.g., interdigital spaces, lip, facial folds).

Apply the tape firmly to the surface of the affected skin multiple times to lift superficial material (e.g., keratin, bacteria, yeast) without disrupting the skin. Either of the 2 staining techniques can be used (see Step 4: Stain the Slides).

Skin Scraping

Skin scrapings are best used for multiple types of lesions, ectoparasites, or deeper follicular conditions. Which procedure to use depends on clinical signs, the appearance/type of lesions, and the area (FIGURE 3).

• Use a dulled scalpel blade or a medical scraping spatula to retrieve material from an area under a crusted, exudative, seborrheic, or scaly lesion or a claw fold.¹
• Scrape in 1 direction, then smear the material onto the slide.^1,3
• If mites are suspected, apply oil to the slide and the dulled blade. Obtain the sample, mix in the oil, and apply a coverslip.
• Use a new slide for each area scraped; a sample that is too thick will be difficult to evaluate.
• For mites living deeper in the follicle (e.g., Demodex), squeeze the area to be scraped to bring any mites closer to the skin surface. Scrape in 1 direction, until a small amount of blood occurs, indicating you have gone deep enough to get material out of the follicles. Scrape all suspicious areas.
• When performing a superficial scraping, scrape a wider area superficially in multiple places.

Figure 3. Collecting a skin scraping from a dog’s leg.

Fine-Needle Aspirate

Fine-needle aspiration is best used for nodules, masses, or thickened areas (FIGURE 4). Fine-needle aspirates collect cells within the lesion that are helpful for differentiating between inflammation, infection, or neoplasia. The technique can be used with or without aspiration.

• For a sterile or clean site, clip fur and wipe the area with alcohol.
• Choose the needle (22 to 25 gauge) and syringe (3 to 6 mL).
• Stabilize the mass with your nondominant hand while using your dominant hand to hold the syringe.

Figure 4. Fine-needle aspiration of a mass.

For hemorrhagic or easily exfoliated lesions, use nonaspiration collection.

• Advance the needle into the lesion, withdraw partway, and redirect multiple times in different directions.
• Attach an air-filled syringe.
• Spray the cells gently onto a slide.

For nonhemorrhagic lesions, use aspiration collection (VIDEO 1).

• Attach the needle to the syringe, advance it into the lesion, pulling the plunger back and quickly releasing it.
• Redirect the needle and repeat in a few different areas of the lesion.
• Before withdrawing the needle, release the suction.
• Separate the syringe from the needle, fill the syringe with air, reattach the needle (bevel side down toward the slide), and expel the sample onto the slide (holding the hub of the needle if it is not a Luer Lock).

For both techniques, distribute the cells so they are not clumped in a single area by putting another slide on top of the slide with the sample and gently pulling them apart (VIDEO 2).

Step 3: Prepare and Fix the Slides

• After the sample is obtained, air dry the slides thoroughly, label them with the patient’s name and lesion location, and heat fix otic or greasy samples if desired.⁴
• Heat fix by holding a flame under the slide for 1 to 2 seconds, and wipe off any residue.

Note: One study that compared heat-fixed slides with air-dried slides from dogs with confirmed Malassezia otitis externa infection found no significant difference in yeast counts between the 2 methods, suggesting that heat-fixing is not necessary to adhere cells to the slide and may be safely omitted without compromising diagnostic accuracy.⁵

Step 4: Stain the slides

For dry slide preparations, Diff-Quik is commonly used for routine cytology, highlighting cellular detail with a fast processing time.

• Dip the slide in fixative for 5 to 10 seconds or drop in for 30 seconds. Thicker smears require longer processing times. Drain excess on an absorbent towel.⁴
• Dip into #2 and #3 stains for ten 1-second dips or up to 60 seconds each, draining in between.
• Rinse thoroughly with tap or distilled water, not rinsing directly over the area.
• Either air dry or use a low-heat dryer to speed the process.

Note: An evidence-based study in which mast cell granules were stained with the fixative at varying times (5 seconds, 30 seconds, 1 minute, and 2 minutes) found no significant difference in the quality, indicating that it is up to the individual to do what works for them to get the best quality and most accurate results. Change the stains on a regular basis to preserve the quality of the staining.⁶

For wet preparations, new methylene blue is mainly used and is ideal for identifying nuclear detail showing presence of bacteria.

Gram staining is used for differentiating types of bacteria, distinguishing gram-positive from gram-negative organisms, and is used more for guiding antibiotic decisions.

For tape preparations, 1 of 2 staining techniques can be used.

• Place the adhesive side of the tape on the slide and lift the edge of the tape, add 1 to 2 drops of either the #3 Diff-Quik stain or new methylene blue stain to the slide and reapply the tape.²
• Dip the tape into the #3 Diff-Quik stain, rinse, gently blot dry, and place the tape with a drop of immersion oil (adhesive side down) onto a slide. Look under the microscope within a short time.^1,4

Step 5: Evaluate the Slides

Determine the best area of the slide to evaluate by starting at low power (4×, 10×) and scanning for mites, acantholytic cells, or fungal spores. When looking for mites, increase the contrast by lowering the microscope condenser. For more detailed evaluation of inflammatory cells (e.g., neutrophils, eosinophils, lymphocytes, mast cells) and to identify microorganisms (e.g., cocci, rods, Malassezia; FIGURE 5), increase the power to 40× and 100× (immersion oil). Look for intracellular bacteria (indicating a true infection) and be able to differentiate artifacts, such as melanin granules, from microorganisms and nuclear streaming, which can be caused by poor sample and slide handling.⁷

Figure 5. Malessezia and bacteria on a slide. Diff-Quik stain, 100× magnification.

Step 6: Document and Communicate Findings

Record results clearly and consistently in the patient’s record, and discuss the results with the veterinarian to help guide decisions for future testing and treatment.

Summary

In-house cytology empowers veterinary teams to obtain rapid, cost-effective diagnostic information that will help guide immediate treatment decisions, determine the need for further testing, and support timely follow-up care. The accuracy of cytologic interpretation depends on proper sample collection, handling, staining, and the ability to distinguish true cellular and microbial findings from artifacts. An educated veterinary nurse who is able to perform cytology confidently and competently can improve diagnostic accuracy and efficiency and can contribute to improved patient outcomes.