The art of the aspirate

By Jamie Morgan, MAEd, CVT, RVT, CPEP, FFCP

Fine-needle aspiration (FNA) is one of the most valuable diagnostic tools available in small animal practice. It is fast, inexpensive, minimally invasive, and can provide diagnostic information within minutes. The ability to perform an FNA, prepare quality slides, and recognize common errors can dramatically improve the likelihood of obtaining a diagnostic sample.

Early tumor detection is especially important in veterinary medicine because many neoplastic processes are subtle in the early stages. A small cutaneous mass, an enlarged lymph node, or an oral lesion may appear benign on initial examination; however, early cytologic evaluation can identify inflammatory lesions and malignant neoplasia before it progresses further.

Why it matters

"Every lump gets aspirated" is a common phrase in veterinary practice. The size and appearance of a mass do not reliably predict whether it is benign or malignant. FNA offers several advantages, including:

• Minimal patient discomfort
• Usually no need for sedation
• Rapid sample collection
• Low cost when compared to tissue biopsies
• Ability to evaluate multiple lesions
• Useful for skin masses, lymph nodes, internal organs, and body cavity lesions

FNA is not intended to replace histopathology in every case. Some tumors do not exfoliate well, and cytology may not provide a definitive diagnosis. However, a properly collected aspirate frequently provides enough information to determine whether a lesion is inflammatory, cystic, benign, or suspicious
for malignancy.

Indications for performing an FNA

So, which lesions are appropriate for aspiration? Common indications include:

• Any new skin or subcutaneous mass
• Enlarged peripheral lymph nodes
• Persistent swellings or nodules
• Splenic, hepatic, or other internal lesions identified on imaging
• Fluid-filled structures or cystic masses

A general guideline in practice is that any mass present for more than one month, or any mass larger than 1 cm, should be aspirated. Smaller lesions (less than 1 cm) may warrant aspiration if they are rapidly changing in size, shape, or texture.

Patient preparation

Most cutaneous and subcutaneous masses can be aspirated with little restraint and no sedation. Calm handling is important because excessive movement may cause improper sample collection and increased patient anxiety.

Cutaneous and subcutaneous areas should generally not be aggressively scrubbed and prepped. Doing so may alter the cytology sample and make the lesion difficult to palpate. If a lesion is visibly dirty, a small amount of alcohol can be used to clean the surface. Hair clipping may be necessary if the mass is obscured by a thick coat.

If microbiologic tests are to be performed or if a body cavity is to be penetrated (e.g., the peritoneal and thoracic cavities and joints), then the area of aspiration should be surgically prepped.

Before starting, the lesion should be palpated and the following noted:

• Size
• Shape
• Consistency
• Mobility
• Whether it feels fluid-filled or firm
• Any pain elicited

This assessment helps determine which technique is
most appropriate.

Collection techniques

There are two primary FNA methods used in veterinary practice: the aspiration technique and the non-aspiration, or capillary, technique. The type utilized depends on the type of lesion.

Aspiration technique (Figure 1)

The aspiration technique uses negative pressure from a syringe to draw your sample into the needle. Steps include:

1. Stabilize the mass firmly with one hand.
2. Attach the needle to the syringe.
3. Insert the needle, with syringe attached, into the center of the lesion.
4. Pull back on the syringe plunger to approximately three fourths of the volume of the syringe to create negative pressure.
5. While maintaining negative pressure, redirect the needle several times within the mass without exiting the skin.
6. Release negative pressure before withdrawing the needle.
7. Remove the needle from the syringe.
8. Fill the syringe with air, reattach the needle, and expel the sample onto a clean microscope slide.

The aspiration method is often useful for:

• Firm masses
• Fibrous lesions
• Lymph nodes
• Some internal organs

It's important to note too much negative pressure may cause excessive blood contamination, particularly in vascular tissues and some soft tissue tumors.

Non-aspiration (capillary) technique

The non-aspiration technique relies on capillary action rather than negative pressure. Steps include:

1. Stabilize the mass firmly with one hand.
2. Insert the needle (typically 22-gauge) into the center of the mass without attaching a syringe or removing the plunger from the syringe to facilitate handling (Figure 2).
3. Redirect the needle several times within the lesion (Figure 3).
4. Withdraw the needle.
5. Attach an air-filled syringe.
6. Expel the material onto a slide.

   

   

This method will often provide a cleaner sample with less blood contamination. It is especially useful for:

• Highly vascular masses
• Soft or friable tumors
• Thyroid masses
• Splenic lesions
• Mast cell tumors

Clinicians often prefer the capillary technique for superficial skin masses because it frequently provides better cellular detail. It is important to note this technique generally yields a small sample, so the procedure may need to be repeated to ensure adequate slide numbers and to evaluate the
mass areas.

Common mistakes in FNA collection

Even experienced staff occasionally obtain non-diagnostic samples. Recognizing common mistakes helps technicians improve success rates.

Using too much suction

Excessive negative pressure often results in slides that contain mostly blood. Red blood cells can obscure diagnostic cells, making interpretation difficult. If repeated samples contain blood, switch to a smaller needle or use the capillary technique.

Failing to release suction before withdrawing the needle

If the syringe plunger is pulled back to create negative pressure when the needle exits the mass, the collected material may be aspirated into the syringe barrel rather than remaining in the needle hub. This can significantly reduce the amount of sample available for slide preparation and interpretation.

Proper slide preparation

Collecting a diagnostic sample is only half of the process. An excellent aspirate can become unstable if the slide is
prepared incorrectly.

Immediately after sample collection, the material should be expelled near one end of a clean glass slide. The sample should then be spread on the slide using an appropriate technique based on the sample consistency.

Squash or compression technique (Figure 4)

This method is commonly used for thicker or more particulate samples and can yield excellent cytologic smears.

1. Place a small amount of the sample in the middle of the slide.
2. Gently place a second slide (spreader slide) over the aspirate horizontally, at a right angle to the first slide (the prep slide).
3. The spreader slide is then quickly and smoothly slid across the prep slide.

The slides should never be pressed downward forcefully. Excessive pressure ruptures cells and creates artifacts.

Line smear technique (Figure 5)

When a sample is low in cellularity, the line smear technique may be used to concentrate cells.

1. Place a small drop near one end of the slide.
2. Using the same technique as a blood smear, pull the spreader slide backward into the sample.
3. Push the spreader slide forward smoothly to create a thin film.
4. About three-fourths of the way through the smear, raise the spreading slide directly upward – this creates a line that contains a much higher concentration of cells than the rest of the slide.

   

This technique works well for fluids of low cellularity, but it often does not sufficiently spread cells from highly cellular fluids. In general, translucent fluids are of low to moderate cellularity, whereas opaque fluids usually have high cellularity.

Starfish technique (Figure 6)

The starfish technique is useful for thick, sticky, or highly cellular samples that do not spread well. This technique tends not to damage fragile cells, but does allow a thick layer of fluid to remain around the cells. This can help prevent the cells from spreading and interfering with the evaluation of cell detail.

1. Place the aspirate sample in the center of the microscope slide.
2. The tip of a needle is placed in the aspirate and moved peripherally to pull a tail of the sample with it.
3. Repeat the procedure in several directions, resulting in multiple projections.

This method often preserves cellular detail in samples such as mast cell tumors or sebaceous lesions.

Air drying and staining

Slides should be air-dried immediately after preparation. Avoid blowing on the slide or waving it in the air, as doing so can introduce contaminants or distort cells. Heat should also be avoided. Hair dryers and warming devices can create artifacts that interfere with interpretation.

Once dry, the slide can be stained using a cytology stain (Figure 7). Many stains incorporate a cellular fixative. The preferred fixative for FNA specimens is 95 percent methanol. Prepared slides should remain in fixative for at least two to five minutes before staining.

Proper staining technique is important. Each stain has a unique recommended procedure. These procedures should be followed in general but should be adapted to the type and thickness of the smear being stained. The thinner the smear, the less time the stain needs. The thicker the smear, the more time needed in the stain.

Under-stained slides may appear pale and difficult to interpret; overstained ones may be too dark, obscuring cellular details.

Conclusion

FNA is a simple procedure. When performed with care and precision, it provides rapid insight into the nature of a mass and supports early, informed decision-making. Mastering this skill is an opportunity to directly impact patient outcomes.

Every well-prepared slide represents more than just a sample—it represents an opportunity to detect disease earlier, guide treatment, and improve quality of life for our patients.

Jamie Morgan is a veterinary technologist certified in both Illinois and North Carolina. She has more than 20 years of teaching experience in AVMA-accredited veterinary technology programs. Sample collection by aspiration technique.