ABSTRACT: “Is it a lipoma?” This is one of the most frequently asked questions from the vet to the nurse, who is looking at a fine-needle aspirate (FNA) down the microscope. The vet will have an inclination as to what the superficial mass might be, but a confirmation is essential before offering a definitive diagnosis to our clients.

While simple lipomas are benign in nature, they can be difficult to distinguish from infiltrative forms and liposarcomas, without microscopy.

Lipomas consist of adipose tissue and the supportive tissues from the region in which it is found. Any unusual cells seen can indicate that this is not a straightforward benign lipoma. Adipose tissue is made up of a large accumulation of adipocytes (fat cells), which naturally occur in the body and also store energy; but excess adipose tissue can result in a lipoma.

Fine-needle aspirates

There are a number of ways to sample a palpable mass, but I will concentrate on fine-needle aspirate techniques. Fine-needle aspirates are a simple, inexpensive way to diagnose ‘lump’ types, as long as they are performed correctly and the cells are examined by a competent person. This could be at an external laboratory or ‘in house’ by a confident, experienced member of staff – and especially for lipomas, a competent VN.

In the majority of cases, fine-needle aspirates can be taken from a conscious patient, although sedation may be required for fractious animals or if the lump is in an awkward location, such as in the axilla. The size of the lump also needs to be taken into account.

A single aspirate from a large mass will leave a lot of potentially diagnostic material behind; therefore, aspirates from various locations within the lump are recommended. It is also worth trying to get a peripheral and central aspirate. The following sampling and preparation techniques can be used on any lump that requires testing.

The main thing to remember is not to be afraid of your microscope, because it can be one of the most interesting and informative pieces of diagnostic equipment you will come across.

Sampling

The superficial mass can be clipped and cleaned, but this is not always necessary – an alcohol swab is sufficient. The mass is first located and then fixed. This can be performed on your own but having an extra pair of hands to keep the mass fixed in one place will help at this stage.

There are two main sampling techniques: either needle-only or suction.

Needle-only

This technique is designed to minimise the risk of dissemination of tumour cells, especially if sampling a suspected malignant mass.

A 21- or 23-gauge sterile needle is preferred, held between your thumb and index finger. Quickly introduce the needle into the mass and move it up, down, and side to side, but never exiting the skin. Spinning the needle at each new position will help extract the cells needed. Once you are confident that enough of the sample has been collected, withdraw the needle from the mass.

Using an air-filled 2.5 ml syringe, attach this to the needle and quickly expel the cellular material on to a clean, labelled slide. The material expelled may appear fatty in nature or alternatively creamy to blood-tinged in colour. Ideally the sampling process is then repeated using a fresh sterile needle.

The material collected is then prepared for examination (detailed later). If no cellular material is collected, it may be worth trying the suction method.

Suction

This method increases the risk of blood contamination and cell damage, but is often essential for firmer masses. This is the same technique as above except the needle is attached to a 2.5ml or 5ml syringe. Once the needle is in the mass, suction is applied by drawing back on the plunger of the syringe. Suction can be applied in two ways, either intermittent or continuous:

Intermittent

•   Place the needle into the mass, apply suction and then release

•   The needle can then be redirected within the mass, suction reapplied and released several times before withdrawing from the mass completely.

Continuous

•   Place the needle as above, although in this method suction is applied and maintained whilst the needle is moved around the mass

•   Once the suction has been released, you can withdraw from the mass

•   If you remove the needle whilst suction is being applied, the cellular material will be sucked into the syringe and you may not be able to expel anything onto a slide.

In both suction methods the syringe must be detached from the needle before depositing the sample on to a slide. This allows air to be drawn into the syringe without sucking the cellular material into the syringe itself.

Sample preparation

Once you have obtained the sample, it should be spread on to a slide, stained and examined. When spreading the aspirate material, it is crucial to have a monolayer so that the cells can easily be identified, and it is important not to damage the cells. As with blood films, only practice will make a good smear.

There are a number of methods of spreading your aspirate material.

Blood film technique

This technique is exactly the same as preparing a blood film, although the slide spreader should be advanced at a slower pace. This technique is very good for more liquid-based aspirates (Figure 1).

Figure 1: Example of a blood film technique; note the rounded edge to the finished smear

Line concentration

This is also similar to preparing a blood film. However, once the aspirate material has been advanced about two thirds of the way down the slide, the slide spreader should be removed. This leaves a line of concentrated cells at one end. This technique is really useful for fluids/lavages, especially if they have a low cellular content (Figure 2).

Figure 2: Example of the line technique; note the abrupt finish of the smear

Squash

This technique normally requires steady hands and lots of practice.

•   Hold slide A (the slide containing the aspirate material) at each end, between your thumb and index finger; or for the very unsteady, place slide A on a flat surface.

•   Using another clean, labelled slide (B), hold this at right angles over slide A

•   Keeping only one end (generally that closer to your hand) over the aspirate material, lower slide B onto the aspirate material and it will naturally spread out

•   
Do not apply any downward pressure once the aspirate has met slide B as the two slides may stick together, damaging the cells

•   Smoothly but briskly move slide B across slide A

•   The majority of the aspirate is now on slide B. However, it is advisable to stain both slides anyway (Figure 3).

Figure 3: Example of the squash technique, pull slide B across slide A

Star

This technique is minimally traumatic, but unfortunately rarely spreads the cells well. It can, however, be good for fluid aspirates, which are grossly turbid in appearance. Here you use the aspirate needle to drag the material into streaks radiating from a central point, hence a star-like appearance (Figure 4).

Figure 4: Example of the star technique

Staining

Aspirates taken from lipomas will appear oily and glisten prior to staining. Most practices use a water-based stain such as Diff Quik, Wrights, or New Methylene Blue. Use whichever staining technique you are used to, as this will be fine for most aspirate material. Do not stain material from any lump that has been sitting in formalin or expose aspirates to formalin fumes. If biopsy samples and aspirate slides are being sent to an external laboratory, they need to be sent separately.

Microscopy

Cytology and histology is an extensive subject, which cannot be covered fully in this article. However, using the following technique will make it easier to identify a lipoma.

Set up the microscope in the normal fashion. Once you have focused on the stained cells at x40 magnification, place a cover slip over the area and apply a drop of oil onto the cover slip, click in the x100 (oil) lens and focus onto the cells.

Most nurses can identify basic cells and also know about cell morphology. Briefly, the nucleus of cells stains purple. Inactive (resting, non-multiplying) cells have nuclei that are usually smooth and dark purple in colour. Active cells have nuclei that appear to have whiter, paler areas in them.

This can give a starting guide as to whether the cells within the lump are in a development stage or if they have stopped growing. If many nuclei are seen within a cell, this could indicate neoplasm.

In general, cells synthesise lipids in order to repair damaged membranes, but also as a means of storing excess energy. Most supporting tissues contain specific cells, which are adapted for storing fat, known as adipocytes (fat cells). As more lipids are accumulated, they fuse into one large lipid droplet.

Under the microscope, blue/purple stained cytoplasm in a circular shape and usually of similar size and appearance can be seen. Once the lipid droplets have fused together, they push the cytoplasm of the cell outwards to form an outer ring.

If the aspirate is lipid-based, you will find that during the staining process the lipids are removed and you will be left with an obvious unstained/clear area within the cell. The nucleus is also compressed and pushed to the side. If there are other cells present on the aspirate, then the lipid drops inside these cells will also appear as clear circles (Figure 5).

Figure 5: A sketched example of a FNA of a benign lipoma showing a washed away appearance to the cytoplasm; the occasional nucleus can be seen

Benign lipomas are slow-growing and usually feel soft and spongy. The skin will move freely over the mass and you may also find that it is not connected to fascia or muscle.

Infiltrative lipomas do exactly what they say: infiltrate local tissues, usually muscles. They are slow-growing and feel soft to firm but may also appear more aggressive in nature. They are rarely seen but because they invade the muscle, they can cause pain and result in lameness. Under the microscope an infiltrative lipoma may look very similar to a normal lipoma but the adipocytes are usually arranged in single rows between muscle cells. This row formation is only occasionally seen and, therefore, a definitive diagnosis is only made after a histological evaluation.

Liposarcomas are very difficult to diagnose, even with fine-needle aspirates. It may be easier to send a biopsy off to an external laboratory for histopathological evaluation. Most malignant lipomas will show different sizes of adipocytes (this can be normal in any lipoma though) with cells containing numerous nuclei of varying shapes and sizes.

Malignant liposarcomas have not been included, as these are not usually identified through FNAs.

With lots of practice and a good text book you will become more confident at identifying cells. If you find any unusual cells within the suspected lipoma, do not be afraid to ask. If you cannot say it is a simple lipoma at the outset, it is advisable to take a biopsy and send it for histopathological examination.

Enjoy practising your techniques on any lump that is removed. If you do not ask, it may never happen!

Author

Christine Jameison RVN MBVNA

Christine Jameison has been nursing for 12 years. She qualified in 2003 and carried out locum work for one year, gaining valuable experience. She enjoys all aspects of nursing, but is especially keen on laboratory work. Christine currently works on a part-time basis, having become a 'mum' for the first time!

References

BERGER, B. G., LATIMER, K. S., LEROY, B. E. and BAIN, P. J. Liposarcomas in the Dog and Cat College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7388.

YOUNG, B. and HEATH, J. W. Wheater's Functional Histology 4th Edition p74, Elsevier and Harcourt

• VOL 25 • No10 • October 2010 • Veterinary Nursing Journal