ABSTRACT: The aim of these articles is to provide veterinary nurses with a basic overview of some cytological sampling and preparation techniques and provide a few pictures of common cells seen in cytology. The field of cytology is far too vast to cover completely but following a few simple procedures nurses can aid the veterinary surgeon in rapid diagnosis.

Cytology can be very useful in diagnosing certain conditions as well as ruling out others and narrowing the veterinary surgeons list of differential diagnoses. It can be quick and is minimally invasive; and for superficial masses or fluid samples can sometimes be obtained without sedation, depending on the nature of the patient, thereby reducing the need for a more risky general anaesthetic.

Sampling techniques

Impression smears

This technique can be readily used to diagnose inflammatory lesions, such as eosinophilic granulomas; but with ulcerated skin masses, deeper aspiration should be obtained to provide further information on possible causes.

Impression smears can also be taken from moist skin structures such as the mouth, eyelids and the vagina. To obtain an impression smear of an accessible area, a clean dry microscope slide is placed over the site, allowing the material to naturally stick to the slide.

Care should be taken when diagnosing conditions using impression smears, as there is often a lot of debris on the slide and deeper and often more significant material is not seen.

Impression smears can be made of areas, such as the external ear canal, using a swab or cotton bud moistened with saline. The swab is rubbed in the area and then very gently rolled on to a clean microscope slide. Care should be taken not to drag the swab across the slide, as this can damage the cells.

Both of these preparations should then be left to air dry before staining. The common method of staining in practice would be ‘Diff-Quick’, which is especially good when performing ear cytology.

Common ear cytology

As nurses, ear cytology may be the most common cytology performed and can be done while the vet has the client in the consulting room. Ear cytology is done to identify infectious agents and can direct the vet to what treatment is best. The most common cells seen on ear cytology are squames, which are superficial epithelial skin cells that have either no nucleus – or a very small purple-staining one – within a smooth turquoise cytoplasm.

Ear mites can be seen with the naked eye, but should be identified under the microscope. Malassezia yeasts are next on the list of differentials, and are identified as circular blue/purple-staining structures which are often budding and look like ‘footprints’.

Bacteria can also be identified – both cocci (small blue/purple-staining dots in pairs or chains) and rods (small blue/purple-staining ‘rice grains’). If bacteria are seen, then a culture should be performed; but typically bacterial cocci are Gram positive and rods are Gram negative, and this can aid the vet as to which antibiotic to use. 

Fine-needle aspiration

Fine-needle aspirations (or FNAs) are used to help diagnose superficial or palpable masses. There are various ways to collect and prepare samples ensuring that the harvest of cells is left intact and diagnostic.

Firstly, the area should be clipped and prepared aseptically, especially if a bacterial culture is required. This prevents introducing skin flora to the mass. The mass should then be fixed in place with one hand and then, using one of the techniques below to harvest the cells.

Needle only

A 21G or 23G needle should be used, because larger gauge needles are less likely to damage the cells. It is also best to fill a 2.5ml or 5ml syringe with air, as this will be needed later. Gloves should be worn to maintain sterility.

The needle is then held between the thumb and index finger and quickly introduced to the mass and moved to and fro around the mass but without exiting the skin. Rotating the needle can help obtain a better harvest of cells. The needle is then removed and placed on to the air-filled syringe, which is then used to expel the cells on to a clean and labelled microscope slide. The material is spread (see preparation section) into a thin layer and air dried.

Suction and a needle

This technique is exactly the same as above, but when the needle is in the mass, suction is applied using a 2.5ml syringe. The suction could be either intermittent (when the needle is moved suction is applied and released), or continuous where suction is applied as the needle is moved around the mass. 

Care should be taken not to apply suction while the needle is removed from the mass, as this could contaminate the harvest with skin flora or the harvest of cells may be lost into the syringe.

The harvest of cells is expelled on to a labelled microscope slide and left to air dry.

Preparation of sample

The preparation of the sample is as important as taking the harvest. The cells should be smeared gently in to a thin layer (or monolayer) as anything too thick will prove non-diagnostic. Each method has its ‘pros’ and ‘cons’, and the method chosen will largely depend on the operator’s strengths.

•   Blood film method – as the name suggests, this method follows the same approach as making a blood smear (Figure 1). It is particularly good for fluid samples or blood-contaminated samples. The slide should be left to air dry before staining.

Figure 1: The blood film method

•   Line concentration – is exactly the same as the blood film approach, but half way through the ‘smearing process’, the spreader is stopped, leaving a line of concentrated cells.

The smear is then left to air dry. This can be very useful in fluid which can have very low cell counts such as bronchoalveolar lavage (BAL) samples and other wash preparations, such as nasal flushes and prostatic washes.

•   Squash method – requires very steady hands, as it is very easy to stick the slides together. Place a small amount of sample on a labelled microscope slide, then hold another clean labelled slide at a right angle, with one end of the slide over the droplet of material (Figure 2). Now lower the second slide on the first. As it touches, the material will gently spread out. Without applying any pressure, gently and smoothly pull the slide across the droplet. Too much pressure will stick the slides together and damage all the cells.

Figure 2: The squash method

Staining

There are many different stains available and most practices will have ‘Diff-Quick’ on the shelf. As an external laboratory, we routinely use Modified Wright’s stain as it is a good cytology and haematology stain. The most important thing is that the slide is correctly and completely stained, because incorrect staining can lead to a mis-diagnosis.

Figures 3-5 show how properly stained slides can help accurate diagnosis in cases where neoplasia is suspected.

Figure 3: Mesenchymal cell seen in neoplasias, such as fibrosarcomas

 Figure 4: A mast cell

Figure 5: A mitotic cell

I hope this article will aid nurses in the preparation and the examination of basic cytology samples. In a future article, I shall explain how to prepare body cavity fluid samples, including cell counts and sediment slides.

The field of cytology is too vast to cover fully but I hope this article has whetted your appetite to do some further reading. 

Author

Matthew Garland CertNat Sci(Open) VN MBVNA

After qualifying as a VN in 2004, Matthew worked in small animal practice before moving to Torrance Diamond Diagnostic services in 2006. Now, as laboratory manager of TDDS-Ringwood, he has developed a strong interest in haematology and biochemistry.

To cite this article use either

DOI: 10.1111/j.2045-0648.2012.00174.x or Veterinary Nursing Journal Vol 27 pp 187-188

Further reading

BSAVA Manual of Clinical Pathology (Second Edition) Edited by Elizabeth Villers & Laura Blackwood BSAVA, Gloucester

RASKIN. R E. and MEYER. D. J . Atlas of Canine and Feline Cytology by Published by Saunders.

• VOL 27 • May 2012 • Veterinary Nursing Journal