Sample collection and smear preparation
Cytology samples can be collected from solid lesions by several techniques including:
- Aspiration cytology – fine needle aspiration (FNA): Ideal for cutaneous or subcutaneous masses since it avoids surface contamination.
- Non-aspiration method: Samples often of equal or better quality than those obtained with the aspiration method (see below). Works well with readily exfoliating tissue such as lymph nodes or when the mass is highly vascular or the cells are fragile.
- Aspiration method: Needed in poorly exfoliating masses.
- Exfoliative cytology:
- Impression smears: Useful in exudative skin lesions and preparation of cytology smears from biopsy specimens. Smears may yield only surface contamination and may not be representative of the lesion. In preparing impression smears from biopsy specimens it is critical to make smears before exposure of the tissue to formalin to avoid staining artifacts from formalin fumes.
- Tissue scrapings: Indicated in flat skin lesions that cannot be easily aspirated or on biopsy specimens that are poorly exfoliating. Hints:
- Make and submit multiple slides, “spreader” slides can also be stained or submitted.
- Stain and examine a slide prior to submission to a laboratory to ensure adequate cellularity of the sample, especially if the animal is still in the hospital or under sedation/anesthesia.
- Aspirate solid areas of cystic masses or masses with soft fluctuant areas as the fluid filled areas will often only contain poorly cellular fluid, blood or necrotic debris.
- Be gentle! Use gentle pressure when preparing squash preps and impression smears as cells, particularly neoplastic cells, can rupture easily.
- Avoid thick smears as cells will be impossible to identify if they are not well spread. Using only a small drop of sample will help, especially from tissue that readily exfoliate (ex. lymph nodes) or if peripheral blood contamination has occurred.
Most stains used in veterinary medicine are Romanowsky-type stains such as the Diff-Quik® stain. Slides being stained in-clinic or being submitted to a reference laboratory should simply be air-dried; prior fixation is not needed (including heat fixation) and may interfere with staining quality. In general follow the recommendations of the manufacturer for staining procedure, though the following hints may be of use:
- Shorten staining times for smears that are thin or of low protein content, similarly thick smears or those with high protein may need longer staining times.
- Ensure stain reagents are fresh and well filtered, with time and repeated use precipitates may form in the stain making interpretation difficult, the stain may ‘fatigue’ leading to understaining, and organisms may contaminate the reagents.
- Ensure slides are well dried before staining, water may still be present when the slide appears dry, use a hair dryer or leave for sufficient time for adequate drying.
Note: Some mast cell granules will not stain with Diff-Quik making it easy to mistake mast cells for other round cells or macrophages.