How to prepare diagnostically useful cytology slides!  

Kristiina Ruotsalo

To maximize the diagnostic value of cytology submissions, it is important to submit optimally prepared smears along with a relevant clinical history. The goal of slide preparation is to obtain a monolayer of cells which can be adequately stained and evaluated microscopically, without excessive crush artefact or cell lysis. Helpful hints:

Þ Use good quality glass slides with a frosted end to record patient ID and collection site on each slide (e.g., Fluffy Smith, R leg mass). Use a pencil or ‘sharpie’ marker so that this information is not lost during slide staining.

Þ Have all the materials required close at hand (consider assembling a cytology sample kit) so that no time is lost between sample acquisition and slide preparation. In addition to slides, needles, and syringes, keep a few EDTA and serum tubes close by in case aspiration yields fluid material.

Þ Make sure that there is adequate ‘person power’ (or a small fan) available so that the slides can be quickly air dried immediately after they are made. Dry the slides briskly (think vigorous ‘jazz hands’ while firmly grasping the slides), to preserve cellular morphology. Slow drying results in cellular rounding artefact, making it difficult or impossible to assess morphology. Please refrain from blowing on the slides to dry them; this may add moisture to the preparation and distort cellularity. Store air-dried smears in cardboard or foam slide containers to protect them from environmental contaminants, moisture, and excessive temperatures.

Þ Do NOT heat-fix cytology or hematology slide preparations. Avoid exposure to formalin fumes, which will partially fix the cells and alter their morphology.

Þ Fine-needle aspiration (FNA) biopsies are done utilizing a 21-22 ga needle with or without a 5-12 mL syringe, depending upon whether an aspiration procedure or a capillary technique is used. For more detail:

Raskin & Meyer, Canine and Feline Cytology, 2nd ed.

Valenciano & Cowell, Diagnostic Cytology and Hematology of the Dog and Cat, 4th ed.

Following FNA, the harvested material needs to be applied onto slides immediately so that a monolayer is achieved and there is optimal cellular preservation. Samples containing blood or fluid are best spread using a blood smear technique (Fig. 1). Preparation of direct smears from concentrated cell buttons (sediment smears) following fluid centrifugation will maximize the number of cells available for cytologic evaluation while maintaining cellular integrity; this is particularly helpful if there are inherent delays related to sample transit to the laboratory, or for fluids such as urine when submitted for cytologic evaluation.

Semi-solid or solid material obtained by FNA is best spread using a ‘slide-over-slide’ technique (Fig. 2)illustrated below. When used properly, this is generally the best and most reliable method of preparing slides from FNA or scrapings of solid tissue, and my method of choice. The collected material is expelled near one end of the sample slide. A spreader slide is placed on top of and parallel to the sample slide. The aspirated material will generally spread out between the slides because of the weight of the spreader slide alone, and no additional pressure is typically required. If the sample is thick or granular, very gentle pressure may be applied to the spreader slide but do not ‘squash’ the slides together!! The spreader slide is then lightly drawn across the length of the sample slide. The end result should be 2 slides with material spread into a thin monolayer, and both are suitable for staining and evaluation.

Please do not spread aspirated material onto slides using the needle (i.e., NO ‘starfish’ preparations)! Similarly, material should not simply be sprayed onto the slides with no additional efforts at spreading the sample. Such preparations typically result in thick smears with numerous ruptured cells and a non-diagnostic sample.

Ulcerated or exudative superficial lesions are amenable to impression smears. Remember to remove excess blood or fluid exudate prior to sampling and gently press the slide to the lesion, rolling it off slowly, to ensure that a thin layer of cellular material remains. It is possible that these samples represent only the superficial aspect of the lesion, and further investigation may be required.

Material gathered from swabs (e.g., uterine or vaginal) should be gently rolled onto a slide immediately after acquiring the sample to prevent drying of cellular material. Please do not submit the swab to the lab for cytologic preparation as cellular material will have invariably been rendered uninterpretable because of drying.   AHL

Blood smear technique

Figure 1. Blood smear technique

'Slide-over-slide’ technique - do not squash!

Figure 2. ‘Slide-over-slide’ technique - do not squash!