A minimum of equipment is required, the most basic being a microscope, glass slides, syringes, needles, and stain. More sophisticated equipment and ultrasound guidance may be required to obtain less accessible samples such as bone marrow, cerebrospinal fluid, internal organs, deep masses, and bile. Stains commonly used to examine blood smears, i.e. Romanowsky-type stains, such as Wright-Giemsa and Hema 3 (formerly Diff-Quik), are used for cytologic examination. Many practitioners focus on obtaining good quality samples and preparing good smears to submit to a diagnostic laboratory for analysis. The quality of the result and interpretation is heavily dependent on the quality of the specimen. Smears that are being submitted to a diagnostic laboratory should be air dried only and left unstained. Cytology smears should not be exposed to formalin fumes (i.e. should be kept away from open formalin containers and not shipped with formalin-fixed tissues) or excessive humidity.

Fine Needle Samples

Superficial masses and lymph nodes may be sampled by using a fine needle aspirate (FNA) or fine needle nonaspirate (FNNA) technique. Although not always required, ultrasound guidance can be very useful in the fine needle sampling of organs and deep masses. Nothing larger than a 22 gauge needle should be used; larger needles cause significant trauma and can result in a blood contaminated sample that is nondiagnostic. This rule also holds true for samples obtained from large animals, where there is often a temptation to use larger gauge needles.

For FNA, a syringe is attached to the needle to provide suction (Video: Fine Needle Aspiration and Smear Making). The size of the syringe depends on the tissue to be aspirated- a 3ml syringe is appropriate for softer tissues such as lymph nodes, but a larger syringe (up to 20ml) is appropriate for firm tissues requiring more suction to collect an adequate sample of cells. A 12ml syringe is appropriate if the texture of the tissue is not known. Do not expect to see material entering the syringe when aspirating solid masses or lymph nodes. After aspirating and redirecting the needle 2-4 times in the mass, the suction is released, and the needle and syringe unit is removed. The needle is disconnected from the syringe, and the syringe is filled with air and reconnected to the needle. Then, holding the needle and syringe unit in a near vertical position, the material is quickly sprayed out near the frosted end of the slide. The aspirated material is then smeared out, either using a blood smear technique, or a slide over slide technique (holding the spreader slide at right angles to the sample slide and gently smearing the material out; Video: Fine Needle Aspiration and Smear Making). Great care must be taken not to apply too much pressure when making the smear or to create suction when lifting the spreader slide, particularly with lymph node preparations, as lymphocytes tend to be fragile and rupture easily, especially if neoplastic.

Ideally, several smears are made from more than one or two regions of the mass, depending on the size and heterogeneity of the mass. If a large amount of fluid is aspirated, it is recommended to make smears from the fluid, but also to perform a second sampling of the mass targeting the wall of the mass or any more solid areas. Fluid can be put into an EDTA tube and submitted to the lab, where concentrated preparations can be made using the cytospin centrifuge.

For FNNA, only a needle, without a syringe for suction, is used (Video: Fine Needle Nonaspiration). Once the needle has been redirected a few times within the mass, it is withdrawn and an air-filled syringe is attached to spray the material out onto a slide. The smearing and staining procedure is the same for both types of samples. It is largely the veterinarian’s preference which method is used, although it has been suggested that FNNA may help to limit blood contamination and rupture of cells.

Impression Smears

Impression smears (also called touch preparations or touch imprints) can be made from ulcerated cutaneous masses and masses or tissues that have been surgically removed. This is done by gently pressing the ulcerated or fresh cut surface to the slide, taking care not to damage the cells by using excessive pressure or “wiggling” the tissue (Video: Impression Smear Making). Keep in mind that superficial, ulcerated masses often have significant secondary inflammation and bacterial contamination. Fine needle aspiration from below the superficial aspects of the mass may be required to obtain a diagnostic sample. Tissues should be well blotted, even if not visibly oozing fluid or blood, before making impression smears. This is particularly true when smears are being prepared from biopsies of spleen or liver. Biopsy samples that are firm or fibrous may not exfoliate well. Scraping the surface with a scalpel blade or scoring the surface with the blade can loosen cells and facilitate exfoliation when making impression smears.

Fluid Samples

Fluid samples should always be collected into an EDTA tube. Even samples that contain no visible blood may clot without the addition of anticoagulant, rendering them useless. EDTA also helps to preserve cell morphology and limit growth of bacteria. If culture of the fluid is desired, some fluid should be collected into a red top tube as well. Direct smears from the EDTA tube should be made immediately after collection (Video: Cytology Fluid Handling). Line preparations can also be made with cell-poor fluids; instead of making the smear like a blood smear, the spreader slide is lifted abruptly leaving a line rather than a feathered edge. This provides a strip of concentrated cells at the end of the smear. If submitting to a reference laboratory, the EDTA tube of fluid should be sent along with the smears. Samples that appear to be cell-poor (i.e. are thin, watery, and/or transparent) can be centrifuged at low speed (500 rpm) to concentrate the cells before making smears of the sediment. Most diagnostic laboratories also concentrate cells from fluid samples using a cytospin centrifuge (Video: Cytology Fluid Handling).