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How to perform cytology for fluid analysis in the emergency room | VETgirl Veterinary Continuing Education Videos

In this VETgirl online veterinary continuing education video, we review how to perform sample preparation for cytologic evaluation of body fluids in house. Cytology is an important clinical tool that veterinary professionals must feel comfortable performing, especially in the emergency room setting. While stable patients can have fluid analysis submitted to a veterinary diagnostic laboratory for analysis, emergency cytology often needs to be performed to investigate the presence of acute abdomen (e.g., septic peritonitis, uroabdomen, etc.). Cytologic evidence of septic suppurative inflammation (confirmed by seeing bacteria within neutrophils), plant material, bile pigment, etc., warrant immediate surgical intervention.

To be prepared, effusions should be collected into both purple (EDTA) and red top tubes. EDTA preserves cells and is used for cytologic evaluation, while a red top tube can be used for bacterial culture or chemistry tests on the fluid, such as creatinine, bilirubin, lactate, or glucose, if indicated. A red top tube is also helpful to look for clot formation, which differentiates in vivo hemorrhage (no clot formation) versus tap-related blood contamination (clot formation). EDTA inhibits bacterial growth, therefore it is important to not submit an EDTA tube for culture. Once body fluid has been obtained, initial gross evaluation of the fluid should be performed (e.g., color, turbidity, viscosity). The turbidity will give you a sense of the fluid’s cellularity. For example, fluids that are clear are likely a low cellularity (<3,000 nucleated cells/ul), whereas opaque or flocculent fluids are likely of high cellularity (>30,000 nucleated cells/ul). Next, direct smears of the fluid should be prepared as soon as possible. If the fluid sits for >30 minutes, cells can phagocytize bacteria and red blood cells in vitro, which complicates the diagnosis of septic inflammation and hemorrhage, respectively. If the fluid is of high cellularity, direct smears will suffice. However, if the fluid appears to be of low cellularity, centrifugation to form a sediment smear should be utilized to aid in cell concentration for cytologic evaluation. While the best way to prepare fluids with <3,000 cells/ul is via cytocentrifuge preparation (a specialized centrifuge), this is not readily available in private practice and standard centrifugation can be sufficient, and is actually the preferred method for fluids with nucleated cell counts between 3,000 and 30,000 cells/ul. Fluids are spun for 3 minutes at about 3,750 rpm. After spinning, the total protein should be measured on the supernatant using a refractometer. Transudates will have a TP 2.5 g/dL. Next, the supernatant should be removed by gently pouring it out, leaving a cellular pellet at the bottom of the tube with a small amount of residual supernatant fluid. A clean pipette should be used to resuspend the supernatant with the cells. Once mixed, apply a drop of this fluid onto multiple slides. Then, take another slide to perform a “squash prep” smear (similar to how you would spread a fine needle aspirate cytology sample). The smears should then be rapidly air dried using a hair dryer on the underside of the slides. In general practice, the use of Diff-Quick stain (e.g., Romanowsky) can be used, but ideally a “clean” stain should be used (e.g., not the Diff-Quick stain set that you use for fecal smears and ears).

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  1. How likely is bacterial contamination of your sediment from the centrifuge test tube and pipette if they are not sterile, I always use a sterile red tube for the centrifuge and transport the sediment using a sterile syringe, is this ok practice?

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