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Examination and interpretation of cellular samples is a rapid, inexpensive, and minimally‐invasive tool that shelter practitioners can use to help establish a definitive diagnosis, establish a morphological diagnosis (e.g. mixed septic inflammation), or rule out differential diagnoses (Belford and Lumsden 1998). Cytologic samples can be obtained through a variety of techniques depending on the location or type of lesion and the preliminary or differential diagnosis, most commonly including fine needle aspiration (FNA); impression smears, imprints, or swabs; and skin scrapings. Diagnostic cytology can also be used as a component of the evaluation of fecal samples or bodily fluids such as urine, blood, effusions and secretions (Table 4.3). Once a sample is obtained, it is prepared for evaluation under a microscope. In general, for fine needle aspirates, impression smears, imprints, and swabs, identification and description of cellular characteristics or microbial agents are needed, and samples should be stained prior to evaluation. Identification of external parasites, such as Demodex spp., is usually performed on unstained preparations while evaluation of bodily fluids is often performed on both stained and unstained preparations. Romanowsky stains, such as modified Wright‐Giemsa and Diff‐Quik^® (Siemens Healthcare Diagnostics Ltd., Deerfield, IL), can be utilized for most cytologic preparations. Such stains contain both an acid and basic dye that bind to basic and acidic cellular structures respectively, allowing for visualization and recognition under the microscope (Rosenfeld and Dial 2010a). New methylene blue stain may be useful for preparations with heavy blood contamination, visualization of fungal agents, and analysis of urine sediment.

Table 4.4 ELISA technique variations (Tizard 2013).

Technique	Methodology	Detects	Examples
Indirect	Pre‐coated Ag + Test sample Ab + Enzyme‐linked Ab + Enzyme substrate	Ab in sample	TiterCHEK CDV/CPV (Zoetis, Parsippany‐Troy Hills, NJ) Canine VacciCheck® (Biogal Galed Labs, Kibbutz Galed, Israel) Feline VacciCheck (Biogal Galed Labs, Kibbutz Galed, Israel)
Sandwich	Pre‐coated Ab + Test sample Ag + Detection Ab + Enzyme‐linked Ab + Enzyme substrate	Bound Ag	DiroCHEK (Zoetis, Parsippany‐Troy Hills, NJ) SNAP tests (IDEXX Laboratories, Westbrook, ME) Solo Step testsa (Heska Corporation, Loveland, CO) VetScana (Abaxis, Union City, CA) WITNESS HEARTWORMa (Zoetis, Parsippany‐Troy Hills, NJ)
Labeled antigen	Pre‐coated Ag + Test sample Ab + Enzyme‐linked Ag + Enzyme substrate	Bound Ab	Not commonly used outside diagnostic laboratories in companion animals
Competitive	Pre‐coated Ab + Test sample Ag with Enzyme‐linked Ag + Enzyme substrate	Bound Ag/Inverse of Ag in sample	Not commonly used outside diagnostic laboratories in companion animals

Ag, antigen; Ab, antibody.

^a lateral flow assays.

Though detailed descriptions and definitive diagnoses may require the expertise of a cytopathologist, shelter practitioners should be able to classify cytologic preparations in order to obtain a preliminary diagnosis and establish the next steps in a diagnostic or treatment plan. Three general categories of lesions with distinct cellular characteristics have been described:

 Inflammatory: the presence of white blood cells greater than that expected from blood contamination

 Non‐inflammatory: non‐neoplastic lesion; cyst, sialocele, seroma, hematoma

 Neoplasia: homogenous cell population, characterized as benign or malignant.

Table 4.5 Inflammatory lesions and infectious differential diagnoses (Rakich and Latimer 2011; Rosenfeld and Dial 2010a).

Type	Cell Population	Infectious Differential Diagnoses
Purulent	Neutrophils (>85%)	Bacterial infection, typically Gram +
Pyogranulomatous	Neutrophils (>50%) Macrophages (<50%)	Fungal infections Foreign bodies Mycobacteria Protozoa
Granulomatous	Macrophages (>85%)	Fungal infections Foreign bodies Mycobacteria Protozoa
Eosinophilic	Eosinophils (>50%)	Parasites Pythium spp.
Lymphocytic	Lymphocytes (>85%)	Chronic bacterial infections Acute viral infections Protozoa

Most lesions of an infectious nature will be classified as inflammatory and can be further described as purulent, pyogranulomatous, granulomatous, eosinophilic, or lymphocytic depending on the predominant inflammatory cell type present. Such descriptions can narrow the list of potential causative agents (Table 4.5). Identification of an inflammatory cytologic lesion, particularly those with signs of degeneration, should prompt careful observation for microbial agents. Among those agents typically identified through direct microscopy are: bacteria, mycobacteria, superficial yeasts (Malassezia spp., Candida spp.), fungal organisms (Blastomyces, Cryptococcus, Histoplasma, Coccidioides, Sporothrix), fungal hyphae (Zygomycetes), parasitic oomycetes (Pythium spp.), protozoa (coccidia, Trichomonas spp., Leishmania spp.), and algae (Prototheca spp.) (Rakich and Latimer 2011).