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).
4.3.2.3 Fecal Examination
A fecal examination is a primary tool for the diagnosis of endoparasitism in companion animals and typically consists of both direct fecal smears and flotation concentration techniques. (Note that fecal antigen detection for select pathogens is also commercially available as discussed in the section on ELISA testing found under primary diagnostic testing.) The direct fecal smear—microscopic evaluation of a small particle of feces mixed with saline—is ideal for the evaluation of delicate nematode larvae (e.g. Aelurostrongylus spp., Ancylostoma spp., Filaroides spp., Strongyloides spp.) and protozoan trophozoites (e.g. Giardia spp., Tritrichomonas spp.) (Bowman 2014) (Table 4.3). Direct smears also allow for the evaluation of organism motility and identification of organisms that are easily distorted by flotation solutions. Due to the small sample size evaluated, direct fecal smears have low sensitivity. That is, while a positive fecal smear can confirm endoparasitism, a negative fecal smear does not rule out infection. Fecal smears can also be stained for more detailed examination and parasite identification, though motility will be lost with such preparation (Zajac and Conboy 2012).
In most cases, direct fecal smears should be performed in tandem with fecal flotation techniques. Such techniques allow for the separation of fecal and food material, fats and dissolved pigments, and parasite eggs and cysts based on the density of the suspension liquid. Flotation techniques that involve centrifugation of the sample will result in higher sensitivity than those that rely on passive gravitational suspension of eggs. This is most important when low