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The ELISA is perhaps the most readily available and widely utilized diagnostic test in veterinary medicine. In general, ELISAs provide immediate results, are easy to use, cost‐effective, able to be conducted outside of a laboratory setting, highly accurate, and adaptable for the detection of a wide variety of analytes. In the shelter setting, ELISAs are commonly utilized for the detection of both antigen and antibodies to common pathogens (e.g. Dirofilaria immitis, Giardia spp., and feline leukemia virus [FeLV] antigen; Ehrlichia spp., Anaplasma spp., Borrelia burgdorferi, Leptospirosis spp., and feline immunodeficiency virus [FIV] antibody), assessment of antibody titers against common pathogens (e.g. canine distemper virus, canine parvovirus, canine adenovirus‐2, feline panleukopenia, feline herpesvirus‐1, feline calicivirus), and the detection of hormone levels for example, relaxin and luteinizing hormone (see Table 4.3). Test kits for the detection of metabolic biomarkers are also available. Anticoagulated whole blood, serum, or plasma can typically be utilized in point‐of‐care ELISA test kits depending on manufacturer instructions. Particularly as pertains to feline retrovirus (FeLV and FIV) testing, samples of saliva or tears have also been used in such test kits and while historically have resulted in an unacceptably low sensitivity, newer devices may be more accurate (Westman et al. 2016, 2017).

Table 4.3 Common diagnostic tests, indications, and collection and handling techniques for in‐house laboratories (Belford and Lumsden 1998; Bowman 2014; Rosenfeld and Dial 2010a, b; Zajac and Conboy 2012).

Diagnostic Test	Indications	Collection and Handling Techniques
Cytology
Fine needle aspirate	Evaluate mass lesions in the skin, subcutaneous tissue, thorax, and abdomen	(A) Insert a 21–25 g needle attached to a ≥5 ml syringe into the lesion; apply 3–5 ml of vacuum; move the needle forward and back while altering direction; release the vacuum and withdraw needle; (B) Insert a 21–25 g needle directly into the lesion; move the needle forward and back while altering direction; withdraw the needle.Remove needle (A) and aspirate 3–5 ml of air into the syringe; (A, B) attach the needle to the syringe and depress the plunger to deposit a drop of aspirated material onto the slide.
Impression smears, imprints, or swabs	Superficial lesions, otic cytology, surgical biopsy specimens	Ensure the sample site is clean and dry.Gently press clean glass slide on or rub cotton swab across the surface of the tissue.Roll cotton swab across a clean microscope slide.
Scrapings	Diagnosis of external parasites (e.g., Demodex spp.), pyoderma, cutaneous lesions, surgical biopsy specimens	Hold a sterile scalpel blade at a 90° angle to the surface of the tissue; scrape vigorously across the surfaceThe edge of a glass slide can also be utilized to obtain superficial surface samplesFor diagnosis of demodicosis, utilize a scalpel blade and scrape until capillary bleeding occursFor cutaneous lesions and surgical biopsy specimens, ensure the tissue is clean and dry prior to scraping
Immunoenzyme assays
ELISA	Antigen or antibody detection: infection or exposure (e.g., Anaplasma spp., Borrelia burgdorferi, canine parvovirus, Dirofilaria immitis, Ehrlichia spp., feline panleukopenia, feline leukemia, Giardia), antibody titer assessment (canine parvovirus, canine distemper, feline panleukopenia) Hormonal assays: luteinizing hormone, relaxin	Vary by assay; see individual techniques below and refer to test manufacturer guidelines
Multi‐well test kits (e.g. ASSURE®/FeLV, DiroCHEK®, TiterCHEK® CDV/CPV [Zoetis, Parsippany‐Troy Hills, NJ])		Anticoagulated whole blood, serum or plasma samplesaSamples can be fresh, refrigerated for up to 7 days, or frozen and thawedaStore test kits under refrigeration until the expiration dateAllow samples and test kits to reach room temperature before testing
Single‐well test devices (e.g. SNAP tests [IDEXX Laboratories, Westbrook, ME])		Anticoagulated whole blood, serum, plasma, or fecal samplesaSamples can be fresh, refrigerated up to 1 week, or frozen and thawedaAllow samples and test kits to reach room temperature before testingStore test kits under refrigeration until expiration date OR at room temperature for 90 days (or until expired)Test kits removed from refrigeration for more than 24 hours should be used within 90 days or until expired
Lateral flow assays (e.g., Solo Step® tests [Heska Corporation, Loveland, CO], VetScan® [Abaxis, Union City, CA], WITNESS® tests [Zoetis, Parsippany‐Troy Hills, NJ])		Anticoagulated whole blood, serum, plasma, or fecal samplesaSamples can be fresh, refrigerated for 3–7 days (up to two weeks for feces), or frozen and thawedaRun within 10 minutes of opening test pouchaStore test kits at room temperature until the expiration date
Fecal Examination
Direct fecal smear	Nematode larvae, protozoan trophozoites, heavyeggs, motile organisms	Refrigerate if not examined within 1–2 hours of collectionExamine within 30 minutes if evaluating for protozoan trophozoitesBreak up a small piece of feces in a drop of saline on a glass slide; use a coverslipFeces remaining on rectal thermometer after use is sufficientUse of water instead of saline can result in lysis of fragile organismsDried fecal smears may be stained though motility will be lost
Fecal flotation	Nematode eggs, cestode eggs, protozoan cysts	Approximately 1 teaspoon (4 grams) of fresh feces should be analyzedCentrifugation methods will recover more eggs, species with fewer eggs, and dense eggs; eggs will float in less time but additional steps are requiredGravitational methods are suitable for screeningSodium nitrate solution will float eggs in 10 minutes; immediate evaluation is necessary to reduce distortionSaturated sucrose solution will float eggs in 15–20 minutes
Hematology
Complete blood count	Minimum metabolic database	Use largest bore needle available to minimize hemolysisCollect directly in anticoagulant (e.g. EDTA) and promptly mixEnsure proper anticoagulant to sample ratio to minimize dilution of cell counts
Chemistry	Minimum metabolic database	Use largest bore needle available to minimize hemolysisCollect without anticoagulant; allow to clot for 15–20 minutes (serum)For small volume samples (e.g. puppies, kittens, exotics), collect with lithium heparin and centrifuge immediately (plasma)
Blood glucose	Hypo or hyperglycemia	Process whole blood sample within 30 minutes or separate serum and refrigerate until analysisStress, meals, anemia, sample handling and instrument variation can all lead to erroneous resultsPortable monitors are likely to slightly underestimate the concentration
Urinalysis
Specific gravity	Minimum metabolic database, dehydration, evidence of urinary tract disease	Use refractometer, reagent strip stick readings inaccurate
Reagent strips	Minimum metabolic database, dehydration, evidence of urinary tract disease	Note collection method; cystocentesis and catheterization preferred; midstream voided sample acceptableUse refractometer to measure specific gravity, strip readings inaccurateDrop urine onto each pad, do not dip the strip in the sampleAnalyze immediately or refrigerate for up to 6 hoursAllow refrigerated samples to reach room temperature prior to analysisLeukocyte, nitrite, and specific gravity readings unreliableProtect strips from air, light and moisture; mind expiration dates
Sediment analysis	Minimum metabolic database, dehydration, evidence of urinary tract disease	Note collection method; cystocentesis and catheterization preferred; midstream voided sample acceptableCollect 2–6 ml of urineAllow refrigerated samples to reach room temperature prior to analysisAnalyze within 4 hours or store under refrigeration up to 24 hoursCentrifuge sample for 5 minutes at 1,500 RPM, decant supernatant, resuspend sediment prior to analysisExamine stained and unstained sedimentExamine at 10x for crystals, casts, and epithelial cellsExamine at 40x for microorganisms, red blood cells, white blood cells
Culture	Evidence of urinary tract disease	Note collection method; cystocentesis and catheterization preferred; midstream voided sample acceptableUse a sterile collection method and storage containerAerobic culture is indicated in most circumstancesStore under refrigeration and transport to the diagnostic laboratory within 12 hours

^a Vary widely based on specific tests and kit manufacturer, follow manufacturer's product insert guidelines.

As the name implies, ELISAs are testing methods that rely on the detection of an enzyme‐linked protein within a variety of test samples to indicate either a qualitative or semi‐quantitative result. ELISAs can be direct or indirect, though the former are not routinely used outside of diagnostic laboratories. The basic operational principles of indirect ELISAs for antibody detection are as follows:

1 Test substrates are pre‐coated with the antigen by the manufacturer.

2 A biological sample containing the antibody to be detected is exposed to the substrate.

3 Antibody in the sample binds to the pre‐coated antigen creating an antigen–antibody complex. Unbound antibody is washed away.

4 An enzyme‐labeled antibody (i.e. conjugate) binds to the antigen–antibody complex. Unbound labeled antibody is washed away.

5 An enzyme‐substrate reacts with the enzyme‐labeled antigen–antibody complex, resulting in a color change indicating the test result.

In such tests, the intensity of color change is proportional to the amount of antibody present in the sample. Detection of antigen can be accomplished similarly, however, the well is pre‐coated with antibody allowing for binding and identification of antigen in the test sample.

A sandwich ELISA is a variation on the indirect ELISA commonly used to detect specific antigens. Many of the commercially‐available point‐of‐care test kits utilize this methodology in the form of manually activated bi‐directional flow devices (SNAP^® tests [IDEXX Laboratories, Westbrook, ME]) or lateral flow devices (also known as rapid immunochromatography). The former devices are largely automated and self‐contained; the benefits of which include minimization of background coloration, reduced washing time, and an increased opportunity for binding of the test sample to indicators (O'Connor 2015). Lateral flow assays generally offer more rapid analysis with fewer steps, lower operational costs, and greater environmental stability, however, they may be subject to decreased sensitivity and greater difficulty with interpretation of results, particularly in hemolyzed samples (O'Connor 2015; Sajid et al. 2015). Additional variations on the indirect ELISA include the labeled antigen ELISA and the competitive ELISA, see Table 4.4.