Urinalysis is an important laboratory test that can be readily performed in veterinary practice and is considered part of a minimum database. It is useful to document various types of urinary tract diseases and may provide information about other systemic diseases, such as liver failure and hemolysis. Urine may be collected by cystocentesis, urethral catheterization, or voiding. Urine should be evaluated within 30 min, but if this is not possible, it may be refrigerated for up to 24 hr or submitted to an outside diagnostic laboratory; however, this may result in crystal precipitation. Refrigeration does not alter urine pH or specific gravity. If cytologic evaluation is required and a delay in preparing the slides is anticipated, then a preservative should be added. A few drops of 10% formalin (exact concentration not necessary) is better than boric acid. However, the usual Romanowsky stains are unsuitable with formalin; if submitting to an outside laboratory, it should be communicated that formalin has been added.
Normal urine is typically transparent and yellow or amber on visual inspection. The intensity of color is in part related to the volume of urine collected and concentration of urine produced; therefore, it should be interpreted in context of urine specific gravity. Significant disease may exist when urine color is normal. Abnormal urine color may be caused by presence of endogenous or exogenous pigments, but it does not provide specific information. Interpretation of semiquantitative reagent strips, which are colorimetric tests, requires knowledge of urine color because discolored urine may result in a false-positive result. Equine urine may turn brown after a period of time.
Normal urine has a slight odor of ammonia; however, the odor depends on urine concentration. Some species, such as cats and goats, have pungent urine odor because of urine composition. Bacterial infection may result in a strong odor due to pyuria; a strong ammonia odor may occur if the bacteria produce urease.
Urine must be at room temperature for accurate measurement of urine specific gravity and for chemical analysis. These tests are usually done before centrifugation; however, if urine is discolored or turbid, it may be beneficial to perform these tests on supernatant (see Urine Sediment Urine Sediment Urinalysis is an important laboratory test that can be readily performed in veterinary practice and is considered part of a minimum database. It is useful to document various types of urinary... read more ).
Specific gravity is defined as the ratio of the weight of a volume of liquid to the weight of an equal volume of distilled water; therefore, it depends on the number, size, and weight of particles in the liquid. It is different from osmolality, which depends only on the number of particles in the liquid; measurement of osmolality requires specialized instrumentation.
The urine specific gravity (SG) is determined using a refractometer designed for veterinary samples, which includes a scale calibrated specifically for cat urine. SG for species other than cats should be determined using the scale for dogs. In healthy animals, SG is highly variable, depending on fluid and electrolyte balance of the body. It is normally at least 1.015. Interpretation of SG, therefore, depends on the clinical presentation and serum chemistry findings. (See also Urinary System Introduction Urinary System Introduction read more .) An animal that is dehydrated or has other causes of prerenal azotemia will have hypersthenuric urine with an SG >1.025–1.040 (depending on species). Dilute urine in a dehydrated or azotemic animal is abnormal and could be caused by renal failure, hypo- or hyperadrenocorticism, hypercalcemia, diabetes mellitus, hyperthyroidism, and diuretic therapy. In cases of diabetes insipidus, values <1.010 can be expected. Glucosuria increases the SG despite increased urine volume.
Reagent strips such as Multistix® or Chemstrip ® can be used to perform several semiquantitative chemical evaluations simultaneously. They are used routinely to determine urine pH, protein, glucose, ketones, bilirubin/urobilinogen, and occult blood. Some reagent strips include test pads for leukocyte esterase (for detection of WBCs), nitrite (for detection of bacteria), and SG; these are not valid in animals and should not be used. Reagent strips are adversely affected by moisture and have a limited shelf life. Bottles should be kept tightly capped, and unused strips should be discarded after their expiration date.
Urine pH is typically acidic in dogs and cats and alkaline in horses and ruminants, but varies depending on diet, medications, or presence of disease. Reagent strip colorimetric test pads for pH determination are accurate to within ~0.5 pH units. For example, a reading of 6.5 means the actual pH is likely to be between 6.0 and 7.0. Portable pH meters are more accurate than pH colorimetric test pads. A bacterial urinary tract infection with a urease-producing microbe will result in alkaluria. Urine pH will affect crystalluria because some crystals, such as struvite, form in alkaline urine, whereas other crystals, such as cystine, form in acidic urine.
The protein test pad uses a color indicator (tetrabromophenol blue), which detects primarily albumin in urine. Results range from 10 mg/dL to 1,000 mg/dL. Proteinuria can occur from prerenal (fever, strenuous exercise, seizures, extreme environmental temperature, and hyperproteinemia), renal (primarily glomerular and occasionally tubular disease), or postrenal (inflammation, hemorrhage, and infection) causes. A positive reaction must be interpreted in light of SG, pH, and urine sediment examination. For example, a trace amount of protein in concentrated urine is less significant than a trace amount of protein in dilute urine. Alkaluria will give a false-positive reaction. Likewise, presence of other proteins, such as Bence-Jones proteins, will give false-negative results. Proteinuria can be measured using sulfosalicylic acid precipitation, which detects albumin and globulins; however, it is not accurate in dogs and cats. If proteinuria is present with an inactive urine sediment, its significance can be verified and quantitated by dividing the urine protein concentration by the urine creatinine concentration (urine protein to urine creatinine ratio [UP:UC]). Interpretation of a UP:UC is as follows: <0.5:1 (dogs) and <0.4:1 (cats) is normal, 0.4 or 0.5–1:1 is questionable, and >1:1 is abnormal. With primary renal azotemia, a UP:UC >0.5:1 in dogs and >0.4:1 in cats is considered abnormal. A semiquantitative microalbuminuria test is available to detect urinary albumin in the range of 1–30 mg/dL. It uses ELISA technology specific for canine or feline albumin. Because of minor species differences in albumin, there are different kits for dogs and cats. The microalbuminuria test detects lower concentrations of albumin than a standard dipstick test pad. Hematuria must be macroscopic to increase the microalbuminuria or UP:UC; however, pyuria increases both.
Glucose is detected by a glucose oxidase enzymatic reaction that is specific for glucose. Glucosuria is not present normally because the renal threshold for glucose is >180 mg/dL in most species and >240 mg/dL in cats. With euglycemia, the amount of filtered glucose is less than the renal threshold, and all of the filtered glucose is reabsorbed in the proximal renal tubules. Glucosuria can result from hyperglycemia (due to diabetes mellitus, excessive endogenous or exogenous glucocorticoids, hepatocutaneous syndrome, or stress) or from a proximal renal tubular defect (such as primary renal glucosuria or Fanconi syndrome). If glucosuria is present, blood glucose concentration should be determined. False-negative results can occur with high urinary concentrations of ascorbic acid (vitamin C) or with formaldehyde (a metabolite of the urinary antiseptic, methenamine, which may be used for prevention of bacterial urinary tract infections). False-positive results may occur if the sample is contaminated with hydrogen peroxide, chlorine, or hypochlorite (bleach).
Ketones are produced from fatty acid metabolism and include acetoacetic acid, acetone, and β-hydroxybutyrate. The ketone test pad detects acetone and acetoacetic acid, but not β-hydroxybutyrate (which can be measured in blood and urine using a point-of-care instrument or a biochemistry analyzer). The test pad contains nitroprusside that reacts with acetoacetic acid and acetone to cause a purple color change; it is more sensitive to acetoacetic acid than acetone. Ketonuria is associated with primary ketosis (ruminants), ketosis secondary to diabetes mellitus (small animals), consumption of low-carbohydrate diets (especially in cats), and occasionally with prolonged fasting or starvation. A false-positive reaction can occur with presence of reducing substances in urine.
When hemoglobin is degraded, the heme portion is converted to bilirubin, which is conjugated in the liver and excreted in bile. Some conjugated bilirubin is filtered by the glomerulus and excreted in urine. In dogs, but not cats, the kidney can metabolize hemoglobin to bilirubin and secrete it. Male dogs have a higher secretory ability than females. Dipstick reagent pads use diazonium salts to create a color change and are more sensitive to conjugated bilirubin than unconjugated bilirubin. Bilirubinuria occurs when conjugated bilirubin exceeds the renal threshold as with liver disease or hemolysis. In dogs with concentrated urine, a small amount of bilirubin can be normal. Pigmenturia and phenothiazine may result in a false-positive reaction; false-negative reactions may occur with large amounts of urinary ascorbic acid (vitamin C).
Urobilinogen, formed from bilirubin by intestinal microflora, is absorbed into the portal circulation and excreted renally. A small amount of urinary urobilinogen is normal. Increased urinary urobilinogen occurs with hyperbilirubinemia; a negative test may be seen with biliary obstruction. However, the test is not specific enough to be clinically useful.
The occult blood test pad uses a “pseudoperoxidase” method to detect intact RBCs, hemoglobin, and myoglobin. A positive reaction can be due to hemorrhage (hematuria), intravascular hemolysis (hemoglobinuria), or myoglobinuria. The latter two processes can be distinguished by examination of plasma: plasma will appear pink to red after intravascular hemolysis, whereas myoglobin is rapidly cleared from plasma, resulting in clear plasma. As with other colorimetric test pads, discolored urine may yield false-positive results. A positive result should be interpreted with microscopic examination of urine sediment.
Microscopic examination of urine sediment should be part of a routine urinalysis. For centrifugation, 3–5 mL of urine is transferred to a conical centrifuge tube. Urine is centrifuged at 1,000–1,500 rpm for ~3–5 min. The supernatant is decanted, leaving ~0.5 mL of urine and sediment in the tip of the conical tube. The sediment is resuspended by tapping the tip of the conical tube against the table several times. A few drops of the sediment are transferred to a glass slide, and a cover slip is applied. Examination of unstained urine is recommended for routine samples. Microscopic examination is performed at 100× (for crystals, casts, and cells) and 400× (for cells and bacteria) magnifications. Contrast of the sample is enhanced by closing the iris diaphragm and lowering the condenser of the microscope. Stains such as Sedistain® and new methylene blue can be used to aid in cell identification but may dilute the specimen and introduce artifacts such as stain precipitate and crystals. Use of a modified Wright stain increases the sensitivity, specificity, and positive and negative predictive values for detection of bacteria. For some tests, air dried, stained smears are necessary.
In an unstained preparation, RBCs are small and round and have a slight orange tint and a smooth appearance. Normal urine should contain <5 RBCs/field at 400× magnification. Increased RBCs in urine (hematuria) indicates hemorrhage somewhere in the urogenital system; however, sample collection by cystocentesis or catheterization may induce hemorrhage.
WBCs are slightly larger than RBCs and have grainy cytoplasm. Normal urine should contain <5 WBCs/field at 400× magnification. Increased WBCs (pyuria) can occur due to inflammation, infection, trauma, or neoplasia. Catheterization or collection of voided urine may introduce a few WBCs from the urogenital tract.
Transitional epithelial cells, a common urine contaminant derived from the bladder and proximal urethra, resemble WBCs but are larger. They have a greater amount of grainy cytoplasm and a round, centrally located nucleus. In a voided urine sample, squamous epithelial cells may be seen. They are large, oval to cuboidal in shape, and may or may not contain a nucleus. Occasionally, neoplastic transitional cells may be seen in an animal with a transitional cell carcinoma. Neoplastic squamous cells may be seen in an animal with a squamous cell carcinoma.
Casts are elongated, cylindrical structures formed by mucoprotein congealing within renal tubules and may contain cells. Hyaline casts are pure protein precipitates; they are transparent, have parallel sides and rounded ends, and are composed of mucoprotein. They may occur with fever, exercise, and renal disease. Epithelial cellular casts form from entrapment of sloughed tubular epithelial cells in the mucoprotein; they may be seen with renal tubular disease. Granular casts are thought to represent degenerated epithelial cellular casts. Waxy casts have a granular appearance and are thought to arise from degeneration of longstanding granular casts. They typically have sharp borders with broken ends. Other cellular casts include RBC casts and WBC casts and are always abnormal. RBC casts form because of renal hemorrhage. WBC casts form because of renal inflammation, as with pyelonephritis. Fatty casts are not common but can be seen with disorders of lipid metabolism, such as diabetes mellitus. A few hyaline or granular casts are considered normal. However, presence of cellular casts or other casts in high numbers indicates renal damage, and may be one of the earliest laboratory abnormalities noted with toxic damage to renal epithelial cells (eg, gentamicin, amphotericin B).
The presence of bacteria in urine collected by cystocentesis indicates infection. Small numbers of bacteria from the lower urogenital tract may contaminate voided samples or samples collected by catheterization and do not indicate infection. Bacterial rods are most easily identified in urine sediment. Particles of debris may be mistaken for bacteria. Suspected bacteria can be confirmed by staining urine sediment with Gram stain; however, aerobic culture is best to confirm a bacterial urinary tract infection. Rarely, yeast and fungal hyphae and parasitic ova may be seen in urine sediment. Their presence is not always associated with clinical disease. Parasitic ova observed include those of Stephanurus dentatus, Capillaria plica, C felis, and Dioctophyma renale. Additionally, microfilariae of Dirofilaria immitis may be seen in urine sediment.
Many urine sediments contain crystals. The type of crystal present depends on urine pH, concentration of crystallogenic materials, urine temperature, and length of time between urine collection and examination. Crystalluria is not synonymous with urolithiasis and is not necessarily pathologic. Furthermore, uroliths may form without observed crystalluria. Many urine samples are examined at room temperature, which may alter the number of crystals present, and the result may not represent the situation in vivo.
Struvite crystals are commonly seen in canine and feline urine. Struvite crystalluria in dogs is not a problem unless there is a concurrent bacterial urinary tract infection with a urease-producing microbe. Without an infection, struvite crystals in dogs are not associated with struvite urolith formation. However, some animals (eg, cats) do form struvite uroliths without a bacterial urinary tract infection. In these animals, struvite crystalluria may be pathologic. Struvite crystals appear typically as “coffin-lids” or “prisms”; however, they may be amorphous.
Calcium oxalate crystalluria occurs less commonly in dogs and cats. If persistent, it may indicate an increased risk of calcium oxalate urolith formation, which has been increasing in some countries throughout the past few years. (See also Urolithiasis in Horses Urolithiasis in Horses Obstructive urolithiasis is relatively uncommon in horses and affects primarily older animals. Calcium carbonate stones are the most common type. Clinical signs include dysuria, pollakiuria... read more and see Urolithiasis in Small Animals Urolithiasis in Small Animals Some mineral solutes precipitate to form crystals in urine; these crystals may aggregate and grow to macroscopic size, at which time they are known as uroliths (calculi or stones). Uroliths... read more .) However, calcium oxalate and calcium carbonate crystalluria is common in healthy horses and cattle. Calcium oxalate dihydrate crystals appear as squares with an “X” in the middle, or “envelope-shaped.” Calcium oxalate monohydrate crystals are “dumb-bell” shaped. An unusual form of calcium oxalate crystals is typically seen in association with ethylene glycol toxicity (see Ethylene Glycol Toxicity Ethylene Glycol Toxicity read more ). These crystals are found in neutral to acidic urine. They are small, flat, and colorless and are shaped like “picket fence posts.”
Ammonium acid urate crystals suggest liver disease (eg, portosystemic shunt). These crystals occur in acidic urine and are yellow-brown spheres with irregular, spiny projections; however, they may also be amorphous. Certain species, such as birds and reptiles, and certain breeds of dogs, specifically Dalmatians, can normally have ammonium acid urate crystalluria.
Cystine crystals are six-sided and of variable size. They are seen in acidic urine. Presence of cystine crystals represents a proximal tubular defect in amino acid reabsorption. Cystinuria has been reported in many breeds of dogs and rarely in cats. Dachshunds, Newfoundlands, English Bulldogs, and Scottish Terriers have a high incidence of cystine urolithiasis.
Bilirubin crystals occur with bilirubinuria; however, they may be normal in small numbers in dogs.
The bladder tumor antigen test can be used to screen for transitional cell carcinoma in dogs. The results are not specific for transitional cell carcinoma, and non-neoplastic disease (eg, urinary tract infections, hematuria, etc) can give positive results. A negative test, however, is meaningful in that a transitional cell carcinoma is not likely to be present. This test may be useful for routine screening of dogs at higher risk of developing transitional cell carcinoma (eg, Scottish terriers) that do not have other signs or laboratory findings of lower urinary tract disease. This tests for the presence of basement membrane fragments and is a color reaction using a commercial test kit.