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Urinalysis Interpretation

Gregory F. Grauer, DVM, MS, DACVIM (SAIM) Lisa M. Pohlman, DVM, MS, DACVP Kansas State University

1 d Calcium oxalate dihydrate crystals from the sediment of a dog. Crystals vary in size and can be individualized or in aggregates. volume, odor, or frequency are indica- YOU HAVE ASKED... tions for urinalysis, as are dysuria and What is the role of a complete stranguria. Urinalysis also provides information about kidney function via urinalysis in veterinary practice? assessment of (USG) and . Cellular or granular casts or renal tubular epithe- THE EXPERTS SAY... lial cells in the urine sediment, and A complete urinalysis comprises normoglycemic glucosuria and/or Urinalysis can be physical examination of the urine sam- enzymuria, can indicate renal tubular inexpensive, is ple, biochemical assessment, and micro- damage or disease. Urinalysis can also easily performed scopic assessment. Urinalysis can be help identify disease and disorders of in-house by inexpensive, is easily performed in-house other organ systems, and it provides appropriately by appropriately trained technical staff, information in the follow-up of patients trained technical and requires minimal specialized equip- with , urinary tract infec- ment. In the authors’ opinion, urinalysis tions, proteinuria, mellitus, staff, and requires is underused despite the valuable diag- kidney disease, Cushing’s disease, minimal specialized nostic information it provides. hyperthyroidism, and polydipsia equipment. and/or polyuria. Indications for Urinalysis Any health screening program and Factors Influencing Results preanesthetic patient assessment Method of Collection should include a complete urinalysis, a Bacterial contamination from the lower key element of a minimum database in urinary tract can occur with voided sam- patients with systemic or urinary tract ples; iatrogenic hematuria can occur USG = urine specific gravity disease. Changes in urine color, clarity, with cystocentesis.

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TABLE 1 breakdown. Concentrated urine typically is darker than more dilute urine; however, color CAUSES OF ABNORMAL URINE COLOR should not be used to assess concentration. Abnormal urine colors include pink-red, red-brown, brown-black, yellow-orange, and Color Possible Cause yellow-green (Table 1). The most common Pink-red Hematuria, , abnormal urine color is red—usually associ- ated with hematuria. Centrifugation of the Red-brown Hematuria, hemoglobinuria, myoglobinuria urine sample separates the liquid from the Brown-black Methemoglobin (from hemoglobin or solid components, including red blood cells, ), bile pigments and the supernatant becomes clear. If hemo- globinuria or myoglobinuria is the cause of Yellow-orange Highly concentrated urine, the red discoloration, the urine supernatant Yellow-green , biliverdin remains red after centrifugation.

Fresh, well-mixed normal urine should be Timing of Collection clear or transparent to slightly cloudy. Cloudy A first morning urine sample is likely to be urine usually is associated with presence of more concentrated than a mid-day sample, red blood cells, white blood cells, epithelial and postprandial samples may exhibit tran- cells, crystals, casts, bacteria, lipid, mucus, or sient alkalinuria.1 In addition, previous treat- semen within the urine sample. Clear or ment such as fluid or diuretic therapy can slightly cloudy urine should not obviate micro- decrease USG and alter urinalysis findings. scopic examination of the urine sediment as abnormal findings are still possible.3 Sample Handling Urinalysis should be performed on fresh Specific gravity, the ratio of the weight of a urine. If the sample cannot be evaluated solution to the weight of an equal volume of within 30 minutes, it should be refrigerated distilled water, is estimated using a refrac- in a sealed container, without exposure to tometer. USG reflects the ability of the kid- light. Result accuracy decreases as time neys to concentrate and dilute urine between collection and analysis increases. compared with the specific gravity of plasma Refrigerated samples typically are acceptable (Table 2). USG results must be interpreted in for evaluation for 12 hours and possibly up to light of the patient’s hydration status, serum 24 hours, depending on the test. Urine sam- urea nitrogen and creatinine concentrations, ples must be brought to room temperature and recent fluid therapy and medication his- before analysis, including dipstick, or results tory. Because of species differences, refrac- may be inaccurate.2 tometers should be calibrated specifically for dogs and cats. For example, the USG of feline Physical Examination of Urine urine will likely be falsely high if measured Physical examination of urine includes with a refractometer calibrated for humans. assessment of color, clarity (transparency or Marked proteinuria and glucosuria have the turbidity), and specific gravity. Normal urine potential to increase USG in dogs and cats; color, which varies from colorless or pale yel- for every gram of protein or glucose per dL of low to dark yellow, is associated with the urine, the USG may increase by about 0.005. presence of yellow pigments called uro- Because IV administration of hetastarch4 and chromes, the end products of hemoglobin iohexol can transiently but markedly increase

94 cliniciansbrief.com March 2016 TABLE 2

CLASSIFICATION OF USG

Category USG Interpretation

Hypersthenuria ≥1.030 in dogs; ≥1.035 in cats* h Urine is appropriately concentrated; appropriate renal response to antidiuretic hormone h ≥1/3 of the total nephron population is functional and renal medullary hypertonicity is present

Range of minimal concentration >1.012 – <1.030 in dogs h Urine is inappropriately dilute compared with plasma >1.012 – <1.035 in cats h May be normal in a well-hydrated dog or cat h Inappropriate in the face of dehydration or azotemia

Isosthenuria 1.008 – 1.012 in dogs and cats h Urine has not been concentrated or diluted; specific gravity is similar to that of plasma h May be normal response to recent water intake or fluid therapy h Inappropriate in the face of dehydration or azotemia

Hyposthenuria 1.008 in dogs and cats h Urine is dilute compared with plasma h ≥1/3 of the total nephron population is functional h Inappropriate in the face of dehydration or azotemia

*Cats have greater urine-concentrating capacity than dogs; some authors state that in cats is >1.040 or 1.045.6

USG,5 urine-concentrating ability should not interpretation of reactions for bilirubin and be assessed in dogs and cats that receive these ketones. Results of dipstick biochemical analy- drugs. ses of nitrate, , and uro- bilinogen are unreliable and should be ignored Biochemical Assessment of Urine when assessing urine of dogs and cats.2,5,6 Urine is evaluated semi-quantitatively (nega- tive, trace, 1+ to 4+ scale) using commercial Glucose reagent dipsticks that undergo a color change Glucose is a small molecule freely filtered reaction relative to the concentration of the through the glomerulus. Once filtered, glu- analyte in the sample (Table 3, next page). cose is usually completely reabsorbed from the Well-mixed, uncentrifuged urine at room tem- glomerular filtrate by the proximal convoluted perature is used to inoculate reagent pads; tubular epithelia. The most common cause refrigeration of urine may slow reaction time. of glucosuria is hyperglycemia (eg, diabetes The color change typically is evaluated visually, mellitus, stress [especially in cats], glucose- but automated dipstick readers are available. containing fluids), resulting in glucose con- The color change is time-sensitive, with color centrations in the glomerular filtrate that typically increasing beyond the ideal reaction overwhelm the reabsorptive capacity of the time. Dipstick reagent methodology is not con- proximal tubule. The renal tubular transport sistent across products, so results may vary. maximum measurements for urine glucose in USG = urine specific gravity Grossly discolored urine may affect dipstick the dog and cat are approximately 180 to 220

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TABLE 3 or tubular or renal glucosuria); examples include acute ischemic or toxic kidney injury NORMAL URINALYSIS RESULTS and Fanconi syndrome in basenjis. Bilirubin Variable Normal Result Comments Bilirubin is produced from hemoglobin by the reticuloendothelial system when senescent Glucose Negative Most commonly associated with red blood cells are destroyed. Bilirubin is con- hyperglycemia; may also be renal jugated with glucuronide in the liver and pri- in origin and associated with normoglycemia marily excreted in bile. Conjugated bilirubin in plasma is filtered through the glomerulus Bilirubin Negative Low-level bilirubinuria normal in dogs and excreted in urine. Canine kidneys can also Ketones Negative β-hydroxybutyrate not detected metabolize hemoglobin and conjugate biliru- bin; therefore, the renal threshold for biliru- pH 5.0-7.5 Dipstick results not precise bin in dogs, especially male dogs, is low. Protein Negative False-positive results common— Low-level bilirubinuria in concentrated canine especially in cats urine (eg, trace or 1+ in hypersthenuric urine) is likely normal because of the low renal Heme Negative Can be positive without intact RBCs in sediment (ie, myoglobin, hemoglobin, threshold. A 2+ to 4+ bilirubin reaction in or due to red cell hemolysis in dilute or canine urine, especially less concentrated alkaline urine) urine, is more likely abnormal. In contrast, Erythrocytes 0-5/hpf Iatrogenic microscopic hematuria bilirubinuria is never normal in cats; the renal expected with cystocentesis threshold for bilirubin in the cat is approxi- mately 9 times higher than in the dog.2 Leukocytes 0-5/hpf Usually neutrophils

Casts None Hyaline casts can be observed with The major rule-outs for bilirubinuria in dogs proteinuria and cats are cholestatic disorders and hemo- lysis. Additional rule-outs include fever and Epithelial cells Variable Transitional epithelial cells most common prolonged fasting.

Crystals None Influenced by urine temperature and pH Ketones Bacteria None Air-dried sediment slides stained Ketones (ie, β-hydroxybutyrate, acetoacetate, with Wright or gram stain increases acetone) are produced when normal energy 13,14 detection sensitivity production shifts from carbohydrate to lipid Nitrite Unreliable metabolism as in diabetes mellitus, starva- tion, low-carbohydrate diets, glycogen storage Leukocyte Unreliable diseases, and hypoglycemic disorders. Plasma esterase ketones are excreted in the urine via glomeru- Urobilinogen Unreliable lar filtration and tubular secretion; however, ketone production is normally low, and keton- uria is not seen in healthy dogs and cats. and 260 to 280 mg/dL, respectively.7 Less Because urine dipsticks are more sensitive to commonly, glucosuria may be associated with acetoacetate than to acetone (with an approxi- tubular disease/damage and decreased tubu- mately 10-fold sensitivity difference), and they lar reabsorption of the glucose normally pres- do not detect β-hydroxybutyrate, the magni- ent in the glomerular filtrate (normoglycemic tude of may be underestimated.

96 cliniciansbrief.com March 2016 Ketones are volatile and will diffuse into air if TABLE 4 the urine sample is not sealed and analyzed within 30 minutes.2,5 The most common INTERPRETATION OF URINE PH cause of ketonuria in dogs and cats is diabetic ketoacidosis. Potential Causes of Aciduria Potential Causes of Heme (Occult Blood) Alkalinuria Heme proteins in urine (from intact red blood Meat protein-based diet Plant protein-based diet cells, hemoglobin, or myoglobin) react with peroxide in the dipstick reagent pad and cause Use of acidifying agents Postprandial alkaline tide a positive-reaction color change. Hematuria is (eg, NH4Cl) the most common cause of a positive test, Metabolic acidosis UTI with urease-containing although in some cases of hematuria intact bacteria RBCs will not be observed in the urine sedi- Respiratory acidosis Urine sample exposed to air at ment because of hemolysis. This is more likely room temperature to occur if the urine pH is very alkaline (>8) or the urine is very dilute (USG <1.015). Normal Paradoxical aciduria with Use of alkalinizing agents (eg, canine and feline urine should be negative for alkalosis NaHCO3) heme protein, but iatrogenic microscopic Protein catabolic states Metabolic alkalosis hematuria associated with cystocentesis may Ethylene glycol ingestion Respiratory alkalosis result in a positive reaction for heme. pH Normal urine pH in dogs and cats ranges ence of albumin-like proteins in the urine (eg, from 5.0 to 7.5 and varies with type of diet, cauxin in cats).8 collection time (eg, postfasting vs post- prandial), and systemic acid-base status. In the sulfosalicylic acid (SSA) test, equal Urine dipstick pH measurement approximates parts urine supernatant and 3% to 5% SSA urine pH and is estimated to the nearest 0.5 are mixed in a glass test tube and the turbid- pH unit. A more precise urine pH can be ity resulting from precipitation of protein is obtained using a pH meter. Potential causes of graded on a 0 to 4+ scale. In addition to albu- acidic and alkaline urine are listed in Table 4. min, the SSA test can detect globulins and Bence Jones proteins to a greater extent than Proteinuria the dipstick test can. False-positive results The dipstick primarily measures albumin, but may occur if the urine contains radiographic sensitivity and specificity are relatively low. contrast agents, penicillin, cephalosporins, False-negative results (decreased sensitivity) sulfisoxazole, or thymol (a urine preserva- may occur with Bence Jones proteinuria, low tive), as well as for unknown reasons. The pro- concentrations of albumin in the urine, and/or tein content may also be overestimated with dilute or acidic urine. The lower limit of pro- the SSA test if uncentrifuged, turbid urine is tein detection for the conventional dipstick analyzed. The reported sensitivity of the SSA test is approximately 20 to 30 mg/dL. False- test is approximately 5 mg/dL. Because of the positive results (decreased specificity), relatively poor specificity of conventional dip- SSA = common in both species with the dipstick but stick analysis, many reference laboratories sulfosalicylic acid more frequent in cats, may be associated with will confirm a positive dipstick test result for USG = urine specific gravity highly concentrated alkaline urine or the pres- proteinuria using the SSA test.

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TABLE 5

LOCALIZATION OF PROTEINURIA

Type of Proteinuria Examples Diagnosis

PHYSIOLOGIC OR Change in exercise level UPC usually <0.5 BENIGN PROTEINURIA Seizure activity Compatible history

Fever Proteinuria is intermittent, transient

Exposure to temperature extremes

Stress Variable UPC

PATHOLOGIC PROTEINURIA

Non-Urinary Congestive heart failure History/Physical examination/Echo Hemoglobinuria, myoglobinuria Urine remains red after centrifugation

Dysproteinemia, dysproteinuria serum Electrophoresis

Genital tract inflammation or hemorrhage Physical examination/imaging/urine sediment

Urinary (Non-renal) Lower urinary tract inflammation (eg, bacterial UPC not indicated cystitis, cystoliths, polyps, neoplasia) History/Physical examination Compatible urine sediment Lower urinary tract imaging

Urinary (Renal) Renal parenchymal inflammation (eg, Variable UPC pyelonephritis, renoliths, neoplasia) Compatible urine sediment Renal imaging

Tubular proteinuria UPC usually = 0.5-1 May also see decreased tubular reabsorption of glucose and electrolytes

Glomerular proteinuria Persistent UPC ≥1 Inactive urine sediment (with exception of possible hyaline cats)

It is usually recommended that proteinuria in hypersthenuric urine has often been detected by these semi-quantitative screen- attributed to urine concentration rather than ing methods be interpreted in light of the to abnormal proteinuria. Likewise, a positive USG and urine sediment. For example, a posi- dipstick reading for protein in the presence tive dipstick reading of trace or 1+ proteinuria of hematuria or pyuria is often attributed to

98 cliniciansbrief.com March 2016 urinary tract hemorrhage or inflammation. In TABLE 6 both situations, however, these interpreta- tions may be inaccurate. Because of the limits INTERPRETATION OF UPC of conventional dipstick test sensitivity, any positive result for protein, regardless of urine concentration, may be abnormal. Hematuria UPC Classification and pyuria have an inconsistent effect on <0.2 Normal urine albumin concentrations, and not all >0.2 ≤0.4 (cats), Borderline dogs with microscopic hematuria and pyuria ≤0.5 (dogs) have .9 In patients with gross hematuria and/or microscopic pyuria, the ≥0.4 (cats), ≥0.5 (dogs) Abnormal (either glomerular or source of the hemorrhage and/or inflamma- tubular) tion should be determined and treated before >2.0 Abnormal (glomerular range further assessment of the proteinuria. proteinuria)

Proteinuria can be classified as physiologic or pathologic (Table 5). Physiologic or benign proteinuria can be associated with changes in attempt to verify a positive dipstick reac- exercise level, seizures, and fever and is typi- tion) are sufficient to exclude the existence cally low-level and transient.10 Pathologic of all forms of proteinuria except for low proteinuria can be nonurinary or urinary in concentrations of albumin in the urine origin, and pathologic urinary proteinuria (). Species-specific albu- can be renal or nonrenal in origin. Renal pro- minuria assays (ie, microalbuminuria teinuria arises as a result of glomerular and/ assays; antechdiagnostics.com, heska.com) or tubular lesions and is persistent and usu- can be used as screening tests for detection ally associated with a normal or inactive of early chronic kidney disease. urine sediment. If screening test results show persistent pro- The ACVIM Consensus Statement on protein- teinuria associated with normal urine sedi- uria recommends the following11: ment, urine protein excretion should be quantified. This helps evaluate the severity of h Strong positive dipstick reactions (≥1+; renal lesions and assess treatment response confirmed by SSA) are an indication to pro- or disease progression. The most common ceed with determination of urine protein/ method used to quantitate proteinuria is the creatinine ratio (UPC) either immediately UPC. Although the UPCs from spot urine or at least after repeated testing in 2-4 samples accurately reflect the quantity of pro- weeks verifies persistence of the positive tein excreted in the urine over a 24-hour reactions. period,11 it is ideal to base clinical decisions h Weak positive dipstick reactions (trace; on the average of more than 1 measurement. confirmed by SSA) are an indication at least Classification of proteinuria based on UPC for repeated testing in 2-4 weeks to check findings is presented in Table 6.11 SSA = for persistence of the proteinuria, with sulfosalicylic acid UPC = urine determination of UPC if the positive reac- Based on longitudinal test results in dogs with protein/ tions persist. X-linked hereditary nephropathy, the UPC creatinine ratio h Negative reactions (by dipstick alone, by must change by at least 35% at high UPC val- USG = urine specific gravity SSA alone, or by SSA performed in an ues (near 12) and by 80% at low UPC values

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Microscopic Assessment The urine sample should be agitated gently before pouring off an aliquot for centrifugation because cells, casts, crystals, and bacteria will undergo sedimentation if the urine sample is left undisturbed. Low- speed centrifugation (1000–1500 2 3 rpm) for 3 to 5 minutes (using a standardized technique) will mini- d Struvite crystals from the urine d Granular cast from the urine sediment sediment of a dog. Crystals can vary of a cat. Focusing on larger structures mize destruction of cells and casts. greatly in size. may cause smaller structures to It is ideal to always centrifuge the appear out of focus; therefore, the same urine volume because the con- focus should be moved up and down centration of elements in the sedi- when evaluating urine sediment. ment is volume-dependent.

Similarly, after centrifugation, the sediment should always be resus- pended in the same volume of urine. If normal laboratory procedure is to centrifuge 6 mL of urine but only 3 mL is available, there would only be half of the sediment material in the 3-mL sample if it were resus- 4 5 pended in the normal volume of urine. Obviously, this can affect d Urine sediment from a cat with d Urine sediment from a cat with interpretation of the sediment eval- bacterial cystitis. Note the numerous idiopathic cystitis. Numerous red uation. To avoid this error in white blood cells (arrows) and blood cells (solid arrow), occasional “short” samples, the sediment can bacteria (arrow heads). These white blood cells (arrow head), a small be resuspended in a standard per- findings can be confirmed by cluster of sloughed transitional cells preparing an air-dried smear and (dashed arrow), and background centage of the urine volume centri- staining it with a Romanowsky stain amorphous debris are seen. No fuged. For example, if the sediments such as Diff-Quik. organisms were identified on from 6 and 3 mL of urine are resus- microscopic evaluation or via culture. pended in 1.2 and 0.6 mL of urine, respectively (ie, 20% of the original volume), similar results from the sediment evaluation should be (near 0.5) to demonstrate a signifi- obtained. Individual laboratory ref- cant difference between serial val- erence values are typically derived ues.12 A single measurement was by resuspending the urine sediment found to reliably estimate the UPC pellet in 20% of the original urine when the values were less than 4, volume, which is usually 5 mL to but 2 or more determinations were 6 mL. necessary to reliably estimate the UPC when values were higher One to 2 drops of the resuspended, than 4.12 unstained sediment is placed on a

100 cliniciansbrief.com March 2016 THE WAIT IS slide and a coverslip is applied. The average of 10 fields. If the identity microscope condenser is lowered to of a sediment element is undeter- OVER. reduce illumination, and at least 10 mined, a stain (eg, Sedi-Stain, bd. fields are examined at both 10× (low com) can be added to the wet prepa- A breakthrough in the power) and 40× (high power). Sev- ration, or the slide can be air-dried detection of bacteria in eral urine sediment photos are and stained with a Romanowsky urine. Results in minutes. shown in Figure 1, page 93, & stain (eg, Diff-Quik or other quick Figures 2-5. Sediment results are stains [eg, Wright’s, modified p reported as the number of ele- Wright’s, Giemsa, Gram]) for Direct replacement ments/high power field using the further evaluation.13,14 n for urine culture p 98.5% overall accuracy p A fraction of the cost References of urine culture 1. Stockham SL, Scott MA. Urinary system. In: total protein concentrations in canine urine Stockham SL, Scott MA, eds. Fundamentals of samples. Vet Clin Pathol. 2004;33(1):14-19. Veterinary Clinical Pathology. Ames, IA: Black- 9. Lyon SD, Sanderson MW, Vaden SL, Lappin MR, well; 2002:277-336. Jensen WA, Grauer GF. Comparison of dipstick, 2. Wamsley H, Alleman R. Complete urinalysis. sulfosalicylic acid, urine protein-to-creatinine In: Elliott J, Grauer GF, eds. BSAVA Manual of ratio, and species-specific ELISA methodolo- Canine and Feline Nephrology and Urology. 2nd gies for detection of albumin in urine samples ed. Quedgeley, UK: British Small Animal Veteri- of cats and dogs. JAVMA. 2010;236(8):874-879. nary Association; 2007. 10. Grauer GF, Thomas CB, Eicker SW. Estima- 3. Barlough JE, Osborne CA, Stevens JB. Canine tion of quantitative proteinuria in the dog, and feline urinalysis: Value of macroscopic using the urine protein-to-creatinine ratio and microscopic examinations. JAVMA. from a random, voided sample. Am J Vet Res. 1981;178(1):61-63. 1985;46(10):2116-2119. 4. Smart L, Hopper K, Aldrich J, George J, Kass P, 11. Lees GE, Brown SA, Elliott J, Grauer GE, Vaden Haskins S. The effect of hetastarch (670/0.75) SL, ACVIM. Assessment and management on urine specific gravity and osmolality in the of proteinuria in dogs and cats: 2004 ACVIM dog. JVIM. 2009;23(2):388-391. Forum Consensus Statement (small animal). 5. Fry MM. Urinalysis. In: Bartges J, Polzin DJ, JVIM. 2005;19(3):377-385. eds. Nephrology and Urology of Small Animals. 12. Nabity MB, Boggess MM, Kashtan CE, Lees Ames, IA: Wiley-Blackwell; 2011:46-57. GE. Day-to-day variation of the urine protein: 6. Lefebvre HP. Renal function testing. In: Bart- creatinine ratio in female dogs with stable glo- ges J, Polzin DJ, eds. Nephrology and Urology merular proteinuria caused by X-linked heredi- of Small Animals. Ames, IA: Wiley-Blackwell; tary nephropathy. JVIM. 2007;21(3):425-430. 2011:91-96. 13. Swenson CL, Boisvert AM, Kruger JM, Gib- 7. Osborne CA, Stevens JB, Lulich JP, et al. A cli- bons-Burgener SN. Evaluation of modified nician’s analysis of urinalysis. In: Osborne CA, Wright-staining of urine sediment as a method Finco DR, eds. Canine and Feline Nephrology for accurate detection of in dogs. and Urology. Baltimore, MD: Williams & Wilkins; JAVMA. 2004;224(8):1282-1289. 1995:136-205. 14. Way LI, Sullivan LA, Johnson V, Morley PS. 8. Vaden SL, Pressler BM, Lappin MR, Jensen WA. Comparison of routine urinalysis and urine Effects of urinary tract inflammation and sam- Gram stain for detection of bacteriuria in dogs. ple blood contamination on urine albumin and JVECC. 2013;23(1):23-28.

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