Urinalysis: overview

• Changes in the chemical and physical characteristics of urine occur in many diseases, eg urogenital disease, hepatic disease, hematological disorders, endocrinopathies, neoplasia, and musculoskeletal disease.
• Changes in these characteristics can aid in the diagnosis of many diseases in ill animals and act as a cost-effective screening test in asymptomatic animals, or for monitoring specific disease changes and therapeutic response.

Alone

• Upper (pyelonephritis Kidney: pyelonephritis) and lower urinary tract infection Cystitis.
• Urolithiasis Urolithiasis, ureter (ureterolithiasis) Ureter: urolithiasis.
• Diabetes mellitus Diabetes mellitus and insipidus Diabetes insipidus.
• Acute renal failure Kidney: acute kidney injury (AKI), less use if anuric though.

• Portocaval shunt Congenital portosystemic shunt (CPSS) ammonium biurate or urate crystals.

• Transitional cell carcinoma Bladder: neoplasia. Diagnostic wash or catheter suction aspirates, the latter more accurate but often requires targeted imaging. Direct percutaneous aspiration controversial due to field theory and perceived or unclear to minimal risk of metastatic seeding.
  Wash: empty bladder, infuse 10-20 ml phosphate-buffered saline, massage bladder transabdominally, suction saline off into an EDTA and plain tube, examine sample.
• PUPD: polyuria /polydipsia Polydipsia (non-pathological causes).

In combination

• With serum-plasma urea Blood biochemistry: urea and creatinine test Blood biochemistry: creatinine and albumin too Blood biochemistry: albumin to assess renal function Renal function assessment.
• Alongside liver enzymes and serum (pre + 2 h post prandial) bile acids with possibly ammonia test (Blood biochemistry: ammonia NB very labile, separated frozen-chilled plasma in 20 mins, smoking and disinfectant artifact) - to assess hepatic encephalopathy Hepatic encephalopathy due to portocaval shunt Congenital portosystemic shunt (CPSS).
• With serum glucose test Serial blood glucose and fructosamine Blood biochemistry: fructosamine to confirm diabetes mellitus Diabetes mellitus and prolonged hyperglycemia respectively.

• With ultrasonography Ultrasonography: bladder and urinary tract Ultrasonography: kidney - to assess hematuria and urogenital structures such as the prostate.
• With hematology Hematology: complete blood count (CBC) - in severe (intravascular) hemolytic disease Anemia: immune mediated hemolytic.

• With hematology Hematology: complete blood count (CBC) and biochemistry such as cholesterol Blood biochemistry: cholesterol - to assess endocrinopathy.

• 1-10 ml.

• Cystocentesis Cystocentesis.
• Catheterization Urethral catheterization: female Urethral catheterization: male. 
• Midstream free flow sample Urine collection: freeflow or voided.
• (Compression.)

Precautions

• Collect at least 1 ml to allow most routine analysis and a standard volume for sediment examination Urinalysis: centrifuge sediment.
• Trauma during collection (catheterization, compression or cystocentesis) may contaminate sample (RBCs especially, catheterization epithelial cells also). Note how the sample is taken to allow interpretation and comparison.
• The longer the delay between collection and analysis, the less reliable the results.
• If not analysing in 1-2 hours, the sample should be refrigerated (2-8°C or 35-45°F).
• Return urine to room temperature before analysis (for dipstick Urinalysis: dipstick analysis and refractometer USG Urinalysis: specific gravity).
• Protect sample from exposure to bright light (UV light degrades urobilinogen and bilirubin).

Timing of test

• The composition of urine varies throughout the day.
• Sample for screening test at any time of day but a first morning sample provides the greatest amount of information initially.
• Repeat or serial testing for specific parameters, such as fresh for cells, may be required.

Early morning sample

• Advantage:  most representative and likely to be concentrated with higher yield of cells, casts and bacteria.
• Disadvantage: as the urine is stored in vivo, cells and casts degrade naturally, especially if dilute and alkaline.

Recently formed sample

• Advantage: cytological and cast detail best.
• Disadvantage: may be affected by recent feeding and water intake, with fewer cells or casts exfoliating.

Sample storage

• Sample can be refrigerated at 2-8°C (35-45°F) for a few hours (up to 6-12 h for many urine parameters).
• If sample is to be stored >12 h and is of sufficient volume, it can be split to give a plain sample and a preserved sample at room temperature. If using a preservative, the correct volume must be used to maintain the proper ratio and not distort the urinary material. The preservative must be chosen specifically as it will typically prevent some of the routine analysis otherwise performed on the plain sample, especially chemical based (eg dipsticks).
  • Boric acid (0.8%): for bacterial numbers up to 72 h, fill to the line and invert to gently mix and dissolve. It will also stabilize cells to a degree.
  • (Diluted) Formaldehyde (1 drop 40% per 30 ml): for cells and casts especially but requires special staining and special handling.
  • Refrigerate the separate plain sample/aliquot.

Sample transport

• The longer the delay between collection and analysis, the less reliable the results.
• Temperature >8°C (>45°F) for 1-2 h causes artifacts, even preserved samples unsuitable if exposed to temperature extremes (hot causes degradation, freezing lyses cells).
• Package according to Post Office or shipping regulations Transportation of diagnostic specimen.

Physical appearance

• Color: to check for possible interference and abnormal (non-yellow) colors (eg red-blood).
• Turbidity: macroscopic parameter which results from noted sediment findings (eg lipid droplets in iatrogenic samples).

Chemical analysis

• Centrifuge a set volume (minimum 0.5 ml) at slow speed (eg 500 g - see conversion formula for rpm).
• Decant supernatant for chemical analysis and USG, leaving sediment for microscopy (eg 1 drop following 0.5 ml centrifugation)
• Determine specific gravity Urinalysis: specific gravity by refractometer on the supernatant.

• Perform dipstick analysis on the supernatant, comparing the color change of dipstick to the reference range at the correct time of reading (often 1 min/60 s). The color change is typically enzymatic so time and temperature dependent too.
• Rapidly and completely immerse the dipstick and then tip sideways to blot off excess and prevent cross-reaction between pads. Alternatively if a small volume, use a pasteur pipette to streak ot pipette urine drops onto each pad, before tipping off the excess.

Microscopic examination

• Leave 0.25 ml in the Eppendorf or centrifuge tube.
  • Resuspend the sediment by repeatedly flicking or 'racking'  the centrifuge tube. Repeated (5 times) gently withdrawing and expressing via the pipette can also be used.
  • Using a fine tip pasteur pipette, once evenly resuspended, transfer one drop of sediment to a microscope slide and place a coverslip (18 x 18 mm typically) over it.
  • Lower the substage condenser on the microscope to maximize the diffraction.
  • Systematically examine the entire specimen under the lower power objective (x10-20), initially to check and semi-quantify (occ., +, ++, +++) for large epithelial cells, casts and crystals.
  • Examine the sediment under the high power objective (hpf, x40) to confirm the morphology of the elements (eg small oxalate crystals     , cells) and to detect bacteria. Average the number of red and white cells per hpf by counting at least 5 fields of view.
• 1 drop of Sedistain™ or a supravital stain such as Sternheimer-Malbin Staining techniques: Sternheimer-Malbin stain can be added to the standard sediment volume to highlight some cell features, notably nuclei for WBC identification. It does though obscure crystal colors.

• Can be performed in-house by veterinary clinics with an SOP and appropriate training.
• Access to variable speed centrifugation quickens the analysis but is not essential.
• All external laboratories but potentially greater in vitro change.

• A standard operating procedure (SOP) should always be used on a designated plain and standardized volume of urine to provide accurate, reproducible and consistent results. This allows disease changes and therapy to be monitored over time accurately, no matter who performs the testing. Experience adds to the familiarity and speed over time.

Color

• Amber yellow to straw.
• Very dilute urine tends to be almost colorless, while concentrated urine is dark yellow.

Turbidity

• Transparent and clear.

Specific gravity

• 1.001->1.050 (depending on hydration and water intake) Urinalysis: specific gravity.
• Hyposthenuria Hyposthenuria: <1.008.
• Isosthenuria: 1.008-1.012.
• Hypersthenuria: >1.012.

Dipsticks
pH

• Acidic as carnivorous: 6-7 typically Urinalysis: dipstick analysis.
  Some normal dogs may be outside this range, in vitro storage also potentially affecting pH.

Protein

Must interpret in conjunction with USG and sediment. Myelomatous paraproteins will not cause a positive reaction on standard dipsticks.

• Concentrated urine (USG >1.030) may have trace protein, dilute urine possibly too little for detection though.
• Protein/urine ratio (UPCR), creatinine <0.2 Urinalysis: protein Urinalysis: creatinine.
• Part of the IRIS staging scheme for renal failure Kidney: acute kidney injury (AKI).

Glucose

• Negative Urinalysis: glucose.

Ketones

• Negative Urinalysis: ketone.

Bilirubin

• More common in male dogs, more so if urine well concentrated (USG >1.025). Dilute urine, especially in females with bilirubin ++ or more is often pathological (eg cholestasis, liver disease) Urinalysis: bilirubin.

Urobilinogen

• Trace (due to intact enteroheptic recirculation) Urinalysis: urobilinogen.

Hemoglobinuria-myoglobinuria

• Negative Urinalysis: hemoglobin.

Hematuria

• Cystocentesis Cystocentesis and catheterization : typically a few red cells in low numbers (single figures to low doubles per hpf) depending upon technique and degree of trauma during needle advancement, or trauma and difficulty when catheterized.
• Freeflow/voided sample: <3 red cells/hpf unless during estrus in females.

Nitrite

• Negative Urinalysis: nitrite.

White blood cells (WBCs)

• Convention states <3/hpf but typically less than 1 in health. NB again estrus cycle factors in females for voided or catheterized samples can contain low numbers (<10/hpf typically) Urinalysis: white blood cell.
• Dipstick pads are based on esterase reactions and are human derived. Dogs have very differing amounts so this is unreliable and should not be used alone, the pad being insensitive and missing significant inflammation (pyuria).

Epithelial cells

• Occasional in most samples Urinalysis: epithelial cells.
• Increased numbers of voided sampling in females in estrus.
• Increased numbers of even small sheets of cells stuck together if catheterized.

Bacteria

• Negative in cystocentesis sample, few when catheterized to low numbers if voided, but free flow samples when mid stream collected well in a sterile container are also usually negative Urinalysis: bacteriology.

Casts

• <1 hyaline casts/low power field (x 10-20 lens) Urinalysis: centrifuge sediment.

Crystals

• May have struvite if more alkaline and stored in vitro especially.
• Bilirubin crystals or staining as per dipstick interpretation.
• Other crystals may be seen as breed related associations, eg Dalmation often have uric acid or urate crystals, Bulldogs with cystine.
• Otherwise can be pathological and associated with urolithiasis or risk of deposition, the crystal habit though and appearance varying due to time, temperature, saturation and pH.

Color

• Milky-floccular: pyuria.
• Yellow-orange: bilirubin
• Red: RBCs, hemoglobin, myoglobin.
• Red-brown: methemoglobin, myoglobin, RBCs, hemoglobin.
• Brown-black: methemoglobin (NB aged degenerating samples).
• Artifacts:
  •  Yellow-orange: sulfasalazine.
  • Rust-yellow: metronidazole, sulfonamide.

Turbidity

• Commonly lipid droplets when collected, especially cystocentesis samples from mesenteric fat or if catheterized, from lubricant gels.
• Pyuria also a common cause.
• Crystalluria.
• Mucus, especially during estrus if an intact female and voided.
• Artifacts:
  • Contaminants from the collection (vessel).
  • Contamination with other fluids, eg seminal and prostatic or vaginal.

Specific gravity

• <1.008 or >1.012 suggests normal renal tubular function.
• Hyposthenuria generally suggests that renal failure is not present. If persistent, especially morning samples, it suggests a lack of ADH (physiologically due to increased/excessive water intake or (central) diabetes insipidus OR resistance to ADH (nephrongenic diabetes insipidus).
• Resistance to ADH may be due to hyperadrenocorticism Hyperadrenocorticism , hypercalcemia Blood biochemistry: total calcium , hypokalemia, myeloma, amyloidosis Amyloidosis , pyelonephritis Kidney: pyelonephritis , drug therapy, or may be congenital.
• A specific gravity >1.007 and <1.030-1.035 in a patient that is clinically dehydrated or is azotemic Azotemia indicates possible renal failure Kidney: chronic kidney disease (CKD).
• Artifacts:
  • Incorrect temperature (not at room temperature).
  • Non-calibrated refractometer (use at least deionized water to confirm baseline SG at 1.000).
  • Exogenous drugs or fluid such as dextran and radiographic contrast agents.

pH

• If <7:
  • Respiratory or metabolic acidosis Acid base imbalance.
  • Diabetic ketoacidosis Diabetic ketoacidosis. Glucose and anion gap Anion gap.
  • Primary renal failure Kidney: acute kidney injury (AKI).
  • Severe vomiting Vomiting. Check electrolytes.
  • Severe diarrhea.
  • Pyrexia Pyrexia: overview.
  • (Starvation due to protein-muscle catabolism.)
• If >7:
  • Transiently post-prandial.
  • Urinary tract infection Cystitis caused by urease-producing bacteria ( eg Staphylococcus spp, Klebsiella spp, Proteus spp, etc).
  • Respiratory or metabolic alkalosis.
  • Vomiting Vomiting especially gastric, or upper gastrointestinal obstruction.
• Artifacts:
  • Delayed analysis and evaporation (increase and alkalize by loss of CO₂).
  • Varying post prandial effects (typically increase).
  • Exogenous drugs: furosemide, Ammonium chloride, ascorbic acid, D-L methionine typically decrease (acidify).

Proteinuria

• Urine protein:urine creatinine ratio Urinalysis: protein >0.2 = protein loss. If >3-5, consider glomerular involvement Glomerulonephritis or amyloidosis Amyloidosis.
  Hypertension, congestive heart failure, urinary tract inflammation or hematuria may produce proteinuria , <3 commonly.
• Nephrotoxic drugs, eg sulfonamide, aminoglycoside, cephaloridine.
• Benign sediment and normal globulins or polyclonal gammopathy is usually due to glomerulonephropathies including: systemic lupus erythematosus Systemic lupus erythematosus , sarcoptic mange Skin: sarcoptic mange , various neoplasias, ehrlichiosis, pyometra Pyometra , chronic inflammatory disease, glomerulonephritis Glomerulonephritis.
• Artifacts:
  • Exogenous drugs such as gabapentin, cross react both dipstick and some reagents.
  • Alkaline sample pH>8 causes a false positive dipstick reaction.
  • Contamination with certain detergents, eg chlorhexidine.
  • Contamination with other fluids, eg seminal and postatic.

Glucosuria

• Proximal renal tubular dysfunction due to early aminoglycoside toxicity, early amphotericin-B toxicity Amphotericin B , acute renal failure Kidney: acute kidney injury (AKI) , Fanconi syndrome Fanconi's syndrome , primary renal glucosuria Urinalysis: glucose.
• Artifacts:
  • Certain exogenous drugs such as cephalosporins and salicylates (increase).
  • Exogenous medication or dietary ascorbic acid in quantity (decrease).
  • Other drugs have very varying effects depending on the enzyme or reaction (eg Formaldehyde increases or decreases).

Ketonuria

• Starvation.
• Prolonged fasting.
• Diabetic ketoacidosis Diabetic ketoacidosis.
• Artifacts:
  • Certain drugs such as phthaleins and sulphydryls (color change immediately cf developing).

Bilirubinuria

• Hepatic disease.
• Post-hepatic bile duct obstruction.
• Hemolysis.

Urobilinogen

• As labile and degraded in UV light, often not particularly useful but its lack supports obstructive cholestatic pathology due to blocked entero-fecal recirculation Urinalysis: urobilinogen.

Hemoglobinuria-myoglobinuria

• Check for hemoglobinaemia (intravascular hemolytic disease - red plasma). Check HCT, blood smear and bilirubin.
• Lastly, consider myoglobinuria (acute muscle trauma, hyperthermia and myositis). Measure serum creatine kinase (CK) and aspartate amino transferase (AST).

Hematuria

• Non-septic inflammation.
• Calculi/uroliths/urolithiasis.
• Coagulopathy.
• Cystic prostatic disease Prostate disease. In entire males - assess serum esterase and imaging.
• Trauma.
• Neoplasia.
• Leptospirosis Leptospirosis.
• Renal infarct Kidney: thromboembolism.
• Urinary parasites.
• Violent or very prolonged exercise.
• Glomerulonephritis Glomerulonephritis.
• Toxicity of cyclophosphomide → cystitis.
• Idiopathic benign renal hematuria.
• Artifacts:
  • ​Contamination with certain detergents, such as hydrogen peroxide.

Nitrituria

• Urinary tract infection Cystitis with nitrate reducing bacteria Urinalysis: nitrate.

Pyuria

• Urinary tract infection Cystitis.
• Sterile cystitis.
• Neoplasia.
• Calculi: urethra, ureter Ureter: urolithiasis.
• Fever.
• Prolonged or violent exercise.
• Glomerulonephritis Glomerulonephritis (rare).

Bacteriuria

• Urinary tract infections Cystitis Urinalysis: bacteriology.
• Contamination from external genitalia (free flow).
• If voided and asymptomatic, bacteria can be seen without causing clinical signs and infection that needs treatment.

Casts

• Hyaline casts: associated with proteinuria Proteinuria.
• Epithelial, fatty, granular and waxy casts: associated with conditions causing degeneration and necrosis of tubular epithelial cells, eg dehydration, nephrotoxins, tubular hypoxia, acute oliguric renal failure Kidney: acute kidney injury (AKI).
• Red blood cell casts Urinalysis: red blood cells: hemorrhage into renal tubules or severe glomerular inflammation.
• White blood cell casts: renal inflammation, upper urinary tract infection, eg pyelonephritis Kidney: pyelonephritis.

Crystalluria

The following may be abnormal

• Struvite  (magnesium ammonium phosphate - rectangular bevelled, 'coffin lid').
• Calcium oxalate : dihydrate (maltese cross/pyramids) and monohydrate (hemp seed).
• Calcium phosphate (amorphous clear).
• Ammonium urate (brown irregular to 'thorny apple').
• Bilirubin (yellow filligree or star-like).
• Cystine (clear hexagonal disks).

• Not using a fresh urine sample.
• Incorrect storage of sample, eg temperature.
• Incorrect handling of sample, eg preservative.
• Contamination from collection method or vessel, eg kidney bowl or jar.
• Unknown collection technique.
• Inconsistent use of SOP or method for analysis, eg volume centrifuged.
• Exogenous drugs.

Refereed papers

• Recent references from VetMedResource and PubMed.
• Ling G V, Norris C R, Franti C E, Eisele P H, Johnson D L, Ruby A L & Jang S S (2001) Interrelations of Organism Prevalence, Specimen Collection Method, and Host Age, Sex and Breed among 8,354 Canine Urinary Tract Infections (1969-1995). JVIM 15(4), 341-347 PubMed.

Other sources of information

• Osbourne C A et al (1999) Urinalysis: A Clinical Guide to Compassionate Patient Care. Bayer.