Urinalysis: Collection Techniques

Introduction to Urinalysis

• Urinalysis evaluates kidney excretions, offering insights into renal function, metabolic and systemic diseases.
• It is used for health screenings, pre-anesthetic assessments, and as a key part of the minimum database.
• Urinalysis is inexpensive, easily performed in-house, and requires minimal specialized equipment.

Urine Collection Techniques

• Early morning samples are more concentrated, post-prandial samples are more alkaline, and samples after fluid or diuretic therapy are more dilute.

Voided Sample (Free Catch)

• This involves catching mid-stream flow of urine.
• It is suitable for urinalysis but not for bacterial culture due to potential contamination.
• Advantages include being non-invasive and easily performed by owners.
• Limitations: high risk of contamination and may not represent bladder urine at a specific time.

Urethral Catheterization

• This uses a urinary catheter for sample collection.
• It is suitable for urinalysis and bacterial culture, though there's a risk of introducing bacteria.
• Advantages include directly obtaining a sample from the bladder.
• Limitations: potentially traumatic, risk of infection, more difficult in females.

Cystocentesis

• A needle is inserted directly into the urinary bladder.
• It is suitable for urinalysis, though blood contamination may occur.
• This is the best method for bacterial culture due to sterile collection, avoiding contamination.
• Advantages: sterile sample ideal for bacterial culture, avoids urethral contamination.
• Limitations: invasive, requires restraint, risk of bladder laceration or puncture of other organs.

Sample Handling

• Analyze urine within 60 minutes of collection.
• Delays at room temperature can cause cellular lysis, crystal development, increased pH, cell and cast degeneration, decreased glucose/ketone/bilirubin levels, and bacterial multiplication.
• Refrigerate samples for up to 12 hours if analysis is delayed.
• Use a sterile, opaque, airtight container.
• Warm refrigerated samples to room temperature and re-suspend sediment before analysis.

Gross Inspection: Color

• Pink-Red urine: may indicate hematuria, hemoglobinuria, or myoglobinuria.
• Red-Brown urine: may indicate hematuria, hemoglobinuria, or myoglobinuria.
• Brown-Black urine: may indicate methemoglobinuria or bile pigments.
• Yellow-Orange urine: may indicate highly concentrated urine or bilirubinuria.
• Yellow-Green urine: may indicate bilirubin or biliverdin.
• Pigmenturia (HGB, MGB) retains a red supernatant after centrifugation
• Hemoglobinuria indicates hemolysis, check for anemia and pink/red serum.
• Myoglobinuria indicates muscle injury, check for increased CK and AST.

Gross Inspection: Clarity

• Clarity can be described as clear, slightly cloudy, cloudy, opaque, or flocculent.
• Cloudiness is caused by crystals, cells, bacteria, or lipid.

Urine Specific Gravity (USG)

• USG evaluates dissolved molecules ("solutes").
• Measure at room temperature, as cold fluids falsely increase USG.
• Increased USG is due to significant increases in glucose, protein, sodium, or urea.
• Interpret USG with patient's clinical hydration status.

Urine Dipstick (Chemical Analysis)

• Most analytes are graded semi-quantitatively from negative to 4+.
• Dipstick tests used in veterinary medicine include glucose, bilirubin, ketones, heme/blood, pH, and protein.
• Ignore USG, leukocytes, nitrite, urobilinogen, and ascorbic acid results.

Urine Dipstick: Glucose

• Glucosuria happens when the reabsorption capacity is exceeded by glucose levels (Dog ~220 mg/dL, Cat ~280 mg/dL, Horse ~180 mg/dL, Cattle ~100 mg/dL).
• Hyperglycemic glucosuria (most common) causes include diabetes mellitus, acute pancreatitis, stress (especially in cats), glucose-containing fluids, requires knowing serum glucose for interpretation.
• Normoglycemic glucosuria (less common) causes include acute kidney injury, reversible tubular damage (drugs, toxins), urethral obstruction in cats, and Fanconi syndrome.

Urine Dipstick: Bilirubin

• Bilirubinuria precedes bilirubinemia due to low renal threshold.
• Trace to 1+ bilirubinuria can be normal in dogs.
• Bilirubinuria is always abnormal in cats.
• Differential diagnoses for bilirubinuria include hemolysis and hepatobiliary disease.
• False negatives can occur with delayed processing or light exposure.

Urine Dipstick: Ketones

• Dipsticks detect acetoacetate and acetone.
• Ketones are underestimated by dipsticks as they don't detect β-hydroxybuterate.
• Ruminants primarily produce β-hydroxybuterate.
• Differential diagnoses for ketonuria include diabetic ketosis (DKA), negative energy balance (starvation), and ketosis in cattle.

Urine Dipstick: Heme / Blood

• Detects intact RBCs (hematuria), hemoglobin (hemoglobinuria), or myoglobin (myoglobinuria).
• Hematuria (RBCs): most common cause, supernatant clears with centrifugation- DDx: Inflammation, Infection, Trauma, Coagulopathy.

Urine Dipstick: pH

• Normal pH is 5.0-7.5 in dogs and cats, varies with diet, collection time, and systemic acid-base status.
• Acidic urine is associated with meat-protein diet, acidifying agents, metabolic/respiratory acidosis, protein catabolic states, and ethylene glycol ingestion.
• Alkaline urine is associated with plant protein diet, postprandial alkaline tide, UTI with urease-producing bacteria, sample exposed to air, alkalinizing agents, and metabolic/respiratory alkalosis.

Urine Dipstick: Protein

• Primarily detects albumin (negative charge).
• It has low sensitivity and low specificity.
• False positives occur with alkaline urine, cats (cauxin), and hypersthenuria.
• False negatives: Lower limit of 20-30 mg/dL of albumin, insensitive to globulins and Bence-Jones proteins.
• When interpreting positive protein: 1. Hemorrhage (look for RBCs in sediment), 2. UTI/Cystitis (look for bacteria & WBCs in sediment), 3. Intravascular hemolysis (hemoglobinuria, animal will be anemic).
• Proteinuria caused by renal disease will have a negative blood reaction.
• Measure a Urine Protein:Creatinine Ratio (UPCR).
• Proteinuria with nothing in the sediment often indicates glomerular disease.

Sulphosalicylic Acid Test (SSA)

• It is used to confirm dipstick protein results.
• It reacts with albumin, globulins, and Bence-Jones proteins.
• Evaluated by turbidity. Interpret dipstick and SSA results together with USG, chemistry panel, and clinical signs.

Specific Proteinuria Disorders

• These include Nephrotic syndrome and Fanconi syndrome.
• Fanconi Syndrome: Proximal tubular defect causing impaired reabsorption of glucose, amino acids, and phosphate, resulting in proteinuria and normoglycemic glucosuria. Congenital (Basenjis) or acquired.

Microscopic Examination of Sediment: Procedure

• Centrifuge urine at low speed for 5 minutes.
• Decant urine, leaving ~500 uL for resuspension.
• Re-suspend the sediment.
• Transfer a drop to a glass slide and place a coverslip.
• Lower the condenser on the microscope.
• Examine the entire coverslip, especially the edges.

Microscopic Examination of Sediment: Evaluation

• Evaluate at 10x objective for epithelial cells, crystals, and casts.
• Evaluate at 40x objective for RBCs, WBCs, fat, and microorganisms.

Urine Sediment: Cells

• Squamous epithelial cells: Large, thin, transparent, angular or folded. Seen in free-catch urine, rarely pathologic (e.g., Sertolli cell tumor causing squamous metaplasia).
• Transitional epithelial cells: Round, vary in size, found individually or in clusters. Significance: Hyperplasia associated with inflammation, transitional cell tumors (benign or malignant). Expert diagnosis recommended for clusters.

Urine Sediment: Epithelial Cell Source

• Caudate cells (renal pelvis): RARE, may indicate pyelonephritis.
• Renal cells (renal tubules): RARE, may indicate renal tubular injury.
• Red Blood Cells (RBCs): Round, often yellow-tinged, may be crenated. Enumerated as #/hpf (40x). Significance: Hemorrhage, Inflammation. Remember microscopic hematuria is expected in cystocentesis samples.
• White Blood Cells (WBCs): ~2x larger than RBCs, cytoplasm looks granular. 5 WBCs/hpf = pyuria.

Urine Sediment: WBCs

• General causes of pyuria: Urinary tract infection (pyelonephritis or lower UTI - should culture if suspected even if no bacteria are visible), non-infectious inflammation (cystitis), neoplasia, prostatitis.

Urine Sediment: Bacteria

• Usually needs >10,000 rods/mL or >100,000 cocci/mL to be visible.
• Report as few to many or present vs. absent.
• Expect neutrophils unless immunosuppressed.
• Culture anaerobic organisms, quantitative measurement, sensitivity with MIC.

Urine Sediment: Other infectious organisms

• Significance depends on clinical signs, collection method, sample storage, patient's immune status, and other diagnostic findings.
• Examples include Dioctophyma renale, Capillaria ovum, Dirofilaria immitis.

Urine Sediment: Crystalluria Factors

• In vivo: Concentration and solubility of crystalline material, urine pH, diet, excretion of drugs or diagnostic imaging agents.
• In vitro: Temperature (solubility decreases with decreased temperature), evaporation (increases solute concentration), urine pH (changes with standing urine and bacterial overgrowth).

Urine Sediment: Common Crystals

• Amorphous: Yellow to yellow-brown aggregates of finely granular material, no defining shape. Significance: None.
• Struvite (triple phosphate or magnesium ammonium phosphate): Large, colorless, prism-like (“coffin lids”). Most common crystal in dogs and cats. Formation favored in neutral to alkaline pH. Can be found in clinically normal patients. Urease-positive bacteria promote formation (↑urine pH).
• Bilirubin: Orange to copper granules, usually in small bundles. Dogs: low numbers in concentrated urine can be normal. Abnormal in all other species, indicating icterus (pre-hepatic/hemolysis or hepatic/post-hepatic disease).
• Calcium carbonate: Large spheres with radial striations, variably-sized, colorless to yellow-brown. Normal in horses, rabbits, guinea pigs, and goats. No reports in canine or feline urine.
• Calcium oxalate dihydrate: “Envelope” shape, colorless squares connected by intersecting lines, vary in size. Normal in domestic animals, can be a storage artifact. Pathologic in Miniature Schnauzers (predisposed) and with hypercalcemia causing increased calcium excretion or acute renal failure.

Urine Sediment: Uncommon Crystals

• Ammonium bitrate: “Thorn apples,” spiked spheres, brown or yellow-brown. Normal in Dalmatians and English bulldogs. Pathologic: Suggests liver disease (associated with ↓BUN, ↑NH3, liver failure, congenital or acquired shunts).
• Calcium oxalate monohydrate: Spindle (“picket-fence”), oval (“hemp seed”), or dumbbell shaped, usually small and colorless, vary in size. Can be seen in healthy animals, especially horses. Pathologic: Oxalate urolithiasis, hypercalciuric or hyperoxaluric disorders, ethylene glycol toxicosis (rare).
• Cystine: Flat colorless hexagonal plates, often aggregate in layers. Significance: Inherited disease Cystinuria (defective renal tubular resorption of certain amino acids), occurs almost exclusively in male dogs, many breeds affected, renal function otherwise normal.
• Drug-associated crystals: Correlate with history! Vary in appearance and size, frequently light to dark brown, commonly form needles arranged into sheaves, bundles, fans, and radiating spikes. Most drugs excreted in urine can form crystals (antibiotics like sulfa drugs, ampicillin, ciprofloxacin; anticonvulsants; allopurinol; radiographic contrast media).

Urine Sediment: Casts

• Composed of Tamm-Horsfall protein secreted by the tubular epithelium, formed in loops of Henle, distal tubules, and collecting ducts.
• Formed in acidic urine, may dissolve in alkaline urine.
• Formation depends on urine acidity, solute concentration, and flow rate.
• May be intermittently shed and break down during sedimentation.
• May indicate tubular damage and/or urine stasis within the tubules.
• Increased numbers = cylindruria.

Urine Sediment: Cast Types

• Hyaline casts: >2 hyaline casts/10x objective is significant.
• Cellular casts: Composed of epithelial cells, RBCs, and/or WBCs. Indicate significant tubular damage and/or inflammation.
• Granular casts: Indicate cellular degeneration.
• Waxy casts: Indicate chronicity.

Urine Sediment: Fat Droplets

• Commonly seen (especially in feline urine) and usually insignificant.
• Result from degeneration of epithelial cells.
• Differentiated from RBCs and WBCs by being in a different plane of focus, variable in size but more uniformly round than cells.

Urine Sediment: Mucus

• Commonly seen in equine urine.
• Can resemble hyaline casts but is more irregularly shaped with tapered ends.

Urine Sediment: Environmental Contaminant

• Can mimic parasite larvae or urinary casts (e.g., FIBERS).