9.1 Physical and Chemical Examination of Urine and Reagent Strip Principles

Physical and Chemical Examination of Urine

Quick Answer: The routine urinalysis involves physical examination (color, clarity, specific gravity) and chemical analysis via reagent strips. Understanding the underlying principles, such as the glucose oxidase reaction for glucose or the protein error of indicators for albumin, is critical for identifying false positives and negatives.

Physical Examination

Color and Clarity

Normal urine color ranges from pale yellow to amber, primarily due to the pigment urochrome. Variations in color often provide the first clue to underlying pathology or dietary/medication effects:

• Colorless to Pale Yellow: Dilute urine, diabetes insipidus, or diabetes mellitus.
• Dark Yellow to Amber: Concentrated urine, dehydration, or the presence of bilirubin (which may produce yellow foam when shaken).
• Red or Pink: Hematuria, hemoglobinuria, myoglobinuria, porphyrins, or dietary intake (e.g., beets).
• Brown or Black: Melanin, homogentisic acid (alkaptonuria), or methemoglobin.
• Orange: Pyridium (phenazopyridine), a urinary tract analgesic, which frequently interferes with color-based chemical tests.
• Green or Blue: Pseudomonas infection, amitriptyline, or IV contrast dyes.

Clarity (turbidity) is normally clear. Pathologic turbidity is most commonly caused by WBCs, RBCs, or bacteria. Non-pathologic turbidity can result from squamous epithelial cells, mucus, or normal amorphous crystals (amorphous urates in acid urine, amorphous phosphates in alkaline urine).

Specific Gravity (SG)

Specific gravity measures the kidney's ability to concentrate urine. Normal SG ranges from 1.003 to 1.035.

• Refractometer: Measures the refractive index of light passing through urine, comparing the velocity of light in air to that in the solution. It requires correction for high concentrations of glucose (-0.004 for each gram/dL) and protein (-0.003 for each gram/dL).
• Reagent Strip SG: Based on the pKa change of a polyelectrolyte in relation to ionic concentration. Unlike the refractometer, the reagent strip method is not significantly affected by large non-ionic molecules like glucose or radiographic contrast media.

Exam Trap: If a patient's SG is >1.040 by refractometer but normal by reagent strip, suspect radiographic contrast media.

Chemical Examination: Reagent Strip Principles

Quality control (QC) is mandatory for reagent strips, typically performed daily using positive and negative controls. Strips must be stored in a tightly capped container with a desiccant, protected from light and moisture.

pH

• Principle: Double indicator system using methyl red and bromothymol blue.
• Clinical Significance: Identifies acid-base disorders, assists in crystal identification, and indicates sample age (old unpreserved urine becomes highly alkaline due to urea breakdown by bacteria).
• Normal Range: 4.5 to 8.0.

Protein

• Principle: Protein error of indicators. The reagent pad contains tetrabromphenol blue buffered to an acidic pH (pH 3.0). Proteins (acting as H+ acceptors) alter the indicator's color from yellow to green/blue without changing the pH.
• Specificity: Highly sensitive to albumin but much less sensitive to globulins, Bence Jones proteins, or mucoproteins.
• Interferences: False positives occur with highly alkaline urine (overcomes the buffer), quaternary ammonium compounds (cleansers), or prolonged dipping.

Glucose

• Principle: Double sequential enzyme reaction using glucose oxidase and peroxidase. Glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide. Peroxidase then catalyzes the oxidation of a chromogen by H2O2.
• Specificity: Specific for glucose; does NOT detect other reducing sugars (galactose, fructose). The Clinitest (copper reduction test) is used to screen neonates for galactosemia.
• Interferences: False negatives from high doses of Ascorbic Acid (Vitamin C), which reduces the H2O2 before it can oxidize the chromogen.

Ketones

• Principle: Sodium nitroprusside reaction. Acetoacetic acid reacts with sodium nitroprusside in an alkaline medium to produce a purple color.
• Specificity: Detects acetoacetic acid. Does NOT detect beta-hydroxybutyrate (the most abundant ketone in diabetic ketoacidosis) and only slightly detects acetone.
• Clinical Significance: Diabetic ketoacidosis, starvation, vomiting, or strenuous exercise.

Blood

• Principle: Pseudoperoxidase activity of hemoglobin or myoglobin. Heme catalyzes the oxidation of a chromogen by hydrogen peroxide to form a blue-green color.
• Differentiation:
  • Intact RBCs (Hematuria): Speckled pattern on the pad.
  • Free Hemoglobin (Hemoglobinuria): Uniform color. Serum is typically pink/red (haptoglobin depletion).
  • Myoglobin (Myoglobinuria): Uniform color. Serum is clear; associated with rhabdomyolysis or crush injuries.
• Interferences: False negatives from Ascorbic Acid.

Bilirubin

• Principle: Diazo reaction. Bilirubin reacts with a diazonium salt in an acidic medium to form azobilirubin (tan/pink/violet).
• Clinical Significance: Detects conjugated bilirubin (water-soluble), indicating biliary obstruction or hepatic disease. Unconjugated bilirubin cannot pass through the glomerulus.
• Interferences: False negatives due to light exposure (bilirubin breaks down) or ascorbic acid.

Urobilinogen

• Principle: Ehrlich's aldehyde reaction (p-dimethylaminobenzaldehyde).
• Clinical Significance: Normal amounts are small. Increased in hemolytic anemias and liver disease. Absent in complete biliary obstruction (no bilirubin reaches the gut to be converted).
• Exam Trap: Reagent strips cannot detect an absence of urobilinogen.

Nitrite

• Principle: Greiss reaction. Certain bacteria reduce urinary nitrate to nitrite, which reacts with an aromatic amine to form a diazonium salt, coupling to form a pink azo dye.
• Clinical Significance: Rapid screen for urinary tract infection (UTI).
• Interferences: Requires sufficient time in the bladder (approx. 4 hours) for bacteria to convert nitrate to nitrite. False negatives occur if the pathogen does not possess nitrate reductase (e.g., Gram-positive cocci like Enterococcus or S. saprophyticus).

Leukocyte Esterase (LE)

• Principle: Cleavage of an ester by leukocyte esterase to form an aromatic compound, which couples with a diazonium salt to produce a purple color.
• Clinical Significance: Indicates the presence of granulocytic WBCs (neutrophils, eosinophils, basophils, monocytes, but NOT lymphocytes).
• Interferences: False positives from vaginal contamination; false negatives from high specific gravity (crenates WBCs, preventing enzyme release).

Summary of Common Interferences

Ascorbic acid is a notorious reducing agent that classically causes false negatives for Glucose, Blood, Bilirubin, and Nitrite.

Confirmatory Chemical Tests (AMT Urinalysis Competency)

Reagent-strip positives that are clinically critical or analytically ambiguous should be confirmed with the classic wet-chemistry methods named on the AMT outline:

Exam trap: A positive SSA with a negative or trace strip protein suggests non-albumin proteinuria (e.g., light chains). A positive Clinitest with negative glucose oxidase strip suggests a non-glucose reducing sugar (e.g., galactose in neonates)—investigate, do not ignore.