Urine Chemistry Part 2

What does the urine blood reagent strip test for?

The presence of RBCs (hematuria), free hemoglobin (hemoglobinuria), and/or myoglobin (from muscle disease or damage) (myoglobinuria).

What is hematuria, and what are its causes?

The presence of intact RBCs in urine. Causes include kidney stones (which can cause bloody urine), trauma, tumors, glomerular disease (not filtering the blood correctly), and others.

What is hemoglobinuria, and what causes it?

The presence of free hemoglobin in urine. Caused by intravascular hemolysis or lysis of RBCs within the urinary tract.

What is myoglobinuria, and what causes it?

The presence of myoglobin in urine. Caused by muscle trauma, wasting diseases, prolonged coma, and similar conditions.

What is the expected result for the urine blood test?

Negative.

What is the reagent strip reaction for blood?

The "pseudoperoxidase" activity of hemoglobin catalyzes a peroxidase reaction:

H₂O₂ + chromogen → oxidized chromogen + H₂O.

Color changes from yellow → green → blue.

The reaction is read vs 2 color charts. Free hemoglobin or myoglobin produces a uniform color change; whole RBCs produce a speckled pattern because cells lyse on contact with the reagent pad. The test can detect as few as 5 RBCs/µL.

How are hemoglobinuria and myoglobinuria distinguished on the reagent strip?

They cannot be distinguished by the reagent strip alone — both give a uniform color change. The physician reviews the patient's history and may order additional tests. For hemoglobin (hemolysis): CBC (complete blood count), LD, haptoglobin. For myoglobin (muscle): CK, serum myoglobin.

What causes false positives on the blood reagent strip test?

Menstrual contamination, strong oxidizing detergents, and bacterial enzymes (some organisms have peroxidase activity).

What causes false negatives on the blood reagent strip test?

High levels of ascorbic acid; increased specific gravity with crenated RBCs (crenated RBCs may not lyse on the pad so might not show up well); and improper sample mixing.

How is bilirubin normally produced and processed in the body?

Bilirubin is produced during normal degradation of hemoglobin. It binds to albumin and is transported to the liver for conjugation. Unconjugated bilirubin is not water-soluble and cannot be excreted by the kidneys. Conjugated bilirubin is water-soluble and can be excreted by the kidneys. Normally, conjugated bilirubin passes directly into the intestine and is reduced to urobilinogen and urobilin and excreted.

Why does unconjugated bilirubin not appear in urine?

Because unconjugated bilirubin is not water-soluble and therefore cannot be excreted by the kidneys. it wont unless present in large quantities

What is the expected result for urine bilirubin?

Negative.

What is the clinical significance of urine bilirubin?

It is an early indicator of liver disease (because its more than the liver can process) and assists in determining the origin of clinical jaundice (pre-hepatic, hepatic, or post-hepatic).

What are the urine bilirubin and urobilinogen findings in pre-hepatic jaundice?

Urine bilirubin is negative; urobilinogen is increased. The cause is hemolysis — liver function is normal and bilirubin is unconjugated (not water soluble), so it cannot be excreted in urine. Increased heme breakdown leads to more urobilinogen.

What are the urine bilirubin and urobilinogen findings in hepatic jaundice?

Urine bilirubin is usually positive; urobilinogen is increased. The cause is liver injury (cirrhosis, hepatitis). Liver damage causes conjugated bilirubin to leak into circulation, where it passes into the glomerular filtrate and is excreted in urine.

What are the urine bilirubin and urobilinogen findings in post-hepatic jaundice?

Urine bilirubin is increased; urobilinogen is normal (negative). The cause is bile duct obstruction. Conjugated bilirubin passes through the liver normally but cannot get around the obstruction into the intestine, so it backflows into circulation and is excreted in urine. No bilirubin reaches the intestine, so no urobilinogen is produced.

What is the reagent strip reaction for bilirubin?

An azocoupling (diazo) reaction: bilirubin glucuronide + diazonium salt → azo dye. Produces a tan or pink-to-purple color. Reagent strips are sensitive to levels of approximately 0.5 mg/dL.

What causes false positives on the bilirubin reagent strip test?

Highly pigmented samples; Pyridium (phenazopyridine) and other pigmented medications; indican; and Lodine (a medication).

What causes false negatives on the bilirubin reagent strip test?

Light exposure (bilirubin is photosensitive); ascorbic acid; and really high nitrite levels (nitrite may react with the diazonium salt).

What is the confirmatory test for bilirubin and how does it work?

The Ictotest. Uses tablets and a special mat. The mat causes bilirubin to remain on the surface while other interfering substances are absorbed. It is more sensitive than the reagent strip — detects levels as low as 0.05–0.10 mg/dL.

Urobilinogen

What is the expected result for urine urobilinogen?

0.2–1.0 mg/dL (a small amount is normally excreted in urine).

What is the clinical significance of urobilinogen?

Increased values are seen in pre-hepatic jaundice and liver damage. No urobilinogen (absent) is seen in post-hepatic jaundice, although the dipstick result appears normal (the strip cannot detect the absence of urobilinogen).

Why can't the reagent strip detect absent urobilinogen?

Because the strip can only detect the presence of urobilinogen, not its absence. Therefore, a "normal" dipstick result does not rule out post-hepatic jaundice — absence of urobilinogen must be confirmed by other means.

What are the two reagent strip reactions for urobilinogen and how do they differ?

Multistix (Siemens): urobilinogen + Ehrlich's reagent → red/pink color; measured in Ehrlich Units (EU), which equal mg/dL. Chemstrips (Roche): urobilinogen + diazonium salt → red azo dye; measured in mg/dL. The Roche method is more specific than the Multistix method.

What causes false positives on the urobilinogen reagent strip test?

Ehrlich-reactive compounds (there is a large list of these) and highly pigmented urine.

What causes false negatives on the urobilinogen reagent strip test?

Photo-oxidation (urobilinogen oxidizes to urobilin, which is not detected) and formalin (a preservative that interferes with the reaction).

Nitrite

What does the nitrite reagent strip test detect, and what is its clinical use?

It detects the presence of nitrate-reducing bacteria. It is used as a rapid screening test for UTI. It is particularly useful when combined with leukocyte esterase to determine which samples to send for urine culture. It may also be used to evaluate the effectiveness of antibiotics against gram-negative bacilli.

What is the expected result for urine nitrite?

Negative.

What is the reagent strip reaction for nitrite?

The Greiss reaction — a two-part reaction under acidic conditions. Step a: para-arsanilic acid + NO₂⁻ → diazonium salt. Step b: diazonium salt + tetrahydrobenzoquinoline → azo dye (pink color).

What causes false positives on the nitrite reagent strip test?

Old (improperly preserved) specimens and highly pigmented specimens.

What causes false negatives on the nitrite reagent strip test?

Low or no nitrate in the diet (no green vegetables, starvation, parenteral nutrition); UTI caused by bacteria that do not have nitrate reductase (gram-positive organisms or yeast); urine that has not been in the bladder long enough for bacteria to convert nitrate to nitrite; reduction of nitrite beyond nitrite to N₂ (nitrate → nitrite → N₂); ascorbic acid; and antibiotics.

Why might a UTI result in a negative nitrite test?

Not all UTIs are caused by nitrate-reducing bacteria. Gram-positive organisms and yeast do not produce nitrate reductase, so they will not convert nitrate to nitrite. The urine must also have been in the bladder long enough for the reaction to occur.

Leukocyte Esterase

What does the leukocyte esterase reagent strip test detect?

The presence of granulocyte esterase, an enzyme released by white blood cells (WBCs). It is present in urine whether WBCs are intact or degraded, making it useful even when cells have lysed. It is an indicator of UTI, especially when combined with a positive nitrite. It may also be seen in non-bacterial infections such as Trichomonas, Chlamydia, yeast, and renal inflammation.

What is the expected result for leukocyte esterase?

Negative.

What is the reagent strip reaction for leukocyte esterase?

Leukocyte esterase cleaves an indolcarbonic acid ester → indoxyl + acid. Indoxyl + diazonium salt → purple azo dye.

What causes false positives on the leukocyte esterase reagent strip test?

Highly pigmented urine and oxidizing detergents.

What causes false negatives on the leukocyte esterase reagent strip test?

Immunosuppressed patients (decreased WBC production/function); ascorbic acid; and high concentrations of protein.

Why is leukocyte esterase clinically useful even when WBCs are no longer intact?

Because granulocyte esterase is released from WBCs into the urine whether the cells are intact or have already degraded. The enzyme persists in the sample even after cellular disintegration.

Specific Gravity

What is the clinical significance of urine specific gravity?

It is used to monitor hydration status; assess loss of renal tubular concentrating ability; differentiate diabetes insipidus from diabetes mellitus; and for specimen screening purposes.

What is the expected result for urine specific gravity?

1.003–1.035.

What is the reagent strip reaction for specific gravity?

It measures ionic concentration. A color change occurs due to a change in the pKa (dissociation constant) of a polyelectrolyte. H⁺ ions are released from the polyelectrolyte in proportion to the ions present in the solution, affecting a bromthymol blue indicator. Color starts as blue (1.000) and changes to green, then yellow (1.030).

What causes a false increase in specific gravity on the reagent strip?

High protein concentration.

What causes a false decrease in specific gravity on the reagent strip?

pH greater than 6.5, which interferes with the bromthymol blue indicator. Manufacturers suggest adding 0.005 to the result to correct for this, or confirming with an alternate method (e.g., refractometer).

What is a key limitation of the reagent strip specific gravity test?

It only detects ionic solutes. It does not detect non-ionic substances such as urea, glucose, high-molecular-weight IV solutions, or radiographic dye. Therefore, the reagent strip result may not correlate with the refractometer reading when these substances are pr