Week 3 - Technical Considerations 2

What is the principle behind the Bilirubin test on reagent strips?

Diazo Reaction

What reagent is used for bilirubin testing?

2,4-dichloroaniline diazonium salt

What causes a false positive in bilirubin testing?

Highly pigmented urine, Phenazopyridine, Indican, Lodine metabolites

Why does highly pigmented urine cause a false positive bilirubin result?

It may mask or mimic the test pad color

What is Indican and how does it affect bilirubin test?

A substance from intestinal disorders; reacts with the reagent

What causes false negative bilirubin test?

Photo-oxidation, Ascorbic acid, High nitrite

Why does ascorbic acid cause a false negative bilirubin result?

It is a reducing agent that inhibits the color reaction

How does light affect bilirubin test?

Bilirubin is light-sensitive; photo-oxidation breaks it down

Why does high nitrite interfere with bilirubin testing?

It interferes with the diazo reaction

What is the principle behind the Urobilinogen test?

Diazo Reaction

What reagent is used for urobilinogen testing?

4-methylbenzene-diazonium tetrafluoroborate

What causes false positive urobilinogen test?

Highly pigmented urine

What causes false negative urobilinogen result?

Old sample, Formalin, High nitrite, Photo-oxidation

Why does formalin cause a false negative in urobilinogen testing?

It inhibits the diazo reaction

What effect does light exposure have on urobilinogen in urine?

It causes decomposition → false negative

Why does high nitrite cause false negative in urobilinogen test?

It interferes with the diazo coupling reaction

What is the principle behind Ehrlich’s-based nitrite test?

Ehrlich’s Aldehyde Reaction

What reagent is used in Ehrlich’s-based nitrite testing?

p-dimethylaminobenzaldehyde

What color change occurs in Ehrlich’s aldehyde reaction?

Pink to red Schiff base

What can cause false positives in Ehrlich’s-based nitrite tests?

Porphobilinogen, Indican, Certain drugs, Highly pigmented urine

Which drugs can cause false positive nitrite test?

p-aminosalicylic acid, Sulfonamides, Methyldopa, Procaine, Chlorpromazine

What causes false negative in Ehrlich’s-based nitrite testing?

Old sample, Formalin, Photo-oxidation

What is the principle behind the standard nitrite reagent strip test?

Griess Reaction

What are the reagents used in Griess reaction for nitrite?

p-arsanilic acid and Tetrahydrobenzoquinoline

What happens in the Griess Reaction?

Nitrite forms diazonium salt → reacts to produce colored compound

What causes false positive in Griess reaction nitrite test?

Improper preservation, Bacterial contamination, Highly pigmented urine

Why does bacterial contamination cause false positive nitrite?

Contaminants convert nitrate to nitrite after collection

What causes false negative nitrite test in Griess reaction?

Ascorbic acid, High specific gravity, Short contact time, Low nitrate diet, Nitrite reducers, Antibiotics

Why does a low-nitrate diet cause false negative nitrite result?

No nitrate → no substrate for bacteria to form nitrite

How do antibiotics lead to false negative nitrite test?

They kill or inhibit bacteria that convert nitrate to nitrite

Why does ascorbic acid cause false negative nitrite result?

It inhibits the chemical reaction as a reducing agent

What is the principle behind the Leukocyte Esterase test?

Hydrolysis of Acid Ester

What reagents are used in leukocyte esterase testing?

Derivatized Pyrrole Acid Ester, Diazo Salt

What causes false positive leukocyte esterase results?

Strong oxidizing agents, Formalin, Highly pigmented urine, Nitrofurantoin, Beets, Phenazopyridine

Why does nitrofurantoin cause a false positive leukocyte esterase result?

It colors the urine brown, interfering with interpretation

How do beets and phenazopyridine interfere with leukocyte esterase testing?

They cause red or red-orange urine that mimics the test pad color

What causes false negative leukocyte esterase results?

Ascorbic acid, Oxalic acid, High protein (>500 mg/dL), Glucose 3 g/dL, High specific gravity, Antibiotics, Reading too early

Why does high specific gravity cause false negative leukocyte esterase?

It causes WBC crenation, reducing enzyme release

Why is timing critical for leukocyte esterase test?

It has the longest required reading time (2 minutes)

Why does ascorbic acid cause a false negative in leukocyte esterase testing?

It inhibits the color reaction as a reducing agent

What is the principle behind the Specific Gravity reagent strip test?

pKa change of Polyelectrolyte

What reagents are used in specific gravity testing?

Polyelectrolyte and Bromthymol Blue

How does the reagent strip detect ionic concentration?

Ions interact with polyelectrolyte → release H⁺ → pH indicator changes color

What color change indicates high specific gravity on reagent strip?

More acidic → yellow color

What causes falsely increased specific gravity readings?

Protein >100–500 mg/dL, Ketoacidosis/Ketonuria

How does ketonuria falsely increase SG readings?

Alters pH, making bromthymol blue appear more acidic

What causes falsely decreased specific gravity readings?

Urine pH ≥ 6.5

How is a falsely low SG reading corrected?

Add 0.005 to the observed SG result