Week 8: Chemical UA

• Sensitivity and specificity

• Test principles

• Reagent strip tests

• Back-up confirmatory tests

• Interfering substances: false positives and negatives

Urine Chemistries

Test Performance

• Sensitivity: minimum concentration

• Specificity: discrimination

• Interference factors

pH

• Normal: 4 - 8 (mean 6)

• Control blood pH

• Principle: Methyl red and Bromthymol blue double indicator method

• Report to nearest 0.5 pH unit

• Help identify crystals

Protein

• Normal: negative• Sensitive indicator of renal diseases

• Glomerulonephritis• Pyelonephritis

• Malignant hypertension• Preeclampsia• Severe exercise• Principle: Protein error of pH indicator

• Tetrabromphenyl blue buffered at pH 3.2

• Watch for false positive in alkaline urine

Back-Ups for Protein

• 3% sulfosalicylic acid (SSA)• 0 to 4+ depending

on precipitation

• Trichloroacetic (TCA) acid

• Heat and acetic acid

Microalbuminuria

• > 20 mg/L (30-300 mg/day) albumin in urine• Not detectable with reagent strip (6-15 mg/dL) or

SSA (5 mg/dL)• Predictive of nephropathy and eventual renal

failure in patients with type I diabetes mellitus• Detect patients with increased risk of renal and

cardiovascular disease, associated with insulin resistance and endothelial dysfunction

• Albumin to creatinine ratio corrects for hydration level

Glucose

• Normal: negative• Blood threshold for glucose 175 mg%• Elevated with diabetes (hyperglycemia)• Principle: Glucose oxidase and hexokinase

Glu Glu Ox > Gluconic acid + H2O2

H2O2 + o-tolidine Perosidase > color

• Specific for glucose• Sensitive to redox agents

Back-Ups for Glucose

• Lily TesTape• Specific to glucose

• Clinitest (Benedict’s)• Non-specific for any

• reducing agents

• Cupper sulfate reduction

• Cu++ (blue) + Glu ——> CuO (yellow-red)

Ketone Bodies

• Normal: negative• Elevated with diabetic ketoacidosis,

starvation• Most sensitive to acetoacetate, less sensitive

to acetone, not sensitive to -hydroxy butyrate

• Principle: Sodium nitroprusside Legal’s test• Some have glycine to increase sensitivity to

acetone

Back-Ups for Ketone

• Acetest table test• Same principle as

reagent strip

• Gerhardt’s• FeCl3 non-specific test

Urobilinogen

• Normal: up to 1 Ehrlich unit/dL (not negative)

• Negative in complete obstruction of common bile duct

• Elevated in liver disease and hemolytic anemia

• Principle: p-dimethylaminobenzaldehyde, azocoupling in acid to form pink azo dye

Back-Up for Urobilinogen

• Watson-Schwartz• Extraction with chloroform and butanol

• Not commonly done

Bilirubin

• Normal: negative• Liver disease

• Viral hepatitis• Cirrhosis

• Obstructive jaundice• Bile stone in common bile duct• Pancreatic cancer

• Principle: Diazonium salt reaction, azo coupling in acid to form purple azo dye

Back-Ups for Bilirubin

• Ictotest• Same principle as

reagent strip

• Harrison’s spot test• Fouchet’s reagent (TCA)

Blood

• Normal: negative

• Hematuria, hemoglobinuria, myoglobinuria

• Principle: Hb used as catalyst for o-tolidine or benzidine oxidation (pseudo-peroxidase activity)

• If hematuria, should see red cells

• Menstrual contamination

• Bacterial peroxidase can cause false positive

Nitrite

• Some bacteria reduce nitrate to nitrite

• Diazotization with aromatic amine like p-arsanilic acid or sulfanilamide

Leukocyte Esterase

• Leukocyte (neutrophil) esterase cleave an ester which is azocoupled with aromatic amine

• Correlate with microscopic WBC

Specific Gravity

• Measure ionic solute

• As protons are released from polyelectrolytes, pH decreases that change bromthymol blue indicator

Ascorbic Acid

• Ascorbic acid reduces a dye causing color change

• 2,6-dichlorophenolinedophenol

Quality Control and Quality Assurance

Homework

• Construct a table tests: stix and back ups

Test Principle Sens Spec False Pos False Neg