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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