Week 8: Chemical UA
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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
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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
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Normal: negative
Blood threshold for glucose 175 mg%
Elevated with diabetes (hyperglycemia)
Principle: Glucose oxidase and hexokinase
Glu Glu Ox > Gluconic acid + H2O2
Perosidase
H2O2 + o-tolidine
> 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 b-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