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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 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
