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Special Tests Chemistry

Special Tests Chemistry. Glucose Bilirubin Ketones. Negative. Trace (100 mg/dL). + (250 mg/dL). ++ (500 mg/dL). +++ (1000 mg/dL). ++++ (2000+ mg/dL). Glucose Dipstick :. Chemical Principle. Glucose Oxidase. Glucose + 2 H 2 O + O 2 ---> Gluconic Acid + 2 H 2 O 2. Horseradish Peroxidase.

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Special Tests Chemistry

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  1. Special Tests Chemistry Glucose Bilirubin Ketones

  2. Negative Trace (100 mg/dL) + (250 mg/dL) ++ (500 mg/dL) +++ (1000 mg/dL) ++++ (2000+ mg/dL) GlucoseDipstick: Chemical Principle Glucose Oxidase Glucose + 2 H2O+ O2---> Gluconic Acid + 2 H2O2 Horseradish Peroxidase 3 H2O2 + KI ---> KIO3 + 3 H2O Read at 30 seconds RR: Negative

  3. Uses and Limitations of Urine Glucose Detection Significance • Diabetes mellitus. • Renal glycosuria. Limitations • False Neg: reducing agents, ketones. • False Pos: oxidizing detergents • Only measures glucose and not other sugars (Glucose Oxidase) • Renal threshold must be passed in order for glucose to spill into the urine. Other Tests Clinitest • CuSO4 test for reducing sugars.

  4. Glucose • Clinical significance • Major screening test for diabetes mellitus • Renal threshold is 160 to 180 mg/dL • Higher blood sugar = glycosuria • Gestational diabetes • Placental hormones block action of insulin • High fetal glucose stresses baby’s pancreas • Result is fat baby • Mother prone to type 2 diabetes

  5. GlucoseClinical Significance • Elevated blood glucose, diabetes mellitus • Renal threshold is ~160 to 180 mg/dL • Higher blood sugar = glycosuria • Collection under controlled conditions • Fasting specimen • “Second” collection • 2 h postprandial

  6. Nondiabetic Glycosuria • Hormonal disorders: pancreatitis, pancreatic cancer, acromegaly, Cushing’s syndrome, hyperthyroidism, pheochromocytoma • Hormones: glucagon, epinephrine, cortisol, thyroxine, growth hormone oppose glucose • Insulin: converts glucose to storage glycogen • Hormones: glycogen back to glucose • Epinephrine: inhibits insulin; seen with stress, cerebral trauma, and myocardial infarction

  7. Renal Glycosuria • Tubular reabsorption disorder • End-stage renal disease • Cystinosis • Fanconi syndrome • Temporary lowering of renal threshold in pregnancy

  8. GlucoseReagent Strip Reactions • Glucose oxidase reaction specific for glucose • Glucose oxidase, peroxide, chromogen, buffer on test pad • Double sequential enzyme reaction • Glucose oxidase catalyzes a reaction between glucose and oxygen • Produces gluconic acid and peroxide • Peroxidase catalyzes the reaction between peroxide and chromogen to form an oxidized colored compound • Direct proportion to the concentration of glucose

  9. Glucose Reagent Strip Reactions Glucose oxidase Glucose + O2 (air) → gluconic acid + H2O2 Peroxidase H2O2 + chromogen → oxidized colored chromogen + H2O

  10. GlucoseReagent Strip • Chromogens used • Potassium iodide (green to brown) (Multistix) • Tetramethylbenzidine (yellow to green) (Chemstrip) • Reporting results • Neg, trace, 1+, 2+, 3+, 4+ • 100 mg/dL to 2 g/dL • 0.1% to 2%

  11. GlucoseReaction Interference • False-positive: only peroxide, oxidizing detergents • False-negative: enzymatic reaction interference • Ascorbic acid and strong reducing agents • High levels of ketones (unlikely) • High specific gravity and low temperature *Greatest source of error is old specimens • Subjecting the glucose to bacterial degradation

  12. Glucose and other Reducing SugarsCopper Reduction Test (Clinitest) • Reduction of copper sulfate to cuprous oxide with alkali and heat • Clinitest tablets: copper sulfate, sodium carbonate, sodium citrate, sodium hydroxide • Sodium citrate + NaOH = heat • Sodium carbonate = CO2 blocks room air • Reducing substance + CuSO4 • Color change: negative blue (CuSO4) through green, yellow, and orange/red (Cu2O)

  13. Copper Reduction Test Heat CuSO4 (cupric sulfide) + reducing substance ----- Alkali Cu2O (cuprous oxide) + oxidized substance → color (blue/green to orange/red)

  14. Clinitest Procedure • Pass through • High levels of reducing substance • Color from blue through red back to green-brown: rapid reaction • Repeat with two-drop procedure • 10 drops water • 2 drops urine • Values up to 5 g/L versus 2 g/L • Separate chart must be used • Hygroscopic tablets: strong blue color and excess fizzing = deterioration

  15. Reducing Substances • Not a specific test for glucose • Sensitivity: 200 mg/dL (lower) than strip • Clinitest does not provide a confirmatory test for glucose • Interference from reducing sugars • Galactose, lactose, fructose, maltose, pentoses, ascorbic acid, cephalosporins • Major use is quick screen for “inborn error of metabolism” in children up to 2 years old • Newborn screening programs for galactosemia in all states

  16. KetonesMetabolism

  17. Ketones • Three intermediate products of fat metabolism • Acetone: 2% • Acetoacetic acid: 20% • β-hydroxybutyrate: 78% • Appear in urine when body stores of fat must be metabolized to supply energy

  18. KetonesClinical Significance • Increased fat metabolism = inability to metabolize carbohydrate • Primary causes • Diabetes mellitus • Vomiting (loss of carbohydrates) • Starvation, malabsorption, dieting (↓ intake) • Ketonuria shows insulin deficiency • Monitor diabetes • Diabetic ketoacidosis = increased accumulation of ketones in the blood • Electrolyte imbalance, dehydration, and diabetic coma

  19. KetonesClinical Significance (cont’d) • Ketonuria unrelated to diabetes • Inadequate intake/absorption of carbohydrates • Vomiting • Weight loss • Eating disorders • Frequent strenuous exercise

  20. KetonesReagent Strip Reactions • Primary reagent: sodium nitroprusside • (Nitroferricyanide) • Measure primarily acetoacetic acid • Assumes the presence of β-hydroxybutyrate and acetone • Acetoacetic acid (alkaline) + nitroprusside → purple color

  21. KetonesReagent Strip Reactions (cont’d) • Report qualitatively • Negative • Trace • Small (1+) • Moderate (2+) • Large (3+) • Semiquantitatively • Negative • Trace (5 mg/dL) • Small (15 mg/dL) • Moderate (40 mg/dL) • Large (80 to 160 mg/dL)

  22. Ketones Reagent Strip Reactions acetoacetate (and acetone) + sodium nitroprusside Alkaline + (glycine) ——————> purple color

  23. KetonesReaction Interference • Levodopa in large dosage • Medications containing sulfhydryl groups • May produce atypical color reactions • False-positive results from improperly timed readings • Falsely decreased values in improperly preserved specimens • Breakdown of acetoacetic acid by bacteria

  24. Acetest • Not a urine confirmatory test • Tablet = sodium nitroprusside, glycine, disodium phosphate, lactose (gives better color)

  25. Negative + (weak) ++ (moderate) +++ (strong) BilirubinDipstick: Chemical Principle Acidic Bilirubin + Diazo salt ---------> Azobilirubin Read at 30 seconds RR: Negative

  26. Bilirubin • Urine bilirubin early indicator of liver disease • Normal degradation product of hemoglobin • RBCs destroyed by liver and spleen following 120-day life span • Body recycles iron, protein • Protoporphyrin is broken down into bilirubin • Bilirubin is bound to albumin • Kidneys cannot excrete • Unconjugated bilirubin: water insoluble

  27. Bilirubin (cont’d) • Conjugated bilirubin: water soluble • Unconjugated bilirubin to the liver • Conjugated with glucuronic acid • Forms conjugated bilirubin • From liver to intestines • Reduced to urobilinogen, stercobilinogen, and urobilin by intestinal bacteria • Excreted in feces

  28. Bilirubin Clinical Significance • Conjugated bilirubin appears in urine with bile duct obstruction, liver disease or damage • Obstruction: bilirubin backs up into circulation and is excreted in urine • No urobilinogen is formed • Hepatitis, cirrhosis: conjugated bilirubin leaks back into circulation from damaged liver; some bilirubin passes to intestine • Hemolytic disease: increased unconjugated bilirubin, increased urobilinogen • Unconjugated Bilirubinnot water soluble Not found in Urine

  29. BilirubinReagent Strip Reactions • Principle is a diazo reaction • Report: neg, small (1+), moderate (2+), large (3+) • Colors may be difficult to interpret • Easily influenced by other pigments present in the urine • Atypical colors can be problem for automated readers

  30. Bilirubin Reagent Strip Reactions acid bilirubin glucuronide + *diazonium salt-------- azodye (tan or pink to violet) * diazonium salt- (2,4-dichloroaniline diazonium salt or 2,6-dichlorobenzene-diazonium-tetrafluoroborate)

  31. Bilirubin Reaction Interference • False-positive • Urine pigments • Pyridium (phenazopyridine) • Drugs indican, iodine • False-negative • Old specimens (biliverdin does not react) • Ascorbic acid >25 mg/dL • Nitrite • Combine with diazonium salt and block bilirubin reaction

  32. Bilirubin Ictotest • Confirmatory for bilirubin • Tablets containing p-nitrobenzene-diazonium-p-toluenesulfonate, SSA, sodium carbonate, and boric acid • Use specified mat for test; mat keeps bilirubin on surface for reaction • Positive reaction = blue-to-purple color • Interfering substances are washed into the mat, and only bilirubin remains on the surface

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