MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

1. MLAB 2401: Clinical ChemistryKeri Brophy-Martinez Assessment of Liver Function

2. Liver Panel • Albumin • Bilirubin, total • Bilirubin, direct • AST/SGOT • ALT/SGPT • Alkaline Phosphatase

3. History of Bilirubin Analysis • Ehrlich(1883) • Described the reaction of bilirubin with diazotized sulfanilic acid= DIAZO REACTION • Malloy and Evelyn (1937) • Diazo reaction with 50% methanol as an accelerator • Jendrassik and Grof ( 1938) • Diazo reaction with caffeine-benzoate-acetate as accelerator • Increased sensitivity

4. Measured vs. Calculated • Measured Analytes • Total Bilirubin • Conjugated bilirubin (DIRECT) • Calculated Analytes • Unconjugated bilirubin (INDIRECT)

5. Fractions & Their Characteristics • Conjugated/Direct • Polar • Water-soluble • Found in plasma, unbound or free • Reacts with diazotized sulfanilic acid without an accelerator • Unconjugated/Indirect • Nonpolar • Water-insoluble • Found in plasma, bound to albumin • Reacts with diazotized sulfanilic acid with an accelerator • Delta • Conjugated bilirubin bound to albumin • Observed in hepatic obstructions

6. Specimen Collection and Storage • Serum or plasma preferred • Temperature sensitive • Fasting sample preferred • Lipemia increases bilirubin concentrations • No hemolysis • Hemolysis decreases the reaction of bilirubin with the diazo reagent • Light sensitive • Bilirubin levels decrease by 30-50% per hour.

7. Methods of Bilirubin Analysis • Jendrassik-Grof • Measures Total and Conjugated bilirubin • Principle • Bilirubin pigments in serum react with a diazo reagent which results in the production of azobilirubin( a purple product). Measured at 540 nm. • Caffeine -benzoate accerlerates the coupling of bilirubin with the diazo reagent. • Ascorbic acid stops the reaction. • Alkaline tartrate converts the purple azobilirubin to a blue azobilirubin. • This product is measured spectrophotometrically @ 600 nm.

8. Jendrassik-Grof • Advantages • Not affected by pH changes • Maintains optical sensitivity at low bilirubin concentrations • Insensitive to high protein concentrations • Jendrassik-Grof Animation • http://webcls.utmb.edu/lo/publicdl.asp?616404F6782E84851260BFF8F344F92903AF

9. Reference Ranges for Bilirubin

10. Urine Bilirubin • Presence indicates conjugated hyperbilirubinemia • Detected using urine dipsticks • Have a diazo reagent imbedded in the strip • Follows the Ehrlich principle • (Chemstrip/Multistix) • Fresh urine should be used • Avoid light and oxidation

11. Urobilinogen • End product of bilirubin metabolism • Majority excreted in feces, some reabsorbed and returned to the liver • Increased • Hemolytic disease • Defective liver-cell function • Decreased • Biliary obstruction • Carcinoma

12. Determination of Urobilinogen • Ehrlich’s reaction • Ehrlich’s reagent=p-dimethyl aminobenzaldehyde • Urobilinogen + Ehrlich’s reagent = Red color • Performed on fresh urine • Reference Range • 0.1-1.0 Ehrlich units in two hours

13. Enzymes • Liver damage results in the release of enzymes into the circulation • Differentiate between functional or mechanical causes of disease • Significant enzymes • AST • ALT • ALP • GGT • 5’ nucleotidase • LDH

14. Enzymes • Aminotransferases • ALT and AST rise rapidly in most diseases of the liver and stay elevated for up to 2-6 weeks • Highest levels seen with hepatitis, hepatic ischemia and drug/toxin-induced necrosis • Phosphatases • ALP differentiates hepatobiliary disease from bone disease • 5’-Nucleotidase is elevated in hepatobiliary disease

15. Enzymes • GGT elevated in biliary obstruction and in chronic alcoholism • LDH/LD serves as a nonspecific marker of cellular injury

16. Enzymes: Points to Remember • Elevated Liver enzymes are as easy as ABC • Alcoholism • Biliary Obstruction • Cirrhosis

17. Misc. Liver Function Tests • Prothrombin time • Elevated in liver disease • Ammonia • Elevated in liver disease • Glucose/Galactose Tolerance • Assess the liver’s ability to metabolize carbohydrates

18. Disease States I= Increased N= Normal

19. Hepatitis A Markers • Performed by serological antibodies • IgM indicates acute infection and can persist for 3-6 months • IgG appears shortly after IgM, and confers lifelong immunity.

20. Hepatitis B Markers • HBsAG: Hepatitis B Surface Antigen • Detected prior to onset of symptoms • HBcAG: Hepatitis B Core Antigen • Found in an acute infection • HBeAg: Hepatitis B Envelope Antigen • Found in acute and chronic infections

21. Hepatitis B Virus

22. Hepatitis C Testing • Two methods currently used • Anti-HCV detection by EIA (Screen) • A positive test indicates exposure to HCV, it can not determine a current infection versus a past infection • Quantitative nucleic acid PCR for HCV RNA (Confirmatory)

23. References • Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. • http://www.abbottdiagnostics.co.uk/About_Us/UK/hepatitis_antigen.cfm • http://depts.washington.edu/labweb/Divisions/Viro/Hepatitis_sero.htm • http://tmp.kiwix.org:4201/A/Hepatitis_B.html • Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .