MLAB 2401: Clinical ChemistryKeri Brophy-Martinez

Assessment of Liver Function

Liver Panel

• Albumin• Bilirubin, total• Bilirubin, direct• AST/SGOT• ALT/SGPT• Alkaline Phosphatase

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

Measured vs. Calculated

• Measured Analytes– Total Bilirubin– Conjugated bilirubin (DIRECT)

• Calculated Analytes– Unconjugated bilirubin (INDIRECT)

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

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.

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.

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

Reference Ranges for Bilirubin

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

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

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

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

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

Enzymes

• GGT elevated in biliary obstruction and in chronic alcoholism

• LDH/LD serves as a nonspecific marker of cellular injury

Enzymes: Points to Remember

• Elevated Liver enzymes are as easy as ABC– Alcoholism– Biliary Obstruction– Cirrhosis

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

Disease StatesCondition AST ALT ALP GGT Albumin

Alcoholic hepatits

I I NI III N

Acute Hepatitis

III II I I N

Biliary Obstruction

NI NI I I I

Cirrhosis NI NI NI NI D

Reye’s Syndrome

I I N

I= IncreasedN= Normal

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.

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

Hepatitis B Virus

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)

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 .