MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

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MLAB 2401 Clinical ChemistryKeri Brophy-Martinez

• Assessment of Liver Function

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Liver Panel

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

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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

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Measured vs. Calculated

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

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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

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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.

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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 _at_
  600 nm.

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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?616404F6782
  E84851260BFF8F344F92903AF

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Reference Ranges for Bilirubin
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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

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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

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Determination of Urobilinogen

• Ehrlichs reaction
• Ehrlichs reagentp-dimethyl aminobenzaldehyde
• Urobilinogen Ehrlichs reagent Red color
• Performed on fresh urine
• Reference Range
• 0.1-1.0 Ehrlich units in two hours

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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

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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

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Enzymes

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

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Enzymes Points to Remember

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

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Misc. Liver Function Tests

• Prothrombin time
• Elevated in liver disease
• Ammonia
• Elevated in liver disease
• Glucose/Galactose Tolerance
• Assess the livers ability to metabolize
  carbohydrates

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Disease States
Condition 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
Reyes Syndrome I I N
I Increased N Normal
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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.

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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

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Hepatitis B Virus
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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)

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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/hep
  atitis_antigen.cfm
• http//depts.washington.edu/labweb/Divisions/Viro/
  Hepatitis_sero.htm
• http//tmp.kiwix.org4201/A/Hepatitis_B.html
• Sunheimer, R., Graves, L. (2010). Clinical
  Laboratory Chemistry. Upper Saddle River Pearson
  .