MLAB 2401: Clinical Chemistry
Keri Brophy-Martinez
Assessment of Liver Function
Liver Panel
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•
•
•
•
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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?616404F
6782E84851260BFF8F344F92903AF
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
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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 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
Reye’s
Syndrome
I
I
N
I= Increased
N= 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 .