Lec 17: Liver Function Tests Flashcards
5 Tests that are no longer used and are deemed obsolete.
- Hippuric test
- Urobilinogen quantitation in urine & stool
- Gross test
- Cephalic flocculation test
- Colloidal gold test
Define:
- exogenous compounds
- endogenous compounds
- Iodine staining dyes
Exogenous Compounds
Coming from outside the body which should be administered.
• aminopyrine, caffein
Endogenous Compounds
Not routinely used, for research purposes.
• bile acids
Iodine Staining Dyes
Widely used for radiographic and other imaging procedures
How many lab tests do liver profiles usually have?
4 to 8
What substances are tested for the following:
- Special Chemistry tests
- Immunologic test
- Hematologic test
Special Chemistry tests
• Ceruloplasmin
• Alpha-fetoprotein
• Serum Iron & ferritin
• Serum bile acids
• Ammonia
Immunologic test
• IgM & IgG antibodies to Hep A
• IgM & IgG antibodies to Hep D
• Antibodies to Hep C virus
• Antibodies to HbSAg
• Antimicrobial antibodies
Hematologic test
• CBC
• Determination of abnormal Hgb types
• Coagulation factor studies
• Reticulocyte count
• Red cell enzyme studies
• Prothrombin time
Classifications of Tests and their example
Abnormalities of pigment metabolism
• serum bilirubin & VD Bergh reaction
• Icteric index
• urine bilirubin
• urine & fecal Urobilinogen
• serum & urinary bile acids
Changes in plasma proteins
• estimation of Total plasma proteins, albumin, globulin, A/G ratio
• plasma fibrinogen
• Various flocculation tests
• amino acids in urine
Carbohydrate metabolism
• Galactose tolerance test
• Fructose Tolerance test
Lipid abnormalities
• Cholesterol and cholesterol ester and ratio
• determination of fecal fats
Detoxicating function of liver
• hippuric acid synthesis test
• Amino antipyrine breath test
Excretory function of liver
• Bromsulphalein test
• 131I Rose Bengal test
Prothrombin by liver
• prothrombin time
Amino acid catabolism
• Blood NH3
• CSF glutamine
Serum enzyme activities
Serum Bilirubin
- Uses of bilirubin determination.
- In average plasma conc, give the concentrations of TB, Unconjugated and conjugated bilirubin.
-
a. To detect the breakdown of hemoglobin (hemolysis)
b. To detect decrease in excretion by the liver due to obstruction -
Average plasma conc
Total bilirubin: less than 1 mg/dL
Unconjugated B: less than 0.8 mg/dL
Conjugated B: less than 0.2 mg/dL
Forms of Bilirubin
• contains 1 to 2 attached glucuronic acid molecules
• reacts readily with Aqueous solution (give example)
• found in small amounts in urine
Conjugated Bilirubin
• Aqueous sol: diazotized sulfanilic acid
Forms of Bilirubin
• reacts slowly unless with addition of solubilizing agent
• NOT excreted in urine
Unconjugated Bilirubin
Forms of Bilirubin
• seen only in the presence of significant hepatic obstruction
• conjugated that is covalent bound to albumin
Delta Bilirubin
The concentration of conjugated/unconjugated in total bilirubin based on the type of hyperbilirubinemia:
- conjugated hyperbilirubinemia
- unconjugated hyperbilirubinemia
Conjugated Hyperbilirubinemia
50% of Total bilirubin is conjugated
Unconjugated Hyperbilirubinemia
80% of Total bilirubin is unconjugated
Diazotization Principle
- Product (name & color)
- Accelerator used in Evelyn & Malloy in 1937
- Accelerator used in Jendrassik & Grof in 1938
- (Di)Azobilirubin (purple)
- 50% Methanol
- Caffeine-benzoate
Oxidation Principle
- Bilirubin is converted into what derivative? (Name & color)
- Bilirubin behaves like an indicator. Give the color of the pH.
- Biliverdin (green)
-
a. Acidic: pink-purple
b. Alkaline: blue
EVELYN & MALLOY
- Principle
- Reaction time
- Product (name & color)
- Methanol is added for what form of bilirubin?
- Formula for getting Indirect Bilirubin
- Bilirubin reacts with diazotized sulfanilic acid to form a purple azobilirubin
- 1 to 5 minutes
- Purple azobilirubin
- Indirect bilirubin
- Indirect bilirubin = TB - Direct bilirubin
VAN DEN BERGH
- Principle
- Product (name & color)
- Components of Diazo reagent
- Formula for Direct bilirubin
- Formula for TB
- Bilirubin reacts with Van Den Berg’s diazo reagent to form a purple azo bilirubin
- Purple azobilirubin
-
10mL of Sol. A
• Sulfanilic acid in conc. HCl
0.8mL of Sol. B
• Sodium Nitrite in water - Direct bilirubin = (Test – BLK) ÷ 2
- TB = Test – BLK
Purpose of Methanol
- a dissociating agent
- catalyst
- solubilizing agent
Factors affecting Bilirubin Determination: SAMPLE STABILITY
- How is bilirubin affected by light?
- Samples should be placed in ___ glassware or be wrapped in ___.
- Storage of samples and their stability.
- When exposed to light, Bilirubin becomes Biliverdin by photo-oxidation/dehydrogenation.
• bilirubin conc. is reduced:
—> 10% in 30 minutes
—> 50% in 1 hour -
a. Low actinic glassware
b. carbon paper -
a. 2 days — dark at RT
b. 1 week — dark refrigerator
c. 3 months — freezer
Factors affecting Bilirubin Determination: HGB & LIPEMIA INTERFERENCE
- How does hemolysis affect bilirubin?
- How does Lipemia affect bilirubin?
- How much Hemoglobin can cause direct bilirubin to be low by 10, 15, and 45%?
- This hemoglobin oxidizes bilirubin, and is eliminated by saturation of CO and Ascorbic acid.
- Hemolysis decreases the reaction of bilirubin with diazo reagent producing false low concentrations
- Lipemia causes error in the spectrophotometric measurements
- 100, 250, 1000 hgb/100mL
- Oxyhemoglobin
Factors affecting Bilirubin Determination: TEMPERATURE
- Reaction in the presence of methanol is faster at ___ than at 25°C.
- Temperature affects the rate of ___.
- 37°C
- Color development
Factors affecting Bilirubin Determination: STANDARDIZATION
- Effect of Impurities in commercial preparations of bilirubin.
- Effect of incomplete solubility
- Effect of instability
- What substance is used to stabilize bilirubin? For how long?
- This substance only makes the sample stable for 3 minutes?
- Most commercial preparations of bilirubin are not pure, so impurities can affect the accuracy and consistency of the results.
- Commercial bilirubin tend to have incomplete solubility, so it can lead to difficulties in preparing standardized solutions.
- Instability can change the concentration.
-
a. 20mg/100mg of Chloroform
b. 3 months - Ethyl acetate methanol
Define/describe:
- Immediate direct reaction
- delayed direct response
- no direct reaction is obtained
Immediate direct reaction
Immediate color development
Delayed direct response
Color development after 5-30 minutes
NO direct reaction is obtained
Color development after addition of methanol
Components of Artificial STD used in Indirect Reaction.
Methyl red solution in GAA of pH 4.6–4.7
• closely resembling azobilirubin’s color
