Bilirubin Methodology/Assay

Question 1

Sample needed for Bilirubin Methodology:

Answer 1

serum

Question 2

T/F. It must be stored properly in a dark place (if not, it may cause false decrease in bilirubin by 30-50% per hour)

Answer 2

True

Question 3

If sample is prepared and stored properly:
o Room temperature:
o Refrigerator (4degC):
o Freezer (-20degC):

Answer 3

o Room temperature: 2 days
o Refrigerator (4degC): 1 week
o Freezer (-20degC): indefinite

Question 4

T/F. Fasting sample is preferred (NOT REQUIRED)

Answer 4

True

Question 5

Bilirubin Methodology Interferences:

Answer 5

o Lipemia
o Hemolysis

Question 6

causes increased bilirubin concentration (that’s why fasting is preferred)

Answer 6

Lipemia

Question 7

decrease reaction bilirubin with diazo reagent (decreased bilirubin)

Answer 7

Hemolysis

Question 8

Bilirubin Assay Principle:

Answer 8

Van den Berg Reaction – diazotization of bilirubin to form azobilirubin

Question 9

Only measure conjugated bilirubin and total bilirubin (IB + TB)

Answer 9

Bilirubin Assay

Question 10

T/F. We must add accelerants to measure indirect bilirubin because it is fast reacting

Answer 10

False, slow reacting

Question 11

Bilirubin Assay Two Methods:

Answer 11

Evelyn and Malloy method and Jendrassik and Grof Method

Question 12

Evelyn and Malloy Method Principle:

Answer 12

Van den Berg reaction

Question 13

Evelyn and Malloy Method Coupling accelerator:

Answer 13

Methanol

Question 14

T/F. Disadvantage: Methanol can precipitate proteins → can cause turbidity → can interfere the tests

Answer 14

True

Question 15

Diazo reagent:

Answer 15

Direct bilirubin

Question 16

0.1% sulfanilic acid + HCl

Answer 16

Diazo A

Question 17

0.5% sodium nitrite

Answer 17

Diazo B

Question 18

1.5% HCl

Answer 18

Diazo Blank

Question 19

T/F. Evelyn and Malloy Method Performed at pH near 1.0

Answer 19

True

Question 20

Evelyn and Malloy Method Final reaction

Answer 20

Pink to purple azobilirubin (with maximal absorption at 560 nm)

Question 21

• Popular technique for discreet analyzers
• Safer because it has neutral pH

Answer 21

Jendrassik and Grof Method

Question 22

Jendrassik and Grof Method Main reagent:

Answer 22

Diazo reagent

Question 23

Jendrassik and Grof Method Accelerator:

Answer 23

Caffeine sodium benzoate

Question 24

Jendrassik and Grof Method Buffer:

Answer 24

Sodium citrate

Question 25

terminates the accelerator and destroys excess diazo reagent

Answer 25

Ascorbic acid

Question 26

provides alkaline pH after addition of ascorbic acid

Answer 26

Alkaline tartrate solution

Question 27

Jendrassik and Grof Method Final reaction:

Answer 27

Blue azobilirubin (measured at 600 nm)

Question 28

Increased Indirect Bilirubin

Answer 28

• Hemolytic anemia
• G-6PD deficiency
• Gilbert’s syndrome
• Criggler -Najjar syndrome
• Hepatocellular disease
• Lucey-Driscoll syndrome

Question 29

Increase Direct Bilirubin

Answer 29

• Biliary obstruction (gall stones)
• Pancreatic (head) cancer
• Dubin-Johnson syndrome
• Alcoholic and viral hepatitis
• Biliary atresia
• Hepatocellular disease

Question 30

• Prehepatic =
• Hepatic =
• Post hepatic =

Answer 30

• Prehepatic = Increased IB
• Hepatic = Increased IB, DB
• Post hepatic = Increased DB

Question 31

T/F. Prehepatic, Hepatic and Post hepatic may cause increased in Total bilirubin

Answer 31

True