(F) L3: Liver Function Tests (Bilirubin Determination)

Question 1

Serum bilirubin is measured using what method?

Answer 1

Spectrophotometry

Question 2

Serum Bilirubin

What are the 2 methods of spectrophotometry?

Answer 2

1. Chemical
2. Enzymatic

Question 3

Serum Bilirubin (Spectrophotometry)

What type of Spectrophotometric method?
1. Evelyn-Malloy
2. Jendrassik-Grof

Answer 3

Chemical

Question 4

Serum Bilirubin (Spectrophotometry)

What type of Spectrophotometric method?
Uses the Van den Berg reaction (diazotization)

Answer 4

Chemical

Question 5

Serum Bilirubin (Spectrophotometry)

What type of Spectrophotometric method?
Uses bilirubin oxidase

Answer 5

Enzymatic

Question 6

TOF: Bilirubin (B2), which is water-soluble, in the urine is normal

Answer 6

False (urobilinogen)

Question 7

TOF: Bilirubinuria occurs when there is elevated B1 in the urine due to bile obstruction (post-hepatic) and liver damage (hepatic)

Answer 7

False (B2)

Question 8

This test detects conjugated bilirubin and uses the principle of Ehrlich’s Diazo Reaction

Answer 8

Urine Bilirubin

Question 9

Diazo reaction vs. Diazotization

Which is used in the Van den Berg reaction in chemical spectrophotometric methods?

Answer 9

Diazotization

Question 10

Diazo reaction vs. Diazotization

Which is used in urine bilirubin?

Answer 10

Diazo Reaction

Question 11

• This estimates the presence of hyperbilirubinemia
• Uses a standard solution of 0.01% potassium dichromate (pale yellow)
• Dilute the serum with NSS until it matches the color of the standard solution

Answer 11

Icterus Index

Question 12

• This is only used for neonates and newborns (cannot be used for adults because of carotenoid compounds that can interfere with the test)
• No reagents are used
• Is read at a 455nm wavelength

Answer 12

Direct Spectrophotometry

Question 13

Direct Spectrophotometry for Newborns

• This test is transcutaneous and non-invasive
• 1st wavelength is at 455nm
• 2nd wavelength is at 575nm to cancel out interferences of hemoglobin

Answer 13

Bilirubinometer

Question 14

Specimen Collection

What is the preferred sample?

Answer 14

Serum

Question 15

Specimen Collection

Fasting is not required however if the patient does eat before the test, there is a risk for what kind of sample?

Answer 15

Lipemic sample

Note: Can falsely elevate bilirubin

Question 16

Specimen Collection

A hemolyzed sample will inhibit what reaction?

Answer 16

Diazotization (false decreased result)

Question 17

Specimen Collection

TOF: Bilirubin is oxidized upon light exposure

Answer 17

True

Note: Use an amber bottle or cover with aluminum foil

Question 18

Chemical Methods (Spectrophotometric)

Van den Berg Reaction (Diazo Reagent):
0.1% Sulfanilic Acid and HCl

Answer 18

Diazo A

Question 19

Chemical Methods (Spectrophotometric)

Van den Berg Reaction (Diazo Reagent):
0.5% sodium nitrite

Answer 19

Diazo B

Question 20

Chemical Methods (Spectrophotometric)

Bilirubin + Diazotized Sulfanilic Acid = ?

Answer 20

Azobilirubin

Note: Absorption of azobilirubin is directly proportional to the bilirubin in the sample

Question 21

Chemical Methods (Spectrophotometric)

TOF: Chemical methods are considered kinetic assays

Answer 21

False (endpoint assays)

Question 22

Chemical Methods (Spectrophotometric)

In order to determine if a patient is jaundiced, what 3 concentrations are needed?

Answer 22

1. Total Bilirubin
2. Direct Bilirubin
3. Indirect Bilirubin

Question 23

Chemical Methods (Spectrophotometric)

This kind of bilirubin needs a solubilizer or a coupling accelerator

Answer 23

Indirect Bilirubin (slow-acting, water insolube bilirubin)

It needs to be soluble first before it can react with the Diazo reagent

Question 24

Chemical Methods (Spectrophotometric)

TOF: Direct-reacting bilirubin is always equal to B2

Answer 24

False (small amounts of delta-bilirubins and B1’s can also directly react)

Question 25

# Chemical Methods (Spectrophotometric) TOF: All conjugated bilirubins are direct reacting but not all direct reacting bilirubins are conjugated

Answer 25

True

Question 26

# Chemical Methods (Spectrophotometric) Total Bilirubin - Direct Bilirubin = ? | TB was obtained by the solubilizer

Answer 26

Indirect Bilirubin (unconjugated)

Question 27

# Chemical Methods (Spectrophotometric) Direct Bilirubin: Bilirubin + Diazo reagent = Azobilirubin Total Bilirubin: Bilirubin + Methanol + Diazo reagent = Azobilirubin

Answer 27

Evelyn-Malloy Method

Question 28

# Chemical Methods (Spectrophotometric) Evelyn-Malloy Reaction: - Bilirubin + Diazo reagent = Azobilirubin - Bilirubin + Methanol + Diazo reagent = Azobilirubin What color and pH is produced?

Answer 28

Color: Pink to Purple pH: Acidic

Question 29

# Chemical Methods (Spectrophotometric) This method is read at 560nm

Answer 29

Evelyn-Malloy Method

Question 30

# Chemical Methods (Spectrophotometric) Direct Bilirubin: Bilirubin + Diazo reagent + Ascorbic acid + Alkaline tartrate = Azobilirubin Total Bilirubin: Bilirubin + Diazo reagent + Caffeine-Benzoate + Ascorbic acid + Alkaline tartrate = Azobilirubin

Answer 30

Jendrassik-Grof Method

Question 31

# Chemical Methods (Spectrophotometric) What is ascorbic acid and alkaline tartrate's role in the Jendrassik-Grof method? (2 answers)

Answer 31

1. Ascorbic acid - destroys excess reagent/stops the reaction 2. Alkaline tatrate - intensifies the color of azobilirubin

Question 32

# Chemical Methods (Spectrophotometric) Direct Bilirubin: Bilirubin + Diazo reagent + Ascorbic acid + Alkaline tartrate = Azobilirubin Total Bilirubin: Bilirubin + Diazo reagent + Caffeine-Benzoate + Ascorbic acid + Alkaline tartrate = Azobilirubin What is the end color and pH?

Answer 32

Color: Blue pH: Alkaline (has less interferences)

Question 33

# Chemical Methods (Spectrophotometric) What are the solubilizers/coupling accelerators in the Evelyn-Malloy and Jendrassik-Grof Methods? (2 answers)

Answer 33

1. Evelyn-Malloy - Methanol 2. Jendrassik-Grof - Caffeine-Benzoate

Question 34

# Chemical Methods (Spectrophotometric) Why is Caffeine-Benzoate preferred over Methanol as a solubilizer?

Answer 34

Methanol promotes protein precipitation and increases turbidity

Question 35

# Chemical Methods (Spectrophotometric) Which among Evelyn-Malloy and Jendrassik-Grof Method is preferred?

Answer 35

Jendrassik-Grof (because of its solubilizer)

Question 36

# Chemical Methods (Spectrophotometric) This method is read at 600nm

Answer 36

Jendrassik-Grof Method

Question 37

# Enzymatic Methods (Spectrophotometric) Biliverdin can be converted to bilirubin using (biliverdin reductase/bilirubin oxidase)

Answer 37

Biliverdin reductase

Question 38

# Enzymatic Methods (Spectrophotometric) Bilirubin can be converted back to biliverdin using (biliverdin reductase/bilirubin oxidase)

Answer 38

Bilirubin oxidase

Question 39

# Enzymatic Methods (Spectrophotometric) A decreased absorbance reading at 460nm in the sample is correlated to what?

Answer 39

Decreased bilirubin (because it was converted BACK to biliverdin using bilirubin oxidase) | Note: The relationship is directly proportional

Question 40

# Enzymatic Methods (Spectrophotometric) Conjugated, Unconjugated, and Delta Bilirubin will be converted to Biliverdin using Bilirubin Oxidase at what pH?

Answer 40

8.0 (Total Bilirubin)

Question 41

# Enzymatic Methods (Spectrophotometric) Conjugated Bilirubin will be converted to Biliverdin using Bilirubin Oxidase at what pH?

Answer 41

4.5 (Direct Bilirubin)

Question 42

What is the conversion factor?

Answer 42

17.1

Question 43

# Reference Value Table Adult, Premature Infants, or Full-term Infants? B2: 0.0-0.2 mg/dl B1: 0.2-0.8 mg/dl TB: 0.2-1.0 mg/dl

Answer 43

Adult

Question 44

# Reference Value Table Adult, Premature Infants, or Full-term Infants? TB at 24h: 1-5 mg/dl TB at 48h: 6-8 mg/dl TB at 3-5 days: 10-12 mg/dl

Answer 44

Premature Infants

Question 45

# Reference Value Table Adult, Premature Infants, or Full-term Infants? TB at 24h: 2-6 mg/dl TB at 48h: 6-7 mg/dl TB at 3-5 days: 4-6 mg/dl

Answer 45

Full-term Infants