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Clinical Chemistry (CC 1) > (F) L3: Liver Function Tests (Bilirubin Determination) > Flashcards

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

1
Q

Serum bilirubin is measured using what method?

A

Spectrophotometry

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2
Q

Serum Bilirubin

What are the 2 methods of spectrophotometry?

A
  1. Chemical
  2. Enzymatic
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3
Q

Serum Bilirubin (Spectrophotometry)

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

A

Chemical

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4
Q

Serum Bilirubin (Spectrophotometry)

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

A

Chemical

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5
Q

Serum Bilirubin (Spectrophotometry)

What type of Spectrophotometric method?
Uses bilirubin oxidase

A

Enzymatic

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6
Q

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

A

False (urobilinogen)

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7
Q

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

A

False (B2)

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8
Q

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

A

Urine Bilirubin

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9
Q

Diazo reaction vs. Diazotization

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

A

Diazotization

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10
Q

Diazo reaction vs. Diazotization

Which is used in urine bilirubin?

A

Diazo Reaction

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11
Q
  • 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
A

Icterus Index

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12
Q
  • 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
A

Direct Spectrophotometry

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13
Q

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
A

Bilirubinometer

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14
Q

Specimen Collection

What is the preferred sample?

A

Serum

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15
Q

Specimen Collection

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

A

Lipemic sample

Note: Can falsely elevate bilirubin

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16
Q

Specimen Collection

A hemolyzed sample will inhibit what reaction?

A

Diazotization (false decreased result)

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17
Q

Specimen Collection

TOF: Bilirubin is oxidized upon light exposure

A

True

Note: Use an amber bottle or cover with aluminum foil

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18
Q

Chemical Methods (Spectrophotometric)

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

A

Diazo A

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19
Q

Chemical Methods (Spectrophotometric)

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

20
Q

Chemical Methods (Spectrophotometric)

Bilirubin + Diazotized Sulfanilic Acid = ?

A

Azobilirubin

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

21
Q

Chemical Methods (Spectrophotometric)

TOF: Chemical methods are considered kinetic assays

A

False (endpoint assays)

22
Q

Chemical Methods (Spectrophotometric)

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

A
  1. Total Bilirubin
  2. Direct Bilirubin
  3. Indirect Bilirubin
23
Q

Chemical Methods (Spectrophotometric)

This kind of bilirubin needs a solubilizer or a coupling accelerator

A

Indirect Bilirubin (slow-acting, water insolube bilirubin)

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

24
Q

Chemical Methods (Spectrophotometric)

TOF: Direct-reacting bilirubin is always equal to B2

A

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

25
Q

Chemical Methods (Spectrophotometric)

TOF: All conjugated bilirubins are direct reacting but not all direct reacting bilirubins are conjugated

26
Q

Chemical Methods (Spectrophotometric)

Total Bilirubin - Direct Bilirubin = ?

TB was obtained by the solubilizer

A

Indirect Bilirubin (unconjugated)

27
Q

Chemical Methods (Spectrophotometric)

Direct Bilirubin:
Bilirubin + Diazo reagent = Azobilirubin

Total Bilirubin:
Bilirubin + Methanol + Diazo reagent = Azobilirubin

A

Evelyn-Malloy Method

28
Q

Chemical Methods (Spectrophotometric)

Evelyn-Malloy Reaction:
- Bilirubin + Diazo reagent = Azobilirubin
- Bilirubin + Methanol + Diazo reagent = Azobilirubin

What color and pH is produced?

A

Color: Pink to Purple
pH: Acidic

29
Q

Chemical Methods (Spectrophotometric)

This method is read at 560nm

A

Evelyn-Malloy Method

30
Q

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

A

Jendrassik-Grof Method

31
Q

Chemical Methods (Spectrophotometric)

What is ascorbic acid and alkaline tartrate’s role in the Jendrassik-Grof method?

(2 answers)

A
  1. Ascorbic acid - destroys excess reagent/stops the reaction
  2. Alkaline tatrate - intensifies the color of azobilirubin
32
Q

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?

A

Color: Blue
pH: Alkaline (has less interferences)

33
Q

Chemical Methods (Spectrophotometric)

What are the solubilizers/coupling accelerators in the Evelyn-Malloy and Jendrassik-Grof Methods?

(2 answers)

A
  1. Evelyn-Malloy - Methanol
  2. Jendrassik-Grof - Caffeine-Benzoate
34
Q

Chemical Methods (Spectrophotometric)

Why is Caffeine-Benzoate preferred over Methanol as a solubilizer?

A

Methanol promotes protein precipitation and increases turbidity

35
Q

Chemical Methods (Spectrophotometric)

Which among Evelyn-Malloy and Jendrassik-Grof Method is preferred?

A

Jendrassik-Grof (because of its solubilizer)

36
Q

Chemical Methods (Spectrophotometric)

This method is read at 600nm

A

Jendrassik-Grof Method

37
Q

Enzymatic Methods (Spectrophotometric)

Biliverdin can be converted to bilirubin using (biliverdin reductase/bilirubin oxidase)

A

Biliverdin reductase

38
Q

Enzymatic Methods (Spectrophotometric)

Bilirubin can be converted back to biliverdin using (biliverdin reductase/bilirubin oxidase)

A

Bilirubin oxidase

39
Q

Enzymatic Methods (Spectrophotometric)

A decreased absorbance reading at 460nm in the sample is correlated to what?

A

Decreased bilirubin (because it was converted BACK to biliverdin using bilirubin oxidase)

Note: The relationship is directly proportional

40
Q

Enzymatic Methods (Spectrophotometric)

Conjugated, Unconjugated, and Delta Bilirubin will be converted to Biliverdin using Bilirubin Oxidase at what pH?

A

8.0 (Total Bilirubin)

41
Q

Enzymatic Methods (Spectrophotometric)

Conjugated Bilirubin will be converted to Biliverdin using Bilirubin Oxidase at what pH?

A

4.5 (Direct Bilirubin)

42
Q

What is the conversion factor?

43
Q

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

44
Q

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

A

Premature Infants

45
Q

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

A

Full-term Infants

Clinical Chemistry (CC 1) (33 decks)

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