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QIHC maryjw79 @ gmail email 4 access > EPITOPE ENHAMCEMENT > Flashcards

EPITOPE ENHAMCEMENT Flashcards

1
Q

What is the most common fixation/processing protocol for IHC?

A

Formalin fixed, paraffin embedded (FFPE)

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2
Q

What is the major drawback of FFPE tissue?

A

Formalin induced genetic modification of the tissue can cause may result in the loss of antigen and antibody interaction

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3
Q

How does Heat-induced Epitope Retrieval (HIER) work?

A

Disrupting the formalin crosslinks by heating up to 100C or by a strong alkaline solution

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4
Q

What are the two common categories of epitope retrieval methods?

A

Heat (HIER)

Enzymatic (EIER)

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5
Q

When is epitope retrieval commonly used?

A

To break down hydrogen bonds that are formed during formalin fixation in tissue

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6
Q

What are the 7 advantages of epitope retrieval?

A
  1. Ability to further dilute antibody
  2. Exposure of epitope sites that were not previously detectable
  3. More intense reactions with decreased incubation times
  4. More uniform staining
  5. Decreased background staining
  6. Day-to-day staining consistency
  7. Improved standardization
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7
Q

What is the most important component of HIER?

A

Composition

pH

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8
Q

What are the major factors affecting epitope retrieval?

A
  1. pH
  2. Volume of the retrieval solution
  3. Heating time
  4. Temperature
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9
Q

When would a combination of EIER and HIER be used?

A

If the usage of an enzyme digestion alone reduces the quality of tissue due to a lengthy exposure time

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10
Q

How would a combination of EIER and HIER be used?

A

HIER is performed first, followed by rinsing and finally an enzyme digestion for a short amount of time (1-5 minutes)

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11
Q

How is imidazole used in the PAP technique?

A

Intensifies DAB reaction

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12
Q

What linking antibody would follow a monoclonal kappa primary antibody

A

Anti-mouse
Anti-rabbit

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13
Q

What do negative control slides omit?

A

Primary antibody

Uses nonimmune serum instead

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14
Q

What are the strong bonds called that form between proteins and calcium ions as a result of formalin fixation?

A
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15
Q

What type of epitope retieval is proteolytic-induced epitope retrieval (PIER)?

A
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16
Q

What two major factors require stringent control when performing proteolytic-induced epitope retrieval (PIER) antigen unmasking?

A
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17
Q

How does the epitope retrieval affect the choice of staining method and the staining result?

A
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18
Q

What is the mechanism of retrieval for EIER?

A
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19
Q

What is the advantage of using EIER?

20
Q

What is the disadvantage of using EIER?

21
Q

What is the mechanism of retrieval for HIER?

22
Q

What is the advantage of using HIER?

23
Q

What is the disadvantage of using HIER?

24
Q

Explain formaline-sensitive epitope

25
Q

Explain masked epitope

26
Q

List five enzymes used for EIER

A

1.
2.
3.
4.
5.

27
Q

Describe the effects of over-treatment with enzymes

28
Q

How does pH relate the effectiveness of HIER?

29
Q

How does the volume of fluid used relate the effectiveness of HIER?

30
Q

How does heating time relate the effectiveness of HIER?

31
Q

How does temperature relate the effectiveness of HIER?

32
Q

Explain how a pressure cooker works in HIER

33
Q

Explain how steamer works in HIER

34
Q

Explain how an autoclave works in HIER

35
Q

Explain how a microwave works in HIER

36
Q

Explain how a waterbath works in HIER

37
Q

Explain usage of the various pH solutions for HIER

38
Q

List two commonly used solutions for HIER

39
Q

List the pH for the two commonly used solutions for HIER

40
Q

Describe the effects of using solutions with too high of a pH for HIER

41
Q

Describe the effects of using solutions with too low of a pH for HIER

42
Q

What retrieval method is necessary to maintain morphology in alcohol fixed specimens (e.g. cytology)?

43
Q

What antigen retrieval method would be used at light to med at 37C for AE1/AE3, B72.3, Factor XIIIa, HAM-56?

44
Q

What antigen retrieval method would be used at strong at 37C for CK7, CK20, Pan CK, Lu-5(7’)?

45
Q

What antigen retrieval method would be used at Extra strong at room temp for EGFR (5’)?

46
Q

What retrieval method is used at a very strong strength?

A

Proteinase K

47
Q

Why is pH important in antigen retrieval?

A
  1. Most antigens show no signal variation using retreival solutions with pH ranging 1-10
  2. Nuclear antigens (es: ER) show dramatic decrease in intensity at mid-range pH but optimal at low pH
  3. Some antigens (ex: HMB 45) are weak at low pH (1-2) but excellent in the high range

QIHC maryjw79 @ gmail email 4 access (16 decks)

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