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Histology QIHC Exam > Epitope Enhancement (Ag Retrieval) > Flashcards

Epitope Enhancement (Ag Retrieval) 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

The formalin induced genetic modification of the tissue can cause may result in the loss of Ag-Ab interaction.

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3
Q

What does Heat-induced Epitope Retrieval entail?

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 induced epitope retrieval (HIER) and enzyme induced epitope retrieval (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 Ab
  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

Besides heat, what is the most important component if HIER?

A

The composition of the retrieval solution.

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8
Q

What are the four major factors that affect epitope retrieval?

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

Why would a combination of EIER and HIER be used?

A

If the usage of an enzyme digestion alone with EIER 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 peroxidase techniques?

A

Imidazole is an effective intensification reagent for the DAB reaction product.

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12
Q

What link Ab would most likely follow a monoclonal kappa primary Ab?

A

Anti mouse or anti rabbit.

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13
Q

What do negative control slides that are run with each Ab stain omit?

A

The primary Ab, a nonimmune serum is commonly substituted.

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14
Q

T/F: Prediluted Ab should always be used as provided by the manufacturer.

A

False, they should always be validated for reactivity before use on patient tissue.

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15
Q

T/F: Negative tissue Ag controls may be run by substituting for the primary Ab the diluent in which the Ab is prepared.

A

True, the negative controls omit the primary Ab.

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16
Q

T/F: One standard protocol may be written to cover all specimens.

A

False, a procedure needs to be written for all non-standard tissue protocols.

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17
Q

T/F: Zinc formalin preserves immunoreactivity very well.

A

True.

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18
Q

T/F: Multilink Ab can only be used with monoclonal primary Ab.

A

False, they can be used with both monoclonal and polyclonal Ab.

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19
Q

T/F: Blocking reactions are used to block endogenous activity of the same enzyme used for the enzyme immune complex.

A

True.

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20
Q

T/F: The enzyme label for immunoperoxidase methods contains horseradish peroxidase.

A

True.

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21
Q

T/F: Monoclonal Ab are often preferred over polyclonal Ab because there is little batch-to-batch variability.

A

True.

22
Q

T/F: DAB is an alcohol soluble chromogen.

A

False, and sections may be dehydrated, cleared and mounted with synthetic resin.

23
Q

T/F: Harris hematoxylin may not be used with AEC because of the alcohol present.

A

True.

24
Q

T/F: Regulations regarding predictive marker staining of tissue places responsibility for fixation time documentation on the lab.

A

True, the lab is responsible.

25
Q

T/F: The only method of HIER uses the microwave.

A

False. A pressure cooker, circulating water bath or vegetable steamer may be used.

26
Q

What is Ag Retreival?

A

A specimen ‘pretreatment’ procedure that modifies the molecular conformation of target proteins by exposing slide-mounted material, (sectioned tissue and other cellular preparations), to a heated buffer solution.

27
Q

Why is Ag Retrieval necessary?

A

It reverses protein “cross-linking” caused by aldehyde based fixatives.

28
Q

How can the degree of Ag Retrieval be manipulated?

A

A) the chemical composition of the fixative and fixation time period initially used;
B) the chemical composition of the retrieval buffer;
C) the operating parameters (i.e. physical effectiveness) of the heat-generating device; D) the means by which slides are handles at the conclusion of the heating process.

29
Q

What is the main difference between HIER and EIER?

A

HIER non-discriminately effects nearly all cross-linked protein
‘residues’ within the specimen, while EIER selectively removes specific protein residues in such a way as to improve access to other proteins, thereby producing a desired immunostaining reaction.

30
Q

Is on-line (automatic) HIER “better” than off-line (manual HIER?

A

No, the only possible positive outcome from on-line is the convenience factor.

31
Q

Which of the following is true of a polyclonal antibody?
A. lot to lot consistency
B. sustained production
C. reacts with a specific epitope
D. reacts with multiple epitopes on a single molecule

A

D. Reacts with multiple epitopes on a single molecule.

32
Q

Which one of the following is most correct regarding heat induced epitope retrieval (HIER) techniques?
A. Steamers, pressure cookers, microwaves and waterbaths can all be successfully used for HIER.
B. Lead thiocyanate formic acid and water have all been successfully used as HIER solutions.
C. You cannot “over retieve” tissue using HIER techniques.
D. A & B are correct.

A

D. Both “Steamers, pressure cookers, microwaves and waterbaths can all be successfully used for HIER.” and “Lead thiocyanate formic acid and water have all been successfully used as HIER solutions.” are correct.

33
Q

What marker is considered almost a universal control for over-fixation?

A

Vimentin

34
Q

In immunohistochemical procedures using horseradish peroxidase (HRP), a solution of hydrogen peroxide is used to:

A

Block endogenous peroxidase.

35
Q

Where is peroxidase normally found?

A

Peroxidase is normally present in red blood cells and granulocytes.

36
Q

Fluorochrome-conjugated primary antibodies are an example of:

A

Direct labeling.

37
Q

What is “direct labeling”?

A

A system in which the primary antibody is labeled with a fluorochrome or enzyme is termed direct labeling, since the primary is labeled and the system does not require a secondary antibody to which a label would be attached.

38
Q

A cell antigen that can be demonstrated by immunofluorescent staining of fresh-frozen tissues is not visible by immunoperoxidase staining of formalin-fixed paraffin-processed tissue. The difference in the procedures that most likely accounts for the lack of staining is:

A

Masking of antigen binding sites by fixation.

39
Q

A “hybridoma” is formed when an immunoglobulin-producing spleen cell is fused with a non-immunoglobulin-producing:
A. myeloma cell
B. liver cell
C. mesenchymal cell
D. epithelial cell

A

A myeloma.

40
Q

What is the main plus side of hybridomas?

A

A single hybrid cell, which has characteristics of both parent cells, in that it is producing immunoglobulin to a specific antigenic determinant and is immortal, can be cultured, resulting in production of large amounts of specific antibody.

41
Q

When staining sections for examination by immunofluorescence microscopy:
A. tissue antigens are “stained” by fluorescent-labeled antibodies
B. tissue antibodies are “stained” by fluorescent-labeled antigens
C. fluorescent dyes are sandwiched between antigen and antibody
D. fluorescent dyes are bound to tissue with heavy metal mordants

A

A. Tissue antigens are “stained” by fluorescent-labeled antibodies

42
Q

How does fluorescence “work”?

A

In fluorescence microscopy, antibodies are tagged with fluorescent dyes. The antibodies attach to tissue antigens, then the dye is made to fluoresce by exposure to light of a specific wavelength. Tissue antigen is assumed to be present whenever there is fluorescence.

43
Q

In the indirect immunohistochemistry staining method, the:
A. test slide is first treated with unlabeled antiserum
B. test slide is first treated with enzyme-labeled to the primary antibody
C. second step is treatment with enzyme labeled antiserum to the
primary antigen
D. second step is treatment with unlabeled antiserum to the primary
antibody

A

A. The test slide is first treated with unlabeled antiserum.

44
Q

Keratin belongs to a family called the:

A

Intermediate filaments.

45
Q

Which of the following is a chromogen used in peroxidase histochemistry that generates a product that is soluble in alcohol or xylene but not in water?
A. 9-aminoethylcarbazole (AEC)
B. 3,3’-diaminobenzidine (DAB)
C. alpha-naphthyl phosphate (ANP)
D. hexazonium pararosaniline (HPR)

A

A. 9-aminoethylcarbazole (AEC)

46
Q

What is keratin?

A

Keratin is the structural material used to form the outer layer of human skin, as well as hair and nails. It consists of keratin monomers assemble into bundles to form intermediate filaments, which are tough and insoluble and form strong unmineralized tissues

47
Q

In immuno-alkaline phosphatase staining, which control is useful to determine the level of endogenous alkaline phosphatase?
A. incubate in chromogen only
B. incubate overnight in levamisole
C. apply all steps except the enzyme
D. stain several slides at different time periods

A

A. Incubate in chromogen only.

48
Q

What is the purpose of the negative control in IHC?

A

Determine what steps contribute to any non-specific staining. In other words what all the other steps contribute to the staining. If there is significant staining without the primary antibody used it indicates a problem with some of the other steps.

49
Q

What does the preferred fixative for demonstration of intermediate filaments contain?

A

Alcohol. It has been shown that intermediate filaments are best demonstrated following fixation in absolute ethanol or an alcohol based fixative.

50
Q

As a chromogenic substrate, amino-ethylcarbazole (AEC) would be preferred to diaminobenzidine (DAB):
A. if permanent preparation is desired
B. when staining pigmented lesions
C. to reduce contact with a potential carcinogen
D. for subsequent metallic color enhancement

A

B. When staining pigmented lesions.

51
Q

What temperature is employed during HIER?

A

100 degrees may be optimal for breaking links but 90 degrees for a longer period preserves morphology better.

Histology QIHC Exam (9 decks)

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