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Histology Exam > IHC > Flashcards

IHC Flashcards

1
Q

What is the purpose of IHC methods in pathology?

A

Determine origin of tumors and determine between benign and malignancies.

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2
Q

What are considered normal results when using IHC methods?

A
  • Tissue/cells appear normal and have high level of maturity
  • Cells are appropriate to tissue type
  • No specific tumour antigens present
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3
Q

What are advantages to monoclonal antibodies?

A
  • No batch to batch variability
  • Purer than polyclonal
  • high affinity and specificity
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4
Q

How is IHC used in histology?

A
  • Antibodies bind wherever antigens are present
  • excess can be washed away
  • Antibodies can have fluorescent labels on them for visual demonstration under the microscope
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5
Q

What are two epitope retrieval methods for IHC?

A

HEIR & EIER

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6
Q

What is the best preparation method for lymphocytic surface markers when doing IHC methods?

A

Cryostat

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7
Q

What are the advantages of epitope enhancement?

A
  • Dilute antibodies further
  • Expose epitope sites
  • uniform staining
  • intense reactions with decreased incubation
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8
Q

What are the two most common solutions used for HEIR method?

A
  • Sodium citrate
  • EDTA
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9
Q

What is the purpose of increasing the pH for HEIR method?

A
  • enhances nuclear and cell surface antigens
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10
Q

What are the disadvantages of HEIR?

A
  • Tissue damage when heating strong alkaline solutions
  • Tissue detachment
  • Holes in tissue section
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11
Q

What are the methods of heating HEIR slides?

A
  • Microwave
  • Pressure cooker
  • Vegetable steamer
  • Circulating Water bath
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12
Q

What is used in the EIER method to expose antigenic sites in tissue sections?

A

Proteolytic enzymes

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13
Q

What are the disadvantages of using EIER?

A
  • Non specific staining if used improperly
  • Can weaken specific staining
  • Fragmentation or loss of tissue sections
  • Over digestion - distorted tissue morphology
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14
Q

What is the purpose of combining HEIR and EIER?

A
  • Minimize the extend of enzymatic pretreatment
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15
Q

When combining HEIR and EIER, what order are they done?

A
  1. HEIR
  2. EIER
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16
Q

What is a fluorochrome?

A
  • Dye that absorbs light and emits it’s own light at longer wave lengths
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17
Q

What are the advantages of using fluorochromes?

A
  • Sensitive
  • Specific
  • Simple
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18
Q

What is enzyme IHC?

A

Enzyme demonstrates antibody location in the presence of substrate and chromogens.

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19
Q

What are 4 enzymes used as markers?

A
  • Alkaline phosphatase
  • B-galactosidase
  • Glucose oxidase
  • Horseradish peroxidase
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20
Q

In tissue containing melanin what colour of chromogen should be used when doing enzyme IHC?

A

RED

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21
Q

What counter stain can be used for enzyme IHC methods? Why?

A

Mayer hematoxylin - bc it doesn’t contain alcohol

22
Q

What would happen is Harris hematoxylin was used to counterstain a section undergoing enzyme IHC staining?

A

False negative results because ACE and AP are dissolved in alcoholic hematoxylins

23
Q

What is the principle of direct immunofluorescent techniques?

A

Labeled antibody of known specificity is used to identify antigen’s in patient tissue, which are subsequently reacted with a chromogen.

24
Q

What are advantages to direct IHC methods?

A
  • One antibody
  • Short and quick
25
Q

What are the disadvantages of direct IHC methods?

A
  • Little signal amplification
  • Lacks sensitivity
  • May not demonstrate low levels of antigens in tumours.
26
Q

What is the principle of indirect immunofluorescent techniques?

A
  1. Unlabeled primary antibody reacts to tissue antigens
  2. Labeled secondary antibody reacts to the primary antibody.
27
Q

Compared to the direct method, what are advantages of the indirect method of IHC?

A
  • More sensitive
  • Secondary antibody can usually bind with multiple different antibodies.
28
Q

What is the purpose of using a third antibody in an indirect IHC method?

A
  • Further amplify the signal
29
Q

What is the light microscope method of IHC?

A

3 step method:
- Primary antibody
- Secondary antibody
- soluble enzyme-anti-enzyme complex

30
Q

What is the source of the soluble enzyme in the light microscope method of IHC?

A

From the same source as the secondary antibody.

31
Q

What is the PAP complex?

A

3 molecules of peroxidase and two antibodies.

32
Q

What is the APAAP complex?

A

2 molecules of alkaline phosphatase and one antibody.

33
Q

What are the two avidin- biotin methods?

A
  • ABC (avidin-biotin complex method)
  • LAB (Labeled avidin-biotin)
34
Q

What is the principle of avidin?

A

Avidin irreversibly binds to biotin.

35
Q

What are the steps in the avidin-biotin techniques?

A
  1. Primary antibody
  2. Biotinylated secondary antibody
  3. (ABC) preformed avidin-biotin enzyme complex
  4. (LAB) enzyme labeled avidin
36
Q

What is the purpose of pretreating sections with peroxide.

A

Remove endogenous peroxidase enzyme activity, decreases non specific staining

37
Q

What is a polymer enzyme?

A

secondary antibody fused with HRP or AP

38
Q

What is the purpose of PAP?

A

Localize tissue antigens

39
Q

What fixatives are best for Basic PAP?

A

NBF with epitope retrieval

40
Q

What are positive reactions for basic PAP?

A

Brick red

41
Q

What can you use to coverslip IHC slide with synthetic resin?

A

Crystal mount, unless using DAB instead AEC

42
Q

What are DAB and AEC?

A

Chromogens

43
Q

What are two methods of storing control slides?

A
  • Store at -20C
  • Dip pre-cut slides in paraffin, melt in over before use
44
Q

What are advantages of using pre-diluted antibodies?

A
  • Ready to use
  • Optimal dilution by the vendor
45
Q

What are disadvantages of using pre-diluted antibodies?

A

shelf life is shorter

46
Q

What are advantages of using concentrated antibodies?

A

Have longer shelf life and can be snap frozen with liquid nitrogen.

47
Q

What are disadvantages of using concentrated antibodies?

A

Require dilution to optimal concentration prior to use

48
Q

What are three examples of appropriate containers for storing antibodies?

A
  • Borosilicate
  • Propylene
  • Polycarbonate
49
Q

What can be added to antibodies to prevent loss/ adsorption of proteins to the storage container?

A

Albumin

50
Q

What are the two blocking reactions of immunoperoxidase method?

A
  1. peroxide in absolute methanol = block endogenous peroxidase activity.
  2. human serum - block non specific staining

Histology Exam (8 decks)

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