Laboratory Diagnostic Investigations (II) - IHC Flashcards

1
Q

Principle of IHC

3 general function

A

Detect presence of antigens on tissues

Tissue differentiation
Subtyping of tissues
Expression status of markers for prognosis or treatment

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2
Q

Chemical for embedding and fixation in IHC

A

Embed in paraffin
Fix in Formaldehyde, semi-reversible covalent crosslinking reagent

Formalin-fixed, Paraffin embedded before storage

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3
Q

Paraffin removal in IHC

  • Chemical used
  • Purpose
A

Xylene

Remove paraffin that can mask antigen presentation and prevent antibody binding

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4
Q

Antigen retrieval in IHC

  • 2 types?
  • Direct vs indirect detection?
A

Protease-induced epitope retrieval (Proteinase K, Trypsin, Pepsin)
Heat-induced epitope retrieval (Microwave, autoclave)

Direct detection = use primary Ab conjugated to label
Indirect detection = Secondary Ab against primary Ab. Can amplify signal
Use Fluorophores and enzymes that convert substrate into chromogens

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5
Q

2 types of signal detection system in IHC ?

Which is better?

A

Avidin-biotin peroxidase system
Polymer detection system

Polymer systems is better:

  • Simpler
  • No endogenous biotin
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6
Q

Causes of false positive IHC

A
  • Use wrong primary Ab
  • Non-specific staining
  • Biotin or peroxidase expressed
  • Tissue necrosis
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7
Q

Causes of false negative IHC

A
  • Over-fixation
  • Long incubation time
  • Wrong Ab concentration
  • Wrong developing time
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8
Q

Cause of non-specific staining on IHC?

Solution?

A

Primary and secondary Ab bind with serum proteins

Block endogenous forms of biotin, peroxidase or phosphatases with chemicals or physically

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9
Q

4 examples of using IHC to confirm tissue differentiation

A

Epithelial tumor - cytokeratin
Lymphoid tumor - LCA
Smooth muscle tumor - Actin
Melanocytic tumor - HMB45

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10
Q

2 examples of using IHC to subtype lesion tissue

A

B cell lymphoma - CD20, CD79a, PAX5

Invasive lobular carcinoma of breast - E-cadherin

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11
Q

2 examples of using IHC for surrogate marker and prognosis

A

HER2 in breast cancer

Ki67 in breast cancer (Prognostic)

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12
Q

What is the Ki67 marker used for in IHC?

A

Proliferative index
Stained in Nucleus, stains G1 to M phase cells but not G0

Ki67 index:
% of positive-staining cells/ number of cells = % of actively proliferating cells

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