Laboratory Diagnostic Investigations (II) - IHC

Question 1

Principle of IHC

3 general function

Answer 1

Detect presence of antigens on tissues

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

Question 2

Chemical for embedding and fixation in IHC

Answer 2

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

Formalin-fixed, Paraffin embedded before storage

Question 3

Paraffin removal in IHC

• Chemical used
• Purpose

Answer 3

Xylene

Remove paraffin that can mask antigen presentation and prevent antibody binding

Question 4

Antigen retrieval in IHC

• 2 types?
• Direct vs indirect detection?

Answer 4

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

Question 5

2 types of signal detection system in IHC ?

Which is better?

Answer 5

Avidin-biotin peroxidase system
Polymer detection system

Polymer systems is better:

• Simpler
• No endogenous biotin

Question 6

Causes of false positive IHC

Answer 6

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

Question 7

Causes of false negative IHC

Answer 7

• Over-fixation
• Long incubation time
• Wrong Ab concentration
• Wrong developing time

Question 8

Cause of non-specific staining on IHC?

Solution?

Answer 8

Primary and secondary Ab bind with serum proteins

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

Question 9

4 examples of using IHC to confirm tissue differentiation

Answer 9

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

Question 10

2 examples of using IHC to subtype lesion tissue

Answer 10

B cell lymphoma - CD20, CD79a, PAX5

Invasive lobular carcinoma of breast - E-cadherin

Question 11

2 examples of using IHC for surrogate marker and prognosis

Answer 11

HER2 in breast cancer

Ki67 in breast cancer (Prognostic)

Question 12

What is the Ki67 marker used for in IHC?

Answer 12

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