Laboratory Diagnostic Investigations (II) - IHC Flashcards
Principle of IHC
3 general function
Detect presence of antigens on tissues
Tissue differentiation
Subtyping of tissues
Expression status of markers for prognosis or treatment
Chemical for embedding and fixation in IHC
Embed in paraffin
Fix in Formaldehyde, semi-reversible covalent crosslinking reagent
Formalin-fixed, Paraffin embedded before storage
Paraffin removal in IHC
- Chemical used
- Purpose
Xylene
Remove paraffin that can mask antigen presentation and prevent antibody binding
Antigen retrieval in IHC
- 2 types?
- Direct vs indirect detection?
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
2 types of signal detection system in IHC ?
Which is better?
Avidin-biotin peroxidase system
Polymer detection system
Polymer systems is better:
- Simpler
- No endogenous biotin
Causes of false positive IHC
- Use wrong primary Ab
- Non-specific staining
- Biotin or peroxidase expressed
- Tissue necrosis
Causes of false negative IHC
- Over-fixation
- Long incubation time
- Wrong Ab concentration
- Wrong developing time
Cause of non-specific staining on IHC?
Solution?
Primary and secondary Ab bind with serum proteins
Block endogenous forms of biotin, peroxidase or phosphatases with chemicals or physically
4 examples of using IHC to confirm tissue differentiation
Epithelial tumor - cytokeratin
Lymphoid tumor - LCA
Smooth muscle tumor - Actin
Melanocytic tumor - HMB45
2 examples of using IHC to subtype lesion tissue
B cell lymphoma - CD20, CD79a, PAX5
Invasive lobular carcinoma of breast - E-cadherin
2 examples of using IHC for surrogate marker and prognosis
HER2 in breast cancer
Ki67 in breast cancer (Prognostic)
What is the Ki67 marker used for in IHC?
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