Prompt and adequately formalin fixed tissue samples or formalin fixed paraffin embedded tissue blocks. Rarely frozen section tissue samples used but slide quality is better with formalin fixed paraffin embedded slides.
Tissue sectioning using vibratome is done, sections of 3 microns (4-40microns) are obtained and the following steps carried out.
- ANTIGEN RETRIEVAL to uncover the hidden antigen sites the tissue section is placed in an aqueous solution (eg: citrate buffer, TRIS-EDTA etc.,) along with heat application (heat induced epitope retrieval - HIER) or by protein digestion (proteolytic induced epitope retrieval-PIER)
- Detergent (eg:TritonX-100) is used for better antibody penetration and a blocking buffer used to block background staining
- Primary antibody application
- Peroxidase blocking
- Secondary antibody application
- Ag - Ab interaction and detection, addition of chromagen/substrate
- Counterstain, dehydrate slide, clean with xylene and mount for microscopy
Appropriate positive and negative controls evaluated in parallel with the patient sample .Important steps are appropriate antibody selection, correct interpretation, technical quality and integration of results
IHC helps to arrive at a diagnosis in case of
- Undifferentiated malignant tumours/poorly differentiated /diagnostically challenging cancers (eg:sarcomas)/gynaecological tumours and neural tumours
- To select therapy in tumours by detecting hormone receptors (eg: ER/PR receptors in breast carcinoma),chemical inhibitors(eg: tyrosine kinase) and monoclonal antibodies (eg: HER2/neu,EGFR)
Markers frequently requested;
|Cytokeratins for differentiating carcinomas from sarcomas|
|CD15 and CD30||: Hodgkin's disease|
|Alphafetoprotein||: Hepatocellular carcinoma,yolk sac tumour|
|CD117(KIT)||: Gastrointestinal stromal tumour (GIST)|
|CD10(CALLA)||: Renal cell carcinoma and Acute lymphoblastic leukemia|
|PSA||: Prostatic carcinoma|
|CD19,CD20||: B-cell lymphoma|
|CD3,CD4,CD8||: T-cell lymphoma|
IHC is one of the best diagnostic tools in diagnostically difficult carcinomas and plays a vital role in today's oncological diagnosis.