Immunohistochemistry Basics
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Direct answer
Immunohistochemistry (IHC) detects antigens in tissue sections using a primary antibody, a labelled detection polymer and a chromogen — diaminobenzidine producing the familiar brown signal — counterstained with haematoxylin. Its working purpose is triage of the poorly differentiated tumour: cytokeratins confirm carcinoma, CD45 confirms lymphoid origin, S100/SOX10 and HMB45 point to melanoma, and vimentin flags mesenchymal lineage, after which second-line markers identify the specific entity and often the primary site. Beyond classification, IHC now carries therapeutic weight — ER, PR and HER2 in breast cancer, CD117 and DOG1 in GIST, and Ki-67 for grading neuroendocrine tumours. Proper runs demand positive and negative controls, and pre-analytical variables (fixation, decalcification) explain most failed stains.
What you must remember
- Lineage first-line panel: pan-cytokeratin (AE1/AE3) for epithelial, CD45/LCA for lymphoid, S100/SOX10 for melanocytic and neural crest, vimentin for mesenchymal, CD31/CD34 for vascular.
- Small round cell and spindle cell panels: myogenin and desmin for rhabdomyosarcoma, CD99 for Ewing, synaptophysin and chromogranin for neuroendocrine, TLE1 for synovial sarcoma.
- Site-specific markers: TTF-1 for lung and thyroid primaries, PAX8 for renal and Müllerian, GATA3 for urothelial and breast, PSA for prostate, CDX2 for intestinal differentiation.
- Theranostic anchors: ER/PR and HER2 in breast (score 0 to 3+; equivocal 2+ needs ISH refinement), CD117/DOG1 for GIST (imatinib targets), ALK and PD-L1 in lung adenocarcinoma.
- Proliferation and differentiation: Ki-67 percentage grades neuroendocrine neoplasms (G1 up to 2 per cent, G2 3-20, G3 above 20 per cent on hotspots) and stratifies many tumours.
- Lymphoma dissection: CD3 and CD5 for T cells, CD20 and PAX5 for B cells, CD30 for Hodgkin and ALCL, CD138 with kappa/lambda restriction for plasma cell neoplasms.
- Germ cell and miscellany: OCT4 and SALL4 for germ cell tumours, CD1a for Langerhans cells, chromogranin-synaptophysin pair for neuroendocrine differentiation.
An undifferentiated tumour worked through
A 55-year-old smoker has a single enlarged supraclavicular node; the biopsy shows sheets of poorly differentiated malignant cells. The first panel — pan-cytokeratin, CD45, S100, vimentin — runs overnight. Cytokeratin is strongly positive and CD45 negative, so this is carcinoma, not lymphoma or melanoma. The second panel pursues the primary: TTF-1 nuclear staining plus Napsin A suggests lung adenocarcinoma, and a staging CT finds the peripheral lung primary. Had cytokeratin been negative with CD45 positive, flow cytometry and B- and T-cell markers would follow; had S100 and SOX10 been positive with cytokeratin negative, melanoma markers (HMB45, Melan-A) would complete the diagnosis. Every step narrows the differential by elimination — panel thinking, not single-marker thinking, is what NBE vignettes quietly test.
Where students slip
Vimentin is over-trusted: it marks mesenchymal tissue but also stains many carcinomas and mesotheliomas, so it is a poor lone discriminator. S100 is likewise shared by melanoma, nerve sheath tumours, Langerhans cells and even chondrocytes, which is why melanoma needs HMB45 or Melan-A backup. HER2 scoring trips candidates: 2+ is equivocal and requires in-situ hybridisation, a favourite single-best-answer point. And a control-free stain is uninterpretable — without a known positive control on the same run, a brown reaction proves nothing about the patient and everything about luck.
Frequently asked questions
What is the basic principle of immunohistochemistry?
A primary antibody binds a specific tissue antigen, a labelled secondary or polymer system visualises the binding, and a chromogen such as DAB deposits a brown reaction product at the antigen site.
Which markers confirm gastrointestinal stromal tumour?
CD117 (c-KIT) and DOG1, often with a KIT or PDGFRA mutation — positivity that simultaneously predicts imatinib responsiveness.
How is HER2 interpreted on IHC?
0 and 1+ are negative, 3+ positive, while 2+ is equivocal and requires confirmatory in-situ hybridisation before trastuzumab or related therapy.
What does a high Ki-67 labelling index indicate?
A high fraction of proliferating cells, used most formally to grade gastroenteropancreatic neuroendocrine neoplasms — G3 exceeds 20 per cent in hotspot counts.
Which first-line panel sorts an undifferentiated malignant tumour?
Pan-cytokeratin, CD45, S100 and vimentin — separating carcinoma, lymphoma, melanoma and sarcoma before any second-line marker is chosen.