Technique Guide · Path-iQ Global Pathology Review
Immunohistochemistry (IHC) is the most widely used ancillary technique in diagnostic pathology — applied to classify tumours, detect infectious organisms, quantify prognostic biomarkers, and guide targeted therapy selection. Understanding IHC principles, antibody selection, and result interpretation is essential for every practicing pathologist.
IHC detects specific proteins (antigens) within tissue sections using antibodies. The fundamental steps are: (1) antigen retrieval — reverses formalin-induced crosslinks that mask epitopes; (2) primary antibody incubation — the antibody binds its specific target antigen; (3) secondary detection — an enzyme-linked or polymer-based secondary system amplifies the signal; (4) chromogen development — the enzyme reacts with a substrate to produce a coloured precipitate (DAB = brown, AP-Red = red) at the antigen site; (5) counterstaining — haematoxylin provides blue nuclear background contrast.
Two antigen retrieval methods exist: heat-induced epitope retrieval (HIER) using buffer at high temperature (pressure cooker, microwave, or water bath at pH 6.0 citrate or pH 9.0 EDTA) is used for most antigens; protease-induced epitope retrieval (PIER) with proteinase K or trypsin is used for a small number of antigens (e.g., some collagen-based targets). Automated platforms (Leica BOND, Ventana BenchMark, Dako Autostainer) have standardised HIER protocols, improving inter-laboratory reproducibility.
| Marker | Lineage | Positive Result Suggests | Clone / Notes |
|---|---|---|---|
| AE1/AE3 + CAM5.2 | Epithelial (broad cytokeratin) | Carcinoma (most types); mesothelioma; some sarcomas (synovial, epithelioid) | Pan-CK cocktail; negative in most sarcomas and lymphomas |
| CD45 (LCA) | Haematopoietic | Lymphoma, leukaemia (excludes most carcinomas and sarcomas) | 2B11+PD7/26; rarely lost in ALCL and plasma cell myeloma |
| S100 | Melanocytic, neural, myoepithelial | Melanoma, nerve sheath tumours, Langerhans cell histiocytosis, myoepithelioma | Polyclonal; broad; always combine with more specific marker (SOX10, HMB-45, MelanA) |
| Vimentin | Mesenchymal | Sarcoma, melanoma, lymphoma, renal cell carcinoma | Broad — positive in most poorly differentiated tumours; low specificity alone |
| SOX10 | Neural crest / melanocytic | Melanoma (nuclear), nerve sheath tumours (schwannoma, MPNST), myoepithelial tumours | More specific than S100 for melanocytic lineage; negative in carcinoma |
| CD34 | Endothelial, stromal, progenitor | Vascular tumours (angiosarcoma), GIST, DFSP, solitary fibrous tumour, haematopoietic progenitors | Clone QBEnd-10; also marks dermal dendrocytes |
| Marker | Positive In | Key Negative | Primary Use |
|---|---|---|---|
| TTF-1 (NKX2-1) | Lung adenocarcinoma (~75%); thyroid carcinoma; SCLC | Squamous cell carcinoma of lung; GI, breast, GU carcinomas | Distinguish lung adeno (TTF-1+) from SCC (p40+) in lung biopsy; thyroid origin |
| p40 (ΔNp63) | Squamous cell carcinoma (lung, H&N, oesophagus, cervix); urothelial carcinoma; basal cell | Adenocarcinoma; SCLC; neuroendocrine tumours | Most specific SCC marker; superior to p63 for this purpose |
| Napsin A | Lung adenocarcinoma (~80%); renal clear cell and papillary RCC | Squamous, SCLC, mesothelioma | Combined with TTF-1 for lung adeno; strong cytoplasmic granular staining |
| CDX2 | Colorectal adenocarcinoma (~90%); gastric intestinal-type; appendiceal; small bowel | Lung, breast, gynaecological carcinomas (usually negative) | GI origin marker; nuclear; strongest expression in CRC |
| GATA3 | Breast carcinoma; urothelial carcinoma; paraganglioma; trophoblastic tumours | Lung, GI, prostate (usually negative) | Distinguish breast from other adenocarcinomas; urothelial origin in metastases |
| PAX8 | Renal carcinoma; ovarian (serous, clear cell, endometrioid); thyroid; Müllerian tumours | Breast, lung, GI (usually negative) | Gynaecological and renal origin marker; nuclear |
| PSA / NKX3.1 | Prostate adenocarcinoma (highly specific) | Most other carcinomas | PSA less sensitive in high-grade; NKX3.1 more sensitive for high-grade prostate Ca |
| WT1 | Mesothelioma; serous ovarian carcinoma; Wilms tumour; desmoplastic small round cell tumour | Adenocarcinoma (usually negative); urothelial (negative) | Nuclear WT1: mesothelioma vs adenocarcinoma panel |
| Calretinin | Mesothelioma; adrenal cortical tumour; sex cord-stromal tumours; ganglion cells | Adenocarcinoma of lung, breast, GI | Mesothelioma panel (calretinin + WT1 + D2-40 vs CEA + MOC31 for adeno) |
| Synaptophysin / Chromogranin A | Neuroendocrine tumours (carcinoid, SCLC, Merkel, pancreatic NET, phaeochromocytoma) | Adenocarcinoma; squamous carcinoma | Core NE markers; Syn more sensitive, CgA more specific; SCLC: Syn+CgA+TTF-1 |
| Marker | Lineage / Entity | Pattern | Clinical Use |
|---|---|---|---|
| CD20 | B-cell lymphoma (mature) | Membranous | DLBCL, FL, MCL, MZL, CLL/SLL; rituximab therapy target |
| PAX5 | B-cell lineage (broad) | Nuclear (weak in plasma cells) | B-cell confirmation including CHL Reed-Sternberg cells (weak/focal PAX5) |
| CD3 | T-cell lineage | Membranous / cytoplasmic | T-cell lymphoma; distinguish T from B in nodal biopsy |
| CD10 | Germinal centre B-cells | Membranous | FL (CD10+BCL6+BCL2+); GCB-DLBCL; Burkitt (CD10+ MYC+) |
| BCL2 | Follicular lymphoma (strong); GCB DLBCL (weak/mod) | Cytoplasmic | FL: CD10+BCL6+BCL2+ strong; BCL2 strong in non-GCB DLBCL |
| BCL6 | Germinal centre origin | Nuclear | FL; GCB-DLBCL; combined with CD10/MUM1 for Hans COO algorithm |
| MUM1 (IRF4) | Post-GC / plasma cell differentiation | Nuclear | Non-GCB DLBCL (Hans algorithm); plasma cell neoplasms; MALT lymphoma |
| Cyclin D1 | Mantle cell lymphoma (CCND1-IGH) | Nuclear | MCL diagnostic; also hairy cell leukaemia; rare plasma cell myeloma |
| CD30 | ALCL; CHL; primary cutaneous CD30+ LPD | Membranous / Golgi | ALCL (CD30+ALK±); CHL (CD30+CD15+PAX5 weak); brentuximab vedotin target |
| ALK (D5F3) | ALK-positive ALCL; inflammatory myofibroblastic tumour; ALK+ NSCLC | Cytoplasmic ± nuclear (pattern varies by fusion) | ALK+ ALCL: best prognosis PTCL; ALK TKI therapy (crizotinib, alectinib) in NSCLC |
| Ki-67 (MIB1) | All proliferating cells | Nuclear | Proliferation index: Burkitt ~100%; DLBCL 40–90%; FL varies; NET grading (G1 <3%, G2 3–20%, G3 >20%) |
| Biomarker | Tumour | Test | Scoring System | Clinical Impact |
|---|---|---|---|---|
| ER / PR (oestrogen/progesterone receptor) | Breast carcinoma | IHC (SP1/1E2 for ER; 1E2 for PR) | Allred score (0–8) or H-score (0–300); positive = ≥1% nuclear staining (ASCO/CAP 2020) | Hormone therapy (tamoxifen, aromatase inhibitors) eligibility; strongest predictor of endocrine therapy response |
| HER2 | Breast; gastric; GEJ; CRC; endometrial | IHC (4B5; SP3; CB11) → ISH if 2+ | 0 / 1+ / 2+ / 3+ (ASCO/CAP HER2 guidelines); 3+ or ISH-amplified = positive | Anti-HER2 therapy (trastuzumab, pertuzumab, T-DM1, T-DXd); HER2-low (1+ or 2+/ISH-): T-DXd eligibility |
| PD-L1 | NSCLC; TNBC; gastric; cervical; urothelial; H&N SCC; many others | IHC (22C3; 28-8; SP142; SP263 — clone-specific) | TPS (tumour proportion score) for most; CPS (combined positive score) for gastric/urothelial; IC% for atezolizumab | Checkpoint inhibitor (pembrolizumab, nivolumab, atezolizumab, durvalumab) eligibility and dose selection |
| MLH1 / MSH2 / MSH6 / PMS2 | CRC; endometrial; other Lynch-associated tumours | IHC panel (4 markers simultaneously) | Nuclear loss = abnormal (dMMR); intact nuclear expression = MMR-proficient | MSI-H/dMMR → pembrolizumab; Lynch syndrome reflex testing (MLH1 loss → BRAF V600E to exclude sporadic); adjuvant chemotherapy guidance in stage II CRC |
| p53 | Endometrial; ovarian; breast; CRC; many others | IHC (DO-7) | Aberrant pattern: overexpression (>80% strong nuclear) OR complete absence (null pattern) = TP53 mutation | Endometrial carcinoma molecular subgroup (p53-abnormal = worst prognosis); ovarian serous carcinoma signature (p53+ >96%) |
| Ki-67 | Breast; neuroendocrine tumours; lymphoma | IHC (MIB1; SP6) | % positive nuclei in hot-spot or global counting; NET: G1 <3%, G2 3–20%, G3 >20% | Breast: Oncotype DX proliferation score correlation; NET grading (ENETS/WHO); DLBCL proliferation assessment |
| IDH1 R132H | Glioma (IDH-mutant) | IHC (clone H09) | Any cytoplasmic positivity in tumour cells = positive (very high specificity for R132H) | Confirms IDH-mutant glioma class; negative in ~10% of IDH-mutant gliomas (non-R132H — need sequencing) |
| ATRX | IDH-mutant astrocytoma | IHC (polyclonal) | Nuclear loss in tumour cells (internal positive control: endothelial cells must retain staining) | Loss supports astrocytoma lineage over oligodendroglioma; combined with IDH and 1p/19q FISH for integrated glioma diagnosis |
External quality assurance (EQA) is mandatory for all IHC biomarkers used in clinical decision-making. The two largest IHC EQA programmes are:
The most common sources of IHC failure identified by NordiQC assessments: insufficient antigen retrieval (most frequent for ER, PR, Ki-67); inappropriate antibody dilution; use of non-validated antibody clones; suboptimal fixation (under- or over-fixation); and scanner/digital pathology colour calibration issues affecting quantitative IHC scoring.
| Pitfall | Mechanism | Example | Avoidance |
|---|---|---|---|
| False-negative due to over-fixation | Excessive formalin crosslinking masks epitopes despite antigen retrieval | ER negative in breast biopsy fixed >72h; HER2 1+ instead of 3+ | Fix 6–72h; document fixation time on request form; cold ischaemia <1h |
| False-negative due to under-fixation | Epitopes diffuse or degrade; antigen retrieval may damage fragile proteins | ER weakly positive in unfixed frozen section converted to FFPE | Immerse in formalin immediately at specimen cut-up; 10% NBF preferred |
| Edge artefact | Antibody/reagent pooling at biopsy edges causes intense non-specific staining | PD-L1 appearing strongly positive at core biopsy edges only | Interpret central tumour cells only; disregard edge staining for scoring |
| Crush artefact | Mechanical pressure destroys cell architecture; IHC staining uninterpretable | SCLC bronchial biopsy — neuroendocrine markers uninterpretable | Gentle tissue handling; touch prep/smear as backup; adequate core size |
| Non-specific background | Endogenous biotin, peroxidase, or protein binding; inadequate blocking | Liver biopsy: endogenous biotin creates false-positive background with avidin-biotin systems | Use polymer-based detection (non-biotin) for liver; block endogenous peroxidase |
| Wrong clone for CDx application | Different PD-L1 clones not interchangeable; validated clone required per drug | Using SP142 result to determine pembrolizumab eligibility (requires 22C3) | Strict CDx-analyte mapping; one system per therapeutic agent |
| HER2 equivocal 2+ not reflexed to ISH | IHC 2+ is uninformative without ISH confirmation | Treating HER2 IHC 2+ as positive without ISH — 75% of 2+ cases are ISH-negative | Reflex all IHC 2+ cases to FISH/CISH/DISH; never treat on 2+ IHC alone |
Founder of NordiQC — the world's largest IHC external quality assurance programme. Internationally recognised as the leading authority on IHC standardisation, antibody validation, and the consequences of IHC variability in clinical pathology.
Pioneer in diagnostic antibody development for pathology. Developed many clinically essential antibody clones including anti-ER (SP1), anti-PR (1E2), anti-HER2 (4B5), and anti-p63 (4A4). Founder of Biocare Medical.
Editor of Diagnostic Immunohistochemistry — the leading IHC reference text in anatomic pathology. Expert in breast biomarker IHC, ER/PR/HER2 testing standardisation, and the ASCO/CAP HER2 guideline development.
Expert in IHC for prostate pathology, including AMACR (P504S), p63, and CK5/6 in prostate cancer versus benign mimics. Contributor to CAP prostate cancer reporting protocols and IHC quality standards.
Authority on IHC in haematopoietic tumour pathology, including plasmacytoid dendritic cell neoplasm (BPDCN), blastic NK-cell lymphoma, and the use of novel IHC markers in haematopathology classification.
Pioneer who helped establish IHC as a routine diagnostic tool in the 1970s–80s. Champion of IHC standardisation and co-author of seminal work on the sources of IHC variability and pre-analytical factors.