Immunohistochemistry (IHC)

What it is

Immunohistochemistry (IHC) is a lab test that uses antibodies to detect specific proteins in tissue sections, often on biopsy samples. In cancer care, its most important role is helping pathologists identify the tumour type and, in some cases, guide treatment by showing whether a marker is present or absent. IHC is usually performed on formalin-fixed, paraffin-embedded tissue, often called FFPE tissue, which is the standard way many biopsy and surgery specimens are processed.

A pathologist does not use IHC in isolation. It is interpreted together with the patient's history, imaging, the appearance of the tissue on routine haematoxylin and eosin (H&E) staining, and sometimes other tests such as flow cytometry, FISH, PCR, or next-generation sequencing. In practical terms, IHC can help answer questions such as: Is this cancer epithelial, lymphoid, melanocytic, or mesenchymal? Is a metastatic tumour likely to have started in the lung, breast, colon, prostate, or another organ? Does the tumour express a predictive marker such as estrogen receptor, progesterone receptor, HER2, or PD-L1?

How it works

IHC works by applying an antibody that binds to a target antigen, usually a protein, within cells or tissue. A detection system then makes that binding visible under the microscope, commonly as a brown or red stain in the nucleus, cytoplasm, or cell membrane. The location of staining matters because some markers are expected in a specific cellular compartment.

Typical steps include tissue fixation, paraffin embedding, thin sectioning, antigen retrieval, incubation with the primary antibody, signal detection, and microscopic interpretation. Antigen retrieval is important because formalin fixation can mask epitopes; heat or enzymatic treatment may be needed to unmask them.

Common IHC result patterns include:

Pattern	What it may mean
Nuclear staining	Often seen with markers such as ER, PR, Ki-67, TTF-1, p53
Membranous staining	Often relevant for HER2, E-cadherin, some lymphoid markers
Cytoplasmic staining	Seen with many cytokeratins, vimentin, synaptophysin
Positive/negative	Whether the target protein is detected above the lab's threshold
Intensity and percentage	How strong the stain is and how many tumour cells stain

Some IHC tests are mainly diagnostic, meaning they help classify the tumour. Others are predictive or prognostic, meaning they may help estimate likely behaviour or response to a treatment. For example, ER and PR in breast cancer can support use of endocrine therapy, while HER2 IHC can be part of deciding whether HER2-targeted therapy should be considered.

Evidence and uses

IHC is a core tool in modern surgical pathology and is widely used in cancer diagnosis. It is especially useful when routine microscopy alone does not clearly show the tumour lineage or site of origin. It can also detect small groups of tumour cells that are hard to see on H&E staining.

Common uses include:

1. Classifying tumours: distinguishing carcinoma, lymphoma, melanoma, sarcoma, germ cell tumour, and neuroendocrine tumour.
2. Finding the likely primary site: using marker panels such as CK7, CK20, TTF-1, CDX2, PAX8, GATA3, PSA, or others in metastatic cancer.
3. Measuring treatment-related markers: such as ER, PR, HER2, mismatch repair proteins, and PD-L1 in selected cancers.
4. Estimating proliferation: Ki-67 may help describe how actively tumour cells are dividing, although interpretation varies by tumour type.
5. Detecting micrometastases or residual disease: in lymph nodes, bone marrow, or small biopsy samples.

A key point is that pathologists usually order panels of markers rather than relying on a single stain. One marker alone is rarely definitive. For example, TTF-1 positivity may support lung or thyroid origin, but the final interpretation depends on morphology and the rest of the immunoprofile.

In India, IHC is widely available in tertiary cancer centres and many private pathology laboratories, but the exact antibody clones, scoring systems, turnaround time, and quality controls can vary by lab. For treatment-linked markers such as HER2 or hormone receptors, clinicians often prefer testing in laboratories that follow validated protocols and external quality standards.

Diagnosis / how it's measured

IHC is measured by a pathologist's microscopic assessment, not by a simple machine-read number alone. The report may describe:

• the marker tested
• whether staining is positive, negative, or equivocal
• the staining pattern and intensity
• the percentage of tumour cells stained
• a formal score, if that marker has a validated scoring system

Examples of marker-specific reporting include:

Marker example	How it may be reported
ER/PR	Percentage of positive tumour nuclei, sometimes with intensity or Allred-type scoring
HER2	IHC score 0, 1+, 2+, or 3+
Ki-67	Percentage of tumour nuclei staining positive
Mismatch repair proteins	Intact or lost expression of MLH1, MSH2, MSH6, PMS2
PD-L1	Tumour proportion score, combined positive score, or another assay-specific metric

An equivocal result does not always mean the test failed. It may mean the result falls into an intermediate category where another method is needed. A common example is HER2 IHC 2+, which often requires confirmatory in situ hybridisation testing.

Pre-analytical factors strongly affect accuracy. Delayed fixation, poor fixation, overfixation, tissue decalcification, low tumour content, necrosis, and technical variation can all change staining results. This is why repeat testing may sometimes be recommended if the result does not fit the clinical picture.

When to see a clinician

You should discuss an IHC report with the treating oncologist or pathologist when the diagnosis is unclear, the report uses unfamiliar terms such as equivocal or indeterminate, or the result may affect treatment choice. This is particularly important for breast cancer, lymphoma, lung cancer, colorectal cancer, and cancers of unknown primary, where marker interpretation can change management.

Ask for clarification if the report does not state the specimen type, tumour site, marker panel, scoring method, or whether additional confirmatory testing is needed. If treatment decisions depend on the result, a second pathology review at a cancer centre may be reasonable, especially when the findings and the clinical picture do not match.

Limitations and open questions

IHC is powerful, but it is not perfect. False-positive and false-negative results can occur because of technical issues, antibody selection, fixation problems, background staining, or biologic heterogeneity within the tumour. Different parts of the same tumour may stain differently, and small biopsies may not capture the full picture.

Interpretation also depends on context and expertise. A positive stain does not automatically prove a diagnosis, and a negative stain does not always exclude it. Many markers are sensitive but not specific, or specific only when used in the right panel.

Another limitation is standardisation. Some markers have well-established scoring systems, but others do not. For newer predictive biomarkers, assay choice, cutoffs, and inter-observer variation can affect results. Evidence continues to evolve for how best to use some stains, especially when IHC is being compared with molecular methods.

For patients, the practical takeaway is that IHC is usually one part of the pathology work-up, not the whole answer. When results are borderline, unexpected, or treatment-defining, confirmatory testing or expert review may be needed.