IHC testing, short for immunohistochemistry, is a lab technique that detects specific proteins in a tissue sample using antibodies. It’s one of the most widely used tools in cancer diagnosis, helping pathologists identify what type of cancer is present, where it originated, and which treatments are most likely to work. If your doctor ordered IHC testing, it was almost certainly performed on tissue from a biopsy or surgical removal.
How IHC Testing Works
The core principle is straightforward: antibodies bind to specific proteins the way a key fits a lock. In IHC, pathologists apply specially designed antibodies to a thin slice of your tissue mounted on a glass slide. If the target protein is present in the cells, the antibody locks onto it. A chemical reaction then produces a visible color at that spot, allowing the pathologist to see exactly which cells contain the protein and how much of it they’re producing.
Some versions use an enzyme reaction that deposits a brown or red pigment visible under a standard microscope. Others use fluorescent labels that glow under ultraviolet light. The enzyme-based approach is more common in routine clinical work because it’s simpler and the stained slides can be stored and reviewed later without the signal fading.
What Happens to Your Tissue Sample
Before any staining begins, the tissue goes through several preparation steps. First, it’s preserved in a chemical solution (formalin) to keep the cellular structures intact. It’s then embedded in paraffin wax and sliced into sections roughly 4 to 5 micrometers thick, about one-twentieth the width of a human hair, and placed on glass slides.
Here’s where things get tricky. The preservation process that keeps the tissue intact also locks up the proteins the test needs to detect. So the slides go through a step called antigen retrieval, where they’re heated in a chemical buffer at near-boiling temperatures for about 15 minutes. This reopens the protein structures so antibodies can bind to them. After cooling, the antibodies are applied, left to incubate, and then a detection system makes the results visible.
The entire process typically adds one to two days to your pathology report timeline beyond the standard processing time.
Why IHC Is Used in Cancer Diagnosis
IHC testing serves three main purposes in oncology. The first is identifying the type of cancer. Tumors that look similar under a microscope can behave very differently depending on which proteins they express. IHC helps pathologists distinguish between cancer types and determine the organ where the cancer started, which is especially important when a tumor has spread and the original site isn’t obvious.
The second purpose is guiding treatment decisions. Certain proteins on cancer cells are direct targets for therapy. The most familiar examples in breast cancer include estrogen receptor (ER), progesterone receptor (PR), and HER2. If your tumor tests positive for hormone receptors, hormone-blocking therapies can be effective. If HER2 protein is overexpressed, targeted drugs designed to block that protein become an option. These IHC results directly shape your treatment plan.
The third, and increasingly important, role is determining eligibility for immunotherapy. A protein called PD-L1 is tested by IHC across many cancer types, including lung, bladder, head and neck, esophageal, cervical, gastric, and triple-negative breast cancers. The PD-L1 score from your IHC test determines whether you qualify for specific immunotherapy drugs. The FDA has approved specific IHC tests as companion diagnostics, meaning the drug can only be prescribed if the corresponding test shows a qualifying result.
How Results Are Scored
IHC results aren’t simply positive or negative. They’re scored based on how intensely the cells stain and what percentage of cells show the protein. The scoring system varies depending on the protein being tested.
HER2 scoring is a good example. Results are reported on a scale from 0 to 3+. A score of 0 means no staining is detected. A score of 1+ indicates faint staining on the cell membrane in more than 10% of tumor cells. A score of 2+ shows moderate, complete membrane staining in more than 10% of cells. And 3+ reflects strong, complete membrane staining. Scores of 0 and 1+ are considered negative (no HER2 overexpression), 3+ is positive, and 2+ falls into an equivocal zone that requires further testing to confirm.
For PD-L1, the scoring approach depends on the cancer type and the specific test used. Some use a tumor proportion score that only counts cancer cells. Others use a combined positive score that includes both cancer cells and certain immune cells. The cutoff for treatment eligibility varies by drug and cancer type.
IHC Compared to FISH Testing
If your HER2 IHC result comes back as 2+ (equivocal), your doctor will likely order a second test called FISH, which stands for fluorescence in situ hybridization. While IHC measures the protein on the surface of cancer cells, FISH looks at the gene inside the cell nucleus to see if extra copies are driving overproduction.
IHC is recommended as the first-line test because it’s less expensive, faster, and widely available in pathology labs. FISH is more precise but requires specialized equipment and trained technicians, making it costlier and harder to access. The two tests are complementary: IHC shows what’s happening at the protein level, while FISH reveals what’s happening at the genetic level. Another practical difference is that FISH’s fluorescent signals fade at room temperature, making long-term slide storage difficult, whereas IHC slides can be archived and re-examined.
What Can Affect IHC Accuracy
IHC is a powerful tool, but it’s sensitive to how the tissue is handled before it reaches the lab. One of the biggest variables is cold ischemia time, the gap between when tissue loses its blood supply during surgery and when it’s placed in preservative. The longer this window, the more cellular breakdown occurs, which can alter protein levels and lead to inaccurate results.
Fixation time matters too. If the tissue sits in preservative for too long or not long enough, protein detection can be compromised. Even how long a tissue block has been stored in paraffin or how old the cut slide is before staining can shift results. This is why pathology labs follow strict protocols for tissue handling, and why professional organizations have published detailed guidelines for specific tests like HER2.
Because of these variables, an equivocal or unexpected IHC result sometimes leads to repeat testing on the same tissue or on a new biopsy sample.
Multiplex IHC: Testing Multiple Proteins at Once
Traditional IHC tests for one protein per tissue slide. Newer multiplex techniques can detect multiple proteins simultaneously on a single section. This is particularly valuable when tissue is limited, such as a small needle biopsy, because it extracts more information from less material.
Multiplex IHC also reveals something standard IHC cannot: which proteins appear together on the same cell and how different cell types are arranged relative to each other. For example, a multiplex panel might show a tumor cell’s growth marker alongside the immune checkpoint proteins on neighboring immune cells, all on one slide. This spatial information is becoming increasingly important for understanding how tumors interact with the immune system and for predicting responses to immunotherapy.