What is Immunohistochemistry (IHC) Staining? Its Significance in Tumor Diagnosis

ImmunityQ&AMeongsiljang Veterinary Advisory Board

Immunohistochemical (IHC) staining is a precise pathological test that identifies specific proteins within tissue using antibodies, allowing for accurate determination of a tumor’s identity and origin. It plays a crucial role in distinguishing tumor types, assessing malignancy, and guiding treatment decisions when conventional histopathology alone is insufficient.

What is Immunohistochemistry (IHC) Staining?

A veterinary pathologist analyzing immunohistochemical staining slides under a microscope

Immunohistochemistry (IHC) staining is a precise pathological test that identifies the nature of tumor cells by binding antibodies to specific proteins within tissue samples and producing a visible color reaction. Accurate tumor classification is key. While routine histopathology (H&E staining) may only indicate that a lesion is "suspected to be malignant," adding IHC can reveal the cell type of origin, the degree of malignancy, and which treatments are likely to be most effective.

Why Is Routine Histopathology Insufficient?

Standard histopathology examines only the shape and arrangement of cells, but tumors that look similar can have completely different origins. For example, lymphoma, mast cell tumors, and histiocytomas all appear as round cells, making them difficult to distinguish using only H&E staining. Immunohistochemistry (IHC) identifies specific proteins (markers) unique to each cell type, allowing us to determine exactly which immune cell lineage a round cell originates from. Since treatment options and prognosis vary significantly depending on the tumor type, accurate classification is crucial.

How Is IHC Staining Performed?

Thin tissue sections are placed on slides, and a primary antibody that binds to the target protein (antigen) is applied. A secondary antibody conjugated with a chromogen is then added, producing a color change at the binding site under the microscope. Because staining occurs only where the protein is present, you can easily see which cells are expressing which proteins. Since tumor cells can share markers, it is common practice to use a panel of multiple markers for simultaneous interpretation rather than relying on a single marker.

An illustration of the IHC staining principle, where color is applied through antigen-antibody binding.

Major IHC Markers and Diagnostic Significance

Item	Meaning When Positive	Tumors Commonly Used For
CD3	T-cell lineage lymphoma	T-cell lymphoma
CD20 / CD79a	B-cell lineage lymphoma	B-cell lymphoma
c-KIT (CD117)	Mast cell origin; staining pattern predicts metastatic potential and prognosis	Mast cell tumor
Ki-67	Cell proliferation rate (degree of malignancy)	Prognostic evaluation of nearly all tumors
Melan-A	Melanocytic origin	Melanoma
Vimentin	Mesenchymal cells (sarcoma suspected)	Soft tissue sarcoma

The type of marker and the criteria for positivity may vary depending on the laboratory and tumor type.

When Is IHC Recommended?

According to veterinary pathology textbooks, immunohistochemistry (IHC) is strongly recommended in the following situations:

Differential diagnosis of round cell tumors: When it is difficult to distinguish between lymphoma, mast cell tumor, and plasmacytoma.

Immunophenotyping of lymphoma: To determine whether the tumor is T-cell or B-cell origin, which helps decide treatment drugs and prognosis.

Assessment of malignancy grade: To evaluate the rate of cell proliferation using markers such as Ki-67 and mitotic index.

Tracing the origin of metastatic cancer: To find clues about where the primary tumor originated before spreading.

Guiding prognosis and treatment decisions: In c-KIT (CD117)-positive mast cell tumors, the staining pattern of c-KIT helps estimate the likelihood of metastasis and prognosis, which can inform treatment planning.

Key Points for Interpreting IHC Results

Immunohistochemistry (IHC) results are rarely as straightforward as a simple "positive" or "negative." Instead, markers are graded on a scale such as "strongly positive," "weakly positive," or "partially positive," and a diagnosis is never confirmed based on a single marker alone. Typically, multiple markers are interpreted together as a panel, and the final diagnosis is reached by integrating these findings with H&E staining results, clinical information, and imaging studies. If the report uses phrases like "consistent with" or "suggestive of," it indicates that the diagnosis is not yet definitive. In such cases, it is essential to discuss the results thoroughly with your veterinarian.

Test Costs and Turnaround Time

Immunohistochemistry (IHC) takes more time and costs more than routine histopathology. There is an additional fee for each marker, and since markers are typically ordered in panels of two to five, the cost is added on top of the standard histopathology fee. Because pricing varies significantly between laboratories, it is advisable to confirm the estimated cost with your veterinarian when they submit the external referral. Results usually take about seven to fourteen days for external referrals, and this timeframe may extend if additional markers are required.

A scene in the examination room where a veterinarian explains the IHC test results to the pet owner.

Key Points for Pet Owners to Check in the Report

Immunohistochemistry (IHC) reports can be difficult to understand due to the medical terminology, but there are key items that pet owners should definitely pay attention to.

Final Diagnosis: The exact name and grade of the tumor.

Immunophenotype: The cell origin, such as B cells or T cells.

Ki-67 Index: The proliferation rate (a lower index generally indicates slower growth).

Prognostic Comments: Pathological findings such as "good prognosis," "caution," or "poor prognosis."

Targeted Therapy and Prognostic Markers: Whether markers like c-KIT (CD117) are positive or negative.

If there are any items you do not understand, please be sure to ask your veterinarian to explain them in detail.

Additional Considerations for Cats

In cats, the immune phenotype (T-cell vs. B-cell) of gastrointestinal lymphoma is closely linked to prognosis. According to textbook reports, small-cell lymphomas arising in the feline small intestine are predominantly of T-cell lineage, and small-intestinal T-cell lymphomas are known to have a better long-term prognosis than B-cell tumors. The tumor’s location can also serve as a clue for predicting immune phenotype and prognosis. Because treatment approach and prognosis can vary depending on the immune phenotype even within the same diagnosis of lymphoma in cats, it is important to accurately distinguish between T-cell and B-cell types using immunohistochemistry (IHC). When submitting samples for testing, it is advisable to confirm with your veterinarian in advance which marker panel will be used for interpretation.

Reviewed by a veterinarian

Dr. Tony — Punnawat Phongkittirak

Veterinarian

A veterinarian who majored in veterinary medicine at Khon Kaen University, Thailand, and completed the IVSA program at North Carolina State University in the United States. Drawing on clinical experience at animal hospitals, he works in the pet healthcare field and is dedicated to building a digital care environment that connects pet parents with veterinarians.

Frequently Asked Questions

Is immunohistochemistry (IHC) testing absolutely necessary? Can't a standard histopathology exam suffice?

If a standard biopsy provides a clear diagnosis, treatment can begin without immunohistochemistry (IHC). However, IHC is recommended when the tumor type is unclear, when determining the immunophenotype (T-cell or B-cell) of lymphoma, or when identifying candidates for targeted therapy, as it can significantly influence treatment decisions and prognosis.

Do we need to take additional tissue samples for IHC?

In most cases, we use the original paraffin block from the initial biopsy. This allows us to order immunohistochemistry (IHC) without additional procedures, minimizing stress for both you and your pet. However, if the tissue sample is too small, we may need to collect more tissue.

Does a negative result on immunohistochemistry (IHC) mean the tumor is not cancerous?

Not necessarily. A negative result simply means that the specific protein marker in question wasn’t detected; it doesn’t rule out the presence of a tumor. Other markers may still come back positive, and in some cases, a negative result is actually used to help exclude certain types of tumors.

It takes too long to get the results. Should I delay treatment in the meantime?

In urgent cases, we may start treatment based on standard histopathology results alone and then adjust the plan once immunohistochemistry (IHC) results are available. Your veterinarian will help you decide whether to wait for the IHC results or begin treatment immediately, taking into account the type of tumor and your pet’s overall condition.

Can IHC results vary from one veterinary clinic to another?

Slight variations may occur depending on the testing laboratory, antibody type, and interpretation criteria. If the results are inconclusive, you can request a second opinion by sending the same slide to another specialized pathology lab.

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References

[1] Meuten DJ, Tumors in Domestic Animals, 5th Edition, Wiley-Blackwell, 2017

[2] Withrow & MacEwen's Small Animal Clinical Oncology, 6th Edition, Elsevier, 2019

[3] Veterinary Immunology, 11th Edition, Tizard IR, Elsevier, 2021

[4] Fundamentals of Veterinary Clinical Pathology, 3rd Edition, Stockham SL, Wiley-Blackwell, 2013