Immunohistochemistry Test: Purpose, Test Details & Results

Purpose of Immunohistochemistry Test

Immunohistochemistry (IHC) is much more than a technical laboratory procedure—it's a powerful bridge between molecular science and patient care. This test helps clinicians and pathologists visualize the presence and location of specific proteins (antigens) in tissues, enabling precise diagnoses, guiding treatment decisions, and advancing biomedical research. Whether identifying cancer subtypes, detecting infectious agents, or uncovering the origins of mysterious tumors, IHC has become an indispensable part of modern medicine.

Diagnosing Disease

IHC plays a critical role in diagnosing diseases, particularly cancers. By targeting unique proteins that are either present or abnormally expressed in certain conditions, IHC can differentiate between tumor types, detect metastases, and even pinpoint the tissue of origin in cancers of unknown primary site. For infectious diseases, IHC can identify pathogen antigens in fixed tissues, complementing other diagnostic tools like PCR and culture, especially when pathogens are difficult to grow or detect by other means 1 3 4 5.

Guiding Treatment and Prognosis

Beyond diagnosis, IHC informs treatment strategies. For example, the identification of HER2 protein overexpression in breast cancer directly affects eligibility for targeted therapies, while testing for PD-L1 expression in lung cancer helps determine suitability for immunotherapy agents 2 4. IHC can also provide prognostic information by revealing markers associated with aggressive disease or predicting responses to specific treatments 1 4 7.

Research and Biomarker Discovery

IHC is invaluable in research for studying disease mechanisms and discovering new biomarkers. Advanced multiplex IHC techniques can simultaneously measure multiple proteins in a single tissue section, helping to map complex immune landscapes in tumors and uncover novel therapeutic targets 1 3 10.

Immunohistochemistry Test Details

The IHC test is a sophisticated, multi-step process that combines biology, chemistry, and advanced imaging. From preparing tissue samples to interpreting the final stained slides, every step demands precision and expertise to ensure reliable results. Here's how the process unfolds and what makes it so effective—and challenging.

Tissue Preparation and Fixation

• Sample Collection: Tissues are typically collected via biopsy or surgery and immediately fixed (usually in formalin) to preserve cellular structures and proteins.
• Embedding: The fixed tissue is embedded in paraffin wax to enable thin sectioning for slide preparation.
• Sectioning: Thin slices (usually 4–5 micrometers) are cut and mounted onto microscope slides for further processing 4 6 7.

Proper fixation and handling are crucial—poor technique can destroy antigens or create artifacts, leading to false results.

Antibody Selection and Application

• Primary Antibody: The heart of the test, this antibody specifically binds to the target antigen in the tissue. Monoclonal antibodies offer high specificity, while polyclonal antibodies may provide greater sensitivity 3 6 8.
• Secondary Antibody: Binds to the primary antibody and is typically linked to a detection system (such as an enzyme or fluorophore) to visualize the reaction 9.

Selecting the right antibodies is essential for specificity and minimizing background noise.

Antigen Retrieval

• Unmasking Antigens: Many formalin-fixed tissues require antigen retrieval techniques (heat or enzymatic treatment) to restore antigenicity lost during fixation. This step is critical for many modern IHC applications 4 6 9.

Detection and Visualization

• Detection Systems: The most common are enzyme-based (e.g., horseradish peroxidase) that produce a colored reaction, or fluorescent labels for high-resolution localization 6 9 12.
• Multiplexing: Advanced systems can detect multiple antigens simultaneously, providing a more comprehensive view of tissue biology 10.

Automation and Standardization

• Automated Platforms: Modern pathology labs increasingly rely on automated IHC stainers for high-throughput, reproducible results 4 7.
• Quality Control: Standardization of protocols, controls, and validation procedures is vital to ensure consistency across labs and over time 7 12.

Troubleshooting and Limitations

• Common challenges include non-specific staining, weak signal, or loss of antigenicity. Troubleshooting involves adjusting fixation, antibody concentration, or retrieval methods 6 9 12.
• Interpretation requires skilled pathologists who can distinguish true positive signals from artifacts and background staining 4 7 12.

Immunohistochemistry Test Results & Follow-Up

The interpretation of IHC test results is where science meets clinical decision-making. The colored patterns seen under the microscope are not just images—they translate into diagnoses, treatment plans, and prognostic predictions. Here’s how results are assessed, reported, and acted upon in patient care.

Interpreting Results

• Pattern Recognition: Pathologists assess not only the presence or absence of staining, but also its intensity, distribution (cellular or tissue location), and pattern (membranous, cytoplasmic, nuclear) 1 4 7.
• Quantification: Many tests require scoring the proportion of positive cells or the staining intensity. This is especially important for companion diagnostics, such as PD-L1 or HER2 testing, where thresholds determine eligibility for specific therapies 2 10.

Reporting and Communication

• Standardized Reporting: Reports typically describe the type and intensity of staining, percentage of positive cells, and clinical interpretation. Standardized templates and scoring systems help ensure reliability and comparability 7 12.
• Follow-Up Actions:
  • Positive Results: May confirm a diagnosis (e.g., cancer subtype), trigger genetic counseling (e.g., Lynch syndrome screening 11), or support starting a targeted therapy 2 4.
  • Negative Results: May rule out certain conditions or prompt further testing to clarify the diagnosis 1 4.
  • Ambiguous or Borderline Cases: Sometimes, results are inconclusive due to technical issues or biological variability. In these cases, repeat testing or use of alternative markers may be necessary 7 12.

Limitations and Quality Control

• Interobserver Variability: Interpretation can sometimes be subjective, especially for borderline or low-expressing cases. Training, experience, and standardized protocols help reduce variability 2 7 12.
• Technical Artifacts: Poor tissue processing, inadequate fixation, or suboptimal antibody performance can all affect results, underscoring the need for robust quality assurance programs 6 7 12.

The Evolving Landscape

• Automation and Digital Pathology: Increasing use of automated image analysis and digital pathology platforms is helping to standardize scoring and improve reproducibility 10 12.
• Integration with Molecular Testing: IHC is often used alongside molecular tests (like PCR or genetic sequencing) to provide a comprehensive diagnostic picture 5 11.

Conclusion

Immunohistochemistry is at the crossroads of science and clinical medicine, providing crucial insights that shape diagnosis, treatment, and research. As technology advances, its impact will only deepen, offering hope for more personalized, precise patient care.

Key Points Covered:

• Purpose: IHC enables precise diagnosis, prognosis, therapy guidance, and advances research by visualizing specific proteins in tissues 1 3 4 5 7 10.
• Test Details: The process involves careful tissue preparation, antibody selection, antigen retrieval, detection, and interpretation—each step crucial for accurate results 4 6 7 9 12.
• Results & Follow-Up: Results inform clinical decisions, guide therapy, and sometimes require repeat or additional testing; quality control and standardization are essential for reliability 2 4 7 10 11 12.
• Future Directions: Automation, digital pathology, and multiplexing expand IHC’s potential for personalized medicine and biomarker discovery 10 12.

Whether in the lab or at the bedside, immunohistochemistry remains a vital tool, turning microscopic details into life-changing clinical actions.