Fixation on Histology

Mastering the Art of Immunohistochemistry: Essential Techniques for Reliable Diagnostic Results

  
Immunohistochemistry (IHC) plays a crucial role in modern pathology, offering insights that are invaluable in diagnosing various diseases, especially cancers. IHC combines anatomical, immunological, and biochemical techniques to identify specific antigens in tissues, using labeled antibodies as diagnostic tools. While molecular diagnostic methods are gaining popularity, IHC remains indispensable due to its versatility and ability to address many diagnostic challenges. However, despite its importance, there are numerous technical aspects that must be carefully managed to avoid errors and ensure the reliability of results. 
 

Proper Tissue Handling and Fixation

 
High-quality IHC begins with high-quality tissue samples. The way a tissue is handled and preserved from the moment it is excised can have a profound impact on the accuracy of IHC staining. The most common fixation agent used is formalin, which helps preserve tissue morphology and prevent degradation. It’s essential that tissues are fixed promptly, and the fixation time should be optimized, typically between 6 and 48 hours depending on the tissue type.
 
Under-fixation can result in antigen loss, while over-fixation can mask antigenic sites, reducing staining sensitivity. Thin sections of about 3-4 microns should be cut to allow sufficient antibody penetration, and fixation protocols must be standardized across the lab to avoid inconsistencies in results.
 

Selection of Primary Antibody

 
Choosing the correct primary antibody is critical in achieving specific and sensitive staining. The antibody must be validated for the intended target tissue and tested for specificity and sensitivity. Additionally, one must consider whether to use monoclonal or polyclonal antibodies. Monoclonal antibodies are specific to a single epitope, offering high specificity but sometimes lacking sensitivity. Polyclonal antibodies, on the other hand, target multiple epitopes, providing greater sensitivity but risking cross-reactivity.
 
Before use, each batch of antibodies should be tested on positive and negative controls. This helps in identifying any batch-to-batch variability and ensures that the antibody performs reliably in actual diagnostic cases.
 

Blocking Endogenous Activity

 
A common issue in IHC is background staining due to endogenous enzyme activity or non-specific antibody binding. To avoid this, it’s essential to block endogenous peroxidase and biotin activity before applying antibodies. Failure to block these endogenous molecules can lead to false-positive results.
 
Peroxidase activity, for example, is commonly present in red blood cells and can produce unwanted staining. To address this, hydrogen peroxide is commonly used to block endogenous peroxidase. Additionally, if using an avidin-biotin complex (ABC) detection system, labs must block endogenous biotin, which is found in tissues like the liver, kidney, and brain, to prevent non-specific binding.
 

Choosing the Right Detection System

 
Two main detection methods are commonly used in IHC: the avidin-biotin complex (ABC) system and polymer-based systems. While the ABC method relies on the strong binding between avidin and biotin, polymer-based systems use large polymers that carry numerous enzyme molecules, increasing the signal strength without relying on biotin.
 
Polymer-based detection systems have largely replaced ABC methods in many labs due to their lower risk of background staining caused by endogenous biotin. These systems also tend to provide stronger, more reliable staining results, making them a preferred choice in many diagnostic labs.
 

Interpretation of Stains: True vs. False Positives

 
A true positive stain means that the chromogen is localized to the cells or structures containing the antigen of interest. On the other hand, a false positive occurs when staining appears in areas where the antigen is not present. False positives are often caused by non-specific antibody binding or poor tissue preparation.
 
To distinguish between true and false positives, it is crucial to include control slides with each batch of staining. Positive controls should contain the target antigen, while negative controls should lack it. Proper interpretation of these controls will help ensure that the staining pattern is reliable and specific.
 

Regular Use of Control Slides

 
Control slides are essential for verifying the accuracy and reliability of IHC staining. Positive controls ensure that the staining procedure worked correctly, while negative controls help identify any non-specific binding. Without proper controls, it’s impossible to determine whether a positive result is truly due to the presence of the antigen or simply due to technical errors.
 
Many labs now use multi-tissue control slides, which contain various types of tissues with known antigen expression levels. This approach allows for the simultaneous evaluation of multiple antigens and provides a broader range of control tissue types.
 

Avoiding Artifacts

 
Artifacts in IHC can significantly affect the interpretation of results. One common artifact is caused by endogenous biotin, which can bind to the avidin used in the detection system, leading to false-positive staining. Other artifacts include tissue drying, improper antibody incubation times, and contamination from external sources like desquamated skin cells.
 
To minimize artifacts, ensure that tissues are properly hydrated, antibodies are applied at optimal concentrations, and incubation times are strictly followed. Additionally, maintaining a clean laboratory environment will help reduce contamination from external sources.
 

Continuous Quality Control

 
Quality control in IHC should be a continuous process. Routine evaluations of staining sensitivity, specificity, and reproducibility must be performed. Regular audits of the staining procedures, including fixation, antibody selection, and detection methods, can help identify areas where improvements are needed. The use of multi-tissue control blocks, originally developed by Dr. Hector Battifora, is a widely accepted method to maintain stringent quality control.
 
Immunohistochemistry (IHC) remains a vital tool in accurate disease diagnosis, particularly when executed with precision and adherence to best practices. By focusing on key technical details such as proper tissue handling and careful antibody selection, pathologists can achieve reliable results that enhance diagnostic accuracy and guide appropriate treatments. Ultimately, IHC is a powerful technique, and when skillfully applied, it offers new insights into disease understanding and improves patient care.
 
Written by: Khulood Ayad Majeed; College of Dentistry, University of Kirkuk
References:
 
Miller, R. T. (2019). Avoiding pitfalls in diagnostic immunohistochemistry–important technical aspects that every pathologist should know. Seminars in Diagnostic Pathology, 36(5), 312-335. https://doi.org/10.1053/j.semdp.2019.05.002 .
 
Mebratie, D. Y., & Dagnaw, G. G. (2024). Review of immunohistochemistry techniques: Applications, current status, and future perspectives. Seminars in Diagnostic Pathology, 41(3), 154-160. https://doi.org/10.1053/j.semdp.2024.05.001 .
 
Jensen, K., Krusenstjerna-Hafstrøm, R., Lohse, J., & et al. (2017). A novel quantitative immunohistochemistry method for precise protein measurements directly in formalin-fixed, paraffin-embedded specimens: Analytical performance measuring HER2. Modern Pathology, 30(2), 180–193. https://doi.org/10.1038/modpathol.2016.176 . (ref of Figure) 


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