Selecting the Appropriate Reference Standard for Immunohistochemical Assays: A Comprehensive Guide

Summary

• Selecting the appropriate reference standard for immunohistochemical assays is crucial for accurate and reliable results.
• Control tissue should be chosen based on its known expression of the target antigen and should include positive and negative controls.
• Proper selection and validation of control tissue is essential for ensuring the quality and reliability of newly validated immunohistochemical assays.

Introduction

Immunohistochemical assays play a critical role in the diagnosis and management of various diseases, including cancer. These assays rely on the use of reference standards, also known as external control tissue, to ensure the accuracy and reliability of results. Selecting the appropriate reference standard is essential for obtaining consistent and reproducible data. In this article, we will discuss the different types of reference standards that can be used for immunohistochemical assays and provide guidance on how to select control tissue for newly validated assays.

Types of Reference Standards

There are several types of reference standards that can be used for immunohistochemical assays. The most common types include:

1. Cell lines - Cell lines that are known to express the target antigen can be used as reference standards. These cell lines provide a consistent and reliable source of positive control tissue for immunohistochemical assays.
2. Tissue microarrays (TMAs) - TMAs are a collection of tissue samples that are mounted on a single slide. These samples can include both positive and negative controls for the target antigen, making them a valuable reference standard for immunohistochemical assays.
3. Commercial controls - Commercially available control tissues that are validated for use in immunohistochemical assays can provide a convenient source of reference standards. These controls are typically well characterized and can help ensure the accuracy of assay results.

Selection of Control Tissue

When selecting control tissue for immunohistochemical assays, it is important to consider the following factors:

1. Known expression of the target antigen - Control tissue should be chosen based on its known expression of the target antigen. Positive control tissue that is known to express high levels of the antigen can help validate the sensitivity of the assay.
2. Include negative controls - In addition to positive controls, it is important to include negative control tissue in the assay. Negative controls should be selected based on their lack of expression of the target antigen.
3. Variability in expression - When selecting control tissue, it is important to consider the variability in expression of the target antigen. Including control tissues with varying levels of antigen expression can help validate the specificity of the assay.

Validation of Control Tissue

Proper validation of control tissue is essential for ensuring the quality and reliability of immunohistochemical assays. When validating control tissue for a newly developed assay, the following steps should be taken:

1. Characterization of control tissue - Control tissue should be thoroughly characterized to ensure that it accurately reflects the expression of the target antigen. This may involve conducting additional tests, such as western blotting or PCR.
2. Testing of control tissue - Control tissue should be tested in parallel with the assay to validate its performance. This can help ensure that the control tissue provides consistent and reproducible results.
3. Validation of assay performance - After selecting and validating control tissue, the overall performance of the assay should be validated. This may involve testing the assay on a validation cohort of samples to ensure its accuracy and reliability.

Conclusion

Reference standards play a crucial role in the accuracy and reliability of immunohistochemical assays. By selecting the appropriate control tissue and thoroughly validating its performance, researchers can ensure the quality of their assay results. Proper selection and validation of control tissue is essential for the successful development and validation of newly validated immunohistochemical assays.

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