IHC Quality Control and Troubleshooting Notes

IHC Quality Control and Troubleshooting Notes

Introduction

  • Course Title: IHC Quality Control and Troubleshooting

  • Course Code: MLTD2209 WINT 2024

  • Instructor: Monique Hartlen

Quality Control for Immunohistochemistry (IHC)

  • Definition: Quality control (QC) in IHC involves ensuring that test results are accurate and reliable through systematic checks with positive and negative controls.

  • Controls:

    • Positive Controls:

    • Purpose: To confirm that reagents and methods are functioning correctly by checking for false negatives.

    • Usage: Tissue expressing the antigen of interest is employed, ensuring the test can demonstrate the expected positive reaction.

    • Example: Tissue Micro-Arrays (TMAs) are utilized effectively as they provide a variety of expression levels.

    • Negative Controls:

    • Purpose: To assess the specificity of reagents, helping to identify false positive reactions.

    • Usage: Normal tissue that does not express the target antigen is selected, such as liver tissue for prostate-specific antigens.

    • Non-specific negative controls can include duplicate patient slides processed identically but without the primary antibody.

Importance of Controls

  • Positive and negative controls are critical in ensuring that IHC methods yield reliable results, particularly in cases where patient prognosis and treatment options are contingent upon accurate antigen detection (e.g., HER2 testing).

  • HER2 Testing:

    • Characteristics: This antigen's expression is limited to certain tumors.

    • Significance: Confirmation of HER2 positivity often necessitates more robust techniques like FISH (Fluorescence In Situ Hybridization).

Tissue Micro-Arrays (TMAs)

  • Definition: TMAs are collections of small tissue samples arranged on a slide, allowing for simultaneous testing of multiple samples.

  • Advantages:

    • Best practice in control applications, as they include both positive and negative controls and demonstrate a variety of reaction strengths.

    • Essential for accurate scoring, impacting patient treatment and prognosis.

External Quality Control (QC)

  • External laboratories can engage specialized organizations to validate their QC processes and enhance standardization.

  • External QC Companies:

    • NordiQC: Based in Denmark, focuses on quality assessment for IHC.

    • Canadian Institute of Quality Control (CIQC): Recently split into two branches:

    • CPQA-AQCP: General quality assurance program at https://www.cpqa.ca/

    • CBQA-PCAB: Specific for proficiency testing at https://www.cbqa.ca/

Troubleshooting Immunohistochemistry (IHC)

  • Guidelines:

    • Ensure stains do not dry out during processing.

    • Keep slides level to prevent gradient staining.

    • Measure reagents as accurately as possible.

    • Maintain equipment regularly and monitor reagent consistency (e.g., pH).

    • Change only one variable at a time during troubleshooting to isolate issues.

Common Issues in IHC

General Problems

  • Improper/Delayed Fixation:

    • Consequences: Loss of antigen sites may lead to misdiagnoses as control slides detect staining method issues rather than fixation problems.

    • Importance: Proper fixation ensures reliable readings of cancer-specific antigens and influences treatment protocols.

  • Overfixation:

    • Results: Crosslinking of proteins may cause weak or false negative staining.

    • Antigen Retrieval Techniques: Appropriate length of HIER (Heat Induced Epitope Retrieval) or EIER (Enzyme Induced Epitope Retrieval) is essential to retrieve antigens effectively.

  • Improper Processing / Pre-treatment:

    • Guidance: Minimize the use of decalcifying agents; prefer milder methods like formic acid.

    • Processing Tips: Limit time on hot embedding surfaces, monitor paraffin temperatures, and avoid excessive drying temperatures to preserve antigen sites.

  • Verification of Variables:

    • Conduct thorough checks on potential external variables, such as new reagent lots or staff training, that might affect results.

    • Address pre-analytical issues including labelling errors and reagent preparation.

Falsely Weak or Negative Reactions

  • Causes:

    • Fixation Issues: Improper fixation results, either underfixation or overfixation, can lead to loss or difficulty in retrieving antigen sites.

    • Epitope Retrieval: Incorrect temperature or duration can lead to weaker staining results.

    • Aging of Slides: Older slides may display decreased antigenicity.

    • Drying of Sections: Excessive drying during procedures can diminish staining quality.

    • Antibody Issues: Possibly due to wrong concentration, temperature, or incubation time for both primary and secondary antibodies.

False Positive Reactions

  • Causes:

    • Improper epitope retrieval temperature settings.

    • Blocking issues: Failure to use required hydrogen peroxide and biotin blockers can lead to nonspecific staining.

    • Incorrect adhesive slides: Only positively charged slides should be utilized to avoid background staining.

    • Antibody and incubation problems, including inadequate dilutions or improper conditions.

References

  • Bancroft, J. Theory and Practice of Histological Techniques, 6th ed. Elsevier, 2008.

  • Carson, F., & Cappellano, CH. Histotechnology: A Self Instructional Text, 5th ed. ASCP, 2020.

  • Del Campo, Jesse. What Happened To My IHC Stains & What Caused It? National Society of Histotechnology, 2014 Webinar Series.

  • Ramos, Jason. IHC Troubleshooting. National Society of Histotechnology, 2017 Webinar Series.