Immunohistochemistry: Past - Present - Future

Immunohistochemistry: Past - Present - Future

TAMING THE IHC WILD WEST

ORIGINS

Immunohistochemistry: Past - Present - Future

PRECISION MEDICINE IHC

IN SEARCH OF STANDARDIZATION

Immunohistochemistry: Past - Present - Future

FUTURE: ACHIEVING TRUE ACCURACY

The rapid growth of immunohistochemistry (IHC) brought significant quality challenges, resembling a “wild west” of laboratory medicine. Clinical tests often relied on subpar “Research Use Only” (RUO) reagents. In 1989, the Biological Stain Commission began organizing meetings and issuing recommendations to address these issues. In 1998, the FDA classified clinical IHC reagents as at least Class I in vitro diagnostic (IVD) medical devices.

The phrase “fit-for-purpose” is introduced to immunohistochemistry (IHC) in 20171, as illustrated by this example of GATA3 staining. While GATA3 expression is high in breast cancers (left), it is significantly weaker in a subset of T lymphocytes (right). As a result, a GATA3 IHC assay optimized for breast cancer may have insufficient analytic sensitivity for T lymphocytes. 1 Cheung CC, et al. Evolution of quality assurance for clinical immunohistochemistry in the era of precision medicine: Part 1: Fit-for-purpose approach to classification of clinical immunohistochemistry biomarkers. Appl Immunohistochem Mol Morphol 2017 25:4-11.

Estrogen receptor (ER) expression was initially measured using biochemical ligand binding assays (LBAs). However, LBAs were technically demanding, required radioactive reagents and fresh frozen tissue, and were prone to errors. During the 1990s, numerous studies demonstrated the utility of ER immunohistochemistry for identifying breast cancer patients to receive hormone blocking therapy.

The rapid growth of immunohistochemistry has also been associated with persistently high error rates, between 10 – 30%. To promote standardization, new IHC-specific guidelines emerged.

Dr. Barbarajean Magnani and Dr. Clive Taylor proposed that the continuing high error rates warrant a new approach.1 IHC should incorporate quantitative quality assurance methods like those used in clinical pathology. Their call to action inspired four additional editorials, garnering support from the leadership of the National Society of Histotechnologists, UKNEQAS-ICC/ISH, NordiQC, CBQA, and others. 1 Magnani B, Taylor CR. Immunohistochemistry Should Be Regulated as an Assay. Arch Pathol Lab Med. 2023 147(11):1229-1231.

Mathematical methods of IHC standardization complement traditional QA methods using patient tissue samples. Statistical process control, such as the Levey-Jennings chart shown here, promotes intra-laboratory IHC assay standardization. This method is already widely used in other clinical laboratory disciplines.

The rapid growth of immunohistochemistry has also been associated with persistently high error rates, between 10 – 30%. To promote standardization, new IHC-specific guidelines emerged.

The rapid adoption of immunohistochemistry (IHC) created a need for high-quality reagents. At the time, the IHC industry was largely unregulated, usually relying on “Research Use Only” reagents. Amid these challenges, Dakopatts A/S emerged as a key early provider of reliable IHC reagents. The company, founded by Dr. Niels Harboe, played a pivotal role in supplying standardized and reproducible reagents.

There is a growing tension between the increasing need for accuracy as opposed to the inherent limitations of the ‘art’ of IHC. In this published example1, two accredited laboratories show different test results from serial sections of the same tissue. 1 E Torlakovic, et al., Development and validation of measurement traceability for in situ immunoassays. 2021 Clin. Chem. 2021 67(5):763-771.

The first application of immunohistochemistry (IHC) in surgical pathology using routine formalin-fixed, paraffin-embedded (FFPE) tissues.1 1 Taylor CR, Burns J (1974) The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffin-embedded tissues using peroxidase-labelled antibody. J Clin Pathol 27:14–20

As immunohistochemistry became widely used in surgical pathology, automation development efforts began, driven by entrepreneurial innovation. Dr. David Brigati’s Code-On stainer1 (1988) adapted Fisher Scientific’s H&E slide stainer by introducing a slide holder that created capillary gaps between slides, thereby reducing evaporation. 1 Brigati, D.J., et. al. Immunocytochemistry is automated: Development of a robotic workstation based upon the capillary action principle. J. Histotechnology, 11(3): 165-183.

U.S. and European IHC proficiency testing (PT) programs developed different protocols for evaluating laboratory performance:

Few drugs have been chronicled in both film (left) and literature (right), but the story of trastuzumab (Herceptin®) is groundbreaking. Among its many milestones, the FDA’s September 1998 approval was the first to link treatment eligibility directly to an immunohistochemistry (IHC) test for biomarker expression, paving the way for the now-standard drug-diagnostic codevelopment model.

The phrase “fit-for-purpose” is introduced to immunohistochemistry (IHC) in 20171, as illustrated by this example of GATA3 staining. While GATA3 expression is high in breast cancers (left), it is significantly weaker in a subset of T lymphocytes (right). As a result, a GATA3 IHC assay optimized for breast cancer may have insufficient analytic sensitivity for T lymphocytes. 1 Cheung CC, et al. Evolution of quality assurance for clinical immunohistochemistry in the era of precision medicine: Part 1: Fit-for-purpose approach to classification of clinical immunohistochemistry biomarkers. Appl Immunohistochem Mol Morphol 2017 25:4-11.

The rapid growth of immunohistochemistry (IHC) brought significant quality challenges, resembling a “wild west” of laboratory medicine. Clinical tests often relied on subpar “Research Use Only” (RUO) reagents. In 1989, the Biological Stain Commission began organizing meetings and issuing recommendations to address these issues. In 1998, the FDA classified clinical IHC reagents as at least Class I in vitro diagnostic (IVD) medical devices.

This mathematical representation of IHC stain intensity – an “analytic response curve” – is the standard method for calculating the lower limit of detection (LOD), for inter-laboratory IHC assay standardization. This published graph depicts the analytic response curves for four FDAcleared PD-L1 IHC assays.1 Creating such curves requires IHC calibrators. 1 SR Sompuram, EE Torlakovic, NA t’Hart, K Vani, & SA Bogen. Quantitative comparison of PD-L1 IHC assays against NIST standard reference material 1934. Mod. Pathol. 2022 35(3):326-332.

The rapid adoption of immunohistochemistry (IHC) created a need for high-quality reagents. At the time, the IHC industry was largely unregulated, usually relying on “Research Use Only” reagents. Amid these challenges, Dakopatts A/S emerged as a key early provider of reliable IHC reagents. The company, founded by Dr. Niels Harboe, played a pivotal role in supplying standardized and reproducible reagents.

Heat-induced antigen retrieval was first described by Shi et. al. in 19911, dramatically increasing the number of antibodies useful for FFPE tissues. Antigen retrieval used common household appliances: microwave ovens, pressure cookers, and steam pots. The use of everyday kitchen tools is humorously captured in an article cover designed as a 1950s vintage kitchen advertisement. 1 SR Shi, et. al. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991 39(6):741-8