The current utilization of immunohistochemistry (IHC) in a diagnostic context is discussed. The modern facility requirements, the various roles IHC is tasked with and the key concept of standardization are covered. Common terminologies are addressed and explained within an IHC context. The terms 'validation' and 'verification' provide one example of words which may cause confusion. The present status in terms of protocol set-up, antibody clones and epitope retrieval are offered to emphasize current best practice. A treatise is given concerning IHC’s special relationship with emerging molecular technologies and how these two analytical devices are shaping diagnoses and treatment strategies for patients. Specific examples are taken from melanoma, breast, lung and bowel cancers. The reader should be able to ascertain the role of IHC in today’s pathology laboratories.

This chapter is written for the researcher who may encounter immunohistochemistry (IHC) in a slightly different context when compared to diagnostic applications. There are many moving parts to IHC assays, and this chapter covers all of the important aspects the researcher needs to consider when employing IHC for their projects. This objective is achieved by employing a request form for IHC services. The questions posed on the form build towards piecing together a protocol that is fit for purpose and can be used in many applications. Practical explanations about epitope retrieval, diluting antibodies from concentrates and the use of detection kits are provided. The need to block endogenous enzyme activity is also explained, as is the technique for antibody optimization. Borrowing the basic fundamental IHC protocol used in diagnostic histopathology, the researcher should be able to adopt and change parameters to suit their research applications.

Immunohistochemistry has progressed from humble beginnings as experimental techniques to what is now considered routine and essential analytical tools. Technologies available today in the research sphere may be adopted for standard practice in the near future. Multiplex assays can help pathology facilities extract more information from less tissue and improved amplification methods may reveal ultra-low expressing proteins that have escaped detection before. New forms of 'tagging' antibodies using nucleotides and quantum dots may replace traditional chromogen and fluorochrome protocols. Next-generation immunohistochemistry enlisting mass spectrometry principles to not only localize antigens in tissue but to also quantify the amount present has real clinical potential. Digital pathology and whole slide imaging have made significant progress to the point of being financially viable. All of these developments are standing at the doorway to the future of immunohistochemistry. Whether they are accepted and implemented in diagnostic laboratories remains to be seen. It is exciting to witness the continuing progression of immunohistochemistry.

Leica’s current IHC instrument is the Bond III. A review is conducted by a medical scientist using it on a daily basis so that an honest evaluation is afforded from first-hand experience. Topics of interest include the machinery Leica employs for epitope retrieval and the coverplate technology to assist in reagent delivery. A discussion about proprietary reagents and consumables whilst highlighting the various components of the machine is provided. There are tips and tricks offered to get the most out of the platform. Programming stain protocols for both chromogenic, fluorescence and double labelling IHC are specified along with equipment servicing and maintenance requirements. The reader in essence, gets to appreciate what it is really like to operate and work with the Bond III. The chapter concludes with both good and bad aspects of this form of automation and some opportunities for improvement.

Immunohistochemistry assays can be technically difficult. There are countless opportunities for errors. Some faults can be fixed, and this chapter contains a precise appraisal of common problems encountered along with suggested solutions. Incorporating both pre-analytic conundrums and analytic challenges from a staining perspective, it is written by a medical scientist able to think outside the square and offer resolutions ranging from the practical to the unconventional. All prescribed advice has been tested and proven successful. Suggesting corrective actions is an exercise in giving the worker options and workarounds for certain situations. Doing more with less and less testing material and lifting sections from slides to be used again when the paraffin block is exhausted are two examples of what can be achieved. Ultimately, it is about tuning protocols to make the most of the precious test tissue given whilst eliminating unnecessary wastage.

The immunohistochemistry assay has evolved over many years. Primarily based on an antibody binding to epitopes in tissue sections, the technology has come a long way from its earliest manifestation using direct fluorescence labels. Current polymer technology is the result of many variations and adaptations on technique throughout the years. Indeed, much of its continued development is owed to early pioneers who experimented and overcame technical boundaries. This chapter is a broad discussion about what immunohistochemistry is from the historical perspective, and the individuals who have contributed to its expansion. It includes a journey about protocol adaptations and explanations of their context and applications. This chapter provides a sound basis in which to explore the rest of the book.

Immunohistochemistry robotics and automation as defined by Agilent comes in the form of the Dako Omnis. An appraisal is given by a medical scientist with intimate knowledge of the principles behind the technology, the various machine components and Dako’s proprietary reagents. Daily operations with the instrument allow for an honest review of stain protocols, workflow logistics and maintenance obligations. An explanation of the unique dynamic gap technology is provided along with in-built quality assurance measures. As the Omnis is a new instrument when compared to Leica’s Bond III and Ventana’s BenchMark ULTRA, discussions are based upon the good and bad points of both the hardware and the software aspects. The reader should get an idea of how the Omnis produces stained slides and the capabilities of the machine.

Roche’s answer to current immunohistochemistry automation is the Ventana BenchMark ULTRA. An evaluation of the machine’s design, operation and maintenance is provided from a user’s viewpoint. Technologies which differentiate Ventana from other manufacturers are discussed in detail. The liquid coverslip, hapten-based detection system and random access points are examples of these differences when compared to traditional coverplate and polymer technology as used by other vendors. A commentary about proprietary reagents and staining protocols are offered along with advice on achieving workflow efficiencies. Discovering quality control measures of the instrument is another topic covered. Ultimately, it is hoped the reader understands the intricacies of this platform and the advantages and disadvantages of such a system, and gains a sense of the technology behind the BenchMark ULTRA.

Many parameters are associated with IHC testing assays. With so many variables, it is quite easy to accumulate errors within the system. To make things more manageable, these considerations are categorized into three main groups. Pre-analytic aspects occur before the assay, analytic factors are concerned with the staining protocol and post-analytic elements relate to interpreting of results. It has also come to reason that any one variable can impact the reliability and consistency of the overall IHC assay. In this regard, standardization requirements have been enlisted to assist laboratories achieve optimal results. In addition, monitoring proficiency testing regimens and various organizations are in place to ensure high levels of standards are attained. All these endeavours are known as quality assurance and quality control measures. They are arranged under the overall umbrella of a facility’s quality management system.