This chapter describes how immunocytochemistry (ICC) can be employed in the investigation of pathological states. It begins by explaining malignancy, which is defined by a change in the normal cell biology of cells. It results in an abnormal growth or tumour that can then spread (metastasize) to other body sites. Within histopathology, there are recognized key malignant tumour groups, including carcinoma, lymphoma, melanoma, mesothelioma, and sarcoma. The chapter then looks at the key antibodies used in ICC, before focusing on the investigation of breast cancer, lung cancer, and prostate cancer. It also discusses the use of ICC in the investigation of lymphoma; how ICC can contribute to the identification of tumours of an unknown primary malignancy, the role of ICC in the investigation of autoimmune skin disease, and the role of ICC in the strategy of patient management targeted therapies.
Guy Orchard, David Muskett, and Anne Warren
Guy Orchard, Chantell Hodgson, and Brian Nation
This chapter focuses on key artefacts in histological procedures. Artefacts in histopathological studies can be commonplace and have a profound impact on the outcome of any investigations. Pre-analytical artefacts are largely due to the processes and procedures undertaken prior to specimen receipt in the histopathology laboratory. They relate to the need to ensure that Good Clinical Practice (more precisely Good Surgical Practice) is followed while removing tissue samples from patients, to reduce their impact on viable tissue harvesting. Meanwhile, post-analytical artefacts are produced as a result of laboratory procedures and techniques, and there is greater emphasis on appreciating their impact because quality control procedures will need to define what they represent and facilitate the introduction of measures to reduce their incidence.
Data recording and histopathological dissection
Vanda McTaggert, Sue Pritchard, and Anne Warren
This chapter discusses appropriate data recording and histopathological dissection procedures. It is essential that identification checks are performed, to an agreed standard, throughout the analytic stage, so that the right report is generated for the right patient. The biomedical scientist (BMS) is key in ensuring this is completed. In addition, dissection techniques must be of the highest standard to ensure that the tissue sampled allows all the histological questions being asked by the requesting clinician to be answered. The chapter then looks at the process of accepting specimens for histopathological examination, working safely in the dissecting room, and gathering clinical data relevant to specimen dissection. It also outlines the basics of dissection and the macroscopic report, and considers common organ systems, head and neck pathology, and gynaecological cases.
David N Furness
This chapter evaluates electron microscopy, which allows higher magnification than other types of microscopy and is responsible for the discovery and detailed description of most subcellular organelles. There are two kinds of electron microscopes (EMs): the transmission EM and the scanning EM. The main difference between these two types of microscope is that TEM is used to look at sections of samples, or naturally very thin/small samples, while SEM is used to image the surface and so can handle bulky samples of a size that is only limited by the microscope sample chamber size, although typically, samples are only a few millimetres across. The chapter then outlines the specific preparatory requirements for samples requiring electron microscopy. It also considers the application of transmission and scanning electron microscopy in the field of biomedical science and provides examples of their use in pathological evaluation.
Essentials of laboratory management
Sue Alexander and Patricia Fernando
This chapter assesses the essentials of laboratory management. Good laboratory management is a difficult concept to define absolutely, being made up of many different component parts, but it is essential to the smooth running of a modern histopathology laboratory. The chapter considers how all aspects of work involved in laboratory management interact within a clinical governance framework in histopathology. It also looks at quality management systems and risk management. In addition to defined standards for laboratories undertaking histological investigations on human tissue samples, there are also national laws governing the use of tissue samples, and compliance with such laws and regulations is required, being assessed in a similar way to laboratory standards. The chapter then studies the Human Tissue Act 2004.
Fixation and specimen handling
This chapter investigates the way in which biopsies and resections are first handled in the laboratory. Once specimens arrive in the laboratory, they follow a process to the point of diagnosis. The process for most specimens remains the same until the first slide has been viewed for diagnosis. The chapter begins by looking at specimen collection. Specimens need to be transported from the operating theatres and clinical areas to the laboratory; this should be done safely, in appropriate environmental conditions, and in a timely manner. The chapter then explains the need for accurate specimen reception, the principles of specimen fixation, the need for specimen decalcification and the means by which this can be achieved, and the need for accurate patient details supplied with samples.
Edited by Guy Orchard and Brian Nation
Histopathology first asks: what is histopathology? It then looks at fixation and specimen handling. Topics covered include data recording and histopathology dissection, routine staining, special stains, artefacts, and Mohs procedures. The text moves on to examine immunocytochemical techniques and analytical immunocytochemistry. Next, there is a chapter on in situ hybridization. This is followed by a couple of chapters on molecular diagnostics. Histopathology reporting is dealt with after that. Finally, the text discusses microscopy and digital pathology, electron microscopy in diagnosis, mortuary practice, and the essentials of laboratory management.
Guy Orchard, Chris Ward, and Susan Pritchard
This chapter discusses the process and content of a histopathology report, including the supplementary report. As well as containing all the patient demographical information, the report is divided into specific sections. A histopathology report is a verified document that contains a diagnosis, which is determined following the examination of cells, tissues, and sometimes foreign or infectious agents under the microscope. The chapter then looks at the key microscopic observations seen in the two key areas of histopathology – inflammation and cancer. It highlights the use of diagnostic datasets and the role of tissue pathways in histopathology, as well as the value of molecular/genetic information in histopathology reports.
Merdol Ibrahim and Guy Orchard
This chapter addresses immunocytochemical techniques and their role in diagnostic histopathology. Immunocytochemistry (ICC) is used as a second string of investigations following conventional staining techniques. The role of the primary investigations is to demonstrate the morphological appearance of the cell and tissue architecture. This enables identification of the disease process, and in a substantial number of cases is adequate to formulate a final diagnosis. However, the demonstration of morphological detail alone may not enable a diagnosis to be made in all cases, and in such instances an assessment of the cell marker (antigen) expression on the cell membrane, cytoplasm, or nucleus is required. This provides selective information that can be used to define certain cell types. This is the role of ICC, which, in some instances, is closely allied to molecular diagnostics. The chapter then details the process of tissue preparation and antigen retrieval.
In situ hybridization: key concepts and applications
Anthony Warford and Emanuela Volpi
This chapter explores in situ hybridization (ISH), a method that relies on base pair complementarity to enable specific localization of nucleic acids in cellular pathology samples. It is an integrated experimental approach which combines cell-by-cell microscopic visualization with high-resolution analysis of nucleic acids (DNA and RNA). Today, ISH is widely employed in both diagnostics and research. In the pathology setting, it can be successfully applied as an adjunctive, powerful diagnostic, prognostic, and predictive tool to detect a variety of specific DNA changes within tissue sections and inform ‘personalized’ therapeutic approaches. In research applications, ISH presently complements high-throughput microarray analysis outputs by providing evidence of dysregulation of gene expression, at mRNA and microRNA level, on an individual cell basis.
Light microscopy and digital pathology
This chapter examines light microscopy and digital pathology. The principle behind the light microscope is to provide a magnified image of the sample of interest such that what is happening to the samples can be seen at a scale which the eyes alone could not perceive. It is not enough, however, simply to provide a large image of a specimen, as a simple magnifying lens would. Biomedical scientists are typically interested in quite small and fine detailed structures so it is important that the microscopic image contains sufficient resolution of fine detail. The chapter then looks at the compound microscope, phase contrast microscope, polarization microscope, darkfield microscope, fluorescence microscope, and confocal microscopy.
Guy Orchard and Mohammad Shams
This chapter assesses the process and procedures involved in Mohs techniques. Mohs has a growing application for the surgical removal of cancers. Indeed, its main application has been in the use of surgical removal of skin cancers. Mohs procedures have the benefits of offering unparalleled accuracy for the total skin clearance of tumours without recurrence. The chapter then looks at the applications of Mohs in modern histopathology laboratories and the benefits it can bring to patient care. It also explains what slow Mohs is and when its application is appropriate, before considering the role of rapid immunocytochemical staining to aid in the assessment of frozen and paraffin sections in Mohs procedures.
Molecular diagnostics in action
This chapter identifies the mission of a molecular diagnostic service involved in solid tumour testing. Molecular tests add information on diagnosis, prognosis, and prediction to therapy; targets are chromosomes, genes, or proteins. In routine practice, molecular pathology diagnostic services need to be able to offer the whole range of techniques needed for the patient's management. Nature and number of tests per tumour are guided by clinical relevance. Ultimately, pathologists play a key role in the prescription of the relevant tests and their final interpretation. Appropriate management of tissue is crucial; molecular tests are performed following the diagnostic procedure which includes immunocytochemistry. The chapter then looks at mutation testing, multiplex gene testing, and protein assessment.
Molecular diagnostics: techniques and applications
Brendan O'Sullivan and Philippe Taniere
This chapter studies molecular diagnostics, which is the use of widely varied techniques and platforms to profile variation in a broad array of biomolecules, supporting medical decisions regarding the diagnosis of disease, patient prognosis, and prediction of response to treatments and interventions. The chapter begins by outlining the essential theory and concepts of DNA replication and translation. It then looks at the polymerase chain reaction, before explaining the basic principles, organic or semi-synthetic, which underlie the broad range of techniques utilized in the molecular pathology laboratory. The chapter also considers molecular diagnostic processing and testing. Finally, it discusses nucleic acid extraction and explores the different PCR analysis methods.
This chapter focuses on the functions which may be fulfilled by a mortuary and post-mortem facility. These include receipt and storage of the deceased; investigation of the cause and/or circumstances of death by performing a post-mortem examination of the deceased; demonstration of post-mortem findings to clinical staff and to allow teaching; and viewing and/or identification of a body, if requested or required. The mortuary delivers the last point of care for pathology and can be the final stage of the patient care pathway. The chapter then outlines the naturally occurring processes which occur after death, including rigor mortis and decomposition. It looks at the work of the Human Tissue Authority (HTA) and how it impacts on the mortuary. The chapter also differentiates between consented, or authorized, post-mortem examinations and those instructed by legal process.
Routine processing, embedding and staining
This chapter examines routine processing, embedding, and staining. The aim of tissue processing is to prepare tissue in a supporting medium ready to be cut into thin sections, in order to observe individual cells under the microscope. The tissue blocks prepared during specimen dissection are immersed in a number of different chemical solutions. These solutions act to prepare the tissue for mixing with an embedding medium, in most cases paraffin wax, although resin is sometimes used for hard tissue. Tissues are generally processed together in an overnight batch, but this may result in poor processing of fatty tissue and particularly hard tissue. The chapter then discusses microtomy, cryotechniques and cryotomy, and haematoxylin and eosin staining.
Stains in action
David Muskett and Guy Orchard
This chapter evaluates tissue staining. Fixed and processed tissue lacks contrast, and appears largely colourless despite containing numerous individual entities. Routine staining is most commonly carried out using the haematoxylin and eosin method. There are circumstances where the initial stain will not reveal sufficient information and subsequent demonstration methods are required. Various coloured dyes and stains can be used to emphasize the different structures present. These stains have evolved over many years, and in the modern diagnostic histopathology laboratory there are dozens of methods available to colour many entities. This gives contrasting appearances. Most of these methods rely on chemical principles whereby structures of a specific chemical nature can be stained selectively with the appropriate dyes. Often, two or more dyes can be used to provide a vivid contrast between the various structures within the tissue. The chapter looks at liver, native renal, muscle, and nerve biopsies.
What is histopathology?
David Muskett, Guy Orchard, and Anne Warren
This chapter provides an overview of histopathology, which is a mix of cell biology, biochemistry, chemistry, and genetics. All organs in every human being show the same general histology, and possibly more importantly, each pathology of the same type has a characteristic morphology and pattern. It is the manual visual interpretation of these things that forms the basis of histopathology. The chapter describes the role of histopathology in the diagnosis of disease. It then looks at the different types of biopsy submitted to the histopathology laboratory; outlines the specimen journey through the laboratory; and considers the special staining techniques that can be used to aid diagnosis. The chapter also discusses the concept of consent, the principles of the Human Tissue Act 2004, and the laboratory management systems that support an effective histopathology service.