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8.1 What is meant by the terms sensitivity and specificity in immunocytochemistry (ICC)?
Sensitivity is a measure of the concentration of antibody required to bind an antigen (highly sensitive means that a low concentration is required). Specificity is a measure of how selective an antibody is for a particular antigen (highly specific means that the antibody binds only to the antigen against which it has been raised).
5.2 What is the meaning of the abbreviation CD, and how is it employed in classifying antibody characteristics?
CD stands for ‘Cluster of Differentiation’. It is a protocol used to classify cells based on the epitopes present on their cell membranes, thereby enabling the use of antibodies to those epitopes to define cell populations. The cluster number relates to different antibodies that have been found to have the same specificity as defined by at least two or more antibody-based techniques (eg ICC, fluorescence-activated cell sorting [FACS] or Western blotting).
5.3 What are the key areas of application for ICC in histopathology?
The key areas of application in a diagnostic setting are: a) Defining cell types in tumour pathology b) Defining cell types in Inflammatory cell infiltrates c) Evaluating prognostic indicators d) Identifying infectious pathogens e) Identifying defective proteins and providing evidence at the cellular level of defective gene transcription or translation f) Evaluating immune complex deposition in autoimmune diseases g) Providing indicators for potential target therapy strategies h) Evaluating cell activation and apoptotic pathways.
5.4 What is meant by the term ‘antigen retrieval’ and what are the key methods used to achieve this?
Antigen retrieval is the mechanism by which antigens are rendered accessible by antibodies raised against them. Methods predominantly use heat in a water bath, pressure cooker or microwaveable vessel, combined with a citrate or EDTA buffer, although the earlier enzyme-based techniques still have a role to play with some antigens.
5.5 What is meant by the term ‘chromogen’?
A chromogen is a coloured chemical ‘end product’ used to visualise the localisation of an antibody/antigen reaction using light microscopy.
5.6 List the main ICC labelling systems currently available.
The most common chromogenic labelling systems use diaminobenzidine (DAB) in the presence of peroxidase enzyme, the latter being conjugated either to a polymer backbone or streptavidin which binds to the primary antibody via a linking immunoglobulin. Alternative methods utilise a different chromogen/substrate combination, such as fast red/alkaline phosphatase, or In haemochromatosis iron levels are raised due to one of two means. In primary haemochromatosis a genetic abnormality causes unregulated iron upload. Secondary haemochromatosis may be due to alcoholism. Uncontrolled iron overload can lead to a number of unpleasant symptoms e.g. low sex drive, heart failure, diabetes, and may lead to liver cancer. The amount of iron stained blue in a Perls stain is graded by eye so it is important to ensure that all iron is reacted when performing the test. Reticulin The reticulin stain shows general tissue architecture. Under normal circumstances a fine lace like network of reticulin fibres secreted by fibrobrasts supports the cellular tissue of the liver. In certain disease states such as cirrhosis the number of reticulin fibres is increased and their guage is increased. HVG This stain looks at the amount of collagen within the tissue. Collagen is red. Fibrin is yellow. Collagen is increased in cirrhosis and this stain can be useful in identifying collagen deposition. Orcein The modified Orcein stain is the method of choice for the investigation of Australia antigen found in hepatitis B. Orcein also stains the copper binding protein caeruloprotein. Answers or Hints and Tips for discussion questions link a fluorescent molecule to the primary antibody to enable visualisation. The main staining systems include: • Direct • Indirect • Streptavidin-biotin • Polymer-based systems
5.7 Explain internal quality control and external quality assessment in ICC.
Internal quality control (IQC) involves ensuring that only the relevant molecules in a tissue section are stained appropriately, and to the right degree, following the application of an antibody and its subsequent detection. External quality assessment (EQA) involves an outside body providing objective assessment of material stained by a laboratory so that overall accuracy and comparability of results between centres may be determined. It is not solely a means to establish poor performance. More importantly, it is a tool used to educate and improve performance by learning and implementing change to existing practice to meet good laboratory standards.
5.8 What are the advantages of automated ICC in the modern laboratory?
The key advantages of automation are reproducibility and auditable results, standardisation, the ability to handle a high volume of slides continuously and integration into a laboratory information system (LIMS). Benefits include:
• Slide reading and reagent managemen
• Onboard antigen retrieva
• Integrated IT system
• Continuous-flow automatio
• Options for digital image capture and transfer