4 Immunohistochemistry (IHC)
Staining of cells and sections with antibodies has revolutionised histology, since it allows the identification and localisation of individual molecules. A molecule that binds to and is recognised by an antibody is called an antigen, and in the context of histology, such antigens are often referred to as markers, since they act as ways of recognising a particular cell (Figure 8). The technique is particularly valuable for distinguishing different cell types in the diagnosis of cancer. For example, different classes of lymphocyte appear virtually identical in size and shape (morphology), but they can be distinguished according to their surface markers - all T lymphocytes express CD3, and the two major subpopulations of helper T cells and cytotoxic T cells express CD4 and CD8 respectively. So a T cell lymphoma can be tracked in different tissues using these markers. The CD system identifies surface molecules on different cells and includes more than 250 different proteins. For example, CD3 is the T cell's receptor for antigen.
Another use for immunohistochemistry is in guiding treatment. For example, the drug tamoxifen is used to treat breast cancer; many breast cancers require oestrogen to divide and tamoxifen binds to the oestrogen-receptor on the cells, blocking this proliferative effect. However, this only applies to some cancers; some do not have an oestrogen receptor and are therefore not susceptible to tamoxifen therapy. A histologist can use immunohistochemistry to identify whether a breast cancer removed by surgery expresses the oestrogen receptor (which in this context is a cell surface marker identified by an antibody). The clinician wants to treat the patient so as to prevent any secondary tumours from growing and with this information they can decide whether or not tamoxifen therapy is appropriate.
A standard immunohistochemistry protocol involves treatment of the section with an antibody that recognises the marker. This is referred to as the primary antibody. Then the primary antibody is recognised by a secondary antibody that is linked to an enzyme, or several copies of the enzymes - such reagents are sometimes called conjugates, although this term can mean different things in other contexts. Finally the section is treated with a chromogen, a reagent that is acted upon by the enzyme, to deposit an insoluble coloured compound onto the cell, where the original primary antibody had bound. Extensive washing is required between each of the stages to prevent non-specific binding and so that the previous reagent does not neutralise the next reagent before it has a chance to bind to the cell. The system is outlined in Figure 9. Note that although the conjugate for immunohistochemistry is normally an enzyme, if fluorescence microscopy is used, the secondary antibody would be conjugated to a fluorescent tag.
Non-specific binding can either be due to the antibody binding (weakly) to the cells when no antigen is present, or may be due to the antibody cross-reacting, i.e. binding specifically and strongly to another antigen that has a similar structure to the target antigen. Generally one aims to avoid using antibodies that cross-react, if at all possible. There are also a number of ways of reducing weak non-specific binding, indicated below.
What considerations are important in selecting a primary antibody? Give three points.
It should be specific for the target antigen. It needs to be able to react with tissues that have been fixed (or non-fixed tissue can be used). It has to be recognisable by the secondary antibody.
Primary antibodies are usually raised in mice, rats or rabbits. Antibodies come in a number of different classes (e.g. IgG = immunoglobulin-G, IgM = immunoglobulin-M, etc.) and within each class there may be a number of sublcasses (e.g. IgG1, IgG2a, IgG2b, IgG3 in mice). Most primary antibodies will be IgG, because IgG antibodies typically have a high affinity for their antigen, and are therefore more likely to be highly specific for their target antigen. The species, class and subclass of the primary antibody is an important consideration, since each one is slightly structurally different, and not all secondary antibodies can recognise all primary antibodies. For example a secondary antibody that recognises mouse IgG1 may not recognise rabbit IgG1. Put simply the primary and the secondary antibodies must be matched, otherwise they do not work together.
Give three considerations in selecting a secondary antibody for use in a histology laboratory.
It is advantageous if the secondary can recognise a wide range of primary antibodies since it is more versatile. The level of enzyme conjugated to the antibody affects the sensitivity of the test. This reagent will be used often, so cost is also important.
The correct dilutions of the primary and secondary antibodies need to be carefully checked for each antibody. Too little and the section will not be stained; too much and the section will be dark all over. Typically, a concentration of 5-25µg/ml is used for a primary IHC antibody, but this is only a general guide and it is necessary to establish the optimum for each antibody. Once this has been done, the antibody can be used routinely at this dilution. Antibodies are generally very stable reagents if stored in a fridge with a preservative (years), but they lose activity if subjected to repeated freeze/thaw cycles.
Antibodies are expensive reagents, so everyone aims to use the minimum amount, by localising the drop of antibody that is needed to cover the tissue section. It is the concentration, rather than the volume that is important and volumes of as little as 50µl of diluted antibody can be used.
A variety of enzymes may be conjugated to the secondary antibody, but peroxidase is most commonly used. Peroxidase uses H2O2 as one of its substrates, so the chromogen mixture must contain an optimal amount of the substrate. The enzyme itself can be damaged by H2O2 , if too much is present.
Apart from the substrates, what other considerations are important for the enzyme to work?
Enzymes work at a particular optimum pH and temperature, so the chromogen solution must be buffered at this pH, and the reaction should take place for the correct amount of time, at the right temperature.
Different chromogens are used in particular laboratories, but di-amino-benzidine, which deposits a dark brown stain, is very widely used. (Be aware that this reagent is carcinogenic and requires specific handling and disposal procedures.) It is not usually sufficient to just carry out the IHC step, since the staining must be seen in the context of the rest of the tissue, so the IHC may be followed by a simple histochemical stain, to allow the cells to be seen.