Immunology In The Laboratory
The ability to measure accurately and sensitively different aspects of immunological function is an important part of both experimental and clinical immunology (see Chapter 44). Some of the most commonly used techniques in the immunological laboratory (immunological assays) are shown in the figure. Some techniques, such as the differential blood count, have hardly changed in over a hundred years. Others, such as flow cytometry and the PCR continue to evolve at a rapid rate as new technologies are developed. In all cases, clinical laboratories are making increased use of robotics and sophisticated computational analysis to automate all aspects of the process, to make it faster, cheaper and more reliable. The ability to integrate many different measurements (immunological, haematological, psychological, genetic, etc.) taken from each patient rapidly and reliably is also driving the development of ‘personalized medicine’, where doctors will be increasingly able to tailor each treatment precisely to match the needs of individual patients.
Flow cytometry (Fig. 45.1–45.3) is one of the most powerful techniques in the immunologist’s repertoire. Cells are sucked into a fine jet of liquid so that they pass rapidly across a beam of one or, in more sophisticated machines, several lasers. Cells scatter the incoming beam of light by refraction and reflection. Light scattered through a small angle is called ‘forward scatter’ and is proportional to the size of the cells. Light scattered through a 90° angle is called ‘side scatter’ and depends on the granularity of the cell; e.g. a granulocyte has a much larger side scatter than a lymphocyte (see Fig. 45.1).
Cells can also be mixed with mixed with antibodies that bind to specific molecules on the cell’s surface. Each antibody is linked to a molecule (fluorophore) with the property of absorbing light of one wavelength and re-emitting it at another. Many such molecules exist, some originally isolated from marine organisms. Light emitted by each cell is collected by a series of mirrors and then detected by one of several photomultipliers and stored on a computer. The precise com- position of the mixture of cells can then be determined by analysis of their signals. In Fig. 45.2, cells from the thymus are shown as positive for CD4, CD8, both, or neither. The results can be displayed in the form of a dot plot (Fig. 45.1 and 45.2) in which each cell is represented as a dot, or as a histogram (Fig. 45.3). Results from histogram analyses can be superimposed as in Fig. 45.3, permitting easy comparisons between healthy and disease samples (e.g. blood cells from patients with leukaemia as shown in figure).
In a further refinement, cells binding different antibodies can be collected in separate tubes (fluorescence-activated cell sorting [FACS]), a powerful tool for isolating very pure cell populations from a mixture.
Immunofluorescence (Fig. 45.4), in addition to its role in flow cytometry, can also be applied to histological specimens, commonly to identify autoantibodies or immune complexes, or metastatic cancer cells invading healthy tissues. Figure 45.4 shows a kidney from a patient with systemic lupus erythematosus stained with a fluorescent antibody to IgG, which has bound to the immune complexes along the basement membrane.
ELISA (Fig. 45.5) The enzyme-linked immunoabsorbent assay is one of the most versatile immunological techniques. In direct ELISA, a target antigen, e.g. microbial proteins, or human DNA, is adsorbed on to a plastic surface – typically 96 or 396 small ‘wells’ – allowing many samples to be tested simultaneously. Diluted samples of serum to be tested are added, and any antibodies specific for the target antigen will become bound and immobilized to the plastic surface. Unbound serum components are then washed off and a ‘second’ antibody, e.g. to human Ig, which has been linked to an enzyme is added. An enzyme is chosen that converts a colourless substrate to a coloured product, which can then be measured in a spectrophotometer. Direct ELISA is often used to detect antibodies to microbes in infection (Fig. 45.5 shows the results of testing different human sera for the presence of antibodies to HIV) or to self antigens in autoimmune disease.
Sandwich ELISA In another variant, a specific antibody is first adsorbed to the plastic wells, then the serum or other sample to be tested, and finally the enzyme-linked second antibody, so as to form an antibody–antigen–antibody ‘sandwich’. Complement components, cytokines, etc. can be conveniently assayed in this way. Much effort is being put into improved ELISA-like protocols that have increased sensitivity and can detect many components in a single sample (known as ‘multiplexing’), reducing sample size, speed and cost.
PCR (Fig. 45.6) The PCR is the most recent addition to the immunology laboratory. The basic principle is to replicate any desired piece of DNA or RNA and make enough copies of it to be easily detected, sequenced and characterized. The simple and elegant technique consists of three steps. The target DNA (known as the template) is heated to 94°C so as to separate the two strands of double helix (denaturation), and mixed with two very short pieces of DNA (known as primers), which each have a sequence complementary to one end of the piece of DNA to be amplified. One primer is complementary to a specific sequence on one strand of DNA, while the second matches a sequence on the complementary strand in the opposite direction. The temperature is lowered so the primers bind to their complementary matching sequences on the template (annealing). The DNA is replicated (extension), using a special polymerase (often known as Taq), which was originally isolated from thermophilic bacteria living in deep oceanic hot springs, and which therefore works well at very high temperatures. The polymerase uses the primers as starting points to replicate a few hundred to a few thousand bases of the DNA. The cycle is then repeated, each time doubling the number of copies of the DNA sequence that lie between the two primers. The technique is so sensitive it can be used to detect a single copy of starting DNA. PCR has become the standard approach to HLA typing (see Figs 11 and 44). The technique can be modified so it gives a quantitative measurement of the amount of template in the starting material (qPCR), and is now used routinely to measure levels of virus or bacteria in samples of blood or other tissues.