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Investigating Immunity

Investigating Immunity
As most of the cells and molecules of the immune system spend some or all of their time in the blood, sampling them is usually straightforward, and the standard tests illustrated here account for a substantial part of the routine work of the immunology and haematology laboratories, and often of microbiology and biochemistry too. The assays are mainly automated and a report will usually give the normal values and indicate which results are abnormal. Nevertheless, because of the considerable differences between individuals, and in the same individual with time, interpretation is not always obvious, even to those well versed in other aspects of medicine. The interpretation of tests for autoimmunity is particularly tricky.

Investigating Immunity

Assays of both immunological molecules (e.g. antibody) and cells mainly make use of standard reagents, which are themselves frequently monoclonal antibodies, designed to react with only one molecular feature or cell-surface marker. The use of antibodies, whether monoclonal or not, to detect antigens of any kind is referred to as immunoassay (see Fig. 45). For detection, antibodies may be labelled with a radio-isotope (radioimmunoassay), an enzyme (ELISA) or a fluorescent molecule (immunofluorescence).
Occasionally, something more elaborate may be requested, e.g. a skin test for hypersensitivity, a bone marrow or other organ biopsy, blood or tissue typing before a transfusion or an organ graft, or the analysis of a leukaemia. Immunological tests are also used in epidemiology, e.g. to assess the extent of epidemics or the success of vaccines, and for the diagnosis of infections where the pathogen itself is not easily detected. In general, IgM antibodies denote recent, and IgG past, exposure.
Both the immune system and pathogens are increasingly studied through genes (see Fig. 47). Many diagnostic tests now rely on the PCR (see Fig. 45) to detect specific pathogens. High throughput techniques make it feasible to measure and analyse thousands of different proteins, genes and metabolites in tiny samples of tissue or blood: in the medicine of the future, these technologies will be used increasingly for  diagnosis,  and  the  development  of  ever  more  individualized medical treatments.

Investigating  immunodeficiency  (see  also  Fig. 33)
Antibody deficiencies  Total immunoglobulins and individual classes (e.g. IgG, IgA) can be measured by nephelometry or turbidimetry, ELISA, etc. A complete lack of immunoglobulins can be detected by absence of the γ-globulin arc in gel electrophoresis. Levels of antibody against particular antigens (e.g. a candidate virus) are measured by ELISA.
Lymphocyte deficiencies Total lymphocytes are counted by standard haematological methods. B and T lymphocytes are analysed by staining with fluorescent monoclonal antibodies against characteristic surface markers (e.g. CD3, CD4, CD8 for T cells and subsets, CD20 for B cells) combined with flow cytometry (see Fig. 45). Antigen- specific T-cell responses can be investigated in the clinic using a delayed hypersensitivity skin test, a common example being the Mantoux or Heaf test for prior exposure to tuberculosis, read 48–72 hours after injection. An alternative is to measure the proportion of blood producing production of a cytokine (such as IFNγ) in response to an antigen challenge.

Complement deficiencies Individual components, mainly C3, C4 and C1, are measured as for antibody. The functions of the lytic pathway can be assessed by haemolysis of antibody-coated red cells. Complement consumption (‘fixation’) by immune complexes is still sometimes used in estimating antibody levels in infections.
Phagocyte deficiencies Neutrophils and monocytes are counted as a routine part of a full blood count. Neutrophil function can be studied using the nitroblue tetrazolium (NBT) test, which measures the ‘res- piratory burst’, or by the intracellular killing of selected bacteria.

Investigating allergy (see also Fig. 35)
Serum IgE Total and antigen-specific IgE are measured by a solidphase fluorescent ELISA ‘capture’ assay.
Skin testing Immediate hypersensitivity, a reddened wheal 10–20 minutes after intradermal injection of the allergen, denotes the presence of specific IgE on mast cells in the skin. This is the most widely used initial test for allergies. For contact sensitivity, a patch test is used.

Investigating autoimmunity (see also Fig. 38)
Anti-Ig (rheumatoid factor) is detected by agglutination of Ig-coated red cells or latex particles, or by nephelometry/turbidimetry. Antibodies to cellular antigens are detected by immunofluoresence on sections of various tissues, which often shows a pattern characteristic of particular autoantibodies, e.g. antinuclear, antimitochondrial, antibasement membrane. This can be refined by ELISA using specific antigens, e.g. double-stranded DNA.
Note that the association between particular autoantibodies and particular diseases, though often highly suggestive, is seldom 100% positive. Tissue typing and transplantation (see also Fig. 39)
HLA (see Fig. 11) is extremely polymorphic, and matching the specific alleles carried by a donor and a recipient is a key determinant of success in many types of transplantation. This process is known as ‘tissue typing’ and is now performed routinely by characterizing the specific class I and II gene variants using PCR.
Detection of antibody Patients who have been transfused or have rejected a previous graft may already possess antibodies against HLA antigens. These can be detected by reacting them with a panel of donors, or a single potential donor if one has already been identified.
Blood transfusion Fortunately, red cells do not carry HLA antigens, and normally the only antigens for which matching is essential are A, B, O and RhD. Note that ABO matching is as critical for the survival of an organ graft as it is for a pint of blood.

Tumours of immunological cells
Leukaemias can usually be identified by flow cytometry using paired antibodies specific for surface markers (see Fig. 45). For example, chronic B-cell leukemias often carry both CD5 and CD20, while acute B-cell leukaemias carry CD10 and CD19.
Lymphomas can usually be typed in tissue sections using fluorescent or enzyme-linked antibodies.
Myeloma This tumour of plasma cells can be recognized by the presence of a prominent monoclonal ‘spike’ in serum electrophoresis, and class-specific antisera can be used to identify the heavy and light chain class. The diagnosis can be confirmed by bone marrow biopsy.