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.
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.
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.
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.
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.
