Skin Tests. (A) Prick Test; (B) Intradermal Test.

12.2.2. Urine

Urinalysis is necessary in multiple myeloma, in other diseases where M bands are seen in serum on electrophoresis, in idiopathic hypogammaglobulinemia, and in amyloidosis.

Normal immunoglobulin synthesis is accompanied by the production of excess polyclonal free light chains. These light chains are excreted in the urine and are detectable in trace amounts in all individuals. Patients with renal damage excrete large amounts of polyclonal free light chains in the urine.

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The free monoclonal light chain (i.e. Bence Jones protein) was named after the first person to describe its unique thermophysical property of precipitating when the solution was heated to 56 o C, but dissolving at higher temperatures. However, this classical method of detection only detected about 40% of the free light chains in urine. Both methods

Neither the routine method of total urine protein nor the Clinistix test can detect free light chains. Currently, routine testing for suspected Bence Jones proteinuria involves three steps: (1) urine concentration; (2) cellulose acetate electrophoresis to look for the presence of the M band; and (3) immunofixation or immunoelectrophoresis to confirm that the M band is composed of monoclonal kappa light chains or monoclonal lambda light chains. Total excretion of paraprotein by the damaged kidney can give a false-positive result, so we must look for the nature of the free light chain of the M band to confirm the result.

12.2.3. Cerebrospinal fluid

CSF electrophoresis is a useful test for the diagnosis of multiple sclerosis and other demyelinating diseases. As in serum, CSF immunoglobulins are located in the gamma region. In contrast to serum, IgG usually forms oligoclonal bands; that is, a few discrete bands rather than a diffuse mass. Oligoclonal bands cannot be detected by routine electrophoresis of unconcentrated CSF, so CSF must be concentrated (80-fold) to make the bands visible. The sensitivity of the method can be increased by special stains such as enzyme-labeled antisera or silver-enhanced solutions. However, the most sensitive and reliable method is electrophoresis of undiluted CSF on an acrylamide gel. This gel separates proteins according to molecular weight.

12.3. Antibodies to exogenous antigens

In infections, the immune response to microorganisms is protective, allowing the body to recover from infection, and immunity also helps the body fight against reinfection with that microorganism. However, in addition to these beneficial effects, some microbial antigens cross-react with human antigens, so antibodies to these antigens can react with self-antigens and cause autoimmune diseases. Hypersensitivity responses to exogenous antigens can also cause tissue damage.

12.3.1. Antibacterial antibodies

For many years, detection of antibodies to microorganisms has been used to diagnose infections caused by that microorganism. The presence of circulating antibodies simply indicates that the body has previously encountered the antigen. To diagnose an acute infection, we must see an increase in antibody titer in two blood samples taken two weeks apart. If immediate results are needed, the presence of high titers of specific IgM antibodies indicates a primary response to the microorganism.

Detection of anti-bacterial antibodies is also essential in the investigation of immunodeficiencies. A good guide to susceptibility to infection is the serum total immunoglobulin level. Antibodies to enteric flora such as E. coli can be measured at high titers (<1/32) in most normal individuals but not in those with primary immunodeficiencies. If the patient has been vaccinated, detection of antibodies to tetanus toxoid, diphtheria toxoid, and polio virus is also useful. Detection of antibodies to streptococcal antigens is important in the investigation of patients with post-streptococcal immune disease.

12.3.2 Antibodies against non-reactive antigens

Some antibodies to nonreactive antigens can cause immune damage (hypersensitivity). The type of test used in this case depends on whether the mechanism of damage is type I IgE-mediated, type III IgM-mediated, or type III IgG-mediated.

In extrinsic asthma or allergic rhinitis, the skin test is useful because: (1) it shows that the reaction is an IgE-mediated type I reaction; and (2) it detects the antigen involved. Laboratory tests are often useful in patients with contraindications to skin testing, since many patients are positive on both skin and laboratory tests. The prick test is a type of test in which the substance to be tested is introduced into the skin by means of a needle that penetrates a drop of the substance on the surface of the skin and pricks the skin (Figure 12.8); it is easy to perform. The intradermal test is painful.

more. One thing to keep in mind when doing these and other tests is that the substance tested must be pure and active, then the test will give good results. This has caused a lot of trouble for the skin prick test in the past, although there are now many relatively pure preparations available for bee venom, pollen, dust mites, animal hairs and some food antigens such as eggs, fish, and nuts. However, the clinical interpretation of the results must be considered in comparison with the symptoms. A patient with atopy often has a positive skin prick test for many antigens, although only one antigen may cause clinical symptoms.

Provocation testing, which involves stimulating the nasal or bronchial mucosa with antigens, is a common test. However, this test is quite dangerous and should be performed in a hospital by experienced physicians. Skin prick testing, although safer, is not completely anaphylactic; therefore, it should also be performed under medical supervision.

In developed countries, the determination of total IgE in patients suspected of parasitic infection has been shown to be beneficial . The determination of total IgE also helps to distinguish the disease mechanism with or without the role of IgE. IgE determination is often performed by radioimmunoassay because the normal amount of IgE in serum is extremely low (120-480 ng/ml). The amount of IgE is usually measured in IU (International Units, 1 IU = 2.4 ng IgE). The most commonly used measurement technique for IgE is the “paper radioimmunosorbent technique” (PRIST). This test, although somewhat expensive, is a sensitive, accurate and precise test.

Figure 12.8. Skin tests. (a) Pinch test; (b) Endodermal test.

The radioallergosorbent technique (RAST) (Figure 12.9) allows us to quantify specific IgE antibodies.

antigen labeling. In these techniques, antigens are attached to small paper discs or to insoluble particles; then the test serum is added and only the IgE antibodies that react with the antigen are retained after washing. This specific IgE antibody is detected by a radioactively labeled secondary antibody. The results of the RAST test are completely consistent with those of the skin test but are expensive and are rarely used when skin testing is contraindicated or not useful. Patients suitable for this test include infants with severe dermatitis, infants taking drugs that alter skin reactions such as antihistamines, people who are likely to have a severe reaction to skin testing, and some patients with food allergies.

The precipitating antibodies to specific antigens are usually IgM or IgG. These antibodies are often tested for in the diagnosis of exogenous allergic alveolitis. The precipitation technique is performed by the Ouchterlony method; this is a less sensitive method but much cheaper than the radioimmunoassay technique. Extracts of suspected antigens are placed in the outer wells (Figure 12.10) and the patient's serum is placed in the middle well. After several days, a precipitate is observed. Currently, some commonly used antigens are sold on the market, but there are no standardized products . When a substance is suspected to be the culprit causing the patient's pulmonary symptoms, we can use that substance as an antigen to test by precipitation testing with the patient's serum.

Figure 12.9. Principle of allergen-specific IgE antibody measurement

Figure 12.10. Detection of precipitating antibodies in exogenous allergic alveolitis.

Patients with precipitating antibodies against avian albumin demonstrate avian pneumonia.

12.4. Detection of autoantibodies

12.4.1. In serum

In routine laboratories, four methods are commonly used to detect circulating autoantibodies: immunofluorescence, hemagglutination, radioimmunoassay (or enzyme immunoassay), and countercurrent electrophoresis. Each has its own advantages and disadvantages. Immunofluorescence is the least sensitive of these techniques, and results depend on the subjective ability of the reader. Hemagglutination is more sensitive but is time-consuming. Radioimmunoassay (RIA) requires expensive biological materials; gamma or beta detectors and waste disposal equipment are also costly. Enzyme-linked immunosorbent assay (ELISA) avoids the problem of radiation exposure but requires very specialized equipment. Countercurrent immunophoresis is cheap and easy to perform but is relatively insensitive.

12.4.1.1. Indirect immunofluorescence

This is a commonly used technique for the detection of various autoantibodies in serum. Animal tissue is often used as a substrate if the antigen is present in both human and animal tissues. For autoantibodies that are limited to human tissue or even to a single human cell line, animal tissue cannot be used. The tissue material used for this test is frozen immediately after being removed from the animal body and when used, it is frozen and cut at -20 0 C.

The patient's serum is incubated with the substrate (tissue) for 30 minutes. The antibodies that are not bound to the tissue are then washed away before secondary antibody, which is labeled (usually fluorescent), is added. The labeled antibody binds to the immunoglobulin in the patient's serum that has bound to the antigen on the substrate. The sites of antibody fixation are visible under a fluorescence microscope (Figure 12.11).

Each autoantibody is identified by a unique fluorescence pattern on a specific substrate. Once a serum is positive, it is titrated to see how strong the antibody is. The quantification is expressed as a titer ratio (e.g. 1/4, 1/8, 1/32, etc.) or in IU (international units). Most laboratories use IgG-specific secondary antibodies, which only detect IgG autoantibodies (i.e., clinically significant antibodies); IgM autoantibodies are not as important. However, antinuclear antibodies are an exception. The fluorescence pattern of antinuclear antibodies is useful for clinical research, but is not diagnostic. Currently, “screening” tests for autoantibodies are discouraged. Only tests that are clinically useful are requested.

Figure 12.11. Indirect immunofluorescence

The interpretation of the results of the indirect immunofluorescence test depends on the class of antibodies, their titers, and the age and sex of the patient. Older people, especially women, often produce autoantibodies without any clinical symptoms of autoimmune disease. Conversely, high titers of autoantibodies in young people indicate that a latent disease will appear later.

12.4.1.2. Hemagglutination

Red blood cells are used as indicators because they can “clump” or agglutinate when antibodies cross-react with antigens on their surface. The antigens may be the red blood cell’s own antigens (such as ABO antigens or other blood groups) or other purified antigens that have been attached to the surface of the red blood cells. Whether or not a hemagglutination tests are used depends on the availability of purified antigens. The binding method is generally not very simple except in the case of the rheumatoid factor test.

Rheumatoid factor (an IgM antibody that reacts with IgG as an antigen) reacts more strongly with IgG than with native human IgG. IgG is therefore agglutinated by reacting with a known antigen (sheep red blood cells) or by heating. Most laboratories use latex beads in the routine test for rheumatoid factor: human IgG is heat-agglutinated and attached to latex beads, which agglutinate when exposed to rheumatoid factor. This is a rapid and inexpensive test that can be used as a screening test for rheumatoid factor, but its drawback is that it often gives false-positive results. Positive serum is then repeated with the Waaler-Rose test. In this test, rabbit IgG (which has antigenic determinants in common with human IgG) is used to attach to red blood cells.

sheep. A subagglutinating dose of this antibody is incubated with sheep red blood cells and these “sensitized” indicator red blood cells are used to detect rheumatoid factor, which agglutinates sensitized sheep red blood cells but not native red blood cells. Unsensitized red blood cells are used to detect natural antibodies to sheep red blood cells. The result of the Waaler-Rose reaction is expressed as IU/ml or as a titer ratio. Although it is called rheumatoid factor, it is not diagnostic of rheumatoid arthritis but is useful in monitoring the prognosis of the disease.

12.4.1.3. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA)

These are very sensitive methods for detecting autoantibodies at low concentrations. Many techniques have been used, each with its own drawbacks. Enzymes can also be used as tracers instead of radioisotopes in RIA, and the technique is then called enzyme immunoassay (ELISA). This technique appears to be more sensitive than RIA.

Once we suspect a patient has SLE, we need to look for antibodies to double-stranded DNA. This can be detected in a number of ways; the most common are 14C -DNA, 125I -DNA, or DNA previously labeled with 14C -thymidine in the culture medium for bacterial consumption. This method is so sensitive that it can detect very low levels of DNA binding in patients other than SLE. A less sensitive test can be performed with 125I -DNA, but a positive result usually indicates SLE or active chronic hepatitis. Double-stranded DNA gradually dissociates into single-stranded DNA, so it is important to observe the binding values of each batch to see how much antigen is dissociated. Another test used is immunofluorescence on Crithidia luciliae parasites to detect antibodies to double-stranded DNA; This technique has a very high specificity, but is relatively insensitive because not all SLE sera react in this test. Recently, the World Health Organization published an international standard for antibodies to double-stranded DNA.

A substance derived from snake venom called α -bungarotoxin has been shown to bind strongly to the acetylcholine receptor in extracts of human skeletal muscle. This substance has been used to test for antibodies to the acetylcholine receptor (AChR). Purified α-bungarotoxin is labeled with radioactive iodine and combined with extracts of human muscle. The AChR antibodies react with this substance and can be precipitated with antibodies.

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