An allergy is defined as an acquired hypersensitivity to a substance (allergen) that does not normally cause a reaction. It is essentially a disorder of the immune system that results in an antibody-antigen reaction. The most common manifestations of allergy involve the respiratory tract or the skin.
In vitro allergy testing detects antigen-specific immune globulin E (IgE) antibodies in serum. This type of testing may be required when direct skin testing is not possible for inhalant allergens (e.g., pollens, molds, dust, mites, animal danders), foods, insect stings, or other allergens such as drugs. In vitro allergy testing is appropriate for individuals who have skin reactions/conditions such as severe dermatographism, ichthyosis, or generalized eczema. It is also appropriate for uncooperative individuals (young children or individuals with mental and/or physical handicaps), individuals with a high risk of anaphylaxis from skin testing, or individuals for whom direct skin testing has been inconclusive.
The tools that are used for in vitro allergy testing are all variations of one biomedical technology known as an immunoassay. Immunoassays, used in both research and clinical settings, are used to quantify biological molecules of interest, capitalizing on their properties as antigens or antibodies. This assay can measure the concentration of a particular biomolecule via the antigen-antibody reaction, a naturally occurring event in the human immune response. In this reaction, a part of a pathogen known as an antigen is detected by immune effector cells and eventually bound by an antibody to be neutralized. The antibody involved in allergic response is known as IgE; the antibody-antigen complex is labeled by an anti-IgE molecule that is tagged with a chemical agent for visualization. The quality of an immunoassay depends on the specificity and selectivity of antibody reagents used, as well as the method of visualization employed (e.g., radioisotopes, enzyme-catalyzed colored products, fluorescence).
In vitro allergy tests must be interpreted in the context of each individual's specific clinical history; the diagnosis of an allergic disorder cannot be based solely on a laboratory result. Allergy is a dynamic physiologic response with multiple contributing variables, including, but not limited to, presence of allergen-specific IgE. While most individuals who experience symptoms of hypersensitivity on allergen exposure show measurable levels of allergen-specific IgE, rarely some individuals may not have any allergen-specific IgE detectable with routine testing. Specific IgE tests are important and recommended in the identification of allergy, but it should be noted that alone they are not sufficient to diagnose allergy.
The following blood tests may be used to aid in the diagnosis, evaluation, and treatment of allergy:
The multiple allergen simultaneous test (MAST), along with radioallergosorbent test (RAST®) and fluoroallergosorbent test (FAST™) are in vitro techniques for determining whether an individual's serum contains IgE antibodies against specific allergens of clinical importance. RAST was developed in 1974 by Pharmacia Diagnostics AB (Uppsala, Sweden) as a tool to measure allergen-specific IgE. In this methodology, the allergen is coupled to a paper disk. The individual’s serum is added to the disk, whereby an antigen-antibody complex forms in the case of allergy. Radioactive IgE is added and complexes, and a gamma counter scores the level of binding to inform on intensity of the allergy. RAST is a brand name, but over time has become a catch-all term to (erroneously) refer to any in vitro assay for allergen-specific IgE. Although RAST was initially the in vitro allergy test of choice, it is an older technology that has largely been replaced by the use of other immunoassays such as ImmunoCAP and ELISA testing. FAST, developed in 1987, has a similar mechanism of action as RAST, but instead of radiolabeling, uses fluorescence labeling and detects antigen-antibody activity using a spectrofluorometer. MAST tests are also similar to RAST in that the specific allergen of interest is coupled to a solid phase (typically nitrocellulose instead of paper), antibodies from the added serum complex to the antigen if allergy exists, and a tagged anti-IgE is added to visualize the binding. Unlike RAST and FAST, MAST is not a registered brand name, but rather a category of immunoassays. Some MAST tests use chemiluminescence visualization, others use colored products resulting from the oxidation of various organic substrates.
The enzyme-linked immunosorbent assay (ELISA), also known as enzyme immunoassay, is a plate-based assay technique designed for detecting and quantifying substances such as peptides, antibodies, and hormones. In allergy testing, the antigen is immobilized on a solid surface, then complexed with an antibody, which is linked to an enzyme (such as horseradish peroxidase). The enzyme reacts with a substrate that produces a colorimetric product, which can be quantified.
Created in 1989 by the developer of RAST, ImmunoCAP is described as the next generation of laboratory allergy testing. These assays can be performed singly for just one allergen (ImmunoCAP), or in multiplex format (ImmunoCAP Immuno Solid-phase Allergen Chip [ISAC]). Whereas the singleplex assay determines IgE levels against one chosen allergen, the multiplex assay uses a fixed array of 112 native allergen components (classifying it as a MAST). The ImmunoCAP technology uses a three-dimensional, activated cellulose polymer encased in a capsule that increases the surface area and can bind to more antibodies. ImmunoCAP is lauded for its ability to obtain quantified allergen-specific IgE antibody levels, in addition to the lack of interference from allergen-specific IgG antibodies.
The leukocyte histamine release test is an in vitro method of allergy testing that measures the amount of histamine secreted from basophils, a type of white blood cell, upon exposure to an allergen extract added to the serum sample.
Cytotoxic food testing involves mixing a suspected offending substance with a sample of the individual's blood. The sample is then observed under a microscope for changes in the white blood cells.
Lymphocyte stimulation tests, otherwise known as lymphocyte response assays, lymphocyte proliferation assays, and lymphocyte transformation tests, measure the ability of lymphocytes to respond in vitro to an activation stimulus. This method requires the isolation of peripheral blood mononuclear cells, which are then put in tissue culture with a foreign antigen of interest. Subsequent measurement of the number of dividing cells indicates immune stimulation.
The immune globulin G (IgG) ELISA, indirect method, tests IgG immune reaction (rather than IgE immune reaction) in serum. At the present time, there are insufficient data to support the reliability of allergen-specific IgG assays for routine diagnostic testing.
The basophil activation test (BAT) is a flow cytometry–based assay that measures the expression of activation markers on the surface of basophils after stimulation with an antigen. A positive BAT is described as an in vitro surrogate of an acute allergic reaction in vivo. Because of the large degree of inconsistency among individuals, clinical application of BAT will require increased analytical validation, clinical validation, standardization of procedures, and quality assurance to ensure reproducibility and reliability of results.
The T-cell proliferation assay is a drug assay being developed to identify drug hypersensitivity reactions in individuals. The mechanism of action involves the assessment of drug specific activation of naïve T cells, alongside analysis of the strength of the induced response and the number of cultures that respond. To date, more testing and investigation is needed to optimize this assay for use in predicting the potential for drug hypersensitivity in individuals.
There is insufficient evidence to support the efficacy of the following in vitro tests for the diagnosis and treatment of allergy: the leukocyte histamine release test, cytotoxic food testing, the lymphocyte proliferation test, IgG ELISA, indirect method, basophil activation test, and T-cell proliferation assay.
