10 Tests to Assess Inflamation of Allergy
- The Total Eosinophil Count There has been a recent surge of interest in the role of the eosinophil in allergic diseases. Eosinophils in the nasal secretions of patients with rhinitis and in the airways of asthmatics are regarded as diagnostic hallmarks of an allergic process. Eosinophils are also known to play a role in fibrosis, thrombosis and the activation of mast cells. The beneficial role of eosinophils in the immunological defence against parasites is undisputed. Eosinophils in the airways of asthmatic patients release products with result in severe epithelial and cilial damage. Recent discoveries of the cytokines and adhesion molecules responsible for eosinophil activation and localization have opened up opportunities for the production of molecules which may prevent their accumulation in allergic diseases. The normal eosinophil count in healthy individuals is about 150 cells per cubic millimetre and although some normal individuals may have as many as 800 cells per cubic millimetre, a cut-off of 400 cells per cubic millimetre is usually taken as normal. Eosinophils usually circulate for only a few hours and then migrate into the tissues, where they have a life span of 2-3 days. Activated Eosinophils In the airways and blood of asthmatics, eosinophils exposed to various activators become hypodense. Hypodense eosinophils have a high metabolism and higher numbers of hypodense eosinophils are present in asthmatics with poor lung function and patients with exercise induced asthma. Eosinophilia is reduced by steroid therapy via the immunosuppressive effect of steroids on T cells. Increased chemotactic activity in serum for eosinophils is present early in the pollen season. This chemotactic activity is abolished by steroids and also by immunotherapy. Monitoring Eosinophil Activity in Allergic Diseases A raised eosinophil count (more than 400 cells/mm3) is highly suggestive of parasite infestation or active allergic disease. Patients who have acute allergic reactions (e.g. to drugs) often have an associated transient eosinophilia. In patients who are treated with systemic steroids the eosinophil count is expected to fall if the steroid dose is adequate. The state of activation of the eosinophils, as evidenced by their density or profile of mediator release, rather than the total eosinophil count, correlates with disease activity. Recently, several tests have been developed to measure eosinophil activity. These include the determination of Eosinophil Cationic Protein (ECP) and Eosinophil Peroxidase (Epx) levels. Several studies have shown a good correlation between the presence of elevated levels of serum or bronchial Epx or ECP and ongoing airway inflammation. These tests are being recommended for monitoring ongoing allergic disease. It is currently believe that they may be used to monitor inflammatory activity in asthma and eczema and can assist the clinician to tailor steroid doses in severe asthmatics, or to check patient compliance.
- Nasal Eosinophils A nasal eosinophilia is regarded as a hallmark of nasal allergy. Eosinophils are not normally seen on a stained smear of nasal secretions. Eosinophils in a nasal smear are best stained using Hansels stain (see Appendix XII, on page 214). Eosinophil numbers reduce as the patient improves on topical nasal steroids. A grading scale may be used to monitor the response to treatment. Numbers also reduce on smears studied outside of the pollen season in patients with seasonal allergy. The presence of sheets of neutrophils on a nasal smear indicates an infective rhinitis or infection in the paranasal sinuses.
- Eosinophil Cationic Protein Serum Eosinophil Cationic Protein (ECP) and Eosinophil Derived Neurotoxin (EDN) assays (Pharmacia) are currently showing promise for the ongoing monitoring of allergic inflammation in asthmatic patients and patients with eczema. Levels fall when patients are treated with steroids and rise when patients are exposed to allergens (e.g. house dust mite) or are non-compliant with their steroid medication. Special precautions should be taken when submitting clotted blood samples to the laboratory for Eosinophil Cationic Protein measurements (SST tubes must be used) and serum should be separated within one hour of taking blood.
- Mast Cell Tryptase Mast cells, which are granulocytes found in peripheral tissue, play a central role in inflammatory and immediate allergic reactions. beta-Tryptase is a neutral serine protease and is the most abundant mediator stored in mast cell granules. The release of beta-tryptase from the secretory granules is a characteristic feature of mast cell degranulation. While its biological function has not been fully clarified, mast cell beta-tryptase has an important role in inflammation and serves as a marker of mast cell activation. beta-Tryptase activates the protease activated receptor type 2. It is involved in airway homeostasis, vascular relaxation and contraction, gastrointestinal smooth muscle activity and intestinal transport, and coagulation. Serum mast cell beta-tryptase concentration is increased in anaphylaxis and in other allergic conditions. It is increased in systemic mastocytosis and other haematological conditions. Serum beta-tryptase measurements can be used to distinguish mast cell-dependent reactions from other systemic disturbances such as cardiogenic shock, which can present with similar clinical manifestations. Increased beta-tryptase levels are highly suggestive of an immunologically mediated reaction but may also occur following direct mast cell activation. Patients with increased mast cell beta-tryptase levels must be investigated for an allergic cause. However, patients without increased mast cell tryptase levels should be investigated if the clinical picture suggests severe anaphylaxis Mast cells release a unique tryptase enzyme upon activation. Normally tryptase is not present in the blood. The presence of measurable tryptase is a good index of mast cell activation. Recently the Pharmacia assay for mast cell tryptase have been introduced to South Africa and is available at the Allergology Unit, UCT. The measurement of mast cell tryptase is useful e.g. during anaesthesia when one is unsure if an “anaphylactoid” reaction is “allergic” or due to some other cause (e.g. vasovagal). Clotted blood samples should be taken serially after a suspected anaphylactoid reaction at ½ – 1 hour intervals for 4-6 hours after the reaction and serum stored for mast cell tryptase assay. Samples taken within a few hours post mortem can also be used for mast cell tryptase assay where anaphylaxis has resulted in death and its measurement may have medico-legal significance
- CAST Assays The Cellular Allergen Stimulation Test (CAST) has recently been introduced as a research assay. The CAST test is based on the production of sulphido-leukotrienes LTC4, CTD4 and LTE4 by blood leukocytes upon challenge with allergens. This test represents a technical advance on the basophil histamine release assay and preliminary data has shown good correlation with the results of skin test and RAST tests. The CAST test holds great potential for bridging the gap between the clinical limitations of both skin and RAST testing and is at present being evaluated
- Allergy Cytokine Assays “Cytokine” is a general term used for a diverse group of soluble proteins and peptides which act as regulators under both normal and pathological conditions to modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment. Cytokines differ from hormones in that they act on a wider spectrum of target cells and also, unlike hormones, they are not produced by specialized cells which are organized in specialized glands. This group of proteins includes lymphokines, interferons, colony stimulating factors and chemokines (cytokines with chemotactic activity). Cytokine/Chemokine research plays a significant role in achieving a deeper understanding of disease states such as allergic reactions, cardiovascular disease, metabolic syndrome, sepsis and cancer. Therefore, our MILLIPLEX MAP Human Cytokine/Chemokine panel will enable you to focus on the curative potential of cytokines as well as the modulation of cytokine expression in a variety of therapeutic areas. Coupled with the Luminex® xMAP® platform, you receive the advantage of speed and sensitivity, allowing quantitative multiplex detection of dozens of analytes simultaneously, which can dramatically improve productivity. There are a wide range of assays available on the South African market to measure cytokines important in allergic inflammation. These include assays for Interleukin 3, Interleukin 4, Interleukin 5, gamma interferons, tumour neurosis factor (TNF), soluble interleukin 2 receptors and interleukin 2, etc. The uses of these assays in clinical practice have not yet been defined, although several research studies measuring these cytokines are underway.
- Immunoglobulin Levels Besides the measurement of IgE, measurement of other immunoglobulin isotypes is often informative in the allergic patient. One in 200 allergic patients have IgA deficiency and occasionally allergic patients may have hypgammaglobulinaemia of an IgG subclass. IgG subclasses can be measured at most major centres. Deficiency of IgG2 results in recurrent infections with polysaccharide encapsulated organisms such as Streptococcus pneumonia, Haemophilus influenzae and Neisseria meningitidis. Deficiency of IgG1 or IgG3 may be associated with recurrent viral or bacterial infections. The normal ranges for IgG subclasses depend upon the assays used.
- Antibodies to the IgE IgE Antibodies are a type of immunoglobulin made by the body which are implicated in allergic reactions. Receptor It has recently been shown that some patients with chronic resistant urticaria have auto-antibodies to the high affinity IgE receptor on basophils or mast cells. These patients may respond to high dose steroids or plasmapheresis. Auto-antibodies can be measure to the IgE receptor at the UCT Allergology Unit. (5ml of clotted blood or 3ml of serum is required). Quantitative determination of total serum IgE can be made rapidly and accurately by nephelometry, a technique that measures light scattered from a beam passed through a solution. Specific IgE levels must be measured with more sensitive techniques such as by radioallergosorbent testing (RAST) or allergy skin testing. A large number of substances have been found to have allergic potential. They are known as allergens. Measurable allergen-specific antibodies can be identified by radioallergosorbent tests (RAST). It is recommended that a patient’s serum be first screened with a selected panel of six allergens and then followed, if appropriate, by an extended panel of additional allergens. The purpose of study is to test for reaction to certain respiratory and food allergystimulants. The RAST tests measure the increase and quantity of allergen-specific IgE antibodies. These measurements are used in persons, especially children, with extrinsic asthma, hay fever, and atopic dermatitis (eczema) and are an accurate and convenient alternative to skin testing. Although more expensive, they do not cause hypersensitivity reactions.
- Blocking Antibodies A blocking antibody is an antibody that does not have a reaction when combined with an antigen, but prevents other antibodies from combining with that antigen.The term can also be used for inhibiting antibody, prozone phenomenon and, agglutination reaction. Blocking antibodies have been described as a mechanism for HSV-1 to evade the immune system. Patients who receive immunotherapy develop IgG (particularly IgG4) antibodies to the allergens in the vaccine. It was though that these antibodies (blocking IgG antibodies) could be used to monitor the efficacy of immunotherapy regimens. This is however not the case and there is no correlation between the IgG antibody levels to a particular allergen and the level of protection following immunotherapy. It appears now that immunotherapy acts by altering the cytokine profiles of lymphocytes towards a Th-1, gamma interferon and interleukin 2 profile. Blocking antibody assays are therefore no longer offered by laboratories
- Histamine Assays Histamine is a small biogenic amine known to play an important role in immediate hypersensitivity and inflammation reactions as well as in several atopic diseases such as allergic rhinitis, bronchial asthma and eczema. Based on our patented HTRF® technology, the Histamine kit is a highly specific assay for quantifying an allergic response in cell-based conditions. Histamine is the classical mediator of allergy, and can be measured in blood or urine by radio-immunoassay. Plasma or urinary histamine levels are useful to diagnose scromboid fish poisoning, mastocytosis and specific reactivity to food allergens following challenge. However, plasma histamine has a short half-life and urinary histamine levels (methyl histamine) are easier to measure. Urinary histamine is conveniently measured using the Pharmacia methyl histamine assay. Urine should be acidified if stored or kept at -20° C until assayed. Urinary histamine assays can be done at the UCT Allergology Unit by telephone arrangement. Histamine values are expressed in relation to urinary creatinine levels and a non-acidified sample should be submitted simultaneously for urinary creatinine measurement.
- Buys PJC. A practical approach to the diagnosis of allergy. Cont Med Education 1994; 12: 43-50.
- Position Statement. Allergen skin testing. American Academy, Allergy and Immunology. J Allergy Clin Immunol. 1993; 92:636-7.
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