Asthma, Hayfever, Eczema and Food Allergy – -Investigating the Allergic Patient – Page 3

Recent progress in the understanding of the allergic response and the identification of key molecules involved in the allergic inflammatory process has permitted the development of tests to detect the presence of these inflammatory mediators.

1. Eosinophil Count
2. Nasal Eosinophils
3. Eosinophil Cationic Protein
4. Mast cell Tryptase
5. Histamine Assays
6. CAST Assays
7. Cytokine Assays
8. Immunoglobulin levels
9. Antibodies to the IgE Receptor
10. Blocking Antibodies

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.

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.

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.

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.

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 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.

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.

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 in South Africa.

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.

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. Normal ranges for age for immunoglobulin levels for IgG, IgA and IgM are given in Appendix XIII, on page 216.

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).

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 in South Africa.