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The in vitro serum tests employ specific antisera, and the allergen antibody reactions are amplified as a radioimmunoassay (RAST), fluorescent immunoassay (FAST), or an enzyme-linked immunosorbent assay (ELISA). Each of these techniques is comparable when performed properly. In vitro tests are acceptable substitutes for skin tests in the following circumstances:
1) The patient has abnormal skin, such as dermatographism or extensive dermatitis,
2) The patient cannot or did not discontinue antihistamines or other interfering medications,
3) The patient is very allergic by history, and anaphylaxis is a possible risk, and
4) The patient is noncompliant regarding skin testing. The results of either skin tests or in vitro assays depend very much on the quality of the allergen and the competence with which the
test is performed.
A typical radioimmunoassay:

Although the quality of allergens is improving, there is need for more and better standardization. Both skin testing and in vitro assays have been criticized for lack of good quality control. Skin testing should not be an occasional test for the inexperienced and obviously never should be delegated to an inadequately trained or unsupervised assistant. Board certified allergy and immunology specialists are best qualified to correlate patient histories with tests results. Quality control also has been a major problem for in vitro serum IgE antibody tests.
Compulsory participation in quality control programs, such as that offered by the College of American Pathologists and mandated by the Clinical Laboratory Improvement Act, eventually will lead to better quality and standardization of in vitro serum IgE tests.

Positive tests for allergen-specific IgE do not diagnose allergy; they only indicate the presence of IgE molecules that have a particular immunologic specificity. Whether the specific IgE antibodies are responsible for clinically apparent disease must be determined by a well-trained physician. The ultimate standard for the diagnosis of allergic disease remains the combination of: a positive history, the presence of specific IgE antibodies, and demonstration that the symptoms are the result of IgE-mediated inflammation.
To avoid false-negative skin tests, short-acting antihistamines should be withheld for 36 to 48 hours and long-acting antihistamines (ie, astemizole) for 4 to 6 weeks before skin tests are performed because antihistamines suppress skin testing results. The specifics of skin testing are outlined in standard allergy textbooks. Skin tests with the appropriate allergens are mandatory in all patients prior to initiation of immunotherapy with allergy extracts, and the intensity of the local wheal and flare skin reactions is a guide for determining the initial dose of allergen.
Skin testing by the multiple serial dilution (end-point titration method) is not recommended by this author because multiple skin tests increase the cost of evaluating the patient and the postulated more quantitative results have not been validated. Sublingual challenge with allergen is not a useful diagnostic test for inhalant allergy, and so-called neutralization of allergy via sublingual drops of allergen has not been substantiated. In vitro cytotoxic leukocyte test has not been documented as a useful laboratory test in controlled studies and is not recommended.

Laboratory confirmation of the presence of IgE antibodies to specific allergens such as dust mites, pollens, or animals is very helpful in establishing a specific allergic diagnosis, especially if the history of exposure to a specific allergen is not clear-cut. It may be necessary to test for specific allergens to convince the family and patient of an allergic diagnosis and to reinforce the importance of environmental control.
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Although skin testing might be performed in any child at any age, children less than 1 year of age may not mount a positive reaction. Often, the child who has seasonal respiratory allergy will not manifest a positive test until after two seasons of exposure. Clinicians should use allergens for skin testing selectively and employ only common allergens of potential clinical importance. The most useful allergens for which to test in the child who has perennial inhalant allergy are house dust mites (Dermatophygoides), animal danders, and fungi (molds) (Table 2). Allergens important in the diagnosis of seasonal allergic rhinitis are weeds, grasses, and tree pollens. These allergens vary not only by season of year but by geographic distribution. Therefore, allergens used for skin testing must be individualized and should be selected on the basis of prevalence in the local area and the home and school environment.

IgE antibody can be tested via two methods: in vivo skin testing and in vitro serum testing (Table 3). Their advantages and disadvantages are outlined in Table 4. For most patients, skin tests that are performed properly offer the best available method for detecting the presence of allergen-specific IgE. The prick, also called the puncture or epicutaneous skin test, is preferred; scratch testing has been abandoned as too traumatic. If prick tests are negative and allergy is highly suspect, then intradermal testing, which is more sensitive, may be employed. Skin tests are both 10% to 20% more sensitive and less expensive on a per test basis than are in vitro serum tests.

DIAGNOSTIC TESTS
Many pediatricians believe in the need for a screening test for allergy. Blood eosinophilia and total serum IgE levels have been proposed as screening tests, but they have relatively low sensitivity and should be used selectively (Table 3). The nasal secretions or sputum of patients who have a respiratory allergy contain increased numbers of eosinophils, which forms the basis of a useful nonspecific test, although not one that will identify any specific allergen etiology. Eosinophilia may not be present in patients who have not been exposed to allergens recently or who have a superimposed upper respiratory tract infection. Both systemic and inhaled steroids can reduce eosinophilia
in secretions significantly; antihistamines have no direct effect on eosinophils.
Why do people get Allergies
The usefulness of nasal eosinophilia as a diagnostic test depends in large part on the technique used to obtain the specimens to prepare the slides for examination. Patients should expel nasal secretions onto wax paper or parafilm; secretions then are spread on a microscope slide, stained, and eosinophils counted under a microscope. It is difficult to quantify nasal eosinophilia accurately, although a finding of more than 3% eosinophils on stained smear of expelled nasal or bronchial secretions is considered increased. Because cotton or nylon nasal swabs trap secretions, they are not recommended for collecting secretions, except in the young child who will not or cannot expel secretions by blowing the nose. Peripheral blood eosinophilia is observed in allergic asthma but less commonly in allergic rhinitis. Blood eosinophilia is more frequent in atopic dermatitis and other conditions, such as parasite infection.

Total serum IgE is elevated in about 60% of patients who have allergic asthma but only in 30% of those who have allergic rhinitis.
Unfortunately, commercial laboratories have promoted tests of total serum IgE excessively, but its usefulness in screening for allergy is limited to positive tests only because more than 60% of patients who have nasal allergy will have normal levels of total serum IgE.