General Medical Information

Definitive Testing for Atopy

Richard L. Mabry

Proof that upper respiratory allergy is present necessitates finding allergen-specific IgE in the patient. This can be done by means of intranasal challenge with a known allergen and measurement of increased airway resistance with rhinomanometry or assay of the level of mediator substances in nasal secretions. However, such testing is not practical in clinical practice. The first useful allergy test consisted of introducing the suspected allergen under the skin and allowing it to react with IgE bound to mast cells. The resulting wheal and flare reaction confirmed the presence of allergen-specific atopy. Early skin tests were conducted by means of abrading or scratching the skin and introducing antigens. Because such scratch tests were found to be neither sensitive nor reproducible, they were soon replaced by a prick test. In this test, the skin is pricked through a drop of antigenic extract with a specially designed needle, and approximately 3 µL of extract is introduced into the skin. The wheal and flare reaction is compared with results of negative (diluent) and positive (histamine) controls. The results are expressed as 0 to 4+. Because several tests can be performed easily at one sitting with relatively little discomfort, prick testing is popular for screening.

The prick test is not sensitive enough to show low degrees of atopy. Intradermal tests are more sensitive and reproducible than prick tests, although slightly more painful and much more time-consuming. In the intradermal test, small amounts of antigen (0.01 to 0.05 mL) are injected intradermally. Measurement of the wheal that forms provides a quantitative dimension not found in prick testing.

Another variation of the intradermal test is skin end-point titration (often called intradermal dilutional testing) in which, beginning with a weak and anticipated nonreacting dilution, progressively stronger antigen concentrations are injected intradermally until negative results are obtained at the highest concentration tested or until the point at which progressive positive whealing occurs (end point of titration) is determined. The end point marks the concentration at which immunotherapy can safely be initiated. This bioassay of sensitivity for each antigen allows initiation of immunotherapy at much stronger antigen concentrations than with the conventional empiric method of progressing from very weak dilutions for all antigens.

In intradermal dilutional testing, the end point is defined by the first wheal that initiates progressive positive whealing. Intradermal injection of approximately 0.01 mL of any liquid forms a wheal approximately 4 mm in diameter, which rapidly enlarges by means of physical spreading to 5 mm. If no further enlargement occurs within 10 to 15 minutes, the wheal is considered nonreacting. The wheal is considered positive if it enlarges at least 2 mm beyond a negative wheal—to 7 mm or more in diameter. Application of the next stronger antigen concentration should yield a wheal at least 2 mm larger than the preceding wheal—9 mm or more in diameter. The concentration that produces the 7-mm wheal represents the end-point dilution, whereas the 9-mm wheal is a confirming wheal.

Variants in positive whealing can occur. For example, positive wheals can grow more than 2 mm, such as wheals of 5, 8, and 11 mm. A plateau can occur in which there are two positive wheals before a confirming wheal is noticed, such as 5, 7, 7, and 9 mm. The second 7-mm wheal is considered the end point because it initiates progressive whealing. In some cases, a very large wheal, called a flash response, can follow negative wheals, such as 5, 5, and 13 mm. In such cases, usually due to cross-reacting foods, such as recent watermelon ingestion by a patient tested for ragweed, retesting after 24 hours establishes a clear-cut end point.

Skin tests are the benchmark for confirming the existence of IgE-mediated inhalant allergy. However, there are disadvantages to skin testing, including discomfort, the risk of anaphylactic reaction, and the existence of factors affect skin test results. Thus other testing methods have been developed.

Direct measurement of allergen-specific IgE in patient serum has been possible since the mid 1960s. In these in vitro tests, patient serum is allowed to react with known antigens contained on a matrix of varying sorts. After washing to remove unreacted components, the residual allergen-specific IgE complexes on the matrix are incubated with anti-IgE and a marker. Another washing removes all but the final “sandwich” of antigen–IgE–anti-IgE–marker. The amount of marker present indicates the amount of allergen-specific IgE in the serum. If the marker is a radioactive substance, the test is called a radioallergosorbent test (RAST). Other markers such as fluorescing agents are used in enzyme-linked immunosorbent assay (ELISA) tests. Results usually are expressed as classes. Higher values represent the presence of greater amounts of IgE. Modification of the original RAST scoring system by Nalebuff and Fadal has made the test more sensitive and correlates RAST scores with sensitivity levels obtained by means of intradermal dilutional testing.

In vitro IgE measurements are considered more specific but less sensitive than skin tests. In vitro tests are easier on the patient, which undergoes one venipuncture rather than many skin pricks, and carry no risk of systemic anaphylaxis. In vitro testing takes 1 (ELISA) to 3 (RAST) days, unlike skin tests, which take less than 1 hour. Although intradermal dilutional testing is performed with a bioassay and the same extracts used in making treatment mixes, treatment packages based on in vitro results must be checked initially with a limited skin challenge (vial test) before immunotherapy is begun.

Dipstick tests for allergy are based on ELISA technology. Preselected antigens on a dipstick or in a tube system are sequentially incubated with patient serum, anti-IgE, and a colorimetric marker. These semiquantitative tests are useful for screening; however, immunotherapy should not be based on the results of these screens without confirmatory quantitative in vitro or skin tests. The clinician must also consider whether these tests are more cost-effective or efficient than a properly performed quantitative in vitro assay for a limited number of antigens.

Because the United States contains an estimated 1,358 species of grass, 1,775 species of weeds, and 650 species of trees, the selection of antigens seems to be a formidable task. However, species cross-reactivity and a knowledge of antigens most likely to have clinical importance in a given region allow testing for only a few representative grasses, weeds, and trees. For example, either timothy or rye grass cross-reacts well with most other common grasses except Bermuda grass and Bahia grass. Because Bahia grass grows only in limited areas of the United States, testing with Bermuda and either timothy or rye grass suffices in the diagnosis and management of most grass allergies. Likewise, weed cross-reactivity, such as short ragweed with giant and western ragweed or pigweed with careless weed, allows limited testing and treatment. Cross-reactivity among trees is less marked, and these allergies must be treated individually, depending on local prevalence.

Adding the most important molds plus house dust mite and relevant animal danders brings the number of inhalant antigens in the basic screening test to about 15. Others can be added if positive results are obtained at initial skin or in vitro testing, but if test results for the screening panel are negative, the likelihood of marked inhalant allergy to other antigens is less than 5%.