In Vitro Tests

Allergic diseases are often associated with increased numbers of eosinophils circulating in the peripheral blood and invading the tissues and secretions of target organs. Eosinophilia, defined as the presence of >450 eosinophils/µL in peripheral blood, is the most common hematologic abnormality of allergic patients. Seasonal increases in the number of circulating eosinophils may be observed in sensitized patients after exposure to allergens such as tree, grass, and weed pollens. The number of circulating eosinophils can be suppressed by certain infections and systemic corticosteroids. In certain pathologic conditions, such as drug reactions and eosinophilic pneumonias, significantly increased numbers of eosinophils may be present in the target organ in the absence of peripheral blood eosinophilia. Increased numbers of eosinophils are observed in a wide variety of disorders in addition to allergy (Table 135-1) (Chapter 123).

Nasal and bronchial secretions are often examined for the presence of eosinophils. The presence of eosinophils in the sputum of asthmatic patients is classic. An increased number of eosinophils in a smear of nasal mucus stained with Hansel stain is a more sensitive indicator of nasal allergies than peripheral blood eosinophilia and aids in distinguishing allergic rhinitis from other causes of rhinitis. In young children, nasal eosinophilia is defined as the presence of >4% eosinophils in nasal mucus smears, whereas a finding of >10% eosinophils is required in adolescents and adults. Nasal mucus eosinophilia also has therapeutic implications, predicting a higher probability of responsiveness to topical nasal corticosteroid sprays.

An elevated immunoglobulin (Ig) E value is often found in the serum of allergic patients, because IgE is the primary antibody associated with allergic reactions. IgE values are measured in international units (IU), with 1 IU equal to 2.4 ng of IgE. Maternal IgE does not cross the placenta. Although the fetus is capable of producing IgE as early as the 11th wk of gestation, infants in developed countries produce little IgE in utero, owing to the lack of stimulation by allergens. Serum IgE levels gradually rise over the first years of life to peak in the teen years and decrease steadily thereafter. A variety of factors in addition to age, such as genetic influences, race, gender, certain diseases, and exposure to cigarette smoke and allergens, affects serum IgE levels. Serum IgE levels may increase twofold to fourfold in allergic patients during and immediately after the pollen season, and then gradually decline until the next pollen season. Comparison of total serum IgE levels among patients with allergic disease reveals that those with atopic dermatitis tend to have the highest levels, whereas patients with allergic asthma generally have higher levels than those with allergic rhinitis. Although average total serum IgE levels are higher in populations of allergic patients than in comparable populations without allergic disease, the overlap in levels is such that the diagnostic value of a total serum IgE level is poor. Approximately one half of patients with allergic disease have total serum IgE levels in the normal range. Total serum IgE measurement is indicated when the diagnosis of allergic bronchopulmonary aspergillosis is suspected; total serum IgE concentration >1,000 ng/mL is a criterion for diagnosis of this disorder (Chapter 229.1). Continued monitoring of the total serum IgE in patients with allergic bronchopulmonary aspergillosis is encouraged because serum IgE levels decrease with appropriate therapy and rise again during exacerbations of the disease. The total serum IgE value is also elevated in several nonallergic diseases (Table 135-2).

The presence of IgE specific for a particular allergen can be documented in vivo by skin testing or in vitro by the measurement of allergen-specific IgE (as-IgE) levels in the serum (Table 135-3). The first test for documenting the presence of as-IgE was called the radioallergosorbent test (RAST) because it used a radiolabeled anti-IgE antibody. The RAST has been replaced by an improved generation of as-IgE assays that use enzyme-conjugated rather than radiolabeled anti-IgE. These assays use solid-phase supports to which allergens of an individual allergen extract are bound. A small amount of the patient’s serum is incubated with the allergen-coated support, resulting in binding of the patient’s as-IgE to the allergens on the support. Next, the allergen-coated support to which the patient’s as-IgE is bound is incubated with enzyme conjugated antihuman-IgE that then binds to the patient’s as-IgE. Incubation of this complex with a fluorescent substrate of the conjugated enzyme results in the generation of fluorescence that is proportional to the amount of as-IgE in the serum sample. The amount of as-IgE in the serum sample is calculated by interpolation from a standard calibration curve and reported in arbitrary mass units (kilo-IU of allergen-specific antibody per unit volume of sample [kUa/L]).

Table 135-3 DETERMINATION OF SPECIFIC IMMUNOGLOBULIN E (IgE) BY SKIN TESTING VERSUS IN VITRO TESTING

* Skin testing may be the prick test or intradermal injection. Prick testing tends to be quicker, easier to perform and interpret, and more amenable to testing of infants.

^† Because skin tests are more sensitive, they are more reliable than allergen-specific IgE assays in confirming life-threatening anaphylactic conditions if maximal sensitivity is required, such as for penicillin or Hymenoptera hypersensitivity.

The primary advantages of these assays in comparison with allergen skin testing are their safety and that the results are not influenced by skin disease or medications. Overall, the results of these tests correlate well with those obtained by skin testing and provocation challenges. The as-IgE assays are not as sensitive as the skin test. In patients with histories of life-threatening reactions to foods, insect stings, drugs, or latex, skin testing is still required because of its higher sensitivity even if the as-IgE assay result is negative.

In Vivo Tests

Allergen skin testing is the primary in vivo procedure for the diagnosis of allergic disease. Mast cells with allergen-specific IgE antibodies attached to high-affinity receptors on their surfaces reside in the skin of allergic patients. The introduction of minute amounts of an allergen to which the patient is allergic into the skin results in cross linking by the allergen of allergen-specific IgE antibodies on the mast cell surface, thereby triggering mast cell activation. Once activated, these mast cells release a variety of preformed and newly generated mediators that act on surrounding tissues. Histamine is the mediator most responsible for the immediate wheal and flare reactions observed in skin testing. Examination of the site of a positive skin test result reveals a pruritic wheal surrounded by an area of erythema. The time course of these reactions is rapid in onset, reaching a peak within ≈20 min and usually resolving over the next 20-30 min. In some patients, however, a larger area of less distinctly demarcated edema on an erythematous base develops at the skin test site over the next 6-12 hr. This reaction, the late-phase response, usually resolves by 24 hr. Biopsy of the site of a late-phase response reveals the presence of an inflammatory infiltrate consisting of T cells, neutrophils, and eosinophils. These reactions are thought to be similar to the late-phase responses observed in other organs, such as the nose and lungs, after provocation challenges.

Skin testing in children is usually first performed using the prick/puncture technique. With this technique, a small drop of allergen is applied to the skin surface, and a tiny amount is introduced into the epidermis by lightly pricking or puncturing the skin through the drop of extract with a small needle. When the prick/puncture skin test result is negative and the history is suggestive, selective skin testing using the intradermal technique may be performed. This technique involves using a 26-gauge needle to inject 0.01-0.02 mL of a dilute allergen extract into the dermis of the arm. This technique is more sensitive than the prick/puncture technique, and the allergen extracts used are 1,000- to 100-fold less concentrated than extracts used for prick/puncture testing. Intradermal skin tests are not recommended for use with food allergens because of the risk of triggering anaphylaxis. Irritant rather than allergic reactions can occur with intradermal skin testing if higher concentrations of extracts, such as 1 : 100 weight : volume, are used. Although prick/puncture testing is less sensitive than intradermal skin testing, positive prick/puncture skin test results tend to correlate better with symptoms on natural exposure to the allergen.

Panels of skin tests that include the appropriate allergens for a given geographic area in addition to common indoor allergens are often applied; the number of skin tests performed should be individualized, with the allergens suggested by the history taken into account. A positive and negative control skin test, using histamine and saline, respectively, is performed with each set of skin tests. A negative control is necessary to ensure that the patient is not dermatographic and that reactions caused merely by applying pressure to overly sensitive skin are not interpreted as due to allergen sensitivity. A positive control is necessary to establish the presence of a cutaneous response to histamine. Medications with antihistaminic properties in addition to adrenergic agents such as ephedrine and epinephrine suppress skin test responses and should be avoided for appropriate intervals (≈3-10 days) before the performance of skin tests. Prolonged courses of systemic corticosteroids may suppress cutaneous reactivity by decreasing the number of tissue mast cells as well as their ability to release mediators.

Under certain circumstances, provocation testing is performed to examine the association between allergen exposure and the development of symptoms. Provocation challenges involving exposure of the skin, conjunctiva, nasal mucosa, oral mucosa, gastrointestinal tract, or lungs to allergens are performed in a variety of clinical and research settings. Bronchial provocation challenges are performed by having patients inhale increasingly concentrated solutions of nebulized allergen extracts and monitoring for airways obstruction by clinical observation and the performance of pulmonary function testing. Results of bronchial provocation challenges correlate well with other clinical data obtained by skin testing or in vitro testing. Although a large number of bronchial provocation challenges to allergens have been performed safely, the possibility of a severe reaction and the time, expense, and expertise required for the performance of these tests limit their performance to a research setting.

The bronchial provocation test most frequently performed is to methacholine, which causes potent bronchoconstriction of asthmatic but not of normal airways. Methacholine challenge testing is performed to document the presence and degree of bronchial hyperreactivity in a patient in whom asthma is suspected. After baseline spirometry values are obtained, increasing concentrations of nebulized methacholine are inhaled until a specified drop in lung function, such as a 20% decrease in FEV₁ (forced expiratory volume in the first second of expiration), occurs or the patient is able to tolerate the inhalation of a set concentration of methacholine, such as 25 mg/mL, without a significant decrease in lung function.

Oral food challenges are performed to determine whether a specific food causes symptoms or whether a suspected food can be added to the diet. Food challenges are performed for those foods incriminated by the history and results of skin tests and/or in vitro testing. These challenges may be performed in an open, single-blind, double-blind, or double-blind placebo-controlled fashion and involve the ingestion of gradually increasing amounts of the suspected food at set time intervals until the patient either experiences a reaction or tolerates a normal portion of the food openly. Because of the potential for significant allergic reactions, these challenges should be performed only in an appropriately equipped facility with personnel experienced in the performance of food challenges and the treatment of anaphylaxis, including cardiopulmonary resuscitation.