Overview of in vitro allergy tests

INTRODUCTION — Most tests for "allergy" are actually tests for allergic sensitization or the presence of allergen-specific immunoglobulin E (IgE). The majority of patients who experience symptoms upon exposure to an allergen have demonstrable allergen-specific IgE that recognizes that allergen, making these tests essential tools in the diagnosis of allergic disorders. This topic provides an overview of in vitro tests for IgE-mediated allergic disease. Skin testing for allergic disease is reviewed separately. (See "Overview of skin testing for IgE-mediated allergic disease".)

Laboratory testing in cases of suspected anaphylaxis is reviewed in greater detail separately. (See "Laboratory tests to support the clinical diagnosis of anaphylaxis".)

IMMUNOASSAYS FOR ALLERGEN-SPECIFIC IgE — Immunoassays for immunoglobulin E (IgE) specific to an allergen of interest are widely used in the diagnosis of allergic disease. Immunoassays are based upon interactions between antigens and antigen-specific antibodies [1,2]. In allergic disease, the relevant antigens are usually proteins derived from other living organisms (plants, animals, fungi, insects, micro-organisms). Immunoassays are often incorrectly referred to collectively as "radioallergosorbent tests" ("RAST") because radioactive tests were the earliest immunoassays to be used extensively. The term "immunoassay" is more appropriate.

Immunoassays are available for:

●Foods (see "Diagnostic evaluation of IgE-mediated food allergy", section on 'In vitro testing')

●Insect venoms (see "Diagnosis of Hymenoptera venom allergy")

●Environmental allergens (eg, pollens, molds, animal allergens, dust mite, and cockroach allergens)

●Natural rubber latex (see "Latex allergy: Epidemiology, clinical manifestations, and diagnosis")

●A small number of beta-lactam drugs (see "Penicillin allergy: Immediate reactions")

●Some occupational allergens such as isocyanates and phthalic anhydride (see "Occupational rhinitis", section on 'Testing for allergen-specific IgE' and "Occupational asthma: Clinical features, evaluation, and diagnosis", section on 'Skin and immunologic testing')

Indications — The presence of allergen-specific IgE is correctly interpreted as evidence that the patient is sensitized to that allergen and may react upon exposure. Thus, this type of testing is indicated in general terms when the clinician is assessing the likelihood that a patient's reactions or symptoms were due to exposure to a specific allergen.

In vitro tests are occasionally indicated to confirm negative skin tests. As an example, a patient who experienced a systemic reaction following a Hymenoptera sting and had a negative skin test should have in vitro IgE testing done as well to assure that the skin was not falsely-negative, as this allergy is potentially life-threatening. (See "Diagnosis of Hymenoptera venom allergy".)

Advantages relative to skin testing — In most situations, skin testing for IgE-mediated allergy is preferred over in vitro testing because skin testing is more rapidly obtained, less expensive, and more sensitive [3]. However, in vitro testing has certain advantages over skin testing:

●In vitro testing poses no risk to the patient of an allergic reaction. In vitro testing may be preferable in older adults with cardiovascular disease, patients with suspected sensitivities to allergens associated with severe anaphylactic reactions (such as latex), and patients with histories of severe reactions to minute amounts of the allergen. The risk of an allergic reaction in response to skin testing in such patients, although small, may not be acceptable. (See "Overview of skin testing for IgE-mediated allergic disease", section on 'Safety'.)

●In vitro testing is not affected by medications the patient may be taking, with the possible exception of omalizumab (see next bullet). Patients unable to discontinue antihistamines, some antidepressants, or other medications that may confound the results of skin testing are good candidates for in vitro testing. In vitro tests may also be preferred in those unable to discontinue medications that may inhibit the management of, or physiologic response to, anaphylaxis (ie, beta-blockers and angiotensin-converting enzyme [ACE] inhibitors). (See "Overview of skin testing for IgE-mediated allergic disease", section on 'Medications that should be discontinued'.)

●Omalizumab, a monoclonal antibody to IgE which binds to the Fc portion of the IgE molecule, causes both total IgE and allergen-specific IgE to rise [4]. Omalizumab can interfere with the performance of many immunoassays, although the ImmunoCAP system remains accurate [5]. (See "Anti-IgE therapy", section on 'Other changes with therapy'.)

●It is not reliant upon skin integrity or affected by skin disease:

•The skin of infants less than 12 months old may not yet fully reflect their allergic sensitivities. In contrast, immunoassays are valid for infants as young as six weeks of age and can be performed on capillary blood samples.

•Individuals who have experienced an anaphylactic reaction may have falsely-negative skin test results for several weeks after the event. In vitro testing is not known to be affected and can be performed in the postanaphylactic period if skin testing cannot be reasonably postponed.

•In vitro testing may be better for patients with certain skin conditions, such as severe and widespread atopic dermatitis or dermographism (ie, a condition in which raised welts form upon firmly stroking the skin and skin testing with diluent alone can produce false-positive wheals).

●In vitro testing can be more convenient for the patient because it only requires submitting a blood sample.

●In vitro testing can be a good alternative to skin testing when increased risk of airborne disease is present, such as during pandemics, as skin testing requires significant direct patient contact time.

●It can be superior to skin testing in certain clinical settings. Evolving data suggest, at least for some foods, that the level of specific IgE as measured by one specific commercial system, Phadia ImmunoCAP, may be more predictive than skin testing for diagnosing true clinical reactivity upon ingestion. The studies that demonstrated this were performed in children, and generalizability to adults has not been confirmed. (See "Diagnostic evaluation of IgE-mediated food allergy".)

Technical issues

Types — There are many methodologic variations of immunoassays that use different methods for detection of antigen-antibody complexes. The quality of the different detection systems is comparable:

●Enzyme-linked immunosorbent assays (ELISA): ELISAs make use of antibodies or antigens linked to enzymes (figure 1). A solution containing one reactant is placed in a well in a plastic dish, because some plastics will tightly bind many types of macromolecules. Subsequently, the plate is washed, and a tagged reagent is added; this reagent is linked to an enzyme. After washing again, the amount of antigen-antibody binding is measured based on the output of an enzymatic reaction. This is a direct binding assay. The labeled component may be either the antigen or the antibody.  

When the substrate of the enzyme is added, the reaction generates a colored product. Variations on the basic ELISA technique include fluorescent enzyme immunoassays (FEIA) and chemiluminescence immunoassays, which also make use of antibodies linked to enzymes, although when the substrate of the enzyme is added, the reaction generates a fluorescent or chemiluminescent product.

●Radioallergosorbent tests (RAST): RASTs involve antibodies that are coupled to radioactive tags [6]. This method is seldom used today and, as mentioned previously, the term RAST, when applied to immunoassays in general, is an anachronism.

Immunoassays can also be solid-phase or liquid-phase:

●Solid-phase immunoassays are more commonly used and make use of allergens (the antigen) bound to some form of matrix, such as a plastic plate, disc, bead, or other substrate, which is called an "immunosorbent" or "allergosorbent." Each system has its unique attributes. The type of matrix used also influences assay performance. Paper discs have the lowest assay performance, and microcellulose gels perform somewhat better. The technique of solid-phase ELISA is described and illustrated in the figure (figure 2). The allergen is bound to a matrix. The patient's serum is added, which contains antibodies of various classes (immunoglobulin [Ig]E, IgG, IgM, IgA). The patient's antibodies serve as the primary antibodies. These antibodies bind to the allergen if they recognize epitopes on the allergenic protein, and different classes of antibody may bind. Any unbound components of the serum are washed away, and then bound IgE is singled out from other types of bound immunoglobulins using anti-IgE antibodies (the secondary antibodies). The allergen:IgE complexes are detected by a variety of methods, as described previously.

●Liquid-phase immunoassays are performed with solutions of antigens and antibodies. Liquid-phase binding to paramagnetic particles reduces stoichiometric and conformational issues that can arise with binding of an allergen to a solid-phase substrate.

Immunoassays are both qualitative and quantitative. The specific IgE molecules from the patient's serum bind to the antigen in direct proportion to their concentration in the serum, allowing the quantity of specific IgE to be estimated with reference to a standard curve or by reference to a negative and positive control. The best documented and validated systems are those reporting quantitative results in kUA/L for IgE directed against specific allergens (or kIU/L for total IgE measurements), using calibrators traceable to the World Health Organization (WHO) International Reference Preparation for human IgE. Testing systems that report values in these units are preferable to those reporting results in other units.

Some immunoassays will be reported in nanograms of IgE per mL. The conversion is:

Competitive binding technique — Competitive binding (or inhibition) immunoassays are another variation on the basic immunoassay [7]. In this technique, known quantities of well-characterized antigen (allergen) and antibody (allergen-specific IgE) are mixed together. Then, a sample of unknown reagent, either allergen or antibody, is added, which competes with the known components. This technique has several applications:

●To determine the amount of allergen (or antibody) in an uncharacterized sample – As an example, IgE specific to a certain dust mite allergen can be fixed to a matrix, and then a solution of labeled, purified dust mite allergen is added. To this, a test solution containing unknown amounts of allergen is added. The two solutions of allergen compete for the binding sites on the IgE. The unbound allergen is then washed away, and the amount of label detected is used to quantitate the amount of allergen in the test solution.

●To determine the extent of cross-reactivity between different allergens (or antibodies) – As an example, IgE specific to a certain dust mite allergen can be fixed to a matrix, and then a solution of labeled, purified dust mite allergen is added. To this, a test solution containing unknown amounts of allergen from a different type of mite (eg, storage mite) is added. The two allergen solutions compete for binding to the IgE. If the allergens from the two species are sufficiently similar, then binding of the labeled allergen will be inhibited.

Accuracy — The sensitivity and specificity of immunoassays vary with the system being used and the quality of the allergen. Overall, sensitivity ranges from 60 to 95 percent and specificity from 30 to 95 percent [8-10]. Occasionally, patients who react to an allergen may not have any allergen-specific IgE that is detectable with routine testing [11,12]. In comparison, skin tests (prick/puncture method) generally have high sensitivity and specificity (>85 percent) when standardized inhalant extracts with high potency are used. (See "Overview of skin testing for IgE-mediated allergic disease" and "Overview of skin testing for IgE-mediated allergic disease", section on 'Prick/puncture method'.)

The quality of the allergen and the manner in which it is bound to the immobilizing matrix are the most critical aspects of solid-phase immunoassays. Systems that make use of well-characterized and standardized allergens, with a high level of quality control and quality assurance, provide the most reliable clinical results:

●Values of more than 90 percent sensitivity, specificity, and predictive values have been obtained with pollens of common grasses and trees, dust mites, and cat allergens.

●Very high positive-predictive accuracy in children can be achieved for some of the major food allergens with the Phadia ImmunoCAP system [3,13-15]. The use of this in vitro test in the diagnosis of food allergy is discussed in detail separately. (See "Diagnostic evaluation of IgE-mediated food allergy", section on 'Immunoassays'.)

●More problematic allergens include those from venoms, other foods, weed pollens, latex, drugs, dogs, and molds [16,17].

Concordance among different immunoassay systems is reported to be approximately 75 to 90 percent for well-characterized allergens [8-10,18,19]. Generally, <15 percent intra-assay variability in the test results is considered acceptable. When discordance is noted, it is largely due to differences in the allergens bound on the solid-phase matrix of the various systems, as allergen extract composition may differ between companies. Of note, more variable results may be obtained when using different commercial systems to measure allergen-specific IgE to foods and less common inhalants.

Tests that have been validated according to the Clinical Laboratory Standards Institute (formerly called the National Committee for Clinical Laboratory Standards [NCCLS]) should be used whenever possible [20].

Interpretation

Positive tests — A positive test is defined by the performing laboratory. The presence of IgE to a specific allergen demonstrates that a patient is "sensitized" to that allergen and may have a clinical reaction to it. The definitive diagnosis of an IgE-mediated allergy requires both sensitization and a history of allergic signs and symptoms on exposure to that allergen. Sensitization alone is not sufficient to diagnose an allergy, because individuals can be sensitized to an allergen without having clinical symptoms upon exposure. The clinical response of a sensitized individual to the suspect allergen is best understood as a dynamic physiologic event with multiple variables, of which the presence of allergen-specific IgE is just one. Thus, allergy tests must be interpreted in the context of the patient's specific clinical history, and the diagnosis of an allergic disorder cannot be based solely on a laboratory result. This is true for in vitro assays, as well as for skin testing.

The likelihood that a positive result will correlate with clinical reactivity is influenced by the degree of positivity, the allergen in question, and the patient's clinical history. It is not uncommon to find allergen-specific IgE in the serum of people who do not report allergic symptoms. As examples, venom- and food-specific IgE have been reported in up to 25 and 60 percent of the general population, respectively [21-24]. In food allergy, greater serum levels of allergen-specific IgE better predict anaphylactic reaction as determined by oral food challenge [25].

A typical threshold for positivity is >0.35 kIU/L. The ImmunoCAP has been shown to maintain linearity of results below this threshold and can accurately detect minute quantities of allergen-specific IgE; results can be reported down to 0.15 kIU/L [19]. Patients with higher levels of antibody are more likely to experience symptoms upon exposure to the allergen than patients with lower levels, although strongly positive tests do not necessarily predict that a severe reaction (eg, anaphylaxis) is more likely than a milder type of IgE-mediated reaction (eg, urticaria) [21]. Furthermore, for most allergens, threshold levels above which most patients will react clinically have not been determined. A notable exception to the last statement is the utility of Phadia ImmunoCAP results for certain foods, specifically in pediatric patients, as mentioned previously (see 'Advantages relative to skin testing' above). For these foods, the percentage of children with a consistent clinical history who will react to challenge has been determined over a range of food-specific IgE levels. For adults, however, and for most allergens, the patient's reactivity to the allergen must be determined by the clinical history or by a supervised challenge procedure. (See "Diagnostic evaluation of IgE-mediated food allergy", section on 'Immunoassays'.)

Results reported by class — Positive results of immunoassays are often graded into classes (typically I to IV or I to VI) based upon arbitrary divisions of a reference curve (table 1). There is some limited clinical utility to these divisions, as levels of allergen-specific IgE ≥class III (ie, ≥3.5 kIU/L) have been shown to be more consistently related to symptoms upon exposure, whereas asymptomatic sensitization may occur in some individuals having <class III levels of allergen-specific IgE [13,14]. Levels of class II or greater are generally required as proof of sensitization that is clinically relevant when enrolling subjects in research protocols.

A specific example in which IgE immunoassays appear to augment skin testing results is found in the selection of patients for immunotherapy. Most modern clinical trials of allergen immunotherapy stipulate that subjects must have a class II (ie, >0.7 kU/L) or greater result to the allergen being studied, in addition to a positive skin test to that allergen, to be included in the study population. This is based upon the observation that this group of sensitized patients was most likely to benefit in previous trials [26]. One study of sublingual tablet immunotherapy demonstrated that among skin test-positive patients, only those who also had an allergen-specific IgE >0.1 kU/L benefitted from treatment [27]. This suggests that combining allergen-specific IgE testing with skin testing could be used to select patients who are most likely to respond to allergen immunotherapy.

The following are examples of how the class of a result can be useful:

●If the result is markedly positive (eg, a class VI result), the history suggests a past reaction to the allergen, and the allergen is well-characterized, then the diagnosis of an allergy can usually be made without further evaluation.

●If the result is weakly positive (eg, a class I result), then further evaluation is usually needed. Skin testing and possible challenge may be indicated, based upon the patient's clinical history. Most immunotherapy trials require that subjects have both demonstrable allergen-specific IgE and skin test positivity.

Negative tests — A negative immunoassay result in the setting of a strongly suggestive history does not exclude allergy. In this situation, a skin prick test should be considered (if not contraindicated for one of the reasons reviewed previously). (See 'Advantages relative to skin testing' above.)

False-positives — False-positive results of allergen-specific IgE can theoretically occur in patients with extremely elevated total IgE levels. As an example, patients with hyperimmunoglobulin E syndrome can have a markedly elevated total IgE level (approximately 50,000 kIU/L) and test positive for IgE to allergens to which there is no history of clinical reactivity. This situation is believed to be rare, although the level of total IgE that could have this effect is not well-defined and may vary depending upon the immunoassay system. Failure to thoroughly wash away unbound allergen-specific IgE in performance of the assay can lead to false positives, as the tracer anti IgE will bind to any residual IgE not bound to antigen. (See "Autosomal dominant hyperimmunoglobulin E syndrome".)

TOTAL IgE LEVELS — Patients with allergic conditions, such as asthma, allergic rhinitis, or atopic dermatitis, often have higher serum levels of total immunoglobulin (Ig)E compared with the general population. Although an elevated total IgE may indicate that the patient has an atopic condition, it provides no information about which condition or to what allergens the patient is sensitive. Furthermore, because there is a large degree of overlap between IgE levels in people with and without allergic disease, the utility of total IgE in diagnosing these common conditions is limited [28]. (See "The relationship between IgE and allergic disease" and "The biology of IgE".)

In contrast, specific clinical situations in which the measurement of total IgE is helpful include the following:

●Evaluation of specific disorders that are associated with very elevated levels of total IgE, including parasitic infections, allergic bronchopulmonary aspergillosis, and a small number of rare immune disorders (eg, hyperimmunoglobulin E syndrome or Wiskott-Aldrich syndrome) and malignancies (table 2) [29,30]. (See "Clinical manifestations and diagnosis of allergic bronchopulmonary aspergillosis" and "Autosomal dominant hyperimmunoglobulin E syndrome" and "Atopic dermatitis (eczema): Pathogenesis, clinical manifestations, and diagnosis" and "Wiskott-Aldrich syndrome".)

●Evaluation of patients with moderate-to-severe allergic asthma to determine eligibility for treatment with anti-IgE therapy (ie, omalizumab). Total IgE may also serve as a biomarker for type 2 inflammation in asthma when considering use of monoclonal antibodies that counter the type 2 inflammatory process in asthma. (See "Anti-IgE therapy", section on 'Pretreatment testing'.)

Technique — Total IgE is usually measured by enzyme-linked immunosorbent assay (ELISA) using specific anti-IgE monoclonal antibodies. Laboratory techniques used for the assessment of immunoglobulin concentration for other classes of immunoglobulin (ie, IgG, IgA, IgM), such as radial immunodiffusion, cannot be used to measure IgE concentrations because serum IgE levels are normally too low.

TESTS FOR ANAPHYLAXIS — Anaphylaxis results from massive activation of mast cells and basophils. Serum tryptase and plasma histamine released from these cells may be detectable for minutes to hours after episodes of anaphylaxis and can be used to support the diagnosis of anaphylaxis. Elevations in these mediators are short-lived, however, and blood or other fluids must be collected as soon as possible after the event. Metabolites of mast cell mediators, such as N-methyl histamine and prostaglandin compounds, can be assayed in 24-hour urine samples collected shortly after a clinical event. The use of laboratory tests to support the diagnosis of anaphylaxis is reviewed in detail separately. (See "Laboratory tests to support the clinical diagnosis of anaphylaxis".)

TESTS USED MOSTLY IN RESEARCH — Other tests that are largely used in research protocols include immunoblotting, several tests of basophil activation and function, tests for levels of eosinophil-derived mediators, and microarray testing [3,31-35]. In the United States, these tests are not approved for routine clinical use, although some are commercially available. Their use is more widespread in some European countries.

Immunoblotting — The immunoblot technique, which is more expensive than other assays and not quantitative, is predominantly a research tool. This method is used to detect antibodies directed against multiple proteins within an allergenic substance.

A mixture of allergen proteins are separated according to molecular weight by gel electrophoresis (proteins must be within a range of molecular weights that penetrate readily into gels). The separated proteins are then transferred onto a nitrocellulose membrane. In commercial diagnostic kits, nitrocellulose strips containing already separated proteins are provided. The nitrocellulose membrane is incubated with a test serum and then washed to remove unbound serum components. Labeled anti-human immunoglobulin E (IgE) antibody is used to detect IgE from the test serum that has bound to individual allergenic proteins. The results are compared with reference sera.

Component-resolved diagnostics — Component-resolved diagnostics (CRD) are testing methods that allow for more specific identification of the allergen to which a patient's specific IgE reacts. This is a potentially important advance because standard IgE immunoassays may detect IgE against clinically irrelevant allergens, yielding positive results in patients who are not actually reactive to the allergen in question. CRD involves microarray testing using recombinant allergens and antigenic components of major allergens and nanobiologic techniques to assess the precise antigenic epitopes to which a patient's IgE binds.

CRDs are becoming commercially available to diagnose sensitization to some insect venoms and foods and as a means of predicting cross-reactivity between food and pollen allergens. The more detailed information can inform diagnosis and prognosis. Specifically, CRD is expected to assist in identifying patients who have a higher risk of anaphylaxis because they have IgE against major "anaphylactogenic" allergens versus patients with IgE directed against allergens that generally do not elicit anaphylaxis. In the case of plant foods, these techniques may help to clarify which food-allergic patients are likely to experience "oral allergy syndrome" and which are at risk for more serious systemic reactions to foods. Such discrimination is not possible using only allergen skin testing or conventional allergen-specific IgE assays.

In the example of peanut allergy, patients sensitized to pollen-related components, such as the peanut allergen Ara h 8 (which is related to a major birch pollen allergen Bet v 1), usually experience no or very mild oral symptoms, whereas those who are sensitized to more stable components, such as seed storage proteins (eg, Ara h 2), are more likely to experience systemic reactions to peanut. One system uses a high-throughput, bead-based epitope assay to analyze IgE reactivity to discrete food allergen epitopes and requires only a few microliters of plasma. This test has gained US Food and Drug Administration approval for detecting peanut allergy by identifying IgE to multiple epitopes from Ara h1, Ara h2, and Ara h3 peanut proteins [36]. CRDs for foods in addition to peanut include tree nuts, wheat, vegetables, fruits, milk, and hen's egg. (See "Diagnostic evaluation of IgE-mediated food allergy", section on 'Component testing' and "Component testing for pollen-related, plant-derived food allergies", section on 'Peanut' and "Component testing for pollen-related, plant-derived food allergies", section on 'Allergen-specific use/interpretation'.)

CRD has also been explored to assess timothy grass sensitization profiles by region. The highest mean IgE levels and largest proportion of subjects with positivity to Phl p 1 and Phl p 5 were found in regions where timothy grass is most prevalent. In addition, among patients treated with timothy grass sublingual immunotherapy (SLIT) tablets, the data suggest trends toward higher efficacy and an increased incidence of having adverse events in subjects with detectable and higher pretreatment Phl p 1, Phl p 5, and Phl p 6 IgE [37]. Finally, CRDs of vespid venoms are also becoming available to assist in diagnostic assessment of risk of severe allergy based upon the component to which IgE is directed [38].

A commercially available example of CRD is the ImmunoCAP ISAC (Thermo Fisher Scientific), which allows for detection of IgE against more than 100 individual components (allergens) derived from more than 50 allergen sources. This test allows for detection of IgE to Bet v 1, Bet v 2, and Bet v 4, which are all derived from birch pollen. This can be applied as a multiplex diagnostic tool and/or the results can be searched for the presence of IgE against a single component/allergen (eg, Bet v 2). This tool can also be used to detect IgE against cross-reactive carbohydrate determinants (CCDs). This is unique to in vitro methods, as no in vivo test for detection of CCD-specific IgE is available. Tests that provide CRD with less than 10 allergen components are also commercially available in some countries. Euroimmun immunoblots may serve as an example (figure 3). The test is restricted to several components of birch and timothy grass pollens, including major allergens such as Bet v 1, Phl p 1, and Phl p 5 and minor cross-reactive allergens such as profilins Bet v 2/Phl p 12 and polcalcins Bet v 4/Phl p 7. This can be useful in evaluation of patients with pollen allergy who are being assessed for allergen immunotherapy to optimize the constitution of their immunotherapy vaccines [39].

Basophil tests — Basophils and mast cells both express the high affinity receptor for IgE on the cell surface and are activated when this IgE encounters sufficient specific allergen. Mast cells reside in tissues, while basophils circulate in the vasculature, making them more accessible to collection and study. Several techniques have been developed to examine basophil responses to allergens. Unfortunately, basophils are prone to nonspecific activation by a variety of factors and are difficult to transport and manipulate. Thus, these tests are considered investigational.

Basophil histamine release — The basophil histamine release test measures the release of histamine from human peripheral blood basophils incubated with allergen [3]. When well-characterized allergens are used, this test is similar to skin testing in accuracy [31]. The test relies on living cells and thus requires that blood samples are submitted and tested within 24 hours. Only a few laboratories perform the test. Basophil histamine release is not standardized and is considered an investigative tool for drug, food, and environmental allergens. While not used commonly in the US, basophil activation tests are often employed in Europe and should be noted as a rapidly advancing ex vivo method of assessing for cellular activation by allergen resulting in increased expression of select CD markers CD203c and CD63 [40].

Others — Other tests of basophil function following incubation with allergen include release of leukotriene C₄ (LTC₄) and measurement of the level of activation via expression of surface proteins (such as CD63 or CD203c) by flow cytometry [3,32,41]. Although promising, these tests are not as useful as skin testing and are not approved for diagnostic use in the United States. Use of these basophil activation tests in the diagnosis of food allergy is discussed separately. (See "Future diagnostic tools for food allergy", section on 'Basophil activation testing'.)

Eosinophil cationic protein levels — Eosinophils release a broad range of biologically active mediators upon activation. Eosinophil cationic protein (ECP) is an eosinophil-specific mediator that can be measured in bodily fluids to estimate the extent of eosinophil activation. Many studies have demonstrated that serum ECP levels correlate with severity of allergic diseases such as asthma and increase during parasitic infections [33]. It rises with increased airway inflammation (eg, after allergen exposure) and falls during remissions, either spontaneous or associated with therapy [34,35]. Thus, ECP is a marker of eosinophilic inflammation, although it provides no information about the presence of IgE-mediated allergy.

Measurement of ECP is commercially available, although this test requires further characterization before it can be recommended for routine clinical use.

UNVALIDATED TESTS — Patients may present with allergy tests that have been performed by alternative health care providers, including allergen-specific immunoglobulin G (IgG) and IgG4 tests. These are most often performed for the evaluation of food allergy. These methods typically yield multiple positive results and almost always represent normal immune responses to food. They do not predict true food hypersensitivity.

There are validated uses of IgG and IgG4 tests:

●The presence of allergen-specific IgG4 and IgG "blocking antibodies" is measured in studies of allergen immunotherapy. The formation of these antibodies appears to be one of several immunologic changes that are associated with effective immunotherapy, although their exact functional role requires further study. (See "Allergen immunotherapy for allergic disease: Therapeutic mechanisms", section on 'IgG'.)

●Venom-specific IgG correlates with the adequacy of immunotherapy in patients with venom allergy. This is discussed in more detail separately. (See "Hymenoptera venom immunotherapy: Technical issues, protocols, adverse effects, and monitoring", section on 'Venom-specific IgG'.)

Other types of testing for allergy that are not supported by scientific studies include provocation/neutralization tests, kinesiology, cytotoxic tests, and electrodermal testing. These tests can pose risk to patients both by potentially overlooking true allergies and by leading to inappropriate diagnosis and advice, including dietary restrictions [42].

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Allergy diagnostic testing".)

SUMMARY

●In vitro allergy testing, in general terms, is indicated when the clinician is assessing the likelihood that a patient's reactions or symptoms were due to exposure to a specific allergen. (See 'Indications' above.)

●Skin testing is usually preferred to in vitro testing for the diagnosis of allergic disease. However, in vitro testing has certain advantages: it poses no risk to the patient, is not affected by medications, and requires only a simple blood draw. In a few clinical situations, in vitro testing may be superior to skin testing. (See 'Advantages relative to skin testing' above.)

●Immunoassays, in various forms, are the most commonly used in vitro tests for immunoglobulin E (IgE)-mediated allergy. These tests detect allergen-specific IgE in a patient's serum by incubating the serum with the allergen in question, which has been absorbed on a solid-phase medium. The bound IgE is then detected with an anti-IgE antibody, which, in turn, has a label attached to permit detection. (See 'Immunoassays for allergen-specific IgE' above.)

●Testing for allergen-specific IgE, like all forms of allergy testing, must be interpreted in the context of the patient's specific clinical history. The presence of allergen-specific IgE is correctly interpreted as evidence that the patient is sensitized to that allergen and may react upon exposure. Actual reactivity must be determined by history or supervised challenge. (See 'Interpretation' above.)

●Serum levels of total IgE are of limited utility in the diagnosis of allergic diseases. An elevated total IgE level may indicate that the patient has an atopic condition, although it provides no information about which allergens the patient is sensitive to. Total IgE is used to determine the potential utility of anti-IgE therapy (omalizumab) in patients with allergic asthma. (See 'Total IgE levels' above.)

●Tests used largely in research include immunoblotting, basophil histamine or leukotriene release tests, basophil activation, and levels of eosinophil mediators. These tests are not standardized, are generally not superior to skin testing, and cannot be recommended for routine clinical use. (See 'Tests used mostly in research' above.)

●Allergen-specific immunoglobulin G (IgG) and IgG4 tests, which are believed to correlate with normal immunologic responses to foreign substances, are not useful in the diagnosis of allergy, with the possible exception of monitoring the response to certain forms of allergen immunotherapy. Unreliable testing methods include provocation/neutralization tests, kinesiology, cytotoxic tests, and electrodermal testing. (See 'Unvalidated tests' above.)

ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges Hendrik Nolte, MD, PhD, who contributed to an earlier version of this topic review.