Diagnostic allergy testing should be considered when a clinical scenario suggests an external, usually harmless, substance is causing pathology.
The term “allergy” indicates an abnormally hypersensitive immune reaction. Allergic conditions have a significant impact on health and health care. Allergies are the 6th leading cause of chronic illness in the US with an annual cost in excess of $18 billion. More than 50 million Americans suffer from allergies each year.  The pathophysiology of allergy in many other otolaryngologic, pulmonary, and dermatologic disorders has yet to be fully understood.
Examples of allergic triggers include aeroallergens, medications, food, insect stings, and chemicals (occupational exposure). Conditions may vary from chronic illnesses such as allergic rhinitis and allergic asthma to acute anaphylaxis from venom or medication.
Diagnostic allergy testing should be performed in the context of a history that suggests an allergic trigger. A positive test alone is not sufficient to diagnose an allergic condition. Positive allergy tests demonstrate sensitization but do not always indicate clinical reactivity. There may also be some scenarios in which the allergic condition is clear and further evaluation is not needed, such as seasonal pollen-induced allergic rhinitis controlled with cetirizine.
It is important to diagnose the allergic trigger when avoidance can minimize or prevent the allergic reaction. Pharmacologic treatment may vary depending on whether there is an allergic versus nonallergic trigger, such as rhinitis.
Type I immunoglobulin E (IgE)-mediated allergy testing is evaluated by measuring allergen-specific IgE. This can be done through skin testing (in vivo) testing or with serological tests (in vitro). Another option is a direct challenge. End-organ provocation of the nasal mucosa, bronchial mucosa, or conjunctivae is less commonly used in current clinical practice.
Food and medication challenges are more commonly performed because of the rate of high false-positive sensitization for food and need for medications. Any challenge should be performed in a medically supervised setting with treatment for anaphylaxis readily available.
Type IV delayed-type hypersensitivity allergy is assessed through patch testing.
Specific IgE Testing
Specific IgE testing can be done through skin testing or blood testing. Testing should be based on the clinical scenario. If aeroallergen sensitivity is suspected, a panel of indoor and outdoor allergens is indicated. Trees, grass, and weed pollen should reflect the local botanical flora. Indoors allergens should include dust mite species (Dermatophagoides pteronyssinus and Dermatophagoides farinae), cockroach, select molds, and animal dander (typically cat pelt and dog epithelium).
Reactivity persists throughout the year, even if the patient is not in his or her allergy season. Food, venom, and medication allergy evaluations should be based on a history suggestive of an IgE-mediated reaction. An example of a supportive history would be symptoms of rhinitis, conjunctivitis, urticaria, wheezing, or anaphylaxis within minutes to hours after exposure.
In Vivo (Skin) Testing
Skin testing is generally performed by allergy specialists. Prick/puncture testing remains one of the most common and popular methods for allergy testing. It is relatively easy to perform, is more sensitive than in vitro tests, and is cost effective in the clinical setting. It is an indirect measure of cutaneous mast cell reactivity due to the presence of specific IgE. Mast cells reside in the subepithelial layer of the skin and the respiratory, nasolacrimal, and gastrointestinal tracts. Of all these areas, the skin is the most accessible organ to test.
Skin testing detects allergen-specific IgE bound to mast cells. The allergen cross-links specific IgE bound on the mast cell. This causes degranulation of preformed mediators, including histamine and tryptase. Histamine release is the major mediator that results in a hive at the prick site and surrounding erythema, called a wheal and flare.
Skin testing is usually done on the forearm or back (see the image below).
The prick/puncture method involves a skin testing device pricked through a droplet of allergenic extract. Various skin test devices and extracts are commercially available. The wheal and flare is read in 15-20 minutes. It is measured in millimeters and compared with a positive control (histamine) wheal and flare and a negative control (usually glycerinated saline). A positive test is considered as a wheal equal to or larger to the histamine control (or greater than 3 mm). 
Skin testing can be performed at any age. Infants may have smaller positive tests, but the histamine is correlatively smaller. The sensitivity and specificity of skin testing is dependent on the allergens used. The accuracy of skin testing with commercial, standardized aeroallergen extracts exceeds 85% in terms of sensitivity and specificity.  Skin testing to food and mold extracts is less reliable. Food allergy testing has a very high false-positive rate of 50-60% but has a negative predictive accuracy of greater than 95%. [3, 4]
As there is a small risk of anaphylaxis, skin testing should not be performed on patients at risk for complications if they experience anaphylaxis. This includes pregnancy and unstable medical conditions, such as unstable asthma or reduced lung function, recent stroke, or recent cardiac event.
Oral and nasal antihistamines should be stopped 3-7 days before skin testing. Other medications with an antihistaminic effect may alter skin tests as well, including H2 receptor antagonists (cimetidine, ranitidine), tricyclic antidepressants, and antiemetics. Some medications may make the treatment of anaphylaxis less effective, such as angiotensin-converting enzyme inhibitors and beta-blockers. These medications should be discontinued prior to skin testing. False-positive results can occur with dermatographism or chronic urticaria. Patients with atopic dermatitis should be tested on unaffected skin.
Intradermal testing is useful for Hymenoptera (yellow jacket, wasp, and honey bee) venom anaphylaxis and penicillin allergy. For some aeroallergens, intradermals add little to diagnostic information. [5, 6] Intradermal testing is performed only after a negative prick skin test. Note that there is a higher risk of anaphylaxis. Intradermal testing is more sensitive than prick skin testing, but false-positive results are common due to irritant reactions or intracutaneous bleeding.
Intradermal testing typically involves injecting 0.01-0.02 mL of antigen into the dermis via a 27-gauge syringe to create a 2- to 3-mm intracutaneous bleb, similar to an intracutaneous tuberculosis test. The extracts are diluted to 100-1000 times less than the dilution used for skin tests. The wheal is measured after 10-20 minutes. A response is considered positive if the wheal is 7 mm or greater (see the image below).
In Vitro Testing
In vitro tests assess antigen-specific IgE by testing the patient’s serum. Advantages to this method include the use of a single venupuncture that is not affected by medications. In vitro testing can be performed on patients with affected skin, such as dermatographism or atopic dermatitis. It is also a safer option if the patient is at risk for anaphylaxis. However, these tests are expensive compared with skin testing.
In this method, immunoassays measure interactions between antigens and antigen-specific antibodies. Immunoassays are often referred to as radioallergosorbent (RAST) testing, but that term is outdated because radiation is rarely used today. Current methods include enzyme-linked immunosorbent assay (ELISA), which uses antibodies linked to enzymes, as well as fluorescent enzyme immunoassays (FEIA) and chemiluminescent immunoassays, which use fluorescent generation with an enzyme.
A solid-phase immunoassay has an allergen bound to a matrix. The patient’s serum is added and the antibodies bind to the allergen. All serotypes (IgG, IgM, IgA, and IgE) will bind if they recognize the allergen. A secondary anti-IgE antibody is used to identify if IgE is bound. The report is a quantitative value in kIUA/L or in arbitrary divisions into classes I-VI. Asymptomatic sensitization is common below class III (< 3.5 kIUA/L). 
The panel chosen should be based on the patient's clinical history, as with skin testing. The accuracy of immunoassays varies with the system being used and the quality of the allergen. There is good predictive value (>90%) for pollens of grass, trees, dust mites, and cats, whereas less accurate results may be obtained from venoms, weeds, latex, dogs, and molds. [7, 8] If results are equivocal, further evaluation can be done by means of skin testing and, if indicated, a challenge to the allergen.
Patch testing is used to determine the causative agent for chronic eczematous conditions contributing to a delayed-type hypersensitivity reaction. An example is allergic contact dermatitis to jewelry containing nickel. There also is a role for patch testing with food allergies in eosinophilic esophagitis and some drug allergies. [9, 10]
In patch testing, the allergen is placed on the upper back under an occlusive bandage and removed in 48 hours. The skin is reassessed at 72-96 hours for erythema, papules, and vesicles under the area of contact (see the image below). 
The most common patch techniques are the individual Finn chamber or the thin-layer rapid-use epicutaneous (TRUE) test. The TRUE test contains 26 preloaded ready-to-use testing strips with chemicals found in cosmetics, occupational settings, and topical medications. Other series with more allergens are available and specific variations exist based on the allergen in question.
Other Diagnostic Tests
Total IgE levels are often elevated in patients with allergic conditions (atopic dermatitis, allergic rhinitis, and asthma). Total IgE does not assist in further diagnosis of these conditions. Patients without allergic disease may have mildly elevated levels; therefore, total IgE rarely adds additional information. There are some specific conditions for which total IgE can be helpful. This includes hyper-IgE syndrome and other rare immunodeficiency disorders, allergic bronchopulmonary aspergillosis, and occasionally malignancies. Treatment for allergic asthma with omalizumab, an anti-IgE therapy, is guided by the patient’s total IgE levels.
Anaphylaxis results in massive activation of mast cells in the tissue and basophils in the blood. Release of their mediators is a potential way of diagnosing if anaphylaxis has occurred. This can be helpful in clinical scenarios such as interoperative hypotension and flushing with syncope.
Tryptase and histamine are measurable mediators; however, they are metabolized quickly and have a small window of elevation. A normal level does not rule out mediator release; however, an elevated test is helpful. Tryptase should be measured 60 minutes to 6 hours after the event.  Histamine levels are more transient, rising in 5-10 minutes and returning to baseline by 30-60 minutes.  It may take days to weeks to obtain the results; therefore, these levels are not useful for immediate diagnosis.
Basophil activation tests measure the release of histamine from blood basophils incubated with the allergen. These tests require living cells and must be run within 24 hours. This test is not standardized and is considered to be an investigative tool, but it shows promise for drug allergies. 
Measurements for allergen-specific IgG and IgG4 are often performed by alternative health practitioners in the context of food allergies. The immune system mounts responses to numerous epitopes, including pathogens and nonpathogens. It is normal to have specific IgG to food, so testing will yield multiple positive results.  This test does not predict food hypersensitivity. Other types of testing not supported by scientific studies include provocation/neutralization tests, kinesiology, cytotoxic tests, and electrodermal testing.  These diagnostics result in unnecessary testing and inappropriate advice.
Diagnostic testing for allergy can help to diagnose and treat allergic conditions. Considering the immune mechanism of the allergen and then testing appropriately is of the utmost importance to assure proper diagnoses. Results should be interpreted in the context of a supportive history.