Allergy skin tests, Techniques, and interpretation

Introduction The clinical evaluation of a patient with a suspected allergic disease begins with a detailed history, physical exam and ancillary tests (such as PFT, Xray etc.) intended to define and characterize the patients disease state. Allergy testing helps define triggers of allergic disease. These tests are intended to detect Type 1 ( IgE mediated hypersensitivity) sensitization. Allergy tests, strictly speaking, are not tests for the diagnosis of allergic disease; they simply report the presence of detectable amounts of allergen specific IgE that might in part be responsible for triggering disease symptoms. Specific IgE can be found in patients with allergic disease as well as in about 15% of asymptomatic normal individuals. Allergy testing employs carefully prepared extracts of pollens, fungi, animal products, food, latex etc. Extracts are complex mixtures of allergenic proteins and other substances. Each extract may contain several allergens. For successful testing of a patient, an extract must contain all relevant major and minor allergens. Skin testing Several skin test methods are available. These include patch testing, or the direct introduction of antigen into the skin epicutaneously or intracutaneously. The epicutaneous methods in general use are prick testing and puncture testing. Patch testing Patch test has been used for 100 yrs to diagnose contact allergies. Allergens are applied to the intact skin, under an occlusive dressing. In most cases 1 or 2 days of exposure is used. Studies have shown that protein and glycoprotein antigens as large as 30,000 Daltons can penetrate intact skin. Patch test typically detect delayed allergic reactions due to either the late phase of type 1 immediate hypersensitivity reactions or to type 4 cell mediated hypersensitivity. Advantages include 1. Only method to confirm that a specific contact is causing sensitization 2. solid antigens such as fabrics can be tested 3. not painful disadvantages include 1. less reproducible typical sensitivity ranges from 60 to 70% and specificity 70 to 80% 2. difficult to differentiate irritative reactions from true allergic reactions 3. Generally safe but may cause flares of atopic dermatitis and locally severe skin reactions

Prick testing First described by Lewis and Grant in 1926, and popularized in the 1970s by Pepys. In this method a drop of extract is placed on the skin. A sharp needle 25 gauge is introduced into the skin at an angle, through the antigen droplet and the most superficial layers of the skin ( about 1 mm deep ) are lifted up with no downward pressure on the skin, introducing a minute amount of extract. Must not induce any bleeding. This is the technique of choice for dermatographic patients. A variety of testing devices is available. Because of the possibility of false positive reactions when tests are placed closely together, prick test should be separated by about 2 cm. Fresh prick devices should be used for each antigen to prevent cross contamination. The greater the experience level of the testing personnel, the lower the test variability, so personnel training and monitoring is vital. Blotting away antigen instead of leaving the droplet alone does not influence the test result. Puncture testing Here a drop of extract is placed on the skin and the testing device is placed perpendicular to the skin. The device is placed on the skin with enough downward pressure to form a small indentation in the skin. Because puncture methods to some extent involve downward pressure on the skin, they are less appropriate for dermatographic individuals. However they are thought to be easier to learn and use. Some devices can be dipped in the extract before skin application. Intracutaneous skin testing Mantoux is credited with inventing intradermal or intracutaneous skin testing in 1908. His technique was used in 1911 by Robert Cooke for allergy skin test. Testing is performed with a disposable tuberculin syringe and a small-gauge needle. A small amount ( about 0.02 ml - range 0.01 to 0.05ml) of dilute extract is injected into the superficial layers of the skin to produce a 2 to 3 mm diameter superficial bleb in the skin. The syringe is placed at a 45 degree angle to the skin. Penetration into the sub epidermal capillary bed of the skin should be avoided. Many of the clinicians who employ intradermal test for inhalant allergy testing use a 50 to 100 fold dilution of the stock extract when an epicutaneous test with the stock extract is negative or equivocal, yet clinical suspicion of allergic sensitization is high.

Although this approach does increase the clinical sensitivity for skin testing, it does so with a loss of clinical specificity. Other disadvantages include it is more difficult and time consuming, more painful for the patient and irritant reactions occur commonly. Safety of intradermal tests There is no fundamental difference in safety between intradermal and epicutaneous tests that is not explainable by the amount of absorbed antigen. Fatalities from intradermal skin testing have been reported, both due to use of potent allergens and due to injection of excessive amounts of allergen. As a consequence many allergist recommend screening prick test prior to doing intradermal test. As a general rule the starting dose of intradermal extract solutions in patients with preceding negative prick test should range between 100 to 1000 fold dilutions of the extract used for prick/puncture test. Particular care should be taken in patients treated with B blocking agents, ACEI, or monoamine oxidase inhibitors, which may increase the risk of systemic reactions. Although systemic reactions have been observed no fatalities have been reported in epicutaneous skin testing. Skin End Point Titration This is a quantitative modification of intradermal testing that tests multiple antigen dilutions to establish the minimum amount of antigen required to produce a positive skin test. An end point is determined by sequential intradermal injection of constant volumes of successively stronger antigen dilutions until eventually a skin wheal forms that is clearly more reactive than the negative control. This first positive wheal is then confirmed by injecting one additional, more highly concentrated antigen dilution and determining that the stronger dilution produces a wheal that is even larger than the first reactive wheal. It has been found experimentally that skin wheals in the 4 mm to 15mm size range lie within the linear portion of the dose response curve, where wheal size and antigen dose can be correlated. Currently skin end point titration is used for 1. determining stability of allergenic extracts 2. evaluation of sensitivity to standardized allergenic extracts 3. to determine relative potency of allergenic extracts 4. yield a safe starting dose for immunotherapy generally treatment can be initiated at doses of about 5 to 20 times greater than the SET dose Disadvantages of SET includes increase testing time and complexity making it too difficult for general clinical use.

Comparison between epicutaneous and intradermal test Epicutaneous test are thought to be less sensitive and less reproducible, but more specific than intradermal test. Skin prick/puncture test are recommended as the primary test for the diagnosis of IgE mediated allergic diseases. Control solutions The usual positive control is histamine phosphate used at a concentration of 2.7mg/ml. Mast cell secretagogues such as codeine phosphate ( 9% solution ) may also be used. The negative controls are the diluents used to preserve the allergen extracts. Any reaction at the negative control test site will make interpretation of the allergen sites much more difficult. Measurement of skin test reactions The immediate skin test induces a response that reaches a peak in 8 to 10 minutes for histamine and 15 to 20 minutes for allergens. When reading skin test, both the largest and smallest diameter of the wheal are measured, usually at right angles to each other. Both diameters are summed and divided by 2. Criteria of positivity in epicutaneous skin testing, reactions generally regarded as indicative of clinical allergy are usually over 3 mm in wheal diameter and over 10 mm in flare diameter. Another criterion is a ratio of the size of the test induced by the allergen to the size of that induced by the positive control. Factors affecting skin test 1. Area of the body the back as a whole is more reactive than the forearm. The mid and upper back are more reactive than the lower back. The ulnar side of the arm is more reactive than the radial, the anticubital fossa is the most reactive portion of the forearm. Tests should not be placed in areas 5 cm from the wrist or 3 cm from the anticubital fossa. 2. Age skin test wheals increase in size from infancy( after 3 months ) to adulthood and then decline after the age of 50. 3. Gender no difference 4. Race whealing capacity is grater in darkly pigmented skin than light skin 5. Seasonal variation the skin sensitivity increases after the pollen season and then declines until the next season. 6. Pathologic conditions Eczema diminishes skin reactivity to histamine, patients with chronic renal failure have diminished skin reactivity and patient with peripheral nerve abnormalities also have diminished skin reactivity 7. Drugs - drugs that have an inhibitory effect include H1 antagonist ie antihistamines, Ketotifen, tricyclic antidepressants, phenothazines, long term systemic corticosteroid therapy, topical dermal cortocisteroids.

Drugs that have no effect include H2 antagonist, less than one week of oral steroids, inhaled cromolyn and nedocromil. Drugs that increase the effect include B blockers and ACEI. Diagnostic value of skin test With inhalant allergens, skin test represent the primary and most effective diagnostic method. A positive skin test with a history suggestive of clinical sensitivity strongly incriminates the allergen as the cause of the disease. Interpretation of the skin tests that do not correlate with the clinical history is more difficult. Epicutaneous skin test are generally considered to be the most convenient and least expensive screening method for detecting allergic reactions in most patients. In vitro immunoassays for specific IgE IgE is a molecule present in the serum in nanogram amounts. It was first discovered in 1966 by Johansson in Sweden and Ishizaka in the US. One year later the first invitro assay for the measurement of serum IgE was developed. The assay for measuring serum specific IgE was called the radioallergosorbent test (RAST ). The first RAST assay available was the Phadebas RAST. The lower sensitivity of this test led to the modified RAST test developed by Nalebuff and Fadal in 1979. Currently the Pharmacia CAP system, a fully automated assay is the most commonly used assay for measuring specific IgE in the US and Europe. A typical modern immunoassay uses well characterized allergenic source materials bound to a solid phase matrix. This incubates with patient serum. Following washing, an enzyme labeled anti-human IgE antibody is added. After a second incubation and washing, enzyme substrate is added, response is measured, and results are calculated and reported. Extracts should be well characterized to verify that all major and minor allergens are present in the extract, and that all bind to the matrix. Solid phase materials of plastic, cellulose, or agarose are most commonly used for the immunosorbent matrix. All modern methods report semiquantitative results using a class system often 7 classes with increasing numbers reflecting greater amounts of specific IgE. Modern methods can also report quantitative results of IgE.

Skin testing vs Immunoassays for specific IgE In the US, allergy immunology clinicians utilize skin testing as the primary method for detection of specific IgE. IgE immunoassays are used, as an alternative when skin testing is for some reason is not practical or possible. These include 1. clinical suspicion for allergic sensitization is high but skin testing are negative 2. allergenic substance is not available as a licensed extract for skin testing eg latex 3. Pt taking a medication that interferes with skin testing but cannot be stopped 4. Pts with severe asthma 5. Positive skin test controls are negative 6. Very young children and the elderly 7. Extreme severe dermographism 8. Extensive skin disease 9. Increased risk for systemic reactions eg asthma or hx of anaphylaxis 10. Pregnancy Results of conservatively performed and interpreted skin testing; done under optimal conditions, usually agree with those of modern specific IgE immunoassays. When results are discrepant, the skin testing is usually positive and the immunoassay is negative. This is thought to result as a limitation of the immunoassay rather than a problem with skin testing. There is a sensitivity of about 85 to 95% when immunoassay is compared to epicutaneous skin testing.