3. When the patient is exposed to the allergen again it crosslinks the allergen-specific IgE antibodies on these cells.
4. If enough of the IgE antibodies are crosslinked with allergen, then there is a release of mast cell, basophil, and activated eosinophil contents; this release can result in a type I hypersensitivity reaction.
• Each in vitro allergy test uses basically the same technique:
1. Each test uses specific allergens (antigens) attached to a matrix; the patient’s serum is then added; if there is an IgE in the patient’s serum that is specific to the antigen in the matrix, it will bind; anti-IgE IgG with a marker is then added to determine if there is bound IgE present and to quantify the amount (Figure 7–1).
2. There are eight different in vitro tests currently approved by the FDA.
FIGURE 7–1. In vitro testing diagram. Test antigen and antigen matrix. Patient’s serum IgE bound to antigen. Anti-IgE IgG with label attached bound to patient’s IgE.
• RadioAllergoSorbent Test (RAST)
1. RAST was the first in vitro test available.
– Results interpreted in “Classes” from 0 to 4
– Threshold for a positive result was modified (ie, lowered), improving sensitivity of this test but also decreasing specificity
– Now called the modified RAST, or mRAST, with this change in threshold
2. Serum IgE quantification based on a radioactive tag
3. Based on a whole antigen (not subunits)
4. Results are reported as “Classes”
– Classes scaled 0 to 6 for mRAST
5. The higher the class the more IgE present
6. “Class” does NOT necessarily correlate with skin test results
7. Approximately 90% sensitive and specific for inhaled allergens
• Enzyme Linking Immunosorbent Assay (ELISA)
1. Uses a fluorescent tag instead of the radioactive tag used in RAST
2. Results correlate highly with RAST (r = 0.95)
• Component Resolved Diagnosis (CRD) or Component Resolved Testing (CRT)
1. The “next generation” of in vitro testing
2. Uses recombinant purified allergen proteins
3. Decreases risk of cross reactivity by using pure allergen
4. Can also identify IgE reactivity to different protein subunits within one allergen
– Useful in food allergy, specifically peanut allergy
– Multiple allergen subunits in the peanut: some confer severe reactivity and some confer peanut tolerance
– Helpful in predicting reactions to oral food challenge if a patient is sensitive to one subunit more than another
• Basophil Activation Test (BAT)
1. Based on drawing a blood sample and removing basophils by flow cytometry
2. Isolated basophils are exposed to an allergen and histamine release or activation of basophil surface markers is measured
3. Useful in a patient with strong clinical history but prior negative testing and challenge test is too risky (ie, venoms or nut allergies)
– For peanut allergy, has a 95% PPV and 98% NPV
General Information
• Not based on testing the patient’s serum but, rather, based on a reaction visualized on the skin and interpreted by the size of the “wheal” created
• A patient’s IgE antibodies are directly exposed with allergen through skin prick method or injection; if the patient has IgE antibodies to the allergen, there will be associated mast cell degranulation visible on the skin as a wheal.
Types of In Vivo Testing
• Skin Prick Testing (SPT)
1. SPT uses prepared allergens placed on the skin approximately 2 cm apart (to avoid confusion between allergens tested).
2. A drop of allergen is placed either directly on the skin and the skin is pricked through the allergen drop, or the lancet is dipped into an allergen well first and then applied to the skin.
3. There are multi-test lancets available in order to apply multiple allergens to the skin at once.
4. Lancets do not typically puncture deeper than 1 mm to avoid puncturing the dermis.
5. Skin reactivity is interpreted by the size of the wheal formed.
– Each wheal is measured by its diameter in millimeters (Figure 7–2).
– A positive wheal is one in which the diameter is 3 mm greater than the negative control (glycerin).
– If the wheal is oblong, the short and long diameters may be measured, added, and divided by two to give a mean diameter.
6. Wheals may sometimes have a “flare,” “pseudopod,” or “satellite”
– A flare refers to the erythema that extends from the wheal. This can make measuring the wheal challenging, in which case you can use a clear ruler to help blanch the skin and isolate the wheal for accurate measurement; measure the wheal and the flare separately.
– A pseudopod looks like a linear extension emanating from the edge of the wheal and is typically not measured, as its significance is unclear.
– A satellite is an area of induration adjacent to the wheal but not connected to it; the satellite is not included in measurement.
FIGURE 7–2. Demonstration of diameter measurement. Example of a wheal with a flare. Example of a wheal with satellites. Example of a wheal with pseudopods.
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