In Vitro Basophil Activation Assays

In vivo basophil stimulation and release of the inflammatory compound, histamine, is a primary mediator of immediate-type hypersensitivity allergic reactions.48 Assay methods for measuring the release of histamine in vitro have been available for many years and have been implemented in several clinical studies with the promise of a rapid, specific, and sensitive test that can bridge between in vitro serum IgE tests and in vivo clinical testing.49 51 The mechanisms of mast cell and basophil activation have recently been reviewed by Knol.52 More recently, alternative methods for measuring basophil activation have been described, such as the measurement of sulfidoleukotriene release and allergen-induced expression of surface markers such as CD63.53,54 Many of the newest techniques for measuring basophil activation are flow-cytometric55 and tend to be used with latex and drug allergen compounds; however, the longest history of experimental use with food allergens remains the hista-mine release test.

Basophil histamine release tests (BHR tests) have shown success in clinical studies56 for measuring the biologically relevant potential for allergic reactions. A measure of basophil function may represent a measure of allergy sensitivity that is independent of that represented by levels of circulating IgE.57 If proven reliable, a BHR test for allergens could provide an evaluation of allergenicity without resorting to the practice of implementing in vivo tests such as SPT, DBPCFC, or bronchoprovocation.

The goal of in vitro BHR testing is to be able to predict allergy with basophil cells taken from the patient. Thus, the test is intended to directly measure the current IgE-mediated potential for reactivity to an allergen test material. There has been some success in evaluating environmental allergens,58,59 with specificity and sensitivity values ranging from 83% to 90% and 84% to 87%, respectively. Sainte-Laudy et al.57 showed that specificity and sensitivity for a BHR test can even reach 100% in the case of patients with hymenoptera allergies. The BHR test has also shown biological relevance to allergy when employed with food allergens, with sensitivity and specificity values ranging from 53% to 89% and 78% to 82%, respectively.60,61 More recently, the BHR test has shown promise in describing the prevalence of reactivity to individual food allergen proteins.62 The peanut allergens, Ara hi, h2, and h3, were tested with sera from 10 patients who were allergic to peanuts, and results indicated that the BHR could discriminate positive reactivity to the same allergens as compared to serum IgE immunoblotting.

There are a number of patient and assay specific considerations that should be taken into account when considering the BHR test for allergy studies.63 The test can be set up as either a direct or indirect assay using the patient's basophils. In the direct method, a patient's basophils are placed into an in vitro culture system and stimulated with allergens, as well as positive and negative controls. In the indirect method, a donor's basophils are stripped of endogenous surface IgE with lactic acid63 and repopulated with IgE from allergic patient sera containing allergen-specific IgE. The indirect method allows for more flexibility in sample handling since a patient's serum, rather than whole fresh blood, is transported. In principle, the two methods may be expected to give the same results since the assay depends on the direct antigen-specific binding of the patient's IgE.62 However, a lower sensitivity has been reported for the indirect method, requiring a level of specific IgE equal or greater than approximately (2.7 IU/ml) to obtain acceptable reactivity of passively sensitized basophils.64

Several performance aspects of an in vitro assay should be evaluated in designing a standard protocol for measuring reactivity of patient basophils. The priming condition of the basophil cells by interleukins (e.g., IL-3) can be important in determining basophil release of histamine, and thus it can affect results depending on its inclusion in the protocol.57 The source and preparation of allergens should also be taken into account when assessing assay performance since sensitivity to the test material may vary.61 Finally, criteria for a positive response in a BHR test are important to consider for each allergen tested so that the test clearly measures a biologically relevant level of released histamine. To construct a positive threshold value for each test allergen, patient-specific histamine release for a test allergen can be compared to a positive inducer of histamine release or the maximal release. Although the capacity of a BHR test to predict allergy remains unknown, the IBT Reference Laboratory (Lenexa, KS, and RefLab ApS (Copenhagen, Denmark, offer commercially available versions of a diagnostic BHR test with a limited selection of allergens. Buhlmann Laboratories (Basel, Switzerland) provides antigen-specific positive thresholds for a commercially available test, CASTâ„¢, which is an assay for the release of sulfidoleukotri-enes that may produce similar results to BHR tests.65

The allergen-induced basophil histamine release test, when run under standard conditions with appropriate controls, may represent an opportunity for measuring biologically relevant food allergen potential. However, clinical patient history and the association with diagnostic assay performance is an aspect of in vitro allergy testing for which there are very little data. It is known that with some allergens such as drugs, negative and positive predictive values change based on study patient inclusion crite-ria.66 Additionally, basophils are known to both spontaneously release histamine or to be anergic (unresponsive to stimulation) when used in a BHR test. High levels of spontaneous histamine release may produce poor correlations to clinical allergy if included in the assay protocol, whereas nonresponsiveness (anergy) would cause false negative results. With this in mind, it is important to consider criteria for which patient samples to include in the test protocol and to clearly define acceptance criteria for a valid test result. Similar to IgE-binding tests, validation of the assay in regard to the clinical situation may be required for each allergen as well as for different patient populations.39

Animal models and in vitro biomarker assays have not been established as standard methods that can predict clinical allergy in humans because immunogenic sensitization has yet to be completely understood in the context of human clinical allergy.67 Taken together, the results of these types of studies should be carefully considered when extrapolating to the human allergy condition.

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Allergic To Everything

Allergic To Everything

The human body And Todays chemical infested world. Here is a news flash You are not allergic to pollen, pet dander, or whatever it is that makes your body revolt Rather, your body just can not handle that one thing, what ever it is, anymore, due to the massive barrage of toxic chemicals you and everyone else are ingesting every single day.

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