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Human basophils are important tools for studying immediate-type hypersensitivity reactions since they release a variety of mediators (e.g., histamine, leukotriene C4, IL-4 and IL-13) following allergen triggering. Several diagnostic tools have been introduced that measure either leukotriene production or the upregulation of surface markers (CD63 and CD203c) from these cells after antigen stimulation. However, a broad variability in basophil activity exists between different basophil donors and different antigens within one donor. This manifests itself in terms of their reactivity (maximum secretory response), based on the intracellular signaling of the basophils studied, and in terms of their sensitivity. The latter is governed by the number of IgE receptors per basophil, the ratio of antigen-specific IgE to total IgE, and by the number of cell surface antigen-specific IgE molecules for half-maximal responses, termed ‘intrinsic sensitivity’. These variables give rise to shifts in the dose-response curves which, in a diagnostic setting where only a single antigen concentration is employed, may produce false-negative data. Thus, in order to meaningfully utilize the current basophil activation tests for diagnostic purposes, each allergen should be pre-evaluated separately in order to determine a suitable stimulation range. Additionally, anti-IgE or anti-FcΕRIα antibodies should serve as positive controls, bearing in mind that 10–20% of basophil donors are not responsive to IgE-mediated stimulation. Diagnostic studies using CD63 or CD203c in hymenoptera, food and drug allergy are critically discussed. Basophil-based tests are indicated for allergy testing in selected cases but should only be performed by experienced laboratories.
Human basophils are important tools for studying immediate-type hypersensitivity reactions since they release a variety of mediators (e.g., histamine, leukotriene C4, IL-4 and IL-13) following allergen triggering. Several diagnostic tools have been introduced that measure either leukotriene production or the upregulation of surface markers (CD63 and CD203c) from these cells after antigen stimulation. However, a broad variability in basophil activity exists between different basophil donors and different antigens within one donor. This manifests itself in terms of their reactivity (maximum secretory response), based on the intracellular signaling of the basophils studied, and in terms of their sensitivity. The latter is governed by the number of IgE receptors per basophil, the ratio of antigen-specific IgE to total IgE, and by the number of cell surface antigen-specific IgE molecules for half-maximal responses, termed ‘intrinsic sensitivity’. These variables give rise to shifts in the dose-response curves which, in a diagnostic setting where only a single antigen concentration is employed, may produce false-negative data. Thus, in order to meaningfully utilize the current basophil activation tests for diagnostic purposes, each allergen should be pre-evaluated separately in order to determine a suitable stimulation range. Additionally, anti-IgE or anti-FcΕRIα antibodies should serve as positive controls, bearing in mind that 10–20% of basophil donors are not responsive to IgE-mediated stimulation. Diagnostic studies using CD63 or CD203c in hymenoptera, food and drug allergy are critically discussed. Basophil-based tests are indicated for allergy testing in selected cases but should only be performed by experienced laboratories.
Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1 . In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2 . Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain.Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3 .In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4 , whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1 .The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT. Video LinkThe video component of this article can be found at https://www.jove.com/video/3263/ Protocol 1. Preparation of drug conjugates 1. Dissolve 10 mg DF in 1 ml dH 2 O in a 1.5 ml reaction tube. In the further proceeding use 95 μl of this DF solution. To conjugate PP to HSA dissolve 30.4 mg PP derivative (304 g/mol) in 500 μl 0.3 M sodium hydroxide (NaOH). 2. Add 430.6 μl dH 2 O and 100 μl 0.5 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 6.5 to 95 μl of dissolved DF. Add 33.1 μl dH 2 O and 140 μl of 2 M MES buffer pH 2.9 to the PP sample. 3. Add 57.5 μl of a freshly prepared N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) stock solution dissolved in dH 2 O with a concentration of 100 mg/ml to the DF sample, or 10 μl EDC solution to the PP sample. Vortex the samples for 1 minute. 4. Add 16.9 μl of HSA solution with a concentration of 118...
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