Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Abstract

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...

Chlorpheniramine-induced anaphylaxis diagnosed by basophil activation test

“…The findings should be further evaluated, and the immunogenic or haptenic structure should be clarified. However, there are recent reports which assessed the basophil activation by protein bound drugs [ 13 14 ]. In this regard, we suggest that the conjugation with albumin might be a possible option when considering BAT for chlorpheniramine.…”

The Performance of Flowcytometry-Assisted Basophil Activation Tests in the Diagnosis of Drug-Induced Immediate- Type Hypersensitivity Reactions

“…The activation of the basophils involves complex immunological mechanisms, with intracellular changes followed by the release of the mediators in the systemic circulation. CD63 is a transmembrane protein which is anchored in the basophil membrane and is exposed on the cellular surface upon activation [20,57,58]. The principle of BAT is to quantify, after the basophils come in contact with the allergen, the upregulation of certain activation markers such as CD63 on the cellular surface by staining with fluorescent, specific monoclonal antibodies (Figure 1, adapted from [59]).…”

Emerging Role of Human Basophil Biology in Health and Disease

“…Delineation of molecules which can identify basophils and determine their activation state with high sensitivity and specificity has led to use of an in vitro assay, termed the basophil activation test (BAT), with which to investigate the potential role of basophils in disease states [55, 82–•87]. The BAT is a microfluoremetry-based assay that can be performed on peripheral blood.…”

“…Anaphylaxis is clinically characterized by cardiovascular, cutaneous, respiratory, and gastrointestinal manifestations consequent to basophil and mast cell degranulation, and is usually IgE-dependent [1, 3, 11, 16, 33, •49, 52, 55, 82– •84]. Although specific triggers of anaphylaxis in a given patient may be elusive at the time of presentation, the most common defined causes include allergy to foods, drugs, and insect stings and bites [52, 82–88], as well as non-IgE-mediated inducers of basophil or mast cell degranulation, such as exercise, physical factors, opiates, and the anaphylatoxin C5a that is generated by immune complex activation of the complement cascade [62, 83, 85].…”

“…Although specific triggers of anaphylaxis in a given patient may be elusive at the time of presentation, the most common defined causes include allergy to foods, drugs, and insect stings and bites [52, 82–88], as well as non-IgE-mediated inducers of basophil or mast cell degranulation, such as exercise, physical factors, opiates, and the anaphylatoxin C5a that is generated by immune complex activation of the complement cascade [62, 83, 85]. Efforts to determine the relative roles of basophils and mast cells in an anaphylactic episode have included measurements of histamine, tryptase, and prostaglandin D2 (PGD2), as well as BAT [55, 82–•87]. Elevated tryptase and PGD2 levels suggest mast cell involvement and a positive BAT suggests basophil involvement [16, 33].…”