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.