We would like to thank S. Chirumbolo for his comments on our proof-of-concept paper [1] on flow-assisted diagnosis of IgE-mediated opiate allergy and for providing us the opportunity to clarify some important issues about the basophil activation test (BAT) and HistaFlow® assays in the diagnosis of drug hypersensitivity reactions, including IgE-mediated allergies.

In his letter, the author raises doubts about the accuracy and reliability of BAT/HistaFlow tests in the diagnosis of alleged IgE-mediated allergies to opiates. As a matter of fact, the author particularly calls into question the gating strategy of the cells and states that histamine release by basophils in response to the opiate pholcodine could have resulted from an IgE-independent activation of the cells. First, it appears that our plots could have been misinterpreted. Actually, as displayed in the lower left quadrant of panel C of Figure 2, there are no cells that release histamine without appearance of CD63. Second, the finding of “low” responses to a positive control stimulation is not unusual, neither is the observation that allergens can trigger significant CD63 upregulation exceeding the response to the positive control antibody. Furthermore, in our opinion, activation of the basophils with anti-IgE (positive control) is only applied to exclude non-responsiveness in the BAT [2]. Third, the comment about anti-IgE as a characterizing antibody to identify basophils and its potential to activate basophils is inappropriate. In classical basophil activation assays, cells are exposed to anti-IgE for flow cytometric identification only after allergen stimulation has been stopped and activation of basophils by characterizing anti-IgE is further avoided by fixing and/or cooling the cells between allergen stimulation and addition of the conjugated antibody [3, 4]. Besides, we now focus on the lineage specific marker CD203c as an identification marker before and after activation [5]. In contrast to other frequently used basophil characterization markers like the eotaxin CC chemokine receptor 3 (CCR3) or IL-receptor CD123, these clusters are not lineage specific and are expressed on a variety of inflammatory cells associated with allergic responses such as lymphocytes and eosinophils and resident tissue cells such as airway epithelium [6]. Subsequently, additional markers such as CD3 or HLA-Dr should be applied to differentiate basophils from other confounding cells.

In the second part of his letter, the author argues that histamine release by the basophils could have resulted from an alternative, i.e. IgE-independent, activation pathway. Firstly, it has repeatedly and consistently been demonstrated that opiates, unlike their potency to induce non-specific histamine release from cutaneous mast cells, are not capable to trigger significant histamine release from basophils [7-9]. This issue has recently been comprehensively addressed by Baldo and Pham [10]. Secondly, in the absence of a response toward structurally almost similar compounds such as morphine and codeine, a non-specific release restricted to pholcodine is somewhat hard to explain. It seems theoretically justified that the differences in basophil responses rather result from a different substituted group on the molecule of morphine, codeine, and pholcodine. This hypothesis is endorsed by the negative provocation tests with codeine in our patients and additional BAT and/or HistaFlow experiments with opiates in exposed control individuals and so far no activation of the basophils with pholcodine has been observed (unpublished data).

Finally, the author argues about the discrepancy between the appearance of CD63 in the BAT and the titers of drug-specific IgE antibodies. However, to the best of our knowledge, there are no reports that have shown a correlation between titers of sIgE antibodies and basophil responses. Moreover, on several occasions we have demonstrated that the BAT allows discrimination between clinically relevant and irrelevant sIgE results [3, 11].

In conclusion, the BAT/Histaflow technique constitutes an asset to correctly document pholcodine allergy.

  • J. Leysen*

  • C. Bridts

  • D.G. Ebo

  • Faculty of Medicine and Health Science, Department of Immunology, Allergology and Rheumatology, University Antwerp and Antwerp University Hospital, Antwerpen, Belgium


  1. Top of page