Flow-assisted basophil activation test for sevoflurane hypersensitivity: A dose-finding in-vitro experimental study
Halogenated anaesthetic agents are broadly used worldwide. Sevoflurane is generally considered safe, with few reported cases of occupational non-immediate type reactions in anaesthetic staff and surgeons, and no documented sevoflurane immediate-type hypersensitivity before the case reported by Dahlem et al.1 Sevoflurane is not currently listed in the list of culprit allergens that induce perioperative anaphylaxis, which has an incidence ranging from 1/1250 to 1/18 600 per procedure.2
For each suspected intra-anaesthetic drug-induced immediate-type hypersensitivity reaction, the retrospective diagnosis aims to determine the culprit drug.3 This involves the conduction of skin tests, provocation tests, and in-vitro tests. All exposures should be identified and tested.3 This would lead to the necessity of testing for sevoflurane in each patient who experienced intra-anaesthetic anaphylaxis when sevoflurane exposure was encountered.
For halogenated agents, the conduction of in-vivo and in-vitro allergy diagnostic tests are not standardised. Allergological skin tests for sevoflurane are hampered by volatility and possible false positive irritative results, making in-vitro tests even more important. In this context, the availability of a safe and reliable in-vitro diagnostic test becomes even more important. There are no recommendations referring to optimal sevoflurane concentrations that are to be used in flow cytometric basophil activation tests (BAT).4 In-vitro cellular tests need to be validated for sevoflurane by using dose–response curves.
For sevoflurane, which is a volatile halogenated anaesthetic that is not easily miscible, technical factors might influence the results of the test. The methodology of BAT has not been described by Dahlem et al.1
We describe here an in-vitro experimental study to investigate the response of the basophils from patients who tolerated well sevoflurane under general anaesthesia, by performing BAT for sevoflurane diluted in dimethyl sulfoxide (DMSO).
Five patients with previous uneventful general anaesthesia with sevoflurane agreed to be involved in this study, which was approved by the Ethical Committee of the ‘Iuliu Haţieganu’ University of Pharmacy (no. 150/20.04.2016).
We performed BAT with Flow2CAST technique (Bühlmann Laboratories AG, Schönenbuch, Switzerland). Cell stimulation was done immediately after blood collection. We used stimulation buffer as negative control, and anti-FcεRI (a highly specific monoclonal antibody for the IgE receptor) and FMLP (the chemotactic peptide N-Formyl-Met-Leu) as positive controls. In the remaining five test tubes, 50 μl of sevoflurane solutions were added. The tested sevoflurane concentrations were: c1 (25% sevoflurane and 75% DMSO), c2 (33% sevoflurane and 66% DMSO), c3 (50% sevoflurane and 50% DMSO), c4 (66% sevoflurane and 33% DMSO) and c5 (75% sevoflurane and 25% DMSO). Subsequently, 20 μl staining reagent with anti-CCR3-PE (human chemokine receptor labelled with phycoerythrin) and anti-CD63-FITC (or Gp53, a glycoprotein expressed on activated basophils) were added in each tube. The up-regulation of CD63 marker on the basophils was measured using Cell Quest programme (FACSCalibur; Becton Dickinson, San Jose, California, USA Analyzer 2001). The result was expressed as the stimulation index, calculated as the percentage of activated basophils after stimulation with sevoflurane divided by the number of basophils in the negative control probe. Activated basophils less than 5%, together with a stimulation index less than 2, represent a negative result.
All five healthy controls responded to the positive controls. For sevoflurane in DMSO, the stimulation index varied between the five tested concentrations (Fig. 1). For all five healthy controls, the percentage of activated basophils after stimulation with sevoflurane in DMSO was below 5%, thus all results were negative. For increased concentrations of DMSO, we observed a reduction in the number of gated basophils for each individual control, from mean values of 657 basophils for c5 to 164 for c1, suggesting a cytotoxic effect of the solvent (Fig. 1).
Up to now, sevoflurane testing has not been performed on a routine basis in patients with intra-anaesthetic anaphylaxis.