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Keywords:

  • anaphylaxis;
  • CD63;
  • flow cytometry;
  • methylene blue;
  • patent blue

A 20-year-old man was referred because of an anaphylactic reaction with angio-edema, bronchospasm with cyanosis and profound hypotension (systolic pressure 120 mmHg to immeasurable) during anaesthesia for combined excision of a dermal cyst in the neck and a multifistulated sacral cyst. History revealed no previous allergies. Revision of his anaesthetic report disclosed that the reaction had started 30 min after induction with propofol, sufentanyl and cisatracurium, but within minutes after fistulography with patent blue (Blue patente V®, Guerbet, Aulnay, France).

Laboratory analysis showed a normal blood count, complement profile and protease inhibitor concentrations. Total immunoglobulin E (IgE) was 11 kU/l and IgE for latex, suxamethonium and ethylene oxide were negative (<0.35 kUa/l; Immuno-CAP FEIA method; Pharmacia Diagnostics, Brussels, Belgium). Skin tests included latex (Stallergenes, Genval, Belgium), chlorhexidine-digluconate, serial dilutions of five muscle relaxants: suxamethonium (Myoplegine®), pancuronium (Pavulon®), rocuronium (Esmeron®), atracurium (Tracrium®) and cisatracurium (Nimbex®), the analgesic sufentanyl (Sufenta®), the anaesthetic propofol (Diprivan®), and the dyes patent and methylene blue. Except for patent blue (Table 1) skin tests were negative.

Table 1.  Skin tests and basophil activation tests. Results in patient and control individuals
VariablePatientControl 1Control 2Control 3Normal
  1. ND, not done; IgE, immunoglobulin E.

  2. *Results are shown as wheal/flare size in millimetres read after 15 min.

  3. †Results are shown as percentage CD63-positive basophils.

Skin prick test*
 Patent blue 10−23/7NDNDND<3 mm
 Patent blue 10−15/30NDNDND<3 mm
 Methylene blue 10−3 up to neat solution0/0NDNDND<3 mm
Intradermal test*
 Methylene blue 10−4 up to neat solution0/0NDNDND<5 mm
Basophil activation test†
 Spontaneous CD63 expression2222<10
 Anti-IgE94879489>25
 Patent blue (μg/ml)
  5 × 10−54052<10
  5 × 10−352042<10
  5 × 10−186060<10
  585042<10
 Methylene blue (μg/ml)
  5 × 10−41052<10
  5 × 10−31051<10
  5 × 10−14021<10
  54011<10

Flow cytometric quantification (FACSCalibur, BD, Immunocytometry Systems, San Jose, CA) of activated basophils was performed using phycoerythrin (PE) anti-CD123, peridinin chlorophyll protein (PerCP) anti-human leucocyte antigen (HLA) DR and fluorescein isothiocyanate (FITC) anti-CD63 conjugated antibodies (provided by BD, Biosciences, Erembodegem, Belgium). Basophil activation implied, a negative control, a positive control (anti-IgE), suxamethonium, pancuronium, rocuronium, atracurium and cisatracurium, and a serial dilution of the dyes patent and methylene blue in the patient and patent blue in controls. Basophil activation with muscle relaxants induced almost no increased CD63 expression (<4%). Results of basophil activation with the dyes are summarized in Table 1.

Patent blue is a dye with ubiquitous application in textile and paper industry, agriculture and cosmetics. Patent blue is also employed in medical settings. Beside its former use as antibacterial and antifungal agent, it has long been applied for lymphography. Today, it is the dye of choice for intraoperative sentinel lymph node mapping for breast cancer and melanoma. Hypersensitivity reactions towards patent blue have been reported since 1966 (1). Most patients have shown reactions to the intralymphatic administration of the dye. As in our patient, anaphylaxis may result from alternative routes of administration. Reactions occurred at various doses and are frequently characterized by cutaneous and mucosal symptoms such as pruritus, rash, urticaria, and angio-oedema. However, potentially life-threatening reactions with bronchospasm, hypotension and shock have been described (2–4). Symptoms may be delayed in onset and tend to progress. Currently, skin tests constitute the only confirmatory procedure for patent blue anaphylaxis.

Upon activation, basophils up-regulate the expression of certain membrane markers that can be detected by flow cytometry. Flow cytometry-assisted allergy diagnosis has been proven reliable for the diagnosis of different IgE-mediated allergies including inhalant allergens, food, latex, Hymenoptera venom and drugs (review: 5). One could argue that the technique does not allow to determine whether an allergic reaction was due to the bridging of allergen-specific membrane-bound IgE rather than complement activation, short-sensitizing IgG antibodies or even nonimmunological direct mediator release. This should, however, not be considered as a drawback of the assay. The technique might even offer a confirmatory test in cases where no allergens for specific IgE quantification are available (6). In our patient, flow cytometric analysis completely paralleled skin test reactivity. Both tests not only identified patent blue as the offending compound but also designated methylene blue as a potential safe alternative dye.

Authors would like to thank Ms Christel Mertens for her valuable technical assistance.

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