1Department of Anesthesiology, Emory University Hospital, 1364 Clifton Road, NE, Atlanta, GA 30322, USA. 2Department of Dermatology, Emory University Hospital, 1364 Clifton Road, NE, Atlanta, GA 30322, USA.*Corresponding author
Accepted for publication: July 28, 2000
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Abstract |
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Br J Anaesth 2000; 85: 8449
Keywords: neuromuscular block, rocuronium; neuromuscular block, cisatracurium; allergy
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Introduction |
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Methods |
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Each subject underwent intradermal skin testing (during the first visit only) with the following study medications (using 0.02 ml injections): rocuronium 108, 107, 106, 105, 104, 103, 102 M; cisatracurium besylate 108, 107, 106, 105, 104, 103, 102 M; 0.9% normal saline solution; atracurium besylate 104 M; and histamine 104 M. All study drugs were injected intradermally through a 28 gauge needle into the right and left forearms of each subject. In order to avoid confusion with the injections and the data collection during the study, the first study drug (rocuronium) was administered in all dilutions to the lateral part of the right forearm followed by the second study drug treatment (cisatracurium besylate) in all dilutions on the medial part of the same arm. The controlssaline (negative), atracurium besylate (positive) and histamine (positive)were administered on the left forearm. All solutions were freshly prepared and were preservative-free. After 1015 min, the sizes (diameter in mm) of the weals and flares were evaluated by an assessor who was blinded to the study drugs (the assessor was not present when the injections were made and did not know the order of the drugs injected). A weal was defined as a smooth, elevated area on the skin surface, often redder or paler than the surrounding skin. Positivity was shown by a pale pink weal with a diameter greater than 8 mm. A flare was defined by the red outermost zone of the weal reaction. The two largest diameters of the weals and flares were recorded and the diameters averaged. Punch biopsies from the drug-induced weals were taken during the first testing from six of the 30 subjects who had a positive response (weal
8 mm in diameter or
75% of the diameter of the histamine control weal). If a subject had more than one positive response to the study drug(s), the weal produced by the lowest concentration of the study drug(s) that induced a positive weal response was biopsied. Biopsies of the sites of saline control treatments were also taken from the same subjects. The physician performing the punch biopsies was blinded to the study drugs and the corresponding concentrations that gave positive responses.
A 3 mm punch biopsy specimen was taken after injecting lignocaine without preservative around the site to be biopsied using deep subcutaneous injection. The skin specimens were cut into 1.5 mm pieces and immediately placed in fixative solution for light microscopy, or immersed in Karnofskys fixative and processed for transmission electron microscopy. After processing, semi-thin sections were cut and stained with Giemsa for light microscopy. These were examined for degranulation and to select areas with mast cells for thin sectioning. Thin sections (70.0 nm) were cut and counterstained with lead citrate and uranyl acetate. An assessor who was blinded to the study medications examined these specimens under a Phillips 201 electron microscope. The presence of degranulation, the mast cell morphology, granule architecture and comments on the histology were recorded. Degranulation was recorded as 0, 1+, 2+, 3+ or 4+, where 0 represents normal mast cell morphology without evidence of degranulation and 4+ represents complete degranulation.
Blood samples for in vitro histamine release were collected from each subject at the screening visit, before intradermal drug administration and at the follow-up visit 4 weeks after skin testing. For each subject, 10 ml of venous blood was collected into a vacutainer tube containing sodium heparin. Whole blood was diluted (1:5) with the releasing buffer (supplied as a part of the immunoassay kit used for histamine determination; Beckman-Coulter, Miami, Florida, USA). The potential for rocuronium and cisatracurium to mediate allergic sensitization was assessed by adding predetermined concentrations of either drug to separate plastic tubes containing 200 µl of diluted whole blood preparation and 100 µl of releasing agent. The rocuronium (2.5x105 M) and cisatracurium besylate (4x106 M) concentrations were chosen to correspond to the blood levels that would be seen clinically. The calcium ionophore A23187 (3.33x105 M; Sigma Chemical, St Louis, MI, USA) and anti-IgE (Sigma Chemical) were used as positive controls. Negative control tubes (baseline release) had only diluted whole blood and buffer added to the tubes (no drugs). The tubes were incubated in a shaking water bath for 30 min at 37°C. At the end of the incubation, the tubes were immediately placed on ice and then centrifuged for 10 min at 900 g at 4°C. The supernatant was separated and stored at 70°C until analysed. For total histamine determination, 50 µl of undiluted blood was added to 950 µl of distilled water (1:20 dilution), frozen, and thawed three times in order to lyse the cells. The sensitivity of the method allows histamine quantitation down to 0.5 nM (0.05 ng ml1).
The percentage of histamine release from 1 ml of blood by buffer, anti-IgE, calcium ionophore, rocuronium and cisatracurium besylate treatments was calculated using the formula: 100xD/T=% histamine release, where D=concentration of histamine in the sample containing the drug/buffer and T=total concentration of histamine in the frozen and thawed sample.
The Wilcoxon signed-rank test was used to compare the percentage of positive reactions with the percentage in the control (saline) group. A paired Students t-test was used to compare mean weal and flare diameters with the mean for the saline control. Students t-test was used to compare mean percentage histamine release for rocuronium and cisatracurium between the first visit and the follow-up and with the mean percentage histamine release for the positive control. In all tests P0.05 was considered statistically significant. All data are expressed as mean (SE).
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Results |
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Discussion |
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When patients are skin-tested with cisatracurium, solutions should be diluted to <105 M, approximately a 1000-fold dilution (based on 10 mg ml1 of stock solution 102 M). We also noted that cisatracurium produced mast cell degranulation at a local level, as evidenced by light and electron microscopy (Fig. 4B). Further, because previous reports have suggested the threshold for non-immunological degranulation with neuromuscular blocking drugs is approximately 105 M,3 5 cisatracurium produces positive weal and flare responses at the thresholds previously reported with other benzylisoquinoline-derived agents.3 5 Why cisatracurium, another benzylisoquinoline-derived agent, does not release histamine in clinical studies may be a reflection of its potency and the smaller amount of drug administered.
There was no evidence that allergy to either rocuronium or cisatracurium developed de novo in the 29 volunteers studied, as evidenced by the lack of histamine release in blood samples collected at the follow-up visit and in comparison with the response elicited by anti-IgE or calcium ionophore, which were used as positive controls. Despite the small numbers of patients studied, this is an important finding because of the potential for anaphylaxis during re-exposure to NMBAs.12 Monneret and colleagues suggest that, in some cases, detecting basophil activation by flow cytometry may be a useful additional test, helpful in detecting the identity of the offending agent.10 Additional studies will be needed to validate the use of flow cytometry in allergy testing. NMBAs have several unique molecular features that make them potential allergens. All neuromuscular blocking drugs are functionally divalent, which makes them capable of cross-linking cell-surface IgE and initiating mediator release from mast cells and basophils without binding or haptenizing to larger carrier molecules.13 NMBAs are most often implicated in epidemiological studies of anaesthetic drug-induced anaphylaxis.1417 Fisher evaluated 134 consecutive patients after anaphylactoid reactions. Using intradermal, passive transfer or subsequent exposure criteria, 67 patients (50%) had reactions to NMBAs.16 Of the 67 patients reacting to the NMBAs, 54 were women, and the incidence of allergy, atopy and asthma was significantly greater in these patients than in non-reacting patients.15 Epidemiological data from France suggest that NMBAs are responsible for 6281% of reactions, depending on the time period evaluated.2 1718 The Centre for Allergy and Anaesthesia in Nancy, France, has detected approximately 40 anaphylactic reactions to NMBAs each year since 1982.2 1718 Succinylcholine was previously the NMBA most reported; however, in 1996, atracurium and vecuronium had similar incidences to succinylcholine.18 Reactions to neuromuscular blocking drugs occur more commonly in women.19
We have reported previously that aminosteroidal compounds in addition to benzylisoquinoline-derived agents produce positive weal and flare responses when injected intradermally.3 Our present study defined the clinical concentration for injecting aminosteroidal NMBAs for intradermal testing. Estimates of anaphylactic/anaphylactoid reactions in anaesthesia vary,11 but we believe that false-positive skin tests overestimate the incidence of rocuronium-induced anaphylactic reactions. We evaluated the clinical database on potential anaphylaxis to rocuronium in the USA and noted that reactions were rare, occurring in approximately 1 in 500 000 doses (unpublished data from Organon Inc., West Orange, NJ). The differences noted in the incidence of reactions may reflect the potential for false-positive weal and flare responses.
NMBAs can also produce direct vasodilation by multiple mechanisms, which include calcium channel block.6 The false-positive skin tests that were reported to be biopsy-negative for mast cell degranulation clearly confound the interpretation of skin tests in patients who have had life-threatening cardiopulmonary collapse. Dilute solutions of NMBAs need to be used when skin testing for potential allergic reactions to these agents. However, the exact concentration that should be used is unclear. Since skin-testing procedures are important in evaluating potential drug allergies, the threshold for direct vasodilating and false-positive effects must be determined whenever subjects are skin-tested for a particular drug.
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Acknowledgements |
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References |
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