British Journal of Anaesthesia, 2000, Vol. 85, No. 6 844-849
© 2000 The Board of Management and Trustees of the British Journal of Anaesthesia
Weal and flare responses to intradermal rocuronium and cisatracurium in humans
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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Thirty volunteers underwent intradermal skin testing with increasing concentrations of rocuronium and cisatracurium to evaluate weal and flare responses, and whether either agent would cause mast cell degranulation and sensitization upon re-exposure. We found that intradermal injection of rocuronium and cisatracurium at concentrations >10–4 M resulted in positive weal (>8 mm) responses, and positive flare responses at >10–4 and >10–5 M respectively. Only cisatracurium caused mild to moderate mast cell degranulation, and neither drug caused significant in vitro histamine release from whole blood collected from study subjects 4 weeks after skin testing. Skin testing with rocuronium and cisatracurium should be performed at concentrations <10–4 and <10–5 M respectively to avoid false-positive responses. The ability of these agents to produce positive weal and flare responses at relatively low concentrations may explain the high incidence of potential reactions reported.
Br J Anaesth 2000; 85: 844–9
Keywords: neuromuscular block, rocuronium; neuromuscular block, cisatracurium; allergy
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Introduction |
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Many agents administered in the perioperative period have the potential to produce allergic reactions upon re-exposure.1 2 However, certain drugs may directly affect inflammatory cells and the vascular system to produce adverse reactions that are often falsely labelled as allergic. Drugs that directly release histamine from mast cells can also confound tests that use the skin as an immunological window to assess patients with the potential for allergic reactions. Opioids, neuromuscular blocking agents (NMBAs) and some other drugs (e.g. vancomycin, aprotinin and protamine) are highly charged molecules that have the ability to produce positive weal and flare responses independently of mast cell degranulation.3–5 Steroid-derived NMBAs also have direct vasodilating effects.6 These false-positive cutaneous responses can confuse allergy testing and interpretation, especially when different concentrations of the same drug(s) are used for testing by various investigators.7–9 Unfortunately, the actual threshold concentrations for skin testing have not been determined for the newer neuromuscular blocking drugs. Further, previous reports of perioperative anaphylaxis in France have implicated steroid-derived agents (i.e. rocuronium and vecuronium) as the offending drugs, based on positive results of skin testing.7 We investigated the effects of intradermal injections of increasing concentrations of neuromuscular blocking drugs on weal and flare responses in order to determine the threshold for allergic sensitization and to develop a guideline for using the appropriate concentrations of neuromuscular blocking drugs when skin-testing subjects. We also performed leukocyte histamine release tests using whole blood collected from each volunteer to determine their potential for sensitization.
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Methods |
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Thirty healthy volunteers were recruited for the study after institutional approval and informed consent. Subjects were excluded if they had a history of anaphylaxis or asthma or were taking steroid medications, antihistamines or H1 or H2 blockers. All subjects received all doses of rocuronium first, followed by all doses of cisatracurium besylate. The control substances—saline (negative control), atracurium besylate (positive control) and histamine (positive control)—were administered to all subjects. Rocuronium was used as a preservative-free 10 mg ml–1 solution. Cisatracurium besylate was supplied as a marketed product in 20 ml vials, which contain 10 mg ml–1 solution ready for injection. Atracurium besylate was supplied in 5 ml vials containing atracurium besylate 50 mg in solution (10 mg ml–1) ready for injection. Histamine phosphate was supplied in 5 ml vials which contained 2.75 mg ml –1 solution ready for injection. Saline was supplied in 2 ml vials at 0.9% NaCl. All drugs were diluted with 0.9% saline to obtain appropriate dilutions ready for testing.
Each subject underwent intradermal skin testing (during the first visit only) with the following study medications (using 0.02 ml injections): rocuronium 10–8, 10–7, 10–6, 10–5, 10–4, 10–3, 10–2 M; cisatracurium besylate 10–8, 10–7, 10–6, 10–5, 10–4, 10–3, 10–2 M; 0.9% normal saline solution; atracurium besylate 10–4 M; and histamine 10–4 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 controls—saline (negative), atracurium besylate (positive) and histamine (positive)—were administered on the left forearm. All solutions were freshly prepared and were preservative-free. After 10–15 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 Karnofsky’s 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 (2.5x10–5 M) and cisatracurium besylate (4x10–6 M) concentrations were chosen to correspond to the blood levels that would be seen clinically. The calcium ionophore A23187 (3.33x10–5 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 ml–1).
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 Student’s t-test was used to compare mean weal and flare diameters with the mean for the saline control. Student’s 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 P
0.05 was considered statistically significant. All data are expressed as mean (SE).
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Results |
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Data for the subjects with positive cutaneous reactions are shown in Figs 12–3. Rocuronium produced positive weal responses in 27 volunteers and positive flare responses in 29 volunteers at concentrations >10–4 M, and 20 out of 30 patients had positive cutaneous reactions at 10–3 M (P<0.05). Cisatracurium produced positive weal responses at
10–4 M and positive flare responses at >10–5 M in all volunteers, and 25 out of 30 volunteers had positive cutaneous responses at 10–4 M (P<0.05). There was no evidence that allergy to either rocuronium or cisatracurium had developed de novo (P<0.001) in 29 volunteers 4 weeks after skin testing compared with positive controls (anti-IgE and calcium ionophore). One subject in the follow-up did not return for testing. There were no differences in mean percentage histamine release for rocuronium (P=0.22) or cisatracurium (P=0.34) between the initial and follow-up visits.
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Light and electron microscopy of the skin biopsies of saline and rocuronium weals revealed normal mast cell morphology without evidence of degranulation, and mild to moderate degranulation in the cisatracurium induced weals (Figs 4A and B).
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Discussion |
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Our findings suggest that skin testing for hypersensitivity reactions to rocuronium should be performed with concentrations <10–4 M. As rocuronium is prepared as a 10 mg ml–1 solution that is approximately 10–2 M, rocuronium needs to be diluted at least 100-fold before skin testing to prevent false-positive skin tests. This is important because the high incidence of positive cutaneous responses to rocuronium reported in France occurred in patients who were skin-tested with 1:10 dilutions of stock rocuronium solution (approximately 10–3 M) and may represent false-positive skin tests.7 Bouaziz and Laxenaire suggest that rocuronium used at 1:10 dilution (1000 µg ml–1) for intradermal skin testing in controls will not produce any cutaneous reaction.8 According to the information pamphlet published by the Commission Tripartite de Consensus en Allergologie, skin testing for atracurium and mivacurium hypersensitivity should not be done with drug dilutions less than 1:1000 because of the possibility of false-positive reactions with more concentrated solutions (lower dilutions), but dilution can proceed down to 1:10 with rocuronium.9 This is in direct contrast to our data. We noted that 66% of volunteers in our study had positive skin tests (Fig. 1) when injected with rocuronium solution diluted approximately 10-fold (i.e. 1:10 dilution). Light and electron microscopy of positive test biopsies showed no evidence of mast cell degranulation, further supporting the evidence for false-positive weal and flare responses. In recently published reports describing acute reactions to rocuronium, intradermal testing was done at 1:10 dilution,10 and for prick-testing rocuronium was diluted 1:10 and 1:100.11 We believe that the use of drug concentrations higher than those used in our study may have led to the false-positive results. Additional prick-testing studies are needed to determine the optimal concentrations of NMBAs to be used during allergy testing in order to minimize false-positive reactions.
When patients are skin-tested with cisatracurium, solutions should be diluted to <10–5 M, approximately a 1000-fold dilution (based on 10 mg ml–1 of stock solution 
10–2 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 10–5 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.14–17 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 62–81% of reactions, depending on the time period evaluated.2 17–18 The Centre for Allergy and Anaesthesia in Nancy, France, has detected approximately 40 anaphylactic reactions to NMBAs each year since 1982.2 17–18 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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This study was supported by a grant from Organon Inc., West Orange, NJ, USA.
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References |
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1 Levy JH. Common anaphylactic and anaphylactoid reactions seen by the anesthesiologist. In: Anaphylactic Reactions in Anesthesia and Intensive Care. 2nd edn. Stoneham: Butterworth-Heinemann, 1992; 83–120
2 Birnbaum J, Porri F, Pradal M, et al. Allergy during anesthesia. Clin Exp Allergy 1994; 24: 915–21[Medline]
3 Levy JH, Adelson D, Walker B. weal and flare responses to muscle relaxants in humans. Agents Actions 1991; 34: 302–8[Web of Science][Medline]
4 Booij LH, Krieg N, Crul JF. Intradermal histamine releasing effect caused by Org-NC 45. A comparison with pancuronium, metocurine and d-tubocurarine. Acta Anaesthesiol Scand 1980; 24: 393–4[Web of Science][Medline]
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6 Yamaguchi K, Huraux C, Szlam F, Levy JH. Vascular effects of ORG9487 in human mammary arteries, a new short acting muscle relaxant. Anesth Analg 1998; 86 (4S): SCA 109
7 Laxenaire MC. Epidémiologie des réactions anaphylactoides peranesthésiques. Quatrième enquête multicentrique (juillet 1994–décembre 1996). Ann Fr Anesth Reanim 1999; 18: 796–809[Web of Science][Medline]
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11 Neal SM, Manthri PR, Gadiyar V, Wildsmith JAW. Histaminoid reactions associated with rocuronium. Br J Anaesth 2000; 84: 108–11
12 Royston D, Wilkes RG. True anaphylaxis to suxamethonium chloride: a case report. Br J Anaesth 1978; 50: 611–5
13 Vervloet D, Nizankowska M, Arnaud A, et al. Adverse reactions to suxamethonium and other muscle relaxants under general anesthesia. J Allergy Clin Immunol 1983; 71: 552–8[Medline]
14 Laxenaire MC, Moneret-Vautrin DA, Watkins J. Diagnosis of the causes of anaphylactoid anesthetic reactions. Anaesthesia 1983; 38: 147–8[Medline]
15 Moneret-Vautrin DA, Mouton C. Anaphylaxie aux myorelaxants: valeur prédictive des intradermoréactions et recherche de l’anaphylaxie croisée. Ann Fr Anesth Réanim 1985; 4: 186–91[Medline]
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