DETECTION OF IgE ANTIBODIES TO SUXAMETHONIUM AFTER ANAPHYLACTOID REACTIONS DURING ANAESTHESIA D. G. HARLE
B. A. BALDO M. M. FISHER
Kolling Institute of Medical Research and Intensive Therapy Unit, Royal North Shore Hospital of Sydney, St Leonards, New South
Summary detect from
Choline covalently coupled to an insoluble support was used in a radioimmunoassay to antibodies to suxamethonium in serum samples
IgE who patients experienced life-threatening anaphylactoid reactions after receiving the drug. Direct binding and inhibition experiments and correlations with clinical findings showed that use of choline in such an assay is relevant to the detection of suxamethonium-reactive antibodies. IgE antibodies were found in 9 of 10 patients who reacted to suxamethonium and whose skin-tests to the drug were positive and in 8 other patients who had reactions to other muscle-relaxant drugs. The positive reactions in the latter group were probably due to cross-reacting antibodies that recognise quaternary ammonium groups on more than one muscle relaxant. Introduction
given to the occurrence of muscle-relaxant drugs, which resemble type 1 hypersensitivity and occur during anaesthesia.I-7 In the United Kingdomand Australia,9 the safety of alcuronium has been questioned; in studies in Francel,4,10 and New Zealand7 suxamethonium (succinylcholine) is the drug most commonly implicated. Suxamethonium, like other relaxants, releases histamine by a direct effect on mast cells’and it has been suggested that this effect may cause the adverse reactions.7,12 In some patients, however, passive transfer testing and basophil and leucocyte histamine release studies INCREASING attention has been
suggested an immunological basis.’,’,’,’, 10
We have detected IgE antibodies which bind to alcuronium or d-tubocurarine in some patients who react to these 13-15 We have further shown in several of these patients drugs. that the specificities of the IgE antibodies are directed towards tertiary and quaternary ammonium ion determinants. 14,1’ Unlike alcuronium and d-tubocurarine, suxamethonium and decamethonium do not contain any reactive groups (fig 1) that may be chemically exploited to link these compounds to a protein carrier or insoluble support. However, suxamethonium consists of two molecules of choline linked by a carbon chain derived from succinic acid (fig 1). Hence each end of the molecule should be antigenically similar, if not identical, to choline. We covalently coupled choline to a solid support and used the complex in an assay to detect IgE antibodies reacting with suxamethonium. We describe here the identification of such in antibodies patients with severe reactions to suxamethonium. Patients and Methods Blood samples were taken from 24 patients a month after severe anaphylactoid reactions to muscle relaxants. Intradermal testing 16 showed that 10 of these patients (group I) had reacted to suxamethonium. 1 of these patients (patient 10) had received suxamethonium previously, 1 (patient 7) had had a previous severe anaphylactoid reaction to decamethonium on first exposure to a muscle relaxant, and 1 (patient 5) had had previous uneventful exposure to muscle relaxants other than, but not including,
1—Structures of choline and muscle-relaxant drugs methonium and decamethonium.
Common parts of choline and suxamethomum molecules underlined: close
similarity to underlined part of decamethonium molecule. can be seen. suxamethonium. The other 14 patients reacted to relaxants other than suxamethonium: 5 (group II) had positive skin-tests for suxamethonium and 9 (group III) had negative skin-tests for suxamethonium. Control serum samples were obtained from 10 members of hospital staff (9 women, 1 man) with no history of adverse reactions to anaesthesia, and cord serum samples were obtained from the Department of Obstetrics, Royal North Shore Hospital. Choline chloride (BDH; 60 mg in 6 ml distilled water) was covalently coupled to 500 mg ’Epoxy-activated Sepharose 6B’ (Pharmacia) by adjusting to pH 12.55 with 2.55 mol/1 sodium hydroxide and gently shaking with the activated sepharose at 350C for 20 h. After washing with water, 0 -1 mol/l borate buffer, pH 8, and 0 -1 mol/1 acetate buffer, pH 4, remaining free activated groups were blocked by incubation with 1 mol/1 ethanolamine, pH 9, at room temperature for 4 h. The same procedure was used to prepare the ethanolamine-sepharose complex from 300 mg ethanolamine and 500 mg activated sepharose. For detection of IgE antibodies by radioimmunoassay, 50 µ1 serum was added to 6 mg solid phase (choline or ethanolamine sepharose) and left at room temperature for 3 h. The tubes were then washed and centrifuged three times with phosphate-buffered saline containing ’Tween 20’ and 2-4 x 104 cpm iodine-125-labelled antihuman IgE (Pharmacia) was added. After standing overnight, tubes were washed three times and counted in a Packard ’Auto Gamma
Spectrometer’. For inhibition assays, 50 µ1 serum appropriately diluted was incubated for 1 h with 50 µ1 of a solution of choline or suxamethonium chloride (Wellcome Australia) before addition of the solid phase (6 mg in 100 µl. 3 h later, the tubes were washed and centrifuged three times with phosphate-buffered saline/tween before the addition of 125I-anti-human IgE (2-4x 104 cpm/tube). After overnight incubation at room temperature, the tubes were washed three times then counted.
Results No IgE antibodies to suxamethonium were detected in the cord serum samples or in the samples from the 10 controls (mean percentage uptake of 125I-anti-human IgE 0. 8°7o for cord samples; 0.8±SD 0-3% for control samples). The serum samples of 9 of the 10 patients (group 1), who reacted to suxamethonium and had positive skin-tests, clearly showed the presence of IgE antibodies reacting with choline but not with the control solid supports ethanolamine-sepharose and unmodified sepharose (tables I and II). Patient 10, the only patient with a history of exposure to suxamethonium before the reaction, showed low radioactive uptakes similar to those of the control normal and cord sera (table I). Further data to
931 TABLE I-RESULTS OF TESTS FOR SERUM IgE ANTIBODIES UPTAKE OF 125-ANTI-HUMAN SUXAMETHONIUM
TABLE 11-SPECIFICITY OF
IgE ANTIBODIES TO SUXAMETHONIUM
IN GROUP I PATIENTS
Fig 2-Inhibition by choline chloride and suxamethonium chloride of the binding of IgE antibodies in serum from patients 7 and 9 to choline-sepharose complex. Serum samples used
dilution of 1: 12.
basophil degranulation,4 passive transfer,2and
histamine release testsl,7,10 have been shown in all, patients reacting to muscle relaxants. Whether this is due to low sensitivity and of the tests or to non-immune mechanisms operative in some patients has not, however, been established. We have previously found IgE antibodies reacting with alcuronium and d-tubocurarine in the serum of some patients after anaphylactoid reactions to these drugs 13—15 and have now shown that antibodies reacting with suxamethonium also occur in some patients. That thecholine-sepharose assay used is relevant to the detection of IgE antibodies to suxamethonium was shown by the finding of antibodies in 13 of 15 patients who had positive skin-tests to suxamethonium (table 1). The relevance of the assay was also demonstrated by the results of patients 2, 8, and 13 whose serum reacted with choline-sepharose but not with other muscle-relaxant drug solid supports or control solid supports. We have so far found only .4 patients with reactions to choline-sepharose in the radioimmunoassay who have negative skin-tests to suxamethonium (table I). These subjects have serum antibodies that react with alcuronium, d-tubocurarine, or both and in the light of our previous findings it seems likely that the positive reactions with the choline complex are due to cross-reacting IgE antibodies that recognise quarternary ammonium groups on different muscle relaxants. 14,15 Crossreactivity also seems the most likely explanation for the positive skin-tests and radioimmunoassay results in the 4 subjects (patients 11-14) who had not received suxamethonium (table 1)’ There are several reports of patients who had clinical reactions to suxamethonium and either reacted to other relaxantsl6-188 or showed cross-sensitivity with other relaxants demonstrated by skin-testing or histamine release studies. 5,7,10,17 Patients who react to suxamethonium most commonly show cross-sensitivity to gallamine and decamethonium.5,7,18 The findings and conclusions accord with the structural similarities shown by suxamethonium and decamethonium. Although gallamine contains an aromatic ring, its side-chains are structurally similar to the other two compounds, except that ethyl and not methyl groups are present in the quaternary ammonium ions that terminate each of the three side-chains.
some, but- not
check the specificities of the IgE antibodies were provided by studies showing that the antibodies could be inhibited by both choline and suxamethonium. Fig 2 shows results obtained for patients 7 and 9. For 50% inhibition of the reaction of IgE antibodies with choline-sepharose, the amount of choline needed was 22 nmol in both patients, and the amount of suxamethonium 10 nmol for patient 7, and 10.55 nmol for
patient 9. 4 of the 5 patients in group II, who reacted clinically to muscle relaxants other than suxamethonium and who had positive skin-tests to. suxamethonium, had IgE antibodies reacting with suxamethonium (table I). The other patient (patient 15) had a positive skin-test for suxamethonium but showed radioactive uptakes similar to those of controls (table
r). 4 of the 9 patients who reacted clinically to relaxants other than suxamethonium and had negative skin-tests to suxamethonium (group III) had significant radioactive uptakes I). 2 of these patients (17 and 19) subsequently had minor reactions (tachycardia and transient bronchospasm) to suxamethonium. The other 5 group III patients had IgE antibodies reacting with alcuronium, d-tubocurarine, or both, but not with choline.
Discussion An understanding of the mechanism of anaphylactoid reactions to muscle-relaxant drugs is important, since immune-mediated reactions are likely to reoccur on subsequent exosure to the drug. If, however, the reactions are due to direct histamine release/,12 they may be prevented by premedication with antihistamines or by administering the drug in smaller quantity or more slowly. Although intradermal testing is a specific diagnostic test for the drug reactions, it does not prove a mechanism and merely reflects specific drug-induced release of mediators such as histamine.
Of the patients who had severe reactions to suxamethonium only 1 had received the drug previously. The possibility of sensitivity being acquired from other quaternary ammonium compounds has been suggested,14 and the high frequency of positive prick-tests to ’Cetrimonium’ (ICI), a quaternary ammonium compound, in patients who reacted to suxamethonium in a French series10 is interesting. The incidence of severe anaphylactoid reactions during anaesthesia is 1 in 10 000 cases. Consequently, screening a large population for the antibody is unlikely to be rewarding or a cost-effective preventive manoeuvre. Further studies are also needed to determine the value of this assay in the diagnosis of anaphylactoid reactions during anaesthesia. However, administration of suxamethonium to subjects who have a positive reaction with the assay should be avoided. The occurrence of minor reactions in 2 patients (17 and 19) who had negative skin-tests, but significant uptakes of radioactive anti-IgE, suggests that the assay may add useful information to intradermal testing, that the mechanism of these reactions involves IgE, that subsequent exposure to relaxants incriminated by intradermal testing is contraindicated, and that the cross-sensitivity between relaxants is likely to be a greater problem than previously believed.6 This work was funded by the National Health and Medical Research Council of Australia and the Harry Daly Foundation of the Faculty of Anaesthetists, Royal Australasian College of Surgeons. Correspondence should be addressed to B. A. B. Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, New South Wales 2065, Australia.
IS CYTOMEGALOVIRUS INFECTION A MAJOR CAUSE OF T CELL ALTERATIONS AFTER (AUTOLOGOUS) BONE-MARROW TRANSPLANTATION? LEO F. VERDONCK
Department of Haematology, University Hospital Utrecht, Netherlands
Peripheral-blood T cell subsets and functions were studied in 14 patients with malignancies treated with high-dose chemotherapy and radiotherapy followed by autologous bone-marrow transplantation (BMT). 8 CMV-positive patients (6 with latent infections and 2 with a primary infection) showed an inversion of the OKT4/OKT8 ratio caused by an increase in OKT8 + T cells and a decrease in OKT4 + T cells. This was accompanied by a pronounced increase in the percentage of HLA-DR+ T cells, and functionally by increased suppressor T cell activity and decreased helper T cell activity and T cell proliferation. These alterations in T cell subsets and functions were not observed in 6 patients who were kept CMV-negative by a deliberate transfusion policy. In the 6 patients helper T cell activity and T cell proliferation capacity recovered well after day 30. Differences between the two groups were most striking 60 days after BMT. This study suggests that CMV infection, whether primary or secondary, is a major cause of T cell alterations after (autologous) BMT.
Introduction PROFOUND combined immunological deficiency is found for at least 6 months after allogeneic bone-marrow transplantation (BMT). The resulting increased incidence of opportunistic infections1—4 seems mainly to result from
D, Arnaud A, Vellieux P, Kaplanski S, Charpin J. Anaphylactic reactions toto muscle relaxants under general anaesthesia. J Allergy Clin Immunol 1979; 63: 348-53. 2. Fisher MM. Reaginic antibodies to drugs used in anaesthesia. Anetehesiology 1980; 52: 318-20. 3. Lim M, Churchill-Davidson HC. Adverse effects of neuromuscular blocking drugs. In Thornton JW, ed. Adverse reactions to anaesthetic drugs. Monographs in anaesthesiology, no 8. Amsterdam: Elsevier, 1981: 65-136. 4. Moneret-Vautrin DA, Laxenaire MC, Moeller R. Anaphylaxis due to succinylcholine 1. Vervloet
Clin Allergy 1981; 11: 175-83. MM, Munro I. Life-threatening anaphylactoid reactions to muscle relaxants Anesth Analg (Cleve) 1983; 62: 559-64. Stoetling RK. Allergic reactions during anaesthesia. Anesth Analg (Cleve) 1983; 62:
5. Fisher 6.
Youngman PR, Taylor KM, Wilson JD. Anaphylactoid reactions to neuromuscular blocking agents a commonly undiagnosed condition? Lancet 1983; ii: 597-99. 8. Scowen E. Committee on safety of medicine. Br J Anaesth 1978; 50: 170. 9. Fisher MM, Baldo BA. Adverse reactions to alcuronium. An Australian disease? Med J
Aust 1983; i: 630-32. 10. Vervloet D, Nizankowska E, Arnaud A, Senft M, Alazia M, Charpin J. Adverse reactions to suxamethonium and other muscle relaxants under general anaesthesia. J Allergy Clin Immunol 1983; 71: 552-59. 11. Paton W. Histamine release by compounds of simple chemical structure. Pharmacol Rev 1957; 9: 269-301. 12. Editorial. Histamine and antihistamines in anaesthesia and surgery. Lancet 1981; ii: 74-75. 13. Baldo BA, Fisher MM. Detection of serum IgE antibodies that react with alcuronium and tubocurarine after life-threatening reactions to muscle relaxant drugs. Anaesth Intensive Care 1983, 11: 194-97. 14. Baldo BA, Fisher MM Substituted ammonium ions as allergic determinants in drug allergy. Nature 1983; 306: 262-64. 15. Baldo BA, Fisher MM. Anaphylaxis to muscle relaxant drugs: cross-reactivity and molecular basis of binding of IgE antibodies detected by radioimmunoassay. Molecular Immunol 1983; 20: 1393-1400. 16. Fisher MM. The diagnosis of acute anaphylactoid reactions to anaesthetic drugs Anaesth Intensive Care 1981; 9: 242-47. 17. Assem ESK, Frost PG, Levis RD. Anaphylactic-like reaction to suxamethonium. Anaesthesia 1981; 36: 405-10. 18. Fisher MM, Chan MYC. Anaphylaxis to both decamethonium and suxamethonium. Anaesth Intensive Care 1982; 10: 153-55.
alterations of T cell functions and defective B cell maturation.5-’ Although opportunistic infections are less frequent in syngeneic8 and autologous9,1O BMT, similar immunological alterations are found and thus seem to be independent of the type of graft or the presence of acute graftversus-host disease.7,11,12 The inversion of the OKT4/OKT8 ratio has been explained as being the result of a more rapid recovery of the OKT8 subset.7,13,14 We have studied T cell alterations in CMV-positive patients and CMV-negative patients after autologous BMT. Patients and Methods Patients 8 patients with a non-Hodgkin lymphoma (NHL) of high-grade malignancy and 3 patients with acute leukaemia (AL) were treated with cyclophosphamide (120 mg/kg) and total-body irradiation (800 rad). 3 patients with a testicular carcinoma were treated with cyclophosphamide (6000 mg/m2) and etoposide (1200 mg/m2). Chemotherapy or radiotherapy was followed by autologous BMT. Details of the patients are summarised in table 1. A primary CMV infection, manifesting as interstitial pneumonia or haemorrhagic gastroenteritis, developed in patients 1 and 2. In each case the source of infection was thrombocyte concentrates obtained from a relation who was CMV seropositive. Cultures from both patients were CMV positive. Serological studies of 6 patients
with a latent CMV infection demonstrated reactivation or reinfection (a fourfold or greater titre rise) in only patients 3 and 5 and cultures became CMV positive in patient 3. Prompted by the experience in the first 2 patients with a primary CMV infection, we adopted a deliberate transfusion policy in the 6 CMV-negative patients. Filtered leucocyte-free erythrocytes and thrombocyte concentrates from CMV seronegative donors were used. No granulocyte concentrates were used. These patients remained negative for CMV by serology and culture during follow-up. A fever (without other symptoms or signs) developed 2 months after BMT in patient 13. Antibody titres to Epstein-Barr virus (EBV) became positive, indicating a primary EBV infection.