SPECIFIC ANTIBODIES TO HALOTHANE-INDUCED LIVER ANTIGENS IN HALOTHANE-ASSOCIATED HEPATITIS

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1 Br. J. Anaesth. (198), 59, SPECIFIC ANTIBODIES TO HALOTHANE-INDUCED LIVER ANTIGENS IN HALOTHANE-ASSOCIATED HEPATITIS J. G. KENNA, J. NEUBERGER AND R. WILLIAMS The spectrum of liver damage that may follow halothane anaesthesia varies from the frequent, minor increases in the concentration of serum aminotransferase to the rare instances of fulminant hepatic failure (Wright et al., 195; Pohl and Gillette, 1982; Neuberger and Williams, 1984). Previous studies have demonstrated the presence of circulating antibodies reacting with novel, halothane-induced liver cell membrane antigens (halothane antibodies) in about 0 % of patients with fulminant hepatic failure following halothane (Kenna, Neuberger and Williams, 1984). Whether or not these antigens are implicated in the pathogenesis of the liver damage, demonstration of these antibodies is of diagnostic value, since these antibodies cannot be detected in the serum of patients with other forms of liver disease, in patients with no or minor abnormalities of liver function following halothane anaesthesia or in patients who, following halothane anaesthesia, have developed fulminant hepatic failure attributable to other causes (Davis et al., 1980; Vergani et al., 1980; Neuberger et al., 1983; Kenna, Neuberger and Williams, 1984). However, little is known about the incidence of these antibodies in patients with less severe reactions who do not develop encephalopathy, a group which, in some series, represents up to 5% of patients with presumed halothane hepatitis (Walton et al., 196). Until recently, our personal experience of halothane-associated hepatitis was largely limited to those patients, with fulminant halothane hepa- J. G. KENNA, PH.D.; JAMES NEUBERGER,* D.M., M.R.C.P.; ROGER WILLIAMS, M.D., F.R.C.P.; The Liver Unit, King's College Hospital and School of Medicine, Denmark Hill, London SE5 9RS. Accepted for Publication: March 5, 198. Present address, for correspondence: The Liver Unit, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH. SUMMARY Antibodies to halothane-altered liver cell determinants (halothane antibodies) have previously been detected in serum of patients with fulminant hepatic failure after halothane anaesthesia. However, their diagnostic value has not been reported in patients with non-fulminant hepatitis. Sera from 39patients who developed hepatitis following halothane anaesthesia between January 1983 and December 1985 were tested for antibodies to halothane-induced liver antigens using an ELISA; 22 of these patients had hepatitis without encephalopathy. Nineteen of the sera were from patients anaesthetized during 1985; four of the patients were aged 15 yr or less. All patients had undergone previous anaesthesia 1 days to 13 yr (median 3 yr) earlier. In 19 of the patients the final operation was a minor surgical procedure, lasting less than 45 min. In 13 patients a previous adverse reaction to halothane was documented in the case records. Twelve of the patients died. Halothane antibodies were detected in 12 of the 16 (5%) patients with hepatic encephalopathy and 16 of the 23 (0%) who did not develop encephalopathy, demonstrating that halothane antibodies are detectable in a wider spectrum of halothane-associated liver damage than previously appreciated. titis, referred to the Liver Failure Unit for management. However, with the greater awareness of a diagnostic test for the condition, sera from patients with a wider spectrum of liver damage have been referred for testing. In this report, we describe the findings in a series of 39 patients with halothane-associated hepatitis, with and without encephalopathy, who were anaesthetized in the 3 years between 1983 and 1985.

2 HALOTHANE-ASSOCIATED HEPATITIS 1 Year Total TABLE I. Pattern of referral of patients with halothane hepatitis No. admitted to Liver Unit, KCH No. of sera sent for halothane antibody testing PATIENTS Patients Between January 1, 1983 and December 31, 1985, 11 patients were referred to the Liver Unit with otherwise unexplained hepatitis following halothane, nine had fulminant hepatic failure (table I). In addition, samples of serum from a further patients, of whom only seven had fulminant hepatic failure, were sent for testing. Ninteen of the patients developed the hepatitis in 1985 (table I). In all the patients evidence of hepatic damage had appeared within days of a documented halothane anaesthetic and in none was there any other known cause for the liver damage. Infection with hepatitis viruses A and B, cytomegalovirus and toxoplasma was excluded by serological testing and in no patient was there a history of alcohol excess. Methods Sera were tested for halothane antibodies by ELISA, as described in detail elsewhere (Kenna, Neuberger and Williams, 1984). Sera were absorbed to remove antibodies to normal liver antigens, then tested for binding to control and halothane pre-treated rabbit liver microsoma] fractions which had been bound to wells of microtitre plates. Antibody binding was quantitated by incubation with peroxidase-labelled anti-human IgG, following which peroxidase activity was determined using o-phenylenediamine. Halothane antibodies were calculated as: A 4M diff = A 4M hal A 490 control where A 49O hal and A 490 control represent antibody binding to the halothane altered and control rabbit liver fractions, respectively. RESULTS Sixteen of the 39 patients developed fulminant hepatic failure, according to the criteria of Trey and Davidson (190). Of these, 10 progressed to grade IV encephalopathy and died from liver failure. A further two patients progressed to grade III encephalopathy and both died, one from renal failure and the other from sepsis. Of the remainder, 23 had no evidence of hepatic encephalopathy and all these survived (table II). Six of the patients were male. Ages ranged from 1.5 to 5 yr (median 58 yr) and were similar in those with and without encephalopathy. All of the patients had received at least one previous general anaesthetic (range 1-6, median 3 in those with encephalopathy and range 1-8, median 2 in those who did not develop encephalopathy). In 22 patients, including 11 who did not develop hepatic encephalopathy, it was possible to determine that the patient had been exposed to halothane. In the remaining subjects, it was not possible to determine which anaesthetic agents had been used in the penultimate operation. The interval between the final halothane anaesthetic^ and the most recent previous anaesthetic ranged from 1 days to 13 yr (median 3 yr). However, the median interval between the last two anaesthetics was 6 weeks in those who developed grade III/IV encephalopathy, compared with a median of yr in those with grade I/II encephalopathy and 1 yr in those who did not become encephalopathic. These differences are not statistically significant. In eight patients the interval was less than 3 months; in two of these less than 1 month. Thirteen of the patients had had a documented adverse reaction to halothane before the final exposure to the drug, and five of these developed encephalopathy. This adverse reaction consisted of jaundice in four, unexplained postoperative pyrexia in nine, and nausea, vomiting and malaise in one. The final operation was classified as major in 14 of the patients (cholecystectomy, clipping of berry aneurysm, oesophageal fistula repair, hysterectomy, orthopaedic, mastectomy, thyroid resection, parotid gland removal or rectopexy). The remainder had undergone minor surgery (ophthalmic surgery, dilatation and curettage, hypospadias, orthopaedic, breast biopsy, cystoscopy, colonoscopy, nasal polypectomy, salivary gland surgery or varicose vein stripping). In none of the operations was there any record of major intraoperative or immediate postoperative complications. The interval between exposure to halothane and the onset of signs of hepatic decompensation ranged from 3 to days (median 8 days) and was

3 18 BRITISH JOURNAL OF ANAESTHESIA TABLE II. Clinical details of patients with halothane hepatitis. Median (range) or No. (%) With encephalopathy Grade 111/IV (n - 12) Grade I/II (» = 4) Without encephalopathy (FI = 23) Age (yr) Sex ratio (F:M) Type of operation (major: minor) Previous anaesthetics Intervals between final anaesthetics Previous documented halothane reactions Interval, final anaesthesia to jaundice (days) Peak AST concn (iu litre" 1 ) Survival 58 (3-5) 10:2 :5 3 (1-5) 6wks (1 d-12 yr) 3 (25%) (1-) 900 ( ) 0 (0%) 52 (1-5) 4:0 a 2:2 4 (3-6) yr (5 mo-12yr) 4 (100%) (-10) 8 ( ) 4 (100%) 59 (1-68) 19:4 15:8 2 (1-8) lyr (5 wk-13 yr) 6 (26%) 9 (1-23) 1086 (442^000) 23 (100%) TABLE III. Halothane antibody results. * Upper limit iof normal = (mean + 3x SD of results obtained with 26 normalcontrol sera) Halothane antibodies Incidence Median A 4t0 diff* Range of A 4M diff Interval halothane exposure to testing (days') Median Range With encephalopathy Grade III/IV (» = 12) 8(6%) ( ) (4-6) Grade I/II (n = 4) 4(100%) ( ) 29 (21^9) With nut encephalopathy (FI = 23) 16(0%) ( ) 26 (11-) similar in those who did and did not develop encephalopathy. The interval between exposure to halothane and the taking of the serum for testing ranged from 4 to 6 days (median 20 days) and was similar in all three groups. The interval between anaesthesia and death in the 12 who died ranged from 6 to 4 days (median days). In the 39 patients, the peak recorded serum AST concentration ranged from 294 to > 4000 iu litre" 1 (median 905 iu litre" 1 ) and maximal prolongation of the prothrombin time was in the range s (median 19 s). The development of hepatic encephalopathy and mortality did not correlate with age, sex distribution, number of previous anaesthetics, incidence of previous adverse reactions to halothane, type of surgery (major or minor) or the interval from anaesthetic to the onset of jaundice (table II). In three patients, more than one serum sample was available and repeat testing showed no change in the presence or absence of halothane antibody. Halothane antibodies were detected in the serum of (2%) of the patients. Of the 23 without encephalopathy, antibodies were present in 16 (0%) and of those with fulminant hepatic failure, halothane antibodies were detectable in 12 (5 %). The amounts of antibodies were similar in all three groups (table III). Comparison of those patients with and without detectable concentrations of antibodies also showed that there was no difference with respect

4 HALOTHANE-ASSOCIATED HEPATITIS 19 TABLE IV. Comparison of halothane antibody positive and negative patients. Median (range) or No. (%). *P < 0.05 Age(yr) Sex ratio (F: M) AST concn (iu litre" 1 ) Incidence of encephalopathy Survival Previous anaesthetics Interval halothane exposure to jaundice (days) Interval halothane exposure to testing (days) Antibody positive (n = ) 50 (1-5) 24:4 950 ( ) 16(5%) 20(1%) 4* (1-8) (1-) 26 (6-6) Antibody negative (n=ll) 58 (3-6) 9: ( ) (64%) 8(3%) 1 (1-2) 12 (1-23) (4-3) to age, sex ratio, proportion having major or minor surgery (as denned above), degree of liver damage as assessed by increased concentration of serum aminotransferase, the incidence of encephalopathy (5% antibody positive and 64% antibody negative) and mortality (29 % antibody positive and 2 % antibody negative) (table IV). There was also no difference with respect to the timing of the samples (26 days for the antibody positive patients and days for the antibody negative ones). The only significant feature was that those with detectable concentrations of halothane antibodies had undergone a greater number of anaesthetics (median 4 in those with the antibody compared with median 1 in those without; P < 0.05, Spearman's Rank Test). DISCUSSION Greater awareness of the availability of a diagnostic test has resulted in our now being able to test serum from patients with a wide spectrum of otherwise unexplained liver damage following halothane anaesthesia. The results from the present study have demonstrated that the antibody is found in patients who have developed hepatitis but without encephalopathy, as well as those who have progressed to fulminant hepatic failure, and in a similar proportion of patients in both groups (approximately 0%). The spectrum of liver damage seen in this study is similar to that reported in a retrospective analysis of unexplained hepatitis following halothane between 191 and 193, and reported by Walton and colleagues (196). Halothane antibodies were not detectable in serum from about one-quarter of the patients. It is unlikely that the timing of the samples was the cause of the negative findings, since there was no difference in timing between those with and without antibody. Where repeat samples were obtained, these also were negative. It is possible that antibodies were present, but at titres too low for detection. Furthermore, antibody might be hidden by being bound in the form of antibodyantigen complexes, but disruption of circulating immune complexes, which are increased in these patients (Canalese et al., 1981), has not revealed such complexed antibodies (Kenna, unpublished data). An alternative possibility is that the antibody becomes bound onto antigen on the surface of the hepatocyte in vivo. We have no evidence to suggest that, in the antibody negative patients, causes other than halothane are responsible for the liver damage, although it must be emphasized that the diagnosis of halothane-associated hepatitis remains, especially in the absence of detectable halothane antibodies, largely one of exclusion, and it may be that some of the cases are attributable to the non-a non-b hepatitis viruses for which there are no serological markers, or from other causes. While demonstration of the halothane antibody provides confirmation of the cause of the liver damage, failure to demonstrate the antibody does not preclude the diagnosis. A similar analogy is myasthenia gravis, in which the presumed pathogenetic autoantibodies are detectable in a similar proportion of patients (Lindstrum et al., 196). In only 22 of the patients was it possible to show that the subject had had more

5 1290 BRITISH JOURNAL OF ANAESTHESIA than one exposure to halothane. While it may be that halothane-associated hepatitis may follow a first exposure to the agent, it must be emphasized that halothane was and, quite rightly, remains a frequently used anaesthetic so that it is possible that the previous anaesthetic agent was halothane. Furthermore, recent evidence suggests that enough halothane may be absorbed onto the tubing in the anaesthetic circuitry to sensitize a patient given a "non halothane" anaesthetic (Varma, Whitesell and Iskandarani, 1985). As in other studies, the patients were predominantly middle aged and female, and all had undergone previous anaesthesia. The outcome and severity of the liver damage showed no significant correlation with the number of previous anaesthetics or the interval between the final two anaesthetics. In particular, there was a wide range of intervals between the final two anaesthetics, ranging up to 12 yr in those who developed encephalopathy and up to 11 yr in those without encephalopathy; thus there appears to be no " safe interval" beyond which repeat exposure to halothane will not result in liver damage. Furthermore, these findings do not support the suggestion that a short interval between halothane exposures is associated with more severe liver damage. It is clear that cases of halothane-associated hepatitis continue to occur in the U.K., although little can be deduced about the annual incidence because of the uncertainty concerning rates of referral. However, the number of presumed cases referred to us is greater than that referred to the Committee on Safety of Medicines (Committee on Safety of Medicines, personal communication). It is disturbing to note that in one-third of these patients, there had been a documented previous adverse reaction following halothane. This suggests that, if a full history had been available before the final anaesthetic, a potentially fatal complication might have been avoided. ACKNOWLEDGEMENT This work was generously supported by the Wellcome Trust. J.N. is a Wellcome Senior Clinical Research Fellow. REFERENCES Canalese, J., Wyke, R. J., Vergani, D., Eddleston, A., and Williams, R. (1981). Circulating immune complexes in patients with fulminant hepatic failure. Gut, 21, 895. Davis, M., Eddleston, A. L. W. F., Neuberger, J., and Williams, R. (1980). Halothane hepatitis. N. Engl.J. Med., 303, Kenna, J. G., Neuberger, J., and Williams, R. (1984). An enzyme linked immunosorbent assay for detection of antibodies against halothane-altercd hepatocyte antigens. J. Immunol. Methods, 5, 3. Lindstrum, J. M., Seybold, M. E. Leman, V. A., Whirtingham, S., and Duane, D. D. (196). Antibody to acetylcholine receptor in my asthenia gravis. Prevalence, clinical correlates and diagnostic value. Neurology, 26, Neuberger, J., Gimson, A. E. S., Davis, M., and Williams, R. (1983). Specific serological markers in the diagnosis of fulminant hepatic failure associated with halothane anaesthesia. Br. J. Anaesth., 55, 15. Williams, R. (1984). Halothane anaesthesia and liver damage. Br. Med. J., 4, Pohl, L. R., and Gillette, J. R. (1982). A perspective on halothane-induced hepatotoxicity. Anesth. Analg., 61, 809. Trey. C, and Davidson, C. S. (190). The management of fulminant hepatic failure; in Progress in Liver Disease, Vol. 3 (eds H. Popper and F. Schafmer), p.2. New York: Grune and Stratton. Varma, R. R., Whitesell, R. C, and Iskandarani, M. M. (1985). Halothane hepatitis without halothane: role of inapparent circuit contamination and its prevention. Hepatology, 5, Vergani, D., Mieli-Vergani, G., Alberti, A., Neuberger, J., Eddleston, A. L. W. F., Davis, M., and Williams, R. (1980). Antibodies to the surface of halothane-altered rabbit hepatocytes in patients with severe halothane-associated hepatitis. N. Engl. J. Med., 303, 66. Walton, D., Simpson, B. R., Stranin, L., Doniach, D., Perrin, J., and Appleyard, A. (196). Unexplained hepatitis following halothane. Br. Med. J., 1, 11. Wright, R., Chisholm, M., Lloyd, B., Edwards, J. C, Eade, O. E., Moles, T. M., and Gardner, M. J. (195). A controlled prospective trial of the effect on liver function of multiple exposures to halothane. Lancet, 1, 821.