CLINICAL
IMMUNOLOGY
AND
Leukocyte
A cell-mediated halothane-induced of cell-mediated past
5 years,
14, 30-34
IMMUNOPATHOLOGY
Migration
Inhibition Hepatitis
(1979)
in Halothane-Induced
hypersensitivity reaction has been implicated hepatitis. Previous reports have demonstrated immunity may be useful tools in the diagnosis 1 I patients
have
presented
in the pathogenesis of that it? r~itro parameters of this disease. During the
with
suspected
halothane
subsequently were biopsy-proven halothane ifestations consistent with the disease, and have halothane hepatitis. Halothane-induced
hepatitis. 2 patients leukocyte
4 patients had the clinical were subsequently shown migration inhibition was
strated in 6 of 9 patients with halothane did show a positive test. We conclude diagnosis
of halothane-induced
hepatitis. One that migration
hepatitis.
Five
patienls mannot to demon-
patient without halothane hepatiti\ inhibition may be a help in the
hepatitis.
INTRODUCTION
Halothane-associated hepatitis has been a controversial clinical entity since it was first described in 1958 (1, 2). A cell-mediated hypersensitivity reaction has been implicated in the pathogenesis, as a result of the frequency of this condition after repeated exposures to halothane. It has also been noted that following repeated exposures the latency period between challenge with the agent and the appearance of symptoms, signs, and liver dysfunction shortens (3). In addition, the presence of eosinophilia and autoantibodies (4) (mitochondrial antibody) seen in patients with this condition strengthens the belief that an immune mechanism is operative. Several groups of investigators have performed experiments to prove the hypersensitivity theory. Initially the experiments of Paronetto and Popper (4) appeared promising. These authors demonstrated that the lymphocytes of patients with halothane hepatitis, when incubated with varying concentrations of the anesthetic, demonstrated an increase in the tritiated thymidine uptake in the DNA of stimulated lymphocytes. Subsequent studies by Walton rf al. (5) and Wright (6) failed to demonstrate similar lymphocytic stimulation. However, Jones Williams et al. (7) using the macrophage inhibition test as an index of cell-mediated hypersensitivity showed that when the peripheral lymphocytes of two patients with halothane hepatitis were exposed to increasing concentrations of halothane, migration inhibition factor was produced. Subsequently, they (8) studied 3 additional patients with halothane hepatitis, 22 patients with previous single or multiple exposures to halothane without hepatitis, 5 healthy anesthesiologists who used ’ To whom requests Marshtield. Wis. 54449.
for
reprints
should
be
addressed:
30
0090-1229/79/090030-05$01.00/O Copyright @I 1979 by Academic Presc. Inc All rights of reproduction in any form reserved
Marshfield
Clinic.
1000
North
Oak
Ave..
THE
LIF
IN
HALOTHANE
HEPATITIS
31
halothane in their practice, 10 healthy subjects with no halothane exposure, as well as 9 jaundiced patients, 4 of which had hepatitis A. All but one of the patients with halothane hepatitis showed a positive response, while none of the controls showed a hypersensitivity response when challenged with halothane. The present study is an attempt to confirm those findings in our own series of 11 patients. METHODS
From October of 1973 to June of 1978 we saw 11 patients with suspected halothane hepatitis in whom the leukocyte migration inhibition (LIF) test was obtained. The diagnosis of halothane hepatitis was suspected on clinical grounds whenever there was a suitable history of exposure to the anesthetic followed by unexplained postoperative fever, jaundice, eosinophilia, elevated aspartate aminotransferase (AAT), and a negative test for hepatitis B surface antigen. The halothane hepatitis group consisted of 7 females and 2 males aged 44 to 84 years. Two other patients thought initially to have halothane hepatitis had other illnesses (biliary obstruction secondary to choledocholithiasis and chronic active liver disease), Eleven healthy halothane-unexposed controls were each tested at the same time as the patients. Table 1 presents the patients with halothane hepatitis as to the interval of time from exposure to testing as well as the criteria for diagnosis. All halothane hepatitis patients had at least one previous exposure to halothane, a negative test for hepatitis B surface antigen, elevated bilirubin, alkaline phosphatase, and/or AAT. In 5 of 9 patients who underwent a liver biopsy, the histology was consistent with the diagnosis of halothane hepatitis. The remaining 4 were not biopsied. In addition, 2 patients who had been exposed to halothane and had liver dysfunction not attributable to halothane were also tested. One of these patients had biliary obstruction secondary to choledocholithiasis and the other had chronic active liver disease. Antigen preparation. Halothane was prepared primarily as described by Paronetto and Popper (4). Halothane (fluothane) was diluted in medium 199 with 10% horse serum. The suspension was sonicated at 4°C for 1 min and centrifuged for 10 min at 8OOg. The supernatant was diluted lo-fold with medium 199 for use. This antigen preparation did not appear to have a cytotoxic effect on the leukocytes as determined by trypan blue exclusion. Migratim inhibition. The agarose migration inhibition technique was used as previously described by Marx and Burrell (9). The patient’s leukocytes were isolated by dextran sedimentation and resuspended to 1 x lo8 cells/ml in medium 199 with 10% horse serum. Halothane was added to a concentration of 5,25, or 50 ~1 and incubated 20 min at room temperature. The cells were allowed to migrate under an agarose medium for 24 hr at 37°C. The area of migration of cells incubated with halothane was compared to the area of migration of cells in the absence of halothane and expressed as a percentage. A migration index less than 80% was considered a positive test. Streptokinase-streptodomase was also included as a control antigen and tested at 2500 units/lo8 leukocytes. A normal healthy unexposed individual was tested in a similar manner with each patient. All 11 control individuals were tested and at no time was there evidence of sensitivity to halothane by this technique. Patienfs.
32
NUNEZ-GORNES,
MARX, TABLE
DEFINITION
Patient
Interval between exposure and LIF test
D.B.
4 weeks”
V.F.
8 days
AND
I
OF PAI IENT PUPULAIION
HBsAg -_____ ND”
MOTSZKO
Liver histology Halothane hepatitis’
ND
S-I UUI
GROUP
Bilirubin AAT Alk. Phos.
Diagnosis
1.6” 612 IN
Halothaneinduced hepatitis
0
Halothane induced hepatitis
642 IO00 E.B.
14 days
ND
E.T.
12 days
ND
Halothancinduced hepatitis
Halothane hepatitis I.? 620 143
Halothaneinduced hepatitis
N.C.
4 weeks
ND
-
7, I 225 273
Halothaneinduced hepatitis
E.H.
7 weeks
ND
Halothane hepatitis
3.5 200 X0
Halothaneinduced hepatitis
A.S.
4 weeks
ND
Halothane hepatitis
x.3 820 339
Halothaneinduced hepatitis
E.K.
10 months
ND
Halothane hepatitis
0.9 97 I I?
Haluthaneinduced hepatitis
A.E.
3 years
ND
-
I.2 200
Halothaneinduced hepatitis
‘I Interval of time since last exposure to halothane and LIF test. ‘I Not detectable. ” Pathologic reaction was consistent with halothane hepatitis. ” Normal values = bilirubin 11.2 mg%; AAT 9-40 mU/ml: Alk. Phos. 19-91 mU/ml I’ Not available.
RESULTS
We have recently studied nine patients with halothane hepatitis in whom the LIF test was done (Table 2). Seven of these patients were tested when there was evidence of liver dysfunction. Two patients (E.K. and A. E.) had normal liver function tests at the time of LIF testing. In these two cases one patient was tested after she had recovered from one episode of halothane hepatitis (A.E.) and the second after two episodes of halothane hepatitis (E.K.). Of the seven patients tested during periods of functional impairment of the liver, five had a positive LIF reaction and two a negative reaction. Both of these patients had clinical, biochemical, and histological features consistent with halothane hepatitis.
THE LIF IN HALOTHANE TABLE MIGRATION
INHlBITlON
HEPATITIS
33
2
IN HALOTHANE
HEPATITIS
Halothane Patient D.B. V.F. E.B. E.T. N.C. E.H. A.S. E.K. A.E.
98” 96 82 85 100 76 118 94 81
97 87 84 65 99 66 114 81 84
68.9 49 81 52 67 116 81 73
52.7 74 90 78 124 87 67
Mean
92.2 +- 4.28
86.3 + 5.2
73.5 -t 7.37
81.8 2 8.5
Mean control*
95.5 t 2.87
93.8 + 3.2
101.2 k 4.23
67.3 t 3.83
” (Cell migration with halothaneicell migration without halothane) x 100 = percentage migration. b Mean ~* SE. migration indices for nine normal unexposed individuals tested with each individual patient.
Of the patients tested after functional impairment of the liver had disappeared, one had a positive reaction and the second, a patient with two previous episodes of fever, liver dysfunction, and eosinophilia following halothane, had a negative reaction. Two patients with other liver diseases were tested with the above technique because when we first saw them, the diagnosis of halothane hepatitis was entertained. In one of these two patients the LIF reaction was positive and when explored she was found to have biliary obstruction secondary to choledocholithiasis. It is interesting to note that this patient had had a surgical procedure under halothane anesthesia about 10 days prior to her admission for obstructive jaundice. The positivity of the LIF test raises the question of sensitization to halothane. The second patient with chronic active hepatitis had a negative LIF reaction. DISCUSSION
This study represents an attempt to define the usefulness of the LIF test as an indication of hypersensitivity to halothane. Six of nine patients tested demonstrated LIF production when their lymphocytes were exposed to halothane in vitro. Our experience with the LIF test in halothane-induced hepatitis supports previous observations (8). Our results suggest the LIF test may be helpful in making the diagnosis of halothane hepatitis or in potentially identifying a patient at risk. In light of the lack of LIF production in 33% of our patient population, a negative test does not appear to be useful in excluding the diagnosis. It is interesting to note that at least two out of three patients demonstrating no LIF production also had uncontrolled diagnosed diabetes or multiple episodes of hyperglycemia. It has been suggested that diabetic patients have depressed cellmediated immune responses as evidenced by a decreased response to mitogens
34
NUNEZ-GORNES,
MARX,
AND
MOTSZKO
(10). It is, therefore, possible that the lack of LIF production could be related to a concurrent but unrelated clinical condition. In our experience we have seen only one patient without halothane hepatitis respond with LIF production (one false positive). Since this patient has not been reexposed to halothane, it is possible that he has been sensitized by his first exposure. Subsequent exposures to halothane could result in the development of hepatitis. The antigen used in this study was a crude aqueous sonicate of halothanc in tissue culture medium. This antigenic preparation did not have human serum proteins present until the cells were added. Consequently, the cells were exposed presumably to a haptenic group or to a group attached to a different carrier molecule than was used to sensitize the patient. Further refinements of the technique with more homologous protein carriers may well increase the diagnostic usefulness of this technique. This remains for future study. In summary, it is our contention that LIF production in response to halothane may be helpful in making the diagnosis of halothane hepatitis. Further refinements in the technique and experience in larger groups of patients and controls should yield further information about its diagnostic potential. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO.
Burnap, 1’. K.. Galla, S. J.. and Vandam. L,. D.. Alrt,sl/r[,~i,)/,~p\ 19. Virtue, R. W.. and Pane, K. W.. Alfc,slhcsi,,/~,R~ 19, (62, 19%. Inman. W. H. W.. and Mushin. W. W.. Bri!. Mrd. ./. 1, 5. 1974. Paronetto. F., and Popper, N.. Iv’. Elr~/. ./. hft&. 283. 277. 1970. Walton. B., Dumond, D. C.. Williams. C.. Jones, D., Strunin. G. M.. and Simpson. R.. ./. Amer. Med. Ass. 225, 494. lY73. Wright. B. M.. hi/. Mccl. ./. 2, 69, 1975. Jones Williams, W.. Pioli, E., Horton. J. N.. and Waldron. A.. Hlic. Price. C. D., Gibbs. A. R.. and Jones Williams. W.. J. (‘lin. Pu/hd. Marx Jr.. J. J., and Burrell. R.. ./. /r,~rrtrr,~~~/. 11 I, CYS. IY73. Eliashiv, A.. Olumide. F.. Norton. L.. and Eiseman. B.. ,4,-e h. Srtr(:.
307, IY5X.
Layton,
G. M.. Strunin.
,tiI
IMMUNOLOGY
AND
Leukocyte
A cell-mediated halothane-induced of cell-mediated past
5 years,
14, 30-34
IMMUNOPATHOLOGY
Migration
Inhibition Hepatitis
(1979)
in Halothane-Induced
hypersensitivity reaction has been implicated hepatitis. Previous reports have demonstrated immunity may be useful tools in the diagnosis 1 I patients
have
presented
in the pathogenesis of that it? r~itro parameters of this disease. During the
with
suspected
halothane
subsequently were biopsy-proven halothane ifestations consistent with the disease, and have halothane hepatitis. Halothane-induced
hepatitis. 2 patients leukocyte
4 patients had the clinical were subsequently shown migration inhibition was
strated in 6 of 9 patients with halothane did show a positive test. We conclude diagnosis
of halothane-induced
hepatitis. One that migration
hepatitis.
Five
patienls mannot to demon-
patient without halothane hepatiti\ inhibition may be a help in the
hepatitis.
INTRODUCTION
Halothane-associated hepatitis has been a controversial clinical entity since it was first described in 1958 (1, 2). A cell-mediated hypersensitivity reaction has been implicated in the pathogenesis, as a result of the frequency of this condition after repeated exposures to halothane. It has also been noted that following repeated exposures the latency period between challenge with the agent and the appearance of symptoms, signs, and liver dysfunction shortens (3). In addition, the presence of eosinophilia and autoantibodies (4) (mitochondrial antibody) seen in patients with this condition strengthens the belief that an immune mechanism is operative. Several groups of investigators have performed experiments to prove the hypersensitivity theory. Initially the experiments of Paronetto and Popper (4) appeared promising. These authors demonstrated that the lymphocytes of patients with halothane hepatitis, when incubated with varying concentrations of the anesthetic, demonstrated an increase in the tritiated thymidine uptake in the DNA of stimulated lymphocytes. Subsequent studies by Walton rf al. (5) and Wright (6) failed to demonstrate similar lymphocytic stimulation. However, Jones Williams et al. (7) using the macrophage inhibition test as an index of cell-mediated hypersensitivity showed that when the peripheral lymphocytes of two patients with halothane hepatitis were exposed to increasing concentrations of halothane, migration inhibition factor was produced. Subsequently, they (8) studied 3 additional patients with halothane hepatitis, 22 patients with previous single or multiple exposures to halothane without hepatitis, 5 healthy anesthesiologists who used ’ To whom requests Marshtield. Wis. 54449.
for
reprints
should
be
addressed:
30
0090-1229/79/090030-05$01.00/O Copyright @I 1979 by Academic Presc. Inc All rights of reproduction in any form reserved
Marshfield
Clinic.
1000
North
Oak
Ave..
THE
LIF
IN
HALOTHANE
HEPATITIS
31
halothane in their practice, 10 healthy subjects with no halothane exposure, as well as 9 jaundiced patients, 4 of which had hepatitis A. All but one of the patients with halothane hepatitis showed a positive response, while none of the controls showed a hypersensitivity response when challenged with halothane. The present study is an attempt to confirm those findings in our own series of 11 patients. METHODS
From October of 1973 to June of 1978 we saw 11 patients with suspected halothane hepatitis in whom the leukocyte migration inhibition (LIF) test was obtained. The diagnosis of halothane hepatitis was suspected on clinical grounds whenever there was a suitable history of exposure to the anesthetic followed by unexplained postoperative fever, jaundice, eosinophilia, elevated aspartate aminotransferase (AAT), and a negative test for hepatitis B surface antigen. The halothane hepatitis group consisted of 7 females and 2 males aged 44 to 84 years. Two other patients thought initially to have halothane hepatitis had other illnesses (biliary obstruction secondary to choledocholithiasis and chronic active liver disease), Eleven healthy halothane-unexposed controls were each tested at the same time as the patients. Table 1 presents the patients with halothane hepatitis as to the interval of time from exposure to testing as well as the criteria for diagnosis. All halothane hepatitis patients had at least one previous exposure to halothane, a negative test for hepatitis B surface antigen, elevated bilirubin, alkaline phosphatase, and/or AAT. In 5 of 9 patients who underwent a liver biopsy, the histology was consistent with the diagnosis of halothane hepatitis. The remaining 4 were not biopsied. In addition, 2 patients who had been exposed to halothane and had liver dysfunction not attributable to halothane were also tested. One of these patients had biliary obstruction secondary to choledocholithiasis and the other had chronic active liver disease. Antigen preparation. Halothane was prepared primarily as described by Paronetto and Popper (4). Halothane (fluothane) was diluted in medium 199 with 10% horse serum. The suspension was sonicated at 4°C for 1 min and centrifuged for 10 min at 8OOg. The supernatant was diluted lo-fold with medium 199 for use. This antigen preparation did not appear to have a cytotoxic effect on the leukocytes as determined by trypan blue exclusion. Migratim inhibition. The agarose migration inhibition technique was used as previously described by Marx and Burrell (9). The patient’s leukocytes were isolated by dextran sedimentation and resuspended to 1 x lo8 cells/ml in medium 199 with 10% horse serum. Halothane was added to a concentration of 5,25, or 50 ~1 and incubated 20 min at room temperature. The cells were allowed to migrate under an agarose medium for 24 hr at 37°C. The area of migration of cells incubated with halothane was compared to the area of migration of cells in the absence of halothane and expressed as a percentage. A migration index less than 80% was considered a positive test. Streptokinase-streptodomase was also included as a control antigen and tested at 2500 units/lo8 leukocytes. A normal healthy unexposed individual was tested in a similar manner with each patient. All 11 control individuals were tested and at no time was there evidence of sensitivity to halothane by this technique. Patienfs.
32
NUNEZ-GORNES,
MARX, TABLE
DEFINITION
Patient
Interval between exposure and LIF test
D.B.
4 weeks”
V.F.
8 days
AND
I
OF PAI IENT PUPULAIION
HBsAg -_____ ND”
MOTSZKO
Liver histology Halothane hepatitis’
ND
S-I UUI
GROUP
Bilirubin AAT Alk. Phos.
Diagnosis
1.6” 612 IN
Halothaneinduced hepatitis
0
Halothane induced hepatitis
642 IO00 E.B.
14 days
ND
E.T.
12 days
ND
Halothancinduced hepatitis
Halothane hepatitis I.? 620 143
Halothaneinduced hepatitis
N.C.
4 weeks
ND
-
7, I 225 273
Halothaneinduced hepatitis
E.H.
7 weeks
ND
Halothane hepatitis
3.5 200 X0
Halothaneinduced hepatitis
A.S.
4 weeks
ND
Halothane hepatitis
x.3 820 339
Halothaneinduced hepatitis
E.K.
10 months
ND
Halothane hepatitis
0.9 97 I I?
Haluthaneinduced hepatitis
A.E.
3 years
ND
-
I.2 200
Halothaneinduced hepatitis
‘I Interval of time since last exposure to halothane and LIF test. ‘I Not detectable. ” Pathologic reaction was consistent with halothane hepatitis. ” Normal values = bilirubin 11.2 mg%; AAT 9-40 mU/ml: Alk. Phos. 19-91 mU/ml I’ Not available.
RESULTS
We have recently studied nine patients with halothane hepatitis in whom the LIF test was done (Table 2). Seven of these patients were tested when there was evidence of liver dysfunction. Two patients (E.K. and A. E.) had normal liver function tests at the time of LIF testing. In these two cases one patient was tested after she had recovered from one episode of halothane hepatitis (A.E.) and the second after two episodes of halothane hepatitis (E.K.). Of the seven patients tested during periods of functional impairment of the liver, five had a positive LIF reaction and two a negative reaction. Both of these patients had clinical, biochemical, and histological features consistent with halothane hepatitis.
THE LIF IN HALOTHANE TABLE MIGRATION
INHlBITlON
HEPATITIS
33
2
IN HALOTHANE
HEPATITIS
Halothane Patient D.B. V.F. E.B. E.T. N.C. E.H. A.S. E.K. A.E.
98” 96 82 85 100 76 118 94 81
97 87 84 65 99 66 114 81 84
68.9 49 81 52 67 116 81 73
52.7 74 90 78 124 87 67
Mean
92.2 +- 4.28
86.3 + 5.2
73.5 -t 7.37
81.8 2 8.5
Mean control*
95.5 t 2.87
93.8 + 3.2
101.2 k 4.23
67.3 t 3.83
” (Cell migration with halothaneicell migration without halothane) x 100 = percentage migration. b Mean ~* SE. migration indices for nine normal unexposed individuals tested with each individual patient.
Of the patients tested after functional impairment of the liver had disappeared, one had a positive reaction and the second, a patient with two previous episodes of fever, liver dysfunction, and eosinophilia following halothane, had a negative reaction. Two patients with other liver diseases were tested with the above technique because when we first saw them, the diagnosis of halothane hepatitis was entertained. In one of these two patients the LIF reaction was positive and when explored she was found to have biliary obstruction secondary to choledocholithiasis. It is interesting to note that this patient had had a surgical procedure under halothane anesthesia about 10 days prior to her admission for obstructive jaundice. The positivity of the LIF test raises the question of sensitization to halothane. The second patient with chronic active hepatitis had a negative LIF reaction. DISCUSSION
This study represents an attempt to define the usefulness of the LIF test as an indication of hypersensitivity to halothane. Six of nine patients tested demonstrated LIF production when their lymphocytes were exposed to halothane in vitro. Our experience with the LIF test in halothane-induced hepatitis supports previous observations (8). Our results suggest the LIF test may be helpful in making the diagnosis of halothane hepatitis or in potentially identifying a patient at risk. In light of the lack of LIF production in 33% of our patient population, a negative test does not appear to be useful in excluding the diagnosis. It is interesting to note that at least two out of three patients demonstrating no LIF production also had uncontrolled diagnosed diabetes or multiple episodes of hyperglycemia. It has been suggested that diabetic patients have depressed cellmediated immune responses as evidenced by a decreased response to mitogens
34
NUNEZ-GORNES,
MARX,
AND
MOTSZKO
(10). It is, therefore, possible that the lack of LIF production could be related to a concurrent but unrelated clinical condition. In our experience we have seen only one patient without halothane hepatitis respond with LIF production (one false positive). Since this patient has not been reexposed to halothane, it is possible that he has been sensitized by his first exposure. Subsequent exposures to halothane could result in the development of hepatitis. The antigen used in this study was a crude aqueous sonicate of halothanc in tissue culture medium. This antigenic preparation did not have human serum proteins present until the cells were added. Consequently, the cells were exposed presumably to a haptenic group or to a group attached to a different carrier molecule than was used to sensitize the patient. Further refinements of the technique with more homologous protein carriers may well increase the diagnostic usefulness of this technique. This remains for future study. In summary, it is our contention that LIF production in response to halothane may be helpful in making the diagnosis of halothane hepatitis. Further refinements in the technique and experience in larger groups of patients and controls should yield further information about its diagnostic potential. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO.
Burnap, 1’. K.. Galla, S. J.. and Vandam. L,. D.. Alrt,sl/r[,~i,)/,~p\ 19. Virtue, R. W.. and Pane, K. W.. Alfc,slhcsi,,/~,R~ 19, (62, 19%. Inman. W. H. W.. and Mushin. W. W.. Bri!. Mrd. ./. 1, 5. 1974. Paronetto. F., and Popper, N.. Iv’. Elr~/. ./. hft&. 283. 277. 1970. Walton. B., Dumond, D. C.. Williams. C.. Jones, D., Strunin. G. M.. and Simpson. R.. ./. Amer. Med. Ass. 225, 494. lY73. Wright. B. M.. hi/. Mccl. ./. 2, 69, 1975. Jones Williams, W.. Pioli, E., Horton. J. N.. and Waldron. A.. Hlic. Price. C. D., Gibbs. A. R.. and Jones Williams. W.. J. (‘lin. Pu/hd. Marx Jr.. J. J., and Burrell. R.. ./. /r,~rrtrr,~~~/. 11 I, CYS. IY73. Eliashiv, A.. Olumide. F.. Norton. L.. and Eiseman. B.. ,4,-e h. Srtr(:.
307, IY5X.
Layton,
G. M.. Strunin.
,tiI
