Clin. exp. Immunol. (1977) 29, 272-277.
Lymphocyte-stimulation tests and patch tests in carbamazepine hypersensitivity J. HOUWERZIJL,* G. C. DE GAST, J. P. NATER, MARIET T. ESSELINK & H. 0. NIEWEG Department ofMedicine, Clinical Immunology Unit, Division ofHaematology and Department ofDermatology, University ofGroningen, The Netherlands (Received 28 February 1977)
SUMMARY
Seven cases of severe hypersensitivity to carbamazepine (Tegretole) were described in patients with epilepsy or trigeminal neuralgia. Clinical manifestations consisted of fever, rash, facial oedema, lymphadenopathy, impaired liver function, eosinophilia and atypical lymphocytes in the peripheral blood. Lymphocyte-stimulation tests with carbamazepine in vitro showed positive results in all cases; patch tests with carbamazepine were positive in six cases. In two cases the lymphocyte-stimulation tests with carbamazepine were found to be negative during, and shortly after, the illness. However, when the tests were repeated several months later, they turned out to be positive. Lymphocyte reactivity to PPD and PHA in vitro was also impaired during the acute phase of the disease. Thus false-negative lymphocyte-stimulation tests may be found in the first months following such a hypersensitivity reaction, probably due to impaired lymphocyte reactivity. As carbamazepine is a potent drug and is often prescribed for long periods together with other anticonvulsants, it seems important to prove that the allergic reaction is caused by carbamazepine. If the lymphocyte-stimulation test in vitro or the patch test with carbamazepine is found to be negative during or shortly after the illness, they should be repeated several months later. INTRODUCTION The diagnostic value of lymphocyte-stimulation tests in drug allergies is still a matter of discussion. Many factors may be held responsible for this: the lack of proper antigens, poor solubility or cytotoxicity of the drug, variability of the clinical picture and probably the time of testing (Assem, 1967; Levene & Baker, 1968; Ling, 1972; Mathews, Pan & Wells, 1972). Virolainen (1971) obtained a positive result with lymphocyte stimulation in vitro in one patient with a particular type of hypersensitivity to carbamazepine (CBZ). A similar case due to diphenylhydantoin (DPH) was published earlier (Holland & Mauer, 1964). The aim of this study was to evaluate the usefulness of the lymphocyte-stimulation tests in vitro in seven cases of identical hypersensitivity reactions to CBZ and in one case to DPH as well. As the time of testing might influence the outcome of the test, lymphocyte-stimulation tests in vitro were sequentially performed in two cases and later repeated with cryopreserved lymphocytes. Lymphocyte reactivity to drugs in vitro was compared with reactivity to PPD and PHA. In addition, patch tests were performed, although these are generally believed to be of limited value in diagnosing rashes caused by systemically-administered drugs (Agrup, 1972). Present address: Bonifatius Hospital, Leeuwarden, The Netherlands. Correspondence: Dr G. C. de Gast, University Hospital, Department of Internal Medicine, Oostersingel 59, Groningen, The Netherlands. *
272
Tests in carbamazepine hypersensitivity
273
MATERIALS AND METHODS Patients. The clinical features and some laboratory findings from all seven patients are summarized in Table 1. In all cases treatment with CBZ had been started 14-30 days before the onset of the hypersensitivity reaction. The diseases for which this drug was given were trigeminal neuralgia and temporal epilepsy. Some of the patients had already been treated with phenobarbitone; one patient (no. 1) started treatment with DPH as well as with CBZ 14 days before the development of the allergic reaction. Because an allergic reaction to CBZ or DPH was suspected, these drugs were discontinued and replaced, if necessary, by other anticonvulsants. All patients showed an allergic reaction consisting of fever, rash, facial oedema, lymphadenopathy, impaired liver function, eosinophilia and atypical lymphocytes in the peripheral blood. In addition to these symptoms and signs, one patient (no. 6) had in the acute phase of the disease impaired renal function, and a chest X-ray showed diffusely-arranged nodular densities. In two patients (no. 3 and no. 4), recurrence of the symptoms after accidental ingestion of one tablet CBZ proved this drug to be the causative one. Controls. Ten patients, who had been or were exposed to the same drug without adverse effects, made up a control group. They were numbered from 1 to 10. Controls no. 1 and no. 2 were using both DPH and CBZ at the time of the tests. Controls no. 3 and no. 4 stopped using both drugs about 16 weeks before the tests. Controls no. 6 to no. 10 had not been treated with CBZ for 10-52 weeks prior to testing. In order to avoid sensitization to drugs they needed, controls no. 1 and no. 2 did not undergo patch tests. Patch tests were also performed on ten healthy persons. Lymphocyte stimulation test. Lymphocyte-stimulation tests were performed as described elsewhere (DuBois et al., 1973). 300,000 lymphocytes, which had been isolated from defibrinated blood by Ficoll-Isopaque gradient centrifugation, were cultured in tubes (Nunc, Denmark) containing 1 ml medium RPMI 1640, HEPES buffer, 20% pooled human serum, 100 u of penicillin and 100 pg of streptomycin. CBZ* and DPH were dissolved in pure propylene glycol and subsequently diluted with RPMI 1640. Cultures with phytohaemagglutinin (PHA, Burroughs Wellcome) 5 ul/ml were incubated for 3 days in tubes with tight caps. Cultures with CBZ or DPH and a solvent blank with propylene glycol 0 5%4 as well as cultures with tuberculin (PPD without preservativet) 10 pg/ml were incubated under the same conditions for 6 days. The drugs were added in a volume of 0 05 ml, yielding a final concentration of 3 pg/ml, 10 pg/ml or 30 pg/ml CBZ or DPH and 0 5%4 propylene glycol. Previous experiments showed cytotoxicity with higher concentrations of CBZ and DPH. Cultures were done in quadruplicate: three for thymidine incorporation and one for morphological control. 24 hr before harvesting 0 5 PCi [3H]thymidine (Radiochemical Centre, Amersham, sp. act. 40 mCi/mmol) was added. After harvesting radioactivity was measured in a Packard liquid scintillation counter and expressed as disintegrations/min (d/min) per culture. The arithmetical mean and standard deviation (s.d.) were calculated. The stimulation index (SI) was calculated as follows: d/min in presence of drug SI d/min in presence of solvent An SI of more than 2-0 was regarded as evidence of stimulation. In two cases (no. 1 and no. 2) mononuclear cells were frozen and stored for sequential studies, as described by Leguit et al. (1973), in addition to cultures with fresh lymphocytes. Patch tests. Patch tests were performed with silver patch testers (v.d. Bend, Brielle) and read according to the method of the International Contact Dermatitis Research Group (Malten, Nater & Ketel, 1976). CBZ as well as DPH were tested at concentrations of 10%Y, 20%/ and 40% in vaseline. The skin reactions were read after 48 and 72 hr. =
RESULTS Significant lymphocyte stimulation in vitro by CBZ could finally be demonstrated in all seven cases and by DPH as well in one of these cases (no. 1, Table 2). Maximal stimulation was generally obtained with the highest drug concentration (30 pg/ml) used. SI in the cultures with CBZ varied between 3-6 and 58-0 (median 7.9). In one case (no. 7) a positive lymphocyte stimulation was found even 7 years after the illness. Lymphocyte cultures from the controls showed no stimulation by CBZ or DPH. Follow-up studies were carried out on two cases (no. 1 and no. 2) with fresh and with cryopreserved lymphocytes. The results obtained with the cryopreserved lymphocytes are shown in Table 3. In case no. 1 significant stimulation by CBZ was noted for the first time 21 weeks after the onset of the allergic reaction and the effect continued to increase after that. Lymphocyte stimulation by DPH followed a similar course. In case no. 2, lymphocytes were not stimulated by CBZ during the 8 weeks stay in * Carbamazepine (Tegretolg) was kindly supplied by Ciba-Geigy, Basel, Switzerland. t Rijksinstituut voor de Volksgezondheid Bilthoven, The Netherlands.
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Tests in carbamazepine hypersensitivity TABLE 2. Results of lymphocytc-stimulation tests
Cell source Patient no. 1 (21 w)* 2 (49 w) 3 (5 y) 4 (3 y) 5 (13 w) 6 (5 y) 7 (7 y) Control no. 1 2 3 4 5 6 7 8 9 10
DPH
CBZ
PHA (d/min+ s.d.)
Solvent (d/min+ s.d.)
(d/min+ s.d.)
SI
(d/min+ s.d.)
SI
13,840+1365 35,765+ 1567 42,837+6160
97+24 843+ 117 278+76 226+48 152+38 427+ 113 158+74
5699+1107 6112+ 1074 2209+302 1104+ 178 5522+313 1538+ 299 1272+356
580 7-2 7*9 4-8 36-3 3-6 8-0
7217+512 1036+300 383+ 130 434+ 120 n.d. 324+ 54 n.d.
74.4 1-2 1-3 1.9
60+ 12 144+47 219+ 38 593+57 566+264 245+ 134 400+ 132 80+ 14 388+ 53 548+231
86+30 191+49 310+ 35 650+258 520+ 147 242+ 178 616+ 182 144+ 84 316+ 54 597+48
1-4 1-3 1-4 1.1 09 1.0 1-5 1-8 0-8 10
62+40 178+38 288+ 45 629+292 729+285 248+ 62 512+ 118 142+ 58 277+ 133 612+ 156
08 12 1-3 1.1 1-3 1.0 1-3 1-7 07 1.1
14,943+2615 26,198+5308 83,624+ 4107 25,692+ 1240
26,385+ 104 23,334+1937 27,245+ 2367
22,457+1138 48,093+2611 26,374+ 6627 17,523+ 1147 24,480+ 153 23,612+ 1486 60,999+ 14,167
-
0-7 -
n.d. = Not done. * Time in weeks (w) or years (y) after onset of illness. TABLE 3. Results of sequential lymphocyte stimulation tests in two patients
Cell source
Patient no. 1 5 w* 11 w 21 w 82 w Patient no. 2 4w 8w 49 w 60 w
CBZ (d/min+ s.d.)
SI
DPH (d/min+ s.d.)
77+20 34+ 17 97+24 809+ 148
111+23 108+42 5699+1107 16817+4467
1-4 1-7 580 20-7
122+34 n.d. 7217+512 19321+2813
268+46 112+ 69 843+ 117 676+ 167
510+ 112 179+ 44 6112+ 1074 5077+ 335
1.9 1-6 7-2
Solvent
PHA (d/min+ s.d.)
(d/min+ s.d.) (d/min+ s.d.)
5637+714 10202+233 13840+1365 43510+ 1531
5292+380 4729+468 12401+765 30487+ 1729
6929+ 1187 3350+ 387 35765+ 1567 24255+ 1531
306+ 56 876+ 183 4157+290 4137+ 530
PPD
7.5
n.d. n.d. 1036+300 n.d.
SI 1-6 -
74-4 23-8 -
12 -
* Weeks after onset of illness.
hospital. 40 weeks later, however, a positive result was obtained and was confirmed in a later test. The lymphocyte responses to PHA and PPD in cases no. 1 and no. 2 were parallel to those to the drugs. When there was no stimulation to the drugs, PHA and PPD stimulation were low. Later on when stimulation to drugs was evident, PHA and PPD stimulation were on a higher level. Patch tests with CBZ were positive in six of the seven cases (Table 4). No differences in the skin reactions were observed when patch testing with concentrations of 10%, 20% and 40%0 of CBZ. Control subjects showed no reactions. In case no. 2 patch tests with CBZ showed the same sequence as the lymphocyte stimulation tests, being negative in the initial phase but positive afterwards. A patch test with DPH in case no. 1 was negative.
276
J. Houwerzijl et al. TABLE 4. Rcsults of patch tests (read after 72 hr) Patient no. 1 (23 w)* 2(50 w) 3(5 y) 4 (2- y) 5(14 w) 6 (5 y) 7 (7 y) 18 controlst
CBZ
DPH
+++ ++++
_
+ ++
n.d. n.d.
See Malten et al. (1976). * Time in weeks (w) or years (y) after onset of illness. t Including C3-C0I and ten healthy persons.
DISCUSSION In diagnosing drug hypersensitivity, lymphocyte-stimulation tests often give false negative results (MacKinney & Booker, 1972; Vischer, 1966). Since false positive responses are rare, all patients with positive results have to be regarded as established cases of drug hypersensitivity (Mathews et al., 1972; Sarkany, 1967). The test enabled us to confirm an already proven hypersensitivity to CBZ in cases no. 3 and no. 4, even several years after the last exposure. In five other cases hypersensitivity to CBZ was diagnosed and in one case (no. 1) hypersensitivity to DPH was also diagnosed. One of the factors that plays a role in the outcome of the tests is the poor solubility of many drugs which often gives technical problems. It was necessary to dissolve the drug with propylene glycol. Subsequent dilution with RPMI 1640 medium yielded a concentration at which stimulation of lymphocytes was possible without toxic effects. Another factor of importance appears to be the clinical picture of the hypersensitivity reactions. Lymphocyte stimulation is often successful with drugs, which cause the appearance of blasts in the peripheral blood (Holland & Mauer, 1964; Sarkany, 1967; Virolainen, 1971), as well as with drugs which cause hypersensitivity characterized by fever and rashes (Mathews et al., 1972). In all cases all these features were present. Patch testing is an established method of diagnosing allergic contact dermatitis. It is not usually recommended for diagnosing rashes caused by systemically used drugs (Agrup, 1972). Nevertheless, in some instances promising results have been reported (Felix & Comaish, 1974). This study shows that patch testing can be helpful in detecting hypersensitivity to CBZ. Positive results are often obtained first used externally and afterwards internally (Fischer, 1966; Agrup, 1972). In cases of hypersensitivity reactions to CBZ external exposure seems very unlikely. In case no. 1 the negative patch test with DPH contrasted with the positive lymphocyte stimulation in vitro by DPH. The skin rash of this patient may therefore be due to CBZ only. Alternatively both drugs may be involved, which implies that DPH is not suitable for skin testing. The results of both lymphocyte stimulation tests and patch tests appear to be dependent on the time of testing. In two cases (no 1 and no. 2) sequential testing, performed with cryopreserved lymphocytes to avoid differences in culture conditions, indicated that stimulation in vitro by drugs produced negative results during and shortly after the illness. This probably applies to in vivo tests as well, because the first patch test in case no. 2 was negative. Besides, a transient general impairment of lymphocyte responsiveness in vitro is suggested by impaired responses to PHA and PPD in the acute phase of the disease. This phenomenon has already been observed in cases with active tuberculosis, viral disease and even leucocytosis of any aetiology (Pearman, Lycette & Fitzgerald, 1963; Mangi et al., 1974; Heiss & Palmer, 1974). As far as we know, an association between drug hypersensitivity reactions and impaired lymphocyte
Tests in carbamazepine hypersensitivity
277
reactivity in vitro has not been reported. The basic mechanism of the impaired lymphocyte reactivity is not clear. The impaired response to PHA, a predominantly T-cell mitogen, indicates that T-cell function is surely affected. Our findings show that testing during or shortly after the illness may lead to false negative results. When negative results are obtained, both tests should be repeated within 2 or 3 months after complete recovery. The clinical picture of our patients consisted of fever, rash, facial oedema, lymphadenopathy, eosinophilia, lymphocytosis (including many atypical ones) and in six cases impaired liver function tests. This is very similar to the reported 'serum-sickness-like' reactions due to hydantoins, para-amino salicylic acid, phenylbutazone, iron dextran, cephalothin and cephapirin (Saltzstein & Ackerman, 1959; Schen, 1964; Sanders, Johnson & Taggart, 1974), indicating that CBZ is also able to induce such a clinical picture. Results of in vitro lymphocyte-stimulation tests and patch tests suggest involvement of delayed-type hypersensitivity. The incidence of this type of reaction to CBZ is obviously low. Only three similar cases have been published so far (Steen-Johnson, 1970; Virolainen, 1971; Heber, 1972). Because of its effectiveness in temporal epilepsy and trigeminal neuralgia the use of CBZ is still growing. That is why an increase of hypersensitivity reactions to CBZ may be expected. As CBZ is often prescribed for long periods together with other anticonvulsants, it is important to be able to prove a hypersensitivity reaction to this drug. The lymphocyte-stimulation test and the patch test with CBZ, if performed at the right time, have proven to be of value in diagnosing hypersensitivity to CBZ. This work was supported by the 'Nederlandse Vereniging tot Rheumatiekbestrijding'. We thank Miss T. Woest for technical assistance, Dr T. H. The for advice and criticism and Mrs H. Jellema-Brazier for revising the English.
REFERENCES
AGRUP, G. (1972) Mechanisms in Drug Allergy (ed. by C. H. Dash & H. E. H. Jones), p. 135. Churchill Livingstone, Edinburgh. ASSEM, E.S.K. (1967) Drug allergy. Hosp. Med. 2, 199. DuBois, M.J.G.J., HuismANs, D.R., SCHELLEKENS, P.T.A. & EYSVOOGEL, V.P. (1973) Investigation and standardization of the conditions for microlymphocyte cultures. Tissue Antigens, 3, 402. FELIX, R.H. & COMAISH, J.S. (1974) The value of patch and other skin tests in drug eruptions. Lancet, i, 1027. FISCHER, A.A. (1966) Systemic eczematous 'contact-type' dermatitis medicamentosa. Ann. Allergy, 24, 406. HEBER, W. (1972) Erythrodermische Arzneiexanthem nach Carbamazepin. Therapiewoche, 48, 4245. HEIss, L.I. & PALMER, D.L. (1974) Anergy in patients with leukocytosis. Amer. ]. Med. 56, 323. HOLLAND, P. & MAUER, A.M. (1964) Drug induced in vitro stimulation of peripheral blood lymphocytes Lancet, i, 1368. LEGUIT, P., JR, MEINEsz, A., ZEYLEMAKER, P., SCHELLEKENS, P.T.A. & EYSVOOGEL, V.P. (1973) Immunological studies in burn patients. Int. Arch. Allergy, 44, 101. LEvENE, G. & BAKER, H. (1968) Lymphocyte transformation in vitro and drug hypersensitivity. Brit. J. Derm. 80, 415. LING, N.R. (1972) Mechanisms in Drug Allergy (ed. by C. H. Dash & H. E. H. Jones), p. 171. Churchill Livingstone, Edinburgh. MACKINNEY, A.A. & BOOKER, H.E. (1972) Diphenylhydantoin effects in human lymphocytes in vitro and in vivo. Arch. intern. Med. 129, 988. MALTEN, K.E., NATER, J.P. & VAN KETEL, W.G. (1976) Patch Testing Guidelines (ed. by D. VAN VAN DE Vegt). Nijmegen.
MANGI, R.J., NIEDERMAN, J.C., KELLEHER, J.E., JR, DWYER, J.M., EVANS, A.S. & KANTOR, F.S. (1974) Depression of cell mediated immunity during acute infectious mononucleosis. New Engl. J. Med. 291, 1149. MATHEWS, K.P., PAN, P.M. & WELLS, J.H. (1972) Experience with lymphocyte transformation tests in evaluating allergy to aminosalicylic acid, isoniazid and streptomycin. Int. Arch. Allergy, 42, 653. PEARMAN, G., LYCETTE, R.R. & FITZGERALD, R.H. (1963) Tuberculin induced mitosis in peripheral blood leukocytes Lancet, i, 637. SALTZSTEIN, S.L. & ACKERMAN, L.V. (1959) Lymphadenopathy induced by anticonvulsant drugs and mimicking clinically and pathologically malignant lymphomas.
Cancer, 12, 164. SANDERS, W.E., JR, JOHNSON, J.E. & TAGGART, J.G. (1974) Adverse reactions to cephalotin and cephapirin after prolonged intravenous administration of high doses. New Engl. J. Med. 290, 242. SARKANY, I. (1967) Lymphocyte transformation in drug hypersensitivity. Lancet, i, 743. SCHEN, R.J. (1964) Lymphadenopathy due to drugs. Israel Med. ]. 23, 19. STEEN-JOHNSON, J. (1970) Alvorlige bivirkninger red bruk av karbamazepin. (Tegretol). T. Norske. Laegeforen, 90, 1631. VIROLAINEN, M. (1971) Blast transformation in vivo and in vitro in carbamazepin hypersensitivity. Clin. exp. Immunol. 9, 429. VISCHER, T.L. (1966) Lymphocyte cultures in drug hypersensitivity. Lancet, ii, 743.
Lymphocyte-stimulation tests and patch tests in carbamazepine hypersensitivity J. HOUWERZIJL,* G. C. DE GAST, J. P. NATER, MARIET T. ESSELINK & H. 0. NIEWEG Department ofMedicine, Clinical Immunology Unit, Division ofHaematology and Department ofDermatology, University ofGroningen, The Netherlands (Received 28 February 1977)
SUMMARY
Seven cases of severe hypersensitivity to carbamazepine (Tegretole) were described in patients with epilepsy or trigeminal neuralgia. Clinical manifestations consisted of fever, rash, facial oedema, lymphadenopathy, impaired liver function, eosinophilia and atypical lymphocytes in the peripheral blood. Lymphocyte-stimulation tests with carbamazepine in vitro showed positive results in all cases; patch tests with carbamazepine were positive in six cases. In two cases the lymphocyte-stimulation tests with carbamazepine were found to be negative during, and shortly after, the illness. However, when the tests were repeated several months later, they turned out to be positive. Lymphocyte reactivity to PPD and PHA in vitro was also impaired during the acute phase of the disease. Thus false-negative lymphocyte-stimulation tests may be found in the first months following such a hypersensitivity reaction, probably due to impaired lymphocyte reactivity. As carbamazepine is a potent drug and is often prescribed for long periods together with other anticonvulsants, it seems important to prove that the allergic reaction is caused by carbamazepine. If the lymphocyte-stimulation test in vitro or the patch test with carbamazepine is found to be negative during or shortly after the illness, they should be repeated several months later. INTRODUCTION The diagnostic value of lymphocyte-stimulation tests in drug allergies is still a matter of discussion. Many factors may be held responsible for this: the lack of proper antigens, poor solubility or cytotoxicity of the drug, variability of the clinical picture and probably the time of testing (Assem, 1967; Levene & Baker, 1968; Ling, 1972; Mathews, Pan & Wells, 1972). Virolainen (1971) obtained a positive result with lymphocyte stimulation in vitro in one patient with a particular type of hypersensitivity to carbamazepine (CBZ). A similar case due to diphenylhydantoin (DPH) was published earlier (Holland & Mauer, 1964). The aim of this study was to evaluate the usefulness of the lymphocyte-stimulation tests in vitro in seven cases of identical hypersensitivity reactions to CBZ and in one case to DPH as well. As the time of testing might influence the outcome of the test, lymphocyte-stimulation tests in vitro were sequentially performed in two cases and later repeated with cryopreserved lymphocytes. Lymphocyte reactivity to drugs in vitro was compared with reactivity to PPD and PHA. In addition, patch tests were performed, although these are generally believed to be of limited value in diagnosing rashes caused by systemically-administered drugs (Agrup, 1972). Present address: Bonifatius Hospital, Leeuwarden, The Netherlands. Correspondence: Dr G. C. de Gast, University Hospital, Department of Internal Medicine, Oostersingel 59, Groningen, The Netherlands. *
272
Tests in carbamazepine hypersensitivity
273
MATERIALS AND METHODS Patients. The clinical features and some laboratory findings from all seven patients are summarized in Table 1. In all cases treatment with CBZ had been started 14-30 days before the onset of the hypersensitivity reaction. The diseases for which this drug was given were trigeminal neuralgia and temporal epilepsy. Some of the patients had already been treated with phenobarbitone; one patient (no. 1) started treatment with DPH as well as with CBZ 14 days before the development of the allergic reaction. Because an allergic reaction to CBZ or DPH was suspected, these drugs were discontinued and replaced, if necessary, by other anticonvulsants. All patients showed an allergic reaction consisting of fever, rash, facial oedema, lymphadenopathy, impaired liver function, eosinophilia and atypical lymphocytes in the peripheral blood. In addition to these symptoms and signs, one patient (no. 6) had in the acute phase of the disease impaired renal function, and a chest X-ray showed diffusely-arranged nodular densities. In two patients (no. 3 and no. 4), recurrence of the symptoms after accidental ingestion of one tablet CBZ proved this drug to be the causative one. Controls. Ten patients, who had been or were exposed to the same drug without adverse effects, made up a control group. They were numbered from 1 to 10. Controls no. 1 and no. 2 were using both DPH and CBZ at the time of the tests. Controls no. 3 and no. 4 stopped using both drugs about 16 weeks before the tests. Controls no. 6 to no. 10 had not been treated with CBZ for 10-52 weeks prior to testing. In order to avoid sensitization to drugs they needed, controls no. 1 and no. 2 did not undergo patch tests. Patch tests were also performed on ten healthy persons. Lymphocyte stimulation test. Lymphocyte-stimulation tests were performed as described elsewhere (DuBois et al., 1973). 300,000 lymphocytes, which had been isolated from defibrinated blood by Ficoll-Isopaque gradient centrifugation, were cultured in tubes (Nunc, Denmark) containing 1 ml medium RPMI 1640, HEPES buffer, 20% pooled human serum, 100 u of penicillin and 100 pg of streptomycin. CBZ* and DPH were dissolved in pure propylene glycol and subsequently diluted with RPMI 1640. Cultures with phytohaemagglutinin (PHA, Burroughs Wellcome) 5 ul/ml were incubated for 3 days in tubes with tight caps. Cultures with CBZ or DPH and a solvent blank with propylene glycol 0 5%4 as well as cultures with tuberculin (PPD without preservativet) 10 pg/ml were incubated under the same conditions for 6 days. The drugs were added in a volume of 0 05 ml, yielding a final concentration of 3 pg/ml, 10 pg/ml or 30 pg/ml CBZ or DPH and 0 5%4 propylene glycol. Previous experiments showed cytotoxicity with higher concentrations of CBZ and DPH. Cultures were done in quadruplicate: three for thymidine incorporation and one for morphological control. 24 hr before harvesting 0 5 PCi [3H]thymidine (Radiochemical Centre, Amersham, sp. act. 40 mCi/mmol) was added. After harvesting radioactivity was measured in a Packard liquid scintillation counter and expressed as disintegrations/min (d/min) per culture. The arithmetical mean and standard deviation (s.d.) were calculated. The stimulation index (SI) was calculated as follows: d/min in presence of drug SI d/min in presence of solvent An SI of more than 2-0 was regarded as evidence of stimulation. In two cases (no. 1 and no. 2) mononuclear cells were frozen and stored for sequential studies, as described by Leguit et al. (1973), in addition to cultures with fresh lymphocytes. Patch tests. Patch tests were performed with silver patch testers (v.d. Bend, Brielle) and read according to the method of the International Contact Dermatitis Research Group (Malten, Nater & Ketel, 1976). CBZ as well as DPH were tested at concentrations of 10%Y, 20%/ and 40% in vaseline. The skin reactions were read after 48 and 72 hr. =
RESULTS Significant lymphocyte stimulation in vitro by CBZ could finally be demonstrated in all seven cases and by DPH as well in one of these cases (no. 1, Table 2). Maximal stimulation was generally obtained with the highest drug concentration (30 pg/ml) used. SI in the cultures with CBZ varied between 3-6 and 58-0 (median 7.9). In one case (no. 7) a positive lymphocyte stimulation was found even 7 years after the illness. Lymphocyte cultures from the controls showed no stimulation by CBZ or DPH. Follow-up studies were carried out on two cases (no. 1 and no. 2) with fresh and with cryopreserved lymphocytes. The results obtained with the cryopreserved lymphocytes are shown in Table 3. In case no. 1 significant stimulation by CBZ was noted for the first time 21 weeks after the onset of the allergic reaction and the effect continued to increase after that. Lymphocyte stimulation by DPH followed a similar course. In case no. 2, lymphocytes were not stimulated by CBZ during the 8 weeks stay in * Carbamazepine (Tegretolg) was kindly supplied by Ciba-Geigy, Basel, Switzerland. t Rijksinstituut voor de Volksgezondheid Bilthoven, The Netherlands.
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Tests in carbamazepine hypersensitivity TABLE 2. Results of lymphocytc-stimulation tests
Cell source Patient no. 1 (21 w)* 2 (49 w) 3 (5 y) 4 (3 y) 5 (13 w) 6 (5 y) 7 (7 y) Control no. 1 2 3 4 5 6 7 8 9 10
DPH
CBZ
PHA (d/min+ s.d.)
Solvent (d/min+ s.d.)
(d/min+ s.d.)
SI
(d/min+ s.d.)
SI
13,840+1365 35,765+ 1567 42,837+6160
97+24 843+ 117 278+76 226+48 152+38 427+ 113 158+74
5699+1107 6112+ 1074 2209+302 1104+ 178 5522+313 1538+ 299 1272+356
580 7-2 7*9 4-8 36-3 3-6 8-0
7217+512 1036+300 383+ 130 434+ 120 n.d. 324+ 54 n.d.
74.4 1-2 1-3 1.9
60+ 12 144+47 219+ 38 593+57 566+264 245+ 134 400+ 132 80+ 14 388+ 53 548+231
86+30 191+49 310+ 35 650+258 520+ 147 242+ 178 616+ 182 144+ 84 316+ 54 597+48
1-4 1-3 1-4 1.1 09 1.0 1-5 1-8 0-8 10
62+40 178+38 288+ 45 629+292 729+285 248+ 62 512+ 118 142+ 58 277+ 133 612+ 156
08 12 1-3 1.1 1-3 1.0 1-3 1-7 07 1.1
14,943+2615 26,198+5308 83,624+ 4107 25,692+ 1240
26,385+ 104 23,334+1937 27,245+ 2367
22,457+1138 48,093+2611 26,374+ 6627 17,523+ 1147 24,480+ 153 23,612+ 1486 60,999+ 14,167
-
0-7 -
n.d. = Not done. * Time in weeks (w) or years (y) after onset of illness. TABLE 3. Results of sequential lymphocyte stimulation tests in two patients
Cell source
Patient no. 1 5 w* 11 w 21 w 82 w Patient no. 2 4w 8w 49 w 60 w
CBZ (d/min+ s.d.)
SI
DPH (d/min+ s.d.)
77+20 34+ 17 97+24 809+ 148
111+23 108+42 5699+1107 16817+4467
1-4 1-7 580 20-7
122+34 n.d. 7217+512 19321+2813
268+46 112+ 69 843+ 117 676+ 167
510+ 112 179+ 44 6112+ 1074 5077+ 335
1.9 1-6 7-2
Solvent
PHA (d/min+ s.d.)
(d/min+ s.d.) (d/min+ s.d.)
5637+714 10202+233 13840+1365 43510+ 1531
5292+380 4729+468 12401+765 30487+ 1729
6929+ 1187 3350+ 387 35765+ 1567 24255+ 1531
306+ 56 876+ 183 4157+290 4137+ 530
PPD
7.5
n.d. n.d. 1036+300 n.d.
SI 1-6 -
74-4 23-8 -
12 -
* Weeks after onset of illness.
hospital. 40 weeks later, however, a positive result was obtained and was confirmed in a later test. The lymphocyte responses to PHA and PPD in cases no. 1 and no. 2 were parallel to those to the drugs. When there was no stimulation to the drugs, PHA and PPD stimulation were low. Later on when stimulation to drugs was evident, PHA and PPD stimulation were on a higher level. Patch tests with CBZ were positive in six of the seven cases (Table 4). No differences in the skin reactions were observed when patch testing with concentrations of 10%, 20% and 40%0 of CBZ. Control subjects showed no reactions. In case no. 2 patch tests with CBZ showed the same sequence as the lymphocyte stimulation tests, being negative in the initial phase but positive afterwards. A patch test with DPH in case no. 1 was negative.
276
J. Houwerzijl et al. TABLE 4. Rcsults of patch tests (read after 72 hr) Patient no. 1 (23 w)* 2(50 w) 3(5 y) 4 (2- y) 5(14 w) 6 (5 y) 7 (7 y) 18 controlst
CBZ
DPH
+++ ++++
_
+ ++
n.d. n.d.
See Malten et al. (1976). * Time in weeks (w) or years (y) after onset of illness. t Including C3-C0I and ten healthy persons.
DISCUSSION In diagnosing drug hypersensitivity, lymphocyte-stimulation tests often give false negative results (MacKinney & Booker, 1972; Vischer, 1966). Since false positive responses are rare, all patients with positive results have to be regarded as established cases of drug hypersensitivity (Mathews et al., 1972; Sarkany, 1967). The test enabled us to confirm an already proven hypersensitivity to CBZ in cases no. 3 and no. 4, even several years after the last exposure. In five other cases hypersensitivity to CBZ was diagnosed and in one case (no. 1) hypersensitivity to DPH was also diagnosed. One of the factors that plays a role in the outcome of the tests is the poor solubility of many drugs which often gives technical problems. It was necessary to dissolve the drug with propylene glycol. Subsequent dilution with RPMI 1640 medium yielded a concentration at which stimulation of lymphocytes was possible without toxic effects. Another factor of importance appears to be the clinical picture of the hypersensitivity reactions. Lymphocyte stimulation is often successful with drugs, which cause the appearance of blasts in the peripheral blood (Holland & Mauer, 1964; Sarkany, 1967; Virolainen, 1971), as well as with drugs which cause hypersensitivity characterized by fever and rashes (Mathews et al., 1972). In all cases all these features were present. Patch testing is an established method of diagnosing allergic contact dermatitis. It is not usually recommended for diagnosing rashes caused by systemically used drugs (Agrup, 1972). Nevertheless, in some instances promising results have been reported (Felix & Comaish, 1974). This study shows that patch testing can be helpful in detecting hypersensitivity to CBZ. Positive results are often obtained first used externally and afterwards internally (Fischer, 1966; Agrup, 1972). In cases of hypersensitivity reactions to CBZ external exposure seems very unlikely. In case no. 1 the negative patch test with DPH contrasted with the positive lymphocyte stimulation in vitro by DPH. The skin rash of this patient may therefore be due to CBZ only. Alternatively both drugs may be involved, which implies that DPH is not suitable for skin testing. The results of both lymphocyte stimulation tests and patch tests appear to be dependent on the time of testing. In two cases (no 1 and no. 2) sequential testing, performed with cryopreserved lymphocytes to avoid differences in culture conditions, indicated that stimulation in vitro by drugs produced negative results during and shortly after the illness. This probably applies to in vivo tests as well, because the first patch test in case no. 2 was negative. Besides, a transient general impairment of lymphocyte responsiveness in vitro is suggested by impaired responses to PHA and PPD in the acute phase of the disease. This phenomenon has already been observed in cases with active tuberculosis, viral disease and even leucocytosis of any aetiology (Pearman, Lycette & Fitzgerald, 1963; Mangi et al., 1974; Heiss & Palmer, 1974). As far as we know, an association between drug hypersensitivity reactions and impaired lymphocyte
Tests in carbamazepine hypersensitivity
277
reactivity in vitro has not been reported. The basic mechanism of the impaired lymphocyte reactivity is not clear. The impaired response to PHA, a predominantly T-cell mitogen, indicates that T-cell function is surely affected. Our findings show that testing during or shortly after the illness may lead to false negative results. When negative results are obtained, both tests should be repeated within 2 or 3 months after complete recovery. The clinical picture of our patients consisted of fever, rash, facial oedema, lymphadenopathy, eosinophilia, lymphocytosis (including many atypical ones) and in six cases impaired liver function tests. This is very similar to the reported 'serum-sickness-like' reactions due to hydantoins, para-amino salicylic acid, phenylbutazone, iron dextran, cephalothin and cephapirin (Saltzstein & Ackerman, 1959; Schen, 1964; Sanders, Johnson & Taggart, 1974), indicating that CBZ is also able to induce such a clinical picture. Results of in vitro lymphocyte-stimulation tests and patch tests suggest involvement of delayed-type hypersensitivity. The incidence of this type of reaction to CBZ is obviously low. Only three similar cases have been published so far (Steen-Johnson, 1970; Virolainen, 1971; Heber, 1972). Because of its effectiveness in temporal epilepsy and trigeminal neuralgia the use of CBZ is still growing. That is why an increase of hypersensitivity reactions to CBZ may be expected. As CBZ is often prescribed for long periods together with other anticonvulsants, it is important to be able to prove a hypersensitivity reaction to this drug. The lymphocyte-stimulation test and the patch test with CBZ, if performed at the right time, have proven to be of value in diagnosing hypersensitivity to CBZ. This work was supported by the 'Nederlandse Vereniging tot Rheumatiekbestrijding'. We thank Miss T. Woest for technical assistance, Dr T. H. The for advice and criticism and Mrs H. Jellema-Brazier for revising the English.
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