family history of thyroid disease. No correlation was found between the

Transcription

1 THE CLINICAL SIGNIFICANCE OF THE COMPLEMENT- FIXATION TEST IN THYROTOXICOSIS W. WATSON BUCHANAN, D. A. KOUTRAS, J. CROOKS, W. D. ALEXANDER, W. BRASS, J. R. ANDERSON, R. B. GOUDIE and K. G. GRAY University Department of Medicine, Gardiner Institute and the Department of Pathology, University of Glasgow, Western Infirmary, Glasgow, W. l,and the Statistics Department, University of Aberdeen (Received August 6) SUMMARY The significance of a positive thyroid complement-fixation (c.f.) test in thyrotoxicosis has been investigated by studying the correlation between various features of the disease in patients. A significant correlation was found between the positivity of the c.f. test and () the degree of lymphocytic infiltration in the gland; () incidence of postoperative hypothyroidism; () size of the goitre; () previous treatment with radioiodine, and (5) a family history of thyroid disease. No correlation was found between the results of the test and the incidence of reactions to antithyroid drugs. The results suggest that thyrotoxic patients with positive c.f. tests should be treated initially with antithyroid drugs unless there is a definite indication for surgery. INTRODUCTION Auto-antibodies of thyroglobulin have been reported in a high proportion of patients with thyrotoxicosis (Roitt & Doniach, 58; Anderson, Goudie & Gray, 5; Blizzard, Hamwi, Skillman & Wheeler, 5). In a few patients precipitating auto-antibodies have been found (Goudie, Anderson, Gray, Clark, Murray & McNicol, 5; Roitt & Doniach, 58; Anderson et al. 5; Belyavin & Trotter, 5; Buchanan, Alexander, Crooks, Koutras, Wayne, Anderson & Goudie, 6a), and in these patients histological examination of the thyroid gland has revealed severe and extensive thyroiditis indistinguishable from Hashimoto's disease, in addition to areas typical of thyrotoxicosis (Doniach & Hudson, 5; Doniach, Hudson & Roitt, 6; Buchanan, Crooks, Alexander, Koutras, Wayne & Gray, 66). However, in the majority of patients these thyroid auto-antibodies are of low titre and detected only by tanned red-cell agglutination (t.r.c.) methods (Paine, Terplan, Rose, Witebsky & Egan, 5; Roitt & Doniach, 58; Blizzard et al. 5; Cune, Selenkow & Brooke, 5). It has been suggested that the auto-antibodies detected by the t.r.c. method also bear a relationship to the degree of lymphocytic infiltration (Doniach & Roitt, 5; Blizzard et al. 5; Schade, Owen, Smart & Hall, 6), and Blagg (6) has reported a correlation with the outcome of I therapy. 8 Endoc.,

2 6 W. Watson Buchanan and others In addition to antibodies to thyroglobulin, auto-antibodies to the 'microsomal' antigen can be detected in a large number of patients with thyrotoxicosis by the complement-fixation (c.f.) test (Roitt & Doniach, 58; Anderson et al. 5; Belyavin & Trotter, 5), but the significance of these antibodies is not known. The present study reports a clinical and pathological assessment of the c.f. test in thyrotoxic patients. The problem has been approached in three ways. First, we have studied the c.f. test in relation to the clinical features of thyrotoxicosis and to the response to antithyroid therapy. Secondly, we have carried out serial deter minations of the c.f. test following various forms of antithyroid therapy. Thirdly, we have studied the relation between the results of the c.f. test and the extent of round-cell infiltration found in the thyroid gland after operation. MATERIALS AND METHODS Four hundred and sixty-eight patients with thyrotoxicosis were studied, of whom 5 were untreated ( males, 6 females) and 5 ( males, 65 females) had received specific antithyroid therapy. The diagnosis was based on the clinical criteria described by Crooks, Murray & Wayne (5) and confirmed in every case by radioiodine tests, including measurements of the hr. gland uptake and of the 8 hr. protein-bound radio-iodine (PBI; Wayne, 6). Additional investigations in cluded : basal metabolic rate (b.m.r.) or serum protein-bound iodine (PBI) estimations, thyroxine or triiodothyronine suppression tests, and the response to antithyroid drug therapy. Complement-fixation test The c.f. test was performed as a -tube test as described by Anderson et al. (5). The first tube was tested for anticomplementary activity. In tubes and the serum was tested, undiluted and at a : dilution, respectively, for c.f. antibodies. The following additions were made to the tubes in the order shown : Tube Tube Tube - ml. test serum - ml. test serum - ml. of : test serum in saline - m.h.d. complement -5 m.h.d. complement -5 m.h.d. complement - ml. antigen - ml. antigen After hr. at c -5 ml. of a % suspension of sheep red cells, sensitized with four minimal haemolytic doses (m.h.d.) of immune horse serum (Burroughs Wellcome Ltd), was added to each tube to determine the presence of residual complement. After a further hour at the degree of lysis in each tube was observed visually. Sera effecting any inhibition of lysis in tube were regarded as anticomplementary. Partial or complete inhibition of lysis in tube alone was regarded as a positive ( + ) reaction, and in tubes and as a strongly positive ( ) test. With each set of tests were included known weakly and strongly positive, and negative control sera. Complement consisted of guinea-pig serum which was titrated each day, before use, and was diluted with saline solution to provide the required amount in - ml. The antigen was a sahne extract of surgically removed thyrotoxic gland tissue, and as the potency of the antigen was found to decline with storage, each set of c.f. tests included tests of potency of the antigenic extract. Antigen was used only when it

3 Complement-fixation in thyrotoxicosis exhibited full potency at a dilution which had an anticomplementary activity of < -5 m.h.d. of complement. The specificity of the c.f. reaction for thyroid tissue was checked using saline extracts made from other organs (e.g. liver, adrenal and kidney). All test sera, tissues, tissue extracts and complement were stored at 5 c, and the test sera were usually examined within week of collection, each serum being heated at 55 c for min. before testing. Using this test system we detected (Goudie, Anderson & Gray, 5) CF. anti thyroid in the serum of 6-8% of 86 hospital patients with no clinical evidence of thyroid disease. These antibodies were found over five times more frequently in females over the age of 5 years than in other individuals. The precipitin test for thyroglobulin auto-antibodies was carried out by an agar-diffusion method on Ouchterlony plates as described by Anderson et al. (5). Clinical observations The following clinical criteria were recorded in addition to the age and sex of the patient: the presence of a goitre, its consistency (diffuse or nodular), its size (slightly enlarged: approx. 5 g.; moderately enlarged: 5 g.; very large: g. or more), and its duration (less than year; -5 years; 5 years and more). In all cases the 'clinical diagnostic index' of Crooks et al. (5) was recorded. This index is a quantitative estimate of the symptoms and signs and correlates with the severity of the disease. Values lower than + indicate the euthyroid range; values between + and + the equivocal range; and values exceeding + the thyrotoxic range. The presence of ocular signs was noted in every case, the criteria being those described by Crooks et al. (5). The type of treatment given (if any) before the first c.f. test was noted in each case. Potassium perchlorate was the most frequently used antithyroid drug. In the case of I therapy the doses were prescribed by the method of Crooks, Buchanan, Wayne & Macdonald (6) and the number of doses given recorded. The relationship between the results of the c.f. test and the outcome of treatment was studied. Hypothyroidism following surgery or I was recorded only if permanent (Crooks et al. 6) and was confirmed by cholesterol, b.m.r. or PBI determinations and ECG studies. Resistance to I therapy was defined as persistence of hyperthyroidism after two or more doses. The incidence of drug reactions including skin rashes and haematological complications was noted. A patient was considered to have a family history of thyroid disease if aware of a relative with a thyroid disorder. Serial studies of thyroid auto-antibody tests (both precipitin and c.f.) were done in 5 of the untreated group before and after receiving various forms of therapy. In all, 6 tests were carried out in the group and the patients were followed up for periods varying from to months (mean follow-up 6- months). Histological investigations Histological sections were available for study in fifty-seven cases and the degree of round cell infiltration was related to the result of the c.f. test. Round cell infiltration was assessed by counting the number of low-power fields containing round-cell aggregates out of fifty fields examined for each gland. This examination was done by one of us (R.B.G.) who had no knowledge of the results of the c.f. tests.

4 W. Watson Buchanan and others STATISTICAL METHODS The data are presented in contingency tables showing the distribution of patients with each characteristic by the reaction to the c.f. test. Any apparent variations in the c.f. test results with the other classifications were examined for statistical significance. The usual test of association in contingency tables is insensitive when the groupings are in some natural order of measurement and any relations may be expected to vary consistently with this natural order. Here, tests more sensitive than can be devised to examine the hypothesis that the distribution of patients with a given characteristic changes progressively with increasing positiveness of the c.f. reaction. There are a number of tests of this kind (Williams, 5; Cochrane, 5; Armitage, 55). A simple but reasonably efficient method was used in the present analysis. Negative, positive and double positive c.f. reactions were scored, and, respectively, and the analysis was carried out as if these were scale measurements. It is convenient to make comparisons among different groups in terms of the mean values of the measurements (called 'mean scores'). When the other characteristic considered was a dichotomy, the null hypothesis of no association was examined by a t test of the difference between two mean scores. The test was applied exactly as if the scores were measurements. Thus, the variance was estimated from the pooled sums of squares of the two sets of scores about their means, divided by the degrees of freedom (two less than the number of observations). The ratio of the difference between the two means to its standard error, estimated from the the t value. When the other characteristic sample variance by the usual formula, gave was also an ordered classification it was likewise treated as a scale measurement with the groups at equal intervals apart, and the regression of the c.f. score on this measurement was tested by the standard procedure for analysing quantitative data. In general, only a t test of the linear component of regression was applied, but for one relationship (with age) the quadratic component, which was clearly important, was tested also. The standard criteria of statistical significance are used, i.e. t deviations which would have been exceeded by chance in < 5 % and % of trials on the null hypothesis of no association are called ' significant ' and ' highly significant ', respectively. The probabilities that the observed differences or trends would have occurred by chance, on the null hypothesis of no association in the population, are shown in the tables. RESULTS () Clinical significance of single determination of the c.f. test The basic data are shown in Table. Since no correlation was found between the sex of the patient and the result of the c.f. test, male and female cases were analysed together. On the other hand, antithyroid therapy was found to influence the results of the test (see below), and therefore patients who had received no previous treatment (5 cases) and those previously given antithyroid therapy (5 cases) were analysed separately. For convenience these groups are referred to below as 'untreated' and ' treated ', respectively.

5 ) Complement-fixation in thyrotoxicosis ' (a) Untreated' group (5 patients) Age. There were no significant differences in the mean ages of the groups with a ( test (- years), a ( + ) test (- years), and a ( ) test (- years). How ever, as can be seen from Table, the positivity of the c.f. test was increased in the middle-aged group (-6 years); consequently the quadratic component of the change in mean score with age was highly significant. The mean gland sizes in the Table. Relation between complement-fixation test and clinical features of thyrotoxicosis No. of cases and results of c.f. test ' Untreated ' ' Treated ' Clinical features Age (years) and over Type of goitre Diffuse Nodular Absent Size of goitre (g.) 5 5 and more Duration of goitre (months) and over Duration of symptoms (months) and over 8 8 Severity of symptoms (clinical diagnostic index*) Under and over 5 5 Eye signs Exophthalmos Severe exophthalmos Drug reactions 5 Family history of thyroid 6 disease * See text, p..

6 W. Watson Buchanan and others groups aged 6 years or more (- g.) and less than years of age (6- g.) were lower than in the group aged between and 6 years (- g.), but this did not wholly account for the higher incidence of positive tests in the middle-aged group. Table. Relation between age and complement-fixation Age (years) Under and over 5 6 No. of cases with c.f. test test in 'untreated' cases 8 5 Mean score < - for quadratic component with age. Table. Relation between size and duration of goitre and complement-fixation test in 'untreated' cases No. of cases with c.f. test Size (g.) Absent 5 5 Duration (months) and over Mean score J <-5 <-5 Goitre. The positivity of the C.F. test was directly related to size of the goitre (P < -5) and inversely related to its duration (P < -5). The results of the c.f. test did not vary significantly with the consistency of the goitre. Intensity and duration of symptoms. There was no indication that the results of the c.f. test were related to either the severity of the illness, as assessed by the clinical diagnostic index, or the duration of the symptoms. Eye signs. There was no correlation between the result of the c.f. test and the presence of exophthalmos of Grave's disease or of exophthalmic ophthalmoplegia. This is in contrast to the findings of a relationship of t.r.c. antibody titres to exoph thalmos by Hales, Myhill, Rundle, Mackay & Perry (6). Family history. Patients with a family history of thyroid disease had a higher incidence of positive c.f. tests (P =a» -). The same trend was also found in the group with previous antithyroid treatment, and when the evidence from both 'untreated' and 'treated' groups is considered together the results are significant (P < -5). Drug reactions. Reactions to antithyroid drugs, either mild or severe, did not vary with the results of the c.f. test. Response to treatment. The results of partial thyroidectomy were correlated with

7 ) Complement-fixation in thyrotoxicosis the c.f. test obtained pre-operatively (Table 5). There was a tendency for patients with ( tests to relapse after operation whereas those with ( ) tests had a higher incidence of hypothyroidism (P -5). In the = case of I therapy the reverse effect was found and the test was more positive in the order : hypothyroid, euthyroid with one dose, and resistant (P < -). Further analysis showed that this was partly due to the fact that the mean gland size was smauer in the group with negative tests (5 g.) than in the group with ( + ) (65 g.) or ( ) (8 g.) tests, although the residual trend when gland size had been allowed for was still significant. Table. Relation between family history of thyroid disease and complement-fixation test Family history No. of cases with c.f. test + Mean score ' Untreated ' With Without ' Treated ' With Without < -5 when 'treated' and 'untreated' groups are considered together. Table 5. Relation between outcome of treatment and complement-fixation test No. of cases with c.f. test Response to treatment Surgery Relapse Euthyroid 6 Hypothyroid I therapy Resistant Euthyroid ( dose) Hypothyroid Mean score < - ( ) 'Treated' group (5 patients) There was a significantly higher number of patients with positive c.f. tests in the 'treated' than in the 'untreated' group (P < -). From Table 6 it can be seen that this was largely due to the cases which had been previously treated with mi. The results of the c.f. test for these were substantially more positive than the corresponding measurements for other treated patients (P < -), but the latter did not differ significantly from the values for the untreated cases. Furthermore, there was a significant correlation (P < -5) between the positivity of the c.f. test and increasing doses of I. The 'treated' group as a whole was similar to the 'untreated' group with respect to mean age, consistency of the goitre and clinical severity.

8 W. Watson Buchanan and others () Effect of therapy on the complement-fixation test The effect of therapy on serial measurements of the c.f. test is shown in Table. It can be seen that the incidence of negative tests is higher following treatment with antithyroid drugs and the incidence of positive ( ) tests is higher following I therapy. Neither of these trends is significant when considered separately, but when taken together this difference is significant (P < -5). This is consistent with the finding of a higher incidence of positive tests in patients who had been treated with I therapy. It is possible that had the follow-up period been longer, the trend towards a positive test in those who had received I therapy would have been more apparent. Table 6. Relation between previous treatment and complement-fixation test No. of cases with c.f. test + Mean score ' Untreated ' 'Treated' Drugs + surgery Drugs only J treated No. of doses of I or more treated Ml -88 < - < - Table. Changes in complement-fixation test after I and antithyroid drug therapy Before treatment + I therapy after treatment 6 6 Change in mean c.f. score = +-. Before treatment Antithyroid drugs after treatment Change in mean c.f. score = -5. < -5 for difference in changes in mean c.f. scores.

9 - S Complement-fixation in thyrotoxicosis () Relation between complement-fixation test and degree of round-cell infiltration in thyroid It can be seen from Fig. that there is a direct relation between the degree of round-cell infiltration in the thyroid and the positivity of the c.f. test. There is, however, a wide scatter in the distribution of the infiltration measurements in the three c.f. test categories. To reduce the erratic effects of the few extreme observations roots of the measurements of round-cell the analysis was carried out on the square infiltration. The relation with the c.f. test results was then found to be highly significant (P < -). O Positive Precipitin test v -s H -- O i- S I - -J-- d :» Fig.. Complement-fixation test Relation between c.f. test and round-cell infiltration in thyrotoxic glands. DISCUSSION In the present study a significant correlation has been demonstrated between the results of the c.f. test and the degree of round-cell infiltration in the thyroid gland (Fig. ). This may be of practical importance since Whitesell & Black () and Bartels (5) observed the incidence of postoperative hypothyroidism to be greater with increasing lymphocytic replacement in the thyroid gland. Furthermore, in a clinico-pathological study of thyroid operations Levitt (5) found a % incidence ofpostoperative hypothyroidism in the thyrotoxic gland showing 'epithelial hyperplasia ', but % in those showing ' diffuse lymphoid infiltration '. The difference was even more striking in the series of 5 patients reported by Greene (5) - and 8-%, respectively. These observations have been confirmed in the present study and, moreover, the c.f. test has been shown to correlate with the degree of round-cell infiltration in the thyroid gland (P < -), and so to be of some predictive value in the outcome of surgical treatment (P -5). We conclude that thyrotoxic = patients with a positive c.f. test are best treated initially with a course of antithyroid drugs unless there is a definite indication for surgery. Schade et al. (6) have reported a correlation between the degree of round-cell infiltration in thyrotoxic glands and the level of t.r.c. auto-antibodies, but concluded that there was no relationship with the c.f. test. This conclusion seems unjustified since these authors found a higher incidence of positive c.f. tests in patients with

10 W. Watson Buchanan and others lymphoid infiltration in the thyroid gland ; the absence of statistical significance can be attributed to the small number of cases (ten positive c.f. tests in total). Further more, these authors carried out their serological tests at varying intervals after thyroidectomy which is known to produce a fall in the circulating antibody levels (Owen & Smart, 58). We found a much higher incidence (P < -) of positive c.f. tests in our patients who had received treatment with I (Table 6). Furthermore, serial determinations of the c.f. test in patients receiving I therapy showed a significantly greater increase in positive c.f. tests than in those receiving antithyroid drugs (Table ); but the precipitin test showed no change. The increased positivity of the c.f. test after I therapy may be related to the histological lesions resembling Hashimoto's thyroiditis which have been found by various workers in the thyroid gland after I therapy (Freedberg, Kurland & Blumgart, 5; Dailey, Lindsay & Miller, 5; Lindsay, Dailey & Jones, 5). It has therefore been suggested that destructive forms of antithyroid therapy might predispose the thyroid to the development of auto-immunity (Doniach & Roitt, 5 ; Blizzard et al. 5). Profound morphological changes are found in the thyroid epithelial cells after I therapy (Curran, Eckert & Wilson, 58) which may initiate an auto-immune process due to leakage ofmicrosomal antigen. However, an interesting finding is the absence of correlation between the result of the c.f. test and the success of treatment with I; this may be attributable to uneven irradiation of the gland affected by chronic thyroiditis. Correlation of the results of a positive c.f. test with age shows a striking similarity to the age distribution of ' focal thyroiditis ' found in a large series of thyroid glands by Woolner, McConahey & Beahrs (5). The low incidence of positive c.f. tests in the patients under years of age might conceivably be due to persistence of immunological tolerance to thyroid antigens. In view of the observations of blood group iso-antibodies by Thomson & Kettel () and of antibodies to influenzai viruses by Sabin, Ginder, Matumoto & Schlesinger (), the low incidence of positive c.f. tests in patients over the age of 6 years may be due to a diminished antibody-forming potential. The absence of significant sex difference in the incidence of positive c.f. tests in thyrotoxicosis is in striking contrast to the increased incidence of positive c.f. tests in euthyroid females without overt thyroid disease (Goudie et al. 5). Although there is an increased prevalence of all types of thyroid disease in the female, it appears that there is no immunological difference in affected males and females. Blizzard and his associates (5) reported an increased incidence of penicillin reactions in patients with thyroglobulin auto-antibodies, but we have found no correlation between the results of the c.f. test and the incidence of reactions to antithyroid drugs. Hall, Owen & Smart (6) have produced evidence of a genetically determined predisposition to the formation of thyroid auto-antibodies. We have shown that patients with a family history of thyroid disease have a higher incidence of positive c.f. tests. These findings are comparable to the higher prevalence of rheumatoid arthritis found in relatives of patients with seropositive rheumatoid arthritis than in relatives of those with negative Rose-Waaler tests (Lawrence & Ball, 58). This comparison between the two diseases is particularly relevant in view of the known association between auto-immune thyroiditis and rheumatoid arthritis (Buchanan

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