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HYPOGAMMAGLOBULINÆMIA IN THE UNITED KINGDOM
Saturday HYPOGAMMAGLOBULINÆMIA IN THE UNITED KINGDOM Summary Report of a Medical Research Council
Working-party* Between 1956 and 1966 information was collected about 176 patients in whom primary hypogammaglobulinæmia was diagnosed and in whom the serum-IgG level was shown to be less than 200 mg. per 100 ml. (below 100 mg. per 100 ml. in infants under 6 months). Clinically the condition varied widely between patients, owing partly to changes wrought by previous infections and partly to the heterogeneity of the underlying disorder. Initially it was assumed that prophylactic administration of immunoglobulin would be valuable in reducing the incidence of infections, and trials were conducted to discover the optimal dose. In fact, only a relatively small (though statistically significant) difference in morbidity, and no difference in mortality, were observed between dose levels of 0·05 g. and of 0·025 g. per kg. body-weight per week. Failure to obtain stronger evidence in favour of the larger dose is thought to be due partly to the relatively small difference between the dose levels, and partly to the inclusion of many patients not expected to show substantial benefit from treatment. The lower dose (0·025 g. per kg.) has been used for most patients over the past two years and is recommended as the standard treatment, except for patients who continue to suffer from severe infections. Summary
Introduction
IN 1952 Bruton described the first
case of primary hypogammaglobulinaemia: a young boy suffering from repeated severe bacterial infections, who showed evidence
antibody production and was very greatly by improved injections of immunoglobulin. (In the past of deficient
few years the term " immunoglobulin " has tended to replace y-globulin.) Within a year, Janeway et al. (1953) were able to report 9 further patients with hypogammaglobulinsemia; these authors gained the impression that monthly injections of 0-1 g. per kg. pooled human normal immunoglobulin reduced the incidence of infections, whereas continued administration of antibiotics was only partially successful. In 1955, when the syndrome of hypogammaglobulinaemia had become more widely known in this country (Keidan et al. 1953, Grant and Wallace 1954, Hutchison 1955), it was suggested by the Ministry of Health that the Medical Research Council * Members of the working-party: the late Professor J. R. SQUIRE (chairman until 1966), Prof. P. L. MOLLISON, F.R.S. (chairman from 1966), Dr. W. R. S. DOLL, F.R.S., Dr. J. W. FARQUHAR, Dr. F. V. FLYNN, Dr. D. M. T. GAIRDNER, Prof. P. G. H. GELL, Mr. M. J. R. HEALY, Dr. LISA E. HILL, Prof. R. A. KEKWICK, P.R.S., Dr. F. O. MACCALLUM, Prof. R. G. MACFARLANE, F.R.S., Prof. N. H. MARTIN, Dr. W. D’A. MAYCOCK, Dr. J. F. SOOTHILL, Prof. Sir RONALD TUNBRIDGE, Mr. L. VALLET, Prof. Sir EDWARD WAYNE, Dr. H. W. BUNJÉ (secretary). 7587
25
January 1969
might set up a working-party to study the problems of hypogammaglobulinasmia, including the range of clinical syndromes and various aspects of the therapeutic use of immunoglobulin. Physicians who wished to obtain immunoglobulin for the treatment of their patients, or who suspected a diagnosis of hypogammaglobulinsemia and wished to have it confirmed, were referred to the Medical Research Council and invited to join the study. Physicians could obtain immunoglobulin without joining the study provided that the criteria laid down by the working-party for the diagnosis were satisfied, or that there was clinical evidence of defective immunity despite a serum-IgG level of more than 200 mg. per 100 ml. Material and Methods Criteria for Diagnosis Admission to the study was confined to patients with primary hypogammaglobulinxmia; the investigation therefore excluded patients with generalised hypoproteinxmia or the nephrotic syndrome and those with reticuloses or other malignant disease, and also premature infants with temporary
hypogammaglobulinxmia. The major criterion for admission to the study was a serumIgG level of 200 mg. per 100 ml. or less or, in infants of 6 months
or
below, 100
mg. per 100 ml.
or
less.
Estimations For the first two years of the study serum-immunoglobulin levels were estimated by three methods: free-solution electrophoresis, which estimated total y-globulin, and two methods which estimated only IgG-namely, a method using the inhibition of antiglobulin serum and a double gel-precipitin technique (Kekwick et al. 1961). Subsequently only the gelprecipitin method was used, and in the last year of the study this was replaced by a single diffusion precipitin technique (Fahey and McKelvey 1965). As antisera became available, IgM, IgA, and IgD levels were also estimated. A purified freeze-dried standard of immunoglobulin was used for the estimation of IgG, and results were expressed in mg. per 100 ml.; IgM, IgA, and IgD concentrations were expressed as a percentage of those contained in a reference serum (R.N.S.) obtained from a healthy adult male.
Serum-immunoglobulin
Human Immunoglobulin Used in Treatment Almost all the immunoglobulin used in treatment was prepared by the ether fractionation method from normal large-pool plasma (Kekwick and Mackay 1954) at the Blood Products Laboratory, Lister Institute; a small comparative trial with alcohol-fractionated immunoglobulin produced no evidence of any important differences between the two preparations. All the batches contained only a small proportion of IgA and IgM (mostly 4-5 mg. per ml. and 1-2 mg. per ml. respectively) and little or no IgD. Ether-fractionated immunoglobulin was found to be very stable during storage; at 4°C only very slight alterations were found after four years, and it was thought that these changes could have been due to contamination with traces of plasmin. In an accelerated storage test, in which samples of immunoglobulin were held in aseptic conditions at 37°C for twenty-eight days, the results were similar to those of the long-term storage tests.
164 The titres of certain antibodies (antistreptolysin, antistaphylolysin, diphtheria antitoxin, and neutralising antibodies to polio, herpes, and influenza viruses) were estimated in 39 batches. Assays of one batch were also made at intervals during prolonged storage at 4°C, and these showed that certain antibodies remained stable for very long periods. Design of the Trial Since there was strong presumptive evidence from the United States that therapy with immunoglobulin was of considerable benefit in reducing infections in hypogammaglobulinaemia, it was felt that there could be no question of randomised allocation of patients to different groups, one of which would not be receiving replacement therapy. The basic design of treatment trials was therefore a comparison of two different dose levels. It was not possible to disguise the difference between the
assessment of bias. any particular regimen open At first a dose of 0-025 g. per kg. per week given by intramuscular injection (based on American reports of the efficacy of 0.1 g. per kg. monthly) was compared with one of 0-05 g. per kg. per week, alternate patients starting with a high and with a low dose and the dose being changed every seventeen weeks to cut across seasonal variations in the incidence of infections. Each week at the time of injection the treating physician recorded details of infective illnesses during the preceding week, including febrile episodes, the number of days with symptoms, and the use of antibiotics. At the time of injection the actual dose given was noted and the patient’s two
was
and
doses, and thus the to
recorded.
Although body-temperature weight laboratory investigations (other than serum-immunoglobulin levels) were not specifically asked for, clinicians were given were
the record card to enter results of any that were Once during each four-month course a bloodsample was requested for estimation of immunoglobulin levels. In addition, during the later years of the trial C-reactive protein was estimated from this specimen. After three years no clear difference had been demonstrated between these two dosage levels, and a second trial was undertaken comparing a dose of 0-01 g. per kg. with 0-05 g. per kg. weekly, following the same basic plan as before. After twelve months, suspicion arose that patients on the lower dosage were doing less well than the others; and this second trial was abandoned, although in retrospect the evidence on which it was abandoned was not conclusive. Finally, a dose of 0-025 g. per kg. weekly was once more compared with 0-05 g. per kg., but patients were allowed to remain on each dose for one year. In all the trials, before starting on the weekly injections patients were given a " loading dose," of immunoglobulin-namely, 0-25 g. per kg. in five divided daily doses.
space
on
carried
out.
Other Investigations Since it was known that patients with hypogammaglobulinxmia commonly lack isohsemagglutinins, the ABO group of each patient was determined and the serum tested for anti-A and anti-B. The titres of antibodies against certain bacteria and viruses before and after replacement therapy were estimated, and antibody titres were measured after known bacterial and virus infections. In addition, in selected children a deliberate attempt was made at immunisation against poliomyelitis by feeding live vaccine and estimating the resulting antibodies. Results Classification of Cases and Prevalence of the Syndrome During the ten years of the trial, 176 patients (119 male and 57 female) fulfilled all the criteria demanded and were admitted to the study. Their age and sex distribution on admission are shown in the accompanying figure. In addition there were 24 patients known to the working-party who qualified for admission but for various reasons were not included. An attempt to relate the number of accepted cases to the population of live births from which they were
Distribution of age at entry to the
study.
(or, in the case of those born before 1956, the surviving population) suggests that the proportion of people affected at some time in their life is at least 15 per 1,000,000 for males and 4 per 1,000,000 for females. The true prevalence is doubtless greater because (1) drawn
probably some patients who died before a made; (2) there were probably some affected children who had not yet developed a characteristic clinical picture; (3) not all patients with the disease can have been brought to the notice of the workingparty, and (4) the criteria adopted by the working-party did not cover all cases of hypogammaglobulinaamia or of the antibody-deficiency syndrome. It became obvious during the study that the presenting clinical and pathological features were extremely varied, and that the cases collected were heterogeneous and not easily classifiable. It was not considered satisfactory to divide the cases into " congenital " and " acquired " as
there
were
diagnosis
could be
had been customary in the past, since those which are apparently " acquired " may possibly also have a genetic basis (Fudenberg and Hirschhorn 1965). In the present study the problem was partially avoided by classifying patients into three groups according to their age on admission (infants, under 1 year; children, 1-15 years; adults, 16 years and over), each group being subdivided according to whether they did or did not have affected relatives. Of the 176 patients admitted to the study there were 33 infants, 73 children, and 70 adults. There was a clear family history for 41 patients, including 2 females, who probably had a non-sex-linked form of the disease. There were 135 patients with no known affected relatives at the time of the conclusion of the therapeutic trial. The 39 male patients with family histories consisted of 2 adults, 26 children, and 11 infants; 36 of them had affected male relatives (17 families) admitted or qualifying for admission the study and 3 had possibly affected male relatives (not admitted). Of the patients without affected relatives 67 were adults and may have had the acquired form of the disease. 4 male infants admitted to the study, and 1 female
to
infant with a qualifying serum-IgG level who was not admitted to the study, were considered to have transient hypogammaglobulinxmia. All reached normal IgG levels
165 PRESENTING SYMPTOMS OF PATIENTS ADMITTED TO THE STUDY
the presenting feature; in approximately half the patients it was associated with disorders of the blood or arthritis and in 3 with amyloid disease. Of the patients with respiratory-tract infections almost half had permanent lung damage on admission to the study.
Pneumocystis carinii Pneumonia Interstitial-cell pneumonia due to Pneumocystis carinii found in 7 patients, all children. In 5 the diagnosis was made only at necropsy; but in 2 it was confirmed by lung biopsy during life, and treatment with pentamidine isothianate resulted in recovery (Marshall et al. 1964, Rodgers and Haggie 1964). was
Virus Diseases
(above 600 mg. per 100 ml.) by the age of 20 months subsequently maintained them, but in 3 an abnormally low IgA level persisted. Antibody response to challenging antigens was tested in 3 and was found to be normal in 2, although in 1 of these the rise in antibody and
level
was not
maintained.
3 patients, all women, recovered their ability to produce normal serum levels; in 2 there was a full normal antibody response to challenging antigens, but in the 3rd there appeared to be no recovery of antibody response.
Mortality Of the 176 patients admitted, 51 (29%) died during the period of the study; and, of these, 24 died within six months of entry. The fatality-rate in infants under 1 year was distinctly higher (45%) than the overall average and was the same for both sexes; in this group all deaths occurred within six months of entry to the study. At later ages the overall death-rate was much lower (19%), though this difference was due mostly to the much lower mortality in males at this age than in infancy. The lowest mortality-rate (11%) was in boys between the ages of 1 and 16 years, and among these mortality appeared especially low in those with affected male relatives. For various reasons, including doubt about the diagnosis, 22 of the 176 patients were not treated continuously with immunoglobulin. Among these were 8 who either refused further injections or were advised to stop treatment because of severe reactions and who remained off treatment for more than a year. All were over 2 years of age when treatment was stopped, and none stopped because of the severity of their disease; yet 5 died, giving the highest mortality of any group (63%).
Presenting Symptoms The main presenting symptoms of the patients admitted to the study are shown in the accompanying table. It should be noted that the commonest haematological disorder was neutropenia, either permanent or occurring as a transient response to infection. Half the patients with megaloblastic anaemia also had steatorrhoea; there was, however, no definite pattern of one condition preceding the other or of a definite time relationship with the onset of hypogammaglobulinaemia. Hxmolytic anaemia, thrombocytopenia, and hypoplastic anaemia were found in a small proportion of patients. Nearly 18% of patients showed splenomegaly and in some this was
Although the patients in the study were apparently able to deal normally with most virus infections, recurrent attacks of measles, mumps, herpes zoster, and herpes simplex were reported in the pretreatment histories of a number of them. 3 patients had further attacks of herpes, mumps, and varicella after the start of replacement therapy, but not one case of measles was reported. 5 children had attacks of paralytic poliomyelitis, although only one of these occurred after the start of replacement therapy. Hepatitis was reported in 4 patients (1 of these had at least eight attacks before treatment), and cirrhosis of the liver was found in 3 others. The immunoglobulin used was not considered to have caused the hepatitis. Tuberculosis 4 children and 4 adults (4-5% of all patients) had active tuberculosis, and 2 of the adults died from this
This incidence suggests, contrary reports (Parkes 1958, Barandun 1959), that cause.
hypogammaglobulinxmia
are
more
likely
published patients with to
to
contract
tuberculosis than are normal people. There was, however, evidence in the present study that patients with hypogammaglobulinaernia can convert to the tuberculinpositive state and respond normally to antituberculous drugs. However, B.C.G. and other live vaccines (see below) should be given to patients with hypogammaglobulinxmia only after steps have been taken to ensure that cellular immunity and normal lymphocytes are present.
Neurological Disorders 8 children were found to have neurological abnormalities. In most of these the abnormality was mental retardation, either congenital or following an attack of meningitis; but in 3 children there was a progressive pattern, similar in all 3, of spastic paralysis, ataxia, and optic atrophy, coming on in apparently fit children who had been on replacement therapy for between three and eight years. It is not yet clear whether this is the result of a demyelinating process or of a " slow virus " infection.
Malignant Disease 8 patients, 7 of them women, developed malignant disease which had not been apparent at the time of admission to the study. In 6 out of 8 cases the reticuloendothelial or lymphoid tissues were affected-a proportion in excess of the expected incidence. The incidence of malignant disease affecting other tissues was not in excess of the expected frequency. The Combined Immunity-deficiency
Syndrome (Thymic Alympho-
plasia) 11 infants
(5 admitted to the series and 6 qualifying admitted) showed some evidence of the combined immunity-deficiency syndrome-that is, lack of both humoral and cellular immunity. They all showed progressive lymphopenia, and at necropsy absence or but
not
atrophy of the thymus gland and poorly developed lymphoid tissues (see below). 4 of these died of generalised vaccinia following smallpox vaccination, and 1 developed generalised tuberculosis following B.C.G. inoculation. Arthritis In view of American reports of
a high incidence of hypogammaglobulinxmia (Collins and Dudley 1955, Janeway et al. 1956, Kuschner et al. 1960), 73 patients were examined clinically, radiologically, and serologically for signs of the disease. Of the 55 males,
arthritis in
were considered to have rheumatoid arthritis, whereas of the 18 females none had definite arthritis, though 3 gave a history of previous polyarthritis. On the basis of the experience of the normal population the numbers expected would be 0-40 males and 1-14 females. Thus there would appear to be an abnormally high incidence of arthritis in males with hypogammaglobulinaemia. There was no evidence that arthritis was either induced
4
or
improved by treatment.
Immunoglobulin Levels
By protocol all patients had initial IgG values of 200 mg. per 100 ml. or less, or 100 mg. per 100 ml. or less if under 6 months of age. Some IgG was detectable in the serum of all the patients, although it was frequently less than 50 mg. per 100 ml. IgM was rarely undetectable, and IgA was only occasionally undetectable. IgD was more often absent than in the normal population. There was no significant correlation between levels of the different immunoglobulins in particular patients, and the levels of the immunoglobulins other than IgG varied (even within families with several affected sibs), though in patients with transient hypogammaglobulinaemia IgM was always normal. In the patients with prolonged hypogammaglobulinaemia who had normal initial IgM values, relatively high IgG levels were common after treatment. Follow-up studies revealed a general tendency for levels of immunoglobulins other than IgG to remain in the same range in individual patients over many years, although occasional exceptions were found. The IgG
children on replacement therapy and was apparently safe and produced local resistance to the type of virus given. Passive immunisation by antibodies in the injected immunoglobulin may well further reduce such small ability to form circulating antibodies as these patients have-a phenomenon also seen in normal individuals. Replacement therapy raised the level of antibodies to various infecting organisms to between about/10 and 1/100 of that in the injected immunoglobulin. The amount of protection to the patient afforded by this varied considerably, and it was particularly striking that protection against measles seemed to be complete, whereas there was no protection against varicella, mumps, or herpes simplex.
Isoagglutinins The isoagglutinins anti-A and anti-B were below the normal range in 119 out of the 134 patients in whom they 17 out of 60 group-0 patients and 45 were estimated. out of 67 group-A patients had no demonstrable isoagglutinins. In patients with low IgM concentrations, anti-A and anti-B titres were always low. On the other hand, in patients with normal or high IgM levels the isoagglutinin titres were sometimes similarly normal or high; but in other cases they were low or undetectable, presumably because of a qualitative abnormality of the
IgM. Analysis of Dose-level Effect
Antibodies
Analysis indicated a small but statistically significant difference in favour of the 0-05 g. per kg. dose over the lower doses in regard to both C-reactive-protein levels and frequency of febrile episodes (P==0-05, P<001 respectively). Although there was a marked dose-level effect on the serum-immunoglobulin values, it was found that high blood-levels did not correlate with recorded periods of freedom from febrile episodes, nor did low blood-levels correlate with increased frequency of infections; similarly the level of C-reactive protein was not always positively related to the recorded incidence of febrile episodes. Some doubt must remain as to whether the recorded incidence of all such episodes is always a good criterion of the effect of treatment, especially in patients with permanent tissue damage. Moreover, an accurate record of febrile episodes required the concentrated attention of a large number of physicians over many years, and, as the physician always knew which schedule the patient was having, his attention may have been biased by this knowledge. On the other hand an analysis of certain well-defined illnesses (pneumonia, diarrhoea, otitis media, and skin infections) did show a decreased incidence of illnesses on the higher dosage, which was significant in the case of pneumonia p<û,05 respectively). and diarrhoea (P<0-01, No significant difference in mortality was found between patients on the different dosage levels.
Antibodies to the various bacterial and viral antigens were detected in very few indeed of the sera collected from patients before the start of replacement therapy, but it must be said that before treatment examination for antibodies was possible in only some patients. The majority of patients with hypogammaglobulinsemia did not produce detectable titres of antibodies to non-living antigens; and no rise in antibody titre was found after smallpox vaccination or after infection with mumps, poliomyelitis, varicella, or herpes zoster. Immunisation with live polio vaccine was deliberately undertaken in
Reactions Reactions to the injection of immunoglobulin occurred at one time or another in 33 out of 175 (19%) patients. In all there were 85 reactions to about 40,000 injections, given over a period of ten years. Although most reactions were mild, a few were severe and there was 1 death. The features of most of the reactions were as follows: dyspnoea, with tightness in the chest but without wheezing; faintness; hypotension, sometimes with loss of consciousness; flushing and facial swelling; anxiety. There was no apparent correlation between reactions and
the dose level of injected immunoglobulin. Quantitative defects of one immunoglobulin were often associated with qualitative defects of another. levels
were on
Metabolism
the whole related
to
of IgG
The metabolism of IgG was studied in 3 patients, but in only 1 both before and after replacement therapy. Before treatment the findings indicated that the low IgG concentration was due to failure of synthesis rather than to increased catabolism. After low-dosage treatment the rate of catabolism was unchanged, but after high-dosage treatment, not unexpectedly, there was a significant rise in catabolism.
167 of immunoglobulin. There was, significant sparing of boys with affected
an opportunity to gain some information on the prevalence of this syndrome and the relative incidence of accompany male relatives. ing features. For instance, the incidence of arthritis an( in this study was found to be much lowe: steatorrhoea Histopathology that than reported in other studies (Gitlin et al. 1959 Necropsy material was available from just over half and Good Rotstein 1960, Conn and Quintiliani 1966) the patients who died. In all those studied, the lymphoid On the other hand, while isolated cases of malignam tissues showed generally poor differentiation with paucity disease in arising patients with hypogammaglobulinxmia of lymphocytes, absence of plasma cells, and sometimes have occasionally been reported (Page et al. 1963, Greer also hypertrophy of reticuloendothelial cells. Thus in et al. 1966) the incidence of malignancy arising in the the lymph-nodes there was absence or poor differentiation of cortical follicles and germinal centres; in the spleen lymphoreticular system in the present series is striking. Furthermore, it has been shown that the occurrence oi the malpighian follicles were small or absent, and in the combined immunity-deficiency syndrome may be at the gut there was failure of formation of lymphoid follicles. least 6% in children with hypogammaglobulinxmia. The changes in lymphoid tissue were most severe in Although reactions to intramuscular immunoglobulin the children with thymic abnormalities, where in some have been described previously (Kamme et al. 1966) The cases lymphoid structures were unrecognisable. thymus showed failure of development and lack of their frequency seems to be much greater in the present differentiation into cortex and medulla, with absence of study than in previous ones. Experimental work Hassall’s corpuscles, in 7 children; 6 of these died in the (Richerson and Seebohm 1966) has suggested that reactions following intravenous immunoglobulin are due to first year of life, with evidence of the combined immunitythe presence of IgG aggregates which initiate a deficiencv state: the 7th survived until the ase of 8 vears. Thymic atrophy with degenerate and calcified Hassall’ complement-dependent reaction. This conclusion is not corpuscles was found in a further child, and a malignant supported by the present study, and so far no satisfactory thymic tumour in 1 adult. Histological changes in adults explanation has been found. In retrospect it has become clear that there were coming to necropsy showed a marked degree of uniformity. The lymph-node-follicle formation was either several reasons why only small differences in morbidity were likely to be observed in relation to the different absent or rudimentary, and no mature plasma cells were seen. The malpighian corpuscles in the spleen were dosage regimens: first, the syndrome of primary hyposimilarly inconspicuous, and here again no plasma cells gammaglobulinxmia is now known to include several different kinds of immunological deficiency syndromes; were identified in the splenic pulp. Although 6 patients were diagnosed as having died of malignant disease, no second, in the series collected by the working-party some patients had suffered irreversible tissue damage by post-mortem material was made available from these. the time of admission to the study, while others had not; A few patients showed amyloid deposits in the spleen and lymph-nodes, and in 2 patients granulomas of the third, there was difficulty in devising criteria which, when sarcoid type were found. applied by numerous observers, would objectively discriminate between relatively minor differences in Outside the reticuloendothelial system the pathological changes were mainly those associated with recurrent clinical progress; finally, the difference between the infections. 4 of the 5 children who died of Pneumocystis dosages of immunoglobulin used was too small. Nevercarinii pneumonia showed evidence of the combined theless, the difference in favour of the higher dose was statistically significant and, although some of the criteria of immunity-deficiency syndrome. were open to bias in recording, others, such as morbidity Discussion the incidence of pneumonia, were almost certainly not. During the ten years of the study knowledge of the is There further evidence of the value of replacement antibody-deficiency syndrome has greatly increased at least in some patients, in that those who throughout the world, and many of the ideas originally therapy, held have become modified. In retrospect, for instance, presented with such severe infections as septicxmia, the choice of serum levels of IgG of less than 200 mg. meningitis, and osteomyelitis did not usually suffer further attacks once they had started replacement per 100 ml. as a criterion for entry seems rather arbitrary Furthermore, there was the increased fatality therapy. is no evidence of any obvious - firstly, because there in the small group of patients who stopped (63%) discontinuity in the distribution of values below the for more than a year. treatment lower level of the normal range (600 mg. per 100 ml.); in morbidity observed in patients small decrease The secondly, because patients with somewhat higher values on the 0-05 g. per kg. per week dose, as compared with (200-400 mg. per 100 ml.) have also been shown to be 0-025 g. per kg., is taken as evidence of the efficacy of prone to increased infections both by Hobbs (1966) and in the present study, in which over 150 patients were prophylactic immunoglobulin. However, the difference, found to have serum IgG values between 200 and 600 mg. although statistically significant, is not thought to justify per 100 ml. when examined because of a history of the routine use of 0-05 g. per kg. per week in all patients, in view of the added discomfort and expense associated recurrent severe infections (in some instances they even the larger dose. In fact, during the period January, with it and because is required replacement therapy); thirdly, now known that there is a variety of qualitative immuno1966, to June, 1968, most patients in the study were satislogical deficiency states involving quantitative abnormality factorily maintained on a dose of 0-025 g. per kg. per of only one of the immunoglobulins. week. It is therefore recommended that this should be It has also become clear that the syndrome of hypothe normal dose, but that in patients who continue to gammaglobulinxmia is enormously varied, and the group get severe infections on this dose consideration should of cases collected here was quite heterogeneous. Most of be given to increasing it to 0-05 g. per kg. per week. the clinical and pathological findings in this study have It should also be stated that, although the workingalready been reported; but the present series has provided party have no experience of an equivalent dose given at
different
batches
however,
a
168 or monthly intervals, this regimen might prove more convenient for some patients. Live vaccines (polio, vaccinia, and B.C.G.) were given to a third of all patients without apparent ill-effect. The working-party -recommend, however, that immunisation should be undertaken only after careful consideration and after ensuring that normal circulating lymphocytes and normal delayed skin hypersensitivity are present.
fortnightly
The full report of this study is to be published in the Medical Research Council’s Special Report Series. The working-party gratefully acknowledge the help of some two hundred doctors and many other people who supplied information about the patients. Their names will be listed in the forthcoming report of the working-party. Requests for reprints should be addressed to Dr. Lisa Hill, Clinical Research Centre, 164 Tottenham Court Road, London W.l. REFERENCES
Barandun, S. (1959) Das Antikorpermangelsyndrome. Basle. Bruton, O. C. (1952) Pediatrics, 9, 722. Collins, H. D., Dudley, H. R. (1955) New Engl.J. Med. 252, 255. Conn, H. O., Quintiliani, R. (1966) Ann. intern. Med. 65, 528. Fahey, J. L., McKelvey, E. M. (1965) J. Immun. 94, 84. Fudenberg, H., Hirschhorn, K. (1965) Med. Clin. N. Am. 49, 1533. Gitlin, D., Gross, P. A. M., Janeway, C. A. (1959) New Engl.J. Med. 260, 72. Good, R. A., Rotstein, J. (1960) Bull. rheum. Dis. 10, 203. Grant, G. H., Wallace, W. D. (1954) Lancet, ii, 671. Green, I., Litwin, S., Adlersberg, R., Rubin, I. (1966) Archs intern. Med. 118, 592. Hobbs, J. R. (1966) Lect. scient. Basis Med. 106. Hutchison, J. H. (1955) Lancet, ii, 844, 1196. Janeway, C. A., Apt, L., Gitlin, D. (1953) Trans. Ass. Am. Phycns, 66, 200.
Gitlin, D., Craig, J. M., Grice, D. S. (1956) ibid. 69, 93. Kamme, C., Coster, C., Hagelquist, E., Kalen, N., Lindholm, H., Grubb, R. (1966) Acta med. scand. 179, 679. Keidan, S. E., McCarthy, K., Haworth, J. C. (1953) Archs Dis. Childh. 28, —
110.
A., Mackay, M.
R. no. 286.
Kekwick,
E.
(1954) Spec. Rep. Ser. med. Res. Coun.
Vallet, L., Cutbush, M., Mollison, P. L., Thomas, A. R., Gell, H., Soothill, J. F. (1961) J. clin. Path. 14, 470. Kuschner, D. L., Dubin, A., Donlon, W. P., Bronsky, D. (1960) Am. J. Med. 29, 33. Marshall, W. C., Weston, H. J., Bodian, M. (1964) Archs Dis. Childh. 39, 18. Page, A. R., Hansen, A. E., Good, R. A. (1963) Blood, 21, 197. Parkes, R. (1958) Br. med. J. i, 973. Richerson, H. B., Seebohm, P. M. (1966) Archs intern. Med. 117, 568. Rodgers, T. S., Haggie, M. H. K. (1964) Lancet, i, 1042. —
P. G.
A GERMFREE INFANT D. V. I. FAIRWEATHER
R. D. BARNES
JEAN
C. KEANE
HOLLIDAY
ALINA PIESOWICZ
J. F. SOOTHILL
MAUREEN TUFFREY FROM
THE
DEPARTMENTS
HEALTH, AND
OF
HÆMATOLOGY, IMMUNOLOGY,
CHILD
MICROBIOLOGY AT THE INSTITUTE OF CHILD HEALTH AND
CHILDREN, AND THE DEPARTMENT OF OBSTETRICS, UNIVERSITY COLLEGE HOSPITAL, LONDON W.C.1
HOSPITAL FOR SICK
An infant was delivered by cæsarean section and maintained in a " germfree " isolation unit for 6 days during which absence of bacteria was demonstrated by microbiological tests. This procedure was done as part of a programme for the treatment of potential cases of immunity deficiency, but since this infant was not affected with combined immunitydeficiency state, as her brother had been, she was removed from the germfree isolation unit.
Summary
Introduction A SEVERE and fatal form of hypogammaglobulinxmia associated with lymphopenia was first described by Hitzig and Willi (1961). This familial, and possibly inherited,
disease (Hitzig et al. 1968) presents with deficiency of both cellular and humoral immunity-the combined immunitydeficiency syndrome of Soothill (1968a). Foreign-tissue grafts are not rejected. Despite this, attempts at treatment by grafting immunologically competent tissue have been only marginally effective and have not modified the universal mortality in this condition (Hitzig et al. 1965, Harboe et al. 1966, Hong et al. 1968). Failure may have been due to the effects of severe infection at the time the patients were diagnosed and treated, or it may have resulted from the relative maturity of the immunity mechanism and this might; in part, be the effect of the normal antigenic stimulus of infection. In view of this, when the mother of such a child (Thompson 1967) became pregnant again, the child was delivered by caesarean section and maintained in a germfree isolation system while diagnostic tests were done. If the child were affected it was proposed to attempt early grafting of foetal tissue, before the normal bacterial colonisation occurred and whilst the child was, immunologically, relatively " immature ". Methods
Transfer
to
Isolator
The mother, aged 23 was admitted during a normal pregnancy at 38 weeks. Her previous delivery had been normal and clinical examination revealed no abnormality. Elective lower-segment cxsarean section was performed at 39 weeks’ gestation through a sterile, flexible plastic-film surgical isolator described (Barnes, Fairweather, et al. 1968). This isolator was adhered aseptically to the mother’s abdomen which had been previously prepared with an iodine antiseptic solution. The baby, after delivery into the surgical isolator, was then transferred through a connecting tunnel to a mobile plastic transfer isolator for resuscitation. Both isolators, joined (fig. 1) with their enclosed contents (surgical and infant resuscitation requirements), had been sterilised in advance with 4 megarads of gamma irradiation from a cobalt-60 source (U.K. Atomic Energy Authority, Wantage Laboratory). Whilst the infant was being resuscitated in the transfer isolator the connecting tunnel between the two units was sealed and cut between the airtight steel clamps. After resuscitation, the infant was transported in the mobile transfer isolator to the vicinity of the maintenance isolator. The aseptic union, and subsequent incision of the two sterile adhesive membranes incorporated into both the transfer and maintenance isolators formed a common tunnel for the ultimate introduction of the infant into the maintenance isolator. ‘Cornercroft’ type-A human isolator (6g. 2) (Barnes, Tuffrey, and Cook 1968) with minor modification of the sealing of the plastic tent to the consoles was used for the care of the infant. The microbiological integrity of this unit and its sterilisation had been previously confirmed by successful maintenance of germfree mice (Barnes, Holliday, and Cook 1968). Fail-safe devices, similar to those incorporated in the germfree animal isolator (Cook, Tuffrey, and Barnes 1968), were a feature of this isolator and, as a precaution, suction, filtered oxygen i and electricity were also supplied. A metal cot, a sterile plastic film sealed mattress, and a baby incubator were also required I and were introduced into the isolator before sterilisation. ’
Sterilisation The equipment and all supplies likely to be required for the of the infant were sterilised in advance. The method I selected for each item depended upon its ability to withstand : the particular form of sterilisation : care
Gamma irradiation (4 megarads from cobalt-60 source) was used for surgical and connected transfer isolators with enclosed contents and for normal neonatal nursing requirements (instruments &c.) (clothing, bedding, water, sugar, &c.). Gas sterilisation (ethylene oxide) was used to sterilise the air filters, and enclosed incubator in the maintenance isolator; for instruments with glass components, and for the external surfaces of cans of milk.
:
I
Working-party* Between 1956 and 1966 information was collected about 176 patients in whom primary hypogammaglobulinæmia was diagnosed and in whom the serum-IgG level was shown to be less than 200 mg. per 100 ml. (below 100 mg. per 100 ml. in infants under 6 months). Clinically the condition varied widely between patients, owing partly to changes wrought by previous infections and partly to the heterogeneity of the underlying disorder. Initially it was assumed that prophylactic administration of immunoglobulin would be valuable in reducing the incidence of infections, and trials were conducted to discover the optimal dose. In fact, only a relatively small (though statistically significant) difference in morbidity, and no difference in mortality, were observed between dose levels of 0·05 g. and of 0·025 g. per kg. body-weight per week. Failure to obtain stronger evidence in favour of the larger dose is thought to be due partly to the relatively small difference between the dose levels, and partly to the inclusion of many patients not expected to show substantial benefit from treatment. The lower dose (0·025 g. per kg.) has been used for most patients over the past two years and is recommended as the standard treatment, except for patients who continue to suffer from severe infections. Summary
Introduction
IN 1952 Bruton described the first
case of primary hypogammaglobulinaemia: a young boy suffering from repeated severe bacterial infections, who showed evidence
antibody production and was very greatly by improved injections of immunoglobulin. (In the past of deficient
few years the term " immunoglobulin " has tended to replace y-globulin.) Within a year, Janeway et al. (1953) were able to report 9 further patients with hypogammaglobulinsemia; these authors gained the impression that monthly injections of 0-1 g. per kg. pooled human normal immunoglobulin reduced the incidence of infections, whereas continued administration of antibiotics was only partially successful. In 1955, when the syndrome of hypogammaglobulinaemia had become more widely known in this country (Keidan et al. 1953, Grant and Wallace 1954, Hutchison 1955), it was suggested by the Ministry of Health that the Medical Research Council * Members of the working-party: the late Professor J. R. SQUIRE (chairman until 1966), Prof. P. L. MOLLISON, F.R.S. (chairman from 1966), Dr. W. R. S. DOLL, F.R.S., Dr. J. W. FARQUHAR, Dr. F. V. FLYNN, Dr. D. M. T. GAIRDNER, Prof. P. G. H. GELL, Mr. M. J. R. HEALY, Dr. LISA E. HILL, Prof. R. A. KEKWICK, P.R.S., Dr. F. O. MACCALLUM, Prof. R. G. MACFARLANE, F.R.S., Prof. N. H. MARTIN, Dr. W. D’A. MAYCOCK, Dr. J. F. SOOTHILL, Prof. Sir RONALD TUNBRIDGE, Mr. L. VALLET, Prof. Sir EDWARD WAYNE, Dr. H. W. BUNJÉ (secretary). 7587
25
January 1969
might set up a working-party to study the problems of hypogammaglobulinasmia, including the range of clinical syndromes and various aspects of the therapeutic use of immunoglobulin. Physicians who wished to obtain immunoglobulin for the treatment of their patients, or who suspected a diagnosis of hypogammaglobulinsemia and wished to have it confirmed, were referred to the Medical Research Council and invited to join the study. Physicians could obtain immunoglobulin without joining the study provided that the criteria laid down by the working-party for the diagnosis were satisfied, or that there was clinical evidence of defective immunity despite a serum-IgG level of more than 200 mg. per 100 ml. Material and Methods Criteria for Diagnosis Admission to the study was confined to patients with primary hypogammaglobulinxmia; the investigation therefore excluded patients with generalised hypoproteinxmia or the nephrotic syndrome and those with reticuloses or other malignant disease, and also premature infants with temporary
hypogammaglobulinxmia. The major criterion for admission to the study was a serumIgG level of 200 mg. per 100 ml. or less or, in infants of 6 months
or
below, 100
mg. per 100 ml.
or
less.
Estimations For the first two years of the study serum-immunoglobulin levels were estimated by three methods: free-solution electrophoresis, which estimated total y-globulin, and two methods which estimated only IgG-namely, a method using the inhibition of antiglobulin serum and a double gel-precipitin technique (Kekwick et al. 1961). Subsequently only the gelprecipitin method was used, and in the last year of the study this was replaced by a single diffusion precipitin technique (Fahey and McKelvey 1965). As antisera became available, IgM, IgA, and IgD levels were also estimated. A purified freeze-dried standard of immunoglobulin was used for the estimation of IgG, and results were expressed in mg. per 100 ml.; IgM, IgA, and IgD concentrations were expressed as a percentage of those contained in a reference serum (R.N.S.) obtained from a healthy adult male.
Serum-immunoglobulin
Human Immunoglobulin Used in Treatment Almost all the immunoglobulin used in treatment was prepared by the ether fractionation method from normal large-pool plasma (Kekwick and Mackay 1954) at the Blood Products Laboratory, Lister Institute; a small comparative trial with alcohol-fractionated immunoglobulin produced no evidence of any important differences between the two preparations. All the batches contained only a small proportion of IgA and IgM (mostly 4-5 mg. per ml. and 1-2 mg. per ml. respectively) and little or no IgD. Ether-fractionated immunoglobulin was found to be very stable during storage; at 4°C only very slight alterations were found after four years, and it was thought that these changes could have been due to contamination with traces of plasmin. In an accelerated storage test, in which samples of immunoglobulin were held in aseptic conditions at 37°C for twenty-eight days, the results were similar to those of the long-term storage tests.
164 The titres of certain antibodies (antistreptolysin, antistaphylolysin, diphtheria antitoxin, and neutralising antibodies to polio, herpes, and influenza viruses) were estimated in 39 batches. Assays of one batch were also made at intervals during prolonged storage at 4°C, and these showed that certain antibodies remained stable for very long periods. Design of the Trial Since there was strong presumptive evidence from the United States that therapy with immunoglobulin was of considerable benefit in reducing infections in hypogammaglobulinaemia, it was felt that there could be no question of randomised allocation of patients to different groups, one of which would not be receiving replacement therapy. The basic design of treatment trials was therefore a comparison of two different dose levels. It was not possible to disguise the difference between the
assessment of bias. any particular regimen open At first a dose of 0-025 g. per kg. per week given by intramuscular injection (based on American reports of the efficacy of 0.1 g. per kg. monthly) was compared with one of 0-05 g. per kg. per week, alternate patients starting with a high and with a low dose and the dose being changed every seventeen weeks to cut across seasonal variations in the incidence of infections. Each week at the time of injection the treating physician recorded details of infective illnesses during the preceding week, including febrile episodes, the number of days with symptoms, and the use of antibiotics. At the time of injection the actual dose given was noted and the patient’s two
was
and
doses, and thus the to
recorded.
Although body-temperature weight laboratory investigations (other than serum-immunoglobulin levels) were not specifically asked for, clinicians were given were
the record card to enter results of any that were Once during each four-month course a bloodsample was requested for estimation of immunoglobulin levels. In addition, during the later years of the trial C-reactive protein was estimated from this specimen. After three years no clear difference had been demonstrated between these two dosage levels, and a second trial was undertaken comparing a dose of 0-01 g. per kg. with 0-05 g. per kg. weekly, following the same basic plan as before. After twelve months, suspicion arose that patients on the lower dosage were doing less well than the others; and this second trial was abandoned, although in retrospect the evidence on which it was abandoned was not conclusive. Finally, a dose of 0-025 g. per kg. weekly was once more compared with 0-05 g. per kg., but patients were allowed to remain on each dose for one year. In all the trials, before starting on the weekly injections patients were given a " loading dose," of immunoglobulin-namely, 0-25 g. per kg. in five divided daily doses.
space
on
carried
out.
Other Investigations Since it was known that patients with hypogammaglobulinxmia commonly lack isohsemagglutinins, the ABO group of each patient was determined and the serum tested for anti-A and anti-B. The titres of antibodies against certain bacteria and viruses before and after replacement therapy were estimated, and antibody titres were measured after known bacterial and virus infections. In addition, in selected children a deliberate attempt was made at immunisation against poliomyelitis by feeding live vaccine and estimating the resulting antibodies. Results Classification of Cases and Prevalence of the Syndrome During the ten years of the trial, 176 patients (119 male and 57 female) fulfilled all the criteria demanded and were admitted to the study. Their age and sex distribution on admission are shown in the accompanying figure. In addition there were 24 patients known to the working-party who qualified for admission but for various reasons were not included. An attempt to relate the number of accepted cases to the population of live births from which they were
Distribution of age at entry to the
study.
(or, in the case of those born before 1956, the surviving population) suggests that the proportion of people affected at some time in their life is at least 15 per 1,000,000 for males and 4 per 1,000,000 for females. The true prevalence is doubtless greater because (1) drawn
probably some patients who died before a made; (2) there were probably some affected children who had not yet developed a characteristic clinical picture; (3) not all patients with the disease can have been brought to the notice of the workingparty, and (4) the criteria adopted by the working-party did not cover all cases of hypogammaglobulinaamia or of the antibody-deficiency syndrome. It became obvious during the study that the presenting clinical and pathological features were extremely varied, and that the cases collected were heterogeneous and not easily classifiable. It was not considered satisfactory to divide the cases into " congenital " and " acquired " as
there
were
diagnosis
could be
had been customary in the past, since those which are apparently " acquired " may possibly also have a genetic basis (Fudenberg and Hirschhorn 1965). In the present study the problem was partially avoided by classifying patients into three groups according to their age on admission (infants, under 1 year; children, 1-15 years; adults, 16 years and over), each group being subdivided according to whether they did or did not have affected relatives. Of the 176 patients admitted to the study there were 33 infants, 73 children, and 70 adults. There was a clear family history for 41 patients, including 2 females, who probably had a non-sex-linked form of the disease. There were 135 patients with no known affected relatives at the time of the conclusion of the therapeutic trial. The 39 male patients with family histories consisted of 2 adults, 26 children, and 11 infants; 36 of them had affected male relatives (17 families) admitted or qualifying for admission the study and 3 had possibly affected male relatives (not admitted). Of the patients without affected relatives 67 were adults and may have had the acquired form of the disease. 4 male infants admitted to the study, and 1 female
to
infant with a qualifying serum-IgG level who was not admitted to the study, were considered to have transient hypogammaglobulinxmia. All reached normal IgG levels
165 PRESENTING SYMPTOMS OF PATIENTS ADMITTED TO THE STUDY
the presenting feature; in approximately half the patients it was associated with disorders of the blood or arthritis and in 3 with amyloid disease. Of the patients with respiratory-tract infections almost half had permanent lung damage on admission to the study.
Pneumocystis carinii Pneumonia Interstitial-cell pneumonia due to Pneumocystis carinii found in 7 patients, all children. In 5 the diagnosis was made only at necropsy; but in 2 it was confirmed by lung biopsy during life, and treatment with pentamidine isothianate resulted in recovery (Marshall et al. 1964, Rodgers and Haggie 1964). was
Virus Diseases
(above 600 mg. per 100 ml.) by the age of 20 months subsequently maintained them, but in 3 an abnormally low IgA level persisted. Antibody response to challenging antigens was tested in 3 and was found to be normal in 2, although in 1 of these the rise in antibody and
level
was not
maintained.
3 patients, all women, recovered their ability to produce normal serum levels; in 2 there was a full normal antibody response to challenging antigens, but in the 3rd there appeared to be no recovery of antibody response.
Mortality Of the 176 patients admitted, 51 (29%) died during the period of the study; and, of these, 24 died within six months of entry. The fatality-rate in infants under 1 year was distinctly higher (45%) than the overall average and was the same for both sexes; in this group all deaths occurred within six months of entry to the study. At later ages the overall death-rate was much lower (19%), though this difference was due mostly to the much lower mortality in males at this age than in infancy. The lowest mortality-rate (11%) was in boys between the ages of 1 and 16 years, and among these mortality appeared especially low in those with affected male relatives. For various reasons, including doubt about the diagnosis, 22 of the 176 patients were not treated continuously with immunoglobulin. Among these were 8 who either refused further injections or were advised to stop treatment because of severe reactions and who remained off treatment for more than a year. All were over 2 years of age when treatment was stopped, and none stopped because of the severity of their disease; yet 5 died, giving the highest mortality of any group (63%).
Presenting Symptoms The main presenting symptoms of the patients admitted to the study are shown in the accompanying table. It should be noted that the commonest haematological disorder was neutropenia, either permanent or occurring as a transient response to infection. Half the patients with megaloblastic anaemia also had steatorrhoea; there was, however, no definite pattern of one condition preceding the other or of a definite time relationship with the onset of hypogammaglobulinaemia. Hxmolytic anaemia, thrombocytopenia, and hypoplastic anaemia were found in a small proportion of patients. Nearly 18% of patients showed splenomegaly and in some this was
Although the patients in the study were apparently able to deal normally with most virus infections, recurrent attacks of measles, mumps, herpes zoster, and herpes simplex were reported in the pretreatment histories of a number of them. 3 patients had further attacks of herpes, mumps, and varicella after the start of replacement therapy, but not one case of measles was reported. 5 children had attacks of paralytic poliomyelitis, although only one of these occurred after the start of replacement therapy. Hepatitis was reported in 4 patients (1 of these had at least eight attacks before treatment), and cirrhosis of the liver was found in 3 others. The immunoglobulin used was not considered to have caused the hepatitis. Tuberculosis 4 children and 4 adults (4-5% of all patients) had active tuberculosis, and 2 of the adults died from this
This incidence suggests, contrary reports (Parkes 1958, Barandun 1959), that cause.
hypogammaglobulinxmia
are
more
likely
published patients with to
to
contract
tuberculosis than are normal people. There was, however, evidence in the present study that patients with hypogammaglobulinaernia can convert to the tuberculinpositive state and respond normally to antituberculous drugs. However, B.C.G. and other live vaccines (see below) should be given to patients with hypogammaglobulinxmia only after steps have been taken to ensure that cellular immunity and normal lymphocytes are present.
Neurological Disorders 8 children were found to have neurological abnormalities. In most of these the abnormality was mental retardation, either congenital or following an attack of meningitis; but in 3 children there was a progressive pattern, similar in all 3, of spastic paralysis, ataxia, and optic atrophy, coming on in apparently fit children who had been on replacement therapy for between three and eight years. It is not yet clear whether this is the result of a demyelinating process or of a " slow virus " infection.
Malignant Disease 8 patients, 7 of them women, developed malignant disease which had not been apparent at the time of admission to the study. In 6 out of 8 cases the reticuloendothelial or lymphoid tissues were affected-a proportion in excess of the expected incidence. The incidence of malignant disease affecting other tissues was not in excess of the expected frequency. The Combined Immunity-deficiency
Syndrome (Thymic Alympho-
plasia) 11 infants
(5 admitted to the series and 6 qualifying admitted) showed some evidence of the combined immunity-deficiency syndrome-that is, lack of both humoral and cellular immunity. They all showed progressive lymphopenia, and at necropsy absence or but
not
atrophy of the thymus gland and poorly developed lymphoid tissues (see below). 4 of these died of generalised vaccinia following smallpox vaccination, and 1 developed generalised tuberculosis following B.C.G. inoculation. Arthritis In view of American reports of
a high incidence of hypogammaglobulinxmia (Collins and Dudley 1955, Janeway et al. 1956, Kuschner et al. 1960), 73 patients were examined clinically, radiologically, and serologically for signs of the disease. Of the 55 males,
arthritis in
were considered to have rheumatoid arthritis, whereas of the 18 females none had definite arthritis, though 3 gave a history of previous polyarthritis. On the basis of the experience of the normal population the numbers expected would be 0-40 males and 1-14 females. Thus there would appear to be an abnormally high incidence of arthritis in males with hypogammaglobulinaemia. There was no evidence that arthritis was either induced
4
or
improved by treatment.
Immunoglobulin Levels
By protocol all patients had initial IgG values of 200 mg. per 100 ml. or less, or 100 mg. per 100 ml. or less if under 6 months of age. Some IgG was detectable in the serum of all the patients, although it was frequently less than 50 mg. per 100 ml. IgM was rarely undetectable, and IgA was only occasionally undetectable. IgD was more often absent than in the normal population. There was no significant correlation between levels of the different immunoglobulins in particular patients, and the levels of the immunoglobulins other than IgG varied (even within families with several affected sibs), though in patients with transient hypogammaglobulinaemia IgM was always normal. In the patients with prolonged hypogammaglobulinaemia who had normal initial IgM values, relatively high IgG levels were common after treatment. Follow-up studies revealed a general tendency for levels of immunoglobulins other than IgG to remain in the same range in individual patients over many years, although occasional exceptions were found. The IgG
children on replacement therapy and was apparently safe and produced local resistance to the type of virus given. Passive immunisation by antibodies in the injected immunoglobulin may well further reduce such small ability to form circulating antibodies as these patients have-a phenomenon also seen in normal individuals. Replacement therapy raised the level of antibodies to various infecting organisms to between about/10 and 1/100 of that in the injected immunoglobulin. The amount of protection to the patient afforded by this varied considerably, and it was particularly striking that protection against measles seemed to be complete, whereas there was no protection against varicella, mumps, or herpes simplex.
Isoagglutinins The isoagglutinins anti-A and anti-B were below the normal range in 119 out of the 134 patients in whom they 17 out of 60 group-0 patients and 45 were estimated. out of 67 group-A patients had no demonstrable isoagglutinins. In patients with low IgM concentrations, anti-A and anti-B titres were always low. On the other hand, in patients with normal or high IgM levels the isoagglutinin titres were sometimes similarly normal or high; but in other cases they were low or undetectable, presumably because of a qualitative abnormality of the
IgM. Analysis of Dose-level Effect
Antibodies
Analysis indicated a small but statistically significant difference in favour of the 0-05 g. per kg. dose over the lower doses in regard to both C-reactive-protein levels and frequency of febrile episodes (P==0-05, P<001 respectively). Although there was a marked dose-level effect on the serum-immunoglobulin values, it was found that high blood-levels did not correlate with recorded periods of freedom from febrile episodes, nor did low blood-levels correlate with increased frequency of infections; similarly the level of C-reactive protein was not always positively related to the recorded incidence of febrile episodes. Some doubt must remain as to whether the recorded incidence of all such episodes is always a good criterion of the effect of treatment, especially in patients with permanent tissue damage. Moreover, an accurate record of febrile episodes required the concentrated attention of a large number of physicians over many years, and, as the physician always knew which schedule the patient was having, his attention may have been biased by this knowledge. On the other hand an analysis of certain well-defined illnesses (pneumonia, diarrhoea, otitis media, and skin infections) did show a decreased incidence of illnesses on the higher dosage, which was significant in the case of pneumonia p<û,05 respectively). and diarrhoea (P<0-01, No significant difference in mortality was found between patients on the different dosage levels.
Antibodies to the various bacterial and viral antigens were detected in very few indeed of the sera collected from patients before the start of replacement therapy, but it must be said that before treatment examination for antibodies was possible in only some patients. The majority of patients with hypogammaglobulinsemia did not produce detectable titres of antibodies to non-living antigens; and no rise in antibody titre was found after smallpox vaccination or after infection with mumps, poliomyelitis, varicella, or herpes zoster. Immunisation with live polio vaccine was deliberately undertaken in
Reactions Reactions to the injection of immunoglobulin occurred at one time or another in 33 out of 175 (19%) patients. In all there were 85 reactions to about 40,000 injections, given over a period of ten years. Although most reactions were mild, a few were severe and there was 1 death. The features of most of the reactions were as follows: dyspnoea, with tightness in the chest but without wheezing; faintness; hypotension, sometimes with loss of consciousness; flushing and facial swelling; anxiety. There was no apparent correlation between reactions and
the dose level of injected immunoglobulin. Quantitative defects of one immunoglobulin were often associated with qualitative defects of another. levels
were on
Metabolism
the whole related
to
of IgG
The metabolism of IgG was studied in 3 patients, but in only 1 both before and after replacement therapy. Before treatment the findings indicated that the low IgG concentration was due to failure of synthesis rather than to increased catabolism. After low-dosage treatment the rate of catabolism was unchanged, but after high-dosage treatment, not unexpectedly, there was a significant rise in catabolism.
167 of immunoglobulin. There was, significant sparing of boys with affected
an opportunity to gain some information on the prevalence of this syndrome and the relative incidence of accompany male relatives. ing features. For instance, the incidence of arthritis an( in this study was found to be much lowe: steatorrhoea Histopathology that than reported in other studies (Gitlin et al. 1959 Necropsy material was available from just over half and Good Rotstein 1960, Conn and Quintiliani 1966) the patients who died. In all those studied, the lymphoid On the other hand, while isolated cases of malignam tissues showed generally poor differentiation with paucity disease in arising patients with hypogammaglobulinxmia of lymphocytes, absence of plasma cells, and sometimes have occasionally been reported (Page et al. 1963, Greer also hypertrophy of reticuloendothelial cells. Thus in et al. 1966) the incidence of malignancy arising in the the lymph-nodes there was absence or poor differentiation of cortical follicles and germinal centres; in the spleen lymphoreticular system in the present series is striking. Furthermore, it has been shown that the occurrence oi the malpighian follicles were small or absent, and in the combined immunity-deficiency syndrome may be at the gut there was failure of formation of lymphoid follicles. least 6% in children with hypogammaglobulinxmia. The changes in lymphoid tissue were most severe in Although reactions to intramuscular immunoglobulin the children with thymic abnormalities, where in some have been described previously (Kamme et al. 1966) The cases lymphoid structures were unrecognisable. thymus showed failure of development and lack of their frequency seems to be much greater in the present differentiation into cortex and medulla, with absence of study than in previous ones. Experimental work Hassall’s corpuscles, in 7 children; 6 of these died in the (Richerson and Seebohm 1966) has suggested that reactions following intravenous immunoglobulin are due to first year of life, with evidence of the combined immunitythe presence of IgG aggregates which initiate a deficiencv state: the 7th survived until the ase of 8 vears. Thymic atrophy with degenerate and calcified Hassall’ complement-dependent reaction. This conclusion is not corpuscles was found in a further child, and a malignant supported by the present study, and so far no satisfactory thymic tumour in 1 adult. Histological changes in adults explanation has been found. In retrospect it has become clear that there were coming to necropsy showed a marked degree of uniformity. The lymph-node-follicle formation was either several reasons why only small differences in morbidity were likely to be observed in relation to the different absent or rudimentary, and no mature plasma cells were seen. The malpighian corpuscles in the spleen were dosage regimens: first, the syndrome of primary hyposimilarly inconspicuous, and here again no plasma cells gammaglobulinxmia is now known to include several different kinds of immunological deficiency syndromes; were identified in the splenic pulp. Although 6 patients were diagnosed as having died of malignant disease, no second, in the series collected by the working-party some patients had suffered irreversible tissue damage by post-mortem material was made available from these. the time of admission to the study, while others had not; A few patients showed amyloid deposits in the spleen and lymph-nodes, and in 2 patients granulomas of the third, there was difficulty in devising criteria which, when sarcoid type were found. applied by numerous observers, would objectively discriminate between relatively minor differences in Outside the reticuloendothelial system the pathological changes were mainly those associated with recurrent clinical progress; finally, the difference between the infections. 4 of the 5 children who died of Pneumocystis dosages of immunoglobulin used was too small. Nevercarinii pneumonia showed evidence of the combined theless, the difference in favour of the higher dose was statistically significant and, although some of the criteria of immunity-deficiency syndrome. were open to bias in recording, others, such as morbidity Discussion the incidence of pneumonia, were almost certainly not. During the ten years of the study knowledge of the is There further evidence of the value of replacement antibody-deficiency syndrome has greatly increased at least in some patients, in that those who throughout the world, and many of the ideas originally therapy, held have become modified. In retrospect, for instance, presented with such severe infections as septicxmia, the choice of serum levels of IgG of less than 200 mg. meningitis, and osteomyelitis did not usually suffer further attacks once they had started replacement per 100 ml. as a criterion for entry seems rather arbitrary Furthermore, there was the increased fatality therapy. is no evidence of any obvious - firstly, because there in the small group of patients who stopped (63%) discontinuity in the distribution of values below the for more than a year. treatment lower level of the normal range (600 mg. per 100 ml.); in morbidity observed in patients small decrease The secondly, because patients with somewhat higher values on the 0-05 g. per kg. per week dose, as compared with (200-400 mg. per 100 ml.) have also been shown to be 0-025 g. per kg., is taken as evidence of the efficacy of prone to increased infections both by Hobbs (1966) and in the present study, in which over 150 patients were prophylactic immunoglobulin. However, the difference, found to have serum IgG values between 200 and 600 mg. although statistically significant, is not thought to justify per 100 ml. when examined because of a history of the routine use of 0-05 g. per kg. per week in all patients, in view of the added discomfort and expense associated recurrent severe infections (in some instances they even the larger dose. In fact, during the period January, with it and because is required replacement therapy); thirdly, now known that there is a variety of qualitative immuno1966, to June, 1968, most patients in the study were satislogical deficiency states involving quantitative abnormality factorily maintained on a dose of 0-025 g. per kg. per of only one of the immunoglobulins. week. It is therefore recommended that this should be It has also become clear that the syndrome of hypothe normal dose, but that in patients who continue to gammaglobulinxmia is enormously varied, and the group get severe infections on this dose consideration should of cases collected here was quite heterogeneous. Most of be given to increasing it to 0-05 g. per kg. per week. the clinical and pathological findings in this study have It should also be stated that, although the workingalready been reported; but the present series has provided party have no experience of an equivalent dose given at
different
batches
however,
a
168 or monthly intervals, this regimen might prove more convenient for some patients. Live vaccines (polio, vaccinia, and B.C.G.) were given to a third of all patients without apparent ill-effect. The working-party -recommend, however, that immunisation should be undertaken only after careful consideration and after ensuring that normal circulating lymphocytes and normal delayed skin hypersensitivity are present.
fortnightly
The full report of this study is to be published in the Medical Research Council’s Special Report Series. The working-party gratefully acknowledge the help of some two hundred doctors and many other people who supplied information about the patients. Their names will be listed in the forthcoming report of the working-party. Requests for reprints should be addressed to Dr. Lisa Hill, Clinical Research Centre, 164 Tottenham Court Road, London W.l. REFERENCES
Barandun, S. (1959) Das Antikorpermangelsyndrome. Basle. Bruton, O. C. (1952) Pediatrics, 9, 722. Collins, H. D., Dudley, H. R. (1955) New Engl.J. Med. 252, 255. Conn, H. O., Quintiliani, R. (1966) Ann. intern. Med. 65, 528. Fahey, J. L., McKelvey, E. M. (1965) J. Immun. 94, 84. Fudenberg, H., Hirschhorn, K. (1965) Med. Clin. N. Am. 49, 1533. Gitlin, D., Gross, P. A. M., Janeway, C. A. (1959) New Engl.J. Med. 260, 72. Good, R. A., Rotstein, J. (1960) Bull. rheum. Dis. 10, 203. Grant, G. H., Wallace, W. D. (1954) Lancet, ii, 671. Green, I., Litwin, S., Adlersberg, R., Rubin, I. (1966) Archs intern. Med. 118, 592. Hobbs, J. R. (1966) Lect. scient. Basis Med. 106. Hutchison, J. H. (1955) Lancet, ii, 844, 1196. Janeway, C. A., Apt, L., Gitlin, D. (1953) Trans. Ass. Am. Phycns, 66, 200.
Gitlin, D., Craig, J. M., Grice, D. S. (1956) ibid. 69, 93. Kamme, C., Coster, C., Hagelquist, E., Kalen, N., Lindholm, H., Grubb, R. (1966) Acta med. scand. 179, 679. Keidan, S. E., McCarthy, K., Haworth, J. C. (1953) Archs Dis. Childh. 28, —
110.
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Vallet, L., Cutbush, M., Mollison, P. L., Thomas, A. R., Gell, H., Soothill, J. F. (1961) J. clin. Path. 14, 470. Kuschner, D. L., Dubin, A., Donlon, W. P., Bronsky, D. (1960) Am. J. Med. 29, 33. Marshall, W. C., Weston, H. J., Bodian, M. (1964) Archs Dis. Childh. 39, 18. Page, A. R., Hansen, A. E., Good, R. A. (1963) Blood, 21, 197. Parkes, R. (1958) Br. med. J. i, 973. Richerson, H. B., Seebohm, P. M. (1966) Archs intern. Med. 117, 568. Rodgers, T. S., Haggie, M. H. K. (1964) Lancet, i, 1042. —
P. G.
A GERMFREE INFANT D. V. I. FAIRWEATHER
R. D. BARNES
JEAN
C. KEANE
HOLLIDAY
ALINA PIESOWICZ
J. F. SOOTHILL
MAUREEN TUFFREY FROM
THE
DEPARTMENTS
HEALTH, AND
OF
HÆMATOLOGY, IMMUNOLOGY,
CHILD
MICROBIOLOGY AT THE INSTITUTE OF CHILD HEALTH AND
CHILDREN, AND THE DEPARTMENT OF OBSTETRICS, UNIVERSITY COLLEGE HOSPITAL, LONDON W.C.1
HOSPITAL FOR SICK
An infant was delivered by cæsarean section and maintained in a " germfree " isolation unit for 6 days during which absence of bacteria was demonstrated by microbiological tests. This procedure was done as part of a programme for the treatment of potential cases of immunity deficiency, but since this infant was not affected with combined immunitydeficiency state, as her brother had been, she was removed from the germfree isolation unit.
Summary
Introduction A SEVERE and fatal form of hypogammaglobulinxmia associated with lymphopenia was first described by Hitzig and Willi (1961). This familial, and possibly inherited,
disease (Hitzig et al. 1968) presents with deficiency of both cellular and humoral immunity-the combined immunitydeficiency syndrome of Soothill (1968a). Foreign-tissue grafts are not rejected. Despite this, attempts at treatment by grafting immunologically competent tissue have been only marginally effective and have not modified the universal mortality in this condition (Hitzig et al. 1965, Harboe et al. 1966, Hong et al. 1968). Failure may have been due to the effects of severe infection at the time the patients were diagnosed and treated, or it may have resulted from the relative maturity of the immunity mechanism and this might; in part, be the effect of the normal antigenic stimulus of infection. In view of this, when the mother of such a child (Thompson 1967) became pregnant again, the child was delivered by caesarean section and maintained in a germfree isolation system while diagnostic tests were done. If the child were affected it was proposed to attempt early grafting of foetal tissue, before the normal bacterial colonisation occurred and whilst the child was, immunologically, relatively " immature ". Methods
Transfer
to
Isolator
The mother, aged 23 was admitted during a normal pregnancy at 38 weeks. Her previous delivery had been normal and clinical examination revealed no abnormality. Elective lower-segment cxsarean section was performed at 39 weeks’ gestation through a sterile, flexible plastic-film surgical isolator described (Barnes, Fairweather, et al. 1968). This isolator was adhered aseptically to the mother’s abdomen which had been previously prepared with an iodine antiseptic solution. The baby, after delivery into the surgical isolator, was then transferred through a connecting tunnel to a mobile plastic transfer isolator for resuscitation. Both isolators, joined (fig. 1) with their enclosed contents (surgical and infant resuscitation requirements), had been sterilised in advance with 4 megarads of gamma irradiation from a cobalt-60 source (U.K. Atomic Energy Authority, Wantage Laboratory). Whilst the infant was being resuscitated in the transfer isolator the connecting tunnel between the two units was sealed and cut between the airtight steel clamps. After resuscitation, the infant was transported in the mobile transfer isolator to the vicinity of the maintenance isolator. The aseptic union, and subsequent incision of the two sterile adhesive membranes incorporated into both the transfer and maintenance isolators formed a common tunnel for the ultimate introduction of the infant into the maintenance isolator. ‘Cornercroft’ type-A human isolator (6g. 2) (Barnes, Tuffrey, and Cook 1968) with minor modification of the sealing of the plastic tent to the consoles was used for the care of the infant. The microbiological integrity of this unit and its sterilisation had been previously confirmed by successful maintenance of germfree mice (Barnes, Holliday, and Cook 1968). Fail-safe devices, similar to those incorporated in the germfree animal isolator (Cook, Tuffrey, and Barnes 1968), were a feature of this isolator and, as a precaution, suction, filtered oxygen i and electricity were also supplied. A metal cot, a sterile plastic film sealed mattress, and a baby incubator were also required I and were introduced into the isolator before sterilisation. ’
Sterilisation The equipment and all supplies likely to be required for the of the infant were sterilised in advance. The method I selected for each item depended upon its ability to withstand : the particular form of sterilisation : care
Gamma irradiation (4 megarads from cobalt-60 source) was used for surgical and connected transfer isolators with enclosed contents and for normal neonatal nursing requirements (instruments &c.) (clothing, bedding, water, sugar, &c.). Gas sterilisation (ethylene oxide) was used to sterilise the air filters, and enclosed incubator in the maintenance isolator; for instruments with glass components, and for the external surfaces of cans of milk.
:
I