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Clinical correlates of antinuclear antibodies in juvenile rheumatoid arthritis
386
September, 1973 The ]ournal o/ P E D I A T R I C S
Clinical correlates of antinuclear antibodies in juvenile rheumatoid arthritis The presence o/antinuclear antibodies (ANA) was determined by an immunofluorescent technique in 200 children with juvenile rheumatoid arthritis and was compared to that in 80 children with other connective tissue diseases, 164 children with nonconnective tissue diseases, and 90 normal children. The low frequency of positive tests in normal children (3 per cent) and in nonconnective tissue diseases (1 per cent) compared with the high seroposi.tivity in children with systemic lupus erythematosus (100 per cent), polyarteritis (75 per cent), and juvenile rheumatoid arthritis (38.5 per cent) demonstrates that the method used to detect ANA is both sensitive and selective. In juvenile rheumatoid arthritis, ANA were found significantly more #equently in girls, in patients with early onset o/disease or whose present age was young, and in those with polyarticular disease or with monarticular disease and iridocyelitis.
Ross E. Petty, M.D., * James T. Cassidy, M.D., and Donita B. Sullivan, M.D., A n n Arbor, Mich.
DETERMINATIONS of ANA as part of the diagnostic and management program of children with possible connective tissue disFrom the Pediatric Arthritis Clinic and Rehabilitation Service, the Raekham Arthritis Research Unit, and the Departments of Pediatrics and Communicable Diseases, and Internal Medicine, The University o/ Michigan Medical School. Supported by a grant from the Michigan Chapter o/the Arthritis Foundation and a training grant from the National Foundation. The Rackham Arthritis Research Unit is supported in part by a grant from the Horace H. Rackham School o] Graduate Studies. The biostatistieal services were supported by the Medical School Fund for Computing. Presented in part at the annual meeting o/the American Rheumatism Association (Arthritis Rheum. 12: 323, 1969). ~Reprint address: R6056 Kresge Medical Research Building II, The University o] Michigan Medical School, Ann Arbor, Mich. 48104.
Vol. 83, No. 3, pp. 386-389
ease have been of limited value because of the relative paucity of information regarding the significance of ANA in this age group, the wide discrepancies in prevalence of ANA reported in studies of children with juvenile rheumatoid arthritis, 15 and absence of comparable documentation in children with nonconnective tissue disease or in normal children. The purpose of this paper is to report an investigation of ANA in juvenile rheumatoid arthritis and other childhood diseases. M A T E R I A L S AND M E T H O D S The initial serum from each of 200 consecutive children with juvenile rheumatoid arthritis seen from 1961 to 1970 was analyzed. The diagnosis of juvenile rheumatoid arthritis conformed to preliminary criteria
Volume 83 Number 3
Antinuelear antibodies in rheumatoid arthritis
(Classification I) adopted by a subcommittee of the American Rheumatism Association. 6 One hundred forty-four of the patients were girls and 56 were boys. The mean age was 10.1 years and age of onset was through 14 years. Ninety-one per cent had active disease. Type of onset of disease was defined as polyarticular, monarticular or oligoarticular, and systemic. Functional class was based upon recommendations of the American Rheumatism Association. 7 No patient was receiving a drug known to induce ANA. The comparison groups included 80 children with other connective tissue diseases, 90 "normal" children from the Tecumseh Community Health Study, and 164 children who were hospitalized during 1968 with nonconnective tissue diseases. All sera not assayed immediately were frozen at -70 ~ C. Antinuctear antibody determinations, were performed by an indirect immunofluorescent technique. 8 Fresh liver from young C B A / J mice was frozen; cryostat sections were cut and fixed in acetone for 10 minutes at 4 ~ C. Following three 5 minute washes in buffer, the sections were incubated with sera at room temperature in a humidity chamber for 30 minutes. They were then rewashed and incubated with fluorescein-labeled horse antihuman gamma globulin for 30 minutes, washed, and mounted with Elvinol. (The polyvalent antiserum was obtained from Progressive Laboratories, Lot No. 3131. The "reconstituted material was diluted 1:5, had a fluorescein/protein ratio of 3.3," and an antibody nitrogen concentration of 0.6 mg. per milliliter.) Unlabeled monospecific rabbit antihuman IgG, IgA, or IgM prepared in this laboratory, and fluorescein-conjugated sheep antirabbit gamma globulin 1~ were used for determination of immunoglobulin class of ANA. Positive and negative control sera were included in each study. The sections were examined by two observers with a Leitz ultraviolet microscope. Nuclear extracts were prepared from fresh calf thymus? a' 12 Rheumatoid factor was determined by the latex fixation method? a Immunoassay of serum
38 7
Table I. Prevalence of antinuclear antibodies in undiluted serum Diagnosis No. 1. Connective tissue diseases Juvenile rheumatoid arthritis 200 Juvenile ankylosing spondylitis 17 Systemic lupus erythematosus 24 Dermatomyositis 20 Scleroderma 13 Polyarteritis 6 2. Nonconnective tissue diseasese 164 3. Community Health Study 90
No. positive (%) 77 (38.5) 0 24 2 5 4
(0) (loo) (10) (3a.5) (75)
2 (1) 3 (3)
"ZThis group includes 21 newborn infants, 15 children with glomerulonephritls, 9 with nephrotic syndrome, 11 with bacterial infections, 7 with hepatitis, 6 with viral exanthems, 17 with diabetes .mellitus, 9 with congenital heart disease, 4 with allergic diseasesl 4 with myocarditis, 5 with noninfectious enteritis or colitis, 5 with idiopathic or HenochSchoenlein purpura, 6 with hemolytic or iron deficiency anemia, 8 with lymphomas and other tumors, and 37 with surgical disorders.
immunoglobulins was performed by radial diffusion? 4 Nonparametric analyses were performed by the chi square test; parametric tests included analysis of variance and covariance and the Pearson correlation. 15 The Michigan Terminal System on an IBM 360/67 duplex computer was used ~6 and a p value equal to or less than 0.05 was accepted as significant. RESULTS
Prevalence of ANA in juvenile rheumatoid arthritis and comparison groups. Seventyseven (38.5 per cent) of the 200 patients with juvenile rheumatoid arthritis had positive ANA in undiluted sera (Table I). The prevalence of ANA in children with other connective tissue diseases ranged from 100 per cent in systemic lupus erythematosus to 0 per cent in juvenile ankylosing spondylitis. The two children in the hospitalized group of 164 who were positive had chronic active hepatitis. Three girls from the community health study were positive but free of clinically apparent disease. Clinical associations of ANA. ANA were most common in children with polyarticular onset of juvenile rheumatoid arthritis (Table II) and least common in those with
3 8 8 Petty, Cassidy, and Sullivan
Table II. ANA and type of onset of juvenile rheumatoid arthritis
Type of onset Polyarticular Monarticular Systemic
Females Males Posi- Nega- Posi- Negative tire tive tire 36 35 7 18 2 11 28 23 0 18 4 18
systemic onset of disease (p = 0.0002). Sixty-four per cent of those having monarticular onset and iridocyclitis had ANAl7; none of these children had a positive rheumatoid factor test. There was an increased frequency of ANA in girlS (47 per cent) compared to boys (16 per cent) (p ---- 0.0001). The frequency of ANA (Table I I I ) was highest in younger children (p = 0.008), in those with early age of onset (p ---- 0.026), and in those without erosions (p = 0.008). There was no concordance of ANA, functional class, and duration of disease. None of the six patients who subsequently died had positive ANA. There was no association between ANA and rheumatoid factor (p = 1.000). Rheumatoid factor was associated with the older child (p < 0.0000), late onset of disease (p < 0.0000), polyarticular onset (p = 0.0004), and tile presence of rheumatoid nodules (p < 0.0000). ANA was present in half of the 14 children with nodules and in four of the eight children with selective IgA deficiency. Of the 77 undiluted sera which were ANA positive, 19 were positive to a dilution of 1 : 8, 16 t o l : 3 2 , 8 t o 1 : 1 2 8 , 1 t o l : 2 5 6 , a n d l t o 1 : 1024. In no instance did a negative serum become positive on dilution. Ninety-five per cent of the positive reactors had ANA of IgG class, 19 per cent IgA, and 38 per cent IgM. Fifty-seven per cent had ANA solely of the IgG class. Lupus erythematosus cell formation and preclpltin reactions. None of the ANA-positire sera formed lupus erythematosus cells. Three patients had previously had a transiently positive test. These patients have been followed for 14, 8, and 7 years without the appearance of diagnostic features of clinical
The Journal o[ Pediatrics September 1973
Table I I I . Age of onset of juvenile rheumatoid arthritis in ANA-positive group
Age (yr.) 0-4 5-9 10-14
Females Males Posi- ] Nega- Posi- I Negative tire tive tire 2 15 41 29 5 23 18 28 9 19 2 9
systemic lupus erythematosus. Five sera developed precipitin arcs in agarose gel with saline-soluble nuclear extract. These sera had ANA of only the IgG class with a mean titer of 1:32. There were no precipitin reactions with deoxyribonucleic acid. DISCUSSION
Although autoantibodies directed against nuclear constituents are of widespread occurrence in human connective tissue diseases, is the pathogenic significance of ANA in children with juvenile rheumatoid arthritis has been difficult to assess. There have been conflicting reports on frequency of occurrence and a paucity of comparable data in normal children and in those with nonconnective tissue diseases. Earlier investigations of smaller groups of patients have reported frequencies of ANA in juvenile rheumatoid arthritis from 13 p e r cent 1 to 55 per cent. s ANA in juvenile rheumatoid arthritis were demonstrated in 38 per cent of 42 children by Miller and associates ~ and in 22 per cent of 85 children by Kornreich and associates. ~ Both reports pointed out the increased occurrence of ANA in girls, and Kornreich and associates noted a higher frequency of ANA in younger patients. In contrast to the present study, their children who had ANA were more likely to have rheumatoid factor. Bluestone and associates s detected ANA in only 4 per cent of 200 patients with juvenile rheumatoid arthritis, but 60 per cent of their group were in remission at the time of the serologic study. Discrepancies in the frequency of ANA in juvenile rheumatoid arthritis from report to report might be explained by differences in sensitivity and selectivity of the methods as
Volume 83 Number 3
AntinucIear antibodies in rheumatoid arthritis
well as by differences in composition of the study groups. I n some studies 2"5 h u m a n leukocytes were used as nuclear substrates, whereas mouse liver was utilized in the present study. G r e a t e r sensitivity of the latter method m i g h t account for the higher frequency of A N A in our study. O n the other hand, the frequency of A N A in n o r m a l children a n d in children with noneonnective tissue diseases was acceptably low. A l t h o u g h there were m a r k e d differences in prevalence of A N A a m o n g the various subgroups of juvenile r h e u m a t o i d arthritis, a n d A N A a n d r h e u m a t o i d factor were independent variables in the present study, la, 19 this evidence alone is insufficient to consider juvenile r h e u m a t o i d arthritis more than one clinical spectrum of disease. However, these differences do underline the heterogeneity of the clinical disease a n d suggest either differences in host immunologic response, etiologic agents, or pathogenesis. T h e presence of A N A in a child is strongly suggestive of connective tissue disease when a highly sensitive but selective test is utilized as d o c u m e n t e d by the results of the present study. Tests for A N A like those for r h e u m a toid factor are of a d d i t i o n a l clinical usefulness in differentiating juvenile r h e u m a t o i d arthritis from the other connective tissue diseases of children. The authors wish to thank Ann Burt, M.T., for expert technical assistance, and Janice Iravani, B.S., for statistical programing. Dr. P. T. Storm assisted with the initial phase of this study while a sophomore medical student.
5.
6.
7. 8.
9.
10.
11.
12.
13.
14.
15.
REFERENCES
1. Weir, D. M., Holborow, E. J., and Johnson, G. D.: A clinical study of serum antinuelear factor, Br. Med. J. 1: 933, 1961. 2. Barnett, E. V., North, A. F., Jr., Condemi, J. C., Jacox, R. F., and Vaughan, J. H.: Antinuclear factors in systemic lupus erythematosus and rheumatoid arthritis, Ann. Intern. Med. 63: 100, 1965. 3. Miller, J. J,, Henrich, V. L., and Brandstrup, N. E.: Sex difference in incidence of antinuclear factors in juvenile rheumatoid arthritis, Pediatrics 38: 916, 1966. 4. Kornreich, H. K., Drexler, E., and Hanson,
16.
17. 18. 19.
389
V.: Antinuclear factors in childhood rheumatic diseases, J. PEDIATR. 69: 1039, 1966. Bluestone, R., Goldberg, L. S., Katz, R. M., Marchesano, J. M., and Calabro, J. J.: Juvenile rheumatoid arthritis: A serologic survey of 200 consecutive patients, J. PEDIATR. 77: 98, 1970. Brewer, E. H., Bass, J., Cassidy, J. T., Duran, B. S., Fink, C. W., Jacobs, J. C., Markowitz, M., Schaller, J., and Stillman, J. S.: Criteria for the classification of juvenile rheumatoid arthritis, Bull. Rheum. Dis. 23: 712, 1973. Steinbrocker, O., Traeger, C. H., and Batterman, R. C.: Therapeutic criteria in rheumatoid arthritis, J. A. M. A. 140: 659, 1949. Friou, G. J.: The LE cell factor and antinuclear antibodies, in Cohen, A. S., editor: Laboratory diagnostic procedures in the rheumatic diseases, Boston, 1967, Little, Brown & Company, pp. 114-167. Wood, B. T., Thompson, S. H., and Goldstein, G.: Fluorescent antibody staining. III. Preparation of fluorescein-isothiocyanatelabeled antibodies, J. Immunol. 95: 225, 1965. The, T. H., and Feltkamp, T. E. W.: Conjugation of fluorescein isothiocyanate to antibodies. II. A reproducible method, Immunology 18: 875, 1970. Allfrey, V. G., Littau, V. C., and Mirsky, A. E.: Methods for the purification of thymus nuclei and their application to studies of nuclear protein synthesis, J. Cell. Biol. 21: 213, 1964. Tan, E. M., and Kunkel, H. G.: Characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus, J. Immunol. 96: 464, 1966. Cassidy, J. T., and Valkenburg, H. A.: A five year prospective study of rheumatoid factor tests in juvenile rheumatoid arthritis, Arthritis Rheum. 10: 83, 1967. Fahey, J. L., and McKelvey, E. M.: Quantitative determination of serum immunoglobulins in antibody agar plates, J. Immunol. 94: 84, 1965. Dixon, W. J., and Massey, F. J.: Introduction to statistical analysis, ed. 3, New York, 1969, McGraw-Hill Book Company, Inc., pp. 243, 116, and 202. Bartels, R. C. F., Editor: The Michigan terminal system, ed. 3, Ann Arbor, 1971, The University of Michigan Computing Center. Cassidy, J. T., Brody, G. L., and Martel, W.: Monarticular juvenile rheumatoid arthritis, J. PEDIATR. 70: 867, 1967. Beck, J. S.: Auto-antibodies to cell nuclei, Scott. Med. J. 18: 373, 1963. Hanson, V., Drexler, E., and Kornreich, H.: The relationship of rheumatoid factor to age of onset in juvenile rheumatoid arthritis, Arthritis Rheum. 12: 82, 1969.
September, 1973 The ]ournal o/ P E D I A T R I C S
Clinical correlates of antinuclear antibodies in juvenile rheumatoid arthritis The presence o/antinuclear antibodies (ANA) was determined by an immunofluorescent technique in 200 children with juvenile rheumatoid arthritis and was compared to that in 80 children with other connective tissue diseases, 164 children with nonconnective tissue diseases, and 90 normal children. The low frequency of positive tests in normal children (3 per cent) and in nonconnective tissue diseases (1 per cent) compared with the high seroposi.tivity in children with systemic lupus erythematosus (100 per cent), polyarteritis (75 per cent), and juvenile rheumatoid arthritis (38.5 per cent) demonstrates that the method used to detect ANA is both sensitive and selective. In juvenile rheumatoid arthritis, ANA were found significantly more #equently in girls, in patients with early onset o/disease or whose present age was young, and in those with polyarticular disease or with monarticular disease and iridocyelitis.
Ross E. Petty, M.D., * James T. Cassidy, M.D., and Donita B. Sullivan, M.D., A n n Arbor, Mich.
DETERMINATIONS of ANA as part of the diagnostic and management program of children with possible connective tissue disFrom the Pediatric Arthritis Clinic and Rehabilitation Service, the Raekham Arthritis Research Unit, and the Departments of Pediatrics and Communicable Diseases, and Internal Medicine, The University o/ Michigan Medical School. Supported by a grant from the Michigan Chapter o/the Arthritis Foundation and a training grant from the National Foundation. The Rackham Arthritis Research Unit is supported in part by a grant from the Horace H. Rackham School o] Graduate Studies. The biostatistieal services were supported by the Medical School Fund for Computing. Presented in part at the annual meeting o/the American Rheumatism Association (Arthritis Rheum. 12: 323, 1969). ~Reprint address: R6056 Kresge Medical Research Building II, The University o] Michigan Medical School, Ann Arbor, Mich. 48104.
Vol. 83, No. 3, pp. 386-389
ease have been of limited value because of the relative paucity of information regarding the significance of ANA in this age group, the wide discrepancies in prevalence of ANA reported in studies of children with juvenile rheumatoid arthritis, 15 and absence of comparable documentation in children with nonconnective tissue disease or in normal children. The purpose of this paper is to report an investigation of ANA in juvenile rheumatoid arthritis and other childhood diseases. M A T E R I A L S AND M E T H O D S The initial serum from each of 200 consecutive children with juvenile rheumatoid arthritis seen from 1961 to 1970 was analyzed. The diagnosis of juvenile rheumatoid arthritis conformed to preliminary criteria
Volume 83 Number 3
Antinuelear antibodies in rheumatoid arthritis
(Classification I) adopted by a subcommittee of the American Rheumatism Association. 6 One hundred forty-four of the patients were girls and 56 were boys. The mean age was 10.1 years and age of onset was through 14 years. Ninety-one per cent had active disease. Type of onset of disease was defined as polyarticular, monarticular or oligoarticular, and systemic. Functional class was based upon recommendations of the American Rheumatism Association. 7 No patient was receiving a drug known to induce ANA. The comparison groups included 80 children with other connective tissue diseases, 90 "normal" children from the Tecumseh Community Health Study, and 164 children who were hospitalized during 1968 with nonconnective tissue diseases. All sera not assayed immediately were frozen at -70 ~ C. Antinuctear antibody determinations, were performed by an indirect immunofluorescent technique. 8 Fresh liver from young C B A / J mice was frozen; cryostat sections were cut and fixed in acetone for 10 minutes at 4 ~ C. Following three 5 minute washes in buffer, the sections were incubated with sera at room temperature in a humidity chamber for 30 minutes. They were then rewashed and incubated with fluorescein-labeled horse antihuman gamma globulin for 30 minutes, washed, and mounted with Elvinol. (The polyvalent antiserum was obtained from Progressive Laboratories, Lot No. 3131. The "reconstituted material was diluted 1:5, had a fluorescein/protein ratio of 3.3," and an antibody nitrogen concentration of 0.6 mg. per milliliter.) Unlabeled monospecific rabbit antihuman IgG, IgA, or IgM prepared in this laboratory, and fluorescein-conjugated sheep antirabbit gamma globulin 1~ were used for determination of immunoglobulin class of ANA. Positive and negative control sera were included in each study. The sections were examined by two observers with a Leitz ultraviolet microscope. Nuclear extracts were prepared from fresh calf thymus? a' 12 Rheumatoid factor was determined by the latex fixation method? a Immunoassay of serum
38 7
Table I. Prevalence of antinuclear antibodies in undiluted serum Diagnosis No. 1. Connective tissue diseases Juvenile rheumatoid arthritis 200 Juvenile ankylosing spondylitis 17 Systemic lupus erythematosus 24 Dermatomyositis 20 Scleroderma 13 Polyarteritis 6 2. Nonconnective tissue diseasese 164 3. Community Health Study 90
No. positive (%) 77 (38.5) 0 24 2 5 4
(0) (loo) (10) (3a.5) (75)
2 (1) 3 (3)
"ZThis group includes 21 newborn infants, 15 children with glomerulonephritls, 9 with nephrotic syndrome, 11 with bacterial infections, 7 with hepatitis, 6 with viral exanthems, 17 with diabetes .mellitus, 9 with congenital heart disease, 4 with allergic diseasesl 4 with myocarditis, 5 with noninfectious enteritis or colitis, 5 with idiopathic or HenochSchoenlein purpura, 6 with hemolytic or iron deficiency anemia, 8 with lymphomas and other tumors, and 37 with surgical disorders.
immunoglobulins was performed by radial diffusion? 4 Nonparametric analyses were performed by the chi square test; parametric tests included analysis of variance and covariance and the Pearson correlation. 15 The Michigan Terminal System on an IBM 360/67 duplex computer was used ~6 and a p value equal to or less than 0.05 was accepted as significant. RESULTS
Prevalence of ANA in juvenile rheumatoid arthritis and comparison groups. Seventyseven (38.5 per cent) of the 200 patients with juvenile rheumatoid arthritis had positive ANA in undiluted sera (Table I). The prevalence of ANA in children with other connective tissue diseases ranged from 100 per cent in systemic lupus erythematosus to 0 per cent in juvenile ankylosing spondylitis. The two children in the hospitalized group of 164 who were positive had chronic active hepatitis. Three girls from the community health study were positive but free of clinically apparent disease. Clinical associations of ANA. ANA were most common in children with polyarticular onset of juvenile rheumatoid arthritis (Table II) and least common in those with
3 8 8 Petty, Cassidy, and Sullivan
Table II. ANA and type of onset of juvenile rheumatoid arthritis
Type of onset Polyarticular Monarticular Systemic
Females Males Posi- Nega- Posi- Negative tire tive tire 36 35 7 18 2 11 28 23 0 18 4 18
systemic onset of disease (p = 0.0002). Sixty-four per cent of those having monarticular onset and iridocyclitis had ANAl7; none of these children had a positive rheumatoid factor test. There was an increased frequency of ANA in girlS (47 per cent) compared to boys (16 per cent) (p ---- 0.0001). The frequency of ANA (Table I I I ) was highest in younger children (p = 0.008), in those with early age of onset (p ---- 0.026), and in those without erosions (p = 0.008). There was no concordance of ANA, functional class, and duration of disease. None of the six patients who subsequently died had positive ANA. There was no association between ANA and rheumatoid factor (p = 1.000). Rheumatoid factor was associated with the older child (p < 0.0000), late onset of disease (p < 0.0000), polyarticular onset (p = 0.0004), and tile presence of rheumatoid nodules (p < 0.0000). ANA was present in half of the 14 children with nodules and in four of the eight children with selective IgA deficiency. Of the 77 undiluted sera which were ANA positive, 19 were positive to a dilution of 1 : 8, 16 t o l : 3 2 , 8 t o 1 : 1 2 8 , 1 t o l : 2 5 6 , a n d l t o 1 : 1024. In no instance did a negative serum become positive on dilution. Ninety-five per cent of the positive reactors had ANA of IgG class, 19 per cent IgA, and 38 per cent IgM. Fifty-seven per cent had ANA solely of the IgG class. Lupus erythematosus cell formation and preclpltin reactions. None of the ANA-positire sera formed lupus erythematosus cells. Three patients had previously had a transiently positive test. These patients have been followed for 14, 8, and 7 years without the appearance of diagnostic features of clinical
The Journal o[ Pediatrics September 1973
Table I I I . Age of onset of juvenile rheumatoid arthritis in ANA-positive group
Age (yr.) 0-4 5-9 10-14
Females Males Posi- ] Nega- Posi- I Negative tire tive tire 2 15 41 29 5 23 18 28 9 19 2 9
systemic lupus erythematosus. Five sera developed precipitin arcs in agarose gel with saline-soluble nuclear extract. These sera had ANA of only the IgG class with a mean titer of 1:32. There were no precipitin reactions with deoxyribonucleic acid. DISCUSSION
Although autoantibodies directed against nuclear constituents are of widespread occurrence in human connective tissue diseases, is the pathogenic significance of ANA in children with juvenile rheumatoid arthritis has been difficult to assess. There have been conflicting reports on frequency of occurrence and a paucity of comparable data in normal children and in those with nonconnective tissue diseases. Earlier investigations of smaller groups of patients have reported frequencies of ANA in juvenile rheumatoid arthritis from 13 p e r cent 1 to 55 per cent. s ANA in juvenile rheumatoid arthritis were demonstrated in 38 per cent of 42 children by Miller and associates ~ and in 22 per cent of 85 children by Kornreich and associates. ~ Both reports pointed out the increased occurrence of ANA in girls, and Kornreich and associates noted a higher frequency of ANA in younger patients. In contrast to the present study, their children who had ANA were more likely to have rheumatoid factor. Bluestone and associates s detected ANA in only 4 per cent of 200 patients with juvenile rheumatoid arthritis, but 60 per cent of their group were in remission at the time of the serologic study. Discrepancies in the frequency of ANA in juvenile rheumatoid arthritis from report to report might be explained by differences in sensitivity and selectivity of the methods as
Volume 83 Number 3
AntinucIear antibodies in rheumatoid arthritis
well as by differences in composition of the study groups. I n some studies 2"5 h u m a n leukocytes were used as nuclear substrates, whereas mouse liver was utilized in the present study. G r e a t e r sensitivity of the latter method m i g h t account for the higher frequency of A N A in our study. O n the other hand, the frequency of A N A in n o r m a l children a n d in children with noneonnective tissue diseases was acceptably low. A l t h o u g h there were m a r k e d differences in prevalence of A N A a m o n g the various subgroups of juvenile r h e u m a t o i d arthritis, a n d A N A a n d r h e u m a t o i d factor were independent variables in the present study, la, 19 this evidence alone is insufficient to consider juvenile r h e u m a t o i d arthritis more than one clinical spectrum of disease. However, these differences do underline the heterogeneity of the clinical disease a n d suggest either differences in host immunologic response, etiologic agents, or pathogenesis. T h e presence of A N A in a child is strongly suggestive of connective tissue disease when a highly sensitive but selective test is utilized as d o c u m e n t e d by the results of the present study. Tests for A N A like those for r h e u m a toid factor are of a d d i t i o n a l clinical usefulness in differentiating juvenile r h e u m a t o i d arthritis from the other connective tissue diseases of children. The authors wish to thank Ann Burt, M.T., for expert technical assistance, and Janice Iravani, B.S., for statistical programing. Dr. P. T. Storm assisted with the initial phase of this study while a sophomore medical student.
5.
6.
7. 8.
9.
10.
11.
12.
13.
14.
15.
REFERENCES
1. Weir, D. M., Holborow, E. J., and Johnson, G. D.: A clinical study of serum antinuelear factor, Br. Med. J. 1: 933, 1961. 2. Barnett, E. V., North, A. F., Jr., Condemi, J. C., Jacox, R. F., and Vaughan, J. H.: Antinuclear factors in systemic lupus erythematosus and rheumatoid arthritis, Ann. Intern. Med. 63: 100, 1965. 3. Miller, J. J,, Henrich, V. L., and Brandstrup, N. E.: Sex difference in incidence of antinuclear factors in juvenile rheumatoid arthritis, Pediatrics 38: 916, 1966. 4. Kornreich, H. K., Drexler, E., and Hanson,
16.
17. 18. 19.
389
V.: Antinuclear factors in childhood rheumatic diseases, J. PEDIATR. 69: 1039, 1966. Bluestone, R., Goldberg, L. S., Katz, R. M., Marchesano, J. M., and Calabro, J. J.: Juvenile rheumatoid arthritis: A serologic survey of 200 consecutive patients, J. PEDIATR. 77: 98, 1970. Brewer, E. H., Bass, J., Cassidy, J. T., Duran, B. S., Fink, C. W., Jacobs, J. C., Markowitz, M., Schaller, J., and Stillman, J. S.: Criteria for the classification of juvenile rheumatoid arthritis, Bull. Rheum. Dis. 23: 712, 1973. Steinbrocker, O., Traeger, C. H., and Batterman, R. C.: Therapeutic criteria in rheumatoid arthritis, J. A. M. A. 140: 659, 1949. Friou, G. J.: The LE cell factor and antinuclear antibodies, in Cohen, A. S., editor: Laboratory diagnostic procedures in the rheumatic diseases, Boston, 1967, Little, Brown & Company, pp. 114-167. Wood, B. T., Thompson, S. H., and Goldstein, G.: Fluorescent antibody staining. III. Preparation of fluorescein-isothiocyanatelabeled antibodies, J. Immunol. 95: 225, 1965. The, T. H., and Feltkamp, T. E. W.: Conjugation of fluorescein isothiocyanate to antibodies. II. A reproducible method, Immunology 18: 875, 1970. Allfrey, V. G., Littau, V. C., and Mirsky, A. E.: Methods for the purification of thymus nuclei and their application to studies of nuclear protein synthesis, J. Cell. Biol. 21: 213, 1964. Tan, E. M., and Kunkel, H. G.: Characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus, J. Immunol. 96: 464, 1966. Cassidy, J. T., and Valkenburg, H. A.: A five year prospective study of rheumatoid factor tests in juvenile rheumatoid arthritis, Arthritis Rheum. 10: 83, 1967. Fahey, J. L., and McKelvey, E. M.: Quantitative determination of serum immunoglobulins in antibody agar plates, J. Immunol. 94: 84, 1965. Dixon, W. J., and Massey, F. J.: Introduction to statistical analysis, ed. 3, New York, 1969, McGraw-Hill Book Company, Inc., pp. 243, 116, and 202. Bartels, R. C. F., Editor: The Michigan terminal system, ed. 3, Ann Arbor, 1971, The University of Michigan Computing Center. Cassidy, J. T., Brody, G. L., and Martel, W.: Monarticular juvenile rheumatoid arthritis, J. PEDIATR. 70: 867, 1967. Beck, J. S.: Auto-antibodies to cell nuclei, Scott. Med. J. 18: 373, 1963. Hanson, V., Drexler, E., and Kornreich, H.: The relationship of rheumatoid factor to age of onset in juvenile rheumatoid arthritis, Arthritis Rheum. 12: 82, 1969.