JORGE SbCHEZ-GUERRERO. ROBERT A. LEW, ANNE H. FOSSEL, and PETER H. SCHUR

ARTHRITIS & RHEUMATISM Vol. 39, No. 6, June 1996, pp 1055-1061 0 1996. American College of Rlieumatology 1055 UTILITY OF ANTI-Sm, ANTI-RNP, ANTI-Ro/SS-A, AND ANTI-La/SS-B (EXTRACTABLE NUCLEAR ANTIGENS) DETECTED BY ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DIAGNOSIS OF SYSTEMIC LUPUS ERYTHEMATOSUS JORGE SbCHEZ-GUERRERO. ROBERT A. LEW, ANNE H. FOSSEL, and PETER H. SCHUR Objective. To determine the utility of antiextractable nuclear antigen (anti-ena) antibodies detected by enzyme-linked immunosorbent assay as a predictor for the diagnosis of systemic lupus erythematosus (SLE). Methods. Among 2,185 serum samples sent for testing for antinuclear antibodies (ANA) by indirect immunofluorescence, 259 consecutive patients with positive ANA were identified. Medical charts of these patients were reviewed to assess the clinical diagnosis, with the reviewer having no knowledge of the anti-ena result. Clinical data were abstracted for all patients, and diagnoses established using American College of Rheumatology criteria. The utility of ENA antibodies in the diagnosis of SLE was determined by univariate and multivariate analysis among all patients who were positive for ANA, patients who were positive for ANA and for anti-double-stranded DNA (anti-dsdna), and patients who were positive for ANA and negative for anti-dsdna. Clinical differences between SLE patients with and those without anti-ena antibodies were assessed. Supported in part by Diamedix. Dr. Sanchez-Guerrero s work was supported by a Research Fellowship Award from the Fogarty International Center (NIH 5F05-TW04573-02). Jorge Sanchez-Guerrero, MD, MSc (current address: Instituto Nacional de la Nutrici6n Salvador Zubiran. Vasco de Quiroga. Mexico). Robert A. Lew. PhD, Anne H. Fossel: Robert B. Brigham Multipurpose Arthritis and Musculoskeletal Diseases Center, Brigham and Women s Hospital, Boston, MA; Peter H. Schur, MD: Brigham and Women s Hospital. Harvard Medical School, Boston, MA. Dr. Schur has served as a umsultant to biotechnolow companies and as an expert witness on antinuclear antibodies and related matters. Address reprint requests to Peter H. Schur, MD. Brigham and Women s Hospital, Division of Rheumatology and Immunology, 75 Francis Street, Boston, MA 02115. Submitted for publication September 8, 1995; accepted in revised form January 24, 1996. Results. Anti-ENA antibodies, especially anti-ro/ SS-A, showed strong predictive diagnostic value among ANA+/anti-dsDNA- patients, but were of no utility among ANA+/anti-dsDNA+ patients. The only clinical manifestations that were more common among anti-ena+ SLE patients were pleuritis and the use of hydroxychloroquine. Conclusion. The presence of anti-ena antibodies, especially anti-ro/ss-a, is a useful predictor for the diagnosis of SLE, primarily among patients attending a referral rheumatology center who are positive for ANA and negative for anti-dsdna. No major clinical differences were noted among ANA+ SLE patients with versus those without ENA. Systemic lupus erythematosus (SLE) is a disease of unknown etiology in which patients develop distinct immunologic abnormalities, especially autoantibodies. Among the antibodies found in patients with SLE, particular attention has been focused on nuclear, double-stranded DNA (dsdna), phospholipid, and histone antibodies, and to extractable nuclear antigens (ENA). Anti-ENA antibodies are believed to be markers of particular clinical manifestations of SLE, and may hold clues to its pathogenesis (1,2). Most of the current clinical knowledge about these antibodies is based on data from studies in which immunodiffusion techniques were used for their detection (3). Recently, enzyme-linked immunosorbent assays (ELISAs) for anti-ena antibodies have been developed. ELISA has been proven to be a sensitive and specific detection method, having advantages over immunodiffusion (3-6), and is becoming widely used. In the present crosssectional study, we investigated the utility of anti-ena antibodies detected by ELISA as a predictor for the diagnosis of SLE.

1056 SANCHEZ-GUERRERO ET AL PATIENTS AND METHODS Patients. Between August 1990 and June 1991, 6,313 sera were sent to the Brigham and Women s Hospital (BWH) Clinical Immunology Laboratory for 29.292 tests; 2,185 of these samples were sent for testing for antinuclear antibodies (ANA). Sixty-five percent of the ANA tests were ordered by BWH rheumatologists on patients in whom a rheumatic or autoimmune disorder was suspected. The present study was conducted among all patients who had a positive ANA test result (titer >1:20 on mouse liver substrate and/or?1:40 on HEp-2 cells). Patients with a negative ANA result were not studied, since the probability of SLE in this group is only 0.14% (7), and several studies have failed to detect anti-ena antibodies among these patients (8,9). All ANA-positive sera are automatically tested for antibodies to dsdna, single-stranded DNA, Srn, RNP, Ro/ SS-A, and La/SS-B (ENA). The medical charts of 259 patients who had a positive ANA result during the study period were reviewed by one of the authors (JS-G) to assess the clinical diagnosis according to the attending physician; at the time of the chart review, this author was blinded to the anti-ena antibody results. For 127 patients with a diagnosis of SLE, further data on events occurring ever in the patient s history were abstracted. These events included hemolytic anemia, unintentional weight loss of > 10 Ib, fever, esophageal symptoms (dysphagia, hypomotility), pulmonary symptoms (restrictive disease, decreased carbon monoxide diffusing capacity), myositis (elevated creatinine phosphokinase isoenzyme level, muscle biopsy result consistent with inflammatory myositis), central nervous system (CNS) manifestations (defined as disturbances in mental function, motor disorders, or neuropathy), leukopenia (white blood cell count <4,000/mm3 on 2 or more occasions), lyrnphopenia (lymphocyte count <1.500/mm3 on 2 or more occasions), thrombocytopenia (platelet count < 100,000/mm3 in the absence of causative drugs)? hematuria (urinary sediment with 25 red blood cells/high-power field), and proteinuria (21 + on dipstick examination). Missing or nonexisting data in the medical chart were considered negative. Duration of the disease was defined as the time between the date of diagnosis and the date of the index ENA laboratory test. Clinical and laboratory features were not compared in ANA+/ENA+ versus ANAt/ENA- patients with a diagnosis other than SLE. Diagnoses were made using accepted published criteria (10-14). Patients were classified as ENA+ if they had at least 1 positive ENA result (220 units/ml), and ENA- if they did not have any positive ENA result. One hundred ninety-seven patients (76%) had more than 1 determination of anti-ena antibodies. Because of the retrospective nature of the analysis, it is difficult to assess with certainty whether the attending physician used the results of the anti-ena tests to help establish the diagnosis of SLE. For this reason, we computed the number of American College of Rheumatology (ACR) SLE criteria (10) met by any patient, and report the number of patients in whom the fourth SLE criterion was anti-sm antibodies. ANA, ENA, and dsdna testing. ANA were detected by indirect immunofluorescence, utilizing both cryostat sections of mouse liver and HEp-2 cells as substrate, and fluorescein- Table 1. Distribution of the 259 antinuclear antibody (ANA) positive study subjects, according to extractable nuclear antigen (ENA) status, systemic lupus erythematosus (SLE) diagnosis, and sex ANA+/ENA+ ANA+/ENA- (n = 147 [57%]) (n = 112 [43%]) Other Other SLE diagnoses SLE diagnoses (n = 97 (n = 50 (n = 30 (n = 82 [66%]) [34%])* [27%]) [73%])t Male (n = 22) 2(I%) 6(4%) 0 14 (12%) Female (n = 237) 95 (65%) 44 (30%) 30 (27%) 68 (61%) * Connective tissue diseases (CTD) n = 26 (rheumatoid arthritis 8, juvenile rheumatoid arthritis 6, mixed connective tissue disease 6, Raynaud s phenomenon 3, subacute cutaneous lupus erythernatosus 1, dermatomyositis 1, Sjogren s syndrome 1); probable CTD n = 3, non-autoimmune rheumatic disease n = 3, non-rheumatic diseases n = 18. t CTD n = 32 (Raynaud s phenomenon 10, rheumatoid arthritis 10, systemic sclerosis or CREST syndrome 5, Sjogren s syndrome 2, overlap syndrome 2, antiphospholipid syndrome 1, juvenile rheumatoid arthritis 1, polymyositis 1); non-rheumatic diseases n = 35; other rheumatic diseases n = 15 (fibromyalgia 8, osteoarthritis 5, spinal stenosis 1, rotator cuff syndrome 1). conjugated goat anti-ig (15,16). The presence and levels of anti-sm, anti-rnp, anti-ro/ss-a, and anti-la/ss-b antibodies were determined by a commercially available IgG isotypespecific ELISA method (Diamedk, Miami, FL) using microtiter wells coated with purified Sm, RNP, Ro/SS-A, and La/SS-B, according to the manufacturer s instructions. Test samples with values 220.0 units/ml were considered positive. Antibodies to dsdna were tested by ELISA as described previously (17). Statistical analysis. Data analysis was performed using the SAS statistical computing software package. The factor ENA antibodies had 2 levels, positive (220) and negative (<20). The sensitivity and specificity of combinations of ENA antibodies were tabulated and analyzed using contingency tables. For continuous variables, analysis was performed by t-test. Categorical variables were analyzed by chi-square test, or Fisher s exact test whenever an expected cell size was <5. Multivariate analysis was performed using a logistic regression model. All tests were 2-tailed. P values less than or equal to 0.05 were considered significant. RESULTS Study population. Three hundred sixty-five sera (of 2,185 tested) had a positive ANA result; for 74% of these 365 sera, the testing had been ordered by a rheumatologist. The 365 sera were from 298 patients. The computerized medical records of these 298 patients were reviewed, and only 259 had information on-line: 78% from the Arthritis Center and 22% from other BWH services. These 259 patients were further analyzed. The sex, ENA status, and diagnoses of the patients

ANTI-ENA IN SLE DIAGNOSIS 1057 are shown in Table 1. Two hundred thirty-seven patients (91.5%) were female; 147 patients (57%) were classified as ANA+/ENA+, and 112 patients (43%) as ANA+/ENA-. Among the ANA+/ENA+ patients, there were 97 (66%) with a diagnosis of SLE according to their physician (91 [94%] met 8 4 of the ACR criteria for SLE [lo]). In 7 of these 97 patients, anti-ena (Sm) constituted the fourth classification criterion. Most of these SLE patients (98%) were female. Fifty ANA+/ENA+ patients had a diagnosis other than SLE, including 26 with another connective tissue disease (CTD); 18 patients had a diagnosis of a non-rheumatic disease (Table 1). Among the ANA+/ENA- patients, there were 30 with a diagnosis of SLE (according to their physician); 26 (87%) met 24 criteria for SLE (10). All SLE patients in this group were female. Eighty-two patients had a diagnosis other than SLE, including 32 with another CTD and 35 with a diagnosis of a non-rheumatic disease (Table 1). The difference in the proportion with SLE among ANA+/ENA+ and ANA+/ENA- patients was statistically significant (P < 0.0001). Distribution of ENA antibodies. Among all 259 ANA+ patients, anti-sm was found in 23%, anti-rnp in 27%, anti-roiss-a in 40%, and anti-la/ss-b in 23% (Table 2). When we analyzed only the patients with SLE (n = 127), anti-sm was found in 34%, anti-rnp in 39%, anti-ro/ss-a in 61%. and anti-la/ss-b in 35%. When the analysis was restricted to the 67 SLE patients who were negative for anti-dsdna antibodies, the prevalence of each of the ENA antibodies was similar to that in the group of all SLE patients. Anti-Ro/SS-A antibodies showed the highest prevalence among the entire group of patients as well as the patients with SLE. Significance of antibodies to ENA for the diagnosis of SLE among ANA+ patients. Table 3 shows the sensitivities and specificities of 4 ENA antibodies and 11 combinations of ENA antibodies with respect to the diagnosis of SLE in patients with a positive ANA result (primarily those attending a referral arthritis center). Anti-Ro/SS-A antibodies showed the highest sensitivity Table 2. Distribution of anti-ena antibodies among all ANA+ patients. all SLE patients, and SLE patients without anti-doublestranded DNA (anti-dsdna) antibodies' Srn RNP SS-A SS-B All patients (n = 259) 59 (23) 71 (27) 104 (40) 60 (23) All SLE patients (n = 127) 43 (34) 50 (39) 78 (61) 44 (35) Anti-dsDNA- SLE patients 23 (34) 26 (39) 39 (58) 23 (34) (n = 67) * Values are the number (96). See Figure 1 for other definitions. Table 3. Sensitivity and specificity of ENA antibodies for the diagnosis of SLE in all patients with a positive ANA result and in patients with a positive ANA result and a negative anti-double-stranded DNA (anti-dsdna) result' ANA+/dsDNA - All ANA+ patients patients (n = 259) (n = 184) Sensitivity Specificity Sensitivity Specificity Srn 34 88 34 90 RNP 39 84 39 86 Ro 61 80 58 82 La 35 88 34 89 Sm. RNP 24 94 25 96 Srn, Ro 25 95 22 96 Sm, La 20 94 17 95 RNP, Ro 27 95 24 97 RNP. La 19 96 19 98 Ro, La 32 93 30 94 Sm. RNP, Ro 18 98 15 99 Sm, RNP. La 14 97 13 98 Sni, Ro, La 17 95 12 97 RNP. Ro, La 17 98 15 99 Srn, RNP, Ro, La 12 98 9 100 See Figure I for other definitions. + (61%), but also the lowest specificity (80%). Anti-Sm antibodies had high specificity (88%), but low sensitivity (34%). As expected, the specificity of ENA antibodies increased as more of them were added, but the sensitivity decreased. By univariate analysis, the presence of each of the ENA antibodies as well as the 11 combinations were statistically significant for the diagnosis of SLE (P 4 0.001). By multivariate analysis, after controlling for 2 significant factors (anti-ro/ss-a [P = 0.00011 and anti-rnp [P = 0.0015]), no other factors were associated with the diagnosis of SLE. The combination of these 2 antibodies had an odds ratio (OR) of 7.68 (95% confidence interval [95% CI] 3.1-10.04) and a receiver operating characteristic (ROC) curve value of 0.75 for the diagnosis of SLE. Anti-Ro/SS-A had an OR of 6.5 (95% CI 3.8-11.1) and an ROC curve value of 0.71; anti-rnp had an odds ratio of 3.4 (95% CI 1.9-6.1) and an ROC curve value of 0.61 for the diagnosis of SLE. Significance of anti-dsdna antibodies for the diagnosis of SLE. Of the 259 ANA+ patients, 251 were tested for anti-dsdna antibodies. Of these 251 patients, 67 (27%) tested positive. Sixty of the 67 (90%) had a diagnosis of SLE; the diagnoses in the other 7 patients were mixed connective tissue disease in 3, rheumatoid arthritis in 2, Sjogren's syndrome in 1, and congestive heart failure in 1. The anti-dsdna+ SLE patients

~ 1058 SANCHEZ-GUERRERO ET AL constituted 47% of the SLE group as a whole (60 of 127). The presence of anti-dsdna antibodies in those with a positive ANA result had a sensitivity of 49% and a specificity of 95% for the diagnosis of SLE. The OR for the diagnosis of SLE in the presence of dsdna antibodies was 16.9 (95% CI 7.3-39.0), and the ROC curve value was 0.72. Among the 67 patients with anti-dsdna antibodies, none of the ENA antibodies alone or in any of the 11 possible combinations were of additional aid for the diagnosis of SLE, either in the univariate or the multivariate analysis. Significance of ENA antibodies for the diagnosis of SLE among patients without dsdna antibodies. Among the 184 ANA+/dsDNA- patients, the specificity of each ENA antibody and of the 11 combinations for the diagnosis of SLE was slightly greater compared with that shown when all 259 ANA+ patients were studied (Table 3). Among the ENA antibodies, anti-ro/ss-a showed the highest sensitivity (58%) and specificity (82%) for the diagnosis of SLE. All other ENA antibodies and their possible combinations showed lower sensitivity (139%), but higher specificity (286%). By univariate analysis, all ENA antibodies and the 11 different combinations were statistically significant for the diagnosis of SLE. By multivariate analysis, only 2 factors reached statistical significance: anti-ro/ SS-A was the strongest (P = O.OOOl), and the combination Sm and RNP was the next strongest predictor (P = 0.0013). This combination had an ROC curve value of 0.75. Anti-Ro/SS-A alone had an OR of 6.5 (95% CI 3.8-11.1) and an ROC curve value of 0.70 for the diagnosis of SLE. Clinical manifestations. The ANA+/ENA+ and ANA+/ENA- SLE patient groups did not show any major difference in terms of clinical manifestations (Table 4). Pleuritis (P = 0.039) and hydroxychloroquine use (P = 0.018) were more frequent among ANA+/ ENA+ patients. Other clinical manifestations, such as renal, hematologic, and CNS involvement and skin manifestations (malar rash, photosensitivity), were not significantly different between groups. The prevalence of dsdna antibodies, dosage of prednisone, and use of immunosuppressive agents were also similar in the 2 groups. Nine patients in the ANA+/ENA+ group received treatment with intravenous cyclophosphamide versus none in the ANA+/ENA- group; this difference was not significant (P = 0.11). The mean age of the patients at the time of the index test and the duration of disease were similar in the 2 groups, although MA+/ ENA- patients had disease of shorter duration (9.27 years versus 11.23 years). We did not compare clinical Table 4. Clinical manifestations among ANA+/ENA+ and ANA+/ ENA- SLE patients* ENAt ENApatients patients Clinical manifestation (n = 97) (n = 30) P Leukopenia Lymphopenia Thrombocytopenia Hematuria Proteinuria Autoimmune hemolytic anemia Arthritis Art hralgias Weight loss Fatigue Fever Raynaud s phenomenon Myalgias Myositis Rash Discoid lupus Alopecia Photosensitivity Mouth sores Pleuritis Pericarditis Seizures Psychosis CNS other dsdna Treatment Hydroxychloroquine Prednisone Immunosuppressive drugs IV cyclophosphamide Prednisone dosage, mean f SD mglday Disease duration, mean 2 SD years Age, mean 5 SD years 29 (30) 41 (42) 13 (13) 41 (42) 37 (38) 5 (5) 72 (74) 79 (81) 11 (11) 51 (53) 24 (25) 56 (58) 5 (5) 1(1) 67 (69) 10 (10) 40 (41) 40 (41) 15 (15) 50 (52) 24 (25) 12 (12) 3 (3) 7 (7) 46 (47) 53 (55) 55 (57) 14 (15) 9 (9) 16.98 f 14.46 11.23? 7.97 40.26 f 11.89 7 (23) NS 7 (23) NS 3 (10) NS 10 (33) NS 10 (33) NS 25 (83) NS 28 (93) NS 2 (7) NS 12 (40) NS 5 (17) NS 15 (50) NS 18 (60) NS 2 (7) NS 13 (43) NS 10 (33) NS 4 (13) NS 9 (30) 0.039 4 (13) NS 2 (7) NS 4 (13) NS 14 (47) NS 9 (30) 0.018 12 (40) NS 3 (10) NS 15.66 t 12.56 NS 9.27 t 8.56 NS 40.44 -+ 12.22 NS * Except where otherwise indicated, values are the number ( 31). NS = not significant; CNS = central nervous system; dsdna = doublestranded DNA; IV = intravenous. and laboratory features in ANA+/ENA+ and ANA+/ ENA- patients with diagnoses other than SLE. DISCUSSION In many cases, a diagnosis of systemic lupus erythematosus has a level of uncertainty. The ANA test, the best screening study for SLE, is positive in 99% of SLE patients (7,lO) and should be performed whenever SLE is suspected. However, a positive ANA test result has a positive predictive value for SLE of 19-35% (7,18); for this reason, after ANA testing, patients may need further tests in order to help confirm a diagnosis of

ANTI-ENA IN SLE DIAGNOSIS 1059 SLE. We studied the utility of anti-ena antibodies detected by ELISA as a predictor for the diagnosis of SLE in patients with a positive ANA result seen in a referral hospital. In our study, the prevalence of anti-ena antibodies among all of the patients with positive ANA, and the percentage of SLE patients who tested positive for anti-ena antibodies, were consistent with reported data (2,3,5,6,19). The distribution of anti-ena antibodies among SLE patients fell within the range described (2-4,6,19,20) except for anti-la, which showed a higher frequency. We noted a diagnosis of SLE more commonly among ANA+/ENA+ patients than among MA+/ ENA- patients (P < 0.0001). In our study, which primarily included patients attending a referral rheumatology center, the positive predictive value increased from 49% when a patient had a positive ANA result to 66% when a patient had both a positive ANA and a positive ENA result. Anti-dsDNA antibodies have high specificity for the diagnosis of SLE (10). In our study, this specificity was 95%. It has been suggested that if anti-dsdna antibodies are positive in a patient with SLE symptoms and positive ANA, it is diagnostically irrelevant whether that same serum is positive for other antibodies (8,21). This statement was confirmed in our study. When we studied the utility of anti-ena antibodies for the diagnosis of SLE among patients with anti-dsdna antibodies, none of them was additionally useful. The prevalence of anti-dsdna antibodies in our SLE patients (47%) falls within the reported range of 20-91% (7,8). The distribution of anti-dsdna antibodies was identical among ANA+/ENA+ and MA+/ ENA- SLE patients. This result is consistent with the findings of previous studies in which the prevalence of anti-dsdna antibodies was similar between patients with and without anti-sm (22) or anti-ro (23) antibodies. However, a higher prevalence of anti-dna antibodies has been reported among patients with anti- Ro antibodies compared with patients with both anti-ro and anti-la antibodies (24). We did not evaluate the frequency of anti-dsdna antibodies in relation to specific anti-ena antibodies. Among ANA+/dsDNA- patients, all anti-ena antibodies and 11 different combinations were statistically significant predictors for the diagnosis of SLE. Anti-Sm or any combination of anti-ena antibodies showed a specificity of 290% for the diagnosis of SLE. In previous studies, a substantial percentage of patients with other CTDs have been shown to have anti-sm antibodies (2,3,25). Whether the specificity for SLE of anti-sm antibodies detected by ELISA is similar to or lower than the specificity of these antibodies detected by immunodiffusion is controversial (2,19). Anti-Ro/SS-A antibodies are a common finding in the serum of SLE patients, suggesting a high degree of sensitivity for SLE (26). Testing for these antibodies has been recommended as a diagnostic study for SLE among patients with persistently negative ANA results (26). In our study population, anti-ro/ss-a was the strongest predictor for the diagnosis of SLE. No major clinical differences were found between ANA+/ENA+ and ANA+/ENA- SLE patients. Only pleuritis and use of hydroxychloroquine were significantly higher among SLE ANA+/ENA+ patients. We do not know with certainty why ANA+/ENA+ SLE patients in our study were receiving hydroxychloroquine more frequently than were ANA+/ENA- patients. Several studies have shown an association between specific anti-ena antibodies and clinical manifestations (21), including photosensitivity (26,27), malar rash (2), Raynaud s phenomenon (2,28), lung fibrosis, and pericarditis (29); nevertheless, these associations have not been consistent (9,19,22,30). Although we did not specifically analyze the association of any clinical manifestations and antibodies, our results are consistent with those of another study in which the clinical association of anti-ena antibodies was studied among 94 SLE patients with a mean followup 11.8 years (30). Some limitations of our study must be considered. The study was conducted in a referral rheumatology center; this is reflected in the high percentage of patients with CTD (71%), especially SLE (49%). The high prevalence of SLE among our clinic population leads to overestimation of the predictive value of ENA antibodies (31), and may not reflect the predictive value in the general population. It may also reflect the selectiveness of rheumatologists in deciding when to order an ANA test. The strong female predominance in the study population (91.5%) suggests that the study may be biased toward SLE or that rheumatologists order ANA tests mostly on SLE patients and not on all CTD patients. A potential bias arises if attending physicians used the results of the anti-ena antibody tests to establish the diagnosis of SLE in their patients. Because of the retrospective nature of the analysis, it is impossible to know with certainty if this situation occurred. However, we consider it unlikely, since in only 7 of the 97 SLE ANA+/ENA+ patients did anti-sm constitute the fourth ACR classification criterion.

1060 SANCHEZ-GUERRERO ET AL Our cross-sectional study represents prevalent, not incident, findings. The duration of the disease in the ANA+/ENA+ and ANA+/ENA- SLE groups was 11 years and 9 years, respectively. If long-term survival accounts for the frequencies of anti-ena antibodies and the clinical manifestations in our population, then anti-ena antibody status may be only an epiphenomenon. We did not study the relationship between anti-ena antibodies and SLE activity. In the patients studied, we did not ascertain race, which has been associated with differences in the frequency of anti-sm and anti-rnp antibodies (32). In our experience, ELISA testing for anti-ena is morq sensitive than immunodiffusion testing. Furthermore, we have observed variability in ELISA anti-ena kits from different manufacturers. Therefore, the results of the present investigation cannot necessarily be extrapolated to derive predictive values of anti-ena determined by methods not used in this study. Based on the results of this study, we may conclude that testing for anti-ena antibodies, especially anti-ro/ss-a, improves the ability to diagnose SLE, primarily among patients attending a referral rheumatology center who are positive for ANA. This improvement is significant among patients without anti-dsdna antibodies. The prevalence of anti-dsdna antibodies seems to be independent of the presence of anti-ena antibodies. We did not find major clinical differences among SLE patient groups divided according to ENA status. ACKNOWLEDGMENTS The authors thank the staff of the Clinical Immunology Laboratory, Brigham and Women s Hospital for their help and support, and Matthew H. Liang, MD, MPH for reviewing the manuscript. REFERENCES Ben-Chetrit E. The molecular basis of the SSNRo antigens and the clinical significance of their autoantibodies. Br J Rheumatol 32:396-402, 1993 Gripenberg M, Teppo AM, Friman C: Antibodies to Sm and SSA demonstrated by enzyme immunoassay: correlation to clinical manifestations and disease activity in patients with systemic lupus erythematosus. Rheumatol Int 11:209-213, 1991 Maddison PJ, Skinner RP, Vlachoyiannopoulos P, Brennand DM, Hough D: Antibodies to nrnp, Sm, Ro (SSA) and La (SSB) detected by ELISA: their specificity and inter-relations in connective tissue disease sera. Clin Exp Immunol 62:337-345, 1985 Manoussakis MN, Kistis KG, Liu X, Aidinis V, Guialis A, Moutssopoulos HM: Detection of anti-ro (SSA) antibodies in autoimmune diseases: comparison of five methods. Br J Rheumato1 32:449-455, 1993 5. Chretien P, Soulie E, Johanet C, Abuaf N: Comparisons of double immunodiffusion, ELISA, western blot and cape blot for the detection of anti-ssa antibody. Study of anti-ssa prevalence in systemic lupus erythematosus. J Autoimmun 7:379-388, 1994 6. Reeves WH, Fisher DE, Lahita RG, Kunkel HG: Autoimmune sera reactive with Sm antigen contain high levels of RNP-like antibodies. J Clin Invest 75580-587, 1985 7. Griner PF, Mayewski RJ, Mushlin AI, Greenland P: Selection and interpretation of diagnostic test and procedures. Ann Intern Med 9437-592, 1981 8. Juby A, Johnston C, Davis P: Specificity, sensitivity and diagnostic predictive value of selected laboratory generated autoantibody profiles in patients with connective tissue diseases. J Rheumatol 18~354-358, 1992 9. Hamburger M, Hodes S, Barland P: The incidence and clinical significance of antibodies to extractable nuclear antigens. Am J Med Sci 273:21-28, 1977 10. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, Schaller JG, Tala1 N, Winchester RJ: The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 25:1271-1277, 1982 11. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS, Medsger TA Jr, Mitchell DM, Neustadt DH, Pinals RS, Schaller JG, Sharp JT, Wilder RL, Hunder GG: The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315-324, 1988 12. Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee: Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 23:581-590, 1980 13. Bohan A, Peter JB: Polymyositis and dermatomyositis. N Engl J Med 292344-347, 1975 14. Fox RI, Robinson C, Curd J, Kozin F, Howell FV: First international symposium on Sjogren s syndrome: suggested criteria for classification. Scand J Rheumatol Suppl S61:28-30, 1986 15. Gonzalez EN, Rothfield N: Immunoglobulin class and pattern of nuclear fluorescence in SLE. N Engl J Med 274:1333-1338, 1966 16. Schur PH, DeAngelis D, Jackson JM: Immunological detection of nucleic acids and antibodies to nucleic acids and nuclear antigens by counterimmunoelectrophoresis. Clin Exp Immunol17:209-218, 1974 17. Eaton RB, Schnneider G, Schur PH: Enzyme immunoassay for antibodies to native DNA specificity and quality of antibodies. Arthritis Rheum 2652-62, 1983 18. Shiel WC Jr, Jason M: The diagnostic associations of patients with antinuclear antibodies referred to a community rheumatologist. J Rheumatol 16:782-785, 1989 19. Field M, Williams DG, Charles P, Maini RN Specificity of anti-sm antibodies by ELISA for systemic lupus erythematosus: increased sensitivity of detection using purified peptide antigens. Ann Rheum Dis 47320-825, 1988 20. Delpech A, Gilbert D, Daliphard S, le Laet X, Godin M, Tron F: Antibodies to Sm, RNP and SSB detected by solid-phase ELISAs using recombinant antigens: a comparison study with counter irnmunoelectrophoresis and immunoblotting. J Clin Lab Anal 7:197-202, 1993 21. Thompson D, Juby A, Davis P: The clinical significance of autoantibody profiles in patients with systemic lupus erythemato- SUS. LUPUS 211.5-19, 1993 22. Barada FA Jr, Andrews BS, Davis JS IV, Taylor RP: Antibodies to Sm in patients with systemic lupus erythematosus: correlation of Sm antibody titers with disease activity and other laboratory parameters. Arthritis Rheum 241236-1244, 1981 23. Bell DA, Komar R, Chodirker WB, Block J, Cairns E: A compar-

ANTI-ENA IN SLE DIAGNOSIS 1061 ison of serologic reactivity among SLE patients with or without anti-ro (SS-A) antibodies. J Rheuinatol 11:315-317, 1984 24. Wasicek CA, Reichlin M: Clinical and serological differences between systemic lupus erythematosus patients with antibodies to Ro versus patients with antibodies to Ro and La. J Clin Invest 69:835-843, 1982 25. Munves EF, Schur PH: Antibodies to Sm and RNP. Arthritis Rheum 26:848-853, 1983 26. Maddison P, Mogavero H, Provost TT, Reichlin M: The clinical significance of autoantibodies to a soluble cytoplasmic antigen in systemic lupus erythematosus and other connective tissue diseases. J Rheumatol 6:189-195, 1979 27. Mond CB, Peterson MGE, Rothfield NF: Correlation of anti-ro antibody with photosensitivity rash in systemic lupus erythematosus patients. Arthritis Rheum 32:202-204, 1089 28. Winn DM, Wolfe JF, Lindberg DA, Fi-istoe FH, Kingsland L, Sharp GC: Identification of a clinical subset of systemic lupus erythematosus by antibodies to the Sm antigen. Arthritis Rheuin 22:1334-1337, 1979 29. Yasuma M, Takasaki Y, Matsumoto K, Kodama A, Hashimoto H, Hirose SI: Clinical significance of IgG anti-sm antibodies in patients with systemic lupus erythematosus. J Rheumatol 17:469-475, 1990 30. Gulko PS, Reveille JD, Koopman WJ, Burgard SL, Bartolucci AA, Alarcon GS: Survival impact of autoantibodies in systemic lupus erythematosus. J Rheumatol 21:224-228, 1994 31. Hennekens CH, Buring JE: Screening: Epidemiology in Medicine. Edited by SL Mayrent. Boston, Little, Brown and Company, 1987 32. Arnett FC, Hamilton RG, Roebber MG, Harley JB, Reichlin M: Increased frequencies of Sm and nrnp autoantibodies in American blacks compared to whites with systemic lupus erythematosus. J Rheumatol 15:1773-1776, 1988