Detection of antinuclear antibodies by use of an enzyme immunoassay with nuclear HEp-2 cell extract and recombinant antigens: comparison with immunofluorescence assay in 307 patients.
Enzyme immunoassay (EIA) methods that allow detection of individual disease-specific ANAs have recently been developed by use of antigens extracted from HEp-2 cells and/or specific purified or recombinant antigens (6, 7). However, large differences in sensitivity and response to disease-specific ANAs have been reported, depending on the antigens used (8). A new EIA method uses beads (rather than microtiter plates) on which antigens extracted from HEp-2 cells and multiple recombinant antigens are immobilized (9). In this study, we evaluated the usefulness of this new EIA method as a screening test for ANAs, in comparison with the conventional IF-ANA, using several hundred sera from patients diagnosed with CTDs and strictly selected healthy individuals.
Materials and Methods
MEASUREMENT OF ANAs
The COBAS[R] Core HEp2 ANA EIA (COBAS-ANA; Roche Diagnostics, Mannheim, Germany), an EIA reagent set for ANA detection, was used to detect ANAs with a COBAS Core II (Roche Diagnostics), an automated analyzer for EIAs. In principle, this is a two-step EIA in which specimens are incubated with polystyrene beads coated with a mixture of a soluble HEp2 cell extract and seven recombinant proteins (60-kDa SSA/Ro, 52-kDa SSA/Ro, SSB/ La, Scl-70, Jo-1, dsDNA, and CENP-B, which is a major autoantigen corresponding to anti-centromere antibodies) (9). After removal of unbound antibody, the antigen-coated beads are allowed to react with peroxidase-labeled anti-human IgG mouse monoclonal antibody, and sandwich complexes are formed. [H.sub.2][O.sub.2]- 3,3',5,5'-tetramethylbenzidine is added to initiate enzymatic reactions after washing, and the absorbance is measured. The results are shown as an index calculated using the following formula: index = (absorbance of a test sample)/(mean absorbance of negative control + 0.1). In accordance with the operating manual, the assessment criteria were set as follows: positive if [greater than or equal to] 1.1 index negative if index <0.9, and equivocal if [greater than or equal to] 0.9 but <1.1. All serum samples were analyzed in duplicate. The intra- (n = 20) and interassay (20 days) CVs of the COBAS-ANA were 4.5-5.9% and 3.2-5.6%, respectively.
The IF-ANA method used was the FANAwell (DIAIATRON, Tokyo, Japan), a reagent set that uses HEp-2 cells as substrates and a mixture of fluorescein isothiocyanate-labeled mouse anti-human immunoglobulins (IgG, IgA, and IgM) as secondary antibody. Specimens were diluted 20- and 40-fold with the buffer solution included in the reagent set, and if 40-fold-diluted specimens were still positive in the IF-ANA, samples were further diluted up to 1:80-1:5120 for reanalysis. The antibody titer was expressed as the final dilution, regardless of the type of staining. Slides were stained according to the instructions in the operating manual, and they were examined with a BV-excitation type, epifluorescent microscope at X40 (dry) magnification. All slides were examined under the microscope by two laboratory technologists. For quality control, negative and positive controls with known antibody titers were used for each assay to check accuracy.
We analyzed eight disease-specific ANAs, using EIA-based reagent sets with the indicated antigens: antiU1RNP antibody [MESACUP[R] RNP-II TEST; Medical & Biological Laboratories (MBL), Nagoya, Japan; antigens were recombinant 70-kDa A-peptide and C-peptide]; anti-Sm antibody (MESACUP Sm TEST; MBL; purified Sm antigens); anti-SSA/Ro antibody (MESACUP SSA/Ro TEST; MBL; recombinant 60-kDa SSA/Ro antigen and purified SSA/Ro protein); anti-SSB/La antibody (MESACUP SS-B/La TEST; MBL; recombinant SSB/La protein); anti-Scl-70 antibody (MESACUP Scl-70 TEST; MBL; recombinant Scl-70 protein); anti-centromere antibody (MESACUP CENP-B TEST; MBL; recombinant CENP-B protein); anti-Jo-1 antibody (MESACUP Jo-1 TEST; MBL; recombinant Jo-1 protein); and anti-dsDNA antibody (MESACUP DNA-II TEST; MBL, [lambda]-phage-derived purified double-stranded DNA).
SELECTION OF PATIENTS AND CONTROLS
It is important to establish the inclusion criteria for healthy individuals carefully so that the reference interval for ANAs can be set correctly. In other words, the reference interval may not be determined accurately if all ANA-positive individuals are excluded from the analysis because some healthy individuals are known to be positive for ANAs (4). The healthy individuals in this study were selected from volunteers by the following procedures. (a) Individuals who did not exhibit any abnormal finding on any of the test items during a routine physical examination were selected from the hospital staff and the residents of a certain town in Hyogo Prefecture, Japan. The test items were urinalysis, routine hematologic study, blood chemistry tests, and a plain chest film, but ANAs were not included. (b) The results obtained by COBASANA were analyzed in these individuals. For individuals positive in the COBAS-ANA, although most of them were also positive in the IF-ANA, a survey was conducted by questionnaire, and further examinations were performed, if needed. (c) After persons who were diagnosis as having a CTD or were suspected of having CTD were excluded, we assumed that all other individuals were healthy. These healthy individuals were divided into 12 groups based on gender and age. To standardize the groups, whenever >45 individuals were enrolled in a group, 45 were selected at random. As a result, a total of 492 healthy individuals were included in this study, and the genders and ages of the healthy individuals were as follows: 43 women and 44 men 21-30 years of age, 45 women and 44 men 31-40 years of age, 45 women and 45 men 41-50 years of age, 45 women and 36 men 51-60 years of age, 45 women and 41 men 61-70 years of age, and 34 women and 25 men 71-80 years of age. The ratio of women to men among the healthy individuals was 1.1:1.
We examined sera from 307 CTD patients who were followed at the outpatient clinic of Kobe University Hospital. Serum samples were collected without conscious bias mostly at patients' first visits during 1996 to 1999. The patients' diseases had been defined by the established criteria for each disease (10-16). The numbers and genders of the patients were as follows: 111 patients with SLE (median age, 35 years; range, 17-72 years; 104 women, 7 men); 36 patients with Sjogren syndrome (SS; median age, 59 years; range, 21-77 years; 35 women, 1 man); 39 patients with systemic sclerosis (SSc; median age, 52 years; range, 35-73 years; 35 women, 4 men); 39 patients with dermatomyositis and polymyositis (DM/PM; median age, 54 years; range, 16-79 years; 27 women, 12 men); 33 patients with mixed connective tissue disease (MCTD; median age, 39 years; range, 16-65 years; 30 women, 3 men); and 49 patients with rheumatoid arthritis (RA; median age, 50 years; range, 25-76 years; 44 women, 5 men).
[FIGURE 1 OMITTED]
Each sample was obtained with informed consent. After separation of the serum by centrifugation, the serum samples were stored frozen at below -40 [degrees]C. All samples collected from CTD patients and healthy individuals were randomly numbered and were blindly examined for ANAs by both the COBAS-ANA and the IF-ANA and were also analyzed for the eight disease-specific ANAs by each EIA-based reagent set.
ROC analyses were performed for each patient group, using healthy women as the reference group (17,18). The sensitivity and specificity of the IF-ANA were determined at cutoff dilutions of 1:40 and 1:160, and those of the COBAS-ANA at cutoff indexes of 0.6 and 0.9. For computation and analysis of ROC curves, we used the software program MedCalc, Ver. 6.01 (MedCalc Software).
The Mann-Whitney test was used to assess differences in COBAS-ANA antibody titers among CTD patients, whereas gender-based differences in titers among healthy individuals were tested by the two-sample Mest with the Welch correction. In addition, age-related differences in the titers were tested by the Kruskal-Wallis test. Correlations between COBAS-ANA and IF-ANA positivity among CTD patients were tested by the [chi square] test. Concordance between the two methods was determined by the is statistic (19).
[FIGURE 2 OMITTED]
The present report followed the guidelines proposed by Bruns et al. (20) for reporting of studies of diagnostic accuracy of medical tests.
COBAS-ANA AND IF-ANA POSITIVITY IN HEALTHY INDIVIDUALS
COBAS-ANA antibody indexes were 0.47 [+ or -] 0.45 (mean [+ or -] SD) in the 492 healthy individuals, 0.51 [+ or -] 0.58 in the women, and 0.43--0.25 in the men, demonstrating significantly higher antibody titers in women than men (P = 0.042, t-test with Welch correction). Stratification of the COBAS-ANA antibody titers by age (every 10 years) showed significant differences among the six age groups in women but not in men (P = 0.016 for women and 0.38 for men, Kruskal-Wallis test).
The IF-ANA was positive in 34.6% of the women and 18.3% of the men on the basis of a cutoff dilution of 1:40, showing a significant difference between women and men (P <0.0001, comparison of two proportions). The COBAS-ANA was positive in 6.2% of the women and 3.8% of the men on the basis of a cutoff index of 0.9, showing no significant difference in positivity between women and men (P = 0.23, comparison of two proportions; Table 1).
Thus, there were gender-related differences in COBASANA antibody titers and in IF-ANA positivity among healthy individuals. Because most of the patients in the CTD group were women, healthy women were used as controls in the subsequent studies.
COBAS-ANA ANTIBODY TITERS AND POSITIVITY IN CID PATIENTS
The COBAS-ANA antibody titers of the six CTD patient groups are shown in Fig. 1. The median indexes (interquartile range) were 5.20 (1.93-11.83) in 111 SLE patients, 2.50 (1.10-7.80) in 36 SS patients, 4.00 (1.53-7.28) in 39 SSc patients, 0.80 (0.53-1.75) in 39 DM/PM patients, 4.50 (3.25-6.43) in 33 MCTD patients, and 0.60 (0.50-0.83) in 49 RA patients. The indexes were significantly higher in all patient groups than in healthy women (P <0.0001, Mann-Whitney test).
ROC ANALYSES FOR COBAS-ANA AND IF-ANA
ROC analyses were performed for the COBAS-ANA and IF-ANA in the six groups of CTD patients (patient group) and healthy women (reference group; Fig. 2 and Table 2). The ROC curves for the COBAS-ANA in the patients with SLE, SS, or SSc in Fig. 1 are to the left and above those for the IF-ANA, and the areas under the ROC curves (ADCs) for the COBAS-ANA and IF-ANA were 0.98 [95% confidence interval (95% CI), 0.95-0.99] and 0.93 (95% CI, 0.90-0.95), respectively, in SLE patients and 0.93 (95% CI, 0.89-0.96) and 0.82 (95% CI, 0.77-0.86), respectively, in the SS patients, showing significant differences in AUC (P = 0.008 for SLE; P = 0.012 for SS). The ADCs for the COBAS-ANA and IF-ANA were 0.99 (95% CI, 0.96-1.00) and 0.96 (95% CI, 0.93-0.98), respectively, in the SSc patients, showing no significant differences in AUC (P = 0.31). In the patients with DM/PM, MCTD, or RA, both ROC curves almost overlapped, with no significant difference in AUC. In the RA patients, both ROC curves were situated almost diagonally and linear, suggesting that both the COBAS-ANA and the IF-ANA were hardly able to distinguish RA patients from healthy individuals.
ROC analysis was then performed for the COBASANA and IF-ANA in all of the CTD patients except those with RA. The ROC curve for the COBAS-ANA was situated above the curve for the IF-ANA, and the ADCs for the COBAS-ANA and IF-ANA were 0.94 (95% CI, 0.92-0.96) and 0.90 (95% CI, 0.87-0.93), respectively, showing a significant difference (P = 0.005). As evidenced by a sensitivity of 92% (95% CI, 87-95%) and a specificity of 65% (95% CI, 59-71%), the IF-ANA had high sensitivity but poor specificity at the cutoff dilution of 1:40 that has been widely used to date (Table 3). However, the sensitivity and specificity were better balanced at a cutoff dilution of 1:160 (sensitivity, 81%; 95% CI, 76-86%; specificity, 87%; 95% CI, 82-91%).
The COBAS-ANA had well-balanced sensitivity and specificity at cutoff indexes between 0.6 and 0.9, and its sensitivity and specificity were 84% (95% CI, 79-88%) and 94% (95% CI, 90-96%), respectively, at a cutoff index of 0.9. A cutoff index of 0.8 was found to be suitable for patients with SLE, SS, SSc, or MCTD, whereas a cutoff index of 0.7 was found to be most suitable for patients with DM/PM. The COBAS-ANA yielded a sensitivity of 93% (95% CI, 89-95%) and a specificity of 79% (95% CI, 74-84%) at a cutoff index of 0.6, thus showing that the COBAS-ANA with a cutoff index of 0.6 was more sensitive and more specific than the IF-ANA at a cutoff dilution of 1:40. The COBAS-ANA at a cutoff index of 0.9 yielded a likelihood ratio of 13.5 (95% CI, 10.2-18.0) for the CTD patients, excluding those with RA, whereas the IF-ANA at a cutoff dilution of 1:160 yielded a likelihood ratio of 5.9 (95% CI, 4.6-7.7) for CTD patients, excluding those with RA.
CORRELATION BETWEEN COBAS-ANA AND IF-ANA TITERS IN CID PATIENTS
A significant correlation between COBAS-ANA and IFANA titers was found in the sera of CTD patients ([r.sub.s] = 0.647; P <0.0001, Spearman correlation coefficient; not shown). Comparisons between IF-ANA and COBASANA titers were performed in the CTD patient groups with the COBAS-ANA cutoff indexes set at 0.6 and 0.9 and the IF-ANA cutoff dilutions set at 1:40 and 1:160 (Table 4). All patients with MCTD showed high antibody titers by both the COBAS-ANA and the IF-ANA. Among the patients with SLE, SS, or SSc, there was significant correlation between IF-ANA and COBAS-ANA titers (P = 0.003 for SLE; P = 0.002 for SS; P = 0.021 for SSc), but the [kappa] statistic of agreement was <0.4, except in SS, thus showing relatively poor agreement. Among the patients with DM/PM or RA, there was no significant agreement between the COBAS-ANA and IF-ANA (P = 0.20 for DM/PM; P = 0.25 for RA).
Several samples showed extreme discrepancies between the COBAS-ANA index and IF-ANA titer. In 11 of the 307 patients with CTD, the IF-ANA titer was [greater than or equal to] 1:160 but the COBAS-ANA index was <0.6, whereas in another 10 patients, the IF-ANA titer was <1:40 but the COBAS-ANA index was [greater than or equal to] 0.9. The characteristics of the former 11 CTD patients are listed in Table 5; of these patients, 5 (45%) had RA and 4 (36%) had DM/PM. Four of these patients were positive for at least one disease-specific ANA, including one patient with RA who was positive for both anti-SSA/Ro and anti-dsDNA antibodies. The characteristics of the latter 10 CTD patients are shown in Table 6; of these patients, 3 (30%) had DM/PM and 3 (30%) had SS. Three of these patients were positive for at least one disease-specific ANA, including one patient with DM/PM who was positive for anti-Jo-1 antibody with an index of 85.9.
To determine the correlation between COBAS-ANA antibody titers and IF-ANA staining pattern, we selected 140 sera from CTD patients whose IF-ANA antibody titers were >1:40 and with a single staining pattern, including 36 cases with a homogeneous pattern, 88 cases with a speckled pattern, 6 cases with a nucleolar pattern, and 10 cases with a discrete speckled pattern. At a cutoff index of 0.6, the COBAS-ANA was positive in 78% (28 of 36), 95% (84 of 88), 50% (3 of 6), and 90% (9 of 10) of the cases with the homogeneous, speckled, nucleolar, and discrete speckled patterns, respectively.
SENSITIVITY OF COBAS-ANA AND IF-ANA FOR DETECTION OF DISEASE-SPECIFIC ANAs
Among 492 healthy individuals, 16 samples were demonstrated to be positive for at least one of the eight disease-specific ANAs by each EIA-based reagent set. The COBAS-ANA antibody titers were significantly higher in these 16 individuals than in the 476 specific-ANA-negative healthy individuals (P <0.0001, Mann-Whitney test). In addition, none of the healthy individuals negative for disease-specific ANAs exhibited COBAS-ANA antibody indexes >2.1 (Fig. 1). Among the 307 patients with CTD, a total of 207 patients were positive for at least one of the eight disease-specific ANAs by each EIA-based reagent set. Using the sera from these 207 patients, we determined the sensitivity of the COBAS-ANA and the IF-ANA for detection of disease-specific ANAs. At cutoff dilutions of 1:40 and 1:160, the IF-ANA was positive for 97.6% (202 of 207) and 90.8% (188 of 207), respectively, whereas the COBAS-ANA was positive for 97.1% (201 of 207) and 91.8% (190 of 207) at cutoff indexes of 0.6 and 0.9, respectively.
To investigate the selectivity for the disease-specific ANAs that could be detected with certainty by the COBAS-ANA and IF-ANA, we analyzed CTD patients whose sera were positive for only one disease-specific ANA, including 15 patients with anti-U1RNP antibody, 18 patients with anti-SSA/Ro antibody, 14 patients with anti-centromere antibody, 5 patients with anti-Jo-1 antibody, and 19 patients with anti-dsDNA antibody. At a cutoff dilution of 1:40, the IF-ANA was positive in 100% of the patients with anti-U1RNP antibody, 100% of those with anti-SSA/Ro antibody, 100% of those with anti-centromere antibody, 60% of those with anti-Jo-1 antibody, and 100% of those with anti-dsDNA antibody. On the other hand, at a cutoff index of 0.6, the COBAS-ANA was positive in 100% of the patients with anti-U1RNP antibody, 89% of those with anti-SSA/Ro antibody, 86% of those with anti-centromere antibody, 80% of those with anti-Jo-1 antibody, and 100% of those with anti-dsDNA antibody (Table 7).
The purpose of this study was to evaluate the usefulness of the COBAS-ANA, a new EIA-based screening method for detection of ANAs in comparison with the IF-ANA in patients who had been diagnosed as having CTD and in healthy individuals who had been selected based on strict criteria. This new method has two special features: (a) enhancement of overall reactions to multiple ANAs by the use of both HEp-2 cell-derived native antigens and multiple recombinant antigens; and (b) objective data are provided in the form of an index by use of an automated instrument.
It has been documented that the IF-ANA has poor specificity as evidenced by high false-positive rates in healthy individuals regardless of its high sensitivity for CTD patients (5). In 1997, Tan et al. (4) investigated the reference intervals for the IF-ANA in healthy individuals at 15 institutions worldwide and reported that there was variability within and between laboratories, with average CVs of 0.165 and 0.507, respectively. Because the IF-ANA was positive in 31.7% and 5.0% of healthy individuals at dilutions of 1:40 and 1:160, respectively, in their study, they recommended reporting IF-ANA results with the positivity in healthy individuals at two dilutions, 1:40 and 1:160. Laboratories must make a great effort to follow these recommendations and to keep their ANA data reliable and accurate. For these reasons, the development of a simple method for detecting ANAs without requiring training in laboratory procedures has been anticipated as a substitute for IF-ANA. Intraassay CVs of 4.3-12.4% for the COBAS-ANA have been reported (9). The intra- and interassay CVs for the COBAS-ANA in our study were 4.5-5.9% and 3.2-5.6%, respectively.
Two types of EIA-based ANA screening tests are commercially available at present (6, 8). In one type, whole nuclear extracts from HEp-2 cells are immobilized on 96-well plates as the antigens, whereas mixtures of purified or recombinant specific proteins are immobilized as the antigens in the other type. EIA-based methods are computerized and easier to operate than the IF-ANA, which requires complicated manipulations, but still seem to have several disadvantages (6, 8). In the former method, in which whole nuclear extracts are coated onto the EIA plates, the amounts of nuclear antigens may not be constant because the affinity of each antigen for the solid phase is different. The latter method is useful for screening disease-specific ANAs coated on the plates, but ANAs against nuclear antigens other than the specific antigens used in the method may not be detectable. By contrast, the COBAS-ANA investigated in this study is a hybrid between these two EIA-based assay systems and can react with various types of ANAs, including unknown ANAs, because native antigens are used for this assay, and responses are standardized by the addition of recombinant antigens.
The manufacturer used 50 IF-ANA-positive and 30 IF-ANA-negative sera to verify that the COBAS-ANA detects only the disease-specific ANAs and does not react against the control sera, but its clinical usefulness has not been evaluated. In the present study, we used hundreds of sera preselected by diagnosis to evaluate the performance of the COBAS-ANA for clinical use in comparison with the conventional IF-ANA. The internal validity of this study was guaranteed by the use of sera from CTD patients who had been diagnosed according to the established criteria for each disease and by blind comparisons of the COBAS-ANA with the conventional IF-ANA. It is important to identify a proper threshold for ANA determination to be able to distinguish healthy individuals from CTD patients. Using adequate numbers of samples, we therefore performed ROC analysis to evaluate the ability of the COBAS-ANA to discriminate CTD patients from healthy individuals, by which the diagnostic accuracy could be properly assessed. On the basis of the ROC analysis, a cutoff index of 0.8 was found to be most suitable for discriminating CTD patients, except those with RA, from healthy women, although the criteria for evaluation (positive if [greater than or equal to] 1.1 negative if <0.9) are listed in the operating manual for COBAS-ANA. The sensitivity and specificity of COBAS-ANA at a cutoff index of 0.8 were 88% and 93%, respectively, and were superior to those of the IF-ANA at a cutoff dilution of 1:160 (sensitivity, 81%; specificity, 87%).
In the ROC analysis of the COBAS-ANA performed in six groups of CTD patients (each CTD as a patient group) and healthy women (as reference group), the ADCs were 0.93-0.99 in patients with SLE, SS, SSc, or MCTD, indicating a highly accurate diagnosis. In addition, the ADCs of the COBAS-ANA and IF-ANA for SLE and SS were significantly different (P = 0.008 and 0.012, respectively). These findings indicate that the COBAS-ANA is superior to the IF-ANA for the detection of SLE and SS in terms of the sensitivity and specificity of the assays. There were no significant differences between the detection accuracies of these two methods in patients with SSc, DM/PM, or RA.
As indicated earlier, the is statistic for agreement (between the COBAS-ANA and IF-ANA titers) was <0.4, showing poor agreement between the COBAS-ANA and IF-ANA titers in CTD patients as a whole. This is primarily because the COBAS-ANA and IF-ANA titers were not concordant for many patients with RA or DM/PM, in whom positivity in the IF-ANA was higher than in the COBAS-ANA (Table 4). Tan et al. (4) reported that no cutoff dilution was useful for differentiating patients with RA or soft tissue rheumatism from healthy individuals, and there was no suitable cutoff point for the ROC curves of the COBAS-ANA and IF-ANA in RA patients in the present study as well. At a cutoff index of 0.9, the sensitivity of the COBAS-ANA was 20% in RA patients, suggesting that the COBAS-ANA should not be used to diagnose RA. In the ROC analysis performed for SLE patients (as patient group) and RA patients (as reference group), the ADCs were 0.93 (95% CI, 0.88-0.96) and 0.84 (95% CI, 0.77-0.89) for the COBAS-ANA and IF-ANA, respectively, showing a significantly larger AUC for the COBAS-ANA than for the IF-ANA (P = 0.004). Therefore, the COBAS-ANA may be more useful than the IF-ANA for differential diagnosis between SLE and RA, although it is not useful for differentiating RA patients from healthy individuals. This is an advantage for the COBAS-ANA because the ANA test is frequently requested to screen CTD patients (excluding patients with RA) from patients with RA and soft tissue rheumatism.
The COBAS-ANA assessed in this study seems superior to most commercially available ANA-screening tests with regard to diagnostic accuracy. In the ROC analysis performed by Gniewek et al. (18), the AUC for an EIA-based ANA was not significantly different from that for an IF-ANA. According to the report published by Emlen and O'Neill (8), the positivity of six different EIA methods was 62-90% in SLE patients when the positivity of the IF-ANA at a cutoff dilution of 1:64 was 88%. In the present study, the COBAS-ANA could detect 102 of 111 SLE patients (91.9%) when 90 of the 111 patients (81.1%) were positive in the IF-ANA at cutoff dilution of 1:160 (Table 4). It was difficult to compare the specificity of the six EIA methods and our COBAS-ANA because the false-positive rates (specificity) in the study by Emlen and O'Neill (8) were determined on the basis of reevaluation of sera from consecutive patients for whom ANA testing had been ordered. However, the EIA methods with higher sensitivity seemed to have lower specificity.
We also compared the ability of the COBAS-ANA and IF-ANA to detect the eight disease-specific ANAs. When the positivity (sensitivity for detection of disease-specific ANAs) of the COBAS-ANA was determined in CTD patients who were positive for at least one of eight disease-specific ANAs, the positivity was 91.8% at a cutoff index of 0.9, but rose to 97.1% at a cutoff index of 0.6, which was almost the same as the 97.6% positivity for the IF-ANA at cutoff dilution of 1:40. We therefore concluded that a cutoff index of 0.6 could be used for suspicion of the presence of disease-specific ANAs. In the experiments using specimens from CTD patients that were positive for only one of the eight disease-specific antigens (monospecific antisera), the positivity of the COBAS-ANA at a cutoff index of 0.6 was 100%, 89%,86%,80%, and 100% for anti-UlRNP antibody, anti-SSA/Ro antibody, anti-centromere antibody, anti-Jo-1 antibody, and anti-dsDNA antibody, respectively. These results were almost equal to the positivity of 60-100% for the IF-ANA at a cutoff dilution of 1:40. However, the COBAS-ANA positivity was as low as 50% for specimens that exhibited the nucleolar pattern of IF-ANA staining in the same manner as reported previously (9).
Finally, because performing the IF-ANA on a regular basis is labor-intensive and demands advanced skills in the operator, it is desirable to reduce the labor and costs involved in IF-ANA testing through the introduction of EIA methods (6,21). In a study by Roche Diagnostics to evaluate time consumption and costs for ANA testing, the total costs per determination for the COBAS-ANA were calculated to be 7.04-7.26 DM (US $3.03-3.12), whereas those for the IF-ANA were 5.54-5.57 DM (US $2.38-2.40; Roche Diagnostics, unpublished data). Of course, the cost for additional titration of ANAs in the IF-ANA should be multiplied, whereas the COBAS-ANA provides quantitative data. Introduction of the COBAS-ANA would be of great help for this purpose because (a) the COBAS-ANA is at least as good as the IF-ANA with regard to sensitivity and specificity: (b) COBAS-ANA analysis can be performed easily with a fully automated instrument, and it takes only ~60 min to obtain the data; and (c) clinicians can evaluate the results with various cutoff indexes because quantitative data are printed out. For example, the result will be judged negative for the disease-specific ANAs if the index is <0.6 and will suggest the presence of CTDs (except RA) if the index is [greater than or equal to] 0.9.
The COBAS-ANA cannot tell the ANA staining pattern and may not detect some important antibodies to nuclear antigens, such as proliferating cell nuclear antigens and nucleolar antigens, that can be easily detected by the IF-ANA. Although there is no perfect assay available for screening ANAs, the COBAS-ANA is attractive as a new screening test for ANAs from the standpoints of diagnostic accuracy and effectiveness of ANA tests.
This work was supported in part by a Grant-in-Aid for Scientific Research (B) (10922078) from the Ministry of Education, Science, Sports, and Culture of Japan. We are grateful to Prof. Eng M. Tan (Scripps Research Institute) for helpful suggestions and critical reading of the manuscript. We also thank Dr. Hans-Peter Lehmann, David Panneton, and Syuichi Hayashi (Roche Diagnostics), Hiroshi Suno (MBL), and Tomoyoshi Terakawa (DIA-IATRON) for providing the ANA reagent sets and for helpful discussions.
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NOBUHIDE HAYASHI, [1,2] TOMOKO KAWAMOTO,  MASAHIKO MUKAI,  AKIO MORINOBU,  MASAHIRO KOSHIBA,  SHINICHI KONDO, [1,2] SOICHIRO MAEKAWA,  and SHUNICHI KUMAGAI [1,2] *
 Department of Clinical and Laboratory Medicine, Kobe University School of Medicine, 7-5-1 Kusunoki-cyo, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
 Department of Clinical Laboratory, Kobe University Hospital, 7-5-2 Kusunoki-cyo, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
 Nonstandard abbreviations: ANA, antinuclear antibody; CTD, connective tissue disease; SLE, systemic lupus erythematosus; IF-ANA, immunofluorescence-based method for antinuclear antibodies; EIA, enzyme immunoassay; COBAS-ANA, COBAS Core HEp2 ANA ElA; MBL, Medical & Biological Laboratories; dsDNA, double-stranded DNA; SS, Sjagren syndrome; SSc, systemic sclerosis; DM/PM, dermatomyositis and polymyositis; MCTD, mixed connective tissue disease; RA, rheumatoid arthritis; 95% CI, 95% confidence interval; and AUC, area under the curve.
* Address correspondence to this author at: Department of Clinical and Laboratory Medicine, Kobe University School of Medicine, 7-5-1 Kusunokicyo, Chuo-ku, Kobe, Hyogo 650-0017, Japan. Fax 81-78-382-6209; e-mail email@example.com.
Received February 21, 2001; accepted June 12, 2001.
Table 1. Comparison of positivity of the COBAS-ANA and the IF-ANA in healthy individuals by gender. COBAS-ANA Cutoff index Women Men Total 0.6 21.0 (54/257) 21.7 (51/235) 21.3 (105/492) 0.9 6.2 (16/257) 3.8 (9/235) 5.1 (25/492) IF-ANA Cutoff dilution Women Men Total 1:40 34.6 (a) (89/257) 18.3 (43/235) 26.8 (132/492) 1:160 13.2 (a) (34/257) 2.6 (6/235) 8.1 (40/492) (a) P <0.0001, comparison of two proportions vs men. Table 2. AUCs for ROC curves shown in Fig. 2: Comparison of AUCs of COBAS-ANA and IF-ANA. Patient group Reference group Test AUC 95% CI of AUC P SLE (n = 111) Healthy women COBAS-ANA 0.98 0.95-0.99 (n = 257) IF-ANA 0.93 0.90-0.95 0.008 SS (n = 36) Healthy women COBAS-ANA 0.93 0.89-0.96 (n = 257) IF-ANA 0.82 0.77-0.86 0.012 SSc (n = 39) Healthy women COBAS-ANA 0.99 0.96-1.00 (n = 257) IF-ANA 0.96 0.93-0.98 0.31 DM/PM (n = 39) Healthy women COBAS-ANA 0.78 0.72-0.82 (n = 257) IF-ANA 0.76 0.71-0.81 0.81 MCTD (n = 33) Healthy women COBAS-ANA 0.99 0.98-1.00 (n = 257) IF-ANA 0.99 0.98-1.00 1.0 RA (n = 49) Healthy women COBAS-ANA 0.73 0.67-0.78 (n = 257) IF-ANA 0.70 0.64-0.75 0.61 All except RA Healthy women COBAS-ANA 0.94 0.92-0.96 (n = 258) (n = 257) IF-ANA 0.90 0.87-0.93 0.005 Table 3. Test characteristics of the COBAS-ANA and the IF-ANA for cutoff points. COBAS-ANA Cutoff Sensitivity Patient group index (95% CI), % SLE (n = 111) 0.6 96 (90-98) 0.9 92 (85-96) SS (n = 36) 0.6 92 (78-98) 0.9 78 (61-90) SSc (n = 39) 0.6 100 (100-100) 0.9 92 (79-98) DM/PM (n = 39) 0.6 72 (55-85) 0.9 46 (30-63) MCTD (n = 33) 0.6 100 (100-100) 0.9 100 (100-100) RA (n = 49) 0.6 55 (40-69) 0.9 20 (10-34) All diseases (n = 307) 0.6 87 (83-90) 0.9 74 (69-79) All except RA (n = 258) 0.6 93 (89-95) 0.9 84 (79-88) COBAS-ANA Cutoff Specificity Controls index (95% CI) % Women (n = 257) 0.6 79 (74-84) 0.9 94 (90-96) Men (n = 235) 0.6 78 (73-83) 0.9 96 (93-98) Total subjects (n = 492) 0.6 79 (75-82) 0.9 95 (93-97) IF-ANA Cutoff Sensitivity Patient group dilution (95% CI), % SLE (n = 111) 1:40 97 (92-99) 1:160 81 (73-88) SS (n = 36) 1:40 78 (61-90) 1:160 69 (52-84) SSc (n = 39) 1:40 97 (87-100) 1:160 95 (83-99) DM/PM (n = 39) 1:40 74 (58-87) 1:160 62 (45-77) MCTD (n = 33) 1:40 100 (100-100) 1:160 100 (100-100) RA (n = 49) 1:40 67 (53-80) 1:160 35 (22-50) All diseases (n = 307) 1:40 88 (83-91) 1:160 74 (68-79) All except RA (n = 258) 1:40 92 (87-95) 1:160 81 (76-86) IF-ANA Cutoff Specificity Controls dilution (95% CI) % Women (n = 257) 1:40 65 (59-71) 1:160 87 (82-91) Men (n = 235) 1:40 82 (76-86) 1:160 97 (95-99) Total subjects (n = 492) 1:40 73 (69-77) 1:160 92 (89-94) Table 4. Comparison of the COBAS-ANA and the IF-ANA for measurement of ANAs in patients with CTD. SLE IF-ANA [greater than or COBAS-ANA equal to] 1:160 1:40-1:80 <1:40 Total [greater than or 86 14 2 102 equal to] 0.9 0.6 to <0.9 2 1 1 4 <0.6 2 3 0 5 Total 90 18 3 111 82.0% (a) (0.248; P = 0.003) SS IF-ANA [greater than or COBAS-ANA equal to] 1:160 1:40-1:80 <1:40 Total [greater than or 23 2 3 28 equal to] 0.9 0.6 to <0.9 2 0 3 5 <0.6 0 1 2 3 Total 25 3 8 36 80.6% (a) (0.504; P = 0.002) SSc IF-ANA [greater than or COBAS-ANA equal to] 1:160 1:40-1:80 <1:40 Total [greater than or 35 0 1 36 equal to] 0.9 0.6 to <0.9 2 1 0 3 <0.6 0 0 0 0 Total 37 1 1 39 92.3% (a) (0.361; P = 0.021) DM/PM IF-ANA [greater than or COBAS-ANA equal to] 1:160 1:40-1:80 <1:40 Total [greater than or 13 2 3 18 equal to] 0.9 0.6 to <0.9 7 1 2 10 <0.6 4 2 5 11 Total 24 5 10 39 59.0% (a) (0.194; P = 0.20) MCTD IF-ANA [greater than or COBAS-ANA equal to] 1:160 1:40-1:80 <1:40 Total [greater than or 33 0 0 33 equal to] 0.9 0.6 to <0.9 0 0 0 0 <0.6 0 0 0 0 Total 33 0 0 33 100% (a) (not applicable) RA IF-ANA [greater than or COBAS-ANA equal to] 1:160 1:40-1:80 <1:40 Total [greater than or 5 4 1 10 equal to] 0.9 0.6 to <0.9 7 5 5 17 <0.6 5 7 10 22 Total 17 16 16 49 65.3% (a) (0.153; P = 0.25) All diseases IF-ANA [greater than or COBAS-ANA equal to] 1:160 1:40-1:80 <1:40 Total [greater than or 195 22 10 227 equal to] 0.9 0.6 to <0.9 20 8 11 39 <0.6 11 13 17 41 Total 226 43 38 307 79.5% (a) (0.470; P <0.0001) All diseases except RA IF-ANA [greater than or COBAS-ANA equal to] 1:160 1:40-1:80 <1:40 Total [greater than or 190 18 9 217 equal to] 0.9 0.6 to <0.9 13 3 6 22 <0.6 6 6 7 19 Total 209 27 22 258 82.2% (a) (0.382; P <0.0001) (a) Values are the percentages of samples in which the COBAS-ANA (at a cutoff index 0.9) and the IF-ANA (at a cutoff dilution 1:160) gave the same result (positiveor negative); values in parentheses indicate the is coefficient of agreement and significant difference. Table 5. Characteristics of the patients with CTD who exhibited discrepancies of COBAS-ANA titer and IF-ANA titer (COBAS-ANA <0.6; IF-ANA [greater than or equal to] 1:160). IF-ANA Patient COBAS-ANA no. Disease Sex Index Titer Pattern 1 RA F 0.36 1280 Ho (b) + Sp 2 SLE F 0.59 640 Ho + Nu 3 RA F 0.51 640 Ho 4 RA F 0.56 320 Ho + Sp 5 RA F 0.55 320 Ho 6 DM/PM M 0.34 320 Ho 7 DM/PM M 0.22 320 Sp 8 DM/PM F 0.58 160 Sp + Di 9 SLE F 0.50 160 Ho 10 RA F 0.30 160 Nu 11 DM/PM F 0.26 160 Di Single ANA (a) U1RNP Sm SSA/Ro SSB/La Patient Index Index Index Index no. (15) (15) (20) (25) 1 1.3 0.0 85.9 (c) 5.8 2 0.0 0.0 17.9 1.2 3 5.8 1.1 0.8 1.9 4 5.3 1.4 2.3 1.7 5 1.0 0.0 1.1 1.3 6 0.0 0.1 0.0 0.0 7 0.0 0.0 0.1 0.3 8 0.9 2.4 1.7 1.4 9 1.1 0.3 6.7 8.5 10 3.3 0.2 7.3 1.5 11 2.1 0.0 0.0 0.2 Single ANA (a) Scl-70 Jo-1 Centromere dsDNA, Patient Index Index Index units/mL no. (10) (10) (20) (12) 1 0.0 0.0 0.0 16.5 (c) 2 0.2 0.6 12.9 2.0 3 0.9 0.6 5.5 2.8 4 2.7 1.5 4.2 4.9 5 3.1 0.5 39.7 (c) 4.0 6 0.0 0.3 0.0 1.6 7 0.0 13.4 (c) 0.0 1.6 8 0.0 0.2 35.9 (c) 3.9 9 0.4 0.0 3.7 3.1 10 0.6 0.0 3.3 4.7 11 0.0 0.1 2.4 3.8 (a) Values in parentheses indicate the cutoff index for single ANAs. (b) Ho, homogeneous pattern; Sp, speckled pattern; Nu, nucleolar pattern; Di, discrete speckled pattern. (c) Data are titers demonstrating reactivity for disease-specific ANAs (single ANA). Table 6. Characteristics of patients with CTD who exhibited discrepancies of COBAS-ANA titer and IF-ANA titer (COBAS-ANA [greater than or equal to] 0.9; IF-ANA <1:40). IF-ANA Patient COBAS-ANA no. Disease Sex Index Titer Pattern 1 DM/PM F 2.05 <20 2 DM/PM F 1.88 <20 3 SS F 1.52 20 Ho + Sp 4 SLE F 1.40 20 Sp 5 DM/PM F 1.39 <20 6 RA F 1.38 <20 7 SSc F 1.34 20 Not classified 8 SLE F 1.20 <20 9 SS F 1.19 <20 10 SS F 1.08 <20 Single ANA (a) U1RNP Sm SSA/Ro SSB/La Patient Index Index Index Index no. (15) (15) (20) (25) 1 27.1 (c) 1.4 22.5 (c) 16.1 2 0.0 0.0 0.0 0.0 3 13.4 2.9 6.3 12.3 4 0.0 0.0 0.1 0.8 5 2.2 0.3 1.8 0.7 6 19.8 (c) 1.0 8.0 5.4 7 3.8 0.0 2.4 2.9 8 2.6 0.6 2.3 0.0 9 0.0 0.0 0.0 0.0 10 4.3 0.7 4.0 9.6 Single ANA (a) Scl-70 Jo-1 Centromere dsDNA, Patient Index Index Index units/mL no. (10) (10) (20) (12) 1 20.9 (c) 1.7 54.0 (c) 2.0 2 0.0 85.9 * 0.0 1.2 3 4.7 0.6 17.2 9.2 4 0.0 0.6 7.9 5.0 5 1.4 0.6 2.2 1.4 6 8.7 1.1 21.6 (c) 8.4 7 0.0 2.5 18.3 4.0 8 0.3 0.0 1.0 9.5 9 0.0 0.0 0.0 0.0 10 2.1 0.2 6.0 4.8 (a) Values in parentheses indicate the cutoff index for single ANAs. (b) Ho, homogeneous; Sp, speckled. (c) Data are titers demonstrating reactivity for disease-specific ANAs (single ANA). Table 7. Comparison of COBAS-ANA and IF-ANA, using monospecific antisera. (a) No. (%) of positive sera Monospecific antiserum COBAS-ANA IF-ANA U1RNP (n = 15) (b) 15 (100) 15 (100) SSA/Ro (n = 18) 16 (89) 18 (100) Centromere (n = 14) 12 (86) 14 (100) Jo-1 (n = 5) 4 (80) 3 (60) dsDNA (n = 19) 19 (100) 19 (100) (a) Monospecific antisera reactive with U1RNP, SSA-Ro, centromere, Jo-1, and dsDNA antibodies were tested in IF-ANA at a cutoff dilution of 1:40 and COBAS-ANA at a cutoff index of 0.6. The percentage of sera that tested positive in the assays is shown. (b) Numbers in parentheses indicate number of patient serum samples tested.
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|Title Annotation:||Evidence-based Laboratory Medicine and Test Utilization|
|Author:||Hayashi, Nobuhide; Kawamoto, Tomoko; Mukai, Masahiko; Morinobu, Akio; Koshiba, Masahiro; Kondo, Shin|
|Date:||Sep 1, 2001|
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