Detection of Anti-SSA Antibodies by Indirect Immunofluorescence

Transcription

1 Clinical Chemistry 50: (2004) Clinical Immunology Detection of Anti-SSA Antibodies by Indirect Immunofluorescence Xavier Bossuyt, 1* Johan Frans, 1 Ann Hendrickx, 1 Godelieve Godefridis, 1 René Westhovens, 2 and Godelieve Mariën 1 Background: HEp-2 cells that overexpress the human 60-kDa SSA antigen have been used to improve sensitivity and specificity for the detection of anti-ssa antibodies by indirect immunofluorescence. We describe a survey on the detection of anti-ssa antibodies using a commercial substrate that overexpresses SSA. Methods: The evaluation was done on consecutive samples submitted to the laboratory for detection of anti-nuclear antibodies, from which 188 anti-ssa antibody-containing and clinically documented samples were obtained. The presence of anti-ssa antibodies produced a distinct bright speckled pattern with nucleolar staining in 10 20% of interphase cells. The identity of all anti-ssa antibodies was confirmed by dot-blot analysis. Results: Samples containing anti-ssa antibodies were separated into three main groups: group I, distinctive SSA pattern and other nuclear staining (50%); group II, only the distinctive SSA pattern (29%); group III, nuclear staining but without the distinctive SSA pattern (21%). Anti-SSA antibodies with concurrent SSB antibodies were associated with group I, whereas anti-ssa antibodies with concurrent U 1 -RNP antibodies were associated with group III. Group I included mainly patients with Sjögren syndrome and systemic lupus erythematosus (SLE), whereas group III included patients with mixed connective tissue disease and SLE. Diseases not classically associated with the presence of anti-ssa antibodies were found in group II in >50% of the cases. Conclusions: SSA-positive individuals were identified in a population selected on the basis of HEp-2000 positivity. Our study highlights diseases associated 1 Laboratory Medicine and 2 Internal Medicine, University Hospital Leuven, Leuven, Belgium. *Address correspondence to this author at: Department of Laboratory Medicine, Immunology, University Hospital Leuven, Herestraat 49, B-3000 Leuven, Belgium. Fax ; xavier.bossuyt@uz.kuleuven.ac.be. Received April 21, 2004; accepted September 20, Previously published online at DOI: /clinchem with anti-ssa antibodies and associations between the presence of the distinctive SSA pattern on HEp-2000 and some clinical conditions American Association for Clinical Chemistry Anti-SSA antibodies are clinically important anti-nuclear antibodies in patients with systemic rheumatic diseases. These antibodies are found in 60% of patients with Sjögren syndrome, in 30% of patients with systemic lupus erythematosus, in a majority of patients with subacute cutaneous lupus, and in the neonatal lupus syndrome (1, 2). They are also found in 5 8% of patients with rheumatoid arthritis (2). In many clinical laboratories, the initial screening test for the detection of anti-nuclear antibodies in general, and anti-ssa antibodies in particular, is indirect immunofluorescence using HEp-2 cells. The HEp-2 cell line is more sensitive than rodent tissue for the detection of anti-ssa antibodies (3) and has become the standard substrate for the nuclear antibody test. There are many commercial suppliers of HEp-2 substrates, but production techniques vary and there are no international guidelines on culture condition, fixation, and drying. Much of the discussion about the quality of the HEp-2 substrates has concentrated on how well the SSA antigen is preserved. SSA is present in low abundance, and diffusion of the antigen from the nucleus during fixation and subsequent sample preparation can occur (4). Ethanol and methanol fixation may cause denaturation and leaching of SSA to the cytoplasm (5). Therefore, acetone fixation of the HEp-2 substrate slides has been recommended (6). A more recent approach to improve the sensitivity and specificity for the detection of anti-ssa antibodies is the use of a transfected substrate that overexpresses the human 60-kDa SSA antigen. The HEp-2000 substrate (Immunoconcepts TM ) overexpresses SSA in 10 15% of the cells. The presence in serum of autoantibodies to SSA typically leads to a distinctive bright speckled pattern with prominent staining of the nucleoli in the interphase nuclei of these transfected cells. Since the introduction of this genetically modified commercial substrate, several 2361

2 2362 Bossuyt et al.: Anti-SSA Antibodies clinical laboratories have evaluated its performance for the detection of anti-ssa antibodies. Pollock and Toh (7) reported 160 anti-ssa antibodypositive samples. The HEp-2000 substrate (with a cutoff of 1:200) revealed antibodies in 159 of the 160 samples, of which 145 (91%) displayed the distinctive SSA pattern. Peene et al. (8) tested 91 anti-ssa antibody-positive samples with the HEp-2000 substrate and found the distinctive SSA pattern in 70 samples (77%). In a prospective study, we compared the HEp-2000 substrate with counterimmunoelectrophoresis for the detection of anti- SSA antibodies on 2427 consecutive specimens (9). We found 122 specimens positive for anti-ssa antibodies by counterimmunoelectrophoresis, of which 107 (88%) showed the distinctive SSA pattern. Fifteen samples did not show the distinctive SSA pattern but displayed nuclear or cytoplasmic antibodies. Overall, nuclear antibody testing using the HEp-2000 substrate had a sensitivity of 100% for the detection of anti-ssa antibodies compared with counterimmunoelectrophoresis. Similar results were found by Fritzler et al. (10), who compared the HEp-2000 substrate with immunodiffusion and with ELISA on 2576 samples. They reported 114 samples with anti-ssa antibodies, of which 101 (89%) showed the distinctive pattern. The other samples with anti-ssa antibodies gave a positive anti-nuclear antibody test. Fritzler et al. (10) also described 14 sera that had anti-ssa antibodies detected on HEp-2000 cells but not by conventional immunodiffusion or ELISA. In 12 of these samples, anti-ssa antibodies were detected by more sophisticated testing such as immunoprecipitation and immunoblotting. In a prospective study in which 494 samples were tested with HEp and with a line immunoassay, Hoffman et al. (11) identified anti-ssa antibodies in 19 samples with the line immunoassay and in 17 samples with the HEp-2000 cell substrate. Collectively, all published data indicate that the HEp cell substrate is a highly sensitive substrate for screening for anti-ssa antibodies comparable to immunodiffusion, counterimmunoelectrophoresis, and ELISA. In addition to a high sensitivity, excellent specificity of the distinctive pattern for anti-ssa antibodies has been demonstrated (7 11). The distinctive pattern, however, is absent in a substantial number of anti-ssa antibodycontaining sera, and it is not known whether the presence or absence of the distinctive pattern is associated with distinctive subsets of anti-ssa antibody-containing patients. In this report we present a large survey on the detection of anti-ssa antibodies and further characterize the indirect immunofluorescence assay, using the HEP-2000 for detection of anti-ssa antibodies. The results are correlated with clinical data. The study not only provides valuable information on the repertoire of diseases that correlate with anti-ssa antibodies but also reveals associations between specific patterns on HEp-2000 cells and clinical conditions. In addition, several HEp-2 substrates are compared with the HEp-2000 substrate for detection of anti-ssa antibodies. For all samples, the presence of anti-ssa antibodies was confirmed by an independent assay. Materials and Methods anti-nuclear antibody testing using ssa-transfected cells SSA-transfected HEp-2000 TM cells were from Immunoconcepts. The assay was performed according to the manufacturer s instructions. Serum was incubated at a 1:40 dilution for 30 min at room temperature, and slides were then washed and incubated with fluorescein isothiocyanate-labeled goat anti-human IgG (heavy and light chain) immunoglobulin. Slides were examined under ultraviolet light with a Leitz Wetzler Orthoplan microscope. Specimens that exhibited fluorescence were titered to endpoint (serial twofold dilutions) and classified according to the fluorescence pattern. The presence in serum of anti-ssa antibodies leads to development of a characteristic, so-called distinctive fluorescence pattern in the HEp-2000 cells. This characteristic pattern is a distinct bright speckled pattern with prominent staining of the nucleoli in 10 20% of the interphase nuclei. The chromosome region of metaphase mitotic cells is negative. With the HEp-2000 cells, formalin (in ethanol) fixation is used. The distinctive SSA pattern was not titered. Serum samples that displayed the distinctive SSA pattern or serum samples with anti-nuclear antibodies with titers of 1:80 or higher and/or with cytoplasmic antibodies with titers of 1:40 or higher were screened for the presence of antibodies to extractable nuclear antigens. These cutoff values were determined based on a prospective study in which 5859 samples were screened for the presence of anti-nuclear antibodies. The results of this study are provided as Tables 1 and 2 in the Data Supplement that accompanies the online version of this article at Samples with antibodies to extractable nuclear antigens displayed the distinctive SSA pattern or an anti-nuclear antibody titer of 1:80 or higher. Some anti-ssa antibodies and some Jo-1 antibodies, however, gave a cytoplasmic pattern with a low (1:40) antibody titer on the HEp-2000 cells. We previously demonstrated (9) that no SSA precipitin-positive samples were found in HEp-2000 immunofluorescence-negative samples. Therefore, further testing for anti-ssa antibodies was done only on HEp-2000 anti-nuclear antibody-positive samples (nuclear and/or cytoplasmic staining) and not on HEp-2000 anti-nuclear antibody-negative samples. anti-nuclear antibody testing using conventional HEp-2 substrates The anti-nuclear antibody test was also performed with HEp-2 substrates from Bio-Diagnostics Ltd., Alphadia, Euroimmun, Inova Diagnostics, and Bio-Rad. All assays were performed according to the manufacturers instruc-

3 Clinical Chemistry 50, No. 12, tions. Serum dilutions were 1:40, 1:80, 1:100, 1:40, and 1:40, respectively, for the Bio-Diagnostics Ltd., Alphadia, Euroimmun, Inova Diagnostics, and Bio-Rad products. The second fluorescein isothiocyanate-conjugated antihuman antibody was reactive with IgG for the Euroimmun and Inova assays, with human immunoglobulins for the Bio-Rad assay, and with IgG heavy and light chains (thus common to all immunoglobulin classes) for the Biodiagnostics, Alphadia, and Immunoconcepts assays. Specimens that exhibited fluorescence were classified according to the fluorescence pattern. Samples that displayed only cytoplasmic fluorescence were also considered positive. Immunofluorescence readings were done by an experienced medical technologist with 30 years experience with anti-nuclear antibody testing. antibodies to extractable nuclear antigens Screening of antibodies to extractable nuclear antigens was done by counterimmunoelectrophoresis (9) and identification by dot-blot analysis (Biomedical Diagnostics) (12). In the dot-blot assay, protein A was used to react with IgG antibodies. clinical diagnosis Sjögren syndrome was diagnosed according to the classification criteria proposed by the European Study Group on Diagnostic Criteria for Sjögren syndrome (13). Systemic lupus erythematosus was diagnosed according to the American College of Rheumatology classification criteria revised in 1997 (14). Mixed connective tissue disease was diagnosed according to the criteria proposed by Alarcón-Segovia et al. (15). Subacute cutaneous lupus erythematosus was diagnosed on the basis of typical nonfixed, nonscarring, exacerbating, and remitting skin lesions in areas exposed to the sun. Discoid lupus was diagnosed on a clinical basis with the suspected local lesions usually in the face, scalp, ears, or neck confirmed on biopsy. statistical analysis The significance of the differences between groups was determined by the 2 test included in Analyze-it TM for Microsoft Excel (Ver. 1.62). No correction for multiple comparisons (Bonferroni) was applied. Sensitivities were compared by the McNemar test. The STARD guidelines (16) were taken into account wherever possible. Results staining patterns on HEp-2000 cells of ssa-positive samples Over a 2-year period (2001 and 2002), consecutive samples were submitted to the clinical laboratory to screen for the presence of anti-nuclear antibodies on HEp-2000 cells. All of these samples were included in the study. A total of 5217 samples (28.5%) had an anti-nuclear antibody titer of 1:40 or higher, and 3489 samples had a anti-nuclear antibody titer of 1:80 or higher or displayed the distinctive SSA pattern. Anti-cytoplasmic antibodies were found in 978 samples (5.3%). Serum samples that displayed the distinctive SSA pattern or serum samples with anti-nuclear antibodies with titers of 1:80 or higher and/or with cytoplasmic antibodies with titers of 1:40 or higher were screened for the presence of antibodies to extractable nuclear antigens (see Materials and Methods). Counterimmunoelectrophoresis (9) and dot-blot analysis for detection of antibodies to extractable nuclear antigens revealed 375 samples with anti-ssa antibodies. An overview of the immunofluorescence patterns observed in the SSA positive samples is provided in Table 1. In 295 (79%) of the 375 SSA-positive samples, the characteristic SSA pattern was revealed by immunofluorescence analysis, whereas in 80 samples (21%), the characteristic pattern was not observed. The distinctive SSA pattern in the SSA overexpressing cells was the only pattern present in 112 samples (29% of all SSA-positive samples), whereas in 183 (49%) samples, the distinctive pattern was found in combination with another pattern. In SSA-containing samples in which the distinctive pattern was not observed, the titer of the other pattern varied between 1:80 and 1:1280. It should be mentioned that in four SSAcontaining samples, cytoplasmic staining was the only Table 1. Immunofluorescence patterns on HEp-2000 cells of SSA antibody-positive specimens (n 375). Distinctive SSA pattern present, n Distinctive SSA pattern absent, n Homogeneous pattern with chromosome staining 1:40 titer 3 1:80 titer 7 1 1:160 titer 5 4 1:320 titer :640 titer :1280 titer 3 4 1:1280 titer 1 4 Fine speckled pattern 1:40 titer 21 1:80 titer :160 titer :320 titer 9 1:640 titer 6 1:1280 titer 1 4 1:1280 titer 2 15 Coarse speckled 4 Nucleolar staining 2 2 Nuclear dots combined with other pattern 1 Combined speckled and nucleolar pattern 40 3 Combined homogeneous and 8 10 nucleolar pattern Combined nuclear and cytoplasmic staining 6 5 Cytoplasmic staining Speckled pattern 11 2 Fibrillar pattern 2 2 No staining on untransfected cells 112

4 2364 Bossuyt et al.: Anti-SSA Antibodies indirect immunofluorescent finding observed on HEp cells. Taken together, based on fluorescence patterns on HEp-2000 cells, samples containing anti-ssa antibodies could be divided into three main groups: group I displayed the distinctive SSA pattern and staining for another anti-nuclear antibody; group II displayed only the distinctive SSA pattern; and group III was anti-nuclear antibody-positive but lacked the distinctive SSA pattern. clinical data and correlation with staining patterns The 375 specimens containing anti-ssa antibodies collected over a 2-year period were obtained from 200 different individuals (including 2 external quality-control specimens). In 73 patients with anti-ssa antibodies, the anti-nuclear antibody test was performed on several occasions (two to eight times) over the 2-year study period. In 60 of these patients, the distinctive pattern was consistently present or absent in all samples studied, whereas in 13 patients the distinctive pattern was observed in some but not all samples. These 13 patients were considered a separate group. Medical records of 188 patients of the 200 different individuals with anti-ssa antibodies could be reviewed (Table 2). Fifty-seven (30%) patients had Sjögren syndrome; 50 (26.5%) had systemic lupus erythematosus; 7 (3.7%) had systemic lupus erythematosus and/or Sjögren syndrome; 11 (6%) had subacute cutaneous lupus, discoid lupus, drug-induced lupus, or neonatal lupus; 6 (3%) had mixed connective tissue disease; 15 (8.5%) had dermatomyositis, scleroderma, rheumatoid arthritis, polymyalgia rheumatica, or undifferentiated systemic disease; and 42 (21.3%) had a medical condition other than a systemic (undifferentiated) rheumatic disease (i.e., not Sjögren syndrome and/or any form of lupus, mixed connective tissue disease, dermatomyositis, scleroderma, or rheumatoid arthritis). In Table 2, the clinical information is summarized as a function of the indirect immunofluorescence pattern observed on the HEp-2000 cells. Indirect immunofluorescence analysis revealed (a) the distinctive SSA pattern in combination with another staining pattern (group I) in 93 of the 188 patients (49%), (b) the distinctive SSA pattern without any other pattern (group II) in 56 of the 188 patients (30%), and (c) absence of the distinctive pattern (group III) in 26 of the 188 patients (14%). In 13 patients (7%), the indirect immunofluorescence pattern was not consistent among several determinations (see above). Sjögren syndrome was more prevalent in group I [42 (45%) of 93 patients] than in group II [10 (18%) of 56 patients; ; P ] and group III [2 (8%) of 26 patients; ; P ]. Within group I, Sjögren syndrome was more prevalent in the subgroup in which SS-B antibodies were found in combination with anti-ssa antibodies [27 (59%) of 46 patients] compared with the subgroup in which SS-B antibodies were absent [15 (32%) of 47 patients; ; P 0.017]. In 52 of the 57 patients with Sjögren syndrome, the distinctive SSA pattern was present. In three additional patients with Sjögren syndrome, the distinctive pattern was present in some, but not all, samples. Thus 91 96% of the patients with Sjögren disease displayed the distinctive SSA pattern. In 27 of the 57 (47%) patients with Sjögren syndrome, anti-ssb antibodies were found in combination with anti-ssa antibodies. Anti-SSB antibodies were significantly more prevalent in group I [46 (49.4%) of 93 patients] compared with group II [1 (1.8%) of 56 patients; 2 34; P ] and group III [0 (0%) of 26 patients; 2 18; P ]. We found systemic lupus erythematosus in 24 (26%) of the 93 patients in group I, in 8 (14%) of the 56 patients in group II, and in 11 (42%) of the 26 patients in group III. The difference between group II and group III was statistically significant ( ; P 0.012). The distinctive SSA pattern was present in 32 of 50 patients with systemic lupus erythematosus. In seven additional patients with systemic lupus erythematosus, the distinctive pattern was present in some but not all samples. Thus, in 64 78% of the patients with systemic lupus erythematosus, the distinctive SSA pattern was present. The prevalence of subacute cutaneous lupus tended to be higher in group II [5 (9%) of 56 patients] compared with group I [2 (2%) of 93 patients] and group III [0 (0%) of 26 patients], but this was not statistically significant. Mixed connective tissue disease was more prevalent in group III [6 (23%) of 26 patients] than in group II [0 (0%) of 56 patients; ; P 0.001] and than in group I [0 (0%) of 93 patients; ; P ]. Thus, none of the samples obtained from patients with mixed connective tissue disease displayed the distinctive SSA pattern on indirect immunofluorescence analysis. This is consistent with the finding that anti-rnp antibodies were significantly more prevalent in group III [10 (38%) of 26 patients] compared with group I [1 (1%) of 93 patients; ; P ] and group II [0 (0%) of 56 patients; 2 21; P ]. In two additional patients with anti-rnp antibodies, the distinctive pattern was absent in some of the samples obtained. Finally, medical conditions other than systemic rheumatic disease were significantly more common in group II [27 (48%) of 56 patients] compared with group I [11 (12%) of 93 patients; ; P ] and group III [2 (7%) of 26 patients; ; P ]. indirect immunofluorescence pattern and titer of anti-nuclear antibodies in samples containing anti-ssa antibodies The patterns and titers obtained by indirect immunofluorescence on the HEp-2000 substrate in anti-ssa antibody-containing samples from 188 different patients are shown in Table 3. The results are grouped according to the presence or absence of the distinctive SSA pattern in the overexpressing cells (see above). The speckled pattern was more prevalent in group I

5 Clinical Chemistry 50, No. 12, Table 2. Clinical data of patients with anti-ssa antibodies and correlation with staining patterns. a I SSB (n 46) SSB (n 47) Group II (n 56) III (n 26) IV (n 13) Sjögren syndrome Systemic lupus erythematosus and/or Sjögren syndrome Systemic lupus erythematosus Subacute cutaneous lupus Discoid lupus 1 1 Drug-induced lupus 1 Neonatal lupus 1 Mixed connective tissue disease 6 Dermatomyositis 1 Scleroderma 1 Rheumatoid arthritis Polymyalgia rheumatica 1 Systemic disease undifferentiated Essential cryoglobulinemia 1 Crohn disease 1 Colitis ulcerosa 1 Autoimmune hepatitis 1 Primary biliary cirrhosis 1 1 Hyperthyroidism/goiter 1 2 Diabetes 1 Multiple sclerosis 1 Purpura hyperglobulinemia 1 Hepatitis C 1 Interstitial lung disease 1 Idiopathic pulmonary hemosiderosis 1 Alcoholic liver cirrhosis 1 Nonalcoholic steatohepatitis 1 Erythema annularis centrifugum 1 Guillain Barré 1 Chronic fatigue syndrome 2 CHARGE syndrome 1 B-Cell lymphoma 1 2 Mamma carcinoma 1 Basocellular carcinoma 1 Pseudomyxomata and metastases 1 Polyneuropathy 1 Protein S deficiency 1 Tendonitis 1 Tuberculosis 1 No specific disease a We analyzed 188 anti-ssa antibody-containing samples with HEp On the basis of fluorescence patterns, samples were divided into three groups. Group I displayed the distinctive SSA pattern (speckled fluorescence with prominent staining of the nucleoli in the interphase nuclei of transfected cells) and staining for another anti-nuclear antibody; group II displayed only the distinctive SSA pattern; and group III was anti-nuclear antibody-positive but lacked the distinctive SSA pattern. Group I was divided into two subgroups, one in which anti-ssb antibodies were present and one in which anti-ssb antibodies were absent. A fourth group consisted of patients in whom the distinctive pattern was observed in some but not all samples. The clinical data for patients with anti-ssa antibodies are presented. [70 (75%) of 93 patients] than in group III [10 (38%) of 26 patients; ; P 0.001], whereas the homogeneous pattern was more prevalent in group III [13 (50%) of 26 patients] than in group I [24 (26%) of 93 patients; ; P 0.001]. The majority [37 of 46 (80%)] of the samples in which anti-ssb antibodies were present in combination with anti-ssa antibodies displayed the distinctive SSA pattern in combination with a speckled nuclear staining. The distribution of the titers is also presented in Table 3. In samples in which the distinctive SSA pattern was the only staining pattern present (group II), staining of the SSA-overexpressing cells was strong and the titer was not determined. If an additional staining pattern was present

6 2366 Bossuyt et al.: Anti-SSA Antibodies Table 3. Indirect immunofluorescence pattern and titer of anti-nuclear antibodies in samples containing anti-ssa antibodies and association with antibodies to extractable nuclear antigens. a SSB (n 46) I SSB (n 47) Group II (n 56) III (n 26) Pattern Fine speckled Homogeneous/chromosomes Combination fine/homogeneous 1 2 Cytoplasmic Nucleolar Other 1 Titer 1: : : : : : : IV (n 13) a We analyzed 188 anti-ssa antibody-containing samples with HEp-2000 cells. On the basis of fluorescence patterns, samples were divided into three groups. Group I displayed the distinctive SSA pattern (speckled fluorescence with prominent staining of the nucleoli in the interphase nuclei of transfected cells) and staining for another anti-nuclear antibody; group II displayed only the distinctive SSA pattern; and group III was anti-nuclear antibody-positive but lacked the distinctive SSA pattern. Group I was divided into two subgroups, one in which anti-ssb antibodies were present and one in which anti-ssb antibodies were absent. A fourth group consisted of patients in which the distinctive pattern was observed in some but not all samples. The patterns of the anti-nuclear antibodies and the titers of the antibodies found in each group are given. (group I or groups III and IV), it was titered. Group I was divided in two subgroups: a subgroup in which SSB antibodies were present and a subgroup in which anti- SSB antibodies were absent. In the subgroup in which anti-ssb antibodies were present, the anti-nuclear antibody titer was 1:320 or 1:640 in 50% of the samples. These titers were significantly ( ; P ) higher than the titers found in samples that did not contain anti-ssb antibodies and in which another pattern was present in addition to the distinctive SSA pattern. In these samples, 66% of the samples had a titer of 1:40 or 1:80. Comparison of group I with group III revealed that the highest anti-nuclear antibody titers were in samples from group III ( ; P ). In this group, 50% of the samples had a titer of 1:1280 or higher. It should be mentioned that in samples in which cytoplasmic staining was the only staining observed, this staining could be weak (e.g., titer 1:40). detection of anti-ssa antibodies by conventional HEp-2 substrates: comparison with the HEp-2000 substrate We evaluated the performance of a selection of five commercially available HEp-2 substrates for the detection of anti-ssa antibodies. The evaluation was done on 88 samples by comparing the assays with the HEp-2000 substrate, which has previously been demonstrated to be highly sensitive for the detection of anti-ssa antibodies [see the introduction and Refs. (7 11)]. The samples were from different individuals and were selected such that each of the HEp-2000 subgroups (see above) were represented. In all samples, the identity of all IgG-type anti- SSA antibodies was confirmed by dot-blot analysis. In 70 samples, the distinctive SSA pattern was present on HEp-2000 slides. In 16 samples, the distinctive SSA pattern was combined with another pattern [homogeneous (n 6); speckled (n 10); group I], whereas in 54 samples, the characteristic SSA pattern was the only pattern present (group II). In 18 samples, the distinctive SSA pattern was absent on HEp-2000 cells, but antinuclear staining [fine speckled (n 4), homogeneous with positive staining of the chromosomes (n 10); titer 1:80] or cytoplasmic staining (n 4) was present (group III). In all samples, the presence of anti-ssa antibodies was confirmed by dot-blot analysis. Six control samples that contained no autoantibodies were analyzed as well. The medical conditions of the patients are summarized in Table 4. The control samples were negative in all HEp-2 assays. The results for the SSA-positive samples are presented in Table 5. In samples in which analysis on HEp-2000 cells revealed the characteristic SSA pattern and in which another anti-nuclear pattern was present (group I), the sensitivities of the various HEp-2 assays ranged between 87% and 100% [95% confidence interval (CI), %]. Statistical analysis (McNemar test) revealed no significant difference (P 0.5) between the various assays tested and the HEp-2000 assay. In this group of samples (n 16),

7 Clinical Chemistry 50, No. 12, Table 4. Clinical diagnoses of the patients listed in Table 5. a HEp-2000 Total, n SS, b n SLE, n SCL, n DL, n CTD, n Other, n Distinctive SSA pattern present/other pattern present (group I) Distinctive SSA pattern present/other pattern absent (group II) c Distinctive SSA pattern absent/other pattern present (group III) Nuclear staining Cytoplasmic staining Granular d Fibrillar e a Medical records for 78 patients of the 88 patients represented in Table 5 could be consulted. b SS, Sjögren syndrome; SLE, systemic lupus erythematosus; SCL, subacute cutaneous lupus; DL, discoid lupus; CTD, symptoms suggestive of connective tissue disease, although no definite diagnosis could be made. c Malignant lymphoma (n 3), paraneoplastic polyneuropathy (n 1), basocellular carcinoma (n 1), pseudomyxomata peritonea with metastases (n 1), polymyalgia rheumatica (n 2), purpura hypergammaglobulinemia (n 1), cryoglobulinemia (n 1), photosensitivity (n 1), erythema annularis centrifugum (n 1), Guillain Barré (n 1), protein S deficiency (n 2), tendonitis (n 1), tuberculosis (n 1), no diagnosis (n 2). d Primary biliary cirrhosis and sicca. e CHARGE syndrome. cytoplasmic staining was the only positivity found in 0, 2, 1, 1, and 0 samples by the Euroimmun, Bio-Rad, Alphadia, Inova, and Bio-Diagnostics assays, respectively. In samples in which analysis on HEp-2000 cells revealed nuclear or cytoplasmic staining but in which the distinctive SSA pattern was absent (n 18; group III), anti-nuclear antibody analysis on slides from Euroimmun, Bio-Rad, Alphadia, Inova, and Bio-Diagnostics revealed positive staining in 89% (95% CI, 65 99%), 94% (73 100%), 89% (65 99%), 89% (65 99%), and 100% (81 100%) of the samples, respectively. Statistical analysis (McNemar test) revealed no significant difference between HEp-2000 and the various assays tested. Four samples that displayed only cytoplasmic staining on HEp-2000 cells also displayed cytoplasmic staining on all other substrates. In samples in which the characteristic SSA pattern was the only finding on the HEp-2000 cells (n 54), antinuclear antibodies were detected in 43 (80%; 95% CI, 68 91%), 13 (24%; 95% CI, 13 38%), 40 (74%; 95% CI, 60 85%), 25 (46%; 95% CI, 33 60%), and 47 (91%; 95% CI, 78 97%) samples with slides from Euroimmun, Bio-Rad, Alphadia, Inova, and Bio-Diagnostics, respectively. All assays detected a significantly lower number of anti-ssa antibodies compared with HEp-2000 (McNemar test, P 0.001, P , P , P , and P for the Euroimmun, Bio-Rad, Alphadia, Inova, and Bio- Diagnostics assays, respectively). Cytoplasmic staining on HEp-2 analysis was the only finding in one, five, two, zero, and two samples by the Euroimmun, Bio-Rad, Alphadia, Inova, and Bio-Diagnostics assays, respectively. In 24 of the 54 samples in group II, medical records revealed the presence of systemic lupus erythematosus, Sjögren syndrome, and subacute cutaneous lupus. In this subgroup of samples, anti-nuclear antibodies were detected in 22 (92%), 7 (29%), 19 (79%), 12 (50%), and 24 (100%) samples by the Euroimmun, Bio-Rad, Alphadia, Inova, and Bio-Diagnostics assays, respectively. Compar- Table 5. Detection of anti-ssa antibodies by indirect immunofluorescence. a HEp-2 assay HEp-2000 Total A B C D E Distinctive SSA pattern present/other pattern present (group I) n Sensitivity, % 100 (79 100) (87) (62 98) (100) (79 100) (87) (62 98) (100) (79 100) Distinctive SSA pattern present/other pattern absent (group II) n b 13 b 40 b 25 b 49 Sensitivity, % (81) (68 91) (24) (13 38) (74) (60 85) (46) (33 60) (91) (80 97) Distinctive SSA pattern absent/other pattern present (group III) n Sensitivity, % (89) (65 99) (94) (73 100) (89) (65 99) (89) (65 99) (100) (81 100) a We assayed 88 samples with a positive nuclear antibody test on HEp-2000 cells and with anti-ssa antibodies confirmed by dot-blot analysis (BMD) for the presence of anti-nuclear antibodies with five conventional HEp-2 substrates: A, Euroimmun; B, Bio-Rad; C, Alphadia; D, Inova; E, Bio-Diagnostics. Shown are the number of positive nuclear antibody test results and the sensitivity (95% CI). b Statistically significantly different from HEp-2000 assay ( 2 test, P 0.05).

8 2368 Bossuyt et al.: Anti-SSA Antibodies ison with HEp-2000 (McNemar test) gave the following: P 0.5, P , P 0.065, P , and P 1, respectively. The group of samples in which the distinctive SSA pattern was the only staining pattern observed accounted for 61% of the samples used for the comparative analysis of the different HEp-2 substrates, whereas in reality this group accounts for only 30% of all anti-ssa antibodypositive samples. Therefore, overall sensitivities were not calculated. Discussion In this report we describe an investigation on the detection of anti-ssa antibodies by HEp-2000, a substrate that overexpresses SSA. This substrate is increasingly used in clinical laboratories and has previously been described as very sensitive for the detection of anti-ssa antibodies (7 11). With the HEp-2000 substrate, anti-ssa antibodies characteristically produce a distinctive fluorescence pattern that is typified by a bright speckled pattern with prominent staining of the nucleoli in the interphase nuclei of the transfected cells. This typical SSA pattern is reported to be extremely specific for the presence of anti- SSA antibodies and has been found in 68 91% of the samples in which anti-ssa antibodies have been identified by either counterimmunoelectrophoresis, immunodiffusion, ELISA, or line immunoassay (7 11). Samples containing anti-ssa antibodies but in which the distinctive SSA pattern was absent on HEp-2000 cells displayed other anti-nuclear or cytoplasmic staining (7 11). Using HEp-2000 cells to screen for the presence of anti-nuclear antibodies, we identified 375 anti-ssa antibody-positive samples obtained from 200 different patients over a 2-year period. Using fluorescence patterns on HEp-2000 cells, we segregated the samples containing anti-ssa antibodies into three main groups: group I displayed the distinctive SSA pattern and staining for another anti-nuclear antibody (50% of the SSA-positive samples); group II displayed only the distinctive SSA pattern (29% of the SSA-positive samples); and group III was anti-nuclear antibody-positive but lacked the distinctive SSA pattern (21% of the samples). We observed several marked associations. Samples in which anti-ssb antibodies were simultaneously present with anti-ssa antibodies belonged to group I, whereas samples in which anti-u 1 -RNP antibodies were simultaneously present with anti-ssa antibodies fit in group III. In addition, group I included mainly patients with Sjögren syndrome and systemic lupus erythematosus, whereas group III contained patients with mixed connective tissue disease and systemic lupus erythematosus. Group II included several diseases that are not classically associated with the presence of anti-ssa antibodies. One half of the patients in group II had lupus or Sjögren syndrome, and many of the others had diseases with possible autoimmune pathogenesis. Our finding that 21% of the anti-ssa antibody-positive specimens lacked the distinctive staining pattern on indirect immunofluorescence analysis using the HEp-2000 substrate was comparable to previous studies in which the fraction of anti-ssa antibody-positive samples that lacked the characteristic staining pattern varied between 9% and 32% (7 11). The reason that cells that overexpress SSA fail to detect anti-ssa antibodies in one fifth of patients is unknown. It has been suggested that the absence of the distinctive SSA pattern is attributable to the presence of another strong pattern that masks the typical SSA staining on HEp-2000 cells (7, 9). We found various samples with high anti-nuclear antibody titers in which the distinctive SSA pattern was absent. Titration of these samples did not reveal the distinctive SSA pattern, which argues against the assumption that the distinctive SSA pattern was hidden under another pattern. Moreover, we failed to observe the distinctive SSA pattern in samples in which the anti-nuclear antibodies were present in (very) low titers (1:40, 1:80, and 1:160) or in which the only positive characteristic was cytoplasmic staining. Because the distinctive SSA pattern is characterized by a strong and intense fluorescence, it should be detected during titration even when another bright pattern is present. An alternative hypothesis could be that the SSA molecules in the transfected cells do not display all possible epitopes for reaction with anti-ssa antibodies and, consequently, that some anti-ssa antibodies do not react with the transfected SSA molecules. This conjecture is backed by earlier investigations that found distinct differences in the epitopes bound by sera from patients with systemic lupus erythematosus vs sera from patients with primary Sjögren syndrome [for a review, see Ref. (17)]. This hypothesis is also in accordance with our finding that almost all sera obtained from patients with Sjögren syndrome demonstrated the distinctive SSA pattern on the HEp-2000 cells, whereas a substantial number of sera obtained from patients with systemic lupus erythematosus failed to produce the distinctive SSA pattern. Moreover, none of the sera obtained from patients with mixed connective tissue disease displayed the distinctive SSA pattern on immunofluorescence analysis. One could hypothesize that sera obtained from patients with mixed connective tissue disease and a subset of sera obtained from patients with systemic lupus erythematosus react with epitopes that are not readily available on the SSAtransfected HEp-2 cells and that sera obtained from patients with Sjögren syndrome, on the other hand, react with epitopes that are easily available on the SSA-overexpressing cells. We found the distinctive SSA pattern in almost all samples in which anti-ssb antibodies were present in combination with anti-ssa antibodies, but not in samples in which anti-u 1 -RNP antibodies were present in combination with anti-ssa antibodies. This again might indicate that anti-ssa antibodies present in diseases associated with anti-ssb antibodies react with epitopes different from those with which anti-ssa antibodies present in

9 Clinical Chemistry 50, No. 12, diseases associated with anti-u 1 -RNP antibodies react. This could fit with the observation that anti-ssa antibodies to a 13-kDa carboxyl-terminal V8 protease fragment of 60-kDa SSA were associated with concurrent SSB (17). The difference in pattern from time to time can also theoretically depend on differences in the quality of the HEp-2000 cells. A strength of this study is that the diagnosis of SSA antibody-positive individuals was determined in a population selected on the basis of a positive HEp This relatively randomly selected approach gives valuable information on the repertoire of diseases that may lead to, or correlate with, production of anti-ssa antibodies (Table 2). Moreover, we revealed, for the first time, that some specific patterns on SSA-transfected cells correlated with some clinical conditions. This study also illustrates that when the HEp-2000 assay is positive, other assays must be used to determine the specificities of these antibodies. Looking at the binding pattern in the HEp-2000 assay is not a firm approach to determine specificities. The absence of the distinctive SSA pattern does not mean that anti-ssa antibodies are absent. In this study, we also evaluated the performance of five commercially available and widely used HEp-2 substrates for detection of anti-ssa antibodies by comparing them with the sensitive HEp-2000 substrate. All anti-ssa antibodies included in this evaluation were of IgG class (confirmed by IgG-specific dot-blot assay). Because most manufacturers use acetone for fixation, we expected the current HEp-2 substrates to be highly sensitive for the detection of anti-ssa antibodies. The performance of the various HEp-2 substrates for detecting anti-ssa antibodies was comparable to that of HEp-2000 for samples belonging to groups I and III. The HEp-2 substrates had the most difficulties detecting anti-ssa antibodies that produce only the SSA pattern on HEp-2000, but this is the group in which most nonrheumatologic diseases were found. It should also be mentioned that in this group of samples, no classic anti-nuclear staining was found in the nontransfected cells in the HEp-2000 substrate, whereas positive anti-nuclear staining was found on several conventional HEp2 substrates. With several conventional HEp-2 substrates and with the HEp-2000 substrate, cytoplasmic staining was the only positive characteristic found in some SSA antibody-containing samples, which indicated leakage of the SSA antigen into the cytoplasm during substrate preparation. References 1. Chan EKL, Andrade LEC. Nuclear antibodies in Sjögren s syndrome. Rheum Dis Clin North Am 1992;18: Autoantibodies in systemic autoimmune diseases. A diagnostic reference. In: Conrad K, Schlossler W, Hiepe F, Fritzler M, eds. Autoantigens, autoantibodies, autoimmunity, Vol. 2. Lengerich: PABST Science Publishers, 2002: Dore N, Synkowski D, Provost TT. Nuclear antibody determinations in Ro-SSA-positive nuclear-negative lupus and Sjögren s syndrome patients. J Am Acad Dermatol 1983;8: Harmon CD, Deng JS, Peebles CL, Tan EM. The importance of tissue substrate in the SSA Ro antigen-antibody system. Arthritis Rheum 1984;27: Hollingsworth PN, Pummer SC, Dawkins RL. Nuclear antibodies. In: Peter JB, Shoenfeld Y, eds. Autoantibodies. Amsterdam: Elsevier Science BV, 1996: Kavanaugh A, Tomar R, Reveille J, Solomon DH, Homburger HA. Guidelines for clinical use of the nuclear antibody test and tests for specific autoantibodies to nuclear antigens. Arch Pathol Lab Med 2000;124: Pollock W, Toh BH. Routine immunofluorescence detection of Ro/SSA autoantibody using HEp-2 cells transfected with human 60 kda Ro/SSA. J Clin Pathol 1999;52: Peene I, Van Ael W, Vandenbossche M, Vervaet T, Veys E, De Keyser F. Sensitivity of the HEp-2000 substrate for the detection of SSA Ro60 antibodies. Clin Rheum 2000;19: Bossuyt X, Meurs L, Mewis A, Mariën G, Blanckaert N. Screening for autoantibodies to SSA/Ro by indirect immunofluorescence using HEp-2000 cells. Ann Clin Biochem 2000;37: Fritzler M, Hanson C, Miller J, Eystathioy. Specificity of autoantibodies to SSA/Ro on a transfected and overexpressed human 60 kda Ro autoantigen substrate. J Clin Lab Anal 2002;16: Hoffman E, Peene I, Veys E, De Keyser F. Detection of specific nuclear reactivities in patients with negative nuclear antibody immunofluorescence screening tests. Clin Chem 2000;48: Mewis A, Mariën G, Blanckaert N, Bossuyt X. Evaluation of a dot-blot method for identification of antibodies against extractable nuclear antigens and cytoplasmic antibodies. Clin Chem 1999; 45: Vitali C, Bombardieri S, Moutsopoulos HM, Coll J, Gerli R, Hatron PY, et al. Assessment of the European classification criteria for Sjogren s syndrome in a series of clinically defined cases: results of a prospective multicentre study. The European Study Group on Diagnostic Criteria for Sjogren s Syndrome. Ann Rheum Dis 1996; 5: Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997;40: Alarcon-Segovia D, Villareal M. Classification and diagnostic criteria for MCTD. In: Kasukawa R, Sharp GC, eds. Mixed connective tissue disease and antinuclear antibodies. Amsterdam: Elsevier, 1987: Bossuyt PM, Reitsma JB, Bruns DE, Gatsonis CA, Glasziou PP, Irwig LM, et al. The STARD statement for reporting studies of diagnostic accuracy: explanation and elaboration. Clin Chem 2003;49: Scofield RH, Farris D, Horsfall AC, Harley J. Fine specificity of the autoimmune response to the Ro/SSA and La/SSB ribonucleoproteins. Arthritis Rheum 1999;42: