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Clinical Chemistry 48: 2171-2176, 2002;
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(Clinical Chemistry. 2002;48:2171-2176.)
© 2002 American Association for Clinical Chemistry, Inc.

Detection of Specific Antinuclear Reactivities in Patients with Negative Anti-nuclear Antibody Immunofluorescence Screening Tests

Ilse E.A. Hoffman1a, Isabelle Peene1, Eric M. Veys1 and Filip De Keyser1

1 Department of Rheumatology, Ghent University Hospital, De Pintelaan 185, Ghent 9000, Belgium.

aAuthor for correspondence. Fax 32-9-240-38-03; e-mail Ilse.Hoffman{at}rug.ac.be.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: For detection of anti-nuclear antibodies (ANAs) and antibodies to extractable nuclear antigens (ENAs), samples frequently are screened with indirect immunofluorescence (IIF); further determination of anti-ENA antibodies is performed only when the result is positive. However, because anti-ENA reactivities are found in samples with low fluorescence intensities, we determined anti-ENA antibodies in samples with negative IIF and thus calculated the sensitivity of IIF for specific ANAs.

Methods: We collected 494 samples consecutively referred by rheumatologists for routine ANA testing. IIF on HEp-2 and HEp-2000 (HEp-2 cells transfected with Ro60 cDNA) and line immunoassay (LIA) for the detection of specific ANAs were performed on all samples.

Results: Fluorescence intensities and patterns on HEp-2 were strongly correlated with those on HEp-2000 [Spearman {rho} = 0.852 (P <0.001) and 0.838 (P <0.001), respectively]. Sixty-eight of 494 samples were positive on LIA, of which only 72% (confidence interval, 68–76%) were detected with HEp-2 and 75% (confidence interval, 70–78%) with HEp-2000. Of 291 samples negative on both substrates, 12 were positive on LIA. Connective tissue diseases were diagnosed in four of these patients and suspected in at least three others.

Conclusion: The HEp-2 and HEp-2000 substrates perform comparably for fluorescence intensities and patterns and for detecting specific ANAs, but some patients with negative IIF show reactivity on LIA. We recommend testing for fine reactivities, regardless of the IIF result, when the clinical suspicion for rheumatic connective tissue disease is high.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
The detection of anti-nuclear antibodies (ANAs)1 has become an important tool in rheumatologic practice, as their presence and identification can be helpful in the diagnosis of several connective tissue diseases (1). For detection of ANAs, cascade testing is frequently used (2)(3). Samples are screened with indirect immunofluorescence (IIF), and only when this test is positive are further assays for extractable nuclear antigens (ENAs) performed. In some countries, reimbursement for further assays for ENAs is provided only for samples with positive IIF.

In the past, animal substrates such as mouse kidney/liver or rat liver have been used for IIF, but these substrates lacked sensitivity, especially for anti-SSA antibodies (4). Today, human epithelioid cells are used most frequently (HEp-2 cells, a human larynx epithelioma cell line) (5). These cells have a higher sensitivity than does mouse kidney for detecting systemic lupus erythematosus (SLE) and Sjögren syndrome (6). It has been suggested that the HEp-2000TM substrate, consisting of HEp-2 cells transfected with Ro60 cDNA, could be a valuable alternative because it has a higher sensitivity for detecting anti-SSA/Ro antibodies (7)(8)(9). Further identification of the fine reactivities can be done by immunodiffusion (ID), immunoblotting, ELISA, or line immunoassay (LIA) (10)(11)(12)(13)(14)(15).

It has been suggested that some ENAs can be missed on IIF, such as anti-SSA/Ro (7)(16) and anti-SSB/La antibodies (16). In addition, anti-Jo1 antibodies are not excluded in case of negative nuclear fluorescence because Jo1 is a cytoplasmic antigen (5). Most of these studies have been done on distinct patient groups, such as patients with SLE.

In previous work, we demonstrated in a large and consecutive patient population that serum samples with borderline ANA fluorescence might contain anti-SSA or anti-SSB antibodies (13). The situation is different for anti-double-stranded DNA antibodies, which are classically found in samples with strong nuclear fluorescence on HEp-2 cells (13). We therefore performed the current prospective study, designed to evaluate the sensitivity of IIF for detection of ENAs in a series of samples referred by rheumatologists for ANA testing.


   Patients and Methods
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 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
serum samples
From a previous study (13), we knew that the frequency of anti-SSA and/or anti-SSB antibodies in samples with low fluorescence intensity was 4.4–5.6% and that ANA testing is positive in ~23.5% of a consecutive population referred for ANA testing by various specialists. If the occurrence of ENAs were as high in IIF-negative samples as in samples with low IIF intensity, one could expect to find maximally 15–20 anti-SSA- and/or anti-SSB-positive samples in a group of 500 samples with negative IIF on HEp-2 cells. All samples referred by rheumatologists of the Department of Rheumatology, Ghent University Hospital, for routine ANA testing were collected prospectively from August to December of 2000. We did not take the result of the ANA testing into account when including samples. Initially, we included 560 samples. When more than one sample from a patient was sent to the laboratory during the period of inclusion, only the first sample was used for this study. We thus excluded 59 samples. Seven other samples had to be excluded because the amount of spare serum was insufficient to perform all analyses required. Thus, 494 samples were entered into the study. Serum was obtained by centrifugation after blood clotting. Spare material was aliquoted and stored at -20 °C until further analysis.

iif on HEP-2 and HEP-2000 cells
Sera were screened for ANAs by IIF in parallel on HEp-2 and HEp-2000 cells. Serum diluted 1:40 in phosphate-buffered saline (50 µL of serum in 1950 µL of phosphate-buffered saline) was overlaid on fixed HEp-2 cells (MeDiCa Inc.) or on fixed HEp-2000 cells (Immunoconcepts) for 30 min at room temperature. Slides were washed twice for 5 min each with phosphate-buffered saline, overlaid with fluorescently labeled conjugate (anti-human IgG, heavy and light chain specific; MeDiCa), and incubated for an additional 30 min. After a slide was washed twice, a coverslip was placed over the slide with mounting medium (MeDiCa), and the slides were read with a fluorescence microscope at x40 power. The fluorescence intensity was scored semiquantitatively from 1+ to 5+ relative to the intensity of a negative and a positive (4+) control (5). The HEp-2000 result was reported as SSA-positive if the characteristic speckled and bright nucleolar staining pattern described previously was present (7). If only the specific SSA pattern was seen, the HEp-2000 result was considered positive even if ANA fluorescence was negative. Slides were read by an experienced laboratory technician, who was blinded to the diagnosis and the results of IIF on the other substrate.

lia
All sera were analyzed by LIA (INNO-LIATM ANA Update, K1090; Innogenetics N.V.). This multianalyte assay contains the following antigens: SmB, SmD, RNP-70, RNP-A, RNP-C, Ro52, Ro60, La/SSB, cenp-B, Scl-70, Jo-1, ribosomal P, and histones. For this assay, recombinant or purified native nuclear proteins or peptides are coated as discrete lines on a nylon strip with plastic backing (12)(15). The tests were performed according to the manufacturer’s instructions. Briefly, the patient’s serum, diluted 1:200 (10 µL of serum in 2000 µL of phosphate-buffered saline), was incubated with the strip in a test trough. A goat anti-human IgG labeled with alkaline phosphatase binds to the autoantigen-autoantibody complex if present. A reaction between the enzyme substrate and a chromogen, 5-bromo-4-chloro-3-indolyl phosphatase, produces a dark brown color in proportion to the amount of specific autoantibody in the test sample. Sulfuric acid stops the color development. Each line of the test strip is compared with the respective line on a reference strip, obtained by testing a cutoff control sample in each run. These cutoffs have been validated by the manufacturer at a specificity of at least 98% (12)(15). According to the manufacturer, anti-Sm antibodies are positive when the SmD line is positive, and anti-RNP-antibodies are present when there is reactivity to at least two RNP determinants. For anti-SSA antibodies, the presence of either anti-Ro52 or anti-Ro60 reactivity is sufficient.

Repeatability and reproducibility testing of the INNO-LIA ANA Update was performed by the manufacturer on a panel of 7 samples from patients with a confirmed autoimmune diagnosis ("positive" samples) and 24 control samples (negative on all antigen lines) in different conditions. For intraassay variation (repeatability), all samples were tested five times on one lot during one test run; for interassay variation (intermediate precision), all samples were tested five times on one lot during three different test runs. For interlot variation (intermediate precision), all samples were tested in triplicate on different lots; for interlaboratory variation (reproducibility), all samples were tested in triplicate on one lot by three different persons in three different laboratories. All test results were within acceptable limits (no positive-negative or negative-positive switches were allowed; personal communication).

Study personnel were blinded to the results of the IIF tests and the clinical data.

double id
Precipitating antibodies against ENAs were detected by ID in Ouchterlony plates with thymus/spleen nuclear extract (mammalian extracted nuclear antigen; Immunoconcepts) as the antigen source. Antibody specificity was determined by comparison with an anti-SSA/Ro, anti-SSB/La, anti-Sm, anti-RNP, anti-Scl70, and anti-Jo1 reference serum. This assay was performed on the samples that were negative on IIF with either HEp-2 or HEp-2000 and showed specific reactivity on LIA. The superior sensitivity of LIA vs ID has been shown previously (12)(13). Study personnel were blinded to the clinical data.

elisa
Samples negative on IIF with HEp-2 or HEp-2000 and that showed a specific reactivity on LIA were tested with commercially available specific ELISAs (Quanta-lite; Inova Diagnostics) to confirm the reactivity. The following ELISAs were used: the Sm ELISA, coated with purified Sm antigen; the RNP ELISA, which used affinity-purified RNP/Sm complex; the SSA ELISA, coated with purified Ro60 antigen and recombinant Ro52 antigen; the SSB ELISA, coated with purified SSB antigen; the Jo-1 ELISA, coated with purified Jo1 antigen; and the histone ELISA, coated with purified histone antigens. Study personnel were blinded to the clinical data.

statistics
Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, Inc.). Correlations were computed using the Spearman {rho}, whereas the Cohen {kappa} was used to determine agreement between dichotomous data. A P value <0.05 was considered statistically significant. Sensitivities are given with their confidence intervals (CIs) computed as follows: P ± 1.96 , where p represents sensitivity, q is 1 - p, and n is the sample size (17).

We followed the guidelines for reporting of studies of diagnostic accuracy of medical tests (18). The original data are available at our department on request.


   Results
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 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
correlation of iif results on HEP-2 and HEP-2000
Intensity of fluorescence.
There was a good correlation between the fluorescence intensity on both substrates (Spearman {rho} = 0.852; P <0.001; Table 1 ). When the IIF result was considered as positive or negative, the Cohen {kappa} indicated a good agreement between both substrates ({kappa} = 0.785; P <0.001).


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  		Table 1. Fluorescence intensity on HEp-2 vs HEp-2000.1

Pattern of fluorescence.
The frequencies of the patterns of fluorescence are given in Table 2 . There was a good correlation between the immunofluorescence patterns seen on HEp-2 and HEp-2000 substrate (Spearman {rho} = 0.838; P <0.001). The typical SSA pattern was seen in 9.0% of the samples that were positive by IIF on HEp-2000.


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  		Table 2. Fluorescence pattern on HEp-2 vs HEp-2000.1

detection of a specific ana (lia) according to iif result
The percentages of samples showing antibodies on LIA, according to IIF intensity on HEp-2 or HEp-2000, are shown in Fig. 1 . It is noteworthy that in the group negative for IIF on both HEp-2 and HEp-2000, a sizeable fraction of samples (12 of 291; 4.1%) displayed a specific ANA. Eight other samples revealed monoreactivity to only one RNP determinant or to SmB. The significance of these reactivities is uncertain because these monoreactivities correlate poorly with anti-RNP and anti-Sm reactivities, respectively, as defined by conventional assays (12). Four samples (samples 13–16) had discrepant results on IIF depending on the substrate and displayed autoantibodies on LIA. One of these samples (sample 13) showed only the SSA pattern on HEp-2000, without ANA. The IIF and LIA results are shown in Table 3 .



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  		Figure 1. Percentages of specific ANA reactivities detected by LIA, according to IIF result for HEp-2 () and HEp-2000 ({blacksquare}).


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  		Table 3. Clinical findings and test results for patients with negative IIF on HEp-2 or HEp-2000 but demonstrating reactivity on LIA.

correlation between iif on HEP-2000 and lia for detection of anti-ssa antibodies
The sensitivities of HEp-2000 for the detection of anti-SSA antibodies are shown in Table 4 . All samples showing the typical SSA pattern on HEp-2000 also showed reactivity toward Ro52 and/or Ro60 on LIA, yielding a specificity of 100%. The majority of samples with reactivity toward Ro60 on LIA also showed the SSA pattern on HEp-2000. Also listed in Table 4 is the performance of the HEp-2000 substrate, regardless of the presence of the SSA pattern, and of the HEp-2 substrate for detecting anti-SSA antibodies. The HEp-2000 substrate has a sensitivity of 93% (CI, 91–95%) for detecting anti-SSA antibodies vs a sensitivity of 86% (CI, 83–89%) for the HEp-2 substrate.


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  		Table 4. Sensitivity of the SSA pattern on HEp-2000 and of IIF on HEp-2000 and HEp-2 for detection of anti-SSA antibodies.1

clinical findings in iif-negative patients with reactivity on lia
The diagnoses and clinical findings in patients with negative IIF but reactivity on LIA are given in Table 3Up . Some of these patients had defined rheumatic diseases, such as rheumatoid arthritis (patient 7), SLE (patient 4), Sjögren syndrome (patient 13, who showed the typical SSA pattern on HEp-2000), and polymyositis (patient 5). Others had symptoms suggestive of connective tissue disease (patients 6, 11, and 12), although no definite diagnosis could be made.

confirmation of lia reactivities in iif-negative patients
The ID and ELISA results are given in Table 3Up . As shown in Table 3Up , reactivities toward Jo1 could be confirmed. Four of six samples showing reactivity with SSB on LIA were positive or borderline on ELISA. Two of four samples positive for Ro52 and/or Ro60 could be confirmed with ELISA or ID.

sensitivity of iif for detection of ena
Of all samples tested, 68 showed autoantibodies as detected by LIA. Forty-nine of these 68 samples tested positive by IIF on HEp-2, and 51 tested positive by IIF on HEp-2000. Thus, a sensitivity for detecting ENAs of 72% (CI, 68–76%) for IIF on HEp-2 and of 75% (CI, 70–78%) for IIF on HEp-2000 was reached.


   Discussion
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 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
IIF testing is generally accepted as a screening tool for detecting ANAs (2). Substrates may vary, but most often HEp-2 cells are used, allowing high sensitivity for detecting patients with SLE and Sjögren syndrome (5)(6). It has been suggested that HEp-2 cells transfected with Ro60 cDNA (commercially available as the HEp-2000 substrate) could be a valuable alternative because these cells are better suited to detect anti-SSA/Ro reactivity (7)(8)(9)(19)(20). We designed the present study to compare both substrates in a consecutive, well-defined cohort (all samples were referred by rheumatologists for ANA testing). In addition to IIF on HEp-2 and HEp-2000 cells, we also performed LIA on all samples. LIA is a single, multianalyte test, suited to detect multiple specific ANAs (12). The test combines a high specificity (15) with a higher sensitivity for detection of anti-ENA antibodies compared with ID, especially for anti-SSA antibodies (12)(13). Because the presence of anti-ENA antibodies is more predictive for SLE than the sole finding of ANAs (21) and because anti-ENA antibodies may be associated with connective tissue diseases other than SLE, we were interested in the sensitivity of IIF to detect ENAs. The number of samples was calculated to be large enough to determine and quantify the presence of ENAs in ANA-negative samples.

We demonstrated that the HEp-2 and HEp-2000 substrates perform equally for IIF intensity as well as IIF pattern. The HEp-2000 substrate has the advantage of allowing immediate identification of anti-SSA reactivity in many patients. However, the sensitivity (57%) of this SSA-specific pattern for detection of anti-SSA antibodies, as demonstrated by LIA, indicates that the absence of the specific staining pattern on HEp-2000 does not exclude the presence of anti-Ro60 or anti-Ro52 antibodies. As can be expected, the SSA pattern on the HEp-2000 substrate performs better for the detection of anti-Ro60 antibodies then for detection of anti-Ro52 antibodies (9).

Of 291 samples with a negative IIF on both substrates, 12 showed specific ANAs on LIA. Three of the 12 patients with a positive LIA result had classic connective tissue disease, which is known to be associated with the presence of ANAs. Two patients with negative IIF on HEp-2 and HEp-2000 showed reactivities toward Ro52 and Ro60. ID and ELISA confirmed one of the samples. However, it is interesting to note that two other SSA/Ro reactivities gave a negative IIF result on HEp-2, whereas IIF on HEp-2000 yielded a positive result, with one patient showing the typical SSA pattern. This reactivity was confirmed by ELISA. These findings reflect the higher sensitivity (93% CI, 91–95%) for detecting anti-SSA antibodies of the HEp-2000 substrate compared with the HEp-2 substrate (86% CI, 83–89%).

The finding of anti-SSA/Ro reactivities in IIF-negative samples is not unexpected. Not only are anti-SSA/Ro reactivities seen in samples with low fluorescence intensities on IIF (13), but it has also been suggested that anti-SSA/Ro antibodies should also be analyzed in samples with negative IIF on HEp-2 (16)(22). However, when extrapolating data about anti-SSA antibodies in samples with low fluorescence intensity on HEp-2 cells, we had expected a higher frequency of anti-SSA antibodies in IIF-negative patients (13). We could not confirm all anti-Ro reactivities by ID or ELISA, but it has previously been demonstrated that LIA is not only a sensitive but also a specific assay for detection of anti-SSA/Ro reactivity (12)(13). More specifically, for Ro52, it has been confirmed that the reactivity seen on LIA can mostly be confirmed by both natural and recombinant Ro52 (baculovirus expressed), used in other assays (ELISA and blot) (23). SSB reactivity was seen in five samples. Three of these gave borderline results when tested on ELISA, and none was positive on ID. It is known, however, that LIA is also more sensitive for detection of anti-SSB antibodies than ID (12). Anti-Jo1 reactivity was seen in one patient who had negative nuclear fluorescence by IIF on both substrates; the anti-Jo1 reactivity was confirmed by ELISA. The clinical diagnosis in this patient was polymyositis. Because anti-Jo1 antibodies have a cytoplasmic staining pattern on IIF, these reactivities frequently go undetected or unreported (5).

If we consider LIA as the primary assay for detection of ENA antibodies, IIF reached a sensitivity of only 72% when HEp-2 cells were used as substrate and 74% when HEp-2000 cells were used as substrate. Compared with LIA, the sensitivity of IIF was surprisingly low, especially because some patients who would have gone undetected by IIF alone did have relevant clinical diagnoses. Thus, we argue that when clinical suspicion for rheumatic connective tissue disease is present, testing for ENAs should be performed; it should also be performed when IIF is negative. Otherwise, there is a risk of missing anti-SSA/Ro, anti-SSB, and anti-Jo1 antibodies, which have an established clinical relevance.


   Acknowledgments
 
Ilse Hoffman is supported by a research grant from the "Bijzonder Onderzoeksfonds", Ghent University. We thank M. Vandenbossche for excellent technical assistance; we also thank Lydie Meheus (Innogenetics, N.V., Zwijnaarde, Belgium) for critical reading of the manuscript.


   Footnotes
 
1 Nonstandard abbreviations: ANA, antinuclear antibody; IIF, indirect immunofluorescence; ENA, extractable nuclear antigen; SLE, systemic lupus erythematosus; ID, immunodiffusion; LIA, line immunoassay; and CI, confidence interval.


   References
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 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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