Context.—

Antibodies to U1 ribonucleoprotein (U1RNP) were first described more than 50 years ago, and although clinically relevant for antinuclear antibody–associated connective tissue disease (ANA-CTD), test results are challenging to interpret.

Objective.—

To evaluate the impact of anti-U1RNP analyte diversity in the assessment of patients at risk for ANA-CTD.

Design.—

Two multiplex assays for U1RNP (Smith [Sm]/RNP and RNP68/A) were used to test serum specimens from consecutive patients (n = 498) under evaluation for CTD in a single academic center. Discrepant specimens were further tested for Sm/RNP antibody by enzyme-linked immunosorbent assay and the BioPlex multiplex assay. Data were evaluated for antibody positivity per analyte and their method of detection, correlations between analytes, and impact on clinical diagnoses through retrospective chart review.

Results.—

Of the 498 patients tested, 47 (9.4%) were positive in the RNP68/A (BioPlex) and 15 (3.0%) were positive in the Sm/RNP (Theradiag) immunoassays. U1RNP-CTD, other ANA-CTD, and no ANA-CTD were diagnosed in 34% (16 of 47), 12.8% (6 of 47), and 53.2% (25 of 47) of the cases, respectively. The prevalence of antibody by method in patients with U1RNP-CTD was 100.0% (16 of 16), 85.7% (12 of 14), 81.5% (13 of 16), and 87.5% (14 of 16) for RNP68/A, Sm/RNP BioPlex, Sm/RNP Theradiag, and Sm/RNP Inova, respectively. For other ANA-CTD and no ANA-CTD, the highest prevalence was observed with RNP68/A; all others had comparable performance.

Conclusions.—

In this study, the overall performance characteristics of Sm/RNP antibody assays were comparable; however, the RNP68/A immunoassay was very sensitive but less specific. In the absence of harmonization, reporting the type of U1RNP analyte in clinical testing may be useful in guiding interpretation and interassay correlations.

The antibody to U1 ribonucleoprotein (U1RNP) is an important serologic biomarker for the diagnosis of mixed connective tissue disease (MCTD).1  It has also been described in other antinuclear antibody (ANA)-associated connective tissue diseases (CTDs) such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc), undifferentiated CTD (UCTD), overlap myositis, and overlap syndromes.29  Immunologic characterization studies suggest that anti-U1RNP antibodies are directed toward both discontinuous and linear epitopes that are either contained in the protein sequence or are post-translationally modified with reactivities to RNP68 or RNP70, RNPA (33 kD), and occasionally RNPC (22 kD) proteins that are associated with U1RNP.46,1012 

Originally described by Mattioli et al3  by immunodiffusion assay using calf thymus extract, current solid-phase immunoassays now use diverse analytes (purified or recombinant proteins, synthetic peptides of dominant epitopes) of the 3 main proteins (RNP68 or RNP70, RNPA, and RNPC) either singly or in any various combinations.46,1013  Because of the use of these different antigens and/or combinations thereof, the nomenclature, reporting, and interpretation of anti-U1RNP antibodies remain obscure.

This investigation was undertaken to determine the performance of 2 multiplex immunoassays for detecting antibodies to RNP68/A and Sm/RNP in patients at risk for ANA-CTD and to evaluate the need to establish guidance for laboratory reporting and interpretation.

Study Cohort

Consecutive patient serum specimens (n = 498) received at ARUP Laboratories from the University of Utah Health, Salt Lake City, for CTD testing were investigated. Specimens were tested for ANA by indirect immunofluorescence assay (IFA) using HEp-2 substrate (Inova Diagnostics, San Diego, California) or ANA by enzyme-linked immunosorbent assay (ELISA) (Bio-Rad) with positivity confirmed by HEp-2 IFA. Nuclear patterns (centromere, homogeneous, nuclear dots, nucleolar, and speckled) with titers greater than or equal to 1:80 were reported as positive, and the presence of any cytoplasmic pattern was noted as observed, with endpoint titers reported. Smith/RNP (Sm/RNP), Sm, Ro52, Ro60, ribosomal p (ribo p), topoisomerase I (topo 1 or Scl-70), Jo-1, and centromere B antibodies were determined with a multiplex bead assay (Theradiag, Marne La Vallée, France) in ARUP Laboratories, Salt Lake City, Utah. The manufacturer’s recommended reference intervals for all analytes for the Theradiag assay were as follows: negative: less than or equal to 29 arbitrary units (AU)/mL; equivocal: 30 to 40 AU/mL; and positive: greater than or equal to 41 AU/mL. Based on the product insert, the Sm/RNP is a native purified antigen. For this study, antibody levels greater than 30 AU/mL were considered positive. Following testing, all specimens were de-identified and stored frozen at −20°C until investigated by other methods. Charts of the anti-U1RNP antibody–positive patients (see Results) were reviewed for physicians’ clinical diagnoses, systemic clinical manifestations, and extent of skin and/or muscle involvement by a rheumatologist (D.L.-O.). The study protocol was approved by the institutional review board (IRB no. 00029507) of the University of Utah, Salt Lake City.

Assessment of Anti-U1RNP Antibody by Different Types of Analytes and Methods

All 498 patients previously tested for Sm/RNP at ARUP Laboratories were tested for RNP68/A (recombinant human antigens for RNP68 or RNPA, product insert), Sm, SS-B, ribosomal p, topo 1, centromere B, and Jo-1 antibodies with the BioPlex multiplex bead assay (Bio-Rad) in the Antibody Immunology Laboratory at Mayo Clinic Laboratories, Rochester, Minnesota. The manufacturer’s recommended reference interval for the BioPlex assay for all analytes is less than 1.0 U (negative) and greater than or equal to 1.0 U (positive). Available sera positive in any of the multiplex assays were evaluated for Sm/RNP (native purified antigens, product inserts) antibodies by ELISA (Inova, n = 47) at ARUP Laboratories and multiplex immunoassay (BioPlex, n = 42) at Mayo Clinic Laboratories. In addition, all specimens were tested for Ro52 and Ro60 antibodies in a chemiluminescent immunoassay (Inova). The reference range for the ELISA is less than 20 U (negative) and greater than or equal to 20 U (positive), and the reference range for the Ro52/Ro60 chemiluminescent immunoassay is less than 20 U (negative) and greater than or equal to 20 U (positive).

Of the 498 patients, 47 (9.4%) were positive in the RNP68/A (BioPlex) and 15 (3.0%) in the Sm/RNP (Theradiag) immunoassays. All Sm/RNP-positive patients in the Theradiag assay were also positive in the RNP68/A assay. The frequency of Sm/RNP in the BioPlex and Inova ELISA was 14 of 42 (33.3%) and 17 of 47 (36.2%), respectively.

Results for the 47 RNP68/A antibody–positive patients were stratified by analyte, test method, and agreement with clinical diagnoses based on ANA-CTD or no identifiable ANA-CTD (no ANA-CTD). ANA-CTD was diagnosed in 22 cases (Table 1); the remaining 25 subjects had no ANA-CTD (Table 2). Of the 22 patients with ANA-CTD, 16 had primary or secondary diagnoses (UCTD, MCTD, SLE, SSc, overlap myositis, or overlap syndromes) typically associated with the presence of anti-U1RNP antibody, referred to in this investigation as U1RNP-CTD. MCTD or UCTD alone or with another CTD was diagnosed in 8 of 15, limited SSc in 2 of 15, and SLE alone or with Sjogren syndrome in 5 of 15 patients. Of the remaining 6 cases (other ANA-CTD, Table 1), all were positive for RNP68/A alone, with a mean antibody level of 2.1 U (range, 1.4–3.6 U). Except for 2 patients with polymyositis and dermatomyositis, all patients in the other ANA-CTD group (n = 6) were positive for Ro52 (Table 1). Based on the cohort of positive patients (n = 47), the prevalence of antibody by method in patients with U1RNP-CTD was 100% (16 of 16: RNP68/A), 85.7% (12 of 14: Sm/RNP BioPlex), 81.3% (13 of 16: Sm/RNP Theradiag), and 87.5% (14 of 16: Sm/RNP Inova). For the no ANA-CTD group, the highest prevalence was observed with the RNP68/A (100%); all others had comparable performance (4%–8%, Table 2). Overall, the positive agreement between any anti-Sm/RNP antibody assay and the BioPlex anti-RNP68/A antibody test was 100%. The positive agreements between any 2 anti-Sm/RNP antibody assays ranged from 86.7% to 100% with excellent negative agreements (93.8% to 96.4%), data not shown. The negative agreement between the anti-RNP68/A and anti-Sm/RNP antibody assays for Inova or BioPlex anti-Sm/RNP was poor (0% for both) when compared to the Theradiag Sm/RNP assay (92.4%), possibly due to selection bias and/or sample size.

In addition to the presence of U1RNP antibody by antigen, the limited data demonstrated analyte- and kit-specific semiquantitative correlations for U1RNP-CTD versus other ANA-CTD or no ANA-CTD groups (Figure, A and B). For the BioPlex RNP68/A antibody assay, 68.8% (11 of 16) of patients in the U1RNP-CTD group had RNP68/A antibody results greater than 5.0 U, compared to 0% (0 of 6) and 16% (4 of 25) in the other ANA-CTD or no ANA-CTD group, respectively (data not shown). In addition, the borderline RNP68/A-positive results were associated with negative or lower ANA (nuclear pattern) titers using HEp-2 substrate in this group compared to those in any ANA-CTD. Of the 25 patients with no ANA-CTD, 40% (n = 10) were negative for the nuclear pattern, 48% (n = 12) had nuclear patterns ranging from 1:80 to 1:160, and the remaining 12% (n = 3) had nuclear patterns and titers above 1:320. In contrast, all U1RNP-CTD patients with positive Sm/RNP antibody in any of the assays and available results for HEp-2 IFA had a nuclear pattern and titer greater than or equal to 1:320.

In this investigation, we evaluated 498 patients undergoing testing for U1RNP antibodies in a single academic center and observed analyte-dependent correlations with specific CTD diagnoses. Overall, there were significant correlations between methods to detect anti-Sm/RNP antibody with differential associations between patients positive for RNP68/A and Sm/RNP antibodies. When compared to patients who tested positive for RNP68/A, the majority of the subjects with a positive autoantibody response to Sm/RNP had a diagnosis of CTD typically associated with this biomarker,1,4,5,69  irrespective of the type of solid-phase immunoassay. In addition, the Sm/RNP and RNP68/A analytes that were tested on the same assay (BioPlex) did not appear to have equivalent performance characteristics in the subset of patients evaluated. These data suggest unique attributes of U1RNP analytes with implications for reporting results for clinical use and interassay correlations. It is likely these differences may be related to how epitopes are expressed in the assays, the characteristics of test system calibration, and/or patient populations used in the establishment of the RNP68/A and Sm/RNP antibody assays on the BioPlex platform.2,4,6,8,1013 

Compared to Sm/RNP immunoassays, the BioPlex RNP68/A assay had a significant number of patients with no diagnosis of ANA-CTD. Among these patients, 3 were identified who tested positive for RNP68/A antibody and in at least 1 of the Sm/RNP tests. One patient had seronegative rheumatoid arthritis and the other 2 had no obvious diagnosis at the time of evaluation. The reasons for these observations are unclear but could be related to coexisting antibodies in these patients.79  The rest of the patients in the no ANA-CTD group negative for Sm/RNP antibodies but borderline positive for RNP68/A antibody were likely to be falsely seropositive. The increased prevalence of antibodies to RNP68/A in the no ANA-CTD group may be attributable to the analytical principle of the BioPlex immunoassay, cross-reactivity to other antigens, or the use of recombinant antigens utilized in this platform. Based on information from the product inserts, the Sm/RNP assays utilized native purified antigens, while results for the BioPlex RNP68/A are based on recombinant human antigens for RNP68 or RNPA. It would be of analytical and diagnostic significance to determine optimal conditions for testing U1RNP antibodies.

Using manufacturers’ recommended cut-off values for interpretating semiquantitative results, all Sm/RNP immunoassays demonstrated elevated antibodies in patients with U1RNP-CTD compared to the 2 other groups. A majority of the U1RNP-CTD patients positive for RNP68/A had elevated antibody levels above 5.0 U; however, borderline antibody-positive and lower ANA titers (12 of 25 [48%] of cases had nuclear patterns ranging from 1:80 to 1:160) occurred frequently in patients with no ANA-CTD. This observation may be significant for interpreting and reporting laboratory results. For example, the presence of isolated U1RNP antibody and “high” ANA titer with a speckled pattern has traditionally been associated with a diagnosis of MCTD.14  While this correlation may not be well described for other U1RNP-CTDs such as SSc and myositis, the value of titer-specific information for HEp-2 substrate IFA patterns and ANA-specific autoantibodies as a predictive tool has recently been proposed for use in test interpretation by laboratories.12,15 

This investigation is not without limitations, as the performance characteristics of the RNP68/A antigen were evaluated using a single immunoassay. It is very likely that not all immunoassays utilizing nonpurified extract to detect U1RNP antibodies have similar performance characteristics. Additional studies are underway to evaluate different antigens or combinations of antigens for U1RNP using a variety of immunoassays currently utilized in clinical laboratories. Secondly, the data interpretation may be limited by the number of patients with CTD in the cohort. Despite the relatively low number of cases with CTD in this cohort, the unbiased selection of participants is a strength of this investigation. In addition, the clustering of Sm/RNP antibody–positive patients and the demonstration that patients positive in at least 2 or more methods, irrespective of the analyte, typically have the same clinical outcome was reassuring. Lastly, in the patients with U1RNP-CTD, the correlation between U1RNP antibody positivity and HEp-2 IFA pattern and titers was consistent with published literature including recommendations for interpretation.12,14,15  Nevertheless, given the clinical challenges associated with the presence of U1RNP antibodies, efforts to determine optimal conditions for their detection in the clinical laboratory are needed.

In conclusion, this investigation highlights the need for appropriate nomenclature of analytes in the reporting and interpretation of anti-U1RNP antibodies by clinical laboratories and may also have implications for interlaboratory correlations as well as diagnostic outcomes. In the absence of harmonization, U1RNP antibody results should be interpreted in the context of the analyte, results for ANA IFA using HEp-2 substrate, and the relative antibody concentration for the immunoassay used.

The authors are grateful to Troy Jaskowski, BS, Diana Knapp, BS, and Carri Craig, BS (Autoantibody Immunology Laboratory, ARUP Laboratories, Salt Lake City, Utah) and Shelly Sales, BS (Antibody Immunology Laboratory, Mayo Clinic, Rochester, Minnesota), for technical support.

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Author notes

Snyder is a member of the Strategic Advisory Committee for Werfen Diagnostics. The other authors have no relevant financial interest in the products or companies described in this article.