Anti-RNA Polymerase III Antibodies in Patients With Systemic Sclerosis
Anti-RNA Polymerase III Antibodies in Patients With Systemic Sclerosis
Objectives: To evaluate the analytical performance of an ELISA for the detection of anti-RNA polymerase III antibody (ARA) and to assess IIF as a method for identifying this antibody.
Methods: A commercially available ELISA was used to assess the presence of ARA in sera from 1018 SSc patients. The sera had been divided into sub-populations based on the presence of specific autoantibodies, ANA pattern or the absence of both. Patients with ARA (n = 209) had been identified by characteristic ANA pattern by IIF on HEp-2 cell substrate [and additionally by radio-immunoprecipitation (IP) in 157/209 cases]. The remaining 809 SSc patients acted as a control group.
Results: Of 157 patients in whom ARA had been confirmed by IP, 150 were positive by ELISA providing a sensitivity of 96%. In the group where ARA had only been assessed by IIF, 100% (52/52) were ELISA positive. The ANA patterns indicating the presence of ARA were a fine-speckled nucleoplasmic stain with additional occasional bright dots, with or without concurrent punctate nucleolar staining. In the SSc control group, the ELISA attained a specificity of 98%, ARA being detected in 17/809 patients.
Conclusions: We report the outcome of a study on a large population of SSc patients that shows the ARA ELISA to be of high analytical sensitivity and specificity. We confirm that there is minimal overlap between ARA and other SSc-specific autoantibodies. Additionally, it is demonstrated that the presence of ARA correlates with identifiable patterns by IIF on HEp-2 cell substrate.
Systemic sclerosis (SSc) is a heterogeneous autoimmune rheumatic disease characterized by fibrosis of the skin and other organs. SSc patients have autoantibodies to specific cellular components that are mutually exclusive and correlate with distinct clinical subsets of disease. The best documented of these, and the only ones routinely tested outside specialist centres, are ACA and anti-topoisomerase (anti-Scl-70) (ATA). However, these two specificities are only positive in ~50% of patients with SSc. A third major subset of SSc patients with distinct clinical features has antibodies to RNA polymerases (RNAPs). The prevalence of this antibody is influenced by ethnic origin and varies widely from 4% to 25%. It has been suggested that at least 12% of UK patients are anti-RNAP antibody (ARA) positive. These autoantibodies are highly specific and are predominantly present in patients with dcSSc. They are strongly associated with hypertensive renal crisis, a serious complication that may be the presenting feature of disease. Additionally, a number of reports indicate that the antibody can be demonstrated before the onset of skin involvement. It would therefore be desirable to be able to detect ARA in the routine clinical laboratory.
Until recently the only specific method for the identification of ARA was radio-immunoprecipitation (IP), a technique not well suited to routine use. Using this method the three subtypes of RNAP (I, II and III) can be precipitated by antibodies and there are three recognized patterns of reactivity specific for SSc: most ARA-positive SSc sera precipitate RNAP I and III in combination, some recognize all three polymerases and a small minority precipitate RNAP III alone. Some patients with SSc have antibodies that precipitate only RNAP II, but these do not exhibit disease specificity. The recent identification of a major antigenic epitope on RNAP III recognized by almost all sera positive for ARA by IP has led to the development of an ELISA for ARA. Unlike IP, ELISA is used in the majority of diagnostic laboratories making routine detection of ARA feasible.
The technique of IIF is also widely employed for the detection of autoantibodies. Several studies have noted the association of ARA with positive staining by IIF on HEp-2 cell substrate. Interest in SSc-specific ARA by IIF originally focused on a punctate (speckled) nucleolar pattern consistent with the cellular location of RNAP I. However, speckled nucleoplasmic staining patterns were subsequently described, this time consistent with the cellular location of RNAP III. As a consequence, there remains no consensus on the specific pattern produced by SSc-specific ARA on HEp-2 cell substrate.
In this study, we have assessed the utility of two techniques—IIF on HEp-2 cell substrate and a commercially available ELISA—to identify patients with ARA. We further evaluated the specificity of the ELISA method against a large control group of SSc patients.
Objectives: To evaluate the analytical performance of an ELISA for the detection of anti-RNA polymerase III antibody (ARA) and to assess IIF as a method for identifying this antibody.
Methods: A commercially available ELISA was used to assess the presence of ARA in sera from 1018 SSc patients. The sera had been divided into sub-populations based on the presence of specific autoantibodies, ANA pattern or the absence of both. Patients with ARA (n = 209) had been identified by characteristic ANA pattern by IIF on HEp-2 cell substrate [and additionally by radio-immunoprecipitation (IP) in 157/209 cases]. The remaining 809 SSc patients acted as a control group.
Results: Of 157 patients in whom ARA had been confirmed by IP, 150 were positive by ELISA providing a sensitivity of 96%. In the group where ARA had only been assessed by IIF, 100% (52/52) were ELISA positive. The ANA patterns indicating the presence of ARA were a fine-speckled nucleoplasmic stain with additional occasional bright dots, with or without concurrent punctate nucleolar staining. In the SSc control group, the ELISA attained a specificity of 98%, ARA being detected in 17/809 patients.
Conclusions: We report the outcome of a study on a large population of SSc patients that shows the ARA ELISA to be of high analytical sensitivity and specificity. We confirm that there is minimal overlap between ARA and other SSc-specific autoantibodies. Additionally, it is demonstrated that the presence of ARA correlates with identifiable patterns by IIF on HEp-2 cell substrate.
Systemic sclerosis (SSc) is a heterogeneous autoimmune rheumatic disease characterized by fibrosis of the skin and other organs. SSc patients have autoantibodies to specific cellular components that are mutually exclusive and correlate with distinct clinical subsets of disease. The best documented of these, and the only ones routinely tested outside specialist centres, are ACA and anti-topoisomerase (anti-Scl-70) (ATA). However, these two specificities are only positive in ~50% of patients with SSc. A third major subset of SSc patients with distinct clinical features has antibodies to RNA polymerases (RNAPs). The prevalence of this antibody is influenced by ethnic origin and varies widely from 4% to 25%. It has been suggested that at least 12% of UK patients are anti-RNAP antibody (ARA) positive. These autoantibodies are highly specific and are predominantly present in patients with dcSSc. They are strongly associated with hypertensive renal crisis, a serious complication that may be the presenting feature of disease. Additionally, a number of reports indicate that the antibody can be demonstrated before the onset of skin involvement. It would therefore be desirable to be able to detect ARA in the routine clinical laboratory.
Until recently the only specific method for the identification of ARA was radio-immunoprecipitation (IP), a technique not well suited to routine use. Using this method the three subtypes of RNAP (I, II and III) can be precipitated by antibodies and there are three recognized patterns of reactivity specific for SSc: most ARA-positive SSc sera precipitate RNAP I and III in combination, some recognize all three polymerases and a small minority precipitate RNAP III alone. Some patients with SSc have antibodies that precipitate only RNAP II, but these do not exhibit disease specificity. The recent identification of a major antigenic epitope on RNAP III recognized by almost all sera positive for ARA by IP has led to the development of an ELISA for ARA. Unlike IP, ELISA is used in the majority of diagnostic laboratories making routine detection of ARA feasible.
The technique of IIF is also widely employed for the detection of autoantibodies. Several studies have noted the association of ARA with positive staining by IIF on HEp-2 cell substrate. Interest in SSc-specific ARA by IIF originally focused on a punctate (speckled) nucleolar pattern consistent with the cellular location of RNAP I. However, speckled nucleoplasmic staining patterns were subsequently described, this time consistent with the cellular location of RNAP III. As a consequence, there remains no consensus on the specific pattern produced by SSc-specific ARA on HEp-2 cell substrate.
In this study, we have assessed the utility of two techniques—IIF on HEp-2 cell substrate and a commercially available ELISA—to identify patients with ARA. We further evaluated the specificity of the ELISA method against a large control group of SSc patients.