African-American participants irrespective of disease status have a higher frequency and number of autoantibodies
The overall prevalence of autoantibodies detected by the BioPlex 2200 varied depending upon ethnicity (). Of the 786 AAs, 356 patients (88.8%), 81 unaffected relatives (32.9%), and 29 healthy, population-based controls (20.9%) were positive for at least one tested autoantibody. The mean number (± SD) of autoantibodies present within this group was 4.33 (± 2.30) for patients, 2.38 (± 2.09) for unaffected relatives, and 1.31 (± 0.71) for healthy, population-based controls. Of the 507 HIs, 163 patients (73.1%), 34 unaffected relatives (23.4%), and 19 healthy, population-based controls (13.7%) had at least one positive autoantibody specificity. The mean number (± SD) of autoantibodies within this group was 3.68 (± 2.21), 1.74 (± 1.08), and 1.21 (± 0.71), respectively. Of the 1,872 EAs, 483 SLE patients (67.4%), 177 unaffected relatives (26.5%), and 46 healthy, population-based controls (9.5%) were found to be positive for at least 1 of the 10 autoantibody specificities associated with SLE. The mean number (± SD) of autoantibodies present within positive samples was 2.79 (± 1.79) for patients, 1.54 (± 0.99) for unaffected relatives, and 1.41 (± 1.07) for healthy, population-based controls. For the SLE patient groups, 53.1% of AAs, 34.3% of HIs, and 18.7% of EAs had 4 or more tested autoantibody specificities (). Using this commercially available standard antibody testing platform only 88.8% (AA), 73.1% (HI) and 67.4% (EA) of SLE established cases were positive for autoantibodies. Therefore, approximately 11–33% of these established SLE cases would be ANA-negative by this detection method.
Figure 1 Number of autoantibody specificities for three ethnicities. The number of autoantibody specificities as determined by the bead-based assay ANA screening kit is shown. 53.1% of African-American, 34.3% of Hispanic, and 18.7% of European-American lupus patients (more ...)
Select autoantibody specificities are more prevalent in African-Americans compared with European-Americans and Hispanics
Analysis of individual autoantibody specificities revealed significant differences among ethnic groups (). Compared to EA SLE patients, AA patients displayed a significantly higher prevalence of autoantibodies against dsDNA, chromatin, ribosomal P, 60kD Ro, Sm, Sm/RNP, RNP A, and RNP 68 (χ2 =13.3–129.5, p ≤ 0.001 for all comparisons) (). HI patients also had significantly higher autoantibody prevalence than EA patients for anti-dsDNA, anti-ribosomal P, anti-Sm/RNP, and anti-RNP 68 autoantibody specificities (χ2 =8.7–11.3, p ≤ 0.004 for all comparisons). Compared to HI patients, AA patients had higher autoantibody prevalence for chromatin, Sm, Sm/RNP, RNP A, and RNP 68 specificities (χ2 =11.6–41.5, p ≤ 0.001 for all comparisons). Autoantibody prevalence in unaffected relatives displayed no effect due to self-reported ethnicity (). Interestingly, the prevalence of anti-52kD Ro was significantly higher in AA population-based controls than in EA controls ().
Figure 2 African-American SLE patient sera are autoantibody rich. Antibody prevalence is shown for patients (A), unaffected relatives (B), and healthy, population-based controls (C). Black stars identify autoantibody specificities with significant differences (more ...)
Anti-chromatin responses are the most common autospecificity detected in SLE patients and unaffected, unrelated blood relatives
Using the BioPlex 2200 system, 76% of SLE patients, 28% of SLE unaffected relatives, and 12% of controls were found to have at least one lupus autoantibody specificity. As expected, prevalence of all 10 autoantibodies was significantly higher in SLE patients compared to controls (χ2 =80.9–638.1, p<0.001 for all comparisons). When comparing the SLE patients to unaffected relatives, all specificities except anti-La were significantly more common in the SLE patient subgroup (p<0.005). The mean (± SD) number of detectable autoantibody specificities was 3.58 (± 2.18) for patients, 1.78 (± 1.41) for unaffected relatives, and 1.29 (± 0.83) for healthy, population-based controls.
Prevalence of individual autoantibodies varied among SLE patients; anti-chromatin (55.8%) was the most prevalent while anti-ribosomal P (12.4%) was the least. Unaffected relatives also had varied autoantibody prevalence. Chromatin (12.5%) was also the most prevalent specificity in the SLE-unaffected relative group. Compared to unrelated controls, SLE-unaffected relatives had significantly higher prevalence for dsDNA, chromatin, 60kD Ro, 52kD Ro, Sm, and RNP A autoantibodies (p< 0.005 for all comparisons). Anti-Scl-70 prevalence was 2.2% in SLE patients and 1.2% in both SLE-unaffected relatives and population-based controls (no significant difference). Anti-centromere B responses were detected in 3.7% of SLE patients and less than 1% of both unaffected relatives and controls (p< 0.001), with females more than 4 times more likely to be positive (p< 0.001). Jo-1 antibodies were present in less than 0.5% of all samples. Inclusion of prevalence for Scl-70, centromere B, and Jo-1 did not significantly affect the overall ANA prevalence rates and therefore were not included in subsequent analyses.
Association of the BioPlex 2200 and indirect immunofluorescence results varies across racial groups
Indirect immunofluorescence (IIF) is the historical standard for broad-scale ANA screening. Comparisons of results from the IIF assay to the BioPlex ANA assay indicate differences in the sensitivity and specificity of detection. We found that 88.8% of SLE patients, 34.7% of unaffected relatives, and 18.3% of healthy, population-based controls were ANA positive by IIF at a serum titer of ≥ 1:120, whereas 76.4%, 27.9%, and 12.4%, respectively, were positive by BioPlex 2200. When considering all 13 measured autoantibodies, 78.2% of SLE patients, 28.9% of unaffected relatives, and 13.8% of unrelated controls were ANA positive using the BioPlex 2200 ANA assay, extremely similar to the rates detected when analyzing only the 10 lupus-associated autoantibodies. Differences were most striking in EA patients (). IIF resulted in higher autoantibody prevalence among SLE patients, SLE-unaffected relatives and controls in all ethnic groups, suggesting that the IIF assay is able to detect a more diverse repertoire of autoantibodies than those included within the BioPlex 2200 ANA kit. Interestingly, similar prevalence rates among AA controls were found when comparing the BioPlex assay and IIF.
Assay comparison of overall autoantibody prevalence, positive predictive value, and negative predictive value among ethnic groups
Further comparative analysis between diagnostic efficacy of the BioPlex assay and IIF ANA screening was performed using positive predictive value (PPV) and negative predictive value (NPV) for each assay. PPV and NPV analysis was performed within the combined group of SLE patients and healthy, population-based controls as a whole, as well as within ethnic subgroups (). Overall, the PPV for the IIF assay (89.2%) was similar to that of the BioPlex assay (91.3%), but the NPV proved to be better within the IIF assay (81.1% versus 68.6% for BioPlex). Within the individual ethnic subgroups, AAs had the highest PPV and NPV in both assays.
With established clinical associations and therapeutic implications placed on anti-dsDNA in SLE, we specifically examined the consistency of autoantibody detection between the BioPlex 2200 assay and the current standard IIF detection method. Prevalence of dsDNA detected by IIF was 24.3% of SLE patients, 0.4% of unaffected relatives, and 0.1% of unaffected unrelated controls. Examination of dsDNA based on ethnicity revealed that this specificity was greatest in AA SLE patients (28.2%) and had similar prevalence in both EA (21.5%) and HI (21.1%) SLE patients. However, dsDNA autoantibody specificity had higher prevalence in all three ethnicities using the BioPlex 2200 assay. AAs display the largest difference between IIF and BioPlex 2200 dsDNA results with 28.2% and 35.7% positive respectively. In HIs, 35.0% tested positive for anti-dsDNA antibodies using the BioPlex 2200 assay and 21.1% were positive using IIF, while EAs displayed 23.4% and 21.5% anti-dsDNA autoantibody prevalence respectively.
Autoantibodies detected in SLE patients, blood relatives, and healthy controls vary between traditional and Bio-Rad BioPlex detection methods
When considering all 13 Bio-Rad BioPlex 2200 ANA analytes, 29.7% of EAs, 10.5% of AAs and 25.1% of HI SLE patients were negative. Additionally, we have examined family members and control individuals for false negative results. About 30% from AA, EA, and HI blood relatives and about 20% from each control ethnic groups are positive by IIF but negative by BioPlex 2200 for dsDNA antibodies. Of the remaining autoantibody specificities, only the blood relative individuals were false negative for Ro, La, or nRNP autoantibodies. Here, less than 1% of AA (Ro, La, nRNP) and EA (Ro and nRNP) was false negative. We next examined the presence of false positive results. In SLE individuals, 2.11% AA, 6.3% EA, and 7.6% of HI patients were positive by BioPlex 2200 but negative by IIF. In blood relatives, 11% from each ethnicity received false positive results compared to 11.4% AA, 13.2% EA, and 13% HI of control individuals. To explore the possibility of other potential antigenic targets among the BioPlex 2200 ANA negative samples, we examined the prevalence of aCL antibodies. aCL antibodies were lower in BioPlex 2200 ANA negative samples (10.4%) compared to the BioPlex 2200 ANA positive samples (15.6%, p=0.02).
Sensitivity is similar between bead-based assays and immunodiffusion methods
To reference our findings using the BioPlex 2200 to traditional standards for detection of SLE antibody specificities, we compared the BioPlex 2200 results with the corresponding immunodiffusion results (). Within SLE patients, the sensitivity of the BioPlex 2200 results for the Ro, La, ribosomal P, Sm, and nRNP complex (nRNP A and nRNP 70k) tests were 0.92, 0.92, 0.83, 0.89, and 0.92 respectively. The BioPlex 2200 anti-dsDNA detection had a sensitivity of 0.71 and a specificity of 0.80 when compared to IIF. The BioPlex 2200 exhibited sensitivity of 0.70 and 0.80 and specificities of 0.98 and 0.998 for centromere B and Jo-1 detection, respectively.
SLE patient sera from simplex pedigrees are enriched for dsDNA and chromatin autoantibodies
To evaluate the effect of familial relation on autoantibody prevalence, SLE patients were categorized as simplex (having no known familial relation to other SLE patients) or multiplex (having at least one blood-related family member with SLE). Of the 1,540 SLE patients, 53.8% were classified as multiplex, 26.5% were classified as simplex, and 19.7% were unknown. Interestingly, significantly higher prevalence of anti-dsDNA was found among simplex patients (35.0%) than among multiplex patients (25.1%) (χ2 =14.37, p<0.0002). AA and HI patients are significantly more likely than EA patients to be anti-dsDNA positive, so multiple logistic regression was performed to determine if a simplex SLE patient is more likely to be anti-dsDNA positive, regardless of ethnicity. Using dsDNA positivity as the outcome and both ethnicity (EA, AA, HI, and other) and status (simplex or multiplex) as predictors, we found that simplex patients have a significantly greater dsDNA prevalence than multiplex patients, even after adjusting for ethnicity (OR=1.59, p = 0.0003). Significantly higher prevalence of chromatin autoantibodies are also found among simplex patients (59.1%) compared to multiplex patients when adjusted for ethnicity (50.8%) (χ2 =8.33, p = 0.005). No other differences were noted comparing prevalence of other autoantibody specificities between simplex and multiplex SLE patients.
Ribosomal P, Sm/RNP, and 60kD Ro autoantibody specificities show familial aggregation in SLE patient sibling pairs
Familial aggregation analysis was performed as a measurement of genetic influence on production of certain autoantibody specificities. Autoantibody specificities with evidence of familial aggregation within SLE affected sibling pairs included ribosomal P (OR=5.34, p=0.002), Sm/RNP (OR=2.90, p=0.002) and 60kD Ro (OR=2.56, p=0.004) (). Within sibling pairs consisting of an SLE affected patient and an unaffected sibling, no significant aggregation for individual autoantibody specificity was detected. In examining the relationship between ANA positivity and family aggregation results from the BioPlex 2200 indicated familial aggregation in SLE affected sibling pairs (OR=2.51, p=0.01) as well as in the SLE patients and unaffected sibling pairs (OR=0.38, p<0.001). Immunofluorescent ANA assays showed aggregation of positivity between SLE patients and unaffected sibling pairs (OR=0.49, p=0.004).
Familial aggregation of autoantibodies in sibling pairs
Autoantibody clustering identifies unique racial differences in autoantibody subsets
Hierarchical cluster analysis of antibody profiles detected within all SLE patient samples showed three distinct subgroups of autoantibodies: 60kD Ro, 52kD Ro, and La; Sm, Sm/RNP, nRNP A, and nRNP 68, and chromatin; and dsDNA and ribosomal P (). Upon performing the same analysis with SLE patients sub-grouped on the basis of ethnicity, the three clusters described above appear for the AA patients. However, in EA and HI patients, antibodies against chromatin tended to cluster with dsDNA and ribosomal P. All results accounted for approximately 90% of the variability for each set of patients.
Figure 3 Chromatin autoantibodies cluster with spliceosomal autoantibodies in African-American SLE patients. Autoantibody clustering was performed on the total SLE patient group (A) n=1540, European-American SLE patients (B) n=717, African-American SLE patients (more ...)