Dr. Harley has received consulting fees from IVAX Diagnostics (more than $10,000), owns stock or stock options in IVAX Diagnostics (now Erba Diagnostics), and serves on the Erba Diagnostics Board of Directors. He has licensed intellectual property to Erba Diagnostics for the isolation and purification of select autoantigens.
The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or its relevant institutes.
Replacement of standard immunofluorescence methods with bead-based assays for antinuclear antibody (ANA) testing is a new clinical option. The aim of this study was to evaluate a large, multiethnic cohort of patients with systemic lupus erytematosus (SLE), blood relatives, and unaffected control individuals for familial aggregation and subset clustering of autoantibodies by high-throughput serum screening technology and traditional methods.
Serum samples (1,540 SLE patients, 1,154 unaffected relatives, and 906 healthy, population-based controls) were analyzed for SLE autoantibodies using a bead-based assay, indirect immunofluorescence (IIF), and immunodiffusion. Autoantibody prevalence, sensitivity for disease detection, clustering of autoantibodies, and associations between newer methods and standard immunodiffusion results were evaluated.
The frequencies of ANAs in the sera from African American, Hispanic, and European American patients with SLE were 89%, 73%, and 67%, respectively, by BioPlex 2200 bead-based assay and 94%, 84%, and 86%, respectively, by IIF. When comparing the serum prevalence of 60-kd Ro, La, Sm, nuclear RNP A, and ribosomal P autoantibodies across assays, the sensitivity of detection ranged from 0.92 to 0.83 and the specificity ranged from 0.90 to 0.79. Autoantibody cluster analysis showed associations of autoantibody specificities in 3 subsets: 1) 60 kd Ro, 52-kd Ro, and La, 2) spliceosomal proteins, and 3) double-stranded DNA (dsDNA), chromatin, and ribosomal P. Familial aggregation of Sm/RNP, ribosomal P, and 60-kd Ro in SLE patient sibling pairs was observed (P ≤ 0.004). Simplex-pedigree SLE patients had a greater prevalence of dsDNA (P = 0.0003) and chromatin (P = 0.005) autoantibodies compared to patients with a multiplex SLE pedigree.
The frequencies of ANAs detected by a bead-based assay are lower than those detected by IIF in European American patients with SLE. These assays have strong positive predictive values across ethnic groups, provide useful information for clinical care, and provide unique insights into familial aggregation and autoantibody clustering.
The diverse clinical presentations of systemic lupus erytematosus (SLE) create significant difficulties for diagnosis. However, studies have shown that a common feature of SLE, the presence of autoantibodies, is associated with select clinical features (1). Previous studies have found that autoantibodies are often present in SLE patient sera years before diagnosis and prior to the onset of associated clinical symptoms (2, 3). Detection of autoantibodies is an important tool in SLE classification (4, 5), and some autoantibodies may be used to monitor the potential for disease flare (6, 7).
The prevalence of autoantibodies varies among self-reported ethnic groups. Compared with European American patients with SLE, African American patients with SLE have a higher prevalence of autoantibodies targeting Sm and nuclear RNP (nRNP) proteins (8–11). Autoantibody cluster analysis provides additional information about clinical symptom associations or genetic risk; however, studies to date have either evaluated relatively small patient cohorts (12–15) or used historical antibody data measured by a variety of detection methods (16). A few studies examining the prevalence of autoantibodies in blood relatives of SLE patients have shown that low levels of SLE-specific autoantibodies were detectable in clinically healthy relatives (17–19).
Although autoantibodies remain paramount in lupus diagnosis and management, detection of lupus specificities varies significantly between clinically available assays. To date, detailed evaluations of newer methods in large, multiethnic collections of SLE patients and control subjects are incomplete. The historical autoantibody testing methods of immunofluorescence and immunodiffusion require specially trained laboratory personnel and are becoming less available in many US markets. Based on variability in autoantibody detection across and within select methods, it has been difficult to consistently and accurately measure prevalence of autoantibodies in diverse SLE patient cohorts. Questions remain about the number of SLE patients that would be potentially missed because the newer methods may test fewer autoantibody specificities. Moreover, it remains to be determined whether the frequency of specific autoantibody detection is different across different races, and whether healthy family members of SLE patients might have higher rates of autoantibody specificities when these newer methods are used for detection.
Therefore, the primary objective of our present study was to examine the prevalence, specificity, clustering, and familial aggregation of specific autoantibodies within a large cohort of SLE patients, unaffected relatives, and unaffected controls. In addition, we sought to compare antinuclear antibody (ANA) results between a multiplex bead assay and classic detection methods (indirect immunofluorescence [IIF] and immunodiffusion) in a large, multiethnic cohort.
PATIENTS AND METHODS
Patients and controls.
All experiments were performed in accordance with the Declaration of Helsinki and approved by the Oklahoma Medical Research Foundation and the University of Oklahoma Health Sciences Center Institutional Review Boards. SLE patient and control serum samples were identified, based on serum availability, from the Lupus Family Registry and Repository and the Lupus Genetics Cohorts at the Oklahoma Medical Research Foundation. The study group included 1,540 SLE patients, 1,154 SLE-unaffected relatives, and 906 healthy, population-based controls.
The diagnosis of SLE was defined according to the American College of Rheumatology criteria for classification (i.e., the presence of at least 4 of 11 criteria) (4, 5). Upon enrollment, participants were asked to self-report their ethnicity from a list that included African American, non-Hispanic European American, Hispanic, Gullah, American Indian, Asian/Pacific Islander, other, mixed race, or not reported. Gullah individuals live in the Sea Islands and coastal plains of South Carolina and Georgia. These individuals were grouped with African Americans in this study. The distribution of ethnic groups, according to disease status and sex, is shown in Table 1.
Table 1. Demographic characteristics of the cohort based on disease status, ethnicity, and sex*
Values are the number or number (%) of participants. SLE = systemic lupus erythematosus.
African American and Gullah
Mixed race or not reported
Serologic autoantibody testing.
Autoantibody screening was performed by laboratory staff in the College of American Pathologists–certified clinical immunology laboratory at the Oklahoma Medical Research Foundation (a Clinical Laboratory Improvement Amendments–approved laboratory). Each serum sample was screened for SLE-associated autoantibodies. ANAs and anti–double-stranded DNA (anti-dsDNA) were measured using IIF (using HEp-2 cells for detection of ANAs and Crithidia luciliae for detection of anti-dsDNA; Inova Diagnostics) (2, 3, 20). A positive result was defined as detection of ANAs at a titer of ≥1:120 and anti-dsDNA antibodies at a titer of ≥1:30. The IIF assays were manually read by clinical immunology laboratory personnel, using a Nikon Optiphot fluorescence microscope (HBO bulb 100W mercury lamp, 20× objective). Precipitating levels of autoantibodies directed against Ro/SSA, La/SSB, Sm, nRNP, and ribosomal P were detected by immunodiffusion (21). Anticardiolipin (aCL) antibodies were measured by enzyme-linked immunosorbent assay, with a titer of >20 IgG or IgM phospholipid units being classified as positive (22).
BioPlex 2200 bead-based autoantibody analysis.
The BioPlex 2200 system (Bio-Rad) is a US Food and Drug Administration–approved multiplex technology for fully automated, high-throughput serologic analysis. The BioPlex 2200 ANA kit uses fluorescently dyed magnetic beads for simultaneous detection of 13 autoantibody specificity levels within a single serum sample. This method detects antibodies against dsDNA, chromatin, ribosomal P, SSA 60 (60-kd Ro), SSA 52 (52-kd Ro), SSB (La), Sm, the Sm/RNP complex, RNP A, RNP 68, Scl-70, centromere B, and Jo-1. The manufacturer lists the following sources of antigen: dsDNA synthesized by polymerase chain reaction, affinity-purified 60-kd Ro, La, Sm/RNP complex, Sm, chromatin, and ribosomal P proteins, and recombinantly produced 52-kd Ro, RNP A, RNP 68, Scl-70, centromere B, and Jo-1.
In BioPlex 2200 assays for detection of dsDNA, results are reported in IU/ml; thereby this technique serves as a semiquantitative assay, with a previously determined cutoff for anti-dsDNA positivity of 10 IU/ml. In the BioPlex 2200 system, an antibody index (AI) (range 0–8) is used, depending on the fluorescence intensity of each of the other autoantibody specificities, with a cutoff for positivity set at an AI value of 1.0, as recommended by the manufacturer. The AI scale is standardized relative to the binding values of the calibrators and control samples provided by the manufacturer. Levels of factor XIIIb were tested as a quality control, by serving both as a serum confirmation test and as an indicator of sample integrity. Levels of factor XIIIb (an enzyme involved in blood coagulation) have minimal variation between individuals. Low factor XIIIb levels indicate nonserum or nonplasma samples, inappropriate dilution of samples, or sample degradation. Serum samples were excluded if they contained errors based on low factor XIIIb levels, with low levels determined according to cutoff values defined by the manufacturer.
Two-group comparisons using chi-square statistics and McNemar's tests identified statistically significant differences in the prevalence of autoantibodies in the sera of SLE patients, SLE-unaffected family members, and healthy, population-based controls. In analyses comparing the autoantibody prevalence between patients and unaffected relatives, 1 patient and 1 unaffected relative who was matched to the patient by sex and race were used. McNemar's chi-square and exact tests, which were used when smaller subgroups were examined, were performed using SAS version 9.1.3 (SAS Institute). Data on ANA positivity were provided along with data on 10 of the 13 lupus-associated autoantibody specificities, excluding centromere B, Jo-1, and Scl-70; data for these latter 3 autoantibodies are presented separately. Independent subgroups were used for chi-square analysis when comparing differences in autoantibody prevalence based on race/ethnicity and when comparing unaffected relatives and healthy, population-based controls. The potential association between simplex and multiplex families and these 10 autoantibodies was assessed with logistic regression analyses adjusted for race/ethnicity.
In addition, we analyzed the influence of familial association with SLE by comparing SLE patients with no SLE familial occurrence (simplex pedigree) to SLE patients with at least 1 blood relative affected by SLE (multiplex pedigree). To compensate for multiple testing, a Bonferroni correction was applied using a comparison-wise significance level (α) of 0.005; thus, single-comparison statistical significance was defined as P values less than or equal to 0.005. Hierarchical variable cluster analysis with the centroid method was used to identify related groups of similar autoantibody specificities. Tetrachoric correlations between the autoantibody profiles in SLE patients were determined using SAS version 9.2.
Familial aggregation of autoantibody occurrence within siblings was used to explore potential genetic influence on the production of autoantibodies (23). We categorized each sibling pair as either concordant positive, discordant, or concordant negative for each of the 10 lupus-associated autoantibody specificities. The total numbers of concordant positive (n1), discordant (n2), and concordant negative (n3) sibling pairs were determined to calculate odds ratios (ORs), as follows: 4n1n3/([n2]2 − n2). The ORs were calculated for sibling pairs consisting of 2 SLE patients within a family and for sibling pairs of 1 SLE patient and 1 unaffected sibling.
Higher frequency and number of autoantibodies in African American participants irrespective of disease status.
The overall prevalence of autoantibodies detected in the serum samples by BioPlex 2200 bead-based assay varied depending on ethnicity (Figure 1). Of the 1,030 African Americans in the study, 468 patients with SLE (90%), 89 unaffected relatives (33.7%), and 43 healthy, population-based controls (17.5%) were positive for at least 1 of the autoantibodies tested by BioPlex 2200 assay. The mean ± SD number of autoantibodies detected within this ethnic 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 Hispanics, 163 patients with SLE (73.1% of the patient group), 34 unaffected relatives (23.4% of the unaffected relative group), and 19 healthy, population-based controls (13.7% of the control group) had at least 1 positive result for an autoantibody specificity. Within this ethnic group, the mean ± SD number of autoantibodies detected was 3.68 ± 2.21 for patients, 1.74 ± 1.08 for unaffected relatives, and 1.21 ± 0.71 for healthy, population-based controls. Of the 1,872 European Americans, 483 patients with SLE (67.4% of the patient group), 177 unaffected relatives (26.5% of the unaffected relative group), and 46 healthy, population-based controls (9.5% of the control group) were found to be positive for at least 1 of the 10 tested autoantibody specificities associated with SLE. In this group, the mean ± SD number of autoantibodies present by bead-based assay within the 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.
Among all of the SLE patients in the study, 53.1% of African American patients, 34.3% of Hispanic patients, and 18.7% of European American patients were found to have 4 or more of the tested autoantibody specificities (Figure 1). Overall, when we applied this commercially available standard antibody-testing platform, 88.8%, 73.1%, and 67.4% of the patients with established SLE in the African American, Hispanic, and European American ethnic groups, respectively, were found to be positive for SLE autoantibodies. Therefore, ∼11–33% of these patients with established SLE would be ANA negative by this detection method.
Higher prevalence of select autoantibody specificities in African Americans compared with European Americans and Hispanics.
Analyses of the individual autoantibody specificities that were detected revealed significant differences among the ethnic groups (Figure 2). When compared to European American SLE patients, African American SLE patients displayed a significantly higher prevalence of autoantibodies against dsDNA, chromatin, ribosomal P, 60-kd Ro, Sm, Sm/RNP, RNP A, and RNP 68 (χ2 = 13.3–129.5, P ≤ 0.001 for all comparisons) (Figure 2A). Hispanic SLE patients also had significantly higher autoantibody prevalences compared to European American SLE patients for the 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). When compared to Hispanic SLE patients, African American SLE patients had a higher autoantibody prevalence for the chromatin, Sm, Sm/RNP, RNP A, and RNP 68 specificities (χ2 = 11.6–41.5, P ≤ 0.001 for all comparisons).
Analyses of the autoantibody prevalences in unaffected relatives revealed no effect that could be associated with self-reported ethnicity (Figure 2B). Interestingly, in the healthy, population-based control group, the prevalence of anti–52-kd Ro was significantly higher in African American control subjects than in European American control subjects (Figure 2C).
Detection of antichromatin as the most common autoantibody specificity in SLE patients and unaffected blood relatives.
When a BioPlex 2200 system was used to test the sera, 76% of SLE patients, 28% of SLE-unaffected relatives, and 12% of controls were found to have at least 1 lupus autoantibody specificity. As expected, the 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 group (P < 0.005). The mean ± SD number of all 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.
The prevalence of individual autoantibodies varied among SLE patients. Antichromatin was the most prevalent (55.8% of SLE patients), while anti–ribosomal P was the least prevalent (12.4% of SLE patients). Unaffected relatives also had varied autoantibody prevalence. Similar to the findings in SLE patients, chromatin was the most prevalent specificity in the SLE-unaffected relative group (12.5%). When compared to unrelated control subjects, SLE-unaffected relatives had a significantly higher prevalence of dsDNA, chromatin, 60-kd Ro, 52-kd Ro, Sm, and RNP A autoantibodies (P < 0.005 for all comparisons).
The prevalence of anti–Scl-70 was 2.2% in SLE patients and 1.2% in both SLE-unaffected relatives and healthy, population-based controls (P not significant). Anti–centromere B responses were detected in 3.7% of SLE patients and in <1% of both unaffected relatives and healthy, unrelated controls (P < 0.001), with women being >4 times more likely to be positive (P < 0.001). Jo-1 antibodies were present in <0.5% of all samples. Inclusion of the prevalence of Scl-70, centromere B, and Jo-1 antibodies did not significantly affect the overall ANA prevalence rates, and therefore these were not included in subsequent analyses.
Varying associations of the BioPlex 2200 bead-based assay and IIF results across ethnic groups.
IIF is the historical standard for broad-scale ANA screening. Comparisons of results from the IIF assay to those from the BioPlex ANA assay indicated that there were 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 assay. When all 13 measured autoantibodies were assessed together, 78.2% of SLE patients, 28.9% of unaffected relatives, and 13.8% of unrelated controls were considered ANA positive using the BioPlex 2200 assay; these prevalence rates were extremely similar to those detected when only the 10 lupus-associated autoantibodies were analyzed. Differences in autoantibody prevalence between the 2 tests were most striking in European American patients (Table 2).
Table 2. Comparison of overall autoantibody prevalence, positive predictive value, and negative predictive value between BioPlex 2200 bead-based assay and indirect immunofluorescence (IIF) among patients with systemic lupus erythematosus (SLE), unaffected relatives, and healthy, population-based controls in each ethnic group*
Positive antinuclear antibody results were based on a titer of ≥1:120.
Healthy, population-based controls
Positive predictive value
Negative predictive value
Autoantibody testing by IIF revealed a higher prevalence of autoantibodies among the SLE patients, SLE-unaffected relatives, and healthy controls in all ethnic groups, suggesting that the IIF assay is able to detect a more diverse repertoire of autoantibodies than is offered within the BioPlex 2200 ANA kit. Interestingly, among African American control subjects, the prevalence rates were similar between the BioPlex assay and the IIF detection method.
Further comparative analysis of the diagnostic efficacy between the BioPlex assay and the IIF ANA screening test was performed using the positive predictive value (PPV) and negative predictive value (NPV) for each assay. PPV and NPV analyses were performed within the combined group of SLE patients and healthy, population-based controls as a whole, as well as within ethnic subgroups (Table 2). 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%, compared with 68.6% for the BioPlex assay). Within the individual ethnic subgroups, African Americans had the highest PPV and NPV for both assays.
Because of the established clinical associations and therapeutic implications placed on the detection of anti-dsDNA antibodies in patients with SLE, we specifically examined the consistency of anti-dsDNA autoantibody detection between the BioPlex 2200 assay and the current standard IIF detection method. The prevalence of anti-dsDNA detected by IIF was 24.3% among SLE patients, 0.4% among unaffected relatives, and 0.1% among unaffected, unrelated controls.
Examination of the presence of dsDNA autoantibodies based on ethnicity revealed that this specificity was greatest in African American SLE patients (28.2%), with a similar prevalence in both European American (21.5%) and Hispanic (21.1%) SLE patients. However, dsDNA autoantibody specificity had a higher prevalence in all 3 ethnicities when detected using the BioPlex 2200 assay compared to the IIF. African Americans displayed the largest difference in prevalence of anti-dsDNA between IIF and the BioPlex 2200 test, showing a prevalence of 28.2% by IIF and 35.7% by BioPlex 2200 assay. Among Hispanics, 35% tested positive for anti-dsDNA antibodies when the serum was tested with the BioPlex 2200 assay, compared to 21.1% testing positive using IIF, while European Americans displayed an anti-dsDNA autoantibody prevalence of 23.4% by BioPlex assay and 21.5% by IIF.
Varying detection of autoantibodies in SLE patients, blood relatives, and healthy controls between traditional and BioPlex detection methods.
When all 13 Bio-Rad BioPlex 2200 ANA analytes were assayed in the serum of SLE patients in the 3 ethnic groups, we found that 29.7% of European American patients, 10.5% of African American patients, and 25.1% of Hispanic patients were autoantibody negative. In addition, we examined family members and control individuals for false-negative results. Within these groups, ∼30% of African American, European American, and Hispanic blood relatives and ∼20% from each control ethnic group were found positive for dsDNA antibodies by IIF but negative by BioPlex 2200 assay. Of the remaining autoantibody specificities, only individuals who were blood relatives of the SLE patients had false-negative results for Ro, La, or nRNP autoantibodies; among these individuals, fewer than 1% of African Americans were false negative for Ro, La, or nRNP, and fewer than 1% of European Americans were false negative for Ro and nRNP.
We next examined the presence of false-positive results. Among the SLE patients, 2.11% of African Americans, 6.3% of European Americans, and 7.6% of Hispanics were autoantibody positive by BioPlex 2200 assay but negative by IIF. Among the blood relatives, 11% from each ethnicity had false-positive results, compared to 11.4% of African American controls, 13.2% of European American controls, and 13% of Hispanic controls.
To explore the possibility of other potential antigenic targets among the samples found to be ANA negative by BioPlex 2200 assay, we examined the prevalence of aCL antibodies. The prevalence of aCL antibodies was lower in the BioPlex 2200 ANA–negative samples compared to that in the BioPlex 2200 ANA–positive samples (10.4% versus 15.6%; P = 0.02).
Similar sensitivity between bead-based assay and the IIF and immunodiffusion methods.
To reference our findings from the BioPlex 2200 bead-based assays to those obtained with traditional standards for detection of SLE antibody specificities, we compared the BioPlex 2200 assay results with the corresponding immunodiffusion results. In autoantibody testing of the serum of SLE patients, the sensitivity of the BioPlex 2200 assay, relative to the results from immunodiffusion, for detection of Ro, La, ribosomal P, Sm, and nRNP complex (nRNP A and nRNP 70-kd) antibodies was found to be 0.92, 0.92, 0.83, 0.89, and 0.92, respectively. Anti-dsDNA detection by BioPlex 2200 assay, when compared to IIF, had a sensitivity of 0.71 and a specificity of 0.80. Detection of centromere B and Jo-1 autoantibodies by the BioPlex 2200 assay, when compared to IIF and immunodiffusion, respectively, exhibited respective sensitivity values of 0.70 and 0.80 and respective specificity values of 0.98 and 0.998.
Enrichment of dsDNA and chromatin autoantibodies in SLE patient sera from simplex pedigrees.
To evaluate the effect of familial relationship on autoantibody prevalence, SLE patients were categorized as simplex (having no known familial relationship to other SLE patients) or multiplex (having at least 1 blood-related family member with SLE). Of the 1,540 SLE patients, 53.8% were classified as having a multiplex SLE pedigree, 26.5% were classified as having a simplex pedigree, and 19.7% were unknown. Interestingly, a significantly higher prevalence of anti-dsDNA was found among simplex-pedigree patients (35.0%) than among multiplex-pedigree patients (25.1%) (χ2 = 14.37, P < 0.0002).
African American and Hispanic patients are significantly more likely than European American patients to be anti-dsDNA positive. Therefore, multiple logistic regression was performed to determine whether an SLE patient with a simplex pedigree is more likely to be anti-dsDNA positive, regardless of ethnicity. Using dsDNA positivity as the outcome and both ethnicity (European American, African American, Hispanic, or other) and status (simplex or multiplex) as predictors, we found that simplex-pedigree patients had a significantly greater prevalence of anti-dsDNA than did multiplex-pedigree patients, even after adjusting for ethnicity (OR 1.59, P = 0.0003).
A significantly higher prevalence of chromatin autoantibodies was also found among simplex-pedigree patients (59.1%) compared to multiplex-pedigree patients (50.8%), when analyses were adjusted for ethnicity (χ2 = 8.33, P = 0.005). No other differences in the prevalence of other autoantibody specificities between simplex- and multiplex-pedigree SLE patients were noted.
Familial aggregation of ribosomal P, Sm/RNP, and 60-kd Ro autoantibody specificities in SLE patient sibling pairs.
Familial aggregation analysis was performed as a measurement of genetic influence on the production of certain autoantibody specificities. The 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 60-kd Ro (OR 2.56, P = 0.004) (Table 3). Within sibling pairs that consisted of an SLE-affected patient and an unaffected sibling, no significant aggregation for individual autoantibody specificity was detected.
Table 3. Familial aggregation of autoantibodies in sibling pairs*
SLE = systemic lupus erythematosus; ANA = antinuclear antibody; IIF = indirect immunofluorescence; dsDNA = double-stranded DNA; NA = not applicable.
Values are the number of sibling pairs categorized as either concordant positive (+/+), discordant (+/−), or concordant negative (−/−) for each autoantibody.
In examining the relationship between ANA positivity and family aggregation (Table 3), the results from the BioPlex 2200 bead-based assay indicated that there was familial aggregation in SLE-affected sibling pairs (OR 2.51, P = 0.01) as well as in the SLE patient/unaffected sibling pairs (OR 0.38, P < 0.001). IIF ANA assays showed aggregation of positivity for ANAs in SLE patient/unaffected sibling pairs (OR 0.49, P = 0.004).
Unique ethnic differences in autoantibody subsets revealed by autoantibody clustering.
Hierarchical cluster analysis of antibody profiles detected within all SLE patient samples showed 3 distinct subsets of autoantibodies: 1) 60-kd Ro, 52-kd Ro, and La, 2) Sm, Sm/RNP, nRNP A, nRNP 68, and chromatin, and 3) dsDNA and ribosomal P (Figure 3). When we performed the same analysis with SLE patients subgrouped on the basis of ethnicity, the 3 clusters described above were observed in the African American patients with SLE. However, in the European American and Hispanic patients, antibodies against chromatin tended to cluster with dsDNA and ribosomal P. All results accounted for ∼90% of the variability in antibody profiles in each patient group.
The goal of this study was to characterize autoantibody prevalence, specificity, clustering, and familial aggregation within a large cohort of SLE patients, unaffected blood relatives, and healthy, population-based controls using a Bio-Rad BioPlex 2200 ANA screening test. Our results further confirm the high sensitivity of the BioPlex 2200 ANA assay that has been demonstrated previously by other groups (24–26). Moreover, our study detected variable autoantibody prevalence in SLE patients based on ethnicity, identified a subset of autoantibodies present in unaffected family members, demonstrated an enrichment of anti-dsDNA in the simplex pedigree, established familial aggregation of select autoantibodies, and explored the interrelatedness of 3 subsets of common SLE autoantibodies.
The BioPlex 2200 ANA assay has the potential to serve as an ANA screening and detection method to identify individual antibody specificities. The autoantibody prevalence within SLE patients was found to be higher when the IIF ANA detection method was used as compared to the BioPlex 2200 assay. Differences in autoantibody frequency may be influenced by several factors. First, the BioPlex 2200 assay detected the presence of only 13 defined specificities, whereas IIF detects antibodies against a variety of cellular components, such as Ku, Ki, Su, 4–6S RNA, α-actinin, and single-stranded DNA (27), as well as unknown antigens. Nevertheless, specificities included within the BioPlex 2200 assay allowed identification of autoantibodies in the majority of patients found to be ANA positive by IIF. Interestingly, when the BioPlex 2200 detection method was used, 1 in 5 African American healthy individuals had positive findings for ANAs.
Unfortunately, a number of European American SLE patients who were ANA positive by IIF (32.6%) had no autoantibodies that were detectable on the BioPlex 2200 ANA screening test, thereby suggesting that a number of SLE patients might be found to be ANA negative by this method. When European American healthy control subjects were analyzed, the BioPlex 2200 ANA screening test showed that the prevalence of antibodies was nearly 50% lower than the prevalence detected by IIF ANA screening (at a titer of ≥1:120).
This difference in ANA positivity between screening methods and the overall prevalence of other autoantibodies cannot be explained by medication or changes in ANA positivity over 5 years of disease, as no significant differences were observed. Interestingly, as the length of time since SLE diagnosis increased to 10 years, the prevalence of ANA negativity decreased significantly in African American patients (P = 0.0281) and European American patients (P = 0.0077). Since the overall percentage of healthy, population-based control subjects found to be ANA positive was still higher than optimal, it is likely that the BioPlex 2200 ANA test will result in more false-negative results in SLE patients if used as the sole test for ANAs.
We examined the PPV and NPV of each assay to compare their abilities to identify SLE patients. Of particular note, the PPV for both the BioPlex 2200 and IIF assays was strong in African Americans (92.5% and 93.1%, respectively), while the PPV for the BioPlex 2200 assay in Hispanics (89.6%) and European Americans (91.3%) was slightly better than that for IIF (88.3% and 87.3%, respectively). However, the NPV was better for IIF compared to the BioPlex 2200 assay in the overall cohort (81.1% versus 68.6%) and within the individual ethnic subgroups (African Americans 82.2% versus 71%, Hispanics 76.5% versus 66.7%, and European Americans 79.4% versus 65.3%) (Table 2). Our study used a cutoff titer of 1:120 to indicate a positive result (28). Using this cutoff, the PPVs were decreased in the total cohort (83.7%) and in the individual ethnicities (African Americans 90.8%, Hispanics 82.7%, and European Americans 85.2%). Overall, these results support the premise that the BioPlex 2200 assay has a better PPV, while the IIF assay has a stronger NPV for autoantibody detection among patients with established SLE.
The BioPlex 2200 ANA assay also allowed for identification of autoantibody specificities within unaffected family members of SLE patients. This is of considerable interest as we continue to explore the genetic and environmental influences that may lead to the development of SLE and allows further characterization and serial monitoring of these autoantibody-positive, unaffected relatives. Previous study findings have contributed to the idea that production of specific autoantibodies by SLE patients and family members is the result of complex genetic influences and might indicate genetic susceptibility to autoimmune disease (18, 29). Previous studies that demonstrated familial aggregation of autoantibodies were based on data obtained by immunodiffusion. In those studies, Ro, La, nRNP, and ribosomal P showed significant aggregation within patient/patient sibling pairs, while significant aggregation in patient/unaffected relative pairs was detected for dsDNA, Ro, La, nRNP, and ribosomal P (29). In our study, using a BioPlex 2200 ANA screening test, familial aggregation of autoantibodies was detectable in patient/patient sibling pairs (ANAs, ribosomal P, Sm/RNP, and 60-kd Ro), while within patient/unaffected relative pairs, only ANAs showed familial aggregation, suggesting that the heritability pattern may be more refined.
Previous studies have introduced the concept of antibody cluster analysis to create serologic profiles that may assist with diagnosis, prognosis, and disease subsetting (12–16). In this study, we identified clusters of autoantibodies associated with previously defined physical antigen complexes or other suspected temporal associations (30–32). Our study indicated antibody clustering between Ro and La autoantibodies, Sm and nRNP antibodies, and dsDNA and ribosomal P autoantibodies. These results are in agreement with those from the published literature (12–16), with the exception of an association between nRNP and ribosomal P autoantibodies. Autoantibody cluster analysis of separate ethnic groups revealed variation of clustering within African Americans as compared to the trend established in European Americans and Hispanics. A recent study identified ANA variance in healthy adults across 6 different geographic regions, suggesting that there is an environmental component to autoantibody prevalence (33). Thus, differing genetic or initial environmental influences may alter autoantibody prevalence. These findings suggest the need to analyze racial/ethnic groups separately when examining etiology of disease, potential biomarkers, or any other proposed diagnostic or prognostic application of autoantibodies in SLE. Ongoing efforts to integrate these findings into a diagnostic or prognostic algorithm are currently under way.
In conclusion, we found the BioPlex 2200 ANA screening test to be a highly informative tool for assessment of autoantibody prevalence in SLE patients. Our analysis identified a significant increase in prevalence and total number of autoantibodies in African American SLE patients compared to European American SLE patients. Analysis of unaffected relatives of SLE patients and healthy, population-based controls revealed an increased prevalence for a specific group of autoantibody specificities within unaffected relatives, and therefore this finding requires further examination of the genetic influence of autoantibody production. Cluster analysis of autoantibody prevalence revealed an association between autoantibody specificities, which can be subsequently used to establish more accurate algorithms for interpretation of ANA testing results in a race-specific way, thereby maximizing the informative potential of ANA screening. Autoantibodies play a fundamental role in diagnosis and treatment of SLE and, with further characterization, may provide a unique perspective into pathogenesis.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. James had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Bruner, Guthridge, Lu, Reichlin, Scofield, Harley, James.
Analysis and interpretation of data. Bruner, Guthridge, Lu, Robertson, Neas, Scofield, James.
We thank Aaron Guthridge and David Wiist Jr. for their technical assistance and Tony Prestigiacomo, PhD and Steve Binder, PhD for their expertise with the Bio-Rad BioPlex 2200 instrument and data interpretation. We also thank the clinical immunology laboratory personnel at Oklahoma Medical Research Foundation, particularly Cathy Velte, Camille Anderson, and Sandy Long. We thank the Lupus Family Registry and Repository, including its personnel, participants, and referring physicians.