Antinuclear antibodies (ANA) are a heterogeneous group of autoantibodies  that can be detected in the sera of subjects with systemic autoimmune rheumatic diseases (SARD), such as systemic lupus erythematosus (SLE), Sjögren’s syndrome (SSj), systemic sclerosis (SS), rheumatoid arthritis (RA), idiopathic inflammatory myopathies (IIM) and systemic vasculitides (SV) , , . Autoantibodies are very helpful in the diagnosis, prognosis assessment and monitoring of the clinical evolution of patients with SARD , , , .
Classically, the indirect immunofluorescence (IIF) assay on human epithelial type 2 (HEp-2) cells is the preferred method for ANA detection, it being the established gold standard for ANA screening . IIF allows the detection of a large number of nuclear and cytoplasm antigens, however, the IIF assay has some downsides. It is laborious and time consuming due to the large number of serial dilutions and visual determination of the staining patterns . The assay is subjective as it relies heavily on human interpretation . Heterogeneity in microscopes, light power, lens magnification and Hep-2, substantially contributes to its variability . Its limitations can be minimized using automated microscopes, although not all ANA patterns can be recognized by such systems. Another limitation of IIF is its lack of specificity because these antibodies are present in other autoimmune diseases, infections, tumors and in 25% of apparently healthy individuals , , .
Demand for ANA testing has increased significantly in the last few years, highlighting the need for new faster, time efficient techniques that are preferably automated to ensure consistency and reliability. The BioPlex ANA Screen is a Luminex-based assay using magnetic beads. ANA determination by the BioPlex 2200 ANA Screen has high reproducibility and it is a high throughput analysis. However, at this time, not all antigens can be analyzed by this technique as it simultaneously only detects 13 autoantibodies of clinical significance (i.e. Ro52, Ro60, La, Sm, Sm/RNP, RNP-A, RNP-68, Scl70, centromere B, dsDNA, chromatin, Jo1, ribosomal P proteins) , .
There are several comparative studies that have reported autoantibodies detection by IIF and by the BioPlex ANA Screen. In one of these, the analysis of 510 healthy individuals for autoantibodies concluded that BioPlex is more specific than IIF for ANA screening and that the BioPlex ANA Screen is a useful tool for a high-throughput screening of healthy populations . Another comparison between both techniques in routine samples with the diagnosis of autoimmune diseases showed that there were numerous discrepancies for ANA screening, but that good agreement exists for anti-dsDNA and anti-extractable nuclear antigens detection. However, a study on 236 patients with a systemic rheumatic disease upon diagnosis concluded that the sensitivity of BioPlex is higher than IIF except for SLE and SS , . More studies are required to clarify the discrepancy between both techniques in order to harmonize the ANA detection test.
In recent years, several studies have revealed the importance of early detection of autoantibodies as predictors for autoimmune diseases. The analysis of sera samples in 130 patients before diagnosis of SLE showed that the autoantibodies appeared as early as 10 years prior to the diagnosis of SLE. The same study revealed that 88% of patients had at least one SLE autoantibody present prior to the diagnosis . Others studies have confirmed that the presence of autoantibodies can predict the development of autoimmune diseases before their clinical onset , , , . Detection of predictive autoantibodies is important for disease prevention and mitigating clinical impact , . The BioPlex ANA Screen may be a good candidate for screening and follow-up of high-risk subjects.
This study had two aims. The first was to clarify the clinical significance of the autoantibodies detected by BioPlex ANA Screen. The second aim was to establish the predictive value of BioPlex ANA Screen in subjects with clinical suspicion but without a diagnosis of autoimmune diseases.
Patients and methods
Study design and patients
We performed a 3-year follow-up prospective cohort study of 411 subjects without autoimmune diseases. Sera samples were collected for 1 year (from October 2011 to October 2012) from routine tests in the Autoimmunity Laboratory of Hospital 12 de Octubre (Madrid, Spain). Mean age (±standard deviation) of the patients was 52±16 years. The women to men ratio was 6:1 (85% women, 15% men). More than 95% of the patients were Mediterranean Caucasian. Physicians in the rheumatology department determined the diagnosis of autoimmune diseases and clinical manifestations according to the American College of Rheumatology criteria. This study complies with the Spanish legislation and European Community directives for cross-sectional studies.
Selection criteria were: (1) Autoantibodies detected by the BioPlex ANA Screen were not found by the IIF on HEp-2 cells at the time of patient inclusion in the study, (2) At least one annual review was performed during the study period.
The study was submitted to the Ethics Committee for Clinical Research of the Hospital on the 12th October and received a favorable report (Reference No. CEIC-16/383). No informed consent was required.
Autoantibodies detection by the IIF assay on HEp-2 cells
The assay for ANA was performed using an IIF on Hep-2 cells (Inova Diagnostics, Inc, San Diego, CA, USA). Dilutions of 1:80 and 1:160 were performed with phosphatase-buffered saline for screening test. In brief: 30 μL of each diluted serum was incubated on one well with fixed HEp-2 cells. After the cells were incubated and rinsed off, they were incubated with FITC-IgG-conjugated antibody (Reference No. 508113). The results were analyzed using a Nikon Eclipse fluorescence microscope with a magnification ×400.
IIF negative sera (IIF−) was defined as having a negative result in a dilution of 1:80. Detection of nucleolar, nuclear (homogeneous, coarse speckled, fine speckled, peripheral and centromere) or cytoplasmic patterns (diffuse and fine speckled) at a dilution of 1:160 was considered as a positive result (IIF+). Conversion from IIF− to IIF+ was defined as three or more consecutive positive ANA-IIF sera samples in a dilution of 1:160 or higher.
Autoantibodies detection by BioPlex ANA Screen
The BioPlex 2200 ANA Screen (Bio-Rad Laboratories, Hercules, CA, USA) is an automated assay that employs fluorescently dyed magnetic beads for simultaneous detection of 13 autoantibody specificity levels within a single serum sample. The BioPlex 2200 ANA Screen is run on the BioPlex 2200 System, a fully-automated, random access multiplex testing platform. The BioPlex ANA assay can detect autoantibodies against: dsDNA, chromatin, ribosomal P, Ro52 (SS-A), Ro60 (SS-B), La (SS-B), Sm, Sm-RNP complex, RNP-A, RNP-68, Scl-70, centromere B and Jo-1. The primary antibody isotype detected by this assay is IgG. The BioPlex ANA assay reports an antibody index (AI) value (range 0–8) depending on the fluorescence intensity of the antibody-bound magnetic beads. Antibody concentration is proportional to the fluorescence intensity. All cutoffs were established based on the 99% percentile value for the non-disease population in Spain. The cutoff for autoantibodies detected by BioPlex was 1.0 AI as recommended by the manufacturer. Except for anti-RNP-A, anti-RNP-68 antibodies and anti-dsDNA antibodies (cutoff: 2.0 AI, 2.0 AI and 20 AI, respectively). BioPlex ANA Screen positive serum (BP+) was considered when there was a serum with at least one positive result for the antibodies detected by this assay.
Data from the subjects and the BioPlex calibration group were included in two different randomized databases that were processed and analyzed using MedCalc for Windows version 14.12 (MedCalc Software, Ostend, Belgium).
- Prevalence of autoantibodies detected by BioPlex ANA Screen in ANA-IIF negative subjects. Anti-Ro60 (40.6%) and anti-Ro52 (27.3%) antibodies were the most frequently found in the BP+/IIF-sera samples. Antibodies against RNP-68 (1.5%) and Jo-1 (1.2%) were the least prevalent. The remaining nine antibodies detected by the BioPlex ANA Screen were also frequently found in this cohort (Table 1).
- Multiplex technology (BioPlex ANA Screen) is more sensitive than IIF in the early detection of autoantibodies. After a 3-year follow-up of the 411 subjects with BP+/IIF−, 312 turned into IIF positive (BP+/IIF+) and the remaining 99 continued to be BP+/IIF− (Figure 1). Sixty-five of the subjects that did not fulfill the criteria of IIF+ had at least one or more positive determinations over the 3 years. Thirty-four subjects were permanent ANA negative by IIF and positive by BP. We can conclude that the BioPlex ANA Screen has more sensitivity than the IIF on Hep-2 cells for the detection of autoantibodies that are clinically relevant, as it can detect “pathological” antibodies at least 3 years before IIF. The relation between the specific antibodies detected by the BioPlex and the IIF+ sero-conversion was calculated. Different frequencies of ANA-IIF sero-conversion were observed depending on the existence of autoantibodies as detected by the BioPlex (Table 2).
- Early detection of autoantibodies by the BioPlex can predict autoimmune disease development. After a 3-year follow-up, 87% (358/411) of the subjects were diagnosed with an autoimmune disease. The most prevalent diagnosis was systemic lupus erythematous (37%) followed by Sjögren syndrome (23%) (Table 3A). Fifty-three subjects were not diagnosed with an autoimmune disease, yet 19 of them had clinical manifestations of autoimmune diseases as arthralgia and Raynaud’s phenomenon. The remaining 34 had no clinical manifestations of autoimmunity (Table 3B). Predictive value for the BioPlex ANA Screen was 77.1% (70.1%–82.8% confidence interval). Positive predictive value (PPV) for the diagnosis of autoimmune disease for isolated antibodies detected by BioPlex was 65%–100% (Table 4).
The frequencies of the 13 autoantibodies detected by BioPlex ANA Screen in a cohort of 411 subjects BP+/IIF− without diagnosis of an autoimmune disease.
|Autoantibodies prevalence (BioPlex ANA Screen)|
The relation between specific antibodies detected by BioPlex ANA Screen and the grade of IIF+ conversion after 3 years of follow-up.
|Autoantibodies (BioPlex ANA Screen)||IIF positive|
|Topo I/Scl 70||66%|
The clinical characteristics of the 411 subjects after 3 years of follow-up.
|(A) With autoimmune diseases|
|Subjects with autoimmune diseases (n=358)|
|Systemic lupus erythematosus||134|
|Inflammatory bowel disease||15|
|aUCTD (n=2), MCTD (n=5), PBD (n=1), AH (n=3), celiac disease (n=1), APS (n=2).|
|(B) Without autoimmune diseases|
|Subjects without autoimmune diseases (n=53)|
|Subjects with clinical manifestations of autoimmunity||n=19|
|Subjects without clinical manifestations of autoimmunity||n=34|
The predictive value of isolated autoantibodies detected by BioPlex ANA Screen.
|Autoantibodies||PPV, %||95% CI|
Anti-Sm, anti-chromatin, anti-dsDNA and anti-ribosomal P antibodies had high predictive values for the diagnosis of SLE. The other autoantibodies detected by the BioPlex were also involved as predictors for clinical diagnosis of SLE (Table 5A). The anti-centromere B and anti-Scl70 antibodies could predict development of SS (Table 5B). Anti-SSA (Ro52 and R60) and SSB (La) appear at least 3 years before the diagnosis of Sjögren syndrome (Table 5C). Anti-RNP-A antibodies were associated with development of SLE and RA (Table 5A, D). Anti-RNP-A antibodies were the most prevalent antibodies detected in subjects without any external clinical manifestation for autoimmune disease after the 3 years of follow-up. The anti-Ro60 antibodies were detected in the serum of 22% of subjects without external clinical manifestations of autoimmunity (Table 6).
The positive predictive value (PPV) of the antibodies detected by BioPlex ANA Screen for the diagnosis of autoimmune diseases.
|PPV, %||95% CI|
The frequency of antibodies detected by BioPlex ANA Screen in subjects without any external clinical manifestations of autoimmune diseases after 3 years of follow-up.
The probability of developing an autoimmune disease is directly proportional to the number of antibodies detected by BioPlex prior to the diagnosis. There is a higher predictive value of BioPlex ANA Screen when there are two positive antibodies, and it is almost 100% when three or more autoantibodies are detected by the BioPlex (Table 7).
The predictive value of BioPlex ANA Screen when more than one antibody is positive.
|Number of positive autoantibodies BioPlex ANA Screen||Autoimmune diseases after 3 years of follow-up (%)|
|Three or more||96.8 (152/157)|
The multiplex technology (BioPlex 2200) has many advantages in the detection of autoantibodies, among them the simultaneous detection of 13 autoantibodies of established clinical significance , , , , . Several comparative studies exist between BioPlex ANA Screen and ANA-IIF with discrepant results , , , , . We have performed a follow-up study for the first time on subjects without a diagnosis of autoimmune disease and with discrepant results between BP and IIF for autoantibodies detection.
BioPlex ANA Screen sensitivity in comparison to IIF was reported in healthy subjects  at the time of diagnosis  or in treated patients with autoimmune diseases . We have demonstrated that the BioPlex ANA Screen detects autoantibodies years earlier than the IIF assay. Seventy-six percent of BP+/IIF− subjects became IIF+ over the 3-year follow-up period. It is mandatory to perform a long-term follow-up to confirm how many of the IIF-samples would be sero-converted to IIF+.
The most relevant consequence of the higher sensitivity of the BioPlex ANA Screen is its ability to predict autoimmune diseases by detecting early autoantibodies. Several prospective studies have demonstrated that autoantibodies can be detected in the sera of asymptomatic or pauci-symptomatic subjects even years before the development of the autoimmune diseases , . After the decisive confirmation of the presence of autoantibodies years before the diagnosis of SLE , many studies have reported the presence of other specific autoantibodies that precede the clinical onset of autoimmune diseases , , , , , .
This study confirms the presence of autoantibodies at least 3 years before the diagnosis of an autoimmune disease. The most prevalent diseases diagnosed in this study were SLE, SSj and SS. SLE was mostly associated with predictive autoantibodies detected by BioPlex ANA Screen. Patients with SLE were found to be positive for anti-ds-DNA antibodies, anti-Sm and anti-chromatin antibodies at least 3 years before the diagnosis. Other autoantibodies as anti-ribonucleoprotein P, anti-Ro52 and anti-Ro60 also had a PPV for the eventual development of SLE. In other studies, the detection of anti-chromatin and anti-ds-DNA antibodies was reported as early as 10 years and 2 years, respectively, before the first emergence of SLE . Anti-Ro, anti Sm, anti-ribonucleoprotein P were detected in subjects years before the diagnosis of SLE , , . The presence of these antibodies in asymptomatic subjects may be an incentive to perform a follow-up study on these patients as they would be considered high-risk subjects . Anti-CenB antibodies had a predictive positive value of 57% to develop SS. Similarly, anti-Topo I/Scl70 antibodies are also predictive of this disease. It has been reported that these antibodies are important as diagnostic and prognostic biomarkers . The results of this study confirm that BioPlex ANA Screen can detect the autoantibodies before the clinical diagnosis of SS. Anti-Ro52, anti-Ro60 and anti-La antibodies have a PPV for the development of SSj. The presence of these antibodies as early as 4 years before the diagnosis of SSj was reported previously .
Anti-RNP-A antibodies are the most prevalent antibodies in subjects without diagnosis of SARD after 3 years of follow-up. Shovman et al.  found that the anti-RNP-A were the most prevalent antibodies detected by the BioPlex ANA Screen in healthy subjects. Other studies confirm the high frequency of anti-RNP-A antibodies . Association between antibodies against RNP-A and the diagnosis of SLE, RA and other autoimmune diseases was reported in other studies as well , .
Our results confirm that the number of types of autoantibodies increases towards the time of diagnosis . In fact, the presence of three or more positive autoantibodies detected by the BioPlex ANA Screen had a PPV of 97% for the developing of an autoimmune disease. Our findings demonstrate that the BioPlex ANA Screen is useful for early detection of autoantibodies.
Early detection of autoantibodies has a significant role in the mosaic of autoimmunity as an important risk factor for the development of autoimmune disease , . There are other genetic , , ,  and environmental factors (such as; female gender , , , low levels of vitamin D , , , vaccines , , , infections , ,  and hormones  that help to evoke a pro-inflammatory condition that triggers the pathogenesis of autoimmune diseases.
In summary, autoimmune diseases can be predicted by early detection of the antibodies, their titer, and the different number of the autoantibodies present. The high sensitivity of the BioPlex ANA Screen can perform this task, thus predicting the emergence of autoimmune diseases and, consequently, reducing the severity impact of the diseases.
We thank Margarita Sevilla for her excellent technical assistance and Eitan Israeli for his excellent work of translation and English revision of the paper.
Author contributions: AS and YS designed and integrated the research. DP and BG wrote the manuscript. MS and DP made the ANA determinations. JAMF, DP, OCM and DPL were responsible for the database and the statistical analysis. DP, OCM and LN were responsible for the sera and clinical data collection. EPA, AS, LM and YS revised the manuscript. All authors contributed to the data interpretation and report preparation. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
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