A Multiplex Autoantibody Panel for Early Detection of Autoimmune Disease Activity

Background: Detection of autoantibodies has played a consolidate role in diagnosis of systemic autoimmune disorders. A cascade autoantibody testing is usually performed by employing antinuclear antibodies (ANA) test as a first screening test and the other tests as second level determinations. Here, we present that supplementing extractable nuclear antigens (ENA) tests to the ANA test may capture more autoimmunity and provide critical medical information at an early stage. In this study, we evaluated the clinical significance of a multiplex ANA + ENA panel. Methods: A cohort of 110 subjects, identified as ANA negative but ENA positive, were followed up for two years. The detection of their ANA and anti-ENA autoantibodies was assessed with a multiplex ANA + ENA panel at Vibrant America Clinical Laboratory. Results: During two years of multi-visit follow-up, 23 out of 110 subjects (20.9%) were found to become ANA positive within an average of 385 (±144) days. Histone (50/110, 45.5%) and Chromatin (25/110, 22.7%) antibodies were the most frequently found antibodies at their first visits. The subjects who were positive for RNP (5/8, 62.5%) and SSA (Ro) (10/22, 45.5%) have the highest ratio of conversion to positive ANA. No significant correlation was observed between the conversion frequency and the number of anti-ENA antibodies being carried. Conclusion: This study, which followed up on the subjects who had disparate ANA and ENA test results, showed that anti-ENA antibodies may exist years earlier than ANA. Combining ENA tests with ANA screening may reduce false negatives and improve sensitivity.

tients with autoimmune disorders (e.g., systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, rheumatoid arthritis, multiple connective tissue disorder) [1] [2]. A cascade autoantibody testing is usually performed by employing antinuclear antibodies (ANA) test as a first screening test and the other tests as second level determinations [3]. The possibility of an accurate diagnostic autoimmune disorder is closely related to the completion of ANA screening and following tests. The development of high-throughput quantitative antibody-based assays has been intensively pursued and already replaced most traditional manual qualitative assays [4]. There has been broad interest in standardizing diagnostic tests in order to predict the development of diseases, reduce the cost of repeated confirmatory tests, and avoid unnecessary continued investigations [3] [5].
ANA is a heterogenous group of autoantibodies that can be found in the serum of patients with systemic or organ specific autoimmune diseases and a variety of infections. The gold standard of ANA testing accepted by American college of Rheumatology (ACR) is immunofluorescence assay (IFA) on human epithelial type 2 (HEp-2) cells [6]. IFA identifies a variety of antigenic residues, which react to not only autosomal antibodies but also nuclear or nucleoplasmic targets in sera. While IFA detects many nuclear and cytoplasm antigens, its sensitivity and specificity in diagnosis of autoimmunity have been inherently limited by a few factors [3] [7]. First, ANA by IFA can be subjective due to its heavy dependence on humans' operation (a large number of serial dilutions) and interpretation (visual determination of staining patterns) [8]. Another significant limitation is the existence of false positives due to ANA's presence in other diseases, infections, tumors, and in 25% healthy individuals [9]. Interpretation of ANA pattern also presents a unique challenge to provide accurate and inclusive results for physicians to make decisive diagnosis.
Extractable nuclear antigens (ENA) are analyzed in order to determine the specificity of autoantibodies that have produced a positive ANA with a homogeneous or speckled pattern. Detection of anti-ENA antibodies, as a second-tier test, is usually followed by a positive ANA test to identify and distinguish between different autoimmune diseases, especially connective tissue disorders [10].  [11]. The progressively-developed biomedical industry may contribute technological solutions to improve the process, not only in preparing substrates and slides, but also in providing a comprehensive yet affordable test panel.
Though important, the clinical significance of a multiplex autoantibody panel in diagnosis of autoimmune disorders has been barely investigated [12]. In this study, we defined the cohort of patients to a group of 110 subjects who had dis- to be ENA positive. This comparative study showed that a combined test of ANA and ENA was more sensitive than detecting ANA alone but also recognized autoimmune disease activity at an earlier stage. ± 17 years. The female to male ratio was 1:1 (52% female, 48% male). The interpretation of the results depended on the pattern observed, the titer of the autoantibody, and the age of the patient [12]. A sample was considered ANA negative (ANA-) if specific staining was equal to or less than a negative control (buffer containing preservative and human serum with no IgG antinuclear antibodies). Samples might exhibit various degrees of background staining due to heterophile antibodies or low-level autoantibodies to cytoplasmic constituents any specific staining (homogeneous, centromere, speckled, nucleolar, peripheral) was observed to be greater than the negative control. The elderly, especially women, are prone to develop low-titered autoantibodies in the absence of clinical autoimmune disease. A 1:40 dilution was suggested as a good dilution to screen for ANA [6]. Low-titer positive results might occur in apparently healthy persons; therefore, the ANA results were always interpreted considering the patients' total clinical presentation.

Statistical Analysis
Clinical data from the de-identified subjects were included in a randomized database that was processed and analyzed using Java for Windows version 1.8.45.
Data were expressed as mean ± standard deviation (SD) when the distribution was Gaussian. Two-sample t-test is used to examine the significance of difference in two groups. P < 0.05 was considered significant.

Early Detection of Anti-ENA Antibodies Predicted ANA Conversion
Between

Prevalence of Anti-ENA Antibodies Detected in ANA Negative Subjects
As shown in Table 1

No Correlation between ANA Conversion and Carrying Multiple ENA Antibodies
The patients with more than one anti-ENA antibodies are normally highly suspected to have overlapped autoimmune disorders [14] [15]. Figure 2 however shows that the possibility of conversion from ANA negative to ANA positive was not correlated with the total number of anti-ENA antibodies being carried. Among the 110 subjects, 74 subjects had only one anti-ENA antibody and 15 (20.3%) of them converted to ANA positive. 25 subjects had two types of anti-ENA antibodies and 7 of them (28.0%) became ANA positive. However, only 1 out of 11 subjects (9.1%) that who had three types of anti-ENA antibodies sero-converted to positive ANA. The p-value among these three groups were 0.4 (group 1-group 2), 0.2 (group 2-group 3), and 0.3 (group 1-group 3). Therefore, no significant difference was observed between the conversion frequency and the number of anti-ENA antibodies being carried.

Discussion
Genetic background and environmental factors (e.g., smoking, vitamin D, toxins, diet, infection, antibiotics, dysbiosis) are believed to triggering autoimmunity and inducing pro-inflammatory syndromes [16] [17]. Early detection of autoantibodies has always played an important role in predicting and diagnosing autoimmune disorders, especially for the patients suspected with overlapped syndromes and complex conditions. Improved method such as IFA for ANA screening has already greatly reduced the number of false negatives [5]. From our observation, a significant number of subjects still fall into this category, which should not be overlooked. Simultaneous detection of multiple autoantibodies has become technically and costly feasible owing to the advancement in semiconductor methods and the generation of high-throughput biotechnology.
In this study, a multiplex ANA + ENA panel enabled a long-term follow-up for 110 subjects who had discrepant ANA and ENA results. While comparable studies of multiplex autoantibody panels have been conducted by different research groups [18] [19] [20] [21], we extended the conclusions and validated the assumptions with our microarray platform.
The most relevant consequence of performing a multiplex ANA + ENA panel is its ability to predict autoimmune disease by detecting early autoantibodies.
This study confirms that anti-ENA bodies could be identified prior to the presence of ANA in some subjects. Among 110 subjects with negative ANA and positive ENA serology, 21% developed to positive ANA during the 2.5-year follow-up. This conversion rate is in agreement with the numbers reported in another study where it was ~25% for two years and ~76% for three years [13].
The investigation of the same cohort is still undergoing at Vibrant America Clinical Laboratory and it would be mandatory to perform a long-term follow-up for confirmation. We have also noticed a consistent conversion frequency of 10% per year, which may provide information in predicting the percentage of false negatives in further studies. Even though a decisive confirmation of disease status were not provided for these subjects, the presence of specific autoantibodies is still among the most robust evidence to predict clinical onset of autoimmune diseases.
Our analysis showed no significant correlation between the type of anti-ENA antibody and the possibility of sero-conversion. The most prevalent anti-ENA antibody found in the subjects is anti-Histone antibodies (45.5%) but only 10.0% of them converted to ANA positive in two and a half years. The anti-ENA antibodies associated with the highest conversion frequency are the ones to against RNP (62.5%) but the prevalence of their carrier was only 7.3%. Even though anti-ENA antibodies may exist in healthy people thus have limited clinical value in predicting autoimmune diseases, their presence can always be interpreted with Open Journal of Rheumatology and Autoimmune Diseases symptoms to provide critical information in diagnosis. Our finding demonstrates that detecting ten types of anti-ENA antibodies rather than several specific types is advantageous. An ENA panel of ten autoantibodies provided more inclusive results and higher possibility to capture false negatives that could be missed by ANA screening.
The results from our study indicated that the frequency of ANA sero-conversion is hardly related to the number of types of anti-ENA antibodies being carried. The subjects with two types of anti-ENA antibodies had relatively higher conversion frequency (28%) compared with the ones had either one or three types of anti-ENA antibodies. There has been relevant study showing that the average number of types of autoantibody increased with the time of diagnosis of systemic lupus erythematosus [22]. We hypothesize the difference might be due to the presence of multiple autoimmunity syndromes besides systemic lupus erythematosus in this cohort of patients and the development pattern for those syndromes can be less relevant with time. However, we believe it would be worthwhile extending this study to a larger population of subjects suspected with systemic autoimmune disorders.

Conclusion
In summary, this study showed that a combined test of ANA and ENA has great potential to reduce the number of false negatives and improve early detection of asymptomatic subjects suspected with systemic autoimmune disorders. A microarray-based autoantibody panel can detect and confirm multiple autoantibodies in one step while adhering to the standard IFA methodology and conventional ENA interpretation. Early detection at such platform will provide insights into specificity of autoantibodies present, indications of disease likelihood, and confirmation of clinical suspicion.