INTRODUCTION
An autoimmune disease is a condition where immune cells attack self-antigens as a result of a breach in immunological tolerance.[1] Autoimmune diseases can manifest in severe morbidity and mortality among the affected population. The prevalence of autoimmune diseases is 3%–5% in developing countries with significant female predominance.[2] Studies suggest, more than 75% of women are at higher risk of developing autoimmune diseases in comparison to men. There is a substantial rise in number of cases over the decade in western countries and very less data is available in the developing countries including India.[2,3]
Autoimmune diseases are diverse groups of disorders which can be organ specific with single-organ involvement or systemic with multiple-organ involvement.[1,4] Some of the common organ-specific diseases are Hashimoto’s thyroiditis, myasthenia gravis, and pernicious anemia: systemic autoimmune diseases are systemic lupus erythematosus (SLE), systemic sclerosis (SSc), rheumatoid arthritis (RA), and antiphospholipid syndrome.[1,4] Autoimmune diseases of the nervous system can involve the central and/or peripheral nervous system and neuromuscular junction. They can present with symptoms of neuronal damage, axonal injury, demyelination, or destruction of the neuromuscular junction.[4] Limbic encephalitis, optical retinopathy, myasthenia gravis, polymyositis (PM), dermatomyositis, myelopathy, and sensory neuropathy are some of the neurological autoimmune manifestations.[4]
In the diagnosis of autoimmune diseases, comprehensive clinical examination and appropriate investigations play a vital role.[5] Antinuclear antibodies (ANAs) are the first test ordered in any patient suspected of autoimmune diseases, same holds good in our setup as well. ANA are a specific group of autoantibodies which binds and destroys cellular components of the nucleus within the cell.[6] It was first discovered in the year 1948 in the serum of SLE patient, since then ANA is identified to be associated with many other systemic autoimmune diseases and considered as hallmark in their diagnosis.[7]
The indirect immunofluorescence (IIF) assay performed on substrate slides of cultured human epithelioid (Hep-2) cells is currently considered to be the gold standard for the detection of ANA.[2] Reactive IIF testing does not detect specific autoantibodies. ANA profile by immunoblot assay allows us to detect specific antigens which cannot be identified by IIF.
We receive a high number of blood samples for the ANA screening from neurological and psychiatric services. The clinical importance of thus obtained positive test results among cases presenting to a tertiary neuro-care center is less known. Often screening tests have high sensitivity because of high background, which can lead to erroneous clinical diagnosis and management when ordered unnecessarily. With this background, secondary data analysis was undertaken for 3 years from July 2016 to June 2019 in the department of neuromicrobiology, to determine the final diagnosis of ANA-positive patients in the context of descriptive data.
METHODS
Overview
The present study is a secondary data analysis, which was undertaken for 3 years from July 2016 to June 2019 in the department of neuromicrobiology, tertiary neuro-care center, Bengaluru. There were a total of 7795 serum samples subjected for ANA testing by IIF technique from patients who attended neurological and psychiatric services suspected of autoimmune diseases. The study group included 166 cases positive for ANA by IIF screening technique. Serum samples of these 166 cases were further subjected for specific antigen detection by ANA profiling by immunoblot assay. ANA IIF-positive cases data were collected from the medical records in a semi-structured proforma. The demographic variables included age and gender. The clinical variables included duration of illness, clinical features, and diagnosis.
Ethical statement
The following study is a retrospective study. The data was extracted from the records for analysis. Hence the study was exempted from ethical review. Ethical Committee reference number is NIMHANS/IEC/2019-20.
Antinuclear antibody indirect immunofluorescence procedure
Five milliliters of blood sample were received in our department for ANA testing and were subjected to serum separation as per standard protocol. IIF screening was performed on Hep-2 cell substrate by Immunoconcepts ANA kit (Sacramento, CA). As per the manufacturer’s instructions, the serum samples were diluted 1:100 with buffer solution followed by tagging with fluorescein-labeled antihuman globulin. Under the fluorescent microscope ANA binding to specific antigens can be visualized at 10X and 40X. Each of the sample’s fluorescent intensity and ANA pattern was matched with kit controls and the test was considered positive at a titer of 1:100.
Antinuclear antibody immunoblotting
ANA profile was performed on the same samples in the department of Neuropathology using immunoblot assay by EUROLINE ANA profile 3 (immunoglobulin G [IgG]) kit (Germany), against 15 different nuclear, cytoplasmic and mitochondrial antigens, as per the kit manual.
The patient’s serum samples were diluted 1:101 with buffer solution followed by incubation with ANA blot strips. Antibodies (majorly IgG and also IgA and IgM) will bind to specific antigens coated on the strips. To detect the bound antibodies, incubation was carried out using enzyme-labeled antihuman IgG conjugate to produce color reaction. The strips are studied for the specific bands.
Statistical analysis
Data were entered into Microsoft Excel and analyzed using SPSS (version 27.0; SPSS Inc., Chicago, IL, USA) software version 21. The categorical variables were summarized using absolute frequency and proportions and the quantitative variables were summarized by mean and standard deviation (SD). The difference between the two groups was assessed by Fisher’s exact/Chi-square test for categorical variables. The level of statistical significance was considered as 5%.
RESULTS
Of a total of 7795 serum samples subjected for ANA testing by IIF technique patients suspected of autoimmune diseases 166 cases were positive, hence the overall prevalence rate of ANA for 3 years was 2.12%. On further analysis, 59 serum samples were positive for specific antigen detection by ANA profiling by immunoblot assay with positivity rate of 35.54%.
Among 166 cases, peripheral nervous system involvement was seen in 56 (33.73%) which included inflammatory myositis and/overlap diseases in 30 (13.12%) and immune neuropathies 13 (10.5%) were the most common clinical presentation followed by neurodegenerative disorders 54 (32.53%): central nervous system (CNS) autoimmune disorder 40 (24.09%) with neuromyelitis optica (NMO) 14 (7%) and vasculitis 14 (7%) and others.
Prevalence of antinuclear antibody positivity and staining pattern by indirect immunofluorescence
Of the total 7795 serum samples received for ANA testing by IIF, 166 were positive, hence the overall prevalence rate of ANA for 3 years was 2.12%. The most common clinical manifestations among the patients in study was peripheral nervous system autoimmune disorder 56 (33.73%) which was followed by neurodegenerative disorders 54 (32.53%), CNS autoimmune disorders 40 (24.09%) and others. The most common ANA pattern observed was speckled 73 (43.9%) followed by homogenous 64 (38.5%) and others. There was no statistical significance found between the ANA pattern and the system involvement. Table 1 shows association between clinical diagnoses with various ANA patterns by IIF test.
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Table 1: Association between demographic parameters and clinical diagnosis with various antinuclear antibody patterns by indirect immunofluorescence test (n=166)
Identification of fine antinuclear specificity by line immunoassay/antinuclear antibody profile
On further analysis of the 166 ANA IIF-positive serum samples for a specific antigen, 59 samples were reactive with a positivity rate of 35.54%. Among these 59 cases, reactiveness to Ro52 in 35 (40.22%) cases was the most common followed by anti sjogrens syndrome type A (SSA) 24 (27.58%), nuclear ribonucleoprotein (nRNP) 21 (24.13%), anti sjogrens syndrome type B (SSB) 10 (11.4%), Sm 8 (9.19%), and others. Reactivity to one antigen was observed in 44, two antigens in 26, and three or more antigens in 13 cases, respectively. Table 2 shows the fine antinuclear specificity by ANA profiling. Inflammatory myositis and inflammatory myositis/overlap patients showed multiple antigen presence. The odds of turning ANA profile positive among individuals with homogenous ANA pattern was 10 times less than the odds of turning ANA profile positive among speckled ANA pattern. The association was not statistically significant. The odds of developing ANA profile positive status among the individuals with systemic tissue disorder was 1.84 higher than the odds of developing ANA profile positive status among peripheral nervous system involved individuals and statistically insignificant. Table 3 shows the association of ANA profile status with ANA pattern and clinical diagnosis.
Table 2: Distribution of reactiveness to various antigens by antinuclear antibody immunoblot assay among positive cases
Table 3: Association of antinuclear antibody profile status with antinuclear antibody pattern and clinical diagnosis (n=166)
Demographic features
The presence of antibodies was appreciated in all the age groups ranging from 4 to 78 years in our study. The mean SD of the study participants was 39.4 (15.2) completed years. Max (n = 99) were aged between 30 and 59 years. Females were predominantly (70.5%) affected than males (29.5%) with male-to-female ratio of 1:2.17. There was no association found in ANA pattern with age, sex, and duration of illness. Table 1 shows the association between age and sex with ANA pattern.
Clinical presentation
The patients presented to our institute with complaints as early as 3 days to as late as 5 years. The median (interquartile range) duration of illness in days was 150 (30–730). Most of them were presented with illness of <6 months (n = 92). Table 1 shows the association between duration of illness with ANA pattern. The most common symptoms with which patients came to our setup and reason for ANA testing were paresis/weakness in 102 (51%) followed by paresthesia 45 (20.36%), fever 30 (13.57%) and others. Table 4 shows the clinical symptoms of patients with ANA-positive result. Among the dermatomyositis patients (18/29), skin rashes were the leading symptom. Most of NMO (69.2%) patients presented with loss/diminution of vision. In myasthenia gravis patients’ dysphagia, ptosis, and weakness of limbs were the predominant symptoms.
Table 4: Clinical symptoms of patients with antinuclear antibody positive result
In our study, ANA by IIF was reactive predominantly among the peripheral nervous system autoimmune diseases 56 (35%) which includes inflammatory myositis and/overlap diseases 30 (16.6%), immune neuropathies 13 (10.5%), and myasthenia gravis 7 (5%) and others. It was followed by CNS autoimmune diseases 40 (21.5%) cases, among them demyelination diseases (24) 13%, vasculitis (12) 7%, and autoimmune encephalitis (AE) 3 (1.5%). Systemic autoimmune disorder (SLE) 16 (8%) mainly connective tissue disorders such as SLE 5 (2.2%), Sjogrens 4 (1.8%), and scleroderma 2 (Scl) (0.9%), and one thyroid organ-specific autoimmune disorder cases were the other clinical cases. There were 54 (21%) Neurodegenerative cases like stroke 18 (10%), motor neuron disease (MND) 7 (5%), other neurodegenerative diseases 12 (6%), and others in whom ANA by IIF was reactive.
DISCUSSION
Even though various tests are available for determining ANA, IIF is the most preferred method as it is cost-effective and easy to perform. The ANAs are produced against proteins, protein nucleic acids, nucleic acids present in nucleus and other subcellular organelles. So, this test is not limited for detecting ANAs alone, which would be the predominant reason in our study for the reactivity observed in wide range of neurological and nonneurological cases.[7]
The different patterns of ANA which can be determined by IIF are speckled, homogenous, nucleolar, cytoplasmic membrane, centromere/dotted, ad nuclear membrane. The homogeneous pattern reflects antibodies to dsDNA; nDNA; DNP and histone. The speckled pattern shows antibodies to Sm; RNP; Scl-70; SSA/Ro and SSB/La. The nucleolar pattern shows antibodies to fibrillarin, RNA Polymerase I, nucleolus-organizing regions (NOR) 90, and PM/Scl. Centromere pattern shows antibodies to the chromosomal centromere (Kinetochore). Cytoplasmic staining shows antibodies to anti-Jo-1 or anti-mitochondrial antibodies. These patterns are associated with specific clinical conditions, which aid the clinical diagnosis.[8]
Prevalence of antinuclear antibody positivity and staining pattern by indirect immunofluorescence
Our study demonstrates that the positivity rate of ANA is 2.12%, which is very less compared to any other study. This could be because of sensitive pretest algorithm and also might be because of overuse of test. Other studies, show positivity of 23.5% by Peene etal., 18.9% in Minz et al., Sebastian et al. 38.2% and Gupta etal. 33%.[2,9–11]
A speckled pattern was the most common pattern detected in our study. The speckled pattern is suggestive of SLE (Sm antigen), mixed connective tissue disease (RNP antigen), (Scl-70 antigen), or Sjögren’s syndrome-sicca complex (SSA/Ro or SSB/La antigen).[12] In our study, a wide range of neurological diseases are having this pattern. The second most common pattern was homogenous followed by others. Homogeneous pattern is observed in SLE or other connective tissue diseases (CTDs).[13] Nucleolar pattern is seen in Scl and Sjögren’s syndrome (SS).[14] Centromere pattern is indicative of the progressive SSc.[15]
Antinuclear antibody positivity by line immunoassay/antinuclear antibody profile
ANA profile with immunoblotting can detect antibodies against 15 antigens, such as ds DNA, nucleosomes, histones, Sm, PCNA, SSA, SSB, Ro 52, CENP-B, Scl 70, nRNP/Sm, AMA M2, Jo 1, PM-Scl, and Rib.P-protein.[16] These are recombinant and purified antigens which are coated as discrete lines on the strips, which helps us to identify multiple antigens simultaneously. Reactivity to multiple antigens is seen predominantly in connective tissue disorder and inflammatory myositis and overlap disorders in the study.
Demographic features
Female predominance was observed in our study and other studies as well. Female gender could be a risk factor for an autoimmune disorder.[3] Now, studies have shown a relationship between female sex hormones and occurrence of AID. Sex hormones have a direct effect on immune cells of both innate and acquired immune systems.[17] Middle-aged people were the ones affected most in our study.
Clinical presentation
Inflammatory myositis and inflammatory myositis overlap disease accounted for 30 (18.07%) cases which was the predominant clinical diagnosis among the patients who came to our hospital, it was followed by immune neuropathies in 13 (7.8%) and CTDs 16 (9.6%) which explains the symptoms such as weakness and paresthesia being predominant.
Central nervous system
Among CNS autoimmune disorders demyelination disorders was the predominant presentation. Demyelination disorder is the other common CNS autoimmune disorder where there is loss of myelin with intact axons, which could be because of various reasons such as inflammatory processes, infectious, metabolic variations and hypoxic and ischemic conditions, and focal compression.[18] Under demyelination disorders, NMO spectrum was the predominant. NMO spectrum disorder 14 (7%) is an immune-mediated disease of the brain and spinal cord characterized by inflammation of optic nerve and spinal cord with heterogeneous symptoms of loss of vision, weakness, sensory loss, intractable vomiting, and hiccups. Autoantibodies to aquaporin 4 (anti-AQP4) water channel protein in CNS is demonstrated in these cases.[19] Studies have shown NMO, frequently be associated with CTDs.[20] In our study, the patients did not show any complaints of connective tissue disorder even then the ANA antibodies were present among them. On ANA profiling only 10 patients out of 14 patients showed antibodies. Eleven of patients had diminished vision and limb weakness in four of the patient, urinary bladder symptom in one of them.
Vasculitis was the second most common CNS autoimmune disorder in our study. Vasculitis of CNS can be of two types primary vasculitis and secondary vasculitis. Primary vasculitis is a spectrum of diseases which causes inflammation of the inner walls of blood vessels of CNS and is usually challenging to diagnose. In secondary vasculitis, the involvement of CNS is secondary to systemic vasculitis disease such as microscopic polyarteritis, granulomatosis with polyangiitis and others. These two important secondary vasculitis diseases are associated with autoantibody formation such as anti-neutrophil cytoplasmic antibodies and perinuclear neutrophil cytoplasmic antibodies.[21] In our study, most of the cases were primary vasculitis patients who were positive for ANA.
AE comprised only three patients in our study. It is a group of immune mediated inflammation of the brain parenchyma often involving the cortex with or without involvement of deep grey matter, white matter, meninges and the spinal cord. Antibodies against N-MethylD-Aspartate receptor and leucine-rich glioma inactivated are the specific antibodies detected in these disorders. In our study, only one patient out of three was positive for NMDA antibody. We observed positivity for ANA.[22]
Peripheral nervous system autoimmune disorders
Inflammatory myopathies are a group of acquired inflammatory skeletal muscle disorders which include PM, dermatomyositis, necrotizing myopathy, overlap syndrome with myositis (overlap myositis, [OM]) including anti-synthetase syndrome, and inclusion body myositis[23] These patients are characterized by symptoms such as progressive muscle weakness, difficulty to swallow speak, climbing stairs, reaching things above head.[23] Dermatomyositis is characterized by a skin rash with progressive muscle weakness. In our study, we have dermatomyositis cases and OM patients presenting to our outpatient department with complaints of progressive muscle weakness, join pain, and rashes.
In these diseases, there is the presence of autoantibodies, in OM diseases with SLE and Scl the antibodies such as anti-Jo, Ro/SS-A, SS-B, PM/Scl. In our study patients showed Anti-SS-A/Ro52 and anti-SS-B antibodies positive.[23,24] Dermatomyositis 15 (45.45%) was the predominant clinical form under inflammatory myositis.
Immune neuropathies represent a diverse group of peripheral nerve disorders, with Guillain–Barre syndrome (GBS) as typical acute presentation and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) as chronic presentation; multifocal motor neuropathy and paraproteinemic polyneuropathies as other presentations. In our study, there were 12 (6%) GBS cases followed by ADPR-5 and CIDP-3 respectively.[25]
Myasthenia gravis is the autoimmune mediated neuromuscular junction disorder, in this disease antibodies are formed against acetylcholine receptors in the postsynaptic junction. ANA antibodies are observed in myasthenia gravis cases. Patients present with fatigable weakness of muscles.[26] In our study, we had seven (4.2%) cases with MG. Among these patients also ANA was present.
Neuropathies and lumboplexy with diabetes mellitus were the other peripheral neurological autoimmune disorders in our study.
Systemic autoimmune disorders
CTDs are autoimmune diseases commonly affecting multiple organs and characterized by the presence of autoantibodies. Systemic lupus erythematosus (SLE), SS, SSc, myositis, and RA are the common connective tissue disorders.[27,28] There may be presence of one or more type of ANA in these patients, which will help the clinician in diagnosing the case.
Systemic lupus erythematosus can affect any organ in the body with a wide range of symptoms.[28] Sjogren’s predominantly affects women, and they present with a chief presentation of sicca syndrome. Various organs can be involved in severe forms.[27] SSc is characterized by fibrosis, vasculopathy, and autoimmunity.[27]
Organ specific
Autoimmune thyroiditis (Thyrotoxicosis), studies have shown the presence of antibodies to nucleus, smooth muscles, and DNA in autoimmune thyroiditis patients. Even in our study, we observed the presence of ANA in serum of patients with a thyroid disorder.[29]
Neurodegenerative disorder
Neurodegenerative, MND, and stroke are the other clinical disorders with ANA positive by IIF. The newer studies have demonstrated presence of autoantibodies in this group of diseases.[30] These may have given positive test results in our study. There were substantial number of cases 25 (15%) who did not show any clinical autoimmune pathology yet showed positivity for ANA antibody: several studies have shown the presence of ANA among general population which could be the reason in our study as well.[5] Hence, the ANA tests have to be requested where the probability of autoimmune pathology is high, otherwise it will lead to unnecessary confusion and chaos among both the treating physician and patient.
CONCLUSION
In the present study, we determined the final diagnosis of ANA IIF-positive patients to evaluate the prevalence of ANA and its clinical importance among neuropsychiatric patients. The prevalence of ANA was found to be 2.12%, among them Peripheral nervous system autoimmune disorder such as inflammatory myositis and/overlap diseases 13.12% and immune neuropathies 10.5% were the most common clinical presentation followed by CNS autoimmune disorder such as NMO 7% and vasculitis 7%. Under the ANA IIF test, speckled pattern was the common type observed. Middle aged individuals remarkably women are at more risk of developing the autoimmune disorder. The most common population attending to neurological center would be peripheral nervous system autoimmune disorder patients with neurological complaints of weakness, paresthesia and skin rashes. Our study demonstrates that ANA positivity is sporadically associated with autoimmune disorder. Although ANA IIF test appears to be like generously employed, the test provides us with knowledge about the disease status and it could be cost effective and more importantly aids the decision of opting further ANA profiling. Since ANA IIF test is a highly sensitive test with a high background noise, awareness about the limitations of the test, judicious employment and effective application would help in meaningful interpretation of the test and treatment.
Limitation of study
The main limitation of our study is that the ANA IIF test was not performed at 1:160 titer as recommended by ICAP guidelines. There were very less positive samples to apply several statistical analysis.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
We would like to thank our colleagues in the Department of Neuromicrobiology NIMHANS, Bengaluru, India, for the technical support.
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