Abstract
Autoantibodies are the serological hallmark of autoimmune disease. Though their pathogenic role is debatable, they play an important role in the management of a patient with rheumatic disease. However, due to their presence in the general population as well as in multiple autoimmune diseases, the presence of an autoantibody alone does not make a diagnosis; the result has to be interpreted along with clinical findings. Similarly, the absence of autoantibody does not exclude a disease.
The common autoantibodies used in clinical practice include rheumatoid factor, anti-CCP antibodies, antinuclear antibodies (ANAs), anti-neutrophil cytoplasmic antibodies (ANCA) and anti-phospholipid antibodies. Once an autoantibody to a broad antigen is detected in a patient, sub-specificity analysis can improve the utility of the antibody. Autoantibodies are detected in the serum using different assays and results of which can vary; thus, it is important for a clinician to know the method used, its sensitivity and specificity to help in the proper interpretation of the laboratory results. This review will address these issues.
Introduction
During the development of lymphocytes in the primary lymphoid organs, the body tries to avoid generation of cells directed against self-antigens; however, the deletion process is not perfect, and a small frequency of self-reactive T and B lymphocytes escape to the periphery. In the periphery, they are kept under check by different mechanisms involved in tolerance induction. However, in autoimmune disease due to the failure of these regulatory mechanisms or to an increased load of autoantigens, there is a generation of a significant amount of autoantibodies. The pathological significance of autoantibodies in the causation of disease is limited, but they serve as good serological markers of autoimmunity.
Autoantibodies are useful in diagnosis, prognosis and follow-up of patients with rheumatic disease. However, as is true for any laboratory test, the interpretation of the test is dependent on the clinical situation (pre-test probability), the characteristics of the test (sensitivity, specificity and the likelihood ratios) and the reason for doing the test (confirmation or exclusion of a diagnosis). Autoantibodies can be directed against any component of a cell, that is, nuclear, cytoplasmic or membrane. In addition, antibodies against cytokines, hormones, coagulation proteins, phospholipids, etc. are also known.
Most autoantibodies for clinical usage are detected in the serum, but they can be detected in the cerebrospinal fluid and other body fluids. The various tests used for their detection include indirect immunofluorescence assay (IIF), enzyme-linked immunosorbent assay (ELISA), nephelometry and immunoblotting. In recent years, newer techniques such as bead array and chip array are being explored for the ease of automation and a high throughput.
For each test, kits from various companies are available; however, the results are usually not comparable due to differences in the various reagents used. Though there has been an attempt to standardize kits and express results in international units, it has not been possible for all antibodies. Kits certified by a regulatory body such as Food and Drug Administration (FDA) or European Medicines Agency (EMA) should be used.
The autoantibody testing is different from other laboratory tests because of the presence of multitude of antibodies in one disease, the same antibody in many diseases, occurrence prior to the onset of symptoms and fluctuation in levels during the course of a disease. Autoantibodies are also found in healthy individuals, and their prevalence increases with age especially so in the elderly population, which further makes their interpretation difficult. Thus, their interpretation requires training of physicians so that they are used in the right clinical context.
In this review, commonly used autoantibodies and their clinical significance are discussed.
Rheumatoid factor : Rheumatoid factor (RF) is an autoantibody directed against aggregated immunoglobulin G (IgG). They are produced as a result of polyclonal B-cell activation as well as in response to the modified antigen. Though all subtypes of RF are generated, we usually test for IgM RF. IgG and IgA are present in a proportion of patients with RF. RF is present in many conditions besides RA ( Table 1 ).
| Disease | Prevalence | Disease | Prevalence |
|---|---|---|---|
| Rheumatoid arthritis | 60–70 | Chronic infections | |
| Early undifferentiated arthritis | 45–55 | Endocarditis | 15–20 |
| Juvenile idiopathic arthritis | 10–12 | Hepatitis B/C | 15–40 |
| Other autoimmune diseases | Leprosy | 10–50 | |
| SLE | 15–30 | Tuberculosis | 10–20 |
| Sjögren’s syndrome | 75–85 | ||
| Systemic sclerosis | 20–30 | ||
| Inflammatory myositis | 5–10 | ||
| Interstitial lung disease | 10–40 | Healthy control | 5–20 a |
The presence of RF in a patient with arthritis increases the probability of a diagnosis of rheumatoid arthritis (RA). In a patient with RA, it increases the risk of the development of erosions, extra-articular features and poor outcome. In RA, the presence of RF also increases response rate to B-cell depletion therapy.
In addition to RA, RF is present in many autoimmune diseases such as Sjögren’s syndrome (SS), systemic lupus erythematosus (SLE), juvenile idiopathic arthritis, all of which may present with polyarthritis. Patients with chronic infections such as subacute endocarditis, tuberculosis (TB) and leprosy also have a higher prevalence of RF, and some of them can present with polyarthritis-like features. RF production in them occurs due to chronic B-cell stimulation.
RF should be ordered in a patient with
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Early inflammatory arthritis without any obvious cause
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Clinical diagnosis of RA
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Prior to considering B-cell depletion therapy in a patient with RA
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Patient with Sicca symptoms
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Child with chronic polyarthritis
Indiscriminate use of RF in the primary and secondary health-care setting results in the wastage of money as well as in a large number of false-positive results .
Methods
RF can be measured using latex agglutination, ELISA and nephelometry. Among these, nephelometry is the most commonly used method as it is quick and reproducible, provides quantitative result and can be fully automated for a high throughput. Though latex agglutination is a cheap and easy assay and can be performed quickly and at the point of care in a community, it provides qualitative data and has about 20% coefficient of variation due to problems in setting the assay . ELISA is a good method but it is too sensitive, and samples need to be processed in a batch and thus there is a delay in reports. ELISA can help detect other isotypes of RF such as IgG and IgA RF. IgG RF levels are high in patients with RA vasculitis, and IgA RF at onset heralds a poor prognosis but that is rarely required in clinical practice.
A World Health Organization (WHO) standard is available, and all kits should report the result as international unit (IU). A value above 20 IU is taken as significant, but values above 50 IU have a higher diagnostic value.
Anti-citrullinated peptide antibodies
Due to the low specificity of RF, there has been a quest to have a more specific test for RA. In fact, it is being considered that RF may no longer be needed . Over the last 25 years, anti-citrullinated peptide antibodies (ACPAs) have emerged as a major diagnostic test. Though anti-perinuclear factor was described in 1964, it was only in 2000 that the first ELISA for antibodies to cyclic citrullinated peptide was described. Over the years, many new targets have been identified; thus, now they are named as ACPAs. ACPAs are a group of antibodies directed against citrullinated self-proteins such as filaggrin, vimentin, enolase A, fibrinogen, etc. Due to the high specificity of ACPA, they are being used more often for the diagnosis of RA, and they are included in the new classification criteria for RA .
Like RF, their presence increases the probability of RA in a patient with undifferentiated arthritis. Negative ACPA does not help much in a patient with undifferentiated arthritis. Accumulated data from 164 studies calculated between 2002 and 2019 involving >18,000 patients and 20,000 controls showed that it had a sensitivity of 61.4% and a specificity of 99% as compared to healthy controls and 94% as compared to disease control for a diagnosis of early RA4. Similarly, their presence increases the risk of erosions, cardiovascular risk and mortality . ACPAs are most useful in patients with undifferentiated arthritis where a diagnosis of RA is being considered. The presence of ACPA increases the probability of progression to RA by 16-fold . Even the risk of arthritis is higher in patients with arthralgia if they are ACPA positive . It should not be ordered in other conditions as rarely high-titre antibodies may be seen in reactive arthritis, psoriatic arthritis, myositis, etc. Serial monitoring or ACPA is not recommended as the levels do not correlate with disease activity or with response to therapy and change in only a small proportion of patients .
Methods
ACPA is detected by ELISA. Over time, different generations of ELISA have come in the market. The first-generation kits used synthetic peptides derived from human filaggrin, whereas the second-generation kit anti-cyclic citrullinated peptide (CCP2) used epitopes selected from libraries of citrullinated peptides as antigens. The exact nature of antigen for anti-CCP3 is not known. Anti-CCP2 ELISA had a higher sensitivity as compared to anti-CCP1, but there is no difference in the performance characteristics of anti-CCP3 as compared to anti-CCP2; thus, anti-CCP2 is the most widely used ELISA for the detection of these antibodies . Recently, an international reference standard has been generated and is being evaluated . In view of multiple assays available, the use of a generic term such as ACPA was suggested by the panel on multinational evidence-based recommendations on how to investigate and follow up undifferentiated peripheral inflammatory arthritis 2011 .
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ACPA is more specific than RF for rheumatoid arthritis
- •
CCP2-based ELISA is currently the most widely used assay for ACPA detection
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No need to repeat if once positive
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Titres do not correlate with disease activity
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The presence of ACPA in undifferentiated arthritis increases the probability of development of RA to 70–90%
Anti-citrullinated peptide antibodies
Due to the low specificity of RF, there has been a quest to have a more specific test for RA. In fact, it is being considered that RF may no longer be needed . Over the last 25 years, anti-citrullinated peptide antibodies (ACPAs) have emerged as a major diagnostic test. Though anti-perinuclear factor was described in 1964, it was only in 2000 that the first ELISA for antibodies to cyclic citrullinated peptide was described. Over the years, many new targets have been identified; thus, now they are named as ACPAs. ACPAs are a group of antibodies directed against citrullinated self-proteins such as filaggrin, vimentin, enolase A, fibrinogen, etc. Due to the high specificity of ACPA, they are being used more often for the diagnosis of RA, and they are included in the new classification criteria for RA .
Like RF, their presence increases the probability of RA in a patient with undifferentiated arthritis. Negative ACPA does not help much in a patient with undifferentiated arthritis. Accumulated data from 164 studies calculated between 2002 and 2019 involving >18,000 patients and 20,000 controls showed that it had a sensitivity of 61.4% and a specificity of 99% as compared to healthy controls and 94% as compared to disease control for a diagnosis of early RA4. Similarly, their presence increases the risk of erosions, cardiovascular risk and mortality . ACPAs are most useful in patients with undifferentiated arthritis where a diagnosis of RA is being considered. The presence of ACPA increases the probability of progression to RA by 16-fold . Even the risk of arthritis is higher in patients with arthralgia if they are ACPA positive . It should not be ordered in other conditions as rarely high-titre antibodies may be seen in reactive arthritis, psoriatic arthritis, myositis, etc. Serial monitoring or ACPA is not recommended as the levels do not correlate with disease activity or with response to therapy and change in only a small proportion of patients .
Methods
ACPA is detected by ELISA. Over time, different generations of ELISA have come in the market. The first-generation kits used synthetic peptides derived from human filaggrin, whereas the second-generation kit anti-cyclic citrullinated peptide (CCP2) used epitopes selected from libraries of citrullinated peptides as antigens. The exact nature of antigen for anti-CCP3 is not known. Anti-CCP2 ELISA had a higher sensitivity as compared to anti-CCP1, but there is no difference in the performance characteristics of anti-CCP3 as compared to anti-CCP2; thus, anti-CCP2 is the most widely used ELISA for the detection of these antibodies . Recently, an international reference standard has been generated and is being evaluated . In view of multiple assays available, the use of a generic term such as ACPA was suggested by the panel on multinational evidence-based recommendations on how to investigate and follow up undifferentiated peripheral inflammatory arthritis 2011 .
- •
ACPA is more specific than RF for rheumatoid arthritis
- •
CCP2-based ELISA is currently the most widely used assay for ACPA detection
- •
No need to repeat if once positive
- •
Titres do not correlate with disease activity
- •
The presence of ACPA in undifferentiated arthritis increases the probability of development of RA to 70–90%
Antibodies to nuclear antigens
These are one of the most widely used autoantibody tests. The screening test, that is, ANA, looks at the presence of antibodies to all nuclear antigens by using cells as substrate. These include DNA- and RNA-associated proteins, centromere, nuclear membrane as well as nucleoli. On dividing, cells proliferating cell nuclear antigen, etc. are expressed, and antibodies against them can also be detected when using cell-line substrates . Once ANA is found to be positive, the sub-specificities are identified using different assays .
The indications of ordering ANA includes clinical situation where we suspect SLE or other connective tissue disease as follows:
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Arthritis with fever
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Glomerulonephritis
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Unexplained multisystem disease
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Immune cytopaenias
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Unexplained neurological disease
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Polyserositis
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Sicca syndrome
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Raynaud’s phenomenon
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Systemic sclerosis
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Palpable purpura
Methods
Traditionally, ANA has been detected by IIF for the last 65 years, but in the recent past, solid-phase assays such as ELISA and laser bead assays are competing with IIF. Most laboratories use Hep2 cells or its variants such as Hep2000, which has a higher amount of Ro antigen for IIF assay . The major drawback of IIF assay is that it is a labour-intensive test with multiple steps of incubation and washing, the need for a trained reader and difficult to automate. Recently, attempts to use automated systems for staining slides and reading them have been developed; they are yet to reach a wide applicability for clinical usage .
By contrast, solid-phase assays can be automated for a high throughput, and they are less labour intensive. The major drawback of newer assays is the variability in antigens used, which affects their utility and comparability . The American College of Rheumatology (ACR) still recommends IIF to be the gold standard for the determination of ANA . However, the recent international recommendation suggests that IIF is the preferred method, but alternative methods can be used mentioning their sensitivity and specificity. If the alternative method is negative and the clinical suspicion of autoimmune rheumatic disease is high, then it is mandatory to perform IIF assay .
For IIF, screening dilution should be determined locally, and not more than 5% of healthy controls should be positive at that dilution. As a rough estimate, a screening dilution of 1:100 is good. All test reports should mention the pattern ( Fig. 1 ), the titre (highest dilution at which the test is positive) and substrate used (Hep2 or Hep2000 cells). A positive ANA test should be followed by other tests to define the sub-specificities. The pattern of ANA can give some clue to the antigenic specificity as stated in Table 2 .
| Pattern of staining on cell line | Likely antigens |
|---|---|
| Nuclear | |
| Homogeneous | Histone, dsDNA, nucleosome |
| Rim | Lamin, nuclear-pore complex |
| Speckled | |
| Coarse | Sm, nRNP |
| Fine | Ro, La, topoisomerase-I |
| Nucleolar | RNA polymerase 1, PM-Scl, fibrillarin |
| Centromere | Centromeric proteins A, B, C |
| Cytoplasmic | Ribosomal P protein, t-RNA synthase including Jo-1, mitochondrial, actin, golgi |
| Disease | ANA pattern | Antigenic specificity | Prevalence (%) | Clinical significance |
|---|---|---|---|---|
| SLE | Homogeneous/rim | dsDNA | 50–75 | SLE, correlate with renal activity |
| Homogeneous | Histones | 50–70 | Drug-induced SLE (>95%) | |
| Speckled | Sm | 15–30 | Specific for SLE | |
| UIRNP | 30–40 | Raynaud’s phenomenon | ||
| La | 10–30 | Neonatal lupus (75%) | ||
| Ro | 25–50 | More common in SCLE, neonatal lupus | ||
| Systemic sclerosis | Speckled | Scl-70 | 20–60 | Diffuse SSc (70–76%) |
| Centromere | Centromere proteins A, B and C | 30–35 | Limited SSc (50–80%) | |
| Nucleolar | Fibrillarin, NOR-90, RNA-Pol 1 | <10 | – | |
| Rheumatoid arthritis | Homogeneous | Histones | 5–10 | – |
| Speckled | Ro | 3–10 | Associated with secondary Sjögren’s syndrome | |
| MCTD | Speckled | UIRNP | 100 | Diagnostic criterion |
| Primary Sjögren’s syndrome | Speckled | Ro | 40–70 | – |
| La | 30–60 | – | ||
| Polymyositis | Speckled | UIRNP | 4–15 | Overlap with SLE of MCTD |
| Nucleolar/ homogeneous | Ku | 5–12 | Overlap with systemic sclerosis |
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