Anti-nuclear antibodies: A practical approach to testing and interpretation
Anti-nuclear antibodies (ANAs) are a group of antibodies that target macromolecules which bind DNA, RNA, proteins, and their complexes.[1,2] Although they bind cytoplasmic components too, the term ANA was retained for historical reasons. Characteristically associated with connective tissue diseases (CTD), ANA may also be detected in other conditions and even in general population.
LE cell phenomenon was described by Hargraves, Richmond, and Morton in 1948. Kidney or liver sections from rats or mice were used as substrates initially to detect ANAs with indirect immunofluorescence antibody (IFA) technique, which are now replaced with human epithelial type-2 (HEp-2) cells.[6-8]
Techniques to detect ANA
The methods to detect ANA can be broadly classified as indirect immunofluorescence technique and solid-phase assays.
Indirect IFA technique/fluorescent ANA (FANA)
The method of choice for the detection of ANA is the indirect IFA technique, otherwise known as FANA technique.[2,9-11] It involves incubation of serum or plasma with either a tissue section or a cell line fixed to a glass slide. Large nuclei of HEp-2 cells make the detection of fluorescence staining patterns easier. Serial dilutions of positive samples are tested to obtain an endpoint titer, and dilution prior to this endpoint is reported as ANA titer.[1,2,4,6] Three parameters are evaluated – the pattern of fluorescence, substrate used, and the titer of a positive test [Figure 1].
Interpretation of ANA titer by immunofluorescence
ANA titer is the quantitative expression of ANA concentration, in dilution. There is a lack of consensus regarding recommended initial dilution for screening purpose, although a titer of 1:160 is taken as significant. Lower titers are also suggested by some experts.[5,6,10-13]
The staining patterns of ANA on HEp-2 cells can be broadly divided into nuclear, cytoplasmic, and mitotic patterns [Tables 1 and 2]. The intensity of staining expressed in qualitative scale + to ++++ should also be reported, as fluorescence intensity is considered proportional to antibody concentration, which may predict the severity of the CTD. Although staining patterns may provide a clue to the underlying CTD, they cannot be considered specific.[4,14]
Speckled (fine and coarse)
Cytoskeletal filaments (actin, vimentin, and cytokeratin)
|Mitotic patterns||Mitotic spindle
IFA: Indirect immunofluorescence antibody test, PCNA: Proliferating cell nuclear antigen, NuMA: Nuclear mitotic apparatus, CENP-F: Centromere protein F precursor
|ANA pattern||Antigen||Associated diseases|
|Homogenous/diffuse||dsDNA, nucleosomes, histones||SLE, juvenile idiopathic arthritis, drug-induced LE|
|Speckled/granular||ENA, RNP, Sm, SSA/Ro, SSB/La, Scl-70, Jo-1, ribosomal-P||SLE, mixed CTD, systemic sclerosis, primary Sjogren’s syndrome, polymyositis|
|Peripheral||RNP, Sm, Ro/SSA||SLE, systemic sclerosis|
|Nucleolar||Anti-PM-Scl, anti-RNA polymerase I-III, anti-U3-RNP||Systemic sclerosis, polymyositis|
|Centromere||CENP A-E||Limited cutaneous systemic sclerosis|
IF-ANA: Immunofluorescence antinuclear antibody, SLE: Systemic lupus erythematosus, LE: Lupus erythematosus, CTD: Connective tissue disease, ENA: Extractable nuclear antigen, DNA: Deoxyribonucleic acid, RNP: Ribonucleoprotein, RNA: Ribonucleic acid, PM: Polymyositis, CTD: Connective tissue disease, CENP: Centromere protein
Advantages of IFA technique for ANA
The specificity is low – approximately 43% at 1:40 dilution and 63% at 1:80 dilution
HEp-2 cells may lack some antigens, such as the Ro-60 antigen and the ribosomal-P
It is labor and skill intensive.
A panel of purified autoantigens is prepared, and each antigen is immobilized on a solid surface (microtiter plate, fluorescent microsphere, or membrane). Diluted human serum is incubated with the immobilized antigen and a secondary antibody is used to detect bound autoantibodies. These include:
Enzyme-linked immunosorbent assay [Figure 2]
Multiplex bead-based assays
Advantages of solid-phase assays
They are suitable for high throughput testing
Semi-quantification of results is possible
Automation can increase efficiency and decrease labor cost
Simultaneous identification of the responsible autoantibody.
Disadvantages of solid-phase assays
Many laboratories provide a bouquet of antibody tests in addition to FANA. The components of this panel can be different in different laboratories, but usually include antibodies to single-stranded and double-stranded DNA, ribonucleoprotein antigens, Ro, La, Sm, and topoisomerase I (Scl-70) antigens. ANA profile has higher sensitivity than FANA. ANA profile can also provide additional diagnostic and prognostic information depending on the type of antibody present. Different laboratories use different techniques for the performance of ANA profile, and unfortunately, the performance of many of these tests is not consistent always.
Fallacies of ANA testing
Although rheumatic diseases are quite uncommon in general population, the frequency of ANA positivity determined by IFA in an otherwise healthy population can be high. Most people who are ANA positive will never develop a rheumatic disease.
Just like any other test, the higher the pre-test probability that a patient has a rheumatological disease, the more likely that an ANA test will be of use in establishing the diagnosis. Hence, ANA should not be used as a screening tool for asymptomatic individuals. Indiscriminate testing will cause positive results in 5% at the predetermined screening dilution (usually 1:160). A positive ANA test is important only in conjunction with clinical evaluation. A positive result of ANA or diagnosis of SLE should not lead to all clinical features being interpreted as manifestations of SLE (Greenwald’s law of lupus).
However, screening of patients could be valuable for identifying individuals prone to develop SLE who have a pre-autoimmunity state, where abnormalities of the immune system cause autoreactivity, but clinical disease is yet to manifest.[1,19,20] The titer and binding pattern of ANAs through IFA and the identification of specific ANAs such as anti-dsDNA, anti-Ro60, or anti-La antibodies may be helpful in such situations.[1,17,21]
Conditions other than CTDs which cause positive ANA
|Liver disease||Chronic hepatitis
|Other autoimmune diseases||Type 1 diabetes
ANA testing: Which test to order first
When to test for ANA
ANA test must be performed only when there is a clinical suspicion of an autoimmune CTD. If screening test for ANA is positive, further tests for specific autoantibodies must be ordered, based on the clinical features and possible diagnosis. If ANA screening is negative, patient must be reevaluated and kept under follow-up, and test must be repeated only if clinically indicated. If ANA is negative in a patient with a high degree of clinical suspicion for SLE, ANA may be repeated. If negative again, anti-SSA antibody may assist in diagnosis of ANA-negative SLE.[4,25]
When not to test for ANA
For confirming rheumatoid arthritis or osteoarthritis, as it is not considered helpful
To evaluate fatigue, back pain, or other musculoskeletal pain, unless they are accompanied by clinical features suggestive of a CTD
For screening of asymptomatic individuals
ANA tests do not need to be repeated, as changes in ANA titer are not of value in monitoring disease activity
Negative tests need to be repeated only if there is a strong suspicion of an evolving CTD, or a change in the patient’s illness that suggests a revision in diagnosis.
Several cases of ANA negative lupus have been published, and its prevalence may be as high as 5%–10%. The causes of ANA-negative lupus include antigen-deficient substrate and leaching of antigens, concurrent immunosuppressive treatment, persistent renal loss of proteins, and a false diagnosis of lupus.
ANA testing is an important prong in the diagnosis of rheumatological diseases, but in itself has no value in the absence of clinical correlation. A standalone positive or negative ANA test offers nothing to the patient or clinician, and hence, an awareness regarding the judicious use of the same is imperative in clinical practice.
Declaration of patient consentNot required as there are no patients in this article.
Financial support and sponsorshipNil.
Conflicts of interestDr Kidangazhiathmana Ajithkumar is on the editorial board of the Journal.
- Proc Staff Meet Mayo Clin. 1948;23:25-8.Presentation of two bone marrow elements; the tart cell and the L.E. cell.
- [Google Scholar]
- J Rheumatol. 1985;12:265-9.Clinical comparison of cultured human epithelial cells and rat liver as substrates for the fluorescent antinuclear antibody test.
- [Google Scholar]
- Clin Exp Rheumatol. 1994;12:137-41.The prevalence of antinuclear antibodies in healthy young persons and adults, comparing rat liver tissue sections with HEp-2 cells as antigen substrate.
- [Google Scholar]
- Indian J Med Res. 2007;126:34-8.Antinuclear antibodies by indirect immunofluorescence: Optimum screening dilution for diagnosis of systemic lupus erythematosus.
- [Google Scholar]
- Arthritis Rheum. 1991;34:8.Autoantibody profile testing by immunodiffusion (ID) and hemagglutination (HA): Comparison with routine immunofluorescent antinuclear antibody (FANA) testing.
- [Google Scholar]
- Arch Pathol Lab Med. 2000;124:71-81.Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear antigens, American College of Pathologists.
- [Google Scholar]
- Techniques to detect ANA
- Indirect IFA technique/fluorescent ANA (FANA)
- Interpretation of ANA titer by immunofluorescence
- STAINING PATTERNS
- SOLID-PHASE ASSAYS
- Advantages of solid-phase assays
- Disadvantages of solid-phase assays
- ANA profile
- Fallacies of ANA testing
- Conditions other than CTDs which cause positive ANA
- ANA testing: Which test to order first
- When to test for ANA
- When not to test for ANA
- ANA-negative lupus