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Article by

Ashish Rodricks

Bento Rui Mascarenhas



Despite improved understanding of mechanisms of disease, there is no gold standard for the diagnosis of autoimmune diseases. A clinician therefore needs to use a set of criteria for the diagnosis of anautoimmune disease. This article deals with the methods of detection of antinuclear antibodies, their interpretation and clinical significance. This knowledge will help clinicians in their approach to treating a patient with possible connective tissue disease.


Autoantibodies are a characteristic feature of autoimmune rheumatic diseases. The term anti-nuclear antibodies(ANA) refers to a diverse group of autoantibodies directed against one or more components of the cell nucleus, including nucleic acids themselves and proteins concerned with processing of DNA or RNA. This immune dysregulation occurs in genetically predisposed individuals who are exposed to certain insults such as infection or other unknown trigger factors.

But mere presence of autoantibodies is not synonymous with autoimmune diseases as these can be observed in otherwise healthy individuals. Autoantibodies in the presence of characteristic clinical features helps in the diagnosis of autoimmune disease. In the absence of a gold standard for the diagnosis of rheumatological diseases, they are best used as part of a diagnostic panel rather than as a marker indicating one particular disease[1].

The selection of specific antibody tests and interpretation of results requires an understanding of validity and reliability of the test requested. Validity can be assessed by parameters such as sensitivity, specificity, positive and negative predictive value. It is desirable to have high values for all these parameters. ANA has high sensitivity (>95%) for systemic lupus erythematosus (SLE) but low specificity (10-40%). ANA is therefore a good screening test. A negative ANA almost excludes SLE but a positive result does not confirm it. ANA negative lupus exists but is rare in adults. High ANA titres are of greater diagnostic significance but circulating levels are not associated with disease severity or activity.

The positive (PPV) and negative predictive values (NPV) are affected by pre-test probability and the prevalence of a disease in the population.For example, SLE is an uncommon disease with aprevalence of < 0.001% (1 in 1000 individuals) whereas the ANA, an autoantibody specific for SLE can be present in 5-8% of normal population. Therefore, if the ANA test is performed randomly in a normal individual and if the result is positive, the PPV will be <1% and NPV will be > 99%. But if the test is positive in a young female patient with characteristic clinical features of SLE like fever, rash, arthralgia, oralulcers, serositis, leucopenia etc., then the PPV reaches >90%[2]. ANA may be positive in several diseases as shown in Table 1.[3]

TC-Apr2017-015 - Table 1 - conditions with positive ANA

The nomenclature of ANA is complex. The antigens are named by:

a) Chemical structure e.g. double-stranded(ds)DNA

b) Disease association e.g. SS-A and SS-B in Sjogren’s syndrome

c) The individual in whom they were first described e.g. Ro, La, Sm

d) Their location in the nucleus (Figure 1) e.g. nucleolar, centromere

e) The particle where the antigen is found e.g. U1snRNP

Some antigens have more than one name (e.g. Ro and La are known as SS-A and SS-B respectively).[1]

TC-Apr2017-016 - location of the important nuclear artigens

Figure 1. Showing the location of the important nuclear artigens

Methods of detection of ANA:[2]

(1) Immunofluorescence assay (IF): mouse liver cells or Hep2 (human epithelial) cells fixed on glass slides are used as sources of antigens. Sera containing ANA’s will bind to specific nuclear antigens. The antigen-antibody binding is traced with anti-human immunoglobulin (Ig) tagged with fluorescent dye (isothiocyanate) when viewed under fluorescence microscope, the targeted antigen will appear apple green in colour. The intensity, pattern and dilution at which nuclear fluorescence disappears are noted.

The use of Hep2 cells increases the sensitivity of ANA detection because of the large size of nucleus and a good representation of most of the target antigens. Therefore, laboratories should be aware of the substrate used, i.e. animal tissue or cultured cell lines as this leads to possibility of false negative results in ANA screening. Hep2 cells coated onto slides are available commercially.[1]

(2)Enzyme-linked immunosorbent assay(ELISA): various nuclear and cytoplasmic antigens are separated and purified and coated onto wells of ELISA plates. Sera containing specific antibodies will bind to the target antigens and the binding is traced with enzyme-tagged antihuman Ig antibodies. The intensity of colour change in a positive reaction is proportional to antibody titre and is measured as optical density(OD) values which can be expressed as units of activity. Commercially produced ELISA based ‘ANAscreens’ are increasingly available with high variability between different manufacturers depending on antigens used.

(3)Immunoblot assay: the nuclear and cytoplasmic antigens are separated in a gel per their molecular weights and then transferred to membranes. A principle similar to ELISA is used to view the distinct bands of reactivity. The advantage of this method is that the presence of many autoantibodies in a patient can be simultaneously detected as set of bands (autoantibody profile).

(4) Immunoprecipitation: radiolabelled cell extracts are immunoprecipitated by reaction with test sera and then resolved by electrophoresis and analysed. This technique is restricted to research laboratories.


(1) Type of ANA: The detection of a positive staining pattern should lead to a more specific assay such as ELISA for the relevant antigens to confirm the diagnosis. For e.g., if ANA is negative but there is a strong clinical suspicion of SLE, then anti-dsDNA and anti-Sm antibodies may be estimated. AntidsDNA antibodies are highly specific for SLE(95%) but sensitivity is low(60% with ELISA and IF assay). This is valuable for confirming the diagnosis of SLE since in other autoimmune diseases anti-dsDNA is present in <5% only. ELISA assays are used to quantify antibodies to dsDNA. The antibody concentrations are useful in monitoring disease since very high titres are associated with more severe disease, including renal or central nervous system involvement. An increase in antibody titre may precede relapse, especially of nephritis or vasculitis. Antibodies to Sm are also specific for SLE and assist in confirming the diagnosis.Antibodies to extractabl e nuclear antigens (ENA) area group of antigens that leach from the cell nucleus when extracted with saline. These act as markers for certain connective tissue diseases. For example,antibodies to Ro occur in SLE and Sjogren’s syndrome (along with anti-La antibodies) and antibodies to ribonuclear protein (RNP) occur in mixed connective tissue disease. However, standardisation of these assays is difficult and has limitations. Therefore,specific antibody results must always be interpreted with caution and in the background of an appropriate clinical setting.[1]

(2) Staining Pattern of ANA: The fluorescence staining pattern gives some indication of the specificity of the antibodies in the sample.[1] There are some typical ANA staining patterns while others have little clinical significance

a. Homogenous staining pattern (Figure 2):Antibodies to ssDNA seen in 60% of SLE patients. They lack specificity and are also seen in other autoimmune rheumatic diseases and in viral infections.

TC-Apr2017-017 - Homogenous staining pattern on immunofluorescence as seen in antibodies to ssDNA

Figure 2. Homogenous staining pattern on immunofluorescence as seen in antibodies to ssDNA

b. Diffuse staining pattern: caused by antibodies to histones as seen in 95% of drug-induced lupus erythematosus and to Scl70 (orTopoisomerase-I) as seen in progressive systemic sclerosis.

c. Peripheral (Rim) pattern: antibodies to dsDNA (Figure 3).

TC-Apr2017-018 - Peripheral 'Rim' pattern on immunofluorescence as seen in antibodies to dsDNA

Figure 3. Peripheral ‘Rim’ pattern on immunofluorescence as seen in antibodies to dsDNA

d. Speckled pattern (Figure 4): caused by antibodies to extractable nuclear antigens (such as Sm) seen in 20% of patients with SLE, SS-A (or Ro antigen) seen in 60% of patients with primary Sjogren’s syndrome, SS-B protein seen in 40% of Sjogren’s syndrome and RNP.

TC-Apr2017-019 - Speckled pattern on immunofluorescence seen in antibodies to extractable nuclear antigens

Figure 4 . Speckled pattern on immunofluoroescence seen in antibodies to extractable nuclear antigens

e. Nucleolar pattern (Figure 5): antibodies to nucleolar RNA as seen in scleroderma and PM-SC I antigen as seen in 50% of patients with polymyositis-scleroderma overlap.

TC-Apr2017-020 - Nucleolar pattern on immunofluore-scence as seen in antibodies to nucleolar RNA

Figure 5 . Nucleolar pattern on immunofluorescence as seen in antibodies to nucleolar RNA

f. Centromere (seen as fine dots pattern): antibodies to centromeric proteins on chromosomes seen in limited cutaneous scleroderma and in the CREST variant of scleroderma.

g. Cytoplasmic: fine granular pattern caused by antibodies to Histidyl-t-RNA synthetase (Jo-I antigen) seen in 2% of Polymyositis. These are clearly not ANA’s, but they are considered as part of the ‘ANA group’ as they are seen in the screening test for ANA.

(3) ANA Titres: more than 1:160 dilution is significant but not diagnostic of autoimmune disease. Presence of very high concentrations of antibody (titer >1:640) is strongly suggestive of an autoimmune disorder. ANA titers are of little value in patients with obvious clinical signs of a systemic connective tissue disease.

(4) False positive ANA: seen in 1-5% of population, mostly in women, children and elderly patients. These are present in low concentrations and can be found non specifically in many infections (hepatitis C,tuberculosis, infectious mononucleosis, bacterial endocarditis , H I V, lymphoproliferative disorders, and inflammatory conditions (dermatomyositis).

Case Report:

A 25 – year- old female presents with inflammatory oligoarthritis affecting both knees and the right wrist joint for 2 years. She had a low-grade fever for 1 month prior to the onset of symptoms. There is no history of skin rash, photosensitivity, oral ulcers, Raynaud’s phenomenon or weight loss. She had a history of abortion at 3 months gestation. She has been on treatment with hydroxychloroquine for the past 2 years. Examination reveals synovitis of right wrist joint and no other abnormality.

Laboratory investigations: Blood counts and urine examination were normal, biochemical tests were normal, ESR elevated to 50mm/hr, CRP elevated to 6 mg/dl. Rheumatoid factor was negative, ANA (speckled) was positive, anti dsDNA was negative, and complement levels (C3& C4) were normal.

In retrospect, history of sicca symptoms were obtained and ophthalmological examination confirmed dry eyes. Anti-SSA & anti-SSB antibodies are positive. This patient fulfils the Revised International Classification Criteria for the diagnosis of Sjogren’s syndrome.


Sjogren’s is frequently misdiagnosed, underdiagnosed or diagnosed at late stages of the disease. Clinical diagnosis often takes 6-10 years. SLE, rheumatoid arthritis and systemic sclerosis have overlapping clinical and serological features and some patients with one autoimmune disease may develop additional autoimmune disorders, either concurrently or at a later stage of disease. Serological abnormalities such as presence of ANAs and/or Rheumatoid Factor leads to such misdiagnoses. ANAs are highly prevalent in patients with Sjogren’s syndrome(59-85%).[4]

The coexistence of systemic sclerosis and SLE ranges between 9-19%, up to 34% of SLE patients report sicca symptoms. [4] Furthermore, patients with primary Sjogren’s syndrome may meet many classification criteria for SLE. Some patients with Raynaud’s, sicca symptoms and anti-centromere antibodies may have criteria for both systemic sclerosis and Sjogren’s syndrome.It may be appropriate to term such conditions as ‘Overlap syndrome’.

Important Learning Points:

1. There should be a high index of suspicion of a connective tissue disease before asking for ANA as a screening test.

2. The ELISA method is cheaper and may be used in resource limited settings. If the diagnosis of SLE is strongly suspected and ANA test is negative, repeat the test by IF assay using cultured cell lines (Hep2 cells) or closely follow up the patient.

3. Do not repeat a strongly positive ANA test to confirm the diagnosis or assess response to treatment in a patient with classical clinical features of SLE. Use anti-dsDNA antibody titres to confirm the diagnosis and monitor response to treatment.

4. The intensity of ANA-IF staining does not correlate with severity of disease. Even a 1+ANA in high dilution (1:640) is very significant. However, ANA staining patterns may suggest diseases such as Sjogren’s syndrome.

5. There is little association between concentration of antibody to ENA’s and severity of disease. Do not keep requesting antibodies to ENA’s to assess response.

6. Anti-SSA antibodies is seen in 35% of patients with SLE and is associated with neonatal lupus and foetal heart block.

Ashish Rodricks, M.D. (Medicine) Consultant Physician and Rheumatologist, Menezes Polyclinic, Altinho, Panaji, Goa 403001. Email

Bento Rui Mascarenhas, M.D., F.A.C.P.,F.A.C.R Director,Burke Arthritis Center,New York, USA Visiting Consultant Rheumatologist, Menezes Polyclinic, Altinho, Panaji, Goa 403001. Email id:


1. Sheldon J. Laboratory testing in autoimmunerheumatic diseases. Best Practice and Research Clinical Rheumatology.2004;18(3):249-269th

2. Rao URK. Manual of Rheumatology. 4th edition. Indian Rheumatology Association;2014.

3. Davidson’s Principles and Practice of Medicine.22nd edition.Churchill Livingstone;2014.

4. Astrid R., Lida R., David L, Kiely G., DonaldUS, Erick K., et al. Previous diagnosis of Sjogren’s syndrome as rheumatoid arthritisor systemic lupus erythematosus. Rheumatology. 2016;55:1195-1201.