Antinuclear Factor (ANF), Antinuclear Antibody ANA)
- This test is done on the serum of the patient.
- How to get good serum: Take 3 to 5 ml of blood in the disposable syringe or in vacutainer. Keep the syringe for 15 to 30 minutes at 37 °C and then centrifuge for 2 to 4 minutes to get clear serum.
- No fasting or preparation is required.
Purpose of the test (Indications)
- For the diagnosis of Systemic lupus erythematosus (SLE).
- Positive in other autoimmune diseases.
- Drugs may cause false positive tests like aminosalicylic acid, chlorothiazide, procainamide, hydralazine, acetazolamide, penicillin, phenytoin sodium, and griseofulvin.
- Drugs may cause a false negative test like steroids.
- This test may be positive after the viral infection and some of the chronic infection.
- The antinuclear antibodies are produced in connective tissue diseases (autoimmune diseases ) against various antigens in the nucleus like RNA, DNA, histones, and ribonucleoprotein.
- Autoantibodies are directed against nuclear material (ANA) or against cytoplasmic material called anti-cytoplasmic antibodies.
- The major antinuclear antigens are:
- DNA (double and single-stranded).
- Nuclear proteins.
- 95% of SLE patients shows ANA.
- This ANA is not specific for SLE, so it has to be supplemented by other tests.
- Fluorescent staining under an ultraviolet microscope shows different patterns and increase the specificity of this test.
- Fluorescent patterns show different staining in the nucleus e.g.:
- The homogeneous pattern is seen in SLE and mixed connective tissue disease.
- Peripheral outline only is seen in SLE.
- The speckled pattern has seen other autoimmune diseases like SLE, Sjogren’s syndrome, Scleroderma, Rheumatoid arthritis., and mixed connective tissue disease.
- The nuclear pattern is seen in Scleroderma and Polymyositis.
Table showing the pattern of ANA (immunofluorescence staining) in various diseases:
|Diseases||Homogenous pattern||Peripheral Pattern||Speckled pattern||Nucleolar pattern|
|SLE||+ positive||+ positive||+ positive||+ positive|
|Mixed connective disease||+ positive||+ positive|
|Scleroderma||+ positive||+ positive|
|Rheumatoid arthritis||+ positive|
|Sjogren’s syndrome||+ positive|
|Polymyositis||+ positive||+ positive|
- ANA is gamma globulin and belongs to more than one type of immunoglobulin.
- There are ANA-negative cases of SLE.
- Some believe that negative ANA excludes the SLE.
- Diseases with positive antinuclear antibodies.
|Diseases||Positivity of ANA %||another source positivity %|
|SLE||95||90 to 100|
|Mixed connective tissue disease||100|
- Indirect immunofluorescence, where the patient serum (antibody) has combined with the cells.
- EIA technique may replace the indirect immunofluorescence.
- ANA has a sensitivity of 99%. A negative ANA test almost excludes the active SLE.
- This test may be positive in unrelated diseases of the patients.
- Around 20% of the normal population has a titer of 1:40.
- Around 5% of the normal population may have a titer of 1:160
- When cutoff titer is 1:40 then specificity is around 80%.
- When the cutoff value is 1:160, then specificity is around 95%.
- ANA is nonspecific, individual with increasing age show a false positive result.
- 50% positive by the age of 80 years with a low titer.
- These are negative.
- Negative at 1:20 dilution.
- When done with a dilution of the serum then titer of more than 1:32 is positive.
- Negative by ELIZA and IFA method.
- If positive by IFA, the sample is titrated and the pattern is reported.
- A strong positive result, that is >3 on ELIZA and ≥1:160 by IFA, now needs follow-up of specific autoantibodies.
- A positive test does not confirm the disease because its low titers are seen in old people and in some healthy normal person.
- It helps in the diagnosis of Autoimmune diseases and particularly Systemic lupus erythematosus (98%) but with poor specificity.
- This test is positive 30 to 50% in other autoimmune diseases such as Rheumatoid arthritis, Sjogren’s syndrome (70%) and Polymyositis and other related diseases.
- If ANA negative then SLE can be excluded.
The positive or increased level of ANA is seen in:
- Rheumatoid arthritis.
- Polyarteritis Nodosa
- Sjogren’s syndrome.
- Other autoimmune diseases.
- Chronic hepatitis.
- Multiple sclerosis.
The nucleus is pushed by the antinuclear antibody.