Antinuclear Factor (ANF), Antinuclear Antibody (ANA) and Its Significance
Antinuclear factor (ANF)
- 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 a vacutainer. Keep the syringe for 15 to 30 minutes at 37 °C and then centrifuge for 2 to 4 minutes to get the clear serum.
- No fasting or preparation is required.
Purpose of the test (Indications) for Antinuclear Factor (ANF)
- For the diagnosis of Systemic lupus erythematosus (SLE).
- Positive in other autoimmune diseases.
Precautions for Antinuclear Factor (ANF)
- 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 chronic infections.
Pathophysiology of Antinuclear Factor (ANF)
Definition of Antinuclear factor:
- 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 anti-nuclear antigens are:
- DNA (double and single-stranded).
- Nuclear proteins.
- 95% of SLE patients show ANA.
Some of the commonly used anti-nuclear antibodies (ANA) and their significance:
|Type of the Anti-nuclear antigen and antibody (ANA)||ANA and their diagnostic value|
|Anti-nuclear Smith (sm) antibody||
|Anti-SS-A and Anti-SS-B||
|Histones (anti-histone antibodies)||
|Cytoplasmic antigens (anticytoplasmic Ab RNA and others)||
Significance of anti-nuclear factor (anti-nuclear antibody – ANA):
- This ANA is not a specific test for SLE, so it has to be supplemented by other tests. But this is the most sensitive test detecting the SLE at almost >95% positivity.
- Fluorescent staining under an ultraviolet microscope shows different patterns and increases 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 been seen in 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 of positivity %||Another source of positivity %|
|SLE||95||90 to 100||>95|
|Drug-induced lupus erythematosus||>95|
|Sjogren’s syndrome||60||85||75 to 90|
|Scleroderma||70||88||70 to 90|
|Dermatomyositis||30||40 to 60|
|Mixed connective tissue disease||100||>95|
|CREST syndrome||70 to 90|
Lab diagnostic role of Antinuclear Factor (ANF):
- 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 the 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; individuals with increasing age show a false-positive result.
- 50% positive by the age of 80 years with a low titer.
- Procedure for the ANA:
- Take 5 to 10 mL of the blood of the patient.
- Traumatize the RBCs with the glass rod or glass beads.
- Incubate for 15 to 30 minutes at 37 °C.
- Centrifuge and make the smear from the buffy coat.
- Screen the slide to find the SLE cell (LE cell phenomenon).
Normal Antinuclear Factor (ANF) or ANA
- These are negative.
- Negative at 1:20 dilution.
- When done with a dilution of the serum, then a 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.
Interpretations of Antinuclear Factor (ANF)
- A positive test does not confirm the disease because its low titers are seen in old and healthy normal people.
- It helps diagnose Autoimmune diseases, 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.
- Positive ANA without other S/S is not diagnostic.
- The high titer is often associated with SLE, and titer <1:160 is not diagnostic.
- The titer of <1:40 is considered negative.
- The titer of 1:40 to 1:80 is considered low positive.
- While titer of >1:160 is considered positive.
- 5% of the SLE cases show persistently negative results.
- ANA may become negative in remission of SLE.
- If ANA is 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.
Antinuclear factor (ANF): Le cell phenomenon: Nucleus is pushed to the peripheryQuestions and answers: