Antinuclear Factor (ANF), Antinuclear Antibody (ANA) and Its Significance

Sample

• 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)

1. For the diagnosis of Systemic lupus erythematosus (SLE).
2. Positive in other autoimmune diseases.

Precautions

1. Drugs may cause false-positive tests like aminosalicylic acid, chlorothiazide, procainamide,  hydralazine, acetazolamide, penicillin, phenytoin sodium, and griseofulvin.
2. Drugs may cause a false-negative test like steroids.
3. This test may be positive after the viral infection and some of the chronic infections.

Pathophysiology

1. Antinuclear antibodies are produced in connective tissue diseases (autoimmune diseases ) against various antigens in the nucleus like RNA, DNA, histones, and ribonucleoprotein.
2. Autoantibodies are directed against nuclear material (ANA) or against cytoplasmic material called anti-cytoplasmic antibodies.
3. The major anti-nuclear antigens are:
   1. DNA (double and single-stranded).
   2. Histones.
   3. Nuclear proteins.
   4. RNA
   5. 95% of SLE patients show ANA.
      

      Antinuclear antigens and Antibodies

      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
      ssDNA (Anti-ssDNA)	It is seen in many rheumatic diseases. This has no diagnostic value.
      ds-DNA (Anti-dsDNA)	This is specific to the SLE. SLE has 50% positivity, and titer correlates with the activity of the disease. It is rare in other autoimmune diseases.
      Nuclear proteins Soluble nuclear proteins (-sNP) Anti-basic nuclear protein (Histone) Anti-acidic nuclear proteins. Extractable nuclear antigen (ENA) includes: Smith (sm) Ribonucleoprotein (RNP) Sjogren’s syndrome A and B SS-A SS-B	This is specific to the SLE. It is only seen in 30% to 40% of SLE cases.
      Anti-nuclear Smith (sm) antibody	It is positive in 30% (range 20% to 40%) of the SLE cases. 8% in connective tissue disease. It is negative in other collagen diseases.
      Anti-RNP antibody	It is 100% positive in mixed connective disease. SLE shows in 25% of the cases. Scleroderma shows in 25% of the cases. It has a speckled ANA pattern.
      Antinucleolar antibody	It is seen in 55% of progressive systemic sclerosis. SLE patients show in 25% of the cases. Rheumatoid arthritis patients show in 10% of the cases.
      Anti-sNP antibody	It is reported in 50% of the SLE cases. <10% seen in rheumatoid arthritis. Also seen in <10% of Sjogren’s syndrome and mixed connective tissue disease. It shows a solid homogenous ANA pattern.
      Anti-SS-A and Anti-SS-B	These will react with nuclear antigens extracted from the human B-L. SS-A is found in 70% of Sjogren’s syndrome without RA. SS-B is found in 50% of Sjogren’s syndrome without RA. <5% Sjogren’s syndrome cases are seen with RA.
      Histones (anti-histone antibody)	This may be seen in autoimmune diseases. It is useful to differentiate drug-induced lupus from the idiopathic form. Antihistones antibodies are specific for drug-induced lupus.
      Cytoplasmic antigens (anticytoplasmic Ab RNA and others)	It is seen in Primary biliary cirrhosis. It may also be seen in chronic active hepatitis.
      Anti centromere antibody	This is suggestive of CREST syndrome.

4. 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 almost >95% positivity.
5. Fluorescent staining under an ultraviolet microscope shows different patterns and increases the specificity of this test.
6. Fluorescent patterns show different staining in the nucleus, e.g.:
   1. The homogeneous pattern is seen in SLE and mixed connective tissue disease.
   2. Peripheral outline only is seen in SLE.
   3. The speckled pattern has been seen in other autoimmune diseases like SLE, Sjogren’s syndrome, Scleroderma, Rheumatoid arthritis., and mixed connective tissue disease.
   4. The nuclear pattern is seen in Scleroderma and Polymyositis.
      

      ANA pattern by the fluorescent method

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

7. ANA is gamma globulin and belongs to more than one type of immunoglobulin.
8. There are ANA-negative cases of SLE.
   1. Some believe that negative ANA excludes the SLE.

• Diseases with positive antinuclear antibodies.

Diseases	Positivity of ANA %	another source positivity %	Another source 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
Rheumatoid arthritis	30	55	Rare
Polyarteritis	10
Juvenile arthritis		22
Mixed connective tissue disease		100	>95
CREST syndrome			70 to 90

Lab diagnosis role of ANA:

1. Indirect immunofluorescence, where the patient serum (antibody)  has combined with the cells.
2. EIA technique may replace the indirect immunofluorescence.
   1. ANA has a sensitivity of 99%. A negative ANA test almost excludes the active SLE.
   2. This test may be positive in unrelated diseases of the patients.
   3. Around 20% of the normal population has a titer of 1:40.
      1. Around 5% of the normal population may have a titer of 1:160
   4. When the cutoff titer is 1:40, then specificity is around 80%.
      1. When the cutoff value is 1:160, then specificity is around 95%.
3. ANA is nonspecific; individuals with increasing age show a false-positive result.
   1. 50% positive by the age of 80 years with a low titer.

Normal

Source 2

• 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.

Source 4

• 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.

Significance

1. A positive test does not confirm the disease because its low titers are seen in old and healthy normal people.
2. It helps diagnose Autoimmune diseases, particularly Systemic lupus erythematosus (98%) but with poor specificity.
3. 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.
4. Positive ANA without other S/S is not diagnostic.
5. The high titer is often associated with SLE, and titer <1:160 is not diagnostic.
6. The titer of <1:40 is considered negative.
   1. The titer of 1:40 to 1:80 is considered low positive.
   2. While titer of >1:160 is considered positive.
   3. 5% of the SLE cases show persistently negative results.
7. 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:

1. SLE.
2. Rheumatoid arthritis.
3. Polyarteritis Nodosa
4. Dermatomyositis.
5. Sjogren’s syndrome.
6. Other autoimmune diseases.
7. Cirrhosis.
8. Chronic hepatitis.
9. Leukemia.
10. Scleroderma.
11. Multiple sclerosis.
12. Infections.
13. Malignancies.
14. Fibromyalgia.
    

    Le cell phenomenon (The antinuclear antibody pushes the nucleus)

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