Antinuclear Factor (ANF), Antinuclear Antibody ANA)

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 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)

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 infection.

Pathophysiology

1. The 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 antinuclear antigens are:
   1. DNA (double and single-stranded).
   2. Histones.
   3. Nuclear proteins.
   4. RNA
      1. 95% of SLE patients shows ANA.

4. This ANA is not specific for SLE, so it has to be supplemented by other tests.
5. Fluorescent staining under an ultraviolet microscope shows different patterns and increase 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 seen 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.

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 %
SLE	95	90 to 100
Sjogren’s syndrome	60	85
Scleroderma	70	88
Dermatomyositis	30
Rheumatoid arthritis	30	55
Polyarteritis	10
Juvenile arthritis		22
Mixed connective tissue disease		100

Lab diagnosis

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 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, individual 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 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 people and in some healthy normal person.
2. It helps in the diagnosis of Autoimmune diseases and 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.

• If ANA 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.

The nucleus is pushed by the antinuclear antibody.

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