Antinuclear factor (ANF)

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) for Antinuclear Factor (ANF)

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

Precautions for Antinuclear Factor (ANF)

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 chronic infections.

Pathophysiology of Antinuclear Factor (ANF)

Definition of Antinuclear factor:

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.

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.

Some of the commonly used anti-nuclear antibodies (ANA) and their significance:

Significance of anti-nuclear factor (anti-nuclear antibody – ANA):

1. 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.
2. Fluorescent staining under an ultraviolet microscope shows different patterns and increases the specificity of this test.
3. 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:

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

Diseases with positive antinuclear antibodies:

Lab diagnostic role of Antinuclear Factor (ANF):

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.
4. Procedure for the ANA:
   1. Take 5 to 10 mL of the blood of the patient.
   2. Traumatize the RBCs with the glass rod or glass beads.
   3. Incubate for 15 to 30 minutes at 37 °C.
   4. Centrifuge and make the smear from the buffy coat.
   5. Screen the slide to find the SLE cell (LE cell phenomenon).

Le cell phenomenon: Nucleus is pushed to the periphery

Normal Antinuclear Factor (ANF) or ANA

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.

Interpretations of Antinuclear Factor (ANF)

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.

Antinuclear factor (ANF): Le cell phenomenon: Nucleus is pushed to the peripheryQuestions and answers: