Antinuclear factor (ANF)

What sample is needed for Antinuclear Factor (ANF)?

1. This test is performed on the patient’s serum.
   • How to obtain a good serum sample: Collect 3 to 5 mL of blood in a disposable syringe or a Vacutainer. Keep the syringe at 37 °C for 15 to 30 minutes and then centrifuge for 2 to 4 minutes to obtain a clear serum.
2. No fasting or preparation is required.

What are the Indications for Antinuclear Factor (ANF)?

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

What are the precautions for Antinuclear Factor (ANF)?

1. Drugs may cause false-positive tests, such as 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 yield a positive result after a viral infection or some chronic infections.

How will you define Antinuclear factor (ANF)?

1. Antinuclear antibodies are produced in connective tissue diseases (autoimmune diseases) against various antigens in the nucleus, such as RNA, DNA, histones, and ribonucleoproteins.
2. Autoantibodies are directed against nuclear material (ANA) or cytoplasmic material, known as anti-cytoplasmic antibodies.

What are the major anti-nuclear antigens?

1. DNA (double and single-stranded).
2. Histones.
3. Nuclear proteins.
4. RNA
5. 95% of SLE patients show ANA.

What are the commonly used anti-nuclear antibodies (ANA) and their significance?

What is the significance of anti-nuclear factor (anti-nuclear antibody – ANA)?

1. This ANA is not a specific SLE test, so it must be supplemented by other tests. However, this is the most sensitive test, detecting SLE with a positivity rate of almost 95%.
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. The peripheral outline is only seen in SLE.
   3. The speckled pattern has been observed in other autoimmune diseases, including systemic lupus erythematosus (SLE), Sjögren’s syndrome, Scleroderma, Rheumatoid arthritis, and mixed connective tissue disease.
   4. The nuclear pattern is seen in Scleroderma and Polymyositis.

What is the pattern of ANA (immunofluorescence staining) in various diseases?

4. ANA is a 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 SLE.

What are the diseases with positive (%) antinuclear antibodies?

What is the significance of Antinuclear Factor (ANF) in diagnosis?

1. Indirect immunofluorescence is seen when the patient’s serum (antibody) is combined with the cells.
2. The EIA technique may replace indirect immunofluorescence.
   1. ANA has a sensitivity of 99%. A negative ANA test almost excludes active systemic lupus erythematosus (SLE).
   2. This test may be positive for unrelated diseases in 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 the specificity is around 80%.
      1. When the cutoff value is 1:160, then the 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.

What is the procedure for the Antinuclear Factor (ANF)?

1. Take 5 to 10 mL of the patient’s blood.
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).

What is the normal Antinuclear Factor (ANF) or ANA?

Source 2

• These are negative.
  • Negative at 1:20 dilution.
• When a dilution of the serum is performed, a titer of more than 1:32 is considered 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 of >3 on ELISA and ≥1:160 by IFA now requires follow-up testing for specific autoantibodies.

How will you interpret Antinuclear Factor (ANF)?

1. A positive test does not confirm the disease because its low titers are also seen in older and healthy individuals.
2. It helps diagnose Autoimmune diseases, particularly Systemic lupus erythematosus (98%), but with poor specificity.
3. This test is positive for 30% to 50% of other autoimmune diseases such as Rheumatoid arthritis, Sjogren’s syndrome (70%), 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 a titer of >1:160 is considered positive.
   3. 5% of the SLE cases show persistently negative results.
7. ANA may become negative in the remission of SLE.

• If ANA is negative, then SLE can be excluded.

The positive or increased level of Antinuclear Factor (ANF) is seen in the following:

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.

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