ANA Test

What is being tested?

Antinuclear antibodies (ANA) are a group ofantibodies produced by a person's immune system when it fails to adequately distinguish between "self" and "nonself." These antibodies, known as auto antibodies, attack the body's own healthy cells and cause autoimmune disease.The ANA test identifies the presence of these auto antibodies in the blood that target antigens contained inside the nucleus.

The ANA test is ordered when someone shows signs and symptoms that are associated with a systemic autoimmune disease such as systemic lupus erythematosus (SLE)and Sjogren syndrome , or ruling out other conditions with similar signs and symptoms. When is it ordered?

There are different methods to detect ANA. Two common methods are commonly performed:

Laboratory methods used to detect ANA: -Immunoassays: enzyme linked immunosorbent assay (ELISA). -Indirect immunofluorescent technique (IIF): is considered the gold standard semi-quantitative method.

The procedure is carried out in two basic reaction steps:Step 1 - Human serum is reacted with human epithelial laryngeal carcinoma cells (HEp-2) that are affixed to a slide. Antibodies, if present, will bind to the antigens forming stable antigen-antibody complexes. If no antibodies are present, the complex will not be formed and serum components will be washed away.Step 2 - Fluorescein labeled (FITC) antihuman antibodies are added to the reaction site which bind with the complexes formed in step one. This results in a positive reaction of bright apple-green fluorescence when viewed with a fluorescence microscope. Principle of the IIF technique:

Figure 1: Indirect immunofluorescence technique.

Figure 2: ANA test by IIF ( tissue used is HEp-2 which have been shown to have greater sensitivity than tissue sections and yield sharper pattern recognition).

QUALITY CONTROL:Both a positive and negative control should be included with each assayrun. positive control: is used as a monitor of assay sensitivity and pattern identification. Negative control: there should be no distinct pattern visible or any clear difference between the nucleus and cytoplasm .

Figure 3: Negative control (left): Cells display low level non-specific fluorescence, but no specific nuclear staining. Positive control (right): Cells display apple green nuclear fluorescence.

INTERPRETATION OF RESULTS: Four major staining patterns have been described and associated with different autoimmune disorders.

Figure 4: Diffuse staining of the entire nucleus. Homogeneous (diffuse) pattern:

Homogeneous (diffuse) pattern :

Nuclear antigens: ds DNA and histone or histone alone .Disease association:High titers are suggestive of SLE. Antibody to histone alone has a high association with drug-induced lupus and may be ordered to support the diagnosis.

2. Peripheral (rim) pattern: Figure 5 :Smooth staining primarily around the outer region of the nucleus with weaker staining in the center.

Peripheral (rim) pattern:Nuclear antigens: dsDNA.Disease association: High titers to dsDNA are suggestive of active SLE.

3. Speckled pattern :Figure 6: Fluorescent aggregates throughout the nucleus.

Speckled pattern :Figure 7: Fluorescent aggregates throughout the nucleus.

Nuclear antigens: extractable nuclear antigens [Smith antigen, ribonucleoprotein, Ro, La and Scleroderma-70]. Disease association: Scleroderma. Sjogrens syndrome. Mixed connective tissue disease. Rheumatoid arthritis (RA). SLE.

Speckled pattern :

4. Nucleolar pattern: Figure 8: Fluorescent staining of the nucleoli within the nucleus.

Nucleolar pattern:Figure 9: Fluorescent staining of the nucleoli within the nucleus.

Nuclear antigens: nucleolar antigens.Disease association:Scleroderma.Dermatomyositis and poly myositis.Sjogrens syndrome.Less common in SLE and RA.Nucleolar pattern: