Indirect ImmunoFluorescence (IIF)

Connective tissue diseases (CTD) are autoimmune disorders characterized by a chronic inflammatory process involving different organs. Antinuclear Antibodies (ANA) directed against a variety of nuclear antigens are detectable in the serum of patients with many rheumatic and non-rheumatic diseases.

In last years immunoenzymatic method (ELISA) diagnostic kits for ANA have been introduced; the procedure is relatively simple and may be automated, but this method has shown lesser sensitivity and reliability than IIF ANA test.

The recommended method for ANA testing remains indirect immunofluorescence (IIF). That is considered a powerful, sensitive, and comprehensive test for screening autoantibodies and is still the most used screening assay.

In IIF a serum sample is tested with a substrate containing a specific antigen; the antigen-antibody reaction will be revealed by fluorochrome conjugated anti-human immunoglobulin antibodies. The slide is examined by fluorescence microscope. For appropriate IIF ANA tests, current guidelines recommend the use of tumor cell line (HEp-2) substrate, which increases the method sensitivity and facilitates the identification of specific antibody patterns, when compared to differentiated tissue cells (rodent organ sections, mostly used in the past).

The usefulness of ANA tests depends on the clinical situation. If the clinical history and physical examination reveal symptoms or signs suggestive of CTD, a positive ANA test contributes to the diagnosis. In addition, as many CTD have common clinical manifestations, the laboratory may play a fundamental role in formulating the correct diagnosis.

The fluorescence intensity classification is scored semi-quantitatively from 1+ to 4+ relative to the intensity of a negative and a positive control (4+) contained in each slide, by following the guidelines established by the Centers for Disease Control and Prevention, Atlanta, Georgia (CDC):

• 4+ brilliant green (maximal fluorescence);
• 3+ less brilliant green fluorescence
• 2+ defined pattern but dim fluorescence;
• 1+ very subdued fluorescence;

So ANA presence in serum should be measured by a titre (i.e. 1:80, 1:160, 1:320 etc)

There are more than 30 nuclear antigen-antibody (Ab-Ag) specificities that have been identified. Often the specificity Ag-Ab is associated with a specific staining pattern in IIF, which may have diagnostic value in differentiating between types of CTD.