The gold standard for the determination of ANA is the indirect immunofluorescence test (IIFT) with human epithelial cells (HEp-2), which is known for its high sensitivity and specificity. Positive and negative samples produce a large signal difference. In the microscopic evaluation it is possible to establish precisely how an indicator dye (generally fluorescein) is distributed in the tissue or the cells. A typical fluorescence pattern is produced for every bound autoantibody, depending on the location of the individual autoantigens.

The first international consensus on standardised nomenclature of HEp-2 cell patterns in indirect immunofluorescence (ICAP, www.anapatterns.org) defined fifteen nuclear patterns and nine cytoplasmic patterns which are relevant for the diagnosis of various autoimmune diseases.

Furthermore, the consensus recommends that autoantibodies detected in indirect immunofluorescence be confirmed by additional specific tests (e.g. ELISA, line blot). The exclusive use of these monospecific test methods is inadequate for the determination of autoantibodies against cell nuclei, as not all relevant antigens are available in a purified form as yet. Thus, the corresponding ANA can only be detected by IIFT.