Indirect immunofluorescence is one of the most commonly used tests for ANAs. Typically, HEp-2 cells are used as a substrate to detect the antibodies in human serum. Microscope slides are coated with HEp-2 cells and the serum is incubated with the cells. If the said and targeted antibodies are present then they will bind to the antigens on the cells; in the case of ANAs, the antibodies will bind to the nucleus. These can be visualised by adding a fluorescent tagged (usually FITC or rhodopsin B) anti-human antibody that binds to the antibodies. The molecule will fluoresce when a specific wavelength of light shines on it, which can be seen under the microscope. Depending on the antibody present in the human serum and the localisation of the antigen in the cell, distinct patterns of fluorescence will be seen on the HEp-2 cells. Levels of antibodies are analysed by performing dilutions on blood serum. An ANA test is considered positive if fluorescence is seen at a titre of 1:40/1:80. Higher titres are more clinically significant as low positives (≤1:160) are found in up to 20% of healthy individuals, especially the elderly. Only around 5% of the healthy population have ANA titres of 1:160 or higher.