ANA test was an indirect immunofluorescence method for detection of serum anti-nuclear antibody detection. Serum was diluted in PBS at the ratio of 1:80. Serum was loaded on slides pre coated with monolayer of Hep-2 cells (Binding Site Ltd., Birmingham, UK). After 30 minute serum incubation at room temperature and 10 minutes washing in PBS. Detection agent, goat anti mouse IgG (gamma - chain specific) FITC conjugate (Southern Biotechnology Associates Inc., Birmingham, UK), were added to the slides for additional 30 minutes incubation.  After 2hours the slides were viewed under fluorescence microscope (Nikon Instech Co.Ltd., Kanagawa, Japan) and graded according to the strength.