BACKGROUND AND OBJECTIVE: The objective of this study was to investigate the antibody to RBC in autoimmune disease patients with ANA using sensitive Coombs test based on flow cytometry method. MATERIALS AND METHODS: Antinuclear antibodies (ANA) of autoimmune disease patients were added to red blood cells (RBCs) of blood group O. At the same time, healthy individuals' serums were also checked. The sample tubes were incubated for 30 min at 37°C. After incubation, each sample was analyzed on flow cytometry. RESULTS: The agglutination of antinuclear antibodies in autoimmune patients was observed, while in healthy people, there was no agglutination between RBCs and serum. A significant difference was found between the disease group and healthy group (p <
0.0001) showing that a statistical analysis was conducted to compare the presence of ANA agglutination between the two groups. The reported p-value of less than 0.0001 shows that the observed difference is highly significant. The serum stability test conducted over ten consecutive days demonstrated a CV of 6.90% in the test results, indicating favorable stability. CONCLUSION: In conclusion, this study emphasizes the effectiveness of flow cytometry as a valuable tool for detecting RBC-bound antibodies and new antibodies in autoimmune disease patients. Its high sensitivity and accuracy have the potential to greatly improve diagnostic capabilities in clinical laboratories.