Antinuclear antibodies by indirect immunofluorescence : optimum screening dilution for diagnosis of systemic lupus erythematosus

Abstract

Background & objectives: Antinuclear antibodies (ANA) are serological hallmark of systemic lupus erythematosus (SLE). Conventionally, the test is carried out on human epithelial cells (HEp2) by indirect immunofluorescence (IIF) technique. Since culturing and maintaining HEp2 cells in the laboratory are labour intensive, in-house assays have given way to kits manufactured by commercial companies. The reference screening dilutions provided by the manufacturers are based on different ethnic population than ours. Therefore, it becomes mandatory for every laboratory to have its own screening dilutions for the local population that distinguishes best between healthy and diseased state. As, there is paucity of such data, we aimed to define the optimum screening dilution that distinguishes the patient with SLE from healthy individuals. Methods: Sera of patients fulfilling ACR criteria for diagnosis of SLE, idiopathic inflammatory polymyositis/dermatomyositis (PM/DM) and rheumatoid arthritis (RA), and age and sex matched healthy individuals were tested for ANA by IIF using a commercial kit (Euroimmun, Germany) at 5 dilutions, namely 1: 40, 1: 80, 1:58; 1:160, 1:320 and 1:640. Receiver operator characteristics (ROC) curve were constructed to define the optimum dilution that distinguished healthy sera from the diseased ones. Results: Test was performed on 213 sera from 94 healthy individuals, and 43 SLE, 37 RA and 39 DM/PM patients. In healthy individuals, ANA at dilutions 1:40, 1:80, 1:160, 1:320 and 1:640 was positive in 13.8, 4.3, 2.1, 2.1 and 0 per cent respectively, whereas in SLE it was positive in 95.3, 95.3, 65.1, 53.5 and 23.3 per cent respectively. Interpretation & conclusion: ROC curves analysis showed that at 1:40 dilution, sera of 95.3 per cent of SLE and 13.8 per cent of normal individuals were (ANA) positive, whereas at 1:80 dilution it was 95.3 per cent for SLE and 4.3 per cent for healthy individuals. A fluorescent intensity of ≥ 2 was more specific for SLE. The best discrimination between healthy individuals and the SLE patients was found at screening dilution of 1:80 and fluorescent intensity of ≥ 2 in our laboratory.