Antinuclear antibodies (ANAs) are a serological hallmark in the diagnosis of systemic autoimmune rheumatic diseases (SARD). The indirect immunofluorescence (IIF) assay on HEp-2 cells is a commonly used test for the detection of ANA and has been recently recommended as the screening test of choice by a task force of the American College of Rheumatology. However, up to 20% of apparently healthy individuals (HI) have been reported to have a positive IIF ANA test, primarily related to autoantibodies that target the dense fine speckles 70 (DFS70) antigen. Even more important, the DFS IIF pattern has been reported in up to 33% of ANA positive HI, but not in ANA positive SARD sera. Since the intended use of the ANA HEp-2 test is to aid in the diagnosis and classification of SARD, the detection and reporting of anti-DFS70 antibodies and their associated pattern (DFS) as a positive test significantly reduce the specificity and the positive likelihood of the ANA test. This has significant implications for medical management and diagnostic algorithms involving the detection of ANA. Recently, a novel immunoadsorption method has been developed that specifically blocks anti-DFS70 antibodies and, therefore, significantly increases the specificity of the ANA test for SARD. This immunoadsorption method has the potential to overcome a significant limitation of the ANA HEp-2 assay. The present paper summarizes the current knowledge about anti-DFS70 antibodies and their clinical impact on ANA testing.