BACKGROUND Diagnosis of HCV, HBV, and HIV involves antibody screening followed by confirmation and/or treatment decision using nucleic acid tests. However, minimal data exist evaluating the risk of nucleic acid cross-contamination on serology devices upstream of molecular testing despite the potential clinical and laboratory workflow advantages of single specimen vial testing for both procedures. METHODS We conducted a checkerboard study investigating the potential risk of HCV, HBV, and HIV nucleic acid cross-contamination on 480 negative specimens by a serology screening instrument that uses disposable tips for sample transfer, rather than a fixed needle, before molecular testing. RESULTS Nucleic acid contamination was observed in 0 of 480 negative specimens when processed with alternating high-titer HCV, HBV, or HIV specimens on the serology platform. CONCLUSIONS This study suggests that specimens analyzed by a serology instrument using disposable tips for sample transfer may be suitable for direct primary specimen reflex testing by a sensitive nucleic acid confirmatory test.