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Simultaneous antibody testing during screening for autoimmune conditions is discouraged. The incidence of positive extractable nuclear antigen (ENA) in the setting of a negative antinuclear antibody (ANA) has been reported as low.Our objective was to characterize the frequency of diagnosis of new ANA-associated rheumatic disease (AARD) in the setting of a negative ANA with a positive ENA.This was a 7-year retrospective study from a multicenter tertiary health network in Australia. Clinical information was sought on patients over 18 years who had a negative ANA but positive ENA. Results were extracted from hospital computer systems.From March 19, 2011, to July 23, 2018, ENA testing was ordered simultaneously with an ANA test on 4,248 occasions in 3,484 patients. ANA was positive in 2,520 (59.3%) and ENA positive in 1,980 (46.6%). Among positive ANA, ENA was positive in 1,563 (62.0%). Among 1,728 negative ANA tests, ENA was positive in 417 (24.1%) (p0.001). 328 patients with discordant ANA/ENA results had data available for further analysis, of whom 279 had no pre-established rheumatological condition. A new AARD was diagnosed in 17/279 patients yielding a positive predictive value of 6.09% (95% CI 3.59-9.58) CONCLUSION: Despite the higher-than-expected incidence of positive ENA in the setting of a negative ANA, the yield of newly diagnosed rheumatic diseases was low. Our findings support the stepwise addition of ENA requests when an ANA is positive and clinical suspicion of an AARD is high.