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Antinuclear antibodies (ANA) are determined, among other reasons, to identify samples which need a second test to detect the associated specificities. Our aim was to evaluate the clinical and economic impact generated by using an initial dilution for ANA of 1:160. We analyzed all samples for which ANA, anti-ENA and anti-dsDNA were requested over a 1-year period. ANA were detected by indirect immunofluorescence. Anti-ENA were analyzed with a combination of techniques. Anti-dsDNA were detected by radioimmunoassay. Cost analysis was performed by calculating the difference between two cut-offs (ANA 1:40 and 1:160). A total of 13,233 samples were processed for ANA, of which 59.9% were positive with the 1:40 cut-off and 39.2% with the 1:160 cut-off. At ANA titer 1:40, 0.2% of the samples were anti-ENA-positive and 2.2% were anti-dsDNA positive. Only ANA dilutions of 1:160 and higher showed significantly increased positive predictive value for anti-ENA (1.5 versus 0.2, p=0.029) and anti-dsDNA (8.3 versus 2.2, p<0.001) compared to the 1:40 titer. With the 1:160 cut-off, 16.6% fewer ANA tests, 41.8% fewer anti-ENA determinations and 36.4% fewer anti-dsDNA tests would have been needed. The average saving was 0.87 cost-units per sample (1 unit=2.06euro). We conclude that setting the starting dilution for ANA at 1:160 avoids unnecessary studies, increases the positive predictive values of ANA for anti-ENA and anti-dsDNA, and generates clinical and economic benefits., (Copyright © 2011 Elsevier B.V. All rights reserved.)