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Background and Objectives Conventional serology tests for Trypanosoma cruzi blood banks screening are neither sensitive nor specific enough, and currently no gold standard assay is available. Trans-sialidase inhibition assay (TIA) detects neutralizing antibodies against T. cruzi trans-sialidase. Conventional serology inconclusive, positive and negative blood donor samples were evaluated by employing TIA as a supplementary test. Materials and Methods Three hundred and twenty-one blood donor samples were tested using a combination of assays. Based on the results of testing, these were divided into a number of groups. All samples were tested by TIA. Results In conventional serology inconclusive samples 48·1% were TIA-positive, 1/54 conventional serology positive samples was TIA-negative. All negative samples from donors without epidemiological risks were TIA-negative; 1/48 was positive in those with epidemiological risk. Conclusion Trans-sialidase inhibition assay application in blood banks may be useful to resolve inconclusive samples, and thus improves donor counseling and allows individual re-entry. The use of TIA in samples from negative conventional test donors but positive epidemiological antecedents may contribute to decrease transfusional risk. [ABSTRACT FROM AUTHOR]