Aim To determine the effects of DTT treatment of patient sera on C1 interference in mixed bead, single antigen, C1q and flow cytometric assays. Methods For all experiments, patient sera were treated with 0.005M DTT. C1qrs (C1) interference was confirmed for 10 sera samples by dilution studies. We compared single antigen bead (SAB) results for these sera with and without DTT treatment. 4 Of these same sera known to contain complement binding antibodies were used to evaluate the effects of DTT treatment in the C1q assay. The assay for C1q was followed as per manufacturer’s instructions with the exception of the heat inactivation step. We compared our results to IgG subclass testing. 26 T cell and 24 B cell flow crossmatch assays (XM) were run at RT, 4 °C, and with DTT treatment. Results C1 interference was eliminated from all solid phase assays. Repeat testing of the 4 sera using PE-conjugated monoclonal antibodies to the 4 IgG Subclasses confirmed that IgG1 antibodies were present and were binding to the same specificities that had been blocked in the SAB assay due to C1 interference. A modified C1q procedure in which DTT treatment could be done without interfering with the C1q reagent was developed. Results of the modified procedure correlated very well with results of both the IgG subclass testing and the SAB DTT validation. All XMs that showed decreased event counts/C1 interference at RT (30 of 50) showed higher event counts in 4 °C and DTT XM methods, demonstrating that C1 interference was eliminated in 100% of the affected XMs. 100% specificity was seen in both the 4 °C and DTT methods, however, both the RT and 4 °C XMs had one false negative. Lower MFI values were seen in the RT and 4 °C XMs when compared with DTT treated XMs. On average, positive T cell XM values increased by 920 MFI at 4 °C and 1793 MFI with DTT; positive B cell XM values increased by 4894 MFI at 4 °C and 5036 MFI with DTT. All XMs run using DTT achieved expected positive or negative results based on the presence/absence of DSA. Conclusion We have eliminated false negative reactivity in solid phase PRA and flow cytometry XM by incorporating DTT treatment into all assays.