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Background and aims: In order to determine actual concentration of clinically significant IgG antibodies, there is a need to inactivate IgM antibodies, which can be performed by DTT or heat. The aim of the present study was to compare the effect of DTT treatment and heat inactivation (HI) on ABO isoagglutinin titres performed by column agglutination technology (CAT) and conventional tube technique (CTT). Materials and methods: This was a prospective, observational study conducted from October 2018 to March 2020. All consecutive O group donors were included. Serum from each donor was treated with DTT, and heat and tests were performed by CTT and CAT before and after treatment. Results: A total of 2005 donors were included. 401 (20%) samples had inter‐observer variation by CTT, and 69 (3·4%) had inter‐observer variation by CAT. Median anti‐A and anti‐B titre results were higher with CAT as compared to CTT. Median anti‐A and anti‐B IgG titre results showed a one‐ to twofold difference between CTT and CAT with higher titres reported by CAT. Median IgM titres decreased two‐ to threefold after DTT treatment, whereas onefold decrease was confirmed after HI. Concordance was found to be poor. However, correlation was found to be strong. Conclusion: There is overestimation of ABO IgG antibody titres and hence, estimation of ABO IgG titres after inactivation of IgM antibodies is strongly recommended. DTT treatment was found to be superior to HI in terms of efficiency in elimination of IgM activity. [ABSTRACT FROM AUTHOR]