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This paper investigates issues related to practical implementation of the one-quarter of cycle-based estimation method [here called delayed signal cancellation (DSC)] for online detection of positive and negative sequence components of three-phase quantities. The detection error due to non-ideal sampling frequency is calculated. Furthermore, the effect of the DSC method on estimation when harmonic components are present in the input signal is analyzed. It is shown how harmonics transfer through the DSC and how a non-ideal sampling frequency affects the estimation of the harmonics. Experimental verification of the analytical results is presented.