The present teachings comprise systems and methods for calibrating the background or baseline signal in a PCR or other reaction. The background signal derived from detected emissions of sample wells can be subjected to a normalized statistical metric, and be compared to a threshold or other standard to discard outlier cycles or other extraneous data. According to various embodiments, a relative standard deviation (relativeSTD) for the background component can be generated by dividing the standard deviation by the median of differences across all wells, where the difference is defined as the difference between maximum and minimum pixel values of a well. The relativeSTD as a metric is not sensitive to machine-dependent variations in absolute signal output that can be caused by different gain settings, different LED draw currents, different optical paths, or other instrumental variations. More accurate background characterization can be achieved.