The second filter checks the quality of each positive spot. All proteins are spotted in duplicates on protein microarrays, hence should have very similar signal intensities. ProCAT then uses the difference between duplicate signals as an indicator of the signal qualities. The difference between signals of two duplicate spots (s 1 , s 2 ) is calculated as (s 1 + s 2 )/(|s 1 | + |s 2 |) and then fitted to a normal distribution. Proteins with exceptionally large differences in their duplicate spots are more likely to be biased by certain artifacts, and thus are removed from the positive list. The default threshold for the duplicate spot difference in ProCAT is set at two standard deviations away from the mean.