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This work identifies statistical algorithms which need to be included in analysis of 2D gels for accurate determination of differential spot intensity changes. Two-dimensional electrophoresis is a powerful tool for determining differential protein expression in complex mixtures, but 2D electrophoresis, to date, is not producing sufficiently reliable results due to the degree of gel variability generated by this methodology. The new DIGE procedure, comparing two samples in the same gel, does eliminate some of the variability introduced with gel to gel comparison, but still has variability due to difference in dye binding, charge, and fluorescence. Introducing quality assurance statistical algorithms is necessary to extract meaningful data from the gels. A quality control analysis of replicate gels needs to be performed prior to using the set in the final analysis. Increasing replicates to five from the usual three can only add greater variability. A statistical “replicate quality” gel test needs to be done on the computer gel images, and replicates with greater than 20%–30% variability should not be used. In addition, since spot intensity data is not normally distributed, spot differential determinations cannot be a t test. The Studentized Range has been suggested as a more accurate method for calculating significant difference.