required to a minimal level. In this example, we used a MultiMixTM reagent that consists of CD20 FITC, CD5 RPE and CD19 APC. This combination is used to identify the total T and B lymphocyte populations and the CD5+ sub set of B lymphocytes. Since the MultiMix reagents are pre-titrated, the user simply adds the required volume of the MultiMix reagent to an aliquot of patient’s cells. The fluorochrome combinations, FITC, RPE and APC are chosen specifically to reduce problems with spectral overlap. By adding CD45 Pacific Blue as a drop-in reagent we can easily expand from a 3to a 4-color system. The advantage of using CD45 Pacific Blue is that virtually no compensation is required between the Pacific Blue and the FITC, RPE or APC fluorochromes. Thus, for this four-color combination the only compensation required is between RPE and FITC and FITC and RPE. The FITC and RPE are excited by the blue (488nm) laser, the APC by the red (635nm) laser and the Pacific Blue by the violet (405nm) laser.