Four color compensation

Four-color immunophenotyping can now be routinely performed using either a single laser or dual laser flow cytometer. When a single laser instrument i s used, the fluorochromes evaluated are usually FITC, PE, PE-TR and PE-CY5 (or PerCP). For two-laser excitation APC is generally used in place of PE-T R. Since each tandem dye construct contains PE, three of the four detectors are affected and compensation can be problematic. In this report we show that each tandem conjugated antibody, whether differ ent batches from the same supplier or conjugates from different suppliers a ll require unique compensation. This inconsistency results in erroneous dat a, negates the use of single labeled particles as a method for providing ad equate compensation and requires dual and triple labeled cells of known pat tern to verify compensation. It is also shown that improper compensation ca n reduce or eliminate completely the detection of fluorescence emission fro m PECY5 conjugated antibodies. These problems are caused by a variation in energy transfer between PE and either TR or CY5 because the chemistry involved in preparation and conjugat ion to antibodies is not sufficiently controlled to produce reagents with u niform compensation requirements. The variation in tandem dye compensation can be addressed by either using the same tandem conjugated antibody, by us ing the same second step tandem reagent to an appropriate first step antibo dy or by using software compensation. The latter provides an easy solution because a unique compensation matrix can be produced for each antibody tand em conjugate. Cytometry (C) 1999 Wiley-Liss, Inc.