An alternative multicolor compensation method for the sample-directed immune phenotyping of CD34+ cells via flow cytometry:: A technical note

Background: Accurate electronic compensation of spectral overlapping is important for the determination of cell subsets analyzed by multicolor now cytometry. A modification of the stepwise compensation technique with simultaneous single-step and -screen compensation is described. Materials and Methods: Separate compensation controls were generated from blood, leukapheresis, or selected CD34(+) cell samples by using the following combinations of monoclonal antibodies (MoAb): CD3-FITC, isotype control PE, and PE/CY5; CD3-PE, isotype control FITC, and PE/Cy 5; or CD3-PE/Cy5, isotype control FITC, and PE. Instead of CD3(+) cells, the CD34(+) cells were also labelled separately with either CD34-FITC, CD34-PE, or CD34-PE/Cy5 MoAb. Further on, mixtures of the CD3 or CD34 samples, prestained with the three different conjugates and washed, were generated. One screen compensation was performed on the FACScan(R) flow cytometer. Results: No differences were found comparing the results of the stepwise and the single-step technique. The simultaneous compensation with the mixtures provided a better clarity, was less time-consuming, and saved cells and materials. Conclusions: The advantage of this customized compensation method was the use of the same cell subsets with equal light scatter characteristics and antigen density. Furthermore, the lymphocytes, comprising the leukocyte cell subset with the lowest light scatter and autofluorescence, are the ideal fraction for individual sample-directed adjustment of the flow cytometer. The compensa tion using the CD34 mixture provided an additional internal negative CD34(+) cell control for matching with the external negative isotype controls for the CD34(+) cell subset estimation. Thus, reduction of errors can be achieved and comparable data can be created.