Peripheral blood cell preparation influences the level of expression of leukocyte cell surface markers as assessed with quantitative multicolor flow cytometry.

We have compared the influence of sample preparation upon the level of surface expression of T, B, and NK cell-related antigens as assessed by flow cytometry. Lysed whole blood (WBL), Ficoll-Paque separated peripheral blood lymphocyte (F-PBL), and frozen peripheral blood lymphocyte (Fr-PBL) were analyzed via single- and multicolor flow cytometry. The percentage of positive cells expressing the individual cell surface markers was not affected by the procedure for preparation of WBL, F-PBL, and Fr-PBL. In contrast, the fluorescence intensity level of individual cell surface markers varied depending on cell preparation. By using Quantum Simply Cellular (QSC) microbeads, the antibody binding capacity (ABC) of single-color stained cells was quantified and compared. The amount of monoclonal antibody (MAb) anti-CD3-FITC bound to Fr-PBL (mean ABC = 137,040) was significantly higher (P < 0.001) that the amounts bound to WBL (mean ABC = 112,410) and F-PBL (mean ABC = 107,738). In multicolor analysis, the fluorescence intensity of CD3-FITC and CD4-FITC was significantly higher on Fr-PBL than on WBL and F-PBL; CD8-PE and CD20-PerCP was significantly higher on WBL. Furthermore, the intensity of CD3 and CD4 was different on T-cell subsets. The intensity of CD3 staining in three-color analysis was lower than with single-color staining using the same fluorochrome. We conclude that particularly the method of cell preparation but also the selection of MAb combinations may influence the level of staining of certain lymphocyte antigens. This may be of relevance in the analysis of cellular activation and regulation of differentiation.