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 per ipheral blood lymphocyte (F-PBL), and frozen peripheral blood lymphocy te (Fr-PBL) were analyzed via single- and multicolor flow cytometry. T he percentage of positive cells expressing the individual cell surface markers was not affected by the procedure for preparation of WBL, F-P BL, and Fr-PBL. In contrast, the fluorescence intensity level of indiv idual cell surface markers varied depending on cell preparation. By us ing Quantum Simply Cellular (QSC) microbeads, the antibody binding cap acity (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 am ounts bound to WBL (mean ABC = 112,410) and F-PBL (mean ABC = 107,738) . In multicolor analysis, the fluorescence intensity of CD3-FITC and C D4-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 in tensity of CD3 and CD4 was different on T-celI subsets, The intensity of CD3 staining in three-color analysis was lower than with single-col or staining using the same fluorochrome. We conclude that particularly the method of cell preparation but also the selection of MAb combinat ions may influence the level of staining of certain lymphocyte antigen s. This may be of relevance in the analysis of cellular activation and regulation of differentiation. (C) 1995 Wiley-Liss, Inc.