Rhodamine 123-efflux from hematopoietic subpopulations and leukaemic blast populations marked by PerCP-conjugated monoclonal antibodies.

A representative functional assay for determination of drug transporting proteins (e.g. P-glycoprotein) in leukaemic blasts could help to evaluate effects of chemotherapy combined with chemosensitizers. Since subpopulations of normal peripheral blood or bone marrow cells show distinct P-glycoprotein levels, the presence of these cells in leukaemic samples causes a major problem in determination of rhodamine 123 efflux in these types of malignant cells. Additional staining of blasts with specific monoclonal antibodies (marked with FITC (fluorescein) or PE (phycoerythrin) might ensure a selective analysis of a particular subpopulation by flow cytometry, but the emission spectrum of rhodamine 123 interferes with FITC and PE signals and vice versa. This can be avoided by using monoclonal antibodies (mab) conjugated with the newly developed dye PerCP (peridnine chlorophyll protein; Becton/Dickinson), devoid of interfering with the rhodamine 123 fluorescence emission spectrum. Therefore we established an assay for the determination of rhodamine 123 efflux from peripheral blood CD4+, CD8+ or CD56+ subpopulations by detection with PerCP-conjugated mab, followed by electronic gating. The problems of varying signal intensities or the need to recompensate during measurement which normally occurred using FITC- or PE-conjugated mab did not emerge by the use of PerCP-marked mab. Moreover we could correlate MDR1 gene expression and modulation of rhodamine 123 efflux from the leukaemic blasts by proven P-gp MDR chemosensitizing agents such as SDZ PSC 833, dexverapamil and dexniguldipine. This method gives highly reproducible results of P-gp function in patient samples which should be compared with patient outcome after combined chemotherapy including chemosensitizers.