Reference method for detection of Pgp mediated multidrug resistance in human hematological malignancies: A method validated by the laboratories of the French Drug Resistance Network

Multidrug resistance (MDR) associated with overexpression of the MDR1 gene and of its product, P-glycoprotein (Pgp), plays an important role in limiti ng cancer treatment efficacy. Many studies have investigated Pgp expression in clinical samples of hematological malignancies but failed to give defin itive conclusion on its usefulness, One convenient method for fluorescent d etection of Pgp in malignant cells is flow cytometry which however gives va riable results from a laboratory to another one, partly due to the lack of a reference method rigorously tested, The purpose of this technical note is to describe each step of a reference flow cytometric method, The guideline s for sample handling, staining and analysis have been established both for Pgp detection with monoclonal antibodies directed against extracellular ep itopes (MRK16, UIC2 and 4E3), and for Pgp functional activity measurement w ith Rhodamine 123 as a fluorescent probe. Both methods have been validated on cultured cell lines and clinical samples by 12 laboratories of the Frenc h Drug Resistance Network. This cross-validated multicentric study points o ut crucial steps for the accuracy and reproducibility of the results, like cell viability, data analysis and expression. (C) 1998 Wiley-Liss, Inc.