IMPROVED FLOW-CYTOMETRIC DETECTION OF LOW P-GLYCOPROTEIN EXPRESSION IN LEUKEMIC BLASTS BY HISTOGRAM SUBTRACTION ANALYSIS

Expression of the drug efflux pump P-glycoprotein (PGP) was determined by flow cytometry in human lung cancer cell lines and in leukaemic bl asts derived from 60 patients with acute myeloid leukaemia (AML). Cell s from the PGP-negative parent cell line H69/P and the multidrug resis tant (MDR)-variant H69/LX4 could be clearly distinguished by immunosta ining with the anti-PGP monoclonal antibody MRK16. In leukaemic blasts , the differences in fluorescence intensities between samples incubate d with the idiotypic nonspecific (control sample) and specific antibod y (test sample) were small, resulting in nondisjunct distributions. On ly in a few leukaemia specimens were PGP-expressing cells detectable b y simple subtraction of histograms using a threshold. Therefore, an im proved histogram subtraction analysis, based on curve fitting and a st atistical test, was applied to distinguish antigen-positive from antig en-negative cells. Moreover, a multiparametric staining procedure empl oying propidium iodide (PI) and Hoechst 33342 was used to reduce stain ing artefacts. By this approach, leukaemic cells with low expression o f PGP were detected in 39 out of 60 cases. Subpopulations with strong PGP expression, resulting in disjunct fluorescence distributions, were not observed. Only in 5 out of 60 specimens were PGP expressing cells detected by a conventional subtraction of histograms using a threshol d. Comparison of data obtained with or without the multiparametric gat ing procedure indicated that the increase in sensitivity was mainly du e to the application of the data analysis. However, exclusion of cell debris using PI and Hoechst staining properties reduced the deviation of data from mean values. No relation between PGP expression and cell cycle position was observed in either cell lines or in leukaemic blast s. In relation to clinical status, the median fraction of PGP-positive blasts was elevated 3.5-fold in relapsed patients (n = 28) in compari son to patients at first presentation (n = 29). In newly diagnosed pat ients, the median fraction of PGP expressing blasts was 4-fold higher in specimens from patients who failed to reach complete remission (n = 11) in comparison to responsive patients (n = 18). Refined histogram subtraction analysis thus facilitates the detection of clinically rele vant, low-level PC;P expression in subpopulations of leukaemic blasts. (C) 1994 Wiley-Liss, Inc.