Improved flow‐cytometric detection of low P‐glycoprotein expression in leukaemic blasts by histogram subtraction analysis

Publisher Website
Google Scholar
Abstract
Expression of the drug efflux pump P‐glycoprotein (PGP) was determined by flow cytometry in human lung cancer cell lines and in leukaemic blasts derived from 60 patients with acute myeloid leukaemia (AML). Cells from the PGP‐negative parent cell line H69/P and the multidrug resistant (MDR)‐variant H69/LX4 could be clearly distinguished by immunostaining with the anti‐PGP monoclonal antibody MRK16. In leukaemic blasts, the differences in fluorescence intensities between samples incubated with the idiotypic nonspecific (control sample) and specific antibody (test sample) were small, resulting in nondisjunct distributions. Only in a few leukaemia specimens were PGP‐expressing cells detectable by simple subtraction of histograms using a threshold. Therefore, an improved histogram subtraction analysis, based on curve fitting and a statistical test, was applied to distinguish antigen‐positive from antigen‐negative cells. Moreover, a multiparametric staining procedure employing propidium iodide (PI) and Hoechst 33342 was used to reduce staining artefacts. By this approach, leukaemic cells with low expression of 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 threshold. Comparison of data obtained with or without the multiparametric gating procedure indicated that the increase in sensitivity was mainly due 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 blasts. In relation to clinical status, the median fraction of PGP‐positive blasts was elevated 3.5‐fold in relapsed. patients (n=28) in comparison to patients at first presentation (n=29). In newly diagnosed patients, 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 relevant, low‐level PGP expression in subpopulations of leukaemic blasts.