Müller M R, Lennartz K, Nowrousian M R, Dux R, Tsuruo T, Rajewsky M F, Seeber S
Department of Medical Oncology, West German Cancer Centre, Essen.
Cytometry. 1994 Jan 1;15(1):64-72. doi: 10.1002/cyto.990150111.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
采用流式细胞术检测人肺癌细胞系以及60例急性髓系白血病（AML）患者白血病母细胞中药物外排泵P-糖蛋白（PGP）的表达。用抗PGP单克隆抗体MRK16进行免疫染色，可清晰区分PGP阴性亲本细胞系H69/P和多药耐药（MDR）变体H69/LX4的细胞。在白血病母细胞中，与独特型非特异性（对照样本）和特异性抗体（测试样本）孵育的样本之间荧光强度差异较小，导致分布无明显分离。仅在少数白血病标本中，通过使用阈值对直方图进行简单减法运算可检测到表达PGP的细胞。因此，应用基于曲线拟合和统计检验的改进直方图减法分析来区分抗原阳性和抗原阴性细胞。此外，采用碘化丙啶（PI）和Hoechst 33342的多参数染色程序来减少染色假象。通过这种方法，在60例患者中的39例检测到PGP低表达的白血病细胞。未观察到PGP强表达导致荧光分布分离的亚群。仅在60个标本中的5个中，通过使用阈值对直方图进行常规减法运算检测到表达PGP的细胞。比较采用或不采用多参数门控程序获得的数据表明，灵敏度的提高主要归因于数据分析的应用。然而，利用PI和Hoechst染色特性排除细胞碎片减少了数据与平均值的偏差。在细胞系或白血病母细胞中均未观察到PGP表达与细胞周期位置之间的关系。（摘要截短于250字）
