An optimized whole blood method for flow cytometric measurement of ZAP-70 protein expression in chronic lymphocytic leukemia
An optimized whole blood method for flow cytometric measurement of ZAP-70 protein expression in chronic lymphocytic leukemia
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DOI：
10.1002/cyto.b.20135
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发表时间：
2006-07-15
影响因子：
3.4
通讯作者：
Keeney, Mike
中科院分区：
文献类型：
--
作者：
Shankey, T. Vincent;Forman, Meryl;Keeney, Mike
背景资料：ZAP-70蛋白表达已被提议作为免疫球蛋白重链突变状态的标志物，一些研究已将其与B细胞慢性淋巴细胞白血病（CLL）的病程相关。迄今为止发表的使用流式细胞术测量ZAP-70细胞内蛋白表达水平的研究已经证明了性能较差，如通过已知阳性和阴性淋巴细胞群体中的信号差异所定义的。（Chow S，赫德利DW，Grom P，Magari R，Jacobberger JW，Shankey TV，Cytometry A 2005;67：4-17）测量细胞内磷酸化表位，以提供与阴性B-细胞相比，阳性Tor NK-细胞中ZAP-70表达的最佳分离。外周血样本中的细胞。许多市售的抗ZAP-70抗体偶联物使用这种方法进行了筛选，并选择表现出最佳性能的抗体偶联物开发四色，五抗体测定法，以测量ZAP-70在全血samples.Results水平：使用优化的固定和透化方法，在大多数测试的抗体中，检测性能（信噪比，S/N）的改善。当与这种优化的固定/透化方法结合使用时，定制的SBZAP缀合物给出了最佳的S/N。结合仔细标准化的仪器设置协议，我们获得了两个实验室内和实验室间的再现性分析ZAP-70的表达在全血样品从正常和CLL patients.Conclusions：一个敏感的，特异性和高度可重复的ZAP-70检测的发展代表了任何临床检测的第一个重要步骤。在ZAP-70蛋白质分析可以在临床实验室中常规实施之前，必须建立经验证的数据分析方法的普遍实施和基于方法学的临床结果截止点的建立。(c)2006年国际分析细胞学学会。
Background: ZAP-70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B-cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP-70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations.Methods: A recently published method (Chow S, Hedley DW, Grom P, Magari R, Jacobberger JW, Shankey TV, Cytometry A 2005;67:4-17) to measure intracellular phospho-epitopes was optimized using a design of experiments (DOE) approach to provide the best separation of ZAP-70 expression in positive Tor NK-cells as compared to negative B-cells in peripheral blood samples. A number of commercially available anti-ZAP-70 antibody-conjugates were screened using this methodology, and the antibody-conjugate showing the best performance was chosen to develop a four-color, five antibody assays to measure ZAP-70 levels in whole blood specimens.Results: Using the optimized fixation and permeabilization method, improvement in assay performance (signal-to-noise, S/N) was seen in most of the antibodies tested. The custom SBZAP conjugate gave the best S/N when used in conjunction with this optimized fixation /permeabilization method. In conjunction with carefully standardized instrument set-up protocols, we obtained both intra- and interlaboratory reproducibility in the analysis of ZAP-70 expression in whole blood samples from normal and CLL patients.Conclusions: The development of a sensitive, specific and highly reproducible ZAP-70 assay represents only the first essential step for any clinical assay. The universal implementation of a validated data analysis method and the establishment of methodology-based cutoff points for clinical outcomes must next be established before ZAP-70 protein analysis can be routinely implemented in the clinical laboratory. (c) 2006 International Society for Analytical Cytology.