Best O G, Ibbotson R E, Parker A E, Davis Z A, Orchard J A, Oscier D G
Cytometry B Clin Cytom. 2006 Jul 15;70(4):235-41. doi: 10.1002/cyto.b.20121.
The clinical course of chronic lymphocytic leukaemia (CLL) is variable. ZAP-70 expression is believed to provide prognostic information. The flow cytometric detection of ZAP-70 is difficult because it is an intracellular antigen with weak expression in CLL. Consensus has not been reached as to the best method for measurement.
We analyzed 72 CLL patient samples for ZAP-70 expression and IgVH mutational status. Sensitivity and specificity of ZAP-70 expression against IgVH mutational status were assessed for two clones (2F3.2 and 1E7.2) and for four methods of analysis: percentage positivity (PP), comparing test to isotype control, ratio of geometric means of test and isotype control, and percentage and ratiometric methods comparing test and T/NK cell populations. The effects of anticoagulant, collection times, and time to analysis were also evaluated.
Sensitivity and specificity were 85 and 88%, respectively, for Upstate PP; 70 and 88% for Caltag PP; 89 and 91% for Upstate ratio; 89 and 88% for Caltag ratio. Intraobserver variability was smaller when ZAP-70 expression was assessed using a ratiometric approach rather than the percentage method. By 48 h, we observed an average decrease of 13% in the Caltag ratio in the heparin preserved samples compared to an increase of 3% in those collected in EDTA. Within the first 24-h period, a greater percent variability was observed in those samples collected into EDTA compared with heparin.
Our data support a rapid method for ZAP-70 measurement using commercially available fixation/permeabilization reagents, a conjugated antibody, and a ratiometric method of analysis that minimizes subjective interpretation of the results. This is a method of ZAP-70 assessment that could be included in a routine diagnostic CLL panel; however, the choice of anticoagulant and time of analysis after collection are critical factors in accurate assessment of ZAP-70 expression.
慢性淋巴细胞白血病（CLL）的临床病程具有变异性。ZAP-70表达被认为可提供预后信息。ZAP-70的流式细胞术检测较为困难，因为它是一种细胞内抗原，在CLL中表达较弱。对于最佳测量方法尚未达成共识。
我们分析了72例CLL患者样本的ZAP-70表达和IgVH突变状态。针对两个克隆（2F3.2和1E7.2）以及四种分析方法评估ZAP-70表达相对于IgVH突变状态的敏感性和特异性：阳性百分比（PP）、与同型对照比较检测值、检测值与同型对照几何均值之比，以及检测值与T/NK细胞群体比较的百分比和比例法。还评估了抗凝剂、采集时间和分析时间的影响。
Upstate公司的PP法敏感性和特异性分别为85%和88%；Caltag公司的PP法为70%和88%；Upstate公司的比例法为89%和91%；Caltag公司的比例法为89%和88%。使用比例法评估ZAP-70表达时，观察者内变异性小于百分比法。48小时内，我们观察到肝素保存样本的Caltag比例平均下降13%，而EDTA采集样本的比例平均增加3%。在最初24小时内，与肝素采集样本相比，EDTA采集样本的变异性百分比更高。
我们的数据支持一种使用市售固定/通透试剂、偶联抗体和比例分析方法快速测量ZAP-70的方法，该方法可将结果的主观解读降至最低。这是一种可纳入CLL常规诊断检测项目的ZAP-70评估方法；然而，抗凝剂的选择和采集后分析时间是准确评估ZAP-70表达的关键因素。
