Combined normal donor and CLL: Single tube ZAP-70 analysis.
Combined normal donor and CLL: Single tube ZAP-70 analysis.
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DOI：
10.1002/cyto.b.20622
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发表时间：
2012-03
影响因子：
3.4
通讯作者：
Marti, Gerald E.
中科院分区：
文献类型：
--
作者：
Degheidy, Heba A.;Venzon, David J.;Farooqui, Mohammed Z. H.;Abbasi, Fatima;Arthur, Diane C.;Wilson, Wyndham H.;Wiestner, Adrian;Stetler-Stevenson, M. A.;Marti, Gerald E.
ZAP-70已被确定为慢性淋巴细胞白血病（CLL）的独立预后标志物。基于我们以前的研究，我们开发了一种具有多种内部控制的组合单管技术，以优化ZAP-70评估。使用改良的7色单管测定法检测48例未经治疗的CLL病例的ZAP-70表达。正常供体（ND）全血用⑶ 3 APC-Cy 7和⑶ 19 APC染色。在第二个试管中，用CD 5 PE-Cy 7、CD 19 PerCP-Cy 5.5和CD 20 eFluor 450对患者全血进行染色。在表面染色和固定之后，将这两个管组合。在皂苷透化后，用两种抗ZAP-70克隆（1E7.2/AF 488和SBZAP /PE）对细胞进行染色。将从该改良管获得的结果与同时使用非混合单样品管获得的结果进行比较。评价了五种不同的ZAP-70表达分析方法：使用ND T细胞作为参考的阳性细胞百分比;内部患者T细胞/克隆比率; ND T细胞/克隆比率;克隆/ ND B细胞比率;和改良的Z指数。总体而言，患者和ND混合管组合的性能优于非混合的单个样本管。对于1E7.2和SBZAP克隆，ZAP-70表达和IGHV突变状态之间的最强相关性与阳性ND T细胞百分比、ND T细胞/克隆比率、克隆/ND B细胞比率有关（p<0.0001）。结合ND和患者样本的改良单管法提供了与IGHV突变状态相关的高度可靠的结果。该方法应被视为CLL中ZAP-70测定标准化下一步的一部分。
ZAP-70 has been identified as an independent prognostic marker in chronic lymphocytic leukemia (CLL). Based on our previous studies, we have developed a combined one-tube technology with multiple internal controls to optimize ZAP-70 assessment. Forty-eight untreated CLL cases were examined for ZAP-70 expression using a modified 7-color one-tube assay. Normal donor (ND) whole blood is stained with CD3 APC-Cy7 and CD19 APC. In a second tube, patient whole blood is stained with CD5 PE-Cy7, CD19 PerCP-Cy5.5, and CD20 eFluor450. After surface staining and fixation, these two tubes are combined. After saponin permeabilization, the cells were stained with two anti-ZAP-70 clones (1E7.2/AF488 and SBZAP /PE). The results obtained from this modified tube were compared with those obtained concurrently using the non-mixed single sample tubes. Five different methods of ZAP-70 expression analysis were evaluated: percentage positive cells using ND T-cells as a reference; the internal patient T-cell/clone ratio; ND T-cell/clone ratio; clone/ ND B-cell ratio; and modified Z-index. Overall, the combined patient and ND mix tube performed better than the non-mixed single sample tube. The strongest correlations between ZAP-70 expression and IGHV mutational status were seen with percentage positive ND T-cell, ND T-cell/clone ratio, clone/ND B-cell ratio for both 1E7.2 and SBZAP clone (p<0.0001). The modified one tube method combining the ND and patient sample provides highly reliable results that correlate with the IGHV mutational status. This method should be considered as part of the next step in standardization of the ZAP-70 assay in CLL.
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