Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland, USA.
Cytometry B Clin Cytom. 2012 Mar;82(2):67-77. doi: 10.1002/cyto.b.20622. Epub 2011 Oct 26.
Zeta-chain-associated protein kinase 70 (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 immunoglobulin heavy chain variable (IGHV) mutational status were seen with percentage positive ND T-cell, ND T-cell/clone ratio, and 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.
Zeta 链相关蛋白激酶 70（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。基于我们之前的研究，我们开发了一种带有多个内部对照的联合单管技术来优化 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 CD3 APC-Cy7 和 CD19 APC 染色。在第二管中，患者全血用 CD5 PE-Cy7、CD19 PerCP-Cy5.5 和 CD20 eFluor450 染色。表面染色和固定后，将这两个管组合在一起。在皂素通透化后，用两种抗 ZAP-70 克隆（1E7.2/AF488 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本管获得的结果进行比较。评估了五种不同的 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；患者 T 细胞/克隆内比；ND T 细胞/克隆比；克隆/ND B 细胞比；和改良的 Z 指数。
总体而言，组合患者和 ND 混合管的性能优于非混合单样本管。1E7.2 和 SBZAP 克隆的 ZAP-70 表达与免疫球蛋白重链可变区（IGHV）突变状态之间的最强相关性见于 ND T 细胞阳性百分比、ND T 细胞/克隆比和克隆/ND B 细胞比（均 P <0.0001）。
结合 ND 和患者样本的改良单管方法提供了与 IGHV 突变状态高度相关的高度可靠结果。该方法应被视为 CLL 中 ZAP-70 测定标准化的下一步的一部分。
