Optimization and Validation of an 8-Color Single-Tube Assay for the Sensitive Detection of Minimal Residual Disease in B-Cell Chronic Lymphocytic Leukemia Detected via Flow Cytometry
Optimization and Validation of an 8-Color Single-Tube Assay for the Sensitive Detection of Minimal Residual Disease in B-Cell Chronic Lymphocytic Leukemia Detected via Flow Cytometry
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DOI：
10.1093/labmed/lmw006
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发表时间：
2016-05-01
影响因子：
1.3
通讯作者：
Vandenberghe, Elisabeth
中科院分区：
文献类型：
--
作者：
Dowling, Anita K.;Liptrot, Stuart D.;Vandenberghe, Elisabeth
目的：欧洲慢性淋巴细胞性白血病研究计划（ERIC）设计了一种单管、8色慢性淋巴细胞性白血病CLL-MRD流式细胞术检测方法，用于标准化B-CLL患者的检测。本研究的目的是优化和验证8色CLL-MRD检测，与它正在实施的预期结果在圣詹姆斯医院都柏林和潜在的其他医院世界各地的最准确的基于流量的B-CLL-MRD detection.Method：单管检测纳入8种抗体，即，CD 5，CD 3，CD 19，CD 20，CD 22，CD 43，CD 79 b，和CD 81。我们测试了52个外周血和骨髓标本与抗体的组合，以开发一种顺序设门策略，该策略使用B-CLL细胞的典型表型来计数治疗后的小残留B-CLL群体，有效地将它们与正常的多克隆和再生B细胞区分开来。我们进行了灵敏度测定，通过一组系列稀释液，并比较了目前的国际标准化方法（伊萨）的方法，目前在使用的MRD检测B-CLL.Results：我们分析的52个标本显示MRD水平从0.004%到78.000%。稀释研究表明，疾病检测水平为0.00702%，与当前伊萨方法相比，具有良好的相关性（R-2 = 0.991）。8色CLL-MRD检测通过作为重要的疾病生物标志物，MRD阴性作为达到临床终点的指标。
Objective: The European Research Initiative on chronic lymphocytic leukemia (ERIC) has designed a single-tube, 8-color chronic lymphocytic leukemia CLL-MRD flow cytometric assay to standardize testing in patients with B-CLL. This study aims to optimize and validate the 8-color CLL-MRD assay, with the desired outcome of it being implemented in St. James's Hospital Dublin and potentially other hospitals worldwide as the most accurate flow-based B-CLL-MRD detection currently available.Method: The single-tube assay incorporates 8 antibodies, namely, CD5, CD3, CD19, CD20, CD22, CD43, CD79b, and CD81. We tested a combination of 52 peripheral blood and bone marrow specimens with the antibodies to develop a sequential gating strategy that uses the typical phenotype of B-CLL cells to enumerate small residual B-CLL populations after treatment, effectively distinguishing them from the normal polyclonal and regenerating B-cells. We performed sensitivity assays via a set of serial dilutions and compared the assay with the current International Standardized Approach (ISA) method currently in use for MRD testing in B-CLL.Results: The 52 specimens that we analyzed displayed MRD levels from 0.004% through 78.000%. Dilutional studies demonstrated detection of disease to a level of 0.00702%, and an excellent correlation was achieved against the current ISA method (R-2 = 0.991).Conclusion: The 8-color CLL-MRD assay provides a more informative approach than its predecessors for the assessment of MRD in B-CLL by functioning as an important disease biomarker, with MRD negativity acting as an indicator to achieving a clinical endpoint.