Harmonization of BCR-ABL mRNA Quantification Using an Uniform Control Plasmid in 36 International Laboratories.
Harmonization of BCR-ABL mRNA Quantification Using an Uniform Control Plasmid in 36 International Laboratories.
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36 个国际实验室使用统一对照质粒协调 BCR-ABL mRNA 定量。
DOI：
10.1182/blood.v106.11.1991.1991
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发表时间：
2005
期刊：
影响因子：
20.3
通讯作者：
A. Hochhaus
中科院分区：
文献类型：
--
作者：
M. Mueller;P. Erben;G. Saglio;E. Gottardi;T. Schenk;T. Ernst;Stephanie Lauber;M. Emig;R. Hehlmann;A. Hochhaus
定量测定残留BCR-ABL转录水平已被接受为CML患者管理的组成部分。然而，分子方法的异质性导致不同研究之间缺乏可比性。因此，协调程序和表达结果的需要尚未得到满足。在欧洲白血病网络的一系列共识会议上，已经详细阐述了实现最佳灵敏度和标准化的先决条件清单：使用36小时内处理的至少10 ml外周血;多中心试验的床边RNA稳定化;针对每个平台优化的标准化PCR方案;使用含有靶基因和管家基因的单一质粒以避免稀释错误;使用总ABL和/或β葡糖醛酸糖苷酶（GUS）作为内部对照。为了证实这些论文，在14个国家的36个实验室中启动了一项国际多中心试验。本研究的目的是评估使用PAXgene Blood RNA System ®（PreAnalytiX，洪布雷赫蒂孔，瑞士）进行RNA提取、cDNA合成的个体方案、3种不同PCR平台（TaqMan ®，TM，n=24，LightCycler ®，LC，n=14，Rotorgene ® n=1）和优化的定量RT-PCR条件从不同实验室获得的结果的变异性。为了使结果标准化，将b3 a2 BCR-ABL和GUS序列克隆到pCR 2.1-TOPO vector ®（Invitrogen，卡尔斯巴德，CA）中，将其以连续稀释液分配给所有参与者，作为用于定量BCR-ABL、总ABL和GUS mRNA转录物的外部对照。制备十份含有正常白细胞和阴性对照中b3 a2或b2 a2 BCR-ABL阳性细胞稀释液（10、2、1、0.1%）的样本，设盲并运送给参与者。一式三份测定转录物数目，计算BCR-ABL/ABL和BCR-ABL/GUS比率并以%表示。b3 a2样本的BCR-ABL/ABL中位比值为8.9、1.7、0.85和0.11%; b2 a2样本为9.1、1.6、0.82和0.10%。b3 a2样品的BCR-ABL/GUS中位比值为3.4、0.77、0.37和0.042%; b2 a2样品为2.8、0.48、0.29和0.031%。36名参与者中有4名（11%）在阴性对照样本中检测到低BCR-ABL拷贝数。所有参与者、TM和LC使用者的变异系数（CV）分别为0.62、0.57和0.59（BCR-ABL/ABL比值）; 1.03、0.85和1.22（BCR-ABL/GUS比值）。与BCR-ABL/ABL比值（中位数0.18，范围0.022-2.2，p <0.05）相比，回归线的标准误显著降低了BCR-ABL/GUS比值（中位数0.075，范围0.0046-0.90）。
Quantitative determination of residual BCR-ABL transcript levels has been accepted as integral part of the management of CML patients. However, heterogeneity of molecular approaches results in a lack of comparability between different studies. Thus, there is an unmet need for harmonization of both procedures and expression of results. In a series of consensus meetings within the European LeukemiaNet a list of prerequisites to achieve an optimal sensitivity and standardization has been elaborated: use of at least 10ml peripheral blood processed within 36 hrs; bedside RNA stabilization for multicenter trials; standardized PCR protocols optimized for each platform; use of a single plasmid containing target and housekeeping genes to avoid dilution errors; use of total ABL and/or beta glucuronidase (GUS) as internal controls. To substantiate these theses an international multicenter trial within 36 labs in 14 countries was initiated. The aim of the study was to assess the variability of results obtained from different labs using the PAXgene Blood RNA System ® (PreAnalytiX, Hombrechtikon, Switzerland) for RNA extraction, individual protocols for cDNA synthesis, 3 different PCR platforms (TaqMan ® , TM, n=24, LightCycler ® , LC, n=14, Rotorgene ® n=1), and optimized quantitative RT-PCR conditions. In order to standardize results, b3a2 BCR-ABL and GUS sequences were cloned into a pCR 2.1-TOPO vector ® (Invitrogen, Carlsbad, CA), which was distributed to all participants in serial dilutions as external control for quantification of BCR-ABL, total ABL, and GUS mRNA transcripts. Ten samples containing dilutions (10, 2, 1, 0.1%) of b3a2 or b2a2 BCR-ABL positive cells in normal leukocytes and negative controls were prepared, blinded, and shipped to the participants. Transcript numbers were determined in triplicates, ratios BCR-ABL/ABL and BCR-ABL/GUS were calculated and expressed in %. Median ratios BCR-ABL/ABL for b3a2 samples were 8.9, 1.7, 0.85, and 0.11%; for b2a2 samples 9.1, 1.6, 0.82, and 0.10%. Median ratios BCR-ABL/GUS for b3a2 samples were 3.4, 0.77, 0.37, and 0.042%; for b2a2 samples 2.8, 0.48, 0.29, and 0.031%. Four of 36 participants (11%) detected low BCR-ABL copy numbers in negative control samples. The coefficients of variation (CV) for all participants, TM, and LC users were 0.62, 0.57, and 0.59 for ratios BCR-ABL/ABL; 1.03, 0.85, and 1.22 for ratios BCR-ABL/GUS, respectively. Standard errors to the regression line were significantly lower evaluating ratios BCR-ABL/GUS (median 0.075, range 0.0046–0.90) compared to ratios BCR-ABL/ABL (median 0.18, range 0.022–2.2, p