Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction
Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction
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DOI：
10.1182/blood.v99.12.4618
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发表时间：
2002-06-15
期刊：
影响因子：
20.3
通讯作者：
Quelvennec, E
中科院分区：
文献类型：
--
作者：
Alizadeh, M;Bernard, M;Quelvennec, E
我们已经开发了一种实时定量聚合酶链反应（PCR）测定使用TaqMan技术（应用生物系统公司，福斯特市CA）用于监测供体细胞移植在异基因造血干细胞移植受体。为此，我们选择了19个特定的序列多态性，属于11个人类双等位基因位点位于9个不同的染色体。利用一组专门设计的引物和荧光探针，我们评估了19个标记的信息量的面板上的126个DNA样本从63个受体/供体对。在超过90%的这些对中，受体和供体基因谱之间的歧视是可能的。通过对混合DNA的连续稀释，我们评价了该方法的线性和灵敏度。线性相关系数r> 0.98，灵敏度为0.1%。短串联重复序列聚合酶链反应（STR-PCR）和实时荧光PCR嵌合检测与一组人工细胞混合物进行了比较。与STR-PCR嵌合体检测相比，实时PCR方法的主要优点是不存在PCR竞争和平台偏倚，结果表明实时PCR方法具有更高的灵敏度和线性。此外，不同的样品可以在同一个PCR运行中进行测试，最终结果在不到48小时。最后，我们前瞻性分析了接受同种异体移植的患者，并提出了4种不同的临床情况，说明了我们的方法的信息水平。总之，这种新的检测方法提供了一种准确的混合嵌合体定量评估，可用于指导早期实施额外的治疗造血干细胞移植。(C)2002年，美国血液学会。
We have developed a real-time quantitative polymerase chain reaction (PCR) assay using TaqMan technology (Applied Biosystems, Foster City CA) for monitoring donor cell engraftment in allogenic hematopoietic stem cell transplant recipients. For this purpose, we selected 19 specific sequence polymorphisms belonging to 11 human biallelic loci located on 9 different chromosomes. Using a set of specially designed primers and fluorogenic probes, we evaluated the 19 markers' informativity on a panel of 126 DNA samples from 63 recipient/donor pairs. In more than 90% of these pairs, discrimination between recipient and donor genetic profile was possible. By using serial dilutions of mixed DNAs, we evaluated the linearity and sensitivity of the method. A linear correlation with r higher than 0.98 and a sensitivity of 0.1% proved reproducible. Fluorescent-based PCR of short tandem repeats (STR-PCR) and real-time PCR chimerism assay were compared with a panel of artificial cell mixtures. The main advantage of the real-time PCR method over STR-PCR chimerism assays is the absence of PCR competition and plateau biases, and results evidenced greater sensitivity and linearity with the real-time PCR method. Furthermore, different samples can be tested in the same PCR run with a final result in fewer than 48 hours. Finally, we prospectively analyzed patients who received allografts and present 4 different clinical situations that illustrate the informativity level of our method. In conclusion, this new assay provides an accurate quantitative assessment of mixed chimerism that can be useful In guiding early implementation of additional treatments in hematopoietic stem cell transplantation. (C) 2002 by The American Society of Hematology.