Rapid and reliable quantification of minimal residual disease in acute lymphoblastic leukemia using rearranged immunoglobulin and T-cell receptor loci by LightCycler technology.
Rapid and reliable quantification of minimal residual disease in acute lymphoblastic leukemia using rearranged immunoglobulin and T-cell receptor loci by LightCycler technology.
复制标题
利用 LightCycler 技术重排的免疫球蛋白和 T 细胞受体位点，快速、可靠地定量急性淋巴细胞白血病的微小残留病。
作者：
M. Nakao;J. Janssen;T. Flohr;C. Bartram
使用免疫球蛋白和T细胞受体（TCR）重排作为PCR靶点的微小残留病（MRD）检测为急性淋巴细胞白血病（ALL）的体内治疗有效性提供了重要的预后信息。在这里，我们报告了使用LightCycler技术对25例ALL患者的MRD进行实时定量。我们设计并调整了等位基因特异性寡核苷酸（阿索）-PCR方案，该方案能够检测ALL患者中观察到的>90%的IGH、IGK、TCRD和TCRG重排。在所有患者中，至少可以鉴定出两种合适的标记物（平均，3.4种标记物/患者）。我们应用ASO-PCR与35个免疫球蛋白和TCR重排（11 IGH，6 IGK，12 TCRG，和6 TCRD），并比较的灵敏度和实用性的LightCycler策略与传统的ASO-PCR上的封闭热循环仪，然后用凝胶电泳定量。LightCycler通过用DNA结合染料SYBR绿色I染色PCR产物，在每个循环（实时）测量白血病特异性PCR产物。LightCycler技术在8例病例中显示出比传统方法更高的灵敏度，而其他标志物的灵敏度完全匹配。检测水平在10（-4）和10（-6）个白血病细胞之间变化。此外，我们确定了MRD状态的27个骨髓随访样本从15个ALL患者的两种方法，并揭示了可比的结果。然而，LightCycler也允许在含有相对高水平（>10（-3））残留白血病细胞的样品中进行准确定量。传统的ASO-PCR技术包括各种费力和耗时的PCR实验和PCR后步骤，以确定具有最佳线性和PCR灵敏度的循环数。通过LightCycler技术进行实时定量，避免了这些PCR后步骤，通过软件分析提供了最高的灵敏度，因此代表了一种快速、可靠、灵敏和具有成本效益的技术，用于ALL患者MRD的常规监测。
The detection of minimal residual disease (MRD) using immunoglobulin and T-cell receptor (TCR) rearrangements as PCR targets provides important prognostic information on the in vivo effectiveness of treatment in acute lymphoblastic leukemia (ALL). Here we report on the real-time quantification of MRD in 25 ALL patients using LightCycler technology. We designed and adapted allele-specific oligonucleotide (ASO)-PCR protocols that enabled the detection of >90% of the IGH, IGK, TCRD, and TCRG rearrangements observed in ALL patients. In all patients, at least two suitable markers could be identified (average, 3.4 markers/patient). We applied ASO-PCR with 35 immunoglobulin and TCR rearrangements (11 IGH, 6 IGK, 12 TCRG, and 6 TCRD) and compared the sensitivity and practicability of the LightCycler strategy with conventional ASO-PCR on a block thermocycler followed by quantification with gel electrophoresis. The LightCycler measured leukemia-specific PCR products at each cycle (real-time) by staining the PCR product with the DNA-binding dye SYBR Green I. LightCycler technology showed a higher sensitivity than the conventional method in eight cases, whereas the sensitivity of the other markers matched exactly. The detection level varied between 10(-4) and 10(-6) leukemic cells. Furthermore, we determined the MRD status of 27 bone marrow follow-up samples from 15 ALL patients by both methods and revealed comparable results. However, the LightCycler also allowed accurate quantification in samples containing relatively high levels (>10(-3)) of residual leukemia cells. The conventional ASO-PCR technique comprises various laborious and time-consuming PCR experiments and post-PCR steps to determine the number of cycles with the optimal linearity and sensitivity of the PCR. Real-time quantification through LightCycler technology obviates these post-PCR steps, provides the highest sensitivity via software analysis, and therefore represents a rapid, reliable, sensitive, and cost-effective technique for the routine monitoring of MRD in ALL patients.
影响因子：
2.7
作者：
Wittwer, CT;Ririe, KM;Balis, UJ
通讯作者：
Balis, UJ
影响因子：
2.9
作者：
Ririe, KM;Rasmussen, RP;Wittwer, CT
通讯作者：
Wittwer, CT