Stahl Tanja, Böhme Manja U, Kröger Nicolaus, Fehse Boris
Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.
R&D Department, Biotype Diagnostic GmbH, Dresden, Germany.
Exp Hematol. 2015 Jun;43(6):462-8.e1. doi: 10.1016/j.exphem.2015.02.006. Epub 2015 Mar 18.
Analysis of hematopoietic chimerism after allogeneic stem cell transplantation represents a crucial method to evaluate donor-cell engraftment. Whereas sensitivity of classical approaches for chimerism monitoring is limited to ≥1%, quantitative polymerase chain reaction (qPCR)-based techniques readily detect one patient cell in >1,000 donor cells, thus facilitating application of chimerism assessment as a surrogate for minimal residual disease. However, due to methodologic specificities, qPCR combines its high sensitivity with limited resolution power in the state of mixed chimerism (e.g., >10% patient cells). Our aim was to overcome this limitation by employing a further development of qPCR, namely digital PCR (dPCR), for chimerism analysis. For proof-of-principle, we established more than 10 dPCR assays detecting Indel polymorphisms or Y-chromosome sequences and tested them on artificial cell mixtures and patient samples. Employing artificial cell mixtures, we found that dPCR allows exact quantification of chimerism over several orders of magnitude. Digital PCR results proved to be highly reproducible (deviation <5%), particularly in the "difficult" range of mixed chimerism. Excellent performance of the new assays was confirmed by analysis of multiple retrospective blood samples from patients after allogeneic stem cell transplantation, in comparison with established qPCR (14 patients) and short-tandem repeat PCR (4 patients) techniques. Finally, dPCR is easy to perform, needs only small amounts of DNA for chimerism assessment (65 ng corresponds to a sensitivity of approximately 0.03%), and does not require the use of standard curves and replicate analysis. In conclusion, dPCR represents a very promising method for routine chimerism monitoring.
异基因干细胞移植后造血嵌合体分析是评估供体细胞植入的关键方法。传统的嵌合体监测方法灵敏度限于≥1%，而基于定量聚合酶链反应（qPCR）的技术能轻易检测出1000多个供体细胞中的一个患者细胞，从而便于将嵌合体评估用作微小残留病的替代指标。然而，由于方法学特异性，在混合嵌合体状态下（如患者细胞>10%），qPCR虽灵敏度高但分辨率有限。我们的目标是通过进一步发展qPCR即数字PCR（dPCR）来克服这一局限，用于嵌合体分析。为验证原理，我们建立了10多种检测插入缺失多态性或Y染色体序列的dPCR检测方法，并在人工细胞混合物和患者样本上进行测试。利用人工细胞混合物，我们发现dPCR能在几个数量级上精确量化嵌合体。数字PCR结果证明具有高度可重复性（偏差<5%），尤其是在混合嵌合体的“困难”范围内。通过对异基因干细胞移植后患者的多个回顾性血液样本进行分析，并与既定的qPCR（14例患者）和短串联重复PCR（4例患者）技术比较，证实了新检测方法的优异性能。最后，dPCR操作简便，嵌合体评估仅需少量DNA（65 ng对应约0.03%的灵敏度），且无需使用标准曲线和重复分析。总之，dPCR是一种非常有前景的常规嵌合体监测方法。
