Maas F, Schaap N, Kolen S, Zoetbrood A, Buño I, Dolstra H, de Witte T, Schattenberg A, van de Wiel-van Kemenade E
Central Hematology Laboratory, University Medical Center St Radboud, Nijmegen, The Netherlands.
Leukemia. 2003 Mar;17(3):621-9. doi: 10.1038/sj.leu.2402856.
Analysis of changes in recipient and donor hemopoietic cell origin is extremely useful to monitor the effect of stem cell transplantation (SCT) and sequential adoptive immunotherapy by donor lymphocyte infusions (DLI). We developed a sensitive and accurate method to quantify the percentage of recipient and donor cells by real-time PCR using single nucleotide polymorphisms (SNPs) as markers. Allele-specific PCR of seven SNPs resulted in specific markers for donor or recipient in 97% of HLA-identical sibling pairs. Both, recipient- and donor-derived hemopoietic cells can be simultaneously analyzed in 67% sibling pairs. We expect this can be increased to approximately 99% by developing three additional SNP-PCR. Serial dilution of SNP-positive DNA into either SNP-negative DNA or water revealed a detection limit of 0.1-0.01% depending on the amount of input DNA and start C(t) of the used SNP-PCR. Application of our real-time SNP-PCR method for a CML patient treated by allogeneic SCT and DLI demonstrated its feasibility to follow donor T-cell chimerism and early detection of residual and recurrent autologous hemopoiesis in response to treatment. This detailed monitoring of the genetic origin of hemopoietic cells, in particular immune effector cells and target cells after SCT and DLI, may substantially contribute to understanding of the mechanisms that play a role in the success of treatment.
分析受体和供体造血细胞来源的变化对于监测干细胞移植（SCT）以及通过供体淋巴细胞输注（DLI）进行的序贯过继免疫治疗的效果极为有用。我们开发了一种灵敏且准确的方法，利用单核苷酸多态性（SNP）作为标记，通过实时PCR定量受体和供体细胞的百分比。针对7个SNP的等位基因特异性PCR在97%的HLA匹配同胞对中产生了供体或受体的特异性标记。在67%的同胞对中能够同时分析受体和供体来源的造血细胞。我们预计通过开发另外三种SNP-PCR，这一比例可提高至约99%。将SNP阳性DNA连续稀释到SNP阴性DNA或水中，根据输入DNA的量和所用SNP-PCR的起始C(t)值，检测限为0.1 - 0.01%。将我们的实时SNP-PCR方法应用于一名接受异基因SCT和DLI治疗的慢性粒细胞白血病（CML）患者，证明了其在追踪供体T细胞嵌合以及早期检测治疗反应中残余和复发性自体造血方面的可行性。对造血细胞，特别是SCT和DLI后免疫效应细胞和靶细胞的遗传来源进行这种详细监测，可能会极大地有助于理解在治疗成功中起作用的机制。
