Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
Clin Chim Acta. 2015 May 20;445:101-6. doi: 10.1016/j.cca.2015.03.018. Epub 2015 Mar 20.
Chimerism analysis is important for the evaluation of engraftment and predicting relapse following hematopoietic stem cell transplantation (HSCT). We developed a chimerism analysis for single nucleotide polymorphisms (SNPs), including rapid screening of the discriminable donor/recipient alleles using droplet allele-specific PCR (droplet-AS-PCR) pre-HSCT and quantitation of recipient DNA using AS-quantitative PCR (AS-qPCR) following HSCT.
SNP genotyping of 20 donor/recipient pairs via droplet-AS-PCR and the evaluation of the informativity of 5 SNP markers for chimerism analysis were performed. Samples from six follow-up patients were analyzed to assess the chimerism via AS-qPCR. These results were compared with that determined by short tandem repeat PCR (STR-PCR).
Droplet-AS-PCR could determine genotypes within 8min. The total informativity using all 5 loci was 95% (19/20). AS-qPCR provided the percentage of recipient DNA in all 6 follow-up patients without influence of the stutter peak or the amplification efficacy, which affected the STR-PCR results.
The droplet-AS-PCR had an advantage over STR-PCR in terms of rapidity and simplicity for screening before HSCT. Furthermore, AS-qPCR had better accuracy than STR-PCR for quantification of recipient DNA following HSCT. The present chimerism assay compensates for the disadvantages of STR-PCR and is readily performable in clinical laboratories.
嵌合体分析对于评估造血干细胞移植（HSCT）后的移植物植入和预测复发非常重要。我们开发了一种用于单核苷酸多态性（SNP）的嵌合体分析方法，包括在 HSCT 前使用液滴等位基因特异性 PCR（droplet-AS-PCR）快速筛选可区分的供体/受者等位基因，以及在 HSCT 后使用 AS-定量 PCR（AS-qPCR）定量受者 DNA。
通过 droplet-AS-PCR 对 20 对供体/受者 SNP 进行基因分型，并评估 5 个 SNP 标记用于嵌合体分析的信息量。对 6 名随访患者的样本进行分析，通过 AS-qPCR 评估嵌合体。这些结果与短串联重复 PCR（STR-PCR）的结果进行了比较。
droplet-AS-PCR 可在 8 分钟内确定基因型。使用所有 5 个位点的总信息量为 95%（19/20）。AS-qPCR 可提供所有 6 名随访患者的受者 DNA 百分比，而不受 stutter 峰或扩增效率的影响，这会影响 STR-PCR 的结果。
与 HSCT 前的 STR-PCR 相比，droplet-AS-PCR 在快速性和简单性方面具有优势。此外，AS-qPCR 在定量 HSCT 后受者 DNA 方面比 STR-PCR 更准确。本嵌合体检测方法弥补了 STR-PCR 的不足，易于在临床实验室中进行。
