实时双等位基因多态性-聚合酶链反应用于监测造血干细胞移植复发患者的嵌合状态。

Real-time biallelic polymorphism-polymerase chain reaction for chimerism monitoring of hematopoietic stem cell transplantation relapsed patients.

机构信息

Department of Laboratory Medicine, Chang-Gung Memorial Hospital, Taoyuan County, Taiwan.

出版信息

Clin Chim Acta. 2011 Mar 18;412(7-8):625-30. doi: 10.1016/j.cca.2010.12.018. Epub 2010 Dec 23.

DOI:10.1016/j.cca.2010.12.018

PMID:21185273

Abstract

BACKGROUND

An accurate analysis of chimerism kinetics permits early detection of hematopoietic stem cell transplantation (HSCT) in patients with high risks of graft-versus-host disease or those liable to relapse. Although short tandem repeats-PCR (STR-PCR) is the golden standard for quantitative chimerism analysis in most of the clinical laboratories, it has a relatively low sensitivity of 5% and the detection of low percentage in mixed chimerism is usually delayed. In this study, we developed a real-time PCR for chimerism analysis based on the informative biallelic polymorphisms (BP).

METHODS

The allele frequencies of 19 selective biallelic polymorphic markers were analyzed using the genomic DNA from 100 healthy Taiwanese volunteers. The informative biallelic polymorphic markers with high discrimination power in the Taiwanese population were identified. The TaqMan probe-based real-time BP-PCR for amplification of the informative loci was designed and the detection sensitivity was determined. Clinical application of real-time BP-PCR in chimerism monitoring was evaluated and was compared with the conventional STR-PCR by analyzing the DNA samples obtained at different time points post-HSCT from 4 relapsed and 10 non-relapsed patients.

RESULTS

Allele distribution analysis revealed that the loci of S01a, S03, S04a, S05b, S06, S07b, S08b, S09b, S10b and S11a had a relatively high discrimination power and were the informative BP for chimerism monitoring in the Taiwanese population. Real-time BP-PCRs for these 10 BP loci were set up with the detection sensitivity equivalent to 0.003-0.006%. Real-time BP-PCR of the 4 HSCT patients revealed the presence of recipient-specific DNA at early time point than STR-PCR for 3 of the patients, whereas real-time BP-PCR was as effective as STR-PCR in uncovering the sign of relapse for one of the patients. In addition, the baseline value for the patients with no sign of relapse was 0.127 ± 0.193% of recipient DNA.

CONCLUSION

We conclude that real-time BP-PCR is a sensitive and reliable method for chimerism monitoring and is superior to the STR-PCR in identifying patients who are at high risk for relapse after transplantation.

摘要

背景

准确分析嵌合体动力学可以早期检测造血干细胞移植（HSCT）患者发生移植物抗宿主病（GVHD）或复发的风险。虽然短串联重复序列-PCR（STR-PCR）是大多数临床实验室定量嵌合体分析的金标准，但它的灵敏度相对较低，为 5%，并且通常会延迟对混合嵌合体中低比例的检测。在这项研究中，我们基于信息性双等位基因多态性（BP）开发了一种实时 PCR 用于嵌合体分析。

方法

使用来自 100 名健康台湾志愿者的基因组 DNA 分析了 19 个选择性双等位基因多态性标记的等位基因频率。确定了在台湾人群中具有高鉴别力的信息性双等位基因多态性标记。设计了基于 TaqMan 探针的实时 BP-PCR 来扩增信息性位点，并确定了检测灵敏度。通过分析 4 例复发和 10 例非复发患者 HSCT 后不同时间点获得的 DNA 样本，评估了实时 BP-PCR 在嵌合体监测中的临床应用，并与常规 STR-PCR 进行了比较。

结果

等位基因分布分析显示，S01a、S03、S04a、S05b、S06、S07b、S08b、S09b、S10b 和 S11a 等位点具有相对较高的鉴别力，是台湾人群嵌合体监测的信息性 BP。建立了用于这 10 个 BP 位点的实时 BP-PCR，检测灵敏度相当于 0.003-0.006%。对 4 例 HSCT 患者的实时 BP-PCR 显示，3 例患者在早期时间点就存在受者特异性 DNA，而实时 BP-PCR 在发现 1 例患者复发迹象方面与 STR-PCR 一样有效。此外，无复发迹象患者的基线值为 0.127±0.193%的受体 DNA。