造血干细胞移植后通过序列多态性系统的实时荧光定量聚合酶链反应对造血嵌合体进行定量评估。

Quantitative assessment of hematopoietic chimerism by quantitative real-time polymerase chain reaction of sequence polymorphism systems after hematopoietic stem cell transplantation.

机构信息

Peking University People's Hospital, Peking University Institute of Hematology, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Beijing 100044, China.

出版信息

Chin Med J (Engl). 2011 Aug;124(15):2301-8.

PMID:22029079

Abstract

BACKGROUND

Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT.

METHODS

A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally.

RESULTS

Recipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation <1.85%), which might significantly improve detection accuracy of changes in autologous signals early in the post-transplantation course of follow-up. The main advantage of the real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method.

CONCLUSION

This SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative assessment of mixed chimerism that can be useful in predicting graft rejection and early relapse.

摘要

背景

分析受者和供者造血细胞来源的变化对于监测造血干细胞移植（HSCT）和通过供者淋巴细胞输注进行序贯过继免疫治疗的效果非常有用。我们开发了一种基于序列多态性系统的敏感、可靠和快速实时 PCR 方法，用于定量评估 HSCT 后的造血嵌合体。

方法

我们通过实时 PCR 筛选了 101 例白血病和其他血液系统疾病患者的 29 个选定的序列多态性（SP）标记物。对 8 例处于缓解和复发状态的 HSCT 患者的骨髓样本进行了纵向跟踪。

结果

97.0%（101 例中的 98 例）的患者可以进行受者基因型鉴别，每个受者/供者对平均有 2.5 个（1-7 个）信息性标记物。使用包含特定 SP 标记物的质粒进行连续稀释，证明了线性相关性（r）为 0.99，斜率在-3.2 到-3.7 之间，灵敏度为 0.1%。通过这种方法，可以非常准确地检测到 0.1%到 30%范围内的自体信号。该方法在非常重要的 5%以下自体信号范围内的准确性极高（标准偏差<1.85%），这可能显著提高移植后早期自体信号变化的检测准确性。实时 PCR 方法与短串联重复 PCR 嵌合体分析相比的主要优势是不存在 PCR 竞争和平台偏差，具有更高的灵敏度和线性度。最后，我们前瞻性地分析了接受同种异体移植物并呈现缓解和复发情况嵌合体动力学的 8 例患者的骨髓样本，说明了该方法的灵敏度水平和有前途的临床应用。