Tang Xiao-wen, Wu De-pei, Zhu Zi-ling, Wang Wei, Sun Ai-ning, Qiu Hui-ying, Fu Zheng-zheng, Chang Wei-rong, Ruan Chang-geng
The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Suzhou 215006, China.
Zhonghua Xue Ye Xue Za Zhi. 2004 Feb;25(2):78-81.
To establish multiple short tandem repeat (STR) amplification by fluorescence labeling polymerase chain reaction (PCR) combined with capillary electrophoresis for quantitative determination of chimerism, and to evaluate the status of engraftment and predict the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT).
Thirty-one patients received bone marrow transplantation (BMT) or nonmyeloablative allogeneic stem cell transplantation (NST) were evaluated. Peripheral blood and bone marrow were co-llected before and after transplantation in different period. Nine different STR markers were co-amplified in a single reaction by using a commercial AmpF/STR Profiler Plus PCR amplification kit. Separation of the PCR products and fluorescence detection were performed by ABI prism 310 Genetic Analyzer with capillary electrophoresis. The Genescan and Genotype software were used for size calling and quantification of peak areas. The formula to calculate donor chimerism values was based on the different allelic distribution type between donor and recipient.
48.4% of the patients received sex-matched transplantation and the quantification of donor chimerism could only be performed by STR-PCR method. Comparison of values obtained by FISH analysis with that by STR-PCR in patients transplanted from sex-mismatched donors showed an excellent correlation. The median number of informative alleles was 6.7 (range 2 - 10). The donor's alleles appeared in all the patients on day 7 post-transplant. The median values of donor chimerism in BMT group were inferior to that in NST group on day 7, day 14 and 1 month post-transplant. However the difference disappeared in the midterm or later period of transplant. On day 21, all of the 31 patients had stable engraftment and the percentage of donor chimerism was more than 92%. Median follow-up was 17 (3.5 - 29.0) months after transplantation. Twenty-six of 31 patients had durable engraftment and donor chimerism ratio was more than 90%. So for all of them survived leukemia-freely. Four of the 31 patients had unstable mixed chimerism and relapsed within 6 months post allo-HSCT. Another patient with unstable mixed chimerism appeared graft rejection. Decreasing values of donor chimerism were detected prior to the occurrence of graft rejection and disease relapse. The incidence of GVHD was much higher in the group of full donor chimerism.
Sequential and quantitative monitoring of STR is a valuable tool for studying engraftment dynamics, graft rejection, and relapse and for predicting GVHD. Furthermore it can provide a basis for early intervention of clinical treatment.
建立荧光标记聚合酶链反应（PCR）联合毛细管电泳的多重短串联重复序列（STR）扩增技术用于嵌合体定量测定，评估异基因造血干细胞移植（allo-HSCT）的植入状态并预测其预后。
对31例接受骨髓移植（BMT）或非清髓性异基因干细胞移植（NST）的患者进行评估。在移植前后不同时期采集外周血和骨髓。使用商业AmpF/STR Profiler Plus PCR扩增试剂盒在单一反应中共同扩增9种不同的STR标记。通过ABI prism 310基因分析仪进行毛细管电泳分离PCR产物并进行荧光检测。使用Genescan和Genotype软件进行片段大小测定和峰面积定量。根据供体和受体之间不同的等位基因分布类型计算供体嵌合率的公式。
48.4%的患者接受了性别匹配的移植，供体嵌合率的定量只能通过STR-PCR方法进行。在接受性别不匹配供体移植的患者中，FISH分析与STR-PCR获得的值比较显示出良好的相关性。信息性等位基因的中位数为6.7（范围2 - 10）。移植后第7天所有患者均出现供体等位基因。在移植后第7天、第14天和1个月时，BMT组的供体嵌合率中位数低于NST组。然而，在移植中期或后期差异消失。在第21天，31例患者均有稳定植入，供体嵌合率超过92%。移植后中位随访时间为17（3.5 - 29.0）个月。31例患者中有26例有持久植入，供体嵌合率超过90%。因此他们均无白血病存活。31例患者中有4例有不稳定的混合嵌合体，在allo-HSCT后6个月内复发。另1例有不稳定混合嵌合体的患者出现移植物排斥。在移植物排斥和疾病复发发生前检测到供体嵌合率下降。完全供体嵌合组的移植物抗宿主病（GVHD）发生率更高。
STR的连续定量监测是研究植入动态、移植物排斥和复发以及预测GVHD的有价值工具。此外，它可为临床治疗的早期干预提供依据。
