嵌合体的动态定量检测在异基因造血干细胞移植中的应用

目的　建立荧光标记的多重PCR扩增短串联重复序列 (STR PCR)结合毛细管电泳 ,定量检测供体细胞 (DC)嵌合率的方法 ,并探讨该方法的连续定量检测对异基因造血干细胞移植 (allo HSCT)后转归的预警作用。方法　采集 31例接受骨髓移植 (BMT)或非清髓外周血干细胞移植 (NST)患者移植前、移植后不同时段的外周血或骨髓。DNA样本用ProfilerPlus商品化试剂盒扩增后 ,用ABI 310遗传分析仪进行毛细管电泳 ,确定基因位点及峰面积 ,根据供受体基因型的差异选择嵌合率计算公式。结果　 31例患者中 15例 (48.4 % )为性别相合移植 ,只能通过STR PCR进行嵌合体的定量分析 ;性别不合移植患者用STR PCR和荧光原位杂交两种方法定量测得的DC嵌合率一致 ;31对供受体中能区别出供受差别的STR位点有 6 .7(2～ 10 )个 ,所有患者均在移植后 7天 ( 7天 )出现供体来源的细胞 ,BMT组 7天、 14天和 1个月DC中位数均明显低于NST组 ,而在移植中后期无显著性差异。 2 1天时BMT和NST患者均达稳定嵌合 ,DC在 92 %以上 ;中位随访 17(3.5～ 2 9.0 )个月 ,2 6例患者DC≥90 % ,均获得持久植入 ,至今均为无白血病生存。另有 5例患者出现不稳定混合嵌合 (MC)状态 (DC为2 7.3%～ 6 2 .7% ) ,其中 4例复发 ,1例出现移植物被排斥。上述 5例患者均

Application of sequential and quantitative monitoring of chimerism in allogeneic hematopoietic stem cell transplantation

OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.