The STR Typing System By Fluorescence Labeled Multiplex-PCR Quantitative Detection Technique In Allogeneic Stem Cell Transplantation

Objective Using microsatellites genetic polymorphism , polymerase chain reaction and capillary electrophoresis to establish quantitative detection of chimerism by multiple PCR amplification of short tandem repeats markers and capillary electrophoresis with fluorescence detection for 16 STR locus after Allogeneic hematopoietic stem cell transplantation(Allo-HSCT). The chimerism examination were made for 30 dyas after Bone marrow transplantation(BMT) combined with peripheral blood stem cells transplantation(PBSCT) or singel PBSCT while the chimerism was continuous detected within 30 days after Unrelated Cord blood transplantation(UCBT) to judge transplants implantation. At the same time, the evaluation of diseases and prediction of diseases outcome were made by chimerism examination long-term after Allo-HSCT and observe its practical clinical application value by comparing with other detection means.Method To build the STR typing system by fluorescence labeled multiplex-PCR and capillary electrophoresis technique for personal identification. Amplified fluorescence labeled multiplex-STR with PowerPlex16 personal identification kits from Promage corporation. Specific str locus contains fifteen gene locus such as D3S1358,TH01,D21S11,D18S51,PentaE,D5S818,D13S317,D7S820,D16S539,CSF1PO,PentaD,vWA,D8S1179,TPOX,FGA and one sexual gene locus Amelogenin. The amplified products were electrophoreticed by 3100 genetic analyzer from ABI corporation, and data analysis by GenScan analysis software. At the same time, under the basement of qualitative detection, quantitative detection of chimerism were investigated according to the Proportion of the Peak areas corresponding to DNA signal after electrophoresis. The method was used to detected chimerism at different time after Allo-HSCT for 44 hematologic malignancies patients who received Allo-HSCT in Anhui provincial hospital between 2006-2007. In the all 44 hematologic malignancies patients, chimerism was detected at 30 days to judge grafts implantation after Allo-HSCT for 33 BMT combined with PBSCT and 2 singel PBSCT. While in the 9 UCBT, chimerism was detected four times within 30 days after Allo-HSCT, and compared with peripheral hemogram to observe the rule of grafts implantation or predict the transplantation effect in advance. The grafts implantation permanently and diseases outcome were predicted by the continuous tracking of chimerism detection after Allo-HSCT.Result The chimerism can be detected in all the transplantation recipients. 35 Allo-HSCT obtained hematopoietic reconstitution successfully. 34 PBSCT/BMT transplantation recipients formed FDC at 30 days after Allo-HSCT, 1 63 year old patient received decreased intensity PBSCT and formed MC at 30 days and FDC at 90 days after Allo-HSCT. The chimerism can be detected in all the UCBT recipients, 7 patients who obtained hematopoietic reconstitution successfully have FDC in the peripheral blood hemopoietic cells.other 2 patients formed MC at 7 days after Allo-HSCT and whose hematopoietic reconstitution were failed.it is failed to find donor at the chimerism at 30 days after Allo-HSCT, In the tracking follow-up, 7 bone marrow relapse have the sign of donor decreased in the chimerism while 2 out of bone marrow relapse were formed FDC all the time.conclusion 1.The STR typing system by fluorescence labeled multiplex-PCR and capillary electrophoresis technique can dudge graft accurately at early time after Allo-HSCT. The method can can predict graft implantation permanently and disease prognosis by Chimerism track after Allo-HSCT, provid reliable experimental basis for clinical treatment. 2. Detect the Chimerism continuously within 30 days after UCBT can provide the information of graft implantation, Graft can be detected as early as 7 day after UCBT while peripheral hemogram could not recover and have a good coherence with implantation index ANC, the method can reveal UCBT engraftment kinetics and evaluate UCBT engraftment sensitivity and specificity 3.The method has high sensitivity and specificity and have a good coherence with other detection method , the sampling quantity was low and clinical application value was good.