Clinical Study On Chimerism Of Blood Cell Following Allogeneic Peripheral Blood Stem Cell Transpltation

The continues monitoring of chimerism after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a valuable way for studying engraftment dynamics, graft rejection, disease relapse and for predicting GVHD.Furthermore it can provide a basis for early clinical therapy intervention to the patients after allo-HSCT.STR (short tandem repeats) is piece of specific DNA repeat sequences, with high polymorphism. It can get the accurately individual recognition after PCR.So it is the most sensitive method of chimeric state detection to combine STR-PCR with automatic capillary electrophoresis. Quantitative STR - PCR can accurately detect the chimerical rate, assess the implantation of donor cells .This will provide the theory basis for HSCT follow-up treatment.The aim of this study is to establish the technology of MACS separation, observe the chimerical state of donor cells using fluoresce-labeled multiple STR-PCR combined with automatic capillary electrophoresis method, and dynamically test several kinds of blood cells after transplantation. The correlation between the chimerism and cells implantation, hematopoietic recovery, graft rejection, relapse and GVHD will be the guidance to reasonable use of immune intervention therapy, and after all improving the success rate of the transplantation.Partâ… Technology establishment of quantitative STR - PCR detection of donor cell implantation after allo- HSCT【Objective】To establish the method of quantitative STR- PCR detection.【Method】2 normal cases with different HLA were token as donor and recipient respectively. Anticoagulant fresh blood samples of these two were used to make the artificial mixture chimerism (MC) with different ratio according to the number of white blood cells. After genomic DNA extracting, 15 STR sites and 1 gender site were synchronously expanded with fluoresce-labeled STR-PCR. After automatic capillary electrophoresis and clips analysis, genotype would be read according to the different length of DNA fragment and allele ladder, and we can get a chimeric proportion of donor cells in a proper chimeric rate formula.【Result】Donors'specific STR sites could be detected in all the 5 cases after the transplantation, and the chimeric percentage was 5% at least. On the 7^th day after transplantation, 5 patients got an average rate of 70%, and on the 14^th day, all the chimeric rates were more than 90% except one 88%. On the 21^st and 28^th day, all the patients reached the completely chimeric state.【Conclusion】Quantitative STR - PCR test method was established successfully through 5 cases allo-HSCT patients with accurate observation of dynamic chimeric state changes.Partâ…¡The relationship between aGVHD and mononuclear cells, T cells, neutrophils implant dynamics after Allo- HSCT【Objectives】1. To observe the implant order of donor blood cells after the allo-HSCT.2.To analyze the relations between the implant order and a-GVHD.【Method】Peripheral blood or bone marrow samples were collected from 22 patients diagnosed with hematology diseases and treated with allo-HSCT in our hospital. Every samples of the recipients on the day 7, 14, 21, 28 after transplantation were collected as well as the samples from the donors. MACS technology was used to sort out mononuclear cells, CD3⁺T cells, CD15⁺T cells with high purity. STR - PCR was applied for every sample. The clinical manifestation, including GVHD, blood routine, classification and chromosome of the bone marrow cells were observed at the same time.【Results】Hematopoietic recovery was successful for every patient after transplantation. The implant rate of T cells, granulocytes and MNC was 65%, 40% and 70% respectively. Neutrophils were the main implant cells on the day 14 and 21 after transplantation, T cells implanted slightly later than granulocytes. Within 28 days after transplantation, 8 of 19 patients (42.1 %) who had reached a high percentage of donors'T cells chimerism got a-GVHD, the incidence was significantly higher than + 28d T - MC group.【Conclusions】1. After allo-HSCT, T lymphocytes implanted the earliest to the recipients, but the latest to reach FDC . Granulocytes implanted later, but quickly.2. High T cells chimeric state is closely related to a-GVHD.Partâ…¢The application of dynamic chimeric changes in disease recurrence and graft exclusion【Objectives】1.To observe donors'blood cells dynamic chimeric change after allo-HSCT;2. To analyze the relationship between MNC and disease relapse and GR.【Method】Samples form 3 cases of recurrence and 1 case of GR patients were analyzed with STR - PCR before and after, chimeric rate of MNC was focused on, and the relevance was observed.【Results】All paitient have re-establishment which relapsed after allo-HSCT.CD3⁺ T lymphocytes'chimeric level was low at the beginning, but could get higher gradually after hematopoietic rebuild. 3 patients with DC decrease were proved being relapse in bone marrow morphology. After discontinue or reduce the dose of immunosuppressive drugs, companied with successively DLI, IL - 2 or chemotherapy, one patient received FDC from MC, and to the rest, one patient was effective after treament at initial stage but invalid at later stage, one patient was significant but unfollow-up. MC rise after CSA withdrawal in the GR case.【Conclusions】1. Early T lymphocytes MC state does not imply later recurrence or rejection.2. MC decreasing after transplantation is always the sign of relapse or graft rejection.3. When relapse or graft rejection occurred, reduction or discontinuation of immunosuppressive drugs, or DLI can enhance GVL effect. This may reverse the trend, improving MC to...