| Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapeutic approach in patients with malignant hematological diseases or nonmalignant hematological diseases. However, graft failure, disease relapse and graft-versus-host disease (GVHD) are major obstacles that affect prognosis and survival of patients undergoing allo-HSCT. Serial and quantitative analysis of chimerism has been proposed as an important method in early monitoring these negative events in order to set up the relevant preventive therapeutics.Up to date, various techniques have been used to document chimerism, such as red cell phenorypes, karyotype, fluorescence in situ hybridization (FISH) and variable number tandem repeats(VNTR). Limitations of these techniques include limited degrees of polymorphism, poor sensitivity, long hands-on time and restriction to cases with a sex-mismatched donor and to cases without cytopenia, respectively. For the clinical application, the optimal methodological approach to monitor chimerism should be informative, sensitive, quantitatively accurate and independent on sex-mismatch.In the present study, the technique of multiplex amplification of short tandem repeat (STR) markers by fluorescence labeling polymerase chain reaction (PCR) combined with a capillary electrophoresis for the quantitative determination of chimerism were established. The applicability of this method which has been used for forensic purpose was investigated both for detection and quantification of chimerism according to the proportion of the peak areas corresponding to DNA signal and standard curve. Standardization of the calculation formula was validated to obtain reproducible and accurate chimerism values. For reproducible quantitative results, it is important to calculate the mean value of all type I markers to obtain donor chimerism values in order to compensate for random variability in amplification. By using serial dilutions of artificial mixed cell chimerism, we evaluated the linearity andsensitivity of this method. A linear correlation was found between the proportion of cells mixed and the calculated ratio of donor and recipient according to the peak areas of the corresponding signals with a high correlation coefficient ( r = 0.993). The sensitivity for detecting a minor population range from 1-5%. The medium sensitivity was about 3%. In order to verify the reproducibility of this method, the same sample was repeated three times with a median standard deviation from mean of 2.1%. Moreover the range of chimerism values of the same sample reported by two different laboratories was less than 5%. Furthermore, the nine different STR markers that commercial AmpF/STR profiler plus kit applied were tetranucleotide tandem repeats locus and had the characteristics of high polymorphism, high information and discrimination. With the combination of nine STR markers, the informative peak constellation can be identified in all 54 donor/recipient pairs. The median number of informative STRs was six in the 42 related patients and eight in the 12 unrelated donor/recipient pairs. This study demonstrates that STR-based human identity testing commercial kits are well suitable for chimerism analysis. Our results suggest that this procedure allow the high sensitive, reproducible and sex unlimited accurate quantification of mixed chimerism.On the basis of this method established, the sequential and quantitative chimerism analyses were performed in 54 patients who received allo-HSCT which included standard bone marrow transplantation (BMT), peripheral blood stem cell transplantation (PBSCT), nonmyeloablative stem cell transplantation (NST) and cord blood transplantation (CBT). We investigated the chimeric status of whole blood or bone marrow in study of engraftment kinetics, evaluation of minimal residual disease, prediction of transplant outcome and guide for adoptive immunotherapy post-transplant.Serial and quantitative monitoring of donor chimerism has been performed in the group of 18 patients who received NST. No... |
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