Analysis of engraftment kinetics of donor-derived cells in multi-cord blood transplantation

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Due to its many benefits, since 2003 the number of cord blood transplantations(CBT) being performed has increased to almost equal that of bone marrow transplantation in Japan. CBT, however, can be complicated by graft failure, which must be resolved for safe transplantation. To overcome these problems, double-unit cord blood transplantation(dCBT) has been developed. In Japan, a large scale clinical trial ran from 2006 to 2010 to check the effects of dCBT on the engraftment process. The results from this and other global dCBT clinical trials, however, are controversial. Additionally, basic research are not complete. Therefore, it is very important to investigate engraftment kinetics of dCBT and develop effective protocols on the basis of research data.At the start of this research project, we planned to analyze recipient blood after dCBT. After March 2010, however, there were no dCBT clinical trials in Japan. Therefore, we changed the project style from clinical investigation to analysi … More s of artificial chimerism samples in culture.In 2009, we had many biotinylated commercial allele specific HLA monoclonal antibodies and we discovered red blood cell contamination in the leukocyte fraction of peripheral blood. We found that previous multi-color chimerism analysis systems could not clearly analyze different kinds of HLA-bearing white blood cells from multiple persons.In 2010, we changed the combination of antibodies and fluorescent agents to obtain a good separation of each subpopulation and proper compensations. Finally, we successfully established an 11-color flow cytometry system for accurate analysis. We used PE-Cy5-conjugaed anti-CD235a antibody to get rid of red blood cells from gates on white blood cells for chimerism analysis. In addition, we made an anti-HLA-B62 antibody which stained cells very brightly.In 2011, we used our 11-color flow cytometry system to analyze triple chimerism in CD4^+T cells and CD8^+T cells in a mixed lymphocyte culture to measure frequencies of three donors' white blood cells and analyze mRNA levels of each person's CD4^4 T cells and CD8^+T cells which will be useful to understand engraftment kinetics and molecular basis of allo-reactivity in future dCBT. Less