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

由于它的许多好处，自2003年以来，脐带血移植（CBT）的数量已经增加到几乎等于骨髓移植在日本。然而，CBT可能并发移植失败，这必须解决安全移植。为了克服这些问题，已经开发了双单位脐带血移植（dCBT）。在日本，2006年至2010年进行了一项大规模临床试验，以检查dCBT对植入过程的影响。然而，这项研究和其他全球dCBT临床试验的结果是有争议的。此外，基础研究还不完善。因此，研究dCBT的植入动力学并在研究数据的基础上制定有效的方案是非常重要的。然而，2010年3月之后，日本没有dCBT临床试验。因此，我们将项目类型从临床调查改为分析， ...更多信息 在2009年，我们有许多生物素化的商业等位基因特异性HLA单克隆抗体，我们发现外周血白细胞部分中的红细胞污染。我们发现以前的多色嵌合体分析系统不能清楚地分析来自多个人的不同种类的携带HLA的白色血细胞，在2010年，我们改变了抗体和荧光剂的组合，以获得每个亚群的良好分离和适当的补偿。最后，我们成功地建立了一个11色流式细胞仪系统，用于准确分析。我们使用PE-Cy 5偶联的抗CD 235 a抗体从白色血细胞上的门中除去红细胞用于嵌合体分析。此外，我们还制备了一种抗HLA-B62抗体，它可以将细胞染色得非常明亮。我们使用我们的11色流式细胞术系统分析了混合淋巴细胞培养物中CD 4 ^+T细胞和CD 8 ^+T细胞的三重嵌合体，测量了三名供体白色血细胞的频率，并分析了每个人的CD 4 ^4 T细胞和CD 8 ^+T细胞的mRNA水平。T细胞，这将有助于了解移植动力学和同种异体反应性的分子基础，在未来的dCBT。少

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