ABO不合HSCT术后ABO嵌合体诊断新方法的建立及应用

ABO chimera naturally exists post ABO incompatible hematopoietic stem cell transplantation. It is the direct participator in transplant related immunohematologic complications and can reflect the dynamic ABO transition from recipient to donor, but till now,research on ABO chimera is rare,which can be only analyzed by the phenotypic method that have the limitations such as the low sensitivity and inevitable interference by transfusion. To explore a new fresh and high efficient method for revealing the important theoretical and applicational values of ABO chimera analysis is the current key point need to be resloved. Our proposal will develop a fresh diagnostic method for ABO chimera in the genetic level by establishment of new screen panels of markers based on characteristic nucleotide polymorphism sites derived from intrinsic distinguishing commonly ABO diploid genotypes and determined by real time quantitative PCR based Taqman hydrolysis probes,which can overcome these above limitations of phenotypic methods and express high efficience and prominent applied advantages. Then we will fully reveal the new and important values of ABO chimera acted as a new indicator in clinic recipients post ABO incompatible hematopoietic stem cell transplantation: to accurately explore the mechanism and the predictive effects on the occurrence and progress of immunohematologic complications by monitoring ABO chimera in erythroid precursor cells at the genetic level and pathogenic ABO antibody titration,and to validate of the effects on accurate determination of dynamic ABO types after transplantation by monitoring ABO chimera in peripheral reticulocytes at the mRNA level in order to meliorate the security and feasibility of transfusion policies post ABO incompatible hematopoietic stem cell transplantation.

ABO不合造血干细胞移植术后天然存在ABO嵌合体，尽管它是移植免疫血液学并发症的直接参与者且反映供受者ABO血型动态转变，但迄今罕有ABO嵌合体研究，而其仅依赖的表型学分析方法也存在灵敏度低、无法避免输血干扰等缺陷。探索新的、高性能方法以实现其重要理论和应用价值成为当前迫切需要解决的关键问题。本项目拟在前期基础上建立基因水平ABO嵌合体诊断新方法：确立能天然区分ABO基因常见双体型的核酸多态性位点筛选库及依赖Taqman水解探针实时荧光定量PCR，以克服表型方法诸多缺陷、具备高检测性能和显著应用优势；通过移植群体研究充分揭示ABO嵌合体诊断具有的新价值：证实红系前体细胞基因水平ABO嵌合体及致病ABO抗体效价监测，准确探讨免疫血液学并发症发生发展机制及预示效力；验证外周血网织红mRNA水平ABO嵌合体分析在术后动态ABO血型准确诊断的作用，进而完善ABO不合移植输血策略的安全性和可操作性。

ABO不合造血干细胞移植术后天然存在ABO 嵌合体，迄今罕有针对ABO 嵌合体的研究，当前分析方法也仅限于常规血型检测等血型血清学技术。本项目通过直接扩增及测序分析ABO基因exon6和exon7的序列确立ABO基因相关的NPs作为区分移植供受者信息效力靶点的策略，并成功建立了261mut、261nor、467mut、803mut、803nor位点作为靶点、基于taqman水解探针为基础的实时荧光定量PCR检测方法，确立了其检测的线性范围：261mut和803mut位点可达10-4，261nor和803nor位点可达10-2，467mut位点可达4%。我们将这种全新的NPs确立策略实际应用于15例ABO不合HSCT患者中，经验证完全有效、具有高效性、低成本优势，完全适于所有ABO双倍体基因型移植供受者诊断，不再需要考虑ABO双倍体基因型的种群和地域分布差异。通过15例移植患者移植后动态监测，证实外周血基因组水平的ABO嵌合体分析可以有效预示移植失败、移植复发或良好预后的变化趋势，尤其适合分析微量的ABO嵌合体状态。本研究证实微柱凝胶血型卡检出ABO微嵌合体状态其灵敏度仅为5%，而且依赖主观估计微柱凝胶检测卡内的阴阳细胞分层去推断真实的嵌合体比例并不可靠。而流式细胞术更适于诊断ABO表型嵌合体，结果更加准确和量化。