成人移植双份脐血植入状态的定量监测

背景:脐血因所含的有核细胞数量有限,主要应用于儿童患者,近年来,人们尝试将两份脐血混合输入,用于成人血液系统疾病的治疗。目的:定量监测双份异基因脐血用于成人白血病患者移植后两份脐血的植入状态、嵌合体类型、相对数量的动态变化及演变规律。设计:以脐血干细胞移植的供受者为观察对象,供受者移植前及受者移植后不同时间段的系列血样提取DNA作为检测标本,短串联重复序列遗传位点为观察指标。单位:深圳市血液中心输血医学研究所免疫遗传重点实验室。对象:纳入2005-06在北京大学深圳医院住院治疗的急性髓细胞白血病患者,男性,43岁,体质量75kg。首次化疗完全缓解后6个月移植两份人类白细胞抗原(HLA)各一个位点不合的非血缘脐血,脐血1有核细胞数为2.5×107kg-1;脐血2有核细胞数为1.53×107kg-1。脐血来源于广州脐血库。患者对治疗方案知情同意。方法:采用荧光标记复合扩增短串联重复位点嵌合体定量检测技术,对急性髓细胞白血病成人患者移植两份(脐血1有核细胞数为2.5×107kg-1,脐血2有核细胞数为1.53×107kg-1)HLA各一个位点不相合的异基因脐血前后的序列血样进行了9个短串联重复序列位点的检测,利用供、受者之间的差异位点定性判断脐血是否植入以及嵌合体类型;而后根据377XLDNA测序仪上荧光扫描后两供者差异基因检出峰的峰面积计算脐血植入后患者体内两份脐血的相对数量,定量分析供体细胞植入程度及演变规律。并与采用HLA差异基因对植入状态的分析结果进行对比。主要观察指标:在成人移植双份脐血后,观察患者及两供者9个位点的短串联重复序列基因在患者体内的转变过程,对植入状态进行定量及定性的描述。结果:移植后15d两份脐血植入状态为完全双份供者嵌合体,患者体内脐血1的相对细胞数量占51.3%,脐血2占48.7%;30d时脐血1上升为70.0%,脐血2下降为30.0%。52d时只检测到脐血1的基因,植入状态转为完全单份供者嵌合体,有核细胞数少的一份脐血被排斥,有核细胞数多的一份长期植入。结论:荧光标记复合扩增短串联重复序列嵌合体定量检测可精确地描述两份脐血的植入程度及变化过程,为临床脐血的应用及供者的选择提供了一个准确、可靠的实验依据,证明双份HLA各一个位点不合的脐血同时用于成人的移植是可行的。

Quantitative monitoring after double unit umbilical cord blood transplantation in an adult

BACKGROUND: Umbilical cord blood (UCB) with limited karyocytes is mainly used in child patients. Recently, physicians have tried to mix two units of cord blood in the treatment of adults with hematological system diseases.OBJECTIVE: To monitor quantitatively the dynamic changes and the development rules of engraftment, chimera types and relative amount after allogeneic transplantation of mixed UCB from two units in adults with leukemia.DESIGN: Donors and the recipient were regarded as observational subjects in umbilical cord blood transplantation (UCBT). DNA extracted from blood samples of donors and the recipient before and after transplantation was considered as detecting samples. Short tandem repeat (STR) loci were as observational measures.SETTING: Key Laboratory of Immunology and Genetics of Institute of Transfusion Medicine of Shenzhen Blood Center.PARTICIPANT: A 43-year male patient with acute myeloid leukemia (AML), 75 kg, who was hospitalized at Shenzhen Hospital of Peking University, was enrolled in June 2005. The patient received two units of human leucocyte antigen (HLA), one locus mismatched unrelated UCBT (2.5×107 kg-1 karyocytes in UCB 1, and 1.53×107 kg-1 karyocytes in UCB 2) at month 6 after complete remission from first chemotherapy. UCB was collected from Guangzhou umbilical cord blood bank. The patient signed the informed consent.METHODS: The adult with AML received two units of HLA, one locus mismatched unrelated UCBT (2.5×107 kg-1 karyocytes in UCB 1, and 1.53×107 kg-1 karyocytes in UCB 2). Nine STR loci of the blood sample were determined before and after transplantation by quantitative technique of fluorescence labeling with multiplex polymerase chain reaction (MPCR), while the engraftment and chimera types were qualitatively evaluated by comparing differential loci between the recipient and the donors. The relative amount of two units of UCB was calculated in the patient after transplantation according to the differential gene peak areas of two donors with 377XL DNA sequencer after fluorescence scanning. The engraftment level and the development rules of donors' cells were analyzed quantitatively. In addition, the results were also compared with that of HLA loci distinct analysis for engraftment.MAIN OUTCOME MEASURES: After UCBT, transition process of nine STR loci of the recipient and two donors was observed, and engraftment was quantitatively and qualitatively described.RESULTS: Two units of UCB at day 15 after transplantation were engrafted simultaneously and revealed a complete chimera of the two. The relative amounts of UCB 1 and UCB 2 were 51.3% and 48.7%, respectively. Subsequently, UCB 1 went up to 70.0% and UCB 2 declined to 30.0% at day 30. However, only the genotype of UCB 1 was detected at day 52, and engraftment turned to a complete chimera of a single donor. The one with fewer karyocytes was rejected and the one with more karyocytes was engrafted for a long term.CONCLUSION: To detect quantitatively STR chimera with fluorescence labeling and MPCR can show precisely the engraftment level and the change of two units of UCB. It provides an accurate and reliable experimental basis for clinical UCB application and donor selection. It is proved that adult transplantation at the same time with mixed UCB from two units HLA one locus mismatched unrelated donors is feasible.