脐血造血干细胞库建立及其临床应用

目的 建立脐血造血干细胞库,用于治疗白血病、淋巴瘤、再生障碍性贫血等疾病。方法 采集新生儿脐血,用6％HES离心法分离脐血有核细胞以缩小脐血体积,样本以10％DMSO保存,检测有核细胞数、CD_(34)~+、CFU-GM及HLA,并作病原学捡测。结果 收集590份脐血,合格230份(合格率39％),平均脐血体积(89±20)ml,平均有核细胞数(1.48+0.36)×10~9,分离后有核细胞数(1.25±0.28)×10~9,有核细胞回收率(91+7)％,脐血CD_(34)~+细胞的百分率为(0.37±0.26)％,复苏后CFU-GM回收率为84％,平均每份脐血含CFU-GM(0.9±0.4)×10~6个集落,14％的样本CMV抗体阳性。73人查询,HLA 4个抗原以上相合率为95.7％。至2001年7月,提供3份脐血用于非亲缘脐血移植,1份用于亲缘脐血移植,3例植入,1例排斥。结论 建立脐血库有良好的应用前景。     

脐血库HLA匹配机率分析

对广州脐血库 1998年以来保存的 30 0 0份脐血和 10 6 0名患者进行回溯分析 ,分析患者在脐血库中搜寻非亲缘脐血的HLA匹配结果 ,以探讨患者找到适于移植的非亲缘脐血的可能性。结果表明 ,6 /6HLA全相合的患者有 119人 (11.2 3% ) ,4 /6以上相合的患者高达 992人 (93.5 8% )。其中 ,分别有 6 1.2 9% ,89.79%的患者可以找到总有核细胞 (TNC)剂量≥ 3.7× 10 7/kg ,≥ 2 .0× 10 7/kg的 4 /6以上HLA相合脐血 ,相应的体重最高者分别为 79kg和 175kg。结论 :脐血库容量在 30 0 0份以上就可以有HLA 93%的匹配率。由于不同体重对脐血TNC数量要求更高 ,广州脐血库能够满足 30 % - 80 %成人患者的需要。在为患者寻找造血干细胞移植非亲缘供者时 ,无论成人或儿童患者 ,在寻找骨髓供者的同时 ,都有必要寻找适宜的脐血供者。     

异基因外周血干细胞移植供者CD34细胞及亚群的测定

目的　探讨流式细胞技术测定异基因外周血干细胞移植 (allo PBSCT)中供者细胞表面分化抗原 34 (CD34 )细胞及其亚群变化和意义。方法　应用流式细胞多色分析技术 ,测定 15 1份allo PBSCT供者 ,经细胞因子动员后外周血标本CD34 及其亚群变化及影响因素。结果　在检测的15 1份标本中 ,CD34 细胞占外周血单个核细胞的 (0 .95 4± 0 .46 6 ) % ,含量为 (3.5 5± 2 .41)× 10 9/L ;其中CD34 CD38-亚群含量为 (0 .2 5 3± 0 .2 40 )× 10 9/L ,占CD34 细胞的 6 .78% ;CD34 HLA DR 亚群含量为 (0 .2 73± 0 .310 )× 10 9/L ,占CD34 细胞的 6 .82 % ,两者差异无显著意义 (P >0 0 5 ) ;随着采集次数的增加 ,CD34 细胞及其亚群数量逐渐减少 (P <0 .0 5 ) ;随着供者年龄增加 ,其外周血CD34 细胞数逐渐减少 ,≥ 40岁供者CD34 细胞百分比和含量比 <2 0岁供者分别降低了 47%和 5 0 % ;动员后外周血CD34 细胞数存在性别差异 ,男性供者外周血CD34 细胞数较女性高 2 3%。结论　应用流式细胞多色技术测定外周血造血干祖细胞 ,不仅能确定造血细胞数量 ,而且对造血干祖细胞的质量进行评价 ,为临床干细胞移植治疗提供重要数据。     

胎盘组织及血液中含有丰富的造血干/祖细胞

大量的临床移植表明人脐血 (UCB)可以在造血干细胞移植的儿童中得到造血重建 ,可是在成人中脐血移植 (UCBT)效果并不理想 ,这主要是脐血中所含的细胞数及造血干 /祖细胞数有限 ,而不适宜体重较大的成人。本研究在收集脐血的同时 ,也分别收集胎盘血 (UPB)及胎盘组织 (UPT)中的细胞 ,检测有核细胞数 ,CD34(造血干 /祖细胞的表明标记 )阳性细胞 ,粒单细胞集落 (CFU GM)。结果发现 :来自于胎盘血和胎盘组织中的有核细胞数是脐血细胞的 3- 4倍 ;脐血、胎盘血及胎盘组织细胞的有核细胞数分别为 (8.3± 1.0 4 )× 10 8,(16 .33± 5 .5 4 )× 10 8和(8.0 1± 2 .6 4 )× 10 8;CD34+ 细胞分别为 (0 .77± 0 .0 1)× 10 6,(1.2 5± 0 .5 5 )× 10 6和 (4.2 1± 1.90 )× 10 6;在细胞的长期培养中 ,UPB细胞和UPT细胞能生存更长时间 ,而且这些细胞更易贴壁形成纤维样的细胞 ;冰冻前后UPT细胞活性及回收率无明显差异 ,表明胎盘细胞和脐血细胞一样适合于长期储存 ;而且 ,在UPB和UPT中含有更高比例的T淋巴细胞抑制细胞群 ,可能意味着UPB和UPT具有更强的免疫抑制功能。结论指出 ,有必要建立含有胎盘和脐血细胞的血库 ,以便为大剂量放疗及化疗后的儿童及成年病人进行造血干细胞移植 ,为重建造血创造条件     

无关供者脐血造血干细胞移植治疗一例成人重型再生障碍性贫血

目的　研究无关供者脐血造血干细胞移植 (unrelatedallo CBSCT)治疗成人重型再生障碍性贫血 (再障 )。方法　用HLA配型 6个位点全相合无关供者allo CBSCT治疗 1例成人重型再障患者。脐血由广州市脐血库提供 ,输入的单个核细胞 (MNC)数为 1.89× 10 7/kg(按患者体重 6 0kg计 ) ,CD34 阳性细胞占 0 .0 0 9,CFU GM为 1.8× 10 4 /kg。预处理方案由环磷酰胺 (CTX)和抗淋巴细胞球蛋白 (ALG)组成 ,CTX总用量为 6 0mg/kg;ALG按 12 0mg/kg给予。用甲氨蝶呤 (MTX)和环孢菌素A(CsA)预防移植物抗宿主病 (GVHD) ,CsA用至 8个月逐渐减量停药。结果　输入脐血后白细胞最低值为 0 .6× 10 9/L ,血小板最低值为 12 .0× 10 9/L ,中性粒细胞于移植后第 10天大于 0 .5× 10 9/L ,血小板于移植后第 2 0天大于 5 0 0× 10 9/L。移植后 8个月时出现皮肤局限性GVHD。微卫星法DNA指纹图示稳定的供、受体混合嵌合状态 ,但迄今ABO血型仍为受者型。结论　此例为国内首例采用allo CBSCT治疗成人重型再障 ,并获得成功 ,脐血造血干细胞已长期植入。     

脐血CD34+细胞的分离与纯化

脐血造血干细胞的分离是进行干细胞移植、体外扩增培养的关键.研究采用明胶自然沉降法、Fi-coll分层法分离脐血MNC后,用MACS分离纯化CD34+细胞,计数并用流式细胞仪进行纯度分析.结果显示每份脐血的采集量平均为95±52 ml,3 g/dl明胶自然沉降法所获MNC密度平均为(5.76±0.67)×107/ml;CD34+细胞密度平均为(5.53±1.16)×105/ml,较Ficoll分层法高(P＜0.01).因此,3 g/dl明胶自然沉降法是一种较理想的分离MNC的方法,用明胶沉降法和MACS相结合是分离脐血CD34+细胞的理想方法.     

混合脐血成人移植定量监测植入状态的实验研究

为了定量监测和研究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、供者细胞相对数量的动态变化及演变规律 ,采用荧光标记复合扩增短串联重复 (STR)位点嵌合体定量检测技术 ,对 1例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× 10 7/kg ,脐血 2有核细胞数为 1.5 3× 10 7/kg)HLA各 1个位点不相合的异基因脐血前后的序列血样进行 9个STR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细胞相对数量 ,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。结果表明 :移植后 15天两份脐血同时植入 ,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 5 1.3% ,脐血 2占 4 8.7% ;30天时脐血 1嵌合体细胞上升为 70 .0 % ,脐血2嵌合体细胞下降为 30 .0 %。 5 2天时只检测到脐血 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STR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     

HLA不全相合造血干细胞联合脐血移植治疗急性白血病的临床观察

目的观察HLA不全相合造血干细胞联合脐血移植治疗急性白血病的有效性及安全性。方法 2008年6月～2010年12月本科20名急性白血病患者接受HLA不全相合造血干细胞联合脐血移植,和同期进行的HLA不全相合造血干细胞移植的51名急性白血病患者,进行造血重建、植入,移植相关并发症和疗效方面的比较。结果联合移植组平均输入脐血有核细胞数2.96×107/kg,CD34+细胞2.99×105/kg;1名患者未植入,ANC≥0.5×109/L的平均时间为12.6±2.50 d,Plt≥20×109/L的平均时间为17.2±3.70 d和对照组均无显著差异(P0.05);植入检查均为完全供者型;联合移植组Ⅲ～Ⅳ度的严重aGVHD的发生率为5.0%显著低于对照组的11.7%(P0.05);1年OS联合移植组为80%优于对照组的72.5%(P0.05);两组间cGVHD发生率、复发率和感染并发症的比较无统计学意义(P0.05)。结论 HLA不全相合造血干细胞联合脐血移植降低严重aGVHD的发生率,提高长期生存率,是对HLA不全相合造血干细胞移植方法的改良,临床应用安全有效。     

多份脐血移植13例回顾性分析

为了探索成人多份脐血移植的可行性 ,对广州市脐血库 1998年 6月至 2 0 0 3年 7月提供的 13例非亲缘关系多份脐血移植进行回顾性分析。脐血均采自广州市妇婴医院正常分娩的足月顺产孕妇 ,脐血分离、冻存、复温方法参照纽约脐血库标准及相关文献 ;多份移植脐血HLA匹配为 1 6 - 2 6位点不相合。结果表明 :1998年 6月至2 0 0 3年 7月 ,广州市脐血库向国内 7家移植中心提供非亲缘关系脐血 2 8份 ,治疗患者 13例。受者中位年龄 2 2 (8- 4 1)岁 ,中位体重 5 0 (2 1- 75 )公斤 ,2 8份脐血中TNC总输入量中位数为 2 .91× 10 7 kg。13例中 6例植入 (中性粒细胞 >5× 10 8 L) ,植入率为 4 6 .1% ,植入平均时间为 19天。 1例患者发生GVHD反应 ,1例患者发生自体恢复。生存率 4 6 .2 % (6 13)。结论 :多份脐血移植具有良好的应用前景。     

白消安、环磷酰胺联合抗胸腺细胞球蛋白预处理用于儿童血液病异基因脐血造血干细胞移植——附9例报告

目的评价白消安、环磷酰胺联合抗胸腺细胞球蛋白(ATG)作为预处理方案用于儿童血液病异基因脐血造血干细胞移植的疗效.方法对9例因血液病进行异基因脐血造血干细胞移植的患儿应用白消安、环磷酰胺联合ATG作为预处理方案.其中人类白细胞抗原(HLA)全匹配同胞异基因脐血移植(RDUCBT)治疗β重型珠蛋白生成障碍性贫血2例,HLA全匹配非同胞异基因脐血移植(UDUCBT)治疗β重型珠蛋白生成障碍性贫血1例,HLA不全匹配非同胞脐血移植治疗急性淋巴细胞性白血病(ALL)3例、急性非淋巴细胞白血病(ANNL)3例.输入脐血有核细胞(NC)中位数7.13×107/kg.结果全供者植入8例,全供者植入率8/9.合并症急性移植物抗宿主病(GVHD)Ⅳ度1例,Ⅲ度1例,Ⅱ度3例,Ⅰ度3例,0度1例.晚发性出血性膀胱炎4例.预处理时多伴腹泻、发热、寒颤、皮疹等反应,但未发现肝静脉栓塞综合征、间质性肺炎及继发肿瘤.结论该预处理方案用于儿童血液病异基因脐血造血干细胞移植效果较好,且较安全.     

1963份脐血造血干细胞的处理与保存

本研究目的是建立脐血造血干细胞库的标准工作程序。采用自然沉降法加离心法制备脐血的造血干细胞 ,经CD34 细胞计数、集落培养、微生物检测、传染病指标检测、HLA分型后 ,将造血干细胞贮存在液氮中保存。结果表明 :贮存脐带血造血干细胞有核细胞数平均值为 (10 .94± 2 .74 )× 10 8,回收率为 (79.82± 17.76 ) % ,CD34 细胞数平均值为 (5 1.6 2± 30 .5 3)× 10 5。 8份脐带血造血干细胞冰冻 2年后复苏 ,其有核细胞、CD34 细胞、CFU GM回收率分别为 (91.4± 6 .0 ) %、(84 .6± 2 0 .0 ) %、(85 .8± 14 .9) %。结论 :本方法和程序能有效地保存脐带血造血干细胞。     

54例非亲缘脐血造血干细胞移植回顾性分析

本研究对广州市脐血库1998年6月至2003年7月提供的54例非亲缘脐血移植进行回顾性分析,以研究脐血移植的相关影响因素.脐血均采自广州市妇婴医院正常分娩的足月顺产孕妇,脐血分离、冻存、复温方法参照纽约脐血库标准及相关文献;选择脐血的依据是HLA相合程度及有核细胞数量.结果表明1998年6月至2003年7月间,库存脐血3475份,与21家移植中心合作提供脐血99份,治疗患者85例.其中同胞脐血移植11例.对54例非亲缘关系脐血移植病例进行统计分析显示,在54例受者中,恶性病患者43例,占79.6%,非恶性病患者11例,占20.4%,受者中位年龄9.5(1.2-33)岁,中位体重27(10-60)公斤,TNC中位输入量6.82×107/kg,移植后60天植入43例(ANC＞500/μl的中位时间为17天),植入率为79.6%.脐血移植有核细胞重建时间与输入的有核细胞数以及疾病类型相关,同HLA相合程度不相关.急性Ⅲ-Ⅳ度GVHD发生率为21.6%,慢性GVHD发生率为5.4%.6例患者出现自体恢复(11.1%).存活率42.6%.结论非亲缘性脐血造血干细胞移植尤其在治疗小儿白血病方面具有良好的应用前景.     

Increased Nucleated RBCs in Cord Blood: Not an Exclusion Criterion but a Quality Indicator for Hematopoietic Progenitor Cell Transplantation

Increased nucleated red blood cell (NRBC) counts have been reported to be associated with adverse fetal outcomes, and cord blood units (CBUs) with increased NRBC counts require a 2^nd questionnaire to determine their suitability for transplantation. However, a recent study demonstrated a positive correlation of NRBCs with CD34⁺ cells and total nucleated cells (TNCs). We evaluated the association between the NRBC count and hematopoietic progenitor cell (HPC) content (TNC and CD34⁺ cell counts) in Korean full-term newborn CBUs. In addition, we assessed whether an increased NRBC count is associated with newborn health problems that impair CBU safety. Among the 32,876 units processed from May 2006 to December 2018, a total of 23,385 CBUs with a TNC count ≥ 7 × 10⁸ and reliable perinatal information were analyzed to assess the association of the NRBC count with CBU parameters, and the newborns associated with 457 CBUs that required the 2^nd questionnaire due to an increased NRBC (≥ 15 NRBCs/100 WBCs) were assessed at one year for health problems that threatened CBU safety. The majority of the CBUs that required the 2^nd questionnaire due to an increased NRBC count (96.9%) were determined to be suitable for transplantation. Those with an increased NRBC count showed significantly higher CD34⁺ cell and TNC counts and a higher rate of transplantation (P < 0.001, < 0.001 and 0.025, respectively). NRBCs showed a significant positive correlation with TNCs and CD34⁺ cells and a significant negative correlation with birth weight (all P < 0.001; adjusted r = 0.185, 0.369 and - 0.029, respectively). In the multiple linear regression analysis, NRBCs showed independent and positive correlations with TNCs and CD34⁺ cells after adjustments for birth weight and gestational age (all P < 0.001; β = 0.182, adjusted R² = 0.053 and β = 0.367, adjusted R² = 0.418). An increased NRBC count in full-term normal delivery is a surrogate marker of HPCs in CBUs rather than an exclusion criterion for CBU safety. Moreover, providing the NRBC count together with the NRBC-corrected TNC count will be useful for clinicians to select CBUs for transplantation.     

Cord blood units collected at a remote site: a collaborative endeavor to collect umbilical cord blood through the Hawaii Cord Blood Bank and store the units at the Puget Sound Blood Center

BACKGROUND: Umbilical cord blood is a useful source of hematopoietic stem cells, especially because compared to equivalent HLA-matched stem cells from unrelated adult donors. A network of community collection sites targeted at particular ethnic groups and serviced by a central processing and storage facility can maximize the genetic diversity of banked cord blood units (CBUs) in a cost-effective fashion. STUDY DESIGN AND METHODS: The present study compared CBUs collected near the Puget Sound Blood Center in Seattle, WA, with those collected in Honolulu, HI, and processed in Seattle. Evaluated variables include collection volume, total nucleated cell count, cellular viability, CD34+ cell count, clonogenic activity, and donor race for a total of 1646 CBUs received from July 1998 through November 2002. RESULTS: CBUs from the two sites did not differ with regard to volume or total nucleated cells. Those from Hawaii had significantly longer transit times (p < 0.001) and lower whole cord blood cell viability. However, the numbers of CFU and viable CD34+ cells were not affected by remote collection. CBUs screened from Seattle were largely from Caucasian donors, whereas over 85 percent of those from Honolulu were from donors of Asian-Pacific Islander or mixed ethnicity. CONCLUSION: These studies demonstrate the feasibility of long-distance umbilical cord blood banking. Arrangements such as those described here could be used to help target cost-effective collection from minority populations and increase the HLA and ethnic diversity for CBUs.     

Banking cord blood stem cells: attitude and knowledge of pregnant women in five European countries

BACKGROUND: This study explores pregnant women's awareness of cord blood stem cells and their attitude regarding banking options in France, Germany, Italy, Spain, and the UK. STUDY DESIGN AND METHODS: Questionnaires were distributed in six maternities. This anonymous and self‐completed questionnaire included 29 multiple‐choice questions based on: 1) sociodemographic factors, 2) awareness and access to information about cord blood banking, 3) banking option preferences, and 4) donating cord blood units (CBUs) to research. RESULTS: A total of 79% of pregnant women had little awareness of cord blood banking (n = 1620). A total of 58% of women had heard of the therapeutic benefits of cord blood, of which 21% received information from midwives and obstetricians. A total of 89% of respondents would opt to store CBUs. Among them, 76% would choose to donate CBUs to a public bank to benefit any patient in need of a cord blood transplant. Twelve percent would choose a mixed bank, and 12%, a private bank. A total of 92% would donate their child's CBU to research when it is not suitable for transplantation. CONCLUSION: The study reveals a strong preference for public banking in all five countries, based on converging values such as solidarity. Attitudes of pregnant women are not an obstacle to the rapid expansion of allogeneic banking in these EU countries. Banking choices do not appear to be correlated with household income. The extent of commercial marketing of cord blood banks in mass media highlights the importance for obstetric providers to play a central role in raising women's awareness early during their pregnancy with evidence‐based medical information about banking options.     

两份脐血联合移植治疗成人慢性粒细胞性白血病一例

为了探讨两份非亲缘供者脐血联合移植治疗成人血液系恶性肿瘤患者的造血重建及移植相关性并发症 ,对 1例成人慢性粒细胞性白血病患者进行两份非亲缘供者脐血联合移植。预处理采用白消安 /环磷酰胺 (Bu/Cy)方案。移植物抗宿主病 (GVHD)的预防采用霉酚酸酯 (MMF)联合环孢霉素 (CsA)和短程氨甲喋呤 (MTX )方案。移植总单个核细胞数为 4.63× 10 7/kg ,CD3 4 + 细胞数为 8.3 4× 10 5 /kg。采用PCR法测定患者外周血细胞短串重复序列 (STR)获取植入证据。结果表明 :患者于移植后 2 3天外周血中性粒细胞绝对值 >0 .5× 10 9/L ,移植后 3 3天外周血血小板 >2 0× 10 9/L ,移植后 47天血小板 >50× 10 9/L。移植后 ,患者Ph染色体转阴性 ,bcr/abl融合基因阴性。移植后 3 1天 ,46天及 71天查外周血STR显示其中 1份脐血完全植入。患者于移植后 13天出现Ⅱ度急性GVHD ,经治疗后消失。结论 :脐血联合移植可用于成人恶性血液病的治疗 ,多份脐血联合移植可能是解决单个核细胞数偏低问题的途径之一     

The Cord Blood Apgar: a novel scoring system to optimize selection of banked cord blood grafts for transplantation (CME)

BACKGROUND: Engraftment failure and delays, likely due to diminished cord blood unit (CBU) potency, remain major barriers to the overall success of unrelated umbilical cord blood transplantation (UCBT). To address this problem, we developed and retrospectively validated a novel scoring system, the Cord Blood Apgar (CBA), which is predictive of engraftment after UCBT. STUDY DESIGN AND METHODS: In a single‐center retrospective study, utilizing a database of 435 consecutive single cord myeloablative UCBTs performed between January 1, 2000, to December 31, 2008, precryopreservation and postthaw graft variables (total nucleated cell, CD34+, colony‐forming units, mononuclear cell content, and volume) were initially correlated with neutrophil engraftment. Subsequently, based on the magnitude of hazard ratios (HRs) in univariate analysis, a weighted scoring system to predict CBU potency was developed using a randomly selected training data set and internally validated on the remaining data set. RESULTS: The CBA assigns transplanted CBUs three scores: a precryopreservation score (PCS), a postthaw score (PTS), and a composite score (CS), which incorporates the PCS and PTS values. CBA‐PCS scores, which could be used for initial unit selection, were predictive of neutrophil (CBA‐PCS ≥ 7.75 vs. <7.75, HR 3.5; p < 0.0001) engraftment. Likewise, CBA‐PTS and CS scores were strongly predictive of Day 42 neutrophil engraftment (CBA‐PTS ≥ 9.5 vs. <9.5, HR 3.16, p < 0.0001; CBA‐CS ≥ 17.75 vs. <17.75, HR 4.01, p < 0.0001). CONCLUSION: The CBA is strongly predictive of engraftment after UCBT and shows promise for optimizing screening of CBU donors for transplantation. In the future, a segment could be assayed for the PTS score providing data to apply the CS for final CBU selection.