ABO血型主要不合的供者外周血造血干细胞采集

Collection of peripheral blood hematopoietic stem cells from ABO major incompatible allogeneic donors

目的:应用血细胞分离机采集血型主要不合的供者外周血造血干细胞的效率,观察不去除红细胞进行造血干细胞移植的安全性。方法:实验于1998-10/2006-10在华中科技大学附属协和医院血液科完成。①实验对象:健康异基因造血干细胞供者23例,均与受者经HLA配型证实为全相合,亲缘性供者21例,中华骨髓库无血缘关系供者2例;供者为A型血、患者为O型血15例,供者为B型血、患者为O型血8例。23例受者中,急性淋巴细胞白血病患者9例,急性非淋巴细胞白血病患者8例,慢性粒细胞白血病患者5例,再生障碍性贫血患者1例。实验经医院伦理委员会批准,供、受者均知情同意。②实验方法:23例供者均在采集前5d皮下注射粒细胞集落刺激因子5μg/(kg·d)进行干细胞动员,至第5天供者外周血白细胞计数达20×109L-1、单个核细胞所占比例达10%以上时,应用CobeSpectra血细胞分离机的自动外周血干细胞采集程序进行外周血造血干细胞采集,平均循环血量为9210mL,分离过程中平均采血速度为42mL/min,每例供者均采集2次,共46次,采集时可静脉注射或口服10%葡萄糖酸钙,或采集前3d口服钙片,一定程度上可预防低钙反应、麻木甚至抽搐等不良反应。23例受者每次输注干细胞采集物前给予相关干预措施以防止溶血反应及保护肾功能,每例受者均输注2次,共46次。③实验评估:供者采集完毕后留取标本检查有核细胞数、红细胞比容、CD34+细胞计数和细胞存活率锥虫蓝拒染情况。受者输注后注意观察生命体征、尿液颜色及是否有溶血相关不良反应等。结果:①供者外周血造血干细胞采集时不良反应:46例次的供者采集中,4例次(8.7%)出现复方枸椽酸钠相关低钙反应,表现为口周、四肢麻木感、胃肠道反应、胸闷、头昏等,经加大口服10%葡萄糖酸钙症状均得到控制,每例供者平均每次采集的补钙量为50mL,最多者补充80mL;1例次(2.17%)出现血管迷走神经性面色苍白、出汗症状。②受者外周血造血干细胞输注时不良反应:每例供者采集物中的有核细胞数为(3.76±0.89)×108/kg,CD34+细胞计数为(3.23±0.89)×106/kg,采集物锥虫蓝拒染率均为100%,红细胞混入量即血红蛋白为(18±4)g/L,不去除红细胞,直接输给受者,23例受者46次输注后仅1例次出现较严重的溶血性输血反应,应用地塞米松、静脉滴注5%碳酸氢钠并加大输液量后症状逐渐减轻直至完全消失。所有患者造血功能均获得重建。结论:应用血细胞分离机采集ABO血型主要不合供者的外周血造血干细胞,回输时不去除采集物中的红细胞,可获得足够的干细胞数量并安全用于移植。

AIM： To evaluate the efficiency of the blood cell separator for harvesting of peripheral blood hematopoietic stem cells （PBSC） from ABO major incompatible aUogeneic donors and the safety of PBSC transplantation to the patients without removal of erythrocytes. METHODS： The experiment was done in Department of Hematology, Union Hospital Affiliated to Huazhong University of Science and Technology from October 1998 to October 2006. ①Subjects of the experiment： Twenty-three healthy HLA-matched donor of allogeneic hematopoietic stem cell transplantation were enrolled. Twenty-one of them were related donor and two were unrelated donor from the data bank of Chinese hematopoietic stem cell donor. ABO blood groups of recipient/donor pairs of these twenty-three patients was O/A in fifteen and O/B in eight. Among the patients, 9 were acute lymphocytic leukemia, 8 were acute non-lymphocytic leukemia, 5 were chronic myelocytic leukemia and one was aplastic anemia. The experiment was approved by Medical Ethics Committee and got permission of all the patients. ②Methods of the experiment： All of the 23 donors were given hypodermic granulocyte colony-stimulating factor （G-CSF） 5 μg/kg per day for mobilization. When leukocyte count was more than 20×10^9 L^-1 and the ratio of mononuclear cells was more than 10% at the fifth day, peripheral blood hematopoietic stem cells were collected by Cobe Spectra blood cell separator with auto-peripheral blood stem cell program. The mean circulation volume was 9210 mL and the mean inlet rate was 42 mL/min. Collection in each donor was undergone twice, totally forty-six collections were done. To prevent adverse effects such as hypocalcemia, numbness and spasm, 10% calcium gluconate was given intravenously or orally during collection or calcium tablets were given three days before collection. Prophylactic medicines were given before stem cells infusion to prevent hemolytic reaction and protect renal function in 23 recipients. Each patient received infusion twice, a total of 46 infusions were performed. ③Experimental evaluation： Sample from collection bag for nucleated cell count, hematocrit （HCT）, CD34 positive cell count and trypan blue stain. Vital signs, color of the urine and other hemolysis correlated adverse effects were observed after each infusion. RESULTS： ①Adverse effects during PBSC collection： Among the 46 collections, natrium citricum complex related hypocalcemia occurred in 4 （8.7%）cases, and the manifestations of hypocalcemia including anesthesia of limbs and lips, gastrointestinal tract reactions, chest distress and dizziness, etc. All of the above symptoms could be cured by oral 10% calcium gluconate. An average of 50 mL 10% calcium gluconate was given and 80 mL for maximum. Angio-cranial nerve related symptoms such as pale and sweating occurred in one of the donors （2.17%）. ②Adverse effects of PBSC infusion to the recipients： The mean yield of nucleated cells was （3.76±0.89）×10^8/kg, and CD34^＋ cell was （3.23±0.89）×10^8/kg. Trypan blue exclusion rate was 100% for all the samples. The mean hemoglobin in the collection was （18±4） g/L. The collections were infused into the recipients without removal of erythrocytes. Haemolytic reaction was only observed in one infusion and cured by dexamethasone, 5% NaHCO3 and more infusion solutions. Hematopoietic functions were re-established in all of the patients. CONCLUSION： Enough PBSC can be harvested by blood cell separator from ABO major incompatible allogeneic donors and the PBSC collections can be safely infused to the patients without removal of erythrocytes.