米托蒽醌、阿糖胞苷、粒细胞集落刺激因子的动员方案及大容量采集自体外周血干细胞

背景:外周血干细胞移植成功的首要条件是干细胞的有效动员和采集,选择高效低毒的动员方案,掌握动员和采集时机与动员效果密切相关。目的:探讨米托蒽醌-大剂量阿糖胞苷方案化疗后,单用粒细胞集落刺激因子或粒细胞集落刺激因子与粒-巨噬细胞集落刺激因子合用对恶性血液病和实体瘤患者自体外周血干细胞的动员效果。设计:观察对比实验。单位:徐州医学院附属医院血液科。对象:选择1998-09/2006-12在徐州医学院附属医院血液科收治的42例恶性血液病和实体瘤患者,诊断符合国际白血病分型及世界卫生组织新分类标准。男25例,女17例,年龄7～54岁,平均29岁,体质量(52±18)kg。其中急性髓细胞白血病12例,急性淋巴细胞白血病6例,慢性粒细胞白血病慢性期1例,非霍奇金淋巴瘤15例,霍奇金淋巴瘤4例,多发性骨髓瘤2例,晚期乳癌2例。患者均经常规化疗达到或接近完全缓解,骨髓细胞学检查无肿瘤细胞浸润。心、肺、肝、肾等主要脏器功能正常。动员前化疗疗程平均8次,所有患者均对治疗项目知情同意。方法:患者均采用米托蒽醌10mg/(m2·d)静脉滴注第2～3d后,阿糖胞苷2g/m2静脉滴注第1～2d,1次/12h。当白细胞计数下降至最低点开始回升时,20例患者使用粒细胞集落刺激因子5～7.5μg/(kg·d),连用3～5d,22例患者早6:00给予粒细胞集落刺激因子5～7.5μg/(kg·d),晚6:00给予粒-巨噬细胞集落刺激因子5～7μg/(kg·d)。白细胞计数>2.5×109 L-1,CD34 细胞≥1%时,用CS3000plus血细胞分离机连续2d采集自体外周血干细胞,检测CD34 细胞含量和T淋巴细胞亚群。①单个核细胞与FITC标记的CD34 、CD3和CD8单抗及与CD4PE标记的CD4单抗4℃混合30min,采用流式细胞仪检测CD34 细胞和T细胞亚群,分析5×105个细胞,得出CD3、CD34 细胞含量及CD4/CD8比值。用甲基纤维素法测定粒-巨噬细胞集落形成单位。②观察术后相关不良反应。③针对不同类型疾病给予相应预处理36～48h后回输自体外周血干细胞,并行单个核细胞计数及台盼蓝染色,解冻后检测粒-巨噬细胞集落形成单位和CD34 细胞。主要观察指标:①动员前后CD34 细胞和T细胞亚群变化。②术后相关不良反应。③自体外周血干细胞回输量(单个核细胞计数、粒-巨噬细胞集落形成单位和CD34 细胞数)。结果:纳入患者42例,均进入结果分析。①动员前后CD34 细胞和T细胞亚群变化:患者应用粒细胞集落刺激因子/粒-巨噬细胞集落刺激因子动员后外周血CD34 细胞明显增加(0.054±0.032)%,(1.82±0.76)%,P<0.01]。22例联合应用粒细胞集落刺激因子和粒-巨噬细胞集落刺激因子动员患者CD34 细胞和粒-巨噬细胞集落形成单位分别为(8.76±3.39)×106/kg,(3.52±1.33)×105/kg,明显高于单用粒细胞集落刺激因子的20例患者(6.12±2.11)×106/kg,(2.03±1.07)×105/kg,P<0.05]。单独应用粒细胞集落刺激因子及粒细胞集落刺激因子与粒-巨噬细胞集落刺激因子合用后随CD34 细胞增加,T淋巴细胞亚群变化不明显(P>0.05)。②外周血干细胞动员相关不良反应:全部病例出现Ⅱ～Ⅲ度脱发,血小板均有不同程度的下降,为(54.43±26.14)×109 L-1,21例患者出现感染性发热(37.8～41.0℃),经抗生素治疗感染均在短期内得到控制。13例患者在白细胞快速上升时出现骨骼疼痛(腰骶部为主)。③自体外周血干细胞回输量:自体外周血干细胞非程控冷冻-80℃保存2.0～6.5个月,细胞回收率(88.7±7.4)%,台盼蓝拒染率(92.1±5.5)%,回输的单个核细胞(5.21±2.44)×108/kg,CD34 细胞(6.89±3.55)×106/kg,粒-巨噬细胞集落形成单位(2.58±2.33)×105/kg。④循环血量每次10～16L(终点分血量均在3个TBV上)。无严重毒副反应。26例接受自体外周血干细胞移植者造血功能均获得满意重建。结论:米托蒽醌-大剂量阿糖胞苷方案化疗后单用粒细胞集落刺激因子及粒细胞集落刺激因子与粒-巨噬细胞集落刺激因子合用均能安全、有效动员自体外周血干细胞,但以合用更为有效。大容量采集是提高干细胞产率,减少采集次数的重要手段。

Mobilization of autologous peripheral blood stem cells by mitoxantone, arabinosylcytosin and granulocyte colonystimulating factor regimen and harvesting by large-volume leukapheresis

BACKGROUND: The primary qualification of peripheral blood stem cell transplantation (PBSCT) is the effective mobilization and harvesting of hematopoietic stem cells. The mobilization efficacy is closely related to the selection of high-efficacy low-toxicity regimen, the timing of mobilization and harvesting as well.OBJECTIVE: To investigate the efficacy of mitoxantone (MIT) combined with high-dose arabinosylcytosin (Ara-C),followed by granulocyte colony-stimulating factor (G-CSF) alone or combination of G-CSF and granulocyte-macrophage colony-stimulating factor (GM-CSF) on mobilizing PBSCs in patients with hematological malignancies and solid tumors.DESIGN: Controlled study with observation.SETTTNG: Department of Hematology, the Affiliated Hospital of Xuzhou Medical College.PARTICIPANTS: Forty-two patients with hematological malignancies and solid tumors admitted to Department of Hematology, Xuzhou Medical College from September 1998 to December 2006 were involved in this study. They were diagnosed according to FAB classification criteria and new WHO proposals. The involved patients, 25 male and 17 female, averaged 29 years ranging from 7 to 54 years and weighted (52±18) kg. Among them, 12 were patients with acute myeloblastic leukemia, 6 were patients with acute lymphoblastic leukemia (ALL), 1 was patient with chronic granulocytic leukemia (CGL) at chronic phase, 15 were patients with non-Hodgkin lymphoma (NHL), 4 were patients with Hodgkin lymphoma (HL), 2 was patient with multiple myeloma (MM), 2 were patients with advanced breast cancer. All the patients apprcached to or got complete remission after conventional chemotherapy. No tumor cell infiltration was observed in bone marrow cytological examination. The functions of the main organs such as heart, lung, liver and kidney,and so on, were normal. The patients underwent an average of 8-course chemotherapy before the mobilization. Informed consents of all the patients were obtained.METHODS: MIT was intravenously injected at 10 mg/(m2·d)for 2 to 3 days, then Ara-C was also intravenously injected at 2 g/m2 every 12 hours for 1 to 2 days. When white blood cell (WBC) count recovered from the lowest value, 5 to 7.5 μg/ (kg·d)G-CSF was applied in 20 patients for 3 to 5 days successively. And 5 to 7.5 μg/ (kg·d)G-CSF and 5 to 7 μ g/(kg·d)GM-CSF were applied in another 22 patients at 6:00 in the morning and in the evening, respectively. PBSCs harvesting started when WBC ＞ 2.5×109 L-1, especially when CD34+ cells≥ 1%,WBC was doubly increased. Autologous peripheral blood mononuclear cells (MNCs) were collected with CS3000 plus blood cell separator for detecting the level of CD34+ cells and T lymphocyte subsets. CFU-GM assays were performed in a methyl-cellulose-based clonogenic assay.① MNCs mixed with FITC-labeled CD34+, CD3 and CD8 monoclonal antibodies as well as CD4 PE-labeled CD monoclonal antibody at 4 ℃ for 30 minutes. 5×105 cells were determined, and CD3 and CD34+ levels, CD4/CD8 were determined by flow cytometer.Colony forming unit-granulocyte macrophage (CFU-GM) was determined with methyl cellulose. ② Related adverse reactions were observed after operation. ③ Aiming to different types of diseases,autologous PBSCs were back infused 36 to 48 hours after pre-disposal treatment. MNCs count and trypan-blue drying were done. Levels of CFU-GM and CD34+ cells were determined after unfreezing.MATN OUTCOME MEASURES: ① Changes in CD34+ cells and T lymphocyte subsets before and after mobilization. ② Postoperative related adverse reactions. ③ Back perfusion volume of autologous PBSCs (MNCs count, the number of CFU-GM and CD34+ cells).RESULTS: Forty-two involved patients participated in the final analysis. ① Changes in CD34+ cells and T lymphocyte subsets before and after mobilization: Without using hematopoietic growth factors (HGF), the percentage of CD34+ cells in peripheral blood of the patients was (0.054±0.032)%. After using G-CSF/GM-CSF treatment, it was (1.82±0.76)%,which was obviously increased compared with that of without using HGF (P ＜ 0.001). The CD34+ cells and CFU-GM yields of 22 patients in C-CSF plus GM-CSF combination group (8.76±3.39)×106/kg, (3.52±1.33)×105/kg, respectively]were significantly higher than those of 20 patients in G-CSF alone group (6.12±2.11)×106/kg, (2.03±1.07)×105/kg,respectively (P ＜ 0.05)]. There were no obvious changes of T lymphocyte subsets in the patients when using G-CSF/GM-CSF for some days except that CD34+ cells increased gradually (P ＞ 0.05). ② Postoperative related adverse reactions: Ⅱ to Ⅲ degree hair-loss was seen in all the patients. Blood platelets dropped to (54.43±26.14)×109 L-1 at different degrees. Infective fevers (37.8 ℃ to 41.0 ℃) occurred in 21 patients. But they were controlled in short term after antibiotics treatment. All the side effects of G-CSF and GM-CSF were mild and reversible, easily controlled with paracetamol or steroids. Bone pain (mainly in lumbosacral region) occurred in 13 patients when WBC went up quickly. ③ Back perfusion volume of autologous PBSCs: PBSCs were cryopreserved at -80 ℃ without program control for 2.0 to 6.5 months. The cell recovery rate was (88.7±7.4) %. Trypan blue exclusion rate was (92.1±5.5) %. The back perfusion volume of MNCs, CD34+ cells and CFU-GM yields were (5.21±2.44)×108/kg, (6.89±3.55)×106/kg, (2.58±2.33)×105/kg,respectively. ④Circulation blood volume were 10 to 16 L (end-point separation blood volume were all above trebling TBV). Hematopoiesis was well reconstituted in 40 patients received autologous PBSCT.CONCLUSTON: MIT and high-dose Ara-C chemotherapy combined with both G-CSF alone and G-CSF plus GM-CSF can safely and effectively mobilize autologous PBSCs, while G-CSF plus GM-CSF is superior to G-CSF alone.Large-volume leukapheresis is an important method to enhance the productive rate of stem cells and decrease the times of harvesting.