供者用粒细胞集落刺激因子单倍体骨髓移植的临床研究

目的探讨供者用粒细胞集落刺激因子(G-CSF)和受者联合应用多种免疫抑制剂治疗的单倍体骨髓移植在降低重症移植物抗宿主病(GVHD)和改善无病生存的疗效.方法单倍体骨髓移植治疗白血病13例(单倍体移植组),移植后结果和连续完成的13例白血病HLA匹配异基因移植 (相合移植组) 相比较,单倍体移植方法是供者应用G-CSF 250 μg/d,连用7 d后采髓, 受者GVHD预防除环孢素A(CSA)和甲氨蝶呤(MTX)外,在移植前4～1 d用抗胸腺细胞球蛋白(ATG) 5 mg*kg-1*d-1, 移植后7 d始加服霉酚酸酯(MMF).结果单倍体移植组植入物CD+34细胞中位数6.1×106/kg,是相合移植组输入CD+34细胞中位数2.5×106/kg的2倍多(P＜0.01),单倍体移植组和相合移植组植入物CD+3细胞中位数分别是50.5×106/kg和47.0×106/kg(P＞0.05).移植后无1例发生植入失败,两组造血重建速度无差异(P＞0.05),所有患者经骨髓植活直接证据检测证实为完全供者造血.单倍体移植组发生急性Ⅱ～Ⅳ GVHD 5例(38.5%),可评价的8例中7例发生慢性GVHD(87.5%),为局限性慢性GVHD,这与相合移植组差异无显著性 (P＞0.05).单倍体移植组中位随访453 d(180～690),移植相关死亡5例,无复发死亡病例,剩余8例无病存活(61.5%).相合移植组中位随访510 d(220～810),移植相关死亡2例,复发死亡2例,9例无病存活(69.2%),通过比较两组差异无显著性(P＞0.05).结论本研究单倍体骨髓移植治疗白血病是一种安全和有效方法,在降低重症急性GVHD 及改善无病生存方面和HLA相合同胞供者移植相当.     

供体CD+4CD+25T细胞及调控基因FOXP3在急性移植物抗宿主病中的作用

目的 探讨供体CD+4CD+25T细胞亚群、FOXP3调控基因的表达与受者移植物抗宿主病(GVHD)的相关性.方法 (1)30例异基因造血干细胞移植(allo-HSCT),采用免疫荧光标记和流式细胞术检测并比较供体粒细胞集落刺激因子(G-CSF)动员前外周血、动员后采集物CD+4CD+25T细胞亚群比例,随访异基因移植后GVHD的发生率和严重程度.(2)应用RT-PCR技术检测供体FOXP3基因表达情况,分析其与GVHD、疾病复发的相关性.结果 (1)所有患者均获造血重建,粒细胞绝对数(ANC)≥0.5×109/L的中位时间为14(12～15)d,PLT≥20×109/L为18(15～25)d.30例allo-HSCT,中位随访时间12.8(8～16)个月,Ⅰ～Ⅳ度急性GVHD分别为3、4、3、5例.慢性GVHD 6例.(2)供体G-CSF动员前外周血、动员后采集物CD+4CD+25T细胞亚群分别为(2.67±0.38)%、(5.01±1.33)%,两者相比差异无统计学意义(P＞0.05).(3)移植后无急性GVHD组、Ⅰ～Ⅱ度急性GVHD组、Ⅲ～Ⅳ度急性GVHD组供体CD+4CD+25T细胞亚群分别为(5.05±1.34)%、(4.17±1.73)%、(1.98±1.10)%.其中Ⅰ～Ⅱ度急性GVHD组与Ⅲ～Ⅳ度急性GVHD组相比差异有统计学意义(P=0.04),无急性GVHD组与Ⅲ～Ⅳ度急性GVHD组相比差异有统计学意义(P=0.002).(4)30例allo-HSCT,7例FOXP3基因表达阳性,5/7例移植后无急性GVHD,其中3例移植后复发,另2/7例移植后Ⅰ度急性GVHD,Ⅱ～Ⅳ度急性GVHD患者FOXP3均不表达.结论 (1)供体CD+4CD+25T细胞亚群比例与受者急性GVHD的发生具有一定的相关性,提高供体CD+4CD+25T细胞数量有望减低移植后急性GVHD发生率.(2)供体移植物FOXP3基因表达阳性,与移植后有无严重急性GVHD发生存在一定相关性.     

单倍型相合骨髓移植治疗难治复发恶性淋巴瘤初步观察

目的:探讨应用单倍型未去T细胞的异基因骨髓移植(allo-BMT)治疗难治复发恶性淋巴瘤的疗效、造血重建、并发症及生存情况。方法:采用清髓性未去T细胞亲缘单倍型相合allo-BMT治疗11例难治性晚期淋巴瘤患者。供者用粒细胞刺激集落因子(G-CSF)皮下注射,连续7d后采髓,预处理方案采用环磷酰胺、全身照射及阿糖胞苷(CyTBI加Ara-C)。移植物抗宿主病(GVHD)预防采用联合免疫抑制剂包括环孢素A、短程氨甲碟呤、抗胸腺细胞球蛋白、CD25单克隆抗体及霉酚酸酯。结果:患者移植后均获造血重建,中性粒细胞>0.5×109/L及PLT>20×109/L的平均时间分别为17.2及21.3d,植入证据检测证实为完全供者造血。4例发生急性GVHD(aGVHD),3例发生慢性GVHD(cGVHD),中位随访1136(31～2521)d,2例死于感染,1例死于aGVHD,1例死于复发,无病生存7例。结论:清髓性未去T细胞亲缘单倍型相合allo-HSCT是治疗伴骨髓侵犯以及自体造血干细胞移植后复发难治性晚期恶性淋巴瘤有效方法,如有合适供者,可考虑作为年轻晚期患者的挽救治疗。     

单倍体相合造血干细胞移植在难治高度恶性淋巴瘤中的应用

目的 :探讨应用单倍体相合骨髓移植对难治高度恶性非霍奇金淋巴瘤 (NHL)治疗的可行性。方法 :6例难治高度恶性NHL伴有骨髓浸润患者接受人白细胞抗原 (HLA) 2～ 3个位点不合的单倍体相合移植 ,供者用粒细胞集落刺激因子 (G CSF)促进后采髓 ,急性移植物抗宿主病 (GVHD)预防例 1采用环孢菌素A(CSA)、短程甲氨蝶呤 (MTX)、霉酚酸酯 (MMF)和抗胸腺细胞球蛋白 ,余 5例除上述药物外 ,还加用CD2 5单克隆抗体。结果 :6例移植后均获造血重建 ,粒细胞绝对数 >0 .5× 10 9/L中位天数是 17d ,血小板 >2 0× 10 9/L的中位天数是 2 2d ,骨髓植活直接证据检测证实为完全供者造血。 1例于移植后 2 0d时发生急性Ⅳ度肠道GVHD ,可评价慢性GVHD病例4例 ,均发生慢性GVHD ,其中 1例广泛性慢性GVHD ,口服泼尼松和CSA病情控制。中位随访 2 0 (7～ 4 2 )个月 ,1例重度GVHD于移植后 2个月死亡 ,1例在移植后 4个月并发真菌感染死亡 ,无病存活 4例 ,Karnofshy生存质量评价为 10 0 %。结论 :研究表明单倍体相合未经体外去T细胞骨髓移植对难治高度恶性NHL具有一定治疗价值 ,能够降低急性重症aGVHD发生和减少移植相关死亡。     

非清髓性造血干细胞移植加供者淋巴细胞输注治疗多发性骨髓瘤1例报告并文献复习

目的研究非清髓性造血干细胞移植(NST)治疗多发性骨髓瘤(MM)的疗效,观察移植相关并发症的发生.方法1例42岁MM患者,供者为其胞姐,HLA配型完全相合.动员方案粒细胞集落刺激因子(G-CSF)10 μg·kg-1·d-1×5 d.预处理方案抗胸腺细胞球蛋白(ATG)8 mg·kg-1·d-1×3 d,马法兰(MEL)120 mg/m2×1 d.移植单个核细胞数(MNC) 6.5×108/kg;CD34+细胞 4.4×106/kg.环胞菌素A(CsA)和短程甲氨蝶呤(MTX)预防移植物抗宿主病(GVHD).移植后分别于+41 d、+76 d和+112 d进行3次供者淋巴细胞输注(DLI).结果移植后15 d中性粒细胞计数> 0.5×109/L,21 d血小板计数>50×109/L,24 d性染色体和微卫星法DNA指纹图监测显示为混合嵌合体,随着DLI的进行,逐渐转为供者型完全嵌合体,骨髓瘤细胞和血清M蛋白均逐渐消失,移植后8个月达完全缓解.在+180 d(第三次DLI后68 d)发生II度急性GVHD,经甲泼尼龙和CsA治疗得以控制.现随访36个月,患者情况良好,仍处于完全缓解状态.结论非清髓性造血干细胞移植加供者淋巴细胞输注治疗MM是可行和有效的.     

CD34+CD49d+细胞输入量对儿童急性白血病无关脐血移植的影响

目的研究CD34+CD49在d+细胞输入量在无关脐血移植治疗儿童急性白血病中对造血干细胞植入、中性粒细胞恢复时间的影响.方法用流式细胞术分析复苏后的CD34+CD49d+细胞数,并对17例儿童急性白血病在无关脐血移植后的中性粒细胞恢复时间进行分析.结果移植后17例患者中性粒细胞＞0.5×109/L的时间为11～32 d(中位数为17 d).CD34+CD49d+细胞输入量为(10.20～527.41)×104/kg(中位数为120.01×104/kg).中性粒细胞恢复时间与CD34+CD49d+细胞输入量呈负相关(γ＝-0.631,P＜0.05).结论无关脐血移植的CD34+CD49d+细胞输入量与造血于细胞的植入和造血重建有关.     

血液肿瘤病人HLA匹配的骨髓移植与外周血干细胞移植的疗效对比分析

使用粒细胞集落刺激因子(G-CSF)动员获得的异基因外周血造血干细胞进行移植(PBCT),其造血功能的重建作用明显快于骨髓移植(BMT),但两者急、慢性移植物抗宿主病(GVHD)的发生率、复发率、无病存活率是否存在差异还有待研究。作者对PBCT和BMT进行了比较。     

心房颤动患者CD34+造血祖细胞及相关细胞因子的变化

目的 通过观察心房颤动(房颤)对造血祖细胞及相关细胞因子影响,探讨房颤的发病机制以及重构机制.方法 比较持续性房颤病人、阵发性房颤病人与窦性心律者外周血中CD34+造血祖细胞的百分含量以及血清粒细胞集落刺激因子(G-CSF)、血管细胞黏附因子-1(VCAM-1)、基质细胞衍生因子-1α(SDF-1α)水平间的差别,分析CD34+造血祖细胞与后三者的相关性.结果 持续性房颤患者外周血中CD34+造血祖细胞的百分含量以及G-CSF、SDF-1α、VCAM-1的水平明显高于阵发性房颤患者和窦性心律者(P＜0.05,P＜0.01),而后两组间无差别.血清SDF-1α、VCAM-1水平与外周血CD34+造血祖细胞的百分含量具有明显的相关性(r值分别为0.737、0.530,P＜0.01和P＜0.05).结论 (1)持续性房颤可使患者外周血中CD34+造血祖细胞数量增加,使内源性SDF-1α、VCAM-1、G-CSF水平升高;(2)血清SDF-1α、VCAM-1水平与外周血CD34+造血祖细胞的百分含量具有明显的相关性;(3)SDF-1α以及VCAM-1参与心房损伤时心肌组织的修复过程,并对心肌的重构起一定作用.     

异基因外周血干细胞移植治疗白血病的临床观察

目的:观察同胞供者异基因外周血干细胞移植(allo-PBSCT)治疗白血病的疗效及并发症.方法:分析11例白血病患者行同胞供者HLA相合allo-PBSCT治疗的临床资料.供者干细胞动员用粒细胞集落刺激因子(G-CSF),用CS-3000血细胞分离机采集外周血干细胞.患者用改良马利兰加环磷酰胺(Bu/CY)为预处理方案,用环孢菌素A(CSA)+短程甲氨蝶呤(MTX)+霉酚酸酯(MMF)方案预防移植物抗宿主病(GVHD),用前列腺素E1(凯时)+低分子肝素预防肝静脉闭塞病(HVOD),用美司钠预防出血性膀胱炎(HC).结果:获得单个核细胞数(MNC)的中位数为8.3(4.5～13.0)×108/kg;CD34+细胞计数的中位数为6.2(2.4～12.4)×106/kg.10例患者获得造血重建,白细胞和血小板植活的中位数时间均为15(13 ～ 18)d;1例患者干细胞未植入.并发症:发生感染11例,GVHD 8例,HVOD 3例,HC 1例.中位随访15.5(1 ～39)个月,生存率为63.6％(7例),无病存活率为54.5％(6例);4例死亡.结论:同胞供者allo-PBSCT是治疗白血病一种有效手段,移植相关并发症主要为感染、GVHD、HVOD.     

G-CSF动员后异基因骨髓联合外周血干细胞移植治疗血液病

目的 探讨粒细胞集落刺激因子(G-CSF)动员的异基因骨髓与外周血干细胞混合移植后造血重建、移植物抗宿主病(GVHD)、复发及生存情况.方法 45例血液病患者进行了动员后的异基因骨髓联合外周血干细胞混合移植,人白细胞抗原(HLA)全合37例,1～3个位点不合8例.38例恶性病中32例采用清髓性预处理,6例为减低强度预处理;7例重型再生障碍性贫血(SAA)均采用环磷酰胺联合兔抗人胸腺细胞球蛋白(ATG)及甲泼尼龙预处理.采用环孢素联合霉酚酸酯预防移植物抗宿主病,HLA不全相合患者加用ATG.供者给予G-CSF连续5天皮下注射,注射后第5天采集外周血干细胞,第7天采取骨髓血.结果 45例患者均获得快速造血重建,中性粒细胞绝对计数≥0.5×109/L,血小板≥20×109/L的中位时间分别为移植后的12(8～18)天和16(10～28)天.10例发生了急性GVHD(22%),Ⅱ度以上1例.可评估的42例患者中16例出现了慢性GVHD,7例为广泛型(16%).复发9例,死亡11例,其余34例中位随访时间16月(10～46月),可评估的2年无病生存率为75%.结论 G-CSF动员后的异基因骨髓联合外周血干细胞移植治疗血液病可获快速造血重建,移植相关死亡率及重度急、慢性GVHD的发生率低,复发率不增高.     

Analysis of platelet recovery after autologous transplantation with G-CSF mobilized CD34+ cells purified from leukapheresis products

Abstract. We studied platelet recovery in relation to graft content in CFUs and CD34+ cells in 31 patients with multiple myeloma (21) or non-Hodgkin lymphoma (10) receiving marrow-ablative therapy followed by autologous transplantation with G-CSF mobilized CD34+ cells purified from leukapheresis products. Twelve patients had prolonged post-transplantation thrombopenia (? 14 days): their graft contents in CD34+ cells, CFU-GM and BFU-E were significantly inferior to those of patients with rapid platelet recovery. Although numbers of infused CD34+ cells and CFU-GM or BFU-E were well correlated, the graft content in CD34+ cells was the only parameter predictive of platelet recovery (r = ?0.38, p = 0.04), with a threshold of 2.5 × 106 CD34+ cells/kg. However, because rapid platelet reconstitution was obtained for 4 of 16 patients re-infused with < 2.5 × 106 CD34+ cells/kg, we investigated whether the graft CFU-MK content might be a better predictor of platelet reconstitution than the CD34+ cell content. Eighteen CD34 grafts were studied for CFU-MK content: CD34 and CFU-MK contents were weakly correlated (r = 0.52, p = 0.03), but there was no correlation between numbers of infused CFU-MK and time to platelet recovery. We conclude that, for autologous CD34 grafts, CFU-MK assays, like CFU-GM or BFU-E assays, cannot be used to predict platelet recovery. A CD34+ cell content >= 2.5 × 106/kg remains the only reliable indicator of the platelet reconstitution capacity of a CD34 graft.     

Analysis of platelet recovery after autologous transplantation with G-CSF mobilized CD34+ cells purified from leukapheresis products

Abstract. We studied platelet recovery in relation to graft content in CFUs and CD34+ cells in 31 patients with multiple myeloma (21) or non-Hodgkin lymphoma (10) receiving marrow-ablative therapy followed by autologous transplantation with G-CSF mobilized CD34+ cells purified from leukapheresis products. Twelve patients had prolonged post-transplantation thrombopenia (≫ 14 days): their graft contents in CD34+ cells, CFU-GM and BFU-E were significantly inferior to those of patients with rapid platelet recovery. Although numbers of infused CD34+ cells and CFU-GM or BFU-E were well correlated, the graft content in CD34+ cells was the only parameter predictive of platelet recovery (r = −0.38, p = 0.04), with a threshold of 2.5 × 106 CD34+ cells/kg. However, because rapid platelet reconstitution was obtained for 4 of 16 patients re-infused with < 2.5 × 106 CD34+ cells/kg, we investigated whether the graft CFU-MK content might be a better predictor of platelet reconstitution than the CD34+ cell content. Eighteen CD34 grafts were studied for CFU-MK content: CD34 and CFU-MK contents were weakly correlated (r = 0.52, p = 0.03), but there was no correlation between numbers of infused CFU-MK and time to platelet recovery. We conclude that, for autologous CD34 grafts, CFU-MK assays, like CFU-GM or BFU-E assays, cannot be used to predict platelet recovery. A CD34+ cell content >= 2.5 × 106/kg remains the only reliable indicator of the platelet reconstitution capacity of a CD34 graft.     

Peripheral blood stem cell transplantation as an alternative to autologous marrow transplantation in the treatment of acute myeloid leukemia?

The clinical use of autologous marrow transplantation in acute myeloid leukemia (AML) has been hampered by the inability to collect adequate numbers of cells after remission induction chemotherapy and the notably delayed hematopoietic regeneration following autograft reinfusion. Here we present a study in which the feasibility of mobilizing stem cells was investigated in newly diagnosed AML. Among 96 AML patients, 76 patients (79%) entered complete remission. Mobilization was undertaken with low dose and high dose schedules of G-CSF in 63 patients, and 54 patients (87%) were leukapheresed. A median of 2.0 x 10(6) CD34+ cells/kg (range 0.1-72.0) was obtained in a median of three leukaphereses following a low dose G-CSF schedule (150 microg/m2) during an average of 20 days. Higher dose regimens of G-CSF (450 microg/m2 and 600 microg/m2) given during an average of 11 days resulted in 28 patients in a yield of 3.6 x 10(6) CD34+ cells/kg (range 0-60.3) also obtained following three leukaphereses. The low dose and high dose schedules of G-CSF permitted the collection of 2 x 10(6) CD34-positive cells in 46% and 79% of cases respectively (P = 0.01). Twenty-eight patients were transplanted with a peripheral blood stem cell (PBSC) graft and hemopoietic repopulation was compared with the results of a previous study with autologous bone marrow. Recovery of granulocytes (>0.5 x 10(9)/l, 17 vs 37 days) and platelets (>20 x 10(9)/l; 26 vs 96 days) was significantly faster after peripheral stem cell transplantation compared to autologous bone marrow transplantation. These results demonstrate the feasibility of PBSCT in the majority of cases with AML and the potential advantage of this approach with respect to hemopoietic recovery.     

Megakaryocyte progenitors derived from bone marrow or G-CSF-mobilized peripheral blood CD34+ cells show a distinct phenotype and responsiveness to interleukin-3 (IL-3) and PEG-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF)

In the present study we investigated the proliferative response of megakaryocyte progenitor cells (CFU-MK) derived from peripheral blood stem cell (PBSC) collections of patients with haematological malignancies and normal donors. Highly purified CD34⁺ cells and mononuclear cell fractions were assayed in the presence of recombinant interleukin-3 (IL-3) and pegylated-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), alone or in combination, and megakaryocyte colony formation was evaluated in the plasma clot. In comparison, steady-state bone marrow samples from normal donors were highly enriched in CD34⁺ cells and tested with the cytokines studied. Our results showed that IL-3 was able to stimulate CFU-MK colony formation from bone marrow and peripheral blood CD34⁺ cells. Similarly, PEG-rHuMGDF stimulated, in a dose–response manner, CD34⁺ cells from the bone marrow. However, normal mobilized peripheral blood CD34⁺ cells were not induced to generate CFU-MK colonies by PEG-rHuMGDF. The same lack of response was observed when patients peripheral blood CD34⁺ cells primed with chemotherapy plus G-CSF or with G-CSF alone were assessed. In contrast, PEG-rHuMGDF stimulated CFU-MK growth when mononuclear cells, either from the bone marrow or from mobilized peripheral blood, were grown in plasma clot. Moreover, we analysed by flow cytometry the expression of Mpl receptor on the cell membrane of normal mobilized peripheral blood and normal steady-state bone marrow CD34⁺ cells. Our results showed a reduced expression of Mpl receptor on mobilized peripheral blood progenitor cells in comparison with bone marrow cells.     

The CD34+ cell fraction in bone marrow and blood is not universally predictive of CFU-GM.

We have measured the presence of granulocyte-macrophage colony forming cells (CFU-GM) and CD34+ cells in blood and bone marrow. We have compared these hematopoietic cell assays using regression analysis. We have found that under the limited and specific case of blood cells from an individual recovering from myelo-suppressive chemotherapy, the fraction of cells that is CD34 positive is predictive of the number of granulocyte-macrophage colonies (CFU-GM) which will grow. This result is in agreement with published data. We have found, however, that in bone marrow aspirates, or in the blood of individuals recovering from cyclophosphamide chemotherapy and receiving either granulocyte-macrophage colony stimulating factor (G-CSF) or folinic acid (FA) therapy, there is poor correlation between CD34+ cell fraction and CFU-GM. Accordingly, the use of CD34+ fraction cannot be relied upon to substitute for the CFU-GM assay in assessing the hematopoietic cell content of blood or bone marrow samples.     

CD34+ cell dose and hematologic recovery in allogeneic peripheral blood stem cell transplantation

Allogeneic peripheral blood stem cell transplantation (Allo-PBSCT) has been performed as an alternative to bone marrow transplantation (BMT). Here we report poor mobilization with granulocyte-colony stimulating factor (G-CSF) and engraftment kinetics in Allo-PBSCT. Sixteen patients (aged 6-61 yr, median 34 yr) received allogeneic peripheral blood stem cells from related donors (aged 15-68 yr, median 37 yr) after myeloablative therapy. Nine of the patients had standard-risk disease and 7 had high-risk disease. The donors received G-CSF at a dose of 10 micrograms/kg/day by subcutaneous injection for 4 to 6 days. Peripheral blood stem cells were subsequently collected in 1 to 3 aphereses and infused immediately. All patients received G-CSF after transplantation. Fifteen patients underwent Allo-PBSCT and one underwent Allo-PBSCT plus BMT. The mean number of CD34+ cells infused in the 15 Allo-PBSCT patients was 6.32 x 10(6)/kg (range 1.28-14.20). The outcomes were compared with 9 identically treated patients who underwent Allo-BMT. The median times until engraftment for neutrophils > 500/microliter and platelets > 20,000/microliter were 14 (range 10-17) and 15 (range 11-50) days in the Allo-PBSCT group and 17 (range 13-29) and 20 (range 16-160) days in the Allo-BMT group, respectively (p = 0.0177 and p = 0.003). Three donors were considered to have poor mobilization (< 2 x 10(6) CD34+ cells/kg of the recipient); two of them yielded 1.28 and 1.78 x 10(6) CD34+ cells/kg in 3 apheresis procedures. The patients who received cells from these donors showed prompt neutrophil engraftment, but one showed delayed platelet engraftment and another died of grade IV acute GVHD before reaching 20,000 platelets/microliter. An additional bone marrow harvest was necessary from one donor because of poor mobilization(0.17 x 10(6) CD34+ cells/kg). Thus, Allo-PBSCT results in more rapid engraftment. It will be necessary to clarify the minimum CD34+ cell dose for complete engraftment in a larger series of trials.     

The "G-CSF test": the response to a single dose of granulocyte colony-stimulating factor predicts mobilization of hemopoietic progenitors in patients with hematologic malignancies.

A significant proportion of patients with hematologic malignancies fail to mobilize sufficient hemopoietic progenitor cells (HPC), thereby restricting wider application of autologous transplantation. It would be of considerable use to develop a test that could be used prospectively to assess an individual patient's capacity to mobilize HPC. Twenty-two patients with lymphoma, myeloma, and chronic lymphocytic leukemia were given a single dose of 12 microg/kg SC of granulocyte colony-stimulating factor (G-CSF). Blood colony-forming unit granulocyte-macrophage (CFU-GM) and CD34+ cells were scored prior to the test dose, and 72, 96, and 120 hours later. The patients were then mobilized with a standard cyclophosphamide and G-CSF regimen and had blood stem cells harvested. Patients were categorized as good, poor, or intermediate mobilizers on the basis of the CFU-GM/CD34+ cell harvest content and the number of aphereses required to reach established threshold counts. The outcome of cyclophosphamide/G-CSF mobilization was correlated with the response to the test dose of G-CSF. Good mobilizers had significantly higher peak CFU-GM values and CFU-GM increment in response to the test dose of G-CSF compared to intermediate and poor mobilizers. A peak CFU-GM count of > or = 250/mL identified the good mobilizers; conversely, all poor mobilizers had a peak CFU-GM count of <102/mL. An increment in CD34+ cells counts of > or = 2.5/microL was only observed in good mobilizers. The "G-CSF" test is a reliable test that can be used successfully for the assessment of mobilizable HPC in patients with hematologic malignancies. It can also be used to stratify patients enrolled in trials of mobilizing agents.     

Target cells for granulocyte colony-stimulating factor, interleukin-3, and interleukin-5 in differentiation pathways of neutrophils and eosinophils.

To study the relationship between hematopoietic factors and their responsive hematopoietic progenitors in the differentiation process, both purified factors and enriched progenitors are required. We isolated total CD34+ cells, CD34+,CD33+ cells, and CD34+,CD33- cells individually from normal human bone marrow cells by fluorescence-activated cell sorter (FACS), and examined the effects of granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), and IL-5 on in vitro colony formation of these cells. CD34+,CD33+ cells formed granulocyte colonies in the presence of G-CSF. Both CD34+,CD33+ cells and CD34+,CD33- cells formed granulocyte/macrophage colonies in the presence of IL-3. Eosinophil (Eo) colonies were only formed by CD34+,CD33- cells in response to IL-3, but scarcely formed by CD34+ cells in the presence of IL-5. We performed the two-step cultures consisting of the primary liquid culture for 6 days and the secondary methylcellulose culture, and serially examined changes in phenotypes of ,he cells cultured in the primary culture. CD34-,CD33+ cells derived from CD34+,CD33+ cells by preincubation with G-CSF or IL-3 formed Eo colonies in the presence of IL-5 but not IL-3. CD34-,CD33+ cells derived from CD34+,CD33- cells by preincubation with IL-3 also formed Eo colonies by support of IL-5 as well as IL-3. Both CD34+ cells gradually lost the CD34 antigen by day 6 of incubation with G-CSF or IL-3. Loss of this antigen was well-correlated with acquisition of susceptibility to IL-5. It was concluded that G-CSF supported the neutrophil differentiation of committed colony-forming cells, IL-3 supported that of both committed and multipotent colony-forming cells. G-CSF and IL-3 also supported the early stage of E. differentiation; IL-5 supported the late stage of that.     

Circulating Stem Cell Collection in Lymphoma and Myeloma after Mobilization with Cyclophosphamide and Granulocyte Colony-Stimulating Factor for Autologous Transplantation

We report the results of 72 leukapheresis procedures performed for autologous peripheral blood stem cell collection in 18 patients with lymphoma and myeloma, after combined mobilization with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF). The numbers of mononuclear cells (MNCs), CD34+ cells and granulocyte-macrophage colony-forming units (CFU-GM) either in the peripheral circulation (preleukapheresis sample) or in the product obtained from leukapheresis (leukapheresis sample) were evaluated. A highly superior proportion of CD34+ cells (14-fold) and CFU-GM (5-fold) resulted from the mobilization therapy. CFU-GM and CD34+ cells were highly enriched with respect to all MNCs (relative recoveries: 2.13, range 0.3–41, and 1.08, range 0.2–8.5, respectively) due to an additional mobilization effect by the leukapheresis procedure. Also, a relatively strong linear correlation between the three different parameters was found in the leukapheresis product (CD34+:CFU-GM, r = 0.81; MNCs:CD34, r = 0.69; MNCs:CFU-GM, r = 0.75; CFU-GM:CD34+, and MNCs, r = 0.85). Our data suggest that the number of MNCs and CD34+ cells obtained after combined mobilization with cyclophosphamide and G-CSF can be used as predictor of the number of granulomonocytic progenitors.     

Sustained long-term hematopoiesis after myeloablative therapy with peripheral blood progenitor cell support

A retrospective analysis of long-term hematopoiesis was performed in a group of 145 consecutive patients who had received high-dose therapy with peripheral blood progenitor cell (PBPC) support between May 1985 and December 1993. Twenty-two patients had acute myelogenous leukemia, nine had acute lymphoblastic leukemia, 43 had Hodgkin's disease, 57 had non-Hodgkin's lymphoma, and 14 patients had multiple myeloma. Eighty- four patients were male and 61 female, with a median age of 37 years (range, 16 to 58 years). In 46 patients, PBPC were collected after cytotoxic chemotherapy alone, while 99 patients received cytokines either during steady-state hematopoiesis or post-chemotherapy. Sixty patients were treated with dose-escalated polychemotherapy, and 85 patients had a conditioning therapy including hyperfractionated total body irradiation at a total dose of 14.4 Gy. The duration of severe pancytopenia posttransplantation was inversely related to the number of reinfused granulocyte-macrophage colony-forming units (CFU-GM) and CD34+ cells. Threshold quantities of 2.5 x 10(6) CD34+ cells per kilogram or 12.0 x 10(4) CFU-GM per kilogram became evident and were associated with rapid neutrophil and platelet recovery within less than 18 and 14 days, respectively. These numbers were also predictive for long-term reconstitution, indicating that normal blood counts are likely to be achieved within less than 10 months after transplantation. Conversely, 12 patients were autografted with a median of 1.75 x 10(4) CFU-GM per kilogram resulting in delayed recovery to platelet counts of greater than 150 x 10(9)/L between 1 and 6 years. Our study includes bone marrow examinations in 50 patients performed at a median follow-up time of 10 months (range, 1 to 85 months) posttransplantation. A comparison with normal volunteers showed a 3.2-fold smaller proportion of bone marrow CD34+ cells, which was paralleled by an even more pronounced reduction in the plating efficiency of CFU-GM and burst- forming unit-erythroid. No secondary graft failure was observed, even in patients autografted with relatively low numbers of progenitor cells. This suggests that either the pretransplant regimens were not myeloablative, allowing autochthonous recovery, or that a small number of cells capable of perpetual self-renewal were included in the autograft products.