CD34~+细胞纯化在外周血干细胞移植中的应用

外周血干细胞的纯化方法有多种,其中,利用干细胞和一些肿瘤细胞、T细胞表面不同的抗原表达而设计的CD34阳性/阴性选择已开始应用于临床,可以明显减少干细胞植入时移植物所含的肿瘤细胞和T细胞的数量,从而降低了自身移植后的复发倾向和同种异基因移植后GVHD的发生。     

自体纯化CD34+细胞移植治疗自身免疫性疾病14例的临床观察

目的 探讨自体纯化CD34+细胞移植治疗自身免疫性疾病(AID)的疗效.方法 对14例自身免疫性疾病患者进行自体纯化CD34+细胞移植.采用环磷酰胺(CTX)+G-CSF动员外周血干细胞,通过CliniMACS细胞分选仪分选CD34+细胞并冻存.预处理方案:8例采用氟达拉滨(FDB)+抗胸腺细胞球蛋白(ATG)+CT...     

自体纯化CD34+细胞移植治疗严重自身免疫性疾病的初步研究

目的探讨自体外周血CD34+细胞移植治疗严重自身免疫性疾病的干细胞动员、细胞采集和分选、预处理和并发症处理等问题.方法 10例重度自身免疫性疾病患者接受自体外周血CD34+细胞移植治疗.采用环磷酰胺(CTX)+rhG-CSF方案动员外周血干细胞,并以CliniMACS细胞分选仪分选CD34+细胞,适时用CTX+抗胸腺细胞球蛋白(7例)或CTX+全身照射(3例)两种预处理方案后,进行CD34+细胞回输的方法治疗.结果经CTX+rhG-CSF方案动员并以CliniMACS细胞分选仪分选后,可获得(1.98±0.95)×108的CD34+细胞,其纯度为(91.4±10.6)%,回收率为(60.5±19.8)%.在回输(2.14±1.05)×106/kg的CD34+细胞后,ANC ≥0.5×109/L的时间为(8.6±2.5)d,血小板升至20×109/L的时间为(9.0±5.2)d.在造血恢复后,所有CD3+细胞、CD19+细胞和CD16+CD56+细胞均未恢复至移植前状态.在造血和免疫抑制时,巨细胞病毒感染的发生率较高.2例患者死于移植相关并发症.所有患者近期疗效满意,6例系统性红斑狼疮患者DAI评分由移植前的平均17分降为移植后的4分;类风湿关节炎患者DAS28评分由6.4分降至1.8分;干燥综合征患者的症状和体征均明显缓解.结论对常规治疗无效的严重自身免疫性疾病,自体外周血CD34+细胞移植是可选择的治疗方法之一.     

外周血CD34~+细胞移植(2)

异基因外周血ＣＤ34 细胞移植的临床应用6.1　ＭＬＡ相合同胞移植　比较异基因骨髓移植而言,ＧＣＳＦ动员的外周血白细胞分离产物中含的Ｔ淋巴细胞约多1个对数级。但近年越来越多的临床证实,ＨＬＡ相合同胞间异基因外周血干细胞移植后急性ＧＶＨＤ的发生并不比骨髓移植后的高,但慢性ＧＶＨＤ的发生要高一些,且临床表现有所不同。一般来讲,引起临床急性ＧＶＨＤ发生的Ｔ细胞输注量约为(0.1～1)×106/ｋｇ体重。实践证明,将造血细胞因子动员的外周血白细胞分离产物经分选富集ＣＤ34 细胞后,不仅可获得高纯度足量ＣＤ34 细胞用于临床移植,还可…     

外周血CD4~+CD25~+CD127~(low)调节性T细胞在恶性肿瘤患者免疫功能评估中的作用

目的观察恶性肿瘤患者外周血CD4+CD25+CD127low调节性T细胞(CD4+CD25+CD127lowTreg)的数量变化,探讨外周血CD4+CD25+CD127lowTreg检测在恶性肿瘤患者免疫功能评估中的作用。方法以35例正常人作为对照组,采用流式细胞术检测74例肿瘤患者外周血CD4+CD25+CD127lowTreg在CD4+T细胞的比例,分析不同TNM分期恶性肿瘤患者外周血中CD4+CD25+CD127lowTreg占CD4+T细胞的百分比,采用实时荧光定量RT-PCR技术检测PBMC转录因子Foxp3表达水平。结果肿瘤患者外周血中CD4+CD25+CD127low Treg占CD4+T细胞百分比为(6.19±1.82)%,明显高于对照组(3.12±1.16)%,差异具有统计学意义(P0.05)。其中Ⅲ、Ⅳ期患者外周血CD4+CD25+CD127lowTreg百分比分别为(6.08±2.14)%、(6.88±2.65)%,明显高于Ⅰ-Ⅱ期患者(5.65±1.86)%,P0.05;病理分型为低分化者CD4+CD25+CD127lowTreg百分比为(6.72±2.60)%,明显高于高分化者(5.94±2.11)%,P0.05;有淋巴结转移者CD4+CD25+CD127lowTreg百分比为(6.95±3.12)%,明显高于无淋巴结转移者(5.02±2.09)%,P0.05。结论恶性肿瘤患者外周血CD4+CD25+CD127lowTreg占CD4+T细胞的比例明显升高,监测CD4+CD25+CD127lowTreg有利于评估肿瘤患者的免疫功能和辅助判断肿瘤患者的病情进展及预后。     

外周血CD34~+细胞移植(1)

骨髓移植已成为治愈某些恶性血液病、骨髓造血衰竭、实体瘤、遗传性和免疫缺陷疾病的可靠方法［１］。近年来,由于移植后造血功能重建快、患者易接受和较经济等优点,外周血干细胞移植大有替代骨髓移植的趋势［２］。然而,自体外周血细胞移植物中仍有不少残留肿瘤细胞,...     

外周血干细胞移植中标本放置时间对CD34~+细胞及单个核细胞计数结果的影响

目的探讨标本放置温度及时间对外周血CD34+细胞及单个核细胞(MNC)计数结果的影响。方法抽取10例健康供者或自体外周血干细胞(APBSCT)移者经粒细胞集落刺激因子(G-CSF)动员第5天EDTA-K2抗凝静脉血,各分装2管,分别在室温和4℃条件下保存,对每份外周血标本分别于0、1、2、4、6、8、10、12、24h计数MNC和CD34+细胞。结果室温放置的标本中,MNC和CD34+细胞计数随着标本放置时间的延长而逐渐减低,放置到8h时,CD34+细胞计数和0h相比差异有统计学意义(t=5.04,P<0.05);放置到12h时,MNC计数和0h相比差异有统计学意义(t=3.68,P<0.05)。4℃条件下,标本放置到24h,MNC和CD34+细胞计数结果和0h相比差异均无统计学意义(t分别为0.50、1.24,P>0.05)。结论为确保检测结果的准确性,室温放置的标本CD34+细胞计数应在采血后8h内完成,MNC计数可在采血后10h内完成,外周血标本最好放置于4℃条件下保存。     

CD34+细胞分选及临床应用

CD34+是造血干细胞的免疫表型。采用某种方法选择CD34+细胞,选择后CD34+细胞浓集,其百分率有较大增高,并可以有效去除T细胞及某些恶性肿瘤细胞。选择CD34+细胞移植可以减少异基因移植后急性、重度移植物抗宿主病(GVHD),并可能减少自体移植后原发病复发。本文介绍了造血干细胞的免疫表型,生物学特性及CD34+细胞分选方法、临床应用等方面的研究进展。     

The relationship of CD34+ dosage and platelet recovery following high dose chemotherapy and autologous CD34+ reinfusion in multiple myeloma

Autologous hematopoietic stem cell transplantation (ASCT) is an established treatment for multiple myeloma (MM), yet the impact of transplanted CD34+ cell dose remains unresolved, especially in patients over the age of 65 years. Data was collected from 207 consecutive ASCT patients to determine the relationship between CD34+ infusion count and short-term and long-term platelet recovery. For MM patients under the age of 65 years (n = 155), CD34+ dosage correlates with time to platelet engraftment (p < 0.001) and platelet count at 30 days (p = 0.003), but not with long-term platelet counts at 180 or 360 days from the CD34+ reinfusion. For MM patients aged 65 years or older (n = 46), CD34+ dosage did not correlate with time to platelet engraftment, but did correlate with both short-term and long-term platelet counts at 30 (p < 0.001), 180 (p = 0.021), and 360 days (p = 0.005). Exploratory regression analysis was done to explore platelet stability following the current minimum CD34+ dosage reinfusion. For MM patients under the age of 65 years, the minimum standard CD34+ dosage of 2 × 10⁶ cells/kg was sufficient for a timing to platelet engraftment of <21 days and short-term platelets count ≥150 × 10⁹/L at 30 days. Alternatively, for MM patients aged 65 years or older, the CD34+ dosage of 2 × 10⁶ cells/kg was insufficient for platelet counts ≥150 × 10⁹/L at 30 and only marginally attainable at 360 days suggesting that in elderly MM patients a higher CD34+ dosage may be required for platelet recovery and possibly long-term platelet stability.     

体外诱导骨髓CD34+细胞生成血管内皮细胞的方法

目的：体外分离狗骨髓CD34^ 细胞，建立诱导分化血管内皮细胞的方法，并进行生物学鉴定。方法：利用免疫磁珠法分离狗骨髓CD34^ 细胞，在体外经血管内皮细胞生长因子（VEGF）、内皮细胞生长因子（EGF）、碱性成纤维细胞生长因子（hFGF)诱导后，观察细胞生长状况，以光镜、电镜进行形态学鉴定，应用免疫组化方法检测冯维靳布兰德因子（Von Willebrand factor,vWF)表达。结果：光镜下细胞单层融合生长，呈铺路石样形态，单个核位子中央，细胞群体倍增时间为35h。电镜下可见到Weibel-Palade小体，在细胞胞浆vWF染色阳性。结论：采用免疫磁珠法可从骨髓获得高纯度的CD34^ 细胞，在体外诱导分化成血管内皮细胞。     

选择性CD34+细胞和非选择性自体外周血干细胞移植治疗多发性骨髓瘤的比较研究

目的明确选择性CD34+细胞自体外周血干细胞移植(APBSCT)治疗多发性骨髓瘤(MM)的临床效果.方法对21例接受CD34+细胞APBSCT治疗的MM患者(选择组)和另外21例接受非选择性APBSCT的MM患者(对照组)的造血恢复时间、有效率、生存率、移植相关不良反应、移植费用进行比较.两组病例在年龄、初诊时血清β2微球蛋白水平和移植时疾病状态均具有可比性,诱导治疗及预处理方案基本相同.结果与对照组相比,选择组患者回输的CD34+细胞数显著偏低[对照组和选择组分别为9.4(1.1～15.0)×106/kg和2.2(0.5～14.3)×106/kg](P＜0.001),中性粒细胞恢复至≥0.5×109/L和血小板恢复至≥20×109/L儿的中位时间,在选择组分别为10d和9d,对照组分别为9.5d(P=0.357)和4.5d(P=0.005).两组的治疗有效率相似(选择组85.7%,对照组90.4%),3年无病生存率(选择组和对照组分别为32%和39%)和总生存率(选择组和对照组分别为85%和79%)两组相比差异无显著性,而且使用非选择性APBSCT可明显降低移植费用.结论选择性CD34+细胞APBSCT与非选择性APBSCT相比,并不能改善MM的临床治疗结果,而且移植费用更大.     

The number of CD34(+) cells in peripheral blood as a predictor of the CD34(+) yield in patients going to autologous stem cell transplantation

The number of CD34(+) cells in peripheral blood (PB) is a guide to the optimal timing to harvest peripheral blood progenitor cells (PBPC). The objective was to determine the number of CD34(+) cells in PB that allows achieving a final apheresis product containing > or =1.5 x 10(6) CD34(+) cells/kg, performing up to three aphereses. Between March 1999 and August 2003, patients with hematological and solid malignancies who underwent leukapheresis for autologous bone marrow transplantation were prospectively evaluated. Seventy-two aphereses in 48 patients were performed (mean 1.45 per patient; range 1-3). PBPC were mobilized with cyclophosphamide plus recombinant human granulocyte-colony stimulating factor (G-CSF) (n = 40), other chemotherapy drugs plus G-CSF (n = 7), or G-CSF alone (n = 1). We found a strong correlation between the CD34(+) cells count in peripheral blood and the CD34(+) cells yielded (r = 0.903; P < 0.0001). Using receiver-operating characteristic (ROC) curves, the minimum number of CD34(+) cells in PB to obtain > or =1.5 x 10(6)/kg in the first apheresis was 16.48 cells/microL (sensitivity 100%; specificity 95%). The best cut-off point necessary to obtain the same target in the final harvest was 15.48 cells/microL, performing up to three aphereses (sensitivity 89%; specificity 100%). In our experience, > or =15 CD34(+) cells/microL is the best predictor to begin the apheresis procedure. Based on this threshold level, it is possible to achieve at least 1.5 x 10(6)/kg CD34(+) cells in the graft with < or =3 collections.     

自体外周血CD34^＋胞移植治疗系统性红斑狼疮的护理

对11例系统性红斑狼疮患进行了自体外周血CD34^ 细胞移植治疗，患合并相关并发症22例次，除1例移植后3月合并肺部感染死亡，其他病情均缓解，取得了良好疗效。Eh于患移植前已经合并多脏器功能损害，因此在动员及移植期间应密切观察心、肾功能的变化，如尿量、尿色、中心静脉压等，移植后期注意病毒、真菌、结核杆菌感染的发生，及早发现可提高治疗效果。     

Comparison of CD34+ cell collection efficiency on the COBE Spectra and Fenwal CS-3000 Plus

Optimal collections of mobilized CD34+ cells are important in terms of both patient toxicity and cost. The factors that determine CD34+ collection efficiency (CD34eff) of cell separators have not been well studied. In addition, because several cell separators are available, the type of collection device may also be a significant variable. Previous studies comparing the Baxter-Fenwal CS3000 and the COBE Spectra have not yielded consistent conclusions. Therefore, we retrospectively analyzed the collection outcomes of 163 consecutive donors with a peripheral CD34+ cell concentration (pCD34) of > or =5 cells/microl on the first collection that had been harvested on one or the other device. The CS3000 was found to yield a significantly higher CD34eff (50% vs. 39%, P = 0.006). However, donors were not balanced for several prognostic factors, which may contribute to CD34eff including mobilization with G-CSF vs. chemotherapy+G-CSF, average flow rate, and total volume of peripheral blood processed. When appropriate variables were included in a stepwise multiple variable analysis, cell separator type emerged as a significant independent predictive factor for CD34eff (P = 0.018). Our data indicates that the CS3000 will, on average, show a higher absolute CDeff of 8%. Furthermore, since the two devices differ in mechanism, prognostic factors may also differ. Comparisons suggest that peripheral blood WBC and hematocrit may be more important predictors for the CS3000.     

B淋巴细胞白血病骨髓B细胞CD34+CD38+和CD34+CD38low/-细胞亚群的临床意义

本研究比较发病时免疫表型为CD34+CD38+和CD34+CD38low/-两组B淋巴细胞白血病(B-ALL)的生物学特性,并探讨其临床意义。选取54例初发B-ALL并经多参数流式细胞术检测为CD34+的B-ALL患者,根据CD38表达不同将其分为CD34+CD38+组(n=35)和CD34+CD38low/-组(n=19)。利用实时定量PCR的方法分别进行BCR-ABL,TEL-AML1和WT1基因的检测。随访标本利用7色流式细胞术检测微量残留病(MRD),平均随访时间12个月(1-28个月),平均随访间隔2个月(1-5个月)。结果表明,两组患者发病时WBC,血小板和血红蛋白水平及BCR-ABL,TEL-AML1和WT1基因阳性表达的比例均无统计学差异(P>0.05)。在诱导缓解后,CD34+CD38+组微量残留病阳性(MRD+)为28.57%(10/35),CD38low/-组MRD+为68.42%(13/19),CD34+CD38low/-组MRD+的检出率明显高于CD34+CD38+组(P<0.01)。在复发率上,CD34+CD38+组有2例,分别在第94天和第245天复发,复发率为5.71%(2/35)。CD34+CD38low/-组有7例复发,复发率为36.84%(7/19),中位复发时间263 d(46-468 d),两组间具有明显统计学差异(P<0.01)。本研究以16岁为年龄分界点,CD34+CD38+组16岁以上患者为8人(8/35),CD34+CD38low/-组16岁以上患者为10人(10/19),两组间有统计学差异(P<0.05)。结论:CD34+CD38low/-患者在成人中比例较高,且CD34+CD38low/-组在治疗后更易出现MRD+和复发。     

Impact of apheresis automation on procedure quality and predictability of CD34+ cell yield

Success of peripheral blood stem cell (PBSC) collections depends on patient biological parameters and stable apheresis device performance. We investigated product quality and factors influencing main apheresis procedure outcomes including CD34⁺ collection efficiency (CE), product volume or platelet CE. We also assessed different CD34⁺ cell yield prediction algorithms. Autologous PBSC collections by Spectra Optia from myeloma and lymphoma patients were analyzed. Complete blood count (CBC) from patient preprocedure and from collected products were assessed. (1) Product yield was calculated, (2) Product CBC was correlated with patient preprocedure variables, and (3) Predictions of CD34⁺ yields based on (a) product CD34⁺ cell concentration in samples after two or four chamber flushes or (b) traditional CE2 benchmark, were compared. 62 procedures in 41 patients were analyzed. 84% of all procedures were run without operator intervention. Median CD34⁺ CE2 was 56.9% (48.8%‐65.2%) and quite stable irrespective of patient conditions, with minor influence from patient white blood cell (WBC) precounts (r_s = –.47; P < .001). Platelet loss correlated with WBC precount (r_s = .46; P < .001), product volume (r_s = .71; P < .0001) and number of chambers collected (r_s = .72; P < .0001). CD34⁺ cell yield was better predicted based on (a) product CD34⁺ cell concentration from samples after 2 and 4 chamber flushes, respectively (r_s = .969; P < .0001 and r_s = .9648; P < .0001) than based on (b) CE2 formula (r_s = .8262, P < .0001). Spectra Optia provides good quality PBSC products with stable and predictable yield regardless of starting conditions. CD34⁺ sampling of product after few chamber flushes could be used to predict CD34⁺ yield.