Rapid and effective CD3 T-cell depletion with a magnetic cell sorting program to produce peripheral blood progenitor cell products for haploidentical transplantation in children and adults

BACKGROUND: Effective T-cell depletion is a prerequisite for haploidentical peripheral blood progenitor cell (PBPC) transplantation. This study was performed to investigate the performance of magnetic cell sorting-based direct large-scale T-cell depletion, which is an attractive alternative to standard PBPC enrichment procedures. STUDY DESIGN AND METHODS: PBPCs were harvested from 11 human leukocyte antigen (HLA)-haploidentical donors. T cells labeled with anti-CD3-coated beads were depleted with a commercially available magnetic separation unit (CliniMACS, Miltenyi Biotec) with either the Depletion 2.1 (D2.1, n=11) or the novel Depletion 3.1 (D3.1, n=12) program. If indicated, additional CD34+ selections were performed (n=6). Eleven patients received T-cell-depleted grafts after reduced-intensity conditioning. RESULTS: The median log T-cell depletion was better with the D2.1 compared to the D3.1 (log 3.6 vs. log 2.3, p<0.05) and was further improved by introducing an immunoglobulin G (IgG)-blocking step (log 4.5 and log 3.4, respectively). The D3.1 was superior to the D2.1 (p<0.05) in median recovery of CD34+ cells (90% vs. 78%) and in median recovery of CD3- cells (87% vs. 76%). The median processing times per 10(10) total cells were 0.90 hours (D2.1) and 0.35 hours (D3.1). The transplanted grafts (directly T-cell-depleted products with or without positively selected CD34+ cells) contained a median of 10.5 x 10(6) per kg CD34+, 0.93x10(5) per kg CD3+, and 11.6x10(6) per kg CD56+. Rapid engraftment was achieved in 10 patients. The incidences of acute graft-versus-host disease were less than 10 percent (Grade I/II) and 0 percent (Grade III/IV). CONCLUSION: The novel D3.1 program with IgG blocking enables highly effective, time-saving large-scale T-cell depletion. Combining direct depletion techniques with standard CD34+ selection enables the composition of grafts optimized to the specific requirements of the patients.     

Isolation of highly purified autologous and allogeneic peripheral CD34+ cells using the CliniMACS device.

The CliniMACS CD34+ selection device was used for positive selection of apheresis products for autologous transplantation from 10 patients with malignant diseases and for allogeneic transplantation from 26 healthy donors. A total of 71 separations were performed. In 1 allogeneic donor, CD34+ progenitors were also isolated from bone marrow. Between 0.27 and 8.9 x 10(10) nucleated cells (median 4.9 x 10(10)) containing 0.09%-10.8% (median 0.67%) CD34+ progenitor cells were separated. After separation, a median number of 227 x 10(6) mononuclear cells (MNC) (51-524) were recovered, with a median viability of 99% (22%-100%) and a median purity of 97.0% (68.3%-99.7%) CD34+ cells. Depletion of T cells was extensive, with a median of 0.04% residual CD3+ cells (range <0.01%-0.92%). Residual CD19+ cells were between <0.01% and 17%, including CD34+CD19+ cells. Recovery of CD34+ cells was calculated according to the ISHAGE guidelines and ranged from 24% to 105% (median 71%). We conclude that with the CliniMACS device CD34+ cells with high purity and recovery can be isolated with concomitant effective T cell depletion in the allogeneic setting and with a high purging efficacy in the autologous setting.     

动员外周血造血干细胞移植物中同时高效去除T／B细胞的体外研究

单倍体相合造血干细胞移植时，去除移植物中的T细胞和B细胞可减少移植物抗宿主病和EBV相关的淋巴细胞增殖性疾病的发生。本研究在国内首次应用CliniMACS系统，通过CD3／CD19磁珠抗体同时有效地去除动员外周血造血干细胞中的T细胞和B细胞。用流式细胞术检测移植物中T细胞和B细胞的去除效率，以集落形成实验评价去除后的干细胞功能。结果表明：T细胞和B细胞去除之前单个核细胞总数为4．88×10^10，去除之后移植物中T细胞比例为0．02％，去除4．4Log；B细胞比例不到0．01％，至少去除3．3Log。移植物中除CD34^＋细胞外。还包括NK细胞，单核细胞和粒细胞。CD34^＋细胞纯度为0．98％，计数1．84×10^8，回收率为69．7％；NK细胞计数2．54×10^9，回收率为71．7％。集落形成实验显示CD3／CD19去除对造血干细胞功能无影响。结论：应用CliniMACS系统可有效地同时去除动员外周血干细胞中的T细胞和B细胞，且不影响造血干细胞的生物学功能，此项研究为去除T／B细胞的单倍体相合造血干细胞移植提供了技术平台。     

Analysis of factors associated with low peripheral blood progenitor cell collection in normal donors

BACKGROUND: Predictive factors of the response to rHuG-CSF in normal donors have not been extensively studied. STUDY DESIGN AND METHODS: We analyzed factors influencing CD34+ cell yield in the 1st day of collection in 261 healthy donors from the Spanish National Donor Registry. The median age was 38 years (range, 2-72). The median dose of rHuG-CSF was 10 microg per kg per day (range, 5-20) over 4 days. In 103 donors (40%), <4 x 10(6) per kg CD34+ cells were collected. The variables that were analyzed included age, sex, weight, basal complete blood cell count, dose, type of rHuGCSF and schedule of administration, and maximum WBC count before apheresis. RESULTS: By univariate analysis, the maximum WBC count (<50 vs. >or=50 x 10(9)/L, p = 0.004), advanced age (p = 0.008), and number of daily rHuG-CSF doses (one vs. two; p = 0.01) correlated with the number of CD34+ cells collected. By multivariate analysis, donors age (<38 vs. >or=38 years; p = 0.014) and a single daily dose of rHuG-CSF (p = 0.005) were the two variables that significantly predicted a low CD34+ cell yield. CONCLUSION: Donors' age, with a threshold of 38 years or more, and the rHuG-CSF schedule are the factors that significantly affected CD34+ cell mobilization and collection in healthy donors.     

Impact of donor- and collection-related variables on product quality in ex utero cord blood banking

BACKGROUND: Optimizing product quality is a current focus in cord blood banking. This study evaluates the role of selected donor- and collection-related variables. STUDY DESIGN AND METHODS: Retrospective review was performed of cord blood units (CBUs) collected ex utero between February 1, 2000, and February 28, 2002. Preprocessing volume and total nucleated cell (TNC) counts and postprocessing CD34 cell counts were used as product quality indicators. RESULTS: Of 2084 CBUs, volume determinations and TNC counts were performed on 1628 and CD34+ counts on 1124 CBUs. Mean volume and TNC and CD34+ counts were 85.2 mL, 118.9 x 10(7), and 5.2 x 10(6), respectively. In univariate analysis, placental weight of greater than 500 g and meconium in amniotic fluid correlated with better volume and TNC and CD34+ counts. Greater than 40 weeks' gestation predicted enhanced volume and TNC count. Cesarean section, two- versus one-person collection, and not greater than 5 minutes between placental delivery and collection produced superior volume. Increased TNC count was also seen in Caucasian women, primigravidae, female newborns, and collection duration of more than 5 minutes. A time between delivery of newborn and placenta of not greater than 10 minutes predicted better volume and CD34+ count. By regression analysis, collection within not greater than 5 minutes of placental delivery produced superior volume and TNC count. CONCLUSION: Donor selection and collection technique modifications may improve product quality. TNC count appears to be more affected by different variables than CD34+ count.     

Two versus three day upfront use of granulocyte-colony stimulating factor in healthy bone marrow donors for pediatric bone marrow transplantation

In order to decrease donors’ exposure to granulocyte-colony stimulating factor (G-CSF), we compared the effect of two versus three days of G-CSF priming on CD34+ yield in bone marrow (BM) harvest. Although the number of BM-CD34+ cells was higher in 3 day G-CSF priming, we achieved the same number of CD34+ cells per recipient’s weight in 2 day G-CSF priming group, too. In addition, the number of total nucleated cells (TNC) harvested from BM were similar with two or three day regimen. But mononuclear cells (MNC) of the BM graft was higher in the 3 day G-CSF priming group. Similar to CD34+ cell numbers, BM harvest yielded similar TNC, and MNC numbers per kilogram of the recipient. We also found that, young donors (≤10 year) had more peripheral blood MNC, bone marrow MNC and CD34+ cell numbers. Another interesting finding of this study was obtaining adequate number of peripheral blood stem cells for leukapheresis with three day G-CSF administration. Since engrafment times were also similar in two groups, we concluded that 2-days G-CSF priming was resulted in sufficient mobilization of BM stem cells.     

Donor age-related differences in PBPC mobilization with rHuG-CSF

BACKGROUND: Data on the administration of rHuG-CSF to normal donors <18 years old are very limited. STUDY DESIGN AND METHODS: The results of rHuG-CSF administration to 61 donors <18 years old (Group A) were retrospectively evaluated and compared with results from 353 donors > or = 18 years old (Group B) who are included in the Spanish National Donor Registry. The mean age (range) in Group A and B was 14 (1-17) and 38 (18-71) years, respectively (p<0.001). The mean dose of rHuG-CSF was 10 microg per kg per day (range, 9-16) during a mean of 5 days (range, 4-6). Central venous access was placed more frequently in younger donors (25% vs. 6%; p<0.001). RESULTS: The mean number of CD34+ cells collected was 7.6 and 6.9 x 10(6) per kg of donor's body weight in Group A and B, respectively. Fifty-six percent of Group A donors needed only one apheresis to achieve > or = 4 x 10(6) CD34+ cells per kg versus 39 percent of Group B donors (p = 0.01). Side effects were more common in Group B (71% vs. 41%; p<0.001). CONCLUSION: The administration of rHuG-CSF to donors <18 years old leads to CD34+ cell mobilization in a pattern similar to that observed in adults. Greater age was associated with a more frequent requirement for more than one apheresis to achieve a similar number of CD34+ cells.     

PBPC mobilization with paclitaxel, ifosfamide, and G-CSF with or without amifostine: results of a prospective randomized trial

BACKGROUND: The impact of amifostine on PBPC mobilization with paclitaxel and ifosfamide plus G-CSF was assessed. STUDY DESIGN AND METHODS: Forty patients with a median age of 34 years (range, 19-53) who had germ cell tumor were evaluated for high-dose chemotherapy. Patients were randomly assigned to receive either a single 500-mg dose of amifostine (Group A, n = 20) or no amifostine (Group B, n = 20) before mobilization chemotherapy with paclitaxel (175 mg/m(2)) given over 3 hours and ifosfamide (5 g/m(2)) given over 24 hours (TI) on Day 1. G-CSF at 10 microg per kg per day was given subsequent to TI with or without amifostine from Day 3 until the end of leukapheresis procedures. RESULTS: In 2 (10%) of 20 patients receiving amifostine and 3 (15%) of 20 patients not receiving it, no PBPC separation was performed because of mobilization failure. No significant differences were observed in the study arms with regard to the time from chemotherapy until first PBPC collection or the number of apheresis procedures needed to harvest more than 2.5 x 10(6) CD34+ cells per kg. Furthermore, leukapheresis procedures yielded comparable doses of CD34+ cells per kg (3.4 x 10(6) vs. 3.6 x 10(6); p = 0.82), MNCs per kg (2.7 x 10(8) vs. 2.6 x 10(8); p = 0.18), and CFU-GM per kg (15.9 x 10(4) vs. 19.3 x 10(4); p = 0.20). Patients in Group A had higher numbers of circulating CD34+ cells on Day 10 (103.0/microL vs. 46.8/microL; p = 0.10) and on Day 11 (63.0/microL vs.14.3/microL; p = 0.04) than did patients in Group B. CONCLUSION: Administration of a single dose of amifostine before chemotherapy with TI mobilized higher numbers of CD34 cells in the circulation, but did not enhance the overall collection efficiency in the present trial.     

Prospective flow cytometric evaluation of nucleated red blood cells in cord blood units and relationship with nucleated and CD34(+) cell quantification

BACKGROUND: Cord blood (CB) represents an alternate source of stem cells in transplantation. Nucleated red blood cells (NRBCs) are a physiological subset of CB population. Although it is important to have an accurate estimate of CD34(+) cell number, NRBCs could compromise white blood cell count and interfere with CD34(+) cell quantification. STUDY DESIGN AND METHODS: A total of 826 CB units were analyzed for total nucleated cells (TNCs), NRBCs, and CD34(+) cells by flow cytometry. NRBCs were also counted conventionally by manual microscopy. Percentages of CD34(+) cells corrected by NRBC count (CD34+c) were determined as follows: %CD34+c = CD34(+)/CD45(+) (x10(6))/(TNCs (x10(8)) - NRBCs (x10(8))). RESULTS: The mean percentages of CD34+ cells and NRBCs were 0.27 percent (range, 0.01%-1.25%) and 7.64 percent (range, 0.13%-84%), respectively. Comparison between flow cytometric and microscopic NRBC count showed a regression of y = 0.685 + 0.719x and a coefficient of determination of r(2) = 0.721. When corrected with NRBC count, the mean percentage of CD34(+) c cells was 0.295 percent (p = 0.0008 compared with CD34(+)%) and mean TNCc count was 14.8 x 10(8) (p < 10(-4) compared to TNC count). CONCLUSION: The determination of NRBCs with a flow cytometric method might represent a new strategy for providing satisfactory quality assurance controls of CB products.     

Large-volume leukapheresis using femoral venous access for harvesting peripheral blood stem cells with the Fenwal CS 3000 Plus from normal healthy donors: predictors of CD34+ cell yield and collection efficiency

The current paper reports on the predicting factors associated with satisfactory peripheral blood stem cell collection and the efficacy of large-volume leukapheresis (LVL) using femoral vein catheterization to harvest PBSCs with Fenwal CS 3000 Plus from normal healthy donors for allogeneic transplantation. A total of 113 apheresis procedures in 57 patients were performed. The median number of MNCs, CD3+ cells, and CD34+ cells harvested per apheresis was 5.3 x 10(8)/kg (range, 0.3-11.0 x 10(8)/kg), 3.0 x 10(8)/kg (range, 0.2-6.6 x 10(8)/kg), and 7.9 x 10(6)/kg (range, 0.1-188.9 x 10(6)/kg), respectively. The median collection efficiency of MNCs and CD34+ cells was 49.8% and 49.7%, respectively. A highly significant correlation was found between the collected CD34+ cell counts and the pre-apheresis WBC counts in the donors (P = 0.013), and between the collected CD34+ cell counts and the pre-apheresis peripheral blood (PB) CD34+ cell counts (P<0.001). Harvesting at least >4 x 10(6)/kg CD34+ cells from the 1st LVL was achieved in 44 (77.2%) out of 57 donors and in 19 (90.5%) out of 21 donors with a PB-CD34+ cell count of >40/microl. There was no significant difference in the harvested MNC and CD34+ cell counts between the 1st and 2nd apheresis. The catheter-related complications included catheter obstruction (n = 2) and hematoma at the insertion site (n = 3). Accordingly, LVL using femoral venous access for allogeneic PBSC collection from normal healthy donors would appear to be safe and effective.     

Predictive parameters for granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization

To improve the selection of donors for allogeneic stem cell transplantation, it is important to identify reliable parameters that predict CD34+-cell yields after granulocyte-colony stimulating factor (G-CSF)-induced peripheral blood stem cell (PBSC) mobilization. We retrospectively investigated the peripheral blood (PB) kinetics of white blood cells (WBCs), CD34+ cells, matrix metalloproteinases (MMP)-9 and -2, and tissue inhibitors of metalloproteinases (TIMP)-1 and -2 in 15 healthy donors during their treatment with G-CSF. All donors received 10 microg/kg of recombinant human G-CSF once a day subcutaneously. Leukapheresis was initiated after 4 days of G-CSF treatment, and G-CSF treatment continued until the last day of leukapheresis. WBC and CD34+ cell numbers in the PB rose after 2 and 3 or 4 days of G-CSF treatment, respectively. The PB CD34+ cell numbers on day 4 correlated weakly with the increase in WBC counts from day 1 to day 2 (R(2) = 0.254, P = 0.056). There were also positive correlations between the CD34+ cell numbers in the PBSC products on day 4 and the CD34+ cells in the PB on days 1 and 4 (R(2) = 0.768, P < 0.0001 and R(2) = 0.816, P < 0.0005, respectively). The MMP-9 plasma levels on days 1 and 4 also correlated positively with the day 4 circulating CD34+ cell numbers (R(2) = 0.393, P < 0.05 and R(2) = 0.406, P = 0.01, respectively). In conclusion, the CD34+ cell numbers in the PB steady state may be a useful parameter selecting allogeneic PBSC donors.     

CD34+ cell enrichment for autologous peripheral blood stem cell transplantation by use of the CliniMACs device

Several devices for selection of CD34+ peripheral blood stem cells (PBSC) have been used during the last years for reducing tumor cell contamination of the graft. The new CliniMACS system (magnetic-activated cell separation system by Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany) was recently approved for clinical use in Europe. To evaluate its purging efficiency and engraftment data in the autologous transplant, PBSC from 28 adult patients with various malignant diseases (non-Hodgkin's lymphoma, n = 17; chronic lymphocytic leukemia, n = 5; multiple myeloma, n = 4; acute lymphocytic leukemia, n = 1; medulloblastoma, n = 1) were mobilized by chemotherapy and granulocyte colony-stimulating factor (G-CSF) (10 microg/kg per day). Thirty leukapheresis products from 28 patients with a median of 4.4 x 10(8) nucleated cells/kg body weight (bw)(range 0.6-10.8 x 10(8)/kg bw) and a median of 7.1 x 10(6) CD34+ cells/kg bw (range 2.8 to 18.8 x 10(6)/kg bw) were selected using the Cobe spectra cell separator (Cobe BCT Inc., Lakewood, CO). After the CliniMACS procedure, the median yield of CD34+ selected cells was 4.5 x 10(6)/kg (range 2.2-11.1 X 10(6)/kg bw) with a median recovery of 69.5% (range 46.9-87.3%) and a median purity of 97.7% (range 89.4-99.8%). The procedure did not alter viability of selected cells, which was tested by propidium iodide staining. So far, purified PBSC were used for autologous transplantation in 15 out of 28 patients after total body irradiation and/or high-dose chemotherapy. Median time to reach an absolute neutrophil count > 500/microl was 12 days (range 10-18 days), platelet recovery >50,000/microl occurred at day + 16 (range 11-22). With a median follow-up time of 12 months (range 3-19), 5 patients died of relapse. We confirmed the feasibility and safety of the CliniMACS CD34+ cell enrichment procedure in adult patients with autologous PBSC transplantation.     

Immune reconstitution after autologous selected peripheral blood progenitor cell transplantation: comparison of two CD34+ cell-selection systems

BACKGROUND: Selection of CD34+ PBPCs has been applied as a method of reducing graft contamination from neoplastic cells. This procedure seems to delay lymphocyte recovery, while myeloid engraftment is no different from that with unselected PBPC transplants. STUDY DESIGN AND METHODS: Lymphocyte recovery was studied in two groups of patients who underwent autologous CD34+ PBPC transplant with two different technologies (Ceprate SC, Cellpro [n = 17]; CliniMACS, Miltenyi Biotech [n = 13]). The median number of CD34+ cells transfused was 3.88 x 10(6) per kg and 3.32 x 10(6) per kg, respectively. Residual CD3 cells x 10(6) per kg were 4.97 and 0.58, respectively (p = 0.041). Residual CD19 cells x 10(6) per kg were 1.33 and 0.73, respectively (NS). RESULTS: No differences were found between the two groups in total lymphocyte recovery to >0.5 x 10(9) per L, which achieved a stable count by Day 30. During the study period, the CD4+ cell count remained below 0.2 x 10(9) per L, and the B-cell subset showed a trend toward normalization. CD3/HLA-DR+ and CD16/56 increased markedly in both groups by Day 30. An increase in CMV (13%) and adenovirus (17.4%) infection was found in both groups. CONCLUSION: Both CD34+ cell selection technologies used here determined an excellent CD34+ cell purity and an optimal depletion of T cells. The high rate of viral complications is probably due to the inability of residual T cells left from the CD34+ cell selection to generate, immediately after transplant, an adequate number of virus-specific lymphocytes.     

低剂量rhG-CSF对56例非血缘供者外周造血干细胞动员

本研究观察低剂量人重组粒细胞集落刺激因子(rhG-CSF)对非血缘健康供者的影响,探讨用于中华造血干细胞捐赠者资料库提供的非血缘健康供者外周造血干细胞动员方案。56例非血缘健康供者接受rhG-CSF 5μg/(kg.d)皮下注射,在动员第4、5两天或第5、6两天采集干细胞,观察动员效果及不良反应,检测动员前后血常规指标、CD3+、CD4+、CD8+和CD20+细胞比例;对采集物进行单个核细胞(MNC)和CD34+细胞计数;对所有供者随访至2006年5月31日。结果显示:在rhG-CSF动员过程中出现1级毒副作用(按WHO分级标准):腰背酸痛17.9%(10/56)、焦虑失眠8.9%(5/56)、疲乏4.5%(3/56)等,无需特殊处理,无需终止动员。第4、5两天采集和第5、6两天采集所得的MNC分别是(5.95±1.52)×108/kg和(7.19±2.12)×108/kg;CD34+细胞分别是(3.03±1.09)×106/kg和(7.92±2.50)×106/kg。血红蛋白水平、血小板量、CD3、CD4、CD8、CD20百分比动员前后无变化。结论:5μg/(kg.d)rhG-CSF用于非血缘健康供者的动员是安全而有效的。     

Hematopoietic progenitor cell large volume leukapheresis (LVL) on the Fenwal Amicus blood separator

A technique for large volume leukapheresis (LVL) for hematopoietic progenitor cell (HPC) collection using the Fenwal Amicus is presented. It was compared to standard collections (STD) with regard to CD34+ cell yields and cross-cellular content. Optimal cycle volumes and machine settings were evaluated for LVL procedures. A total of 68 patients underwent 80 HPC collection procedures. Because of differences in CD34+ cell yields associated with peripheral white blood cell counts (WBC), the comparison was divided into groups of 20 with WBC < or =35 x 10(9)/L (< or =35 K) and those >35 x 10(9)/L (>35 K). Baseline CD34+ cell counts (peripheral count when patient started HPC collection) were used (median 18-23 cells/microl). Significantly more whole blood (corrected for anticoagulant) was processed with LVL (LVL 20 l vs. STD 13.5 l). For < or =35 K, median CD34+ x 10(6), WBC x 10(9), RBC ml, Plt x 10(11) yields/collection were 183, 21.2, 14, 0.8, respectively, for STD vs. 307, 22.1, 11, 1.0, respectively, for LVL. For >35 K, median CD34+ x 10(6), WBC x 10(9), RBC ml, Plt x 10(11) yields/collection were 189, 32.7, 15, 1.4, respectively, for STD vs. 69, 40.8, 21, 1.3, respectively, for LVL. We have described a method of LVL using the Amicus that, in patients with pre-procedure WBC < or =35 x 10(9)/L, collects more CD34+ cells than a standard procedure with acceptable cross-cellular content. This method is not recommended when pre-procedure WBC counts are >35 x 10(9)/L.     

自体纯化CD34~+细胞移植治疗中/高危淋巴细胞来源恶性肿瘤的临床研究

目的分析自体纯化CD34+细胞移植治疗中/高危淋巴细胞来源恶性肿瘤的临床疗效。方法 10名中/高危组淋巴细胞来源恶性肿瘤患者行自体纯化CD34+细胞移植治疗,细胞分选采用cliniMACS系统。计算并统计分选纯度和CD34+细胞回收率,观察移植相关并发症及患者生存情况。结果纯化后CD34+细胞纯度为(87.79±3.73)%,回收率达到(65.74±10.37)%。10名患者全部顺利造血重建,感染发生率50%(5/10例),复发率为20%(2/10例)。结论利用CliniMACS系统进行外周血CD34+细胞分选,CD34+细胞纯度、回收率均满意,自体移植后造血功能重建顺利,近期疗效满意。     

人胚胎AGM区基质细胞在体外培养中对脐血CD34+细胞的支持作用

目的探讨人主动脉-性腺-中肾(AGM)区基质细胞对脐血CD34+细胞的造血支持作用.方法采用免疫磁珠法分离人脐血CD34+细胞,接种于底层已制备好的人AGM区基质细胞S1～S5饲养层的24孔板中,同时设无饲养层组及取自同期胚胎的躯干成纤维(hFT)细胞为对照,体外培养28 d,每7 d收获细胞并检测总数,流式细胞术检测CD34+、CD34+CD38细胞含量,并行造血细胞集落培养.结果在未加外源性造血生长因子的条件下,5株人AGM区基质细胞hAGMS1～S5对造血细胞总数、CD34+及CD34+CD38细胞、造血细胞集落均有不同程度的扩增及维持作用,与无饲养层组、hFT细胞组比较差异有统计学意义(P＜0.05).细胞总数在21 d达到峰值[扩增(25.13±4.83)倍],CD34+、CD34+CD38-细胞在扩增14 d达到峰值[(2.68±0.51)倍、(2.38±0.45)倍],高增殖潜能集落形成单位(HPP-CFU)亦在14 d达到峰值[(2.62±0.85)倍].hAGMS1～S5的造血支持作用组间比较差异亦有统计学意义(P＜0.05),其中hAGMS3、S4优于S1、S2及S5.结论人AGM区基质细胞对脐血CD34+细胞具有维持及扩增作用,特别是hAGMS3、S4两株细胞具有更好的维持作用,为胚胎早期造血发生机制和诱导胚胎干细胞造血分化研究提供了重要的模式细胞和基础资料.     

How do I perform hematopoietic progenitor cell selection?

Graft‐versus‐host disease remains the most important source of morbidity and mortality associated with allogeneic stem cell transplantation. The implementation of hematopoietic progenitor cell (HPC) selection is employed by some stem cell processing facilities to mitigate this complication. Current cell selection methods include reducing the number of unwanted T cells (negative selection) and/or enriching CD34+ hematopoietic stem/progenitors (positive selection) using immunomagnetic beads subjected to magnetic fields within columns to separate out targeted cells. Unwanted side effects of cell selection as a result of T‐cell reduction are primary graft failure, increased infection rates, delayed immune reconstitution, possible disease relapse, and posttransplant lymphoproliferative disease. The Miltenyi CliniMACS cell isolation system is the only device currently approved for clinical use by the Food and Drug Administration. It uses magnetic microbeads conjugated with a high‐affinity anti‐CD34 monoclonal antibody capable of binding to HPCs in marrow, peripheral blood, or umbilical cord blood products. The system results in significantly improved CD34+ cell recoveries (50%‐100%) and consistent 3‐log CD3+ T‐cell reductions compared to previous generations of CD34+ cell selection procedures. In this article, the CliniMACS procedure is described in greater detail and the authors provide useful insight into modifications of the system. Successful implementation of cell selection procedures can have a significant positive clinical effect by greatly increasing the pool of donors for recipients requiring transplants. However, before a program implements cell selection techniques, it is important to consider the time and financial resources required to properly and safely perform these procedures.     

Transfusion-associated iron overload as a predictive factor for poor stem cell mobilization in patients with haematological malignancies

Transfusion-associated iron overload is often observed in patients with haematological malignancies. We analysed the effect of iron overload, indicated by high serum ferritin level, on the mobilization of CD34(+) peripheral blood stem cells (PBSCs). We evaluated the association between the serum ferritin level prior to PBSC collection and the number of CD34(+) cells collected through leukapheresis in 51 patients with various haematological malignancies. Patients with serum ferritin level over 1000 ng mL(-1) were defined as high-ferritin group. Comparing the good (> or =1 x 10(6) per kg CD34(+) cells) and poor (<1 x 10(6) per kg CD34(+) cells) mobilizing groups, there was no difference in disease status, previous chemotherapies and white blood cell count at the first day of apheresis. However, there was a significant difference in the median units of red blood cell transfused between the good and poor mobilizer (2 vs. 8 units; P = 0.012). Serum ferritin level was notably higher in the poor mobilizer (1670 +/- 1320 ng mL(-1)) compared with the good mobilizer (965 +/- 705 ng mL(-1), P = 0.035). The cumulative number of CD34(+) cells per kg collected during the whole procedure was significantly lower in the high-ferritin group (5.5 +/- 4.7 x 10(6) per kg vs. 13.1 +/- 9.1 x 10(6) per kg, P = 0.01). Multivariate analysis revealed that serum ferritin level remained as an independent predictive factor for poor PBSC mobilization. Our study indicated that transfusion-associated iron overload is a predictive factor for poor PBSC mobilization. Iron chelation therapy prior to apheresis may be required to collect sufficient numbers of PBSCs in the iron overload patients.     

动员外周血CD34阳性细胞流式细胞术测定中的策略探讨

探索用流式细胞术测定动员外周血中CD34^ 细胞的简便有效的方法，以便在干细胞动员中进行有效的动态监测，及时收取干细胞，把握最佳移植效果，将经药物动员的移植供的外周血用CD34和CD45荧光抗体标记，用红细胞裂解液将红细胞裂解后经流式细胞仪检测，运用恰当的分析窗口，有效的设门分析结合干细胞的生物学特性，确定CD34^ 细胞在窗口中的确切位置，统计CD34^ 细胞在有核细胞中的比例，结果表明，通过本方法可有效地测定0.05%-0.1%的CD34^ 细胞的相对含量，在药物动员的第5或6天，多数患外周血中CD34^ 细胞达到峰值，某些患外周血中CD34^ 细胞可超过1%，结论：通过标记中的有效阻断和多参数分析，本方法可对动员外周血中微量的CD34^ 细胞进行有效的动态监测，对适时采集干细胞进行移植提供了重要的参考。