Semaphorin 7A inhibits platelet production from CD34+ progenitor cells

Background:  The multifunctional protein semaphorin 7A (Sema7A) may have regulatory effects on blood cell differentiation via its receptors β1‐integrin and plexin C1. As thrombocytopenia can be treated with transfusion of ex vivo CD34⁺ cell‐derived megakaryocytes, we investigated the effect of Sema7A on differentiation of CD34⁺ progenitor cells into megakaryocytes and platelets. Methods:  Megakaryocytes and platelets were differentiated with a specific cytokine cocktail (CC) from CD34⁺ progenitor cells in the presence or absence of Sema7A. Expression of cell markers CD41, CD42a and CD61 or detection of the activation of the signal mediator focal adhesion kinase (FAK) was performed by flow cytometry, cytokine secretion by Luminex technology, and megakaryocyte cell density and morphology by microscopic studies. Sema7A levels in vivo were assessed by real‐time PCR and ELISA in hematological patients undergoing chemotherapy. Results:  CD34⁺ progenitor cells expressed the receptors for Sema7A. Expression of CD41, CD42a and CD61 was markedly reduced in the presence of Sema7A, after CC‐dependent platelet production from CD34⁺ progenitor cells. As revealed by microscopic analysis, megakaryocyte cell density was significantly lower in the presence of Sema7A as compared with controls. Blocking of CD29 abrogated the Sema7A‐mediated inhibition. Sema7A activated FAK in CD34⁺ progenitor cells and significantly increased secretion of the proinflammatory cytokines IL‐6, IL‐8 and GM‐CSF. Finally, Sema7A levels were up‐regulated in 50% of patients after chemotherapy. Conclusions:  Sema7A markedly reduces the production rates of megakaryocytes and platelets from CD34⁺ progenitor cells. Hence, up‐regulation of Sema7A may be a major risk factor for a reduced platelet repopulation after hematopoietic stem cell transplantation.     

Albumin-based solution is the ideal post-thawing suspension medium for cord blood hematopoietic stem cells: A stability and proliferative evaluation

Background

Cryopreservation and thawing protocols represent key factors for the efficacy of cellular therapy products, such as hematopoietic stem cells (HSCs). While the HSC cryopreservation has already been standardized, the thawing procedures have been poorly studied. This study aimed to evaluate the thawing and washing protocol of cord blood (CB) derived HSCs or the HPC(CB), by selecting the optimal thawing solution and determining CD34+ cells' stability over time.

Study Design and Methods

Seven cryopreserved CB products were thawed, washed, and resuspended in three different solutions (10% Dextran40 in NaCl equally prepared with 5% human albumin; 5% human albumin in PBS/EDTA; and normal saline) and stored at 4°C (±2°C). Mononuclear cell (MNC) count, CD45+/CD34+ cell enumeration, and cell viability were tested at 0, 1, 2, 4, 6, 8, 12, 24, 36, and 48 h. The protocol with the selected solution was further validated on additional 10 CB samples. The above parameters and the colony-forming unit (CFU) assay were analyzed at time points 0, 2, 4, 6, and 8 h.

Results and Discussion

The results showed that the 5% human albumin was the most suitable thawing solution. MNCs were stable up to 4 h (p = 0.009), viable CD45+ cells were unstable even at 2 h (p = 0.013), and viable CD34+ cells were stable until 6 h (p = 0.019). The CFU assay proved the proliferative potential up to 8 h, although significantly decreased after 4 h (p = 0.013), and correlated with the viable CD34+ cell counts. We demonstrated that the post-thawed and washed HPC(CB) using 5% human albumin is stable for up to 4 h.     

Autologous peripheral blood progenitor cell transplantation

Harvesting of autologous peripheral blood stem cells (PBSCs) has been facilitated by using currently available, efficient apheresis technology at the time of rebound from chemotherapy while patients are receiving recombinant growth factors, i.e., granulocyte (G) or granulocyte-macrophage (GM) colony stimulating factor (CSF). Ideally pheresis should be done before patients have had extensive stem cell toxins, i.e., alkylating agents or nitrosoureas. This strategy has facilitated the use of high dose chemoradiotherapy given as a single regimen or in a divided dose for patients with solid tumors or hematologic malignancies and results in more rapid engraftment than bone marrow transplantation (BMT). Although mere are no assays which measure repopulating stem cells, enumeration of CD34⁺ cells within PBSCs is a direct and rapid assay which provides an index of both early and late long-term reconstitutive capacity, since it correlates with colony-forming unit (CFU)-GMs, as well as pre-progenitor or delta assays and long-term culture-initiating cells (LTC-IC). A threshold of ≥2 × 10⁶ CD34⁺ cells/kg recipient body weight has been reported to be required for engraftment, but may vary depending upon the clinical setting. Strategies for mobilization of normal PBSCs also increase tumor cell contamination within PB in the setting of both hematologic malignancies and solid tumors, but the significance of these tumor cells in terms of patient outcome is unclear. Recently isolation of CD34⁺ cells from PBSCs has been done using magnetic beads or immunoabsorption on columns or rigid plates in order to enrich for normal hematopoietic progenitors and potentially decrease tumor cell contamination. As for other cellular blood components, standards have been developed to assure efficient collection and processing, thawing, and reinfusion, and to maintain optimal PBPC viability. Finally, future directions of clinical research include expansion of hematopoietic progenitor cells ex vivo; use of umbilical cord or placenta as rich sources of progenitor cells; syngeneic hematopoietic stem cell transplantation; related and unrelated allogeneic hematopoietic stem cell transplantation; treatment of infections, i.e., Epstein Barr virus, or tumor relapse after allogeneic BMT using donor PBSC infusions; and gene therapy approaches.     

中青年原发性高血压患者循环内皮祖细胞CD34+水平与颈动脉粥样硬化的相关性研究

目的 探讨中青年原发性高血压患者循环内皮祖细胞(EPCs)CD34+水平与颈动脉内膜中层厚度(IMT)和Framingham心血管危险因素积分标准(FRFC)的相关性及其评估高血压患者早期血管病变的价值.方法选择62例年龄25～45岁原发性高血压患者(高血压组)及20例健康体检者(健康对照组).高血压组患者采用FRFC分层方法分为低危组18例,中危组14例,高危组17例,极高危组13例.测定各组的外周循环EPCs CD34+水平及颈动脉IMT,并对EPCs CD34+水平与FRFC积分及颈动脉IMT进行相关性分析.结果 高血压各亚组患者外周循环EPCs CD34+水平随心血管危险程度的增加逐步下降[低危组(0.12±0.02)%,中危组(0.07±0.03)%,高危组(0.04±0.03)%,极高危组(0.01±0.01)%],各组间比较差异有统计学意义(P＜0.05或P＜0.01),且均明显低于健康对照组[(0.15±0.03)%,均P＜0.01];颈动脉IMT随心血管危险程度增加明显增厚[低危组(0.80±0.07)mm,中危组(1.11±0.08)mm,高危组(1.26±0.10)mm,极高危组(1.45±0.09)mm],各组间比较差异有统计学意义(P＜0.05或P＜0.01),且与健康对照组[(0.73±0.08)mm]比较差异有统计学意义(均P＜0.01).高血压患者外周循环EPCs CD34+水平与FRFC积分呈负相关(r=-0.875,P＜0.01),与颈动脉IMT呈负相关(r=-0.852,P＜0.01).结论 中青年原发性高血压患者循环EPCs CD34+水平与心血管危险因素及颈动脉IMT呈负相关;外周循环EPCs CD34+水平可以作为评估高血压患者早期血管病变的标志之一.     

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.     

中青年原发性高血压患者循环内皮祖细胞CD34+水平与颈动脉粥样硬化的相关性研究

Objective To explore the relationship between the level of circulating endothelial progenitor cells (EPCs) CD34+with the Framingham cardiovascular risk factors, or with the carotid artery intima-madia thickness (IMT), and to evaluate the value of circulating EPCs CD34+level as a cytologicalmarker of early vascular lesion in youth and middle aged essential hypertension (EH) patients.Methods A total of 62 patients with EH aged between 25 to 45 were enrolled as study group and 20 healthy people were enrolled as control group.EH patients were stratified with cardiovascular risk factors according to Framingham risk factors score into low-risk group with 18 cases, mid-risk group with 14 cases, high-risk group with 17 cases, and extremely high-risk group with 13 cases.The level of circulating EPCs CD34+,carotid artery IMT were respectively measured.The relationship between the level of circulating EPCsCD34+ and Framingham cardiovascular risk factors score, carotid artery IMT was analyzed.Results The level of circulating EPCs CD34+ was gradually decreased with an increase of the Framingham risk factors score in each hypertensive subgroup [low-risk group:(0.12±0.02)%, mid-risk group:(0.07±0.03)%,high-risk group:(0.04±0.03)%, extremely high-risk group:(0.01±0.01)%], and they were significantly lower than that in control group [(0.15±0.03)%], and there was a significant difference among hypertensive subgroups (P＜0.05 or P＜0.01).Carotid artery IMT was significantly thicker among hypertensive subgroups [low-risk group:(0.80±0.07)mm, mid-risk group:(1.11±0.08)mm, high-risk group: (1.26±0.10)mm, extremely high-risk group:(1.45±0.09)mm], and there was a significant difference between each hypertensive group and that of control group [(0.73±0.08)mm, all P＜0.01].There was also statistical significance among hypertensive subgroups(P＜0.05 or P＜0.01).There was a negative correlation between the level of circulating EPCs CD34+and Framingham risk factors score (r=-0.875, P＜0.01), and also a negative correlation with carotid artery IMT (r=-0.852, P＜0.01).Conclusion There was a significant correlation between the level of circulating EPCs CD34+with Framingham risk factors score and also carotid artery IMT in EH patients.Circulating EPCs CD34+could be a cytological marker of early vascular lesion in hypertension patients.     

CD41+ and CD42+ hematopoietic progenitor cells may predict platelet engraftment after allogeneic peripheral blood stem cell transplantation

The objective of this study was to quantify subpopulations of CD34+ cells such as CD41+ and CD42+ cells that might represent megakaryocyte (MK) precursors in peripheral blood stem cell (PBSC) collections of normal, recombinant human granulocyte‐colony stimulating factor (rhG‐CSF) primed donors and to determine whether there is a statistical association between the dose infused megakaryocytic precursors and the time course of the platelet recovery following an allogeneic PBSC transplantation. Twenty‐six patients with various hematologic malignancies transplanted from their HLA identical siblings between July 1997 and December 1999 were used. All patients except one with severe aplastic anemia who had cyclophosphamide (CY) alone received busulfan‐CY as preparative regimen and cyclosporine‐methotrexate for GVHD prophylaxis. Normal healthy donors were given rhG‐CSF 10 μg/kg/day subcutaneously twice daily and PBSCs were collected on days 5 and 6. The median number of infused CD34+, CD41+ and CD42+ cells were 6.61 × 10⁶/kg (range 1.47–21.41), 54.85 × 10⁴/kg (5.38–204.19), and 49.86 × 10⁴/kg (6.82–430.10), respectively. Median days of ANC 0.5 × 10⁹/L and platelet 20 × 10⁹/L were 11.5 (range 9–15) and 13 (8–33), respectively. In this study, the number of CD41+ and CD42+ cells infused much better correlated than the number of CD34+ cells infused with the time to platelet recovery of 20 × 10⁹/L in 26 patients receiving an allogeneic match sibling PBSC transplantation (r = ?0.727 and P < 0.001 for CD41+ cells, r = ?0.806 and P < 0.001 for CD42+ cells, r = ?0.336 and P > 0.05 for CD34+ cells). There was an inverse correlation between the number of infused CD41+ and CD42+ cells and duration of platelet engraftment. Therefore, as the number of CD41+ and CD42+ cells increased, duration of platelet engraftment (time to reach platelet count of ≥ 20 × 10⁹/L) shortened significantly. Based on this data we may conclude that flow cytometric measurement of CD41+ and CD42+ progenitor cells may provide an accurate indication of platelet reconstitutive capacity of the allogeneic PBSC transplant. J. Clin. Apheresis. 16:67–73, 2001. © 2001 Wiley‐Liss, Inc.     

中青年原发性高血压患者循环内皮祖细胞CD34+水平与颈动脉粥样硬化的相关性研究

Objective To explore the relationship between the level of circulating endothelial progenitor cells (EPCs) CD34+with the Framingham cardiovascular risk factors, or with the carotid artery intima-madia thickness (IMT), and to evaluate the value of circulating EPCs CD34+level as a cytologicalmarker of early vascular lesion in youth and middle aged essential hypertension (EH) patients.Methods A total of 62 patients with EH aged between 25 to 45 were enrolled as study group and 20 healthy people were enrolled as control group.EH patients were stratified with cardiovascular risk factors according to Framingham risk factors score into low-risk group with 18 cases, mid-risk group with 14 cases, high-risk group with 17 cases, and extremely high-risk group with 13 cases.The level of circulating EPCs CD34+,carotid artery IMT were respectively measured.The relationship between the level of circulating EPCsCD34+ and Framingham cardiovascular risk factors score, carotid artery IMT was analyzed.Results The level of circulating EPCs CD34+ was gradually decreased with an increase of the Framingham risk factors score in each hypertensive subgroup [low-risk group:(0.12±0.02)%, mid-risk group:(0.07±0.03)%,high-risk group:(0.04±0.03)%, extremely high-risk group:(0.01±0.01)%], and they were significantly lower than that in control group [(0.15±0.03)%], and there was a significant difference among hypertensive subgroups (P＜0.05 or P＜0.01).Carotid artery IMT was significantly thicker among hypertensive subgroups [low-risk group:(0.80±0.07)mm, mid-risk group:(1.11±0.08)mm, high-risk group: (1.26±0.10)mm, extremely high-risk group:(1.45±0.09)mm], and there was a significant difference between each hypertensive group and that of control group [(0.73±0.08)mm, all P＜0.01].There was also statistical significance among hypertensive subgroups(P＜0.05 or P＜0.01).There was a negative correlation between the level of circulating EPCs CD34+and Framingham risk factors score (r=-0.875, P＜0.01), and also a negative correlation with carotid artery IMT (r=-0.852, P＜0.01).Conclusion There was a significant correlation between the level of circulating EPCs CD34+with Framingham risk factors score and also carotid artery IMT in EH patients.Circulating EPCs CD34+could be a cytological marker of early vascular lesion in hypertension patients.     

人羊膜上皮细胞的干细胞特性

背景:研究发现人羊膜上皮细胞具有类似胚胎干细胞或多能干细胞的多向分化潜能,说明其可能是未来组织工程重建的一种新型种子细胞.目的:研究体外培养的人羊膜上皮细胞的干细胞特性.方法:取足月剖宫产的人羊膜组织,经酶消化法和差异黏附法获得纯度高的人羊膜上皮细胞,接种于含体积分数为10%胎牛血清的DMEM/F12培养基中进行原代和传代培养,用免疫荧光法和流式细胞仪检测法检测人羊膜上皮细胞表面胚胎干细胞的表面标记蛋白OCT-4和干细胞表面分子标记CD29、CD34、CD44、CD45、CD105的表达.结果与结论:人羊膜上皮细胞在体外培养条件下呈上皮细胞特有的铺路石样外观,其胞浆中有OCT-4免疫荧光表达,其干细胞标记分子CD29、CD34的表达是阳性,但干细胞标记分子CD44、CD45、CD105的表达为阴性.结果表明人羊膜上皮细胞具有干细胞的某些特性,其可能是未来组织工程重建的一种新型种子细胞.     

Prospective,randomized, sequential,crossover trial of large‐volume vs. normal‐volume leukapheresis procedures: Effects on subpopulations of CD34+ cells

Some data exist on the influence of leukapheresis volume on the number of harvested peripheral blood hematopoietic progenitor cells (HPC), but less is known about the influence on the composition of HPC. We therefore performed a prospective, randomized crossover trial to evaluate the effect of large‐volume (LVL) vs. normal‐volume leukapheresis (NVL) on subpopulations of CD34⁺ cells in the harvest product of 15 patients with breast cancer and 8 patients with non‐Hodgkin's lymphoma. Patients were randomly assigned to start either with an LVL on day 1 followed by an NVL on day 2 or vice versa. The number of HPC, the extraction efficiency defined as difference between yield in the harvest and decrease in peripheral blood, and the relative proportion as well as the absolute numbers of CD34⁺ cells coexpressing CD38, CD90, HLA‐DR, CD117, CD7, CD19, CD41, or CD33 were evaluated. There was no significant difference with regard to the percentages of the subsets on comparison of LVL to NVL procedures. Only the absolute median number of CD34⁺HLA‐DR^? cells was significantly (P=0.02) higher in LVL harvests compared with the corresponding NVL components, which can be explained on the basis of the higher yield and the higher extraction efficiency in LVL compared with NVL. LVL results in a higher yield of CD34⁺ cells and leads to an intra‐apheresis recruitment of HPC but the relative composition of the harvested CD34⁺ cells is not changed significantly. In addition, the amount of early, HLA‐DR^?, hematopoietic HPC seems to be increased by an LVL. J. Clin. Apheresis. 16:109–113, 2001. © 2001 Wiley‐Liss, Inc.     

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.     

Collection of peripheral progenitor cells in paediatric patients with a new programme for the collection of mononuclear cells

When harvesting peripheral progenitor cells (PPC) in children, the special situation of their circulatory system has to be taken into account. Therefore, extracorporeal blood volume and product volume should be small to avoid side effects. Nine children (age 2-14 years, weight 12.8-58.5 kg) with malignancies underwent 10 PPC collections with the MNC programme of the Amicus blood cell separator. The disposable kit was primed with red blood cells (RBCs) or human albumin to avoid circulatory side effects. The children were monitored for blood pressure and heart rate during the whole apheresis procedure. A median blood volume of 4,577 ml (range 3,536-8,596 ml) was processed in a separation time of 270 min (range 176-331 min). The median product weight was 81 g (range 53-107 g) and the yield of CD 34 antigen expressing cells was 12.5 x 10(6)/kg body weight (range 1.8-26 x 10(6)/kg body weight). Only one child had to undergo a second apheresis to collect the desired transplantation dose. The median platelet contamination of the product was 0.32 x 10(11) (0.13-0.85 x 10(11)). No circulatory side effects were observed. Blood flow alarms occurred in seven of ten aphereses and one collection had to be terminated due to insufficient flow. PPC can be efficiently collected in children with the MNC programme without circulatory side effects. The platelet contamination of the product was low due to the elutriation principle of the collection process, thereby avoiding thrombocytopenic bleeding episodes in the patients.     

不同标记法及去红细胞法对检测微量脐血CD34+造血干细胞含量的影响

目的 采用ISHAGE(international society of hematotherapy and graft engineering)法和常用的CD34^ 造血干细胞流式检测方法对同一脐血样品进行比较实验，确定更适用于微量脐血CD34 ^ 造血干细胞含量检测的方法。方法 分别采用ISHAGE造血干细胞计数协会推荐方法、低渗NH4C1去红细胞CD45／CD34双色标记法(NH4C1双标法)、OptilyseC溶血剂去红细胞CD34单色标记法(OptilyseC单标法)、低渗NH4C1去红细胞CD34单色标记法(NH4C1单标法)、羟乙基淀粉沉淀去红细胞CD45／CD34双色标记法(HES双标法)检测脐血造血干细胞含量，并比较不同方法测得数据的差异。结果 8份脐血标本用ISHAGE方法测得CD34^ 造血干细胞含量的中位数为0．278％，NH4C1双标法测得结果为0．297％，与ISHAGE方法相比差异无显性。用HES双标、OptilyseC单标法和NH4C1单标法测得的结果分别为5．715％，0．391％和0．741％，与ISHAGE方法相比差异有显性。结论 ISHAGE方法是一种公认的准确性高，重复性好的检测造血干细胞方法。OptilyseC单标法、NH4C1单标法和HES双标法测定结果比实际值偏高。     

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.     

Assessment of stability of CD34+ cell products enriched by immunoselection from peripheral blood mononuclear cells during refrigerated storage

Durable engraftment of transplanted CD34+ cells largely depends on the quality of the cell product. Limited data are currently available about extended storage of immunoselected CD34+ cells. The aim of our study was to assess the stability of CD34+ cell product with the cells stored in high concentration (80 × 10⁶ in 6 mL) in small bags intended for cell implantation. Cell products were prepared by leukapheresis and immunoselection (Clinimacs^plus procedure) from 13 patients with chronic dilated cardiomyopathy. CD34+ cell products were stored at 2–8 °C and analyzed at time 0 (fresh products), 24, 48 h, 4 and 6 days. Product viability, absolute number of viable CD34+ cells and apoptosis were determined by flow cytometry. Microbiological contamination of the cell products was tested by BACTEC system. The mean viability of CD34+ cells decreased by 2.7% within 24 h, by 13.4% within 48 h and by 37.5% within 6 days. The mean recovery of viable CD34+ cells was 91.1%, 74.8%, 66.3% and 56.2% at 24, 48 h, 4 and 6 days, respectively. The mean fraction of early apoptotic cells in fresh and stored products was 4.9 ± 3.5% at 0 h, 5.9 ± 3.8% at 24 h, 4.2 ± 3.1% at 48 h, 6.3 ± 2.6% at 4 days and 9.3 ± 4.6% at 6 days. All products were negative for microbial contamination.     

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.     

人脐血单个核贴壁细胞与胃癌细胞的共培养:最适效靶比筛选

背景:近年来大量研究关注脐血细胞可能对肿瘤细胞有杀伤和抑制作用,人们尝试利用脐血细胞移植抑制和杀伤肿瘤细胞,这为临床医生们提供了抗癌的新途径.目的:探讨人脐血单个核细胞与肿瘤细胞体外共培养后,对肿瘤细胞的抑制作用.方法:采用密度梯度离心的方法分离人脐血单个核细胞,流式检测细胞表面Marker表达,将单个核细胞与胃癌细胞进行共培养,倒置显微镜观察两种细胞共培养后细胞形态的变化,采用了乳酸脱氢酶法检测对肿瘤细胞的抑制作用,找出抗肿瘤细胞效果最好的效靶比.结果与结论:加入脐血的贴壁单个核细胞贴壁细胞2 d后,肿瘤细胞的脱落明显,贴壁细胞实验组肿瘤细胞排列不规整,细胞密度低.酸脱氢酶法显示部分脐血贴壁细胞确有较强的抗肿瘤能力,但个体间抗肿瘤的能力差异较大,最大可相差4倍.脐血单个核贴壁细胞对肿瘤细胞有直接的杀伤作用,其最适的效靶比为2:1.     

Rescue stem cell mobilization with plerixafor economizes leukapheresis in patients with multiple myeloma

While extensive data demonstrated that plerixafor improves stem cell harvest in difficult‐to‐mobilize patients, economic concerns limit a broader application. We retrospectively assessed the effect of an early plerixafor rescue regimen for mobilization in patients with multiple myeloma. Patients were intended for high‐dose chemotherapy followed by autologous peripheral blood stem cell transplantation (ABSCT) and therefore received cyclophosphamide‐based mobilization chemotherapy and consecutive stimulation with granulocyte colony‐stimulating factor (G‐CSF). Fifteen patients with poor stem cell harvest in the first leukapheresis session received plerixafor. Data were compared with a matched historic control group of 45 patients who also had a poor stem cell yield in the first apheresis session, but continued mobilization with G‐CSF alone. Patients in the plerixafor group collected significantly more CD34+ cells in total (median 4.9 vs. 3.7 [range 1.6–14.1 vs. 1.1–8.0] × 10⁶ CD34+ cells /kg bw; P < 0.05), and also more CD34+ cells per leukapheresis procedure (P < 0.001). Consequently, they required a significantly lower number of leukapheresis procedures to achieve the collection goal (median 2.0 vs. 4.0 [range 2–3 vs. 2–9] procedures; P < 0.001). The efficiency of the collected stem cells in terms of hematologic engraftment after ABSCT was found to be equal in both groups. These data demonstrate that rescue mobilization with plerixafor triggered by a low stem cell yield in the first leukapheresis session is effective. Although the actual economic benefit may vary depending on the local leukapheresis costs, the median saving of two leukapheresis procedures offsets most of the expenses for the substance in this setting. An exemplary cost calculation is provided to illustrate this effect. J. Clin. Apheresis 29:299–304 2014. © 2014 Wiley Periodicals, Inc.     

健康供者特征对rhG-CSF预激的骨髓采集物CD34+细胞和T细胞亚群的影响

目的 探讨健康供者特征对rhG-CSF预激的骨髓(G-BM)采集物造血和免疫细胞成分的影响.方法 150名健康供者体内应用rhG-CSF 5 μ·kg-1·d-1连续4～5 d,第4,5天采集骨髓和外周血,用流式细胞术测定G-BM采集物中的CD3+、CD3+CD4+、CD3+CD8+、CD14+、CD34+细胞以及CD3+CD4-CD8-调节性T细胞的数量,并分析供者性别、年龄、体重、是否妊娠等特征对G-BM成分的影响.结果 150名健康供者G-BM所含有核细胞,淋巴细胞,CD3+、CD3+CD4+、CD3+CD8+、CD14+、CD34+细胞以及CD3+CD4-CD8-调节性T细胞的中位值分别为31 050(12 200～58 100)、2122(506～6618)、1344(307～4791)、692(145～3038)、616(112～2575)、986(265～2958)、63(11～505)和83(9～390)/μl.年龄≤38岁的供者G-BM中的有核细胞、CD34+细胞以及CD3+ CD4-CD8-调节性T细胞的数量分别为33800(18600～57100)、76(22～505)和97(11～380)/μl,显著高于年龄＞38岁的供者[分别为28 000(12 200～58 100)、49(11～220)和65(9～389)/μl],P值分别为＜0.05,＜0.001和0.001.外周血单核细胞计数＞0.37×109 /L的供者G-BM中有核细胞的数量[33550(12 200～57 100)/μl]显著高于≤0.37×109 /L的供者[27 150(13 800～58 100)/μl](P=0.005).多因素分析显示年龄和外周血单核细胞是G-BM中有核细胞采集量的影响因素[HR值分别为0.41和2.87;P值分别为0.01和0.003];年龄是G-BM中CD34+细胞采集量的唯一影响因素(HR=0.26;P值=0.001);年龄还是G-BM中CD3+ CD4-CD8-调节性T细胞数量的影响凶素[HR值为0.31;P=0.001].结论 年龄是影响G-BM中有核细胞、CD34+细胞和CD3+ CD4-CD8-调节性T细胞采集数量的因素,年龄≤38岁的供者更易采集满足临床需要的有核细胞和CD34+细胞;外周血单核细胞＞0.37×109 /L的供者采集的G-BM含有较多数量的有核细胞.