不同来源CD34^＋CD38^＋和CD34^＋CD38^—细胞群造血性能的不均一…

利用ＦＡＣＳ将ＣＤ３４＾＋细胞分选为ＣＤ３４＾＋ＣＤ３８＾－两个亚群，并比较了来源于人脐带血、骨髓及外周血两个亚群的分布比例和造血性能的差异。结果有髓中ＣＤ３４＾＋及其亚群细胞所占比例最高，外周血最低；脐带血来源的亚群细胞体外形成各系造血集落及液体培养体系中维持长期造血的能力最强，外周血最弱。     

胎盘造血功能的初步研究

目的 探讨胎盘在整个胎儿时期造血中的作用,寻找造血干／祖细胞的新来源,供临床移植应用。方法 分别测定脐动脉和脐静脉血中ＣＤ３４＾＋细胞,粒－巨噬泵细胞集落（ＣＦＵ－ＧＭ０及造智力在子含量,胎盘绒毛膜组织切片采用ＨＥ染色和ＣＤ３４单抗免疫组化染色。结果 脐静脉血中ＣＥ３４＾＋、ＣＤ３４＾＋／ＣＤ３８＾－细胞及ＣＦＵ－ＧＭ含量均高于脐动脉,脐静脉血中含有较多的造血刺激因子,而造血抑制因子的含量与肮动脉     

人参皂甙对人骨髓造血干细胞的增殖作用

目的：探讨人参皂甙（ＣＳ）对人骨髓ＣＤ３４＾＋造血干细胞的增殖作用。方法：采用Ｄｙｎａｌ Ｍ４５０ ＣＥ３４＾＋免疫磁珠分离法阳性选择获取高纯度的人骨髓ＣＳ３４＾＋造血干细胞,应用多向细胞（ＣＦＵ－Ｍｉｘ）体外培养技术,观察ＣＤ３４＾＋造血干细胞对ＣＳ的敏感性。结果：１经Ｄｙｎａｌ Ｍ０４５０ＣＤ３４＾＋免疫磁珠分离获得骨３髓ＣＤ３４＾＋细胞占骨髓单个核细胞（ＭＮＣ）的０．５２－１．３０％,Ｃ３４     

单个CD34^＋CD38^＋和CD34^＋CD38^—l细胞的增殖与分化性能

为深入探讨ＣＤ３４＾＋细胞亚群的不均一性及单个细胞的增殖分化特性，利用ＦＡＣＳ Ｖａｎｔａｇｅ自动细胞分选系统（ＡＣＤＵ）分选出单个ＣＤ３４＾＋亚群细胞：ＣＤ３４＾＋ＣＤ３８＾＋和ＣＤ３４＾＋ＣＤ３８＾－，在９６孔板无血清培养体系中，经ＳＣＦ、ＩＬ－３、ＩＬ－６、ＧＭ－ＣＳＦ、Ｇ－ＣＳＦ和Ｅｐｏ刺激，１４－１８天后扩增形成原代集藩。单个ＣＤ３４＾＋ＣＤ３８＾＋细胞形成原代集落的比例为３８．０％±９     

不同来源CD_（34）~＋CD_（38）~＋和CD_（34）~＋CD_（38）~－细胞群造血性能的不均一性研究

利用ＦＡＣＳ将ＣＤ３４＋细胞分选为ＣＤ３４＋ＣＤ３８＋和ＣＤ３４＋ＣＤ３８－两个亚群，并比较了来源于人脐带血、骨髓及外周血两个亚群的分布比例和造血性能的差异。结果表明骨髓中ＣＤ３４＋及其亚群细胞所占比例最高，外周血最低；脐带血来源的亚群细胞体外形成各系造血集落及液体培养体系中维持长期造血的能力最强，外周血最弱。从而表明不同细胞亚群造血性能不均一，同一亚群不同来源的细胞也同样具有不均一性。     

不同冻存保护剂对脐血造血细胞生物学特性的影响

目的 探索有效低温保存脐血造血细胞的冻存保护剂。方法 对比４种不同的联合低温保护地脐血有核细胞（ＴＮＣ）的保护作用，并在冻存后１，２，３及４个月分别检测其复苏后脐血ＣＤＥ３４＾＋细胞及集落形成细胞（ＣＦＣ）数。结果 ４种不同联合的低温保护剂对脐血ＴＮＣ，ＣＤ３４＾＋细胞及ＣＦＣ的保护作用判别显，依次为右旋糖酐－４０（Ｄｅｘｔｒａｎ－４０）＋二甲基亚砜（ＤＭＳＯ）〉生理盐水＋ＤＭＳＯ〉ＤＭＳＯ＋自     

肥胖儿童红细胞膜变化与血压的关系

目的：探讨细胞膜结构和功能改变在肥胖引起儿童高血压发病机制中的作用。方法：测定了１０４例「正常对照（ＮＴ）３７例,正常血压肥胖（ＮＯＴ）３４例,高血压肥胖（ＯＨＴ）３３例」１２～１６岁中学儿童的Ｎａ＾＋－ｋ＾＋－ＡＴＰ酶和Ｃａ＾２＋－ＡＴＰ酶活性、膜Ｃａ＾２＋结合力和磷脂含量。结果：ＯＮＴ组和ＯＨＴ组的Ｎａ＾＋－Ｋ＾＋－ＡＴＰ酶和Ｃａ＾２＋－ＡＴＰ酶活性及膜Ｃａ＾２＋结合力显著低于ＮＴ组,ＯＨＴ组     

紫外线照射自血回输对脑梗塞患者外周血淋巴细胞亚群和血浆纤维连…

对２４例脑梗塞患者紫外线照射自血回输（ＵＢＩＯ）治疗前、后外周血淋巴细胞亚群及血浆纤维连接蛋白（Ｆｎ）含量变化进行了观察。结果ＵＢＩＯ治疗前脑梗塞患者外周血中总Ｔ细胞（ＣＤ３＾＋）、Ｔ抑制细胞（ＣＤ８＾＋）数均较正常人明显减少，ＣＤ４／ＣＤ８比值上升；Ｂ＾＋和人类白细胞抗原（ＨＬＡ－ＤＲ＾＋）细胞数增多；白细胞介素ＩＩ受体（ＩＬ－２Ｒ＾＋）和Ｔ辅助（ＣＤ＾＋）细胞数则与正常人无差异。血浆Ｆｎ含量低     

HcFU对进展期胃癌患者T淋巴细胞亚群的影响

我们采用间接免疫荧光法检测了２１例进展有期胃癌患者低剂量化疗药物ＨｃＦＵ诱导前后的Ｔ淋巴细胞亚群，结果发现，与正常对照组相比，进展期胃癌患者ＣＤ３＾３＋，ＣＤ４＾＋细胞明显减少，ＣＤ８＾＋细胞增多，ＣＤ４／ＣＤ８比值显著下降。而在ＨｃＦＵ诱导后ＣＤ３＾３＋、ＣＤ４＾＋细胞增多，ＣＤ８＾＋细胞减少，ＣＤ４／ＣＤ８比值升高，提示低剂量化疗药物ＨｃＦＵ可改善进展期胃癌患者的细胞免疫抑制状态。     

CD34—造血干细胞的研究进展

造血干细胞（ＨＳＣｓ）的表型一直是研究的重要课题。一般认为：ＣＤ３４＾＋是骨髓ＨＳＣｓ的标志之一,但近年研究发现：存在－ＣＤ３４＾－ＨＳＣｓ群,它能长期重建造血并可分化主国ＣＤ３４＾＋细胞,可能是ＣＤ３４＾＋细胞的前身细胞。本对ＣＤ３４＾－ＨＳＣｓ从发现、含量、表面分子表达、体外体内生物学特性及体外培养扩增条件方面的研究进展等作一综述。     

Comparison of volumetric capillary cytometry with standard flow cytometry for routine enumeration of CD34+ cells

BACKGROUND: This study assesses the feasibility of a new volumetric cytometry system for the enumeration of CD34+ cells in apheresis components, peripheral blood, and cord blood samples in routine laboratory work. This system is compared with the following flow cytometry protocols: Milan, ISHAGE, ISHAGE with 7-AAD, and flow-count fluorospheres. STUDY DESIGN AND METHODS: Correlation, linearity, and reproducibility studies were performed for the various methods. Clonogenic cultures were performed, as an external control, to assess the correlation between the number of CD34+ cells per microL and the number of colony-forming units per microL. RESULTS: The linear regression analysis demonstrated that the five methods were comparable (R2 ranged from 0.86 to 0.96 and slopes were close to 1). The CD34+ assay and the flow-count methods showed poor linearity for CD34+ cell counts below 10 cells per microL (R2 = 0.46 and 0.47). The reproducibility assay for a CD34+ count of 10 cells per microL showed a CV of 12 percent and 25 percent for the Milan and CD34+ assay methods, respectively. The mean CV among all five methods for the 46 evaluated samples was 20 percent. There was a strong correlation between the number of CD34+ cells per microL and colony-forming units per microL in cord blood and apheresis samples (r = 0.71-0.81). CONCLUSION: The CD34+ assay is useful in CD34 enumeration in cord blood, leukapheresis samples, and peripheral blood samples and provides comparable results to the Milan, ISHAGE, ISHAGE with 7-AAD, and flow-count methods. Nevertheless, peripheral blood samples with low CD34 absolute counts (below 10 cells/microL) should be analyzed by alternative flow cytometry protocols. Even though the same operator performed the study in a single laboratory, the high inter-method CV suggests that differences in sample preparation and gating strategy are factors that increase variability. Protocols with fewer intermediate steps or fully automated protocols such as the CD34+ assay are expected to reduced intra- and inter-laboratory variability.     

Quality assurance of progenitor cell content of apheresis products: a comparison of clonogenic assays and CD34+ enumeration. The Canadian Apheresis Group and Canadian Bone Marrow

The most common methods used for evaluation of the haematopoietic stem cell content of peripheral blood apheresis products are the colony forming cell assay and the enumeration of CD34+ cells by flow cytometry. The Canadian Apheresis Group and the Canadian Bone Marrow Transplant Group established a multicentre study to compare the reproducibility of colony forming cell assays and CD34+ enumeration by flow cytometry in six transplant centres routinely performing haematopoietic stem cell apheresis. Over a 5-month period in 1996, 31 fresh apheresis samples were shipped by overnight courier for testing at six centres to perform CD34+ enumeration by flow cytometry and clonogenic assays. The mean coefficient of variation and range for the following assays were: cell count 36% (2.6-148%), CFU-GM 82% (46-123%), CD34+ absolute/kg 60% (14-174%) and CD34+ per cent 42% (12-84%). The wide variation in cell count in this pilot study highlights the difficulties related to provision of samples for quality assessment programmes. Results showed poor interinstitutional reproducibility even among selected samples with similar cell counts for both CFC and CD34+ assays demonstrating the need for development and implementation of an interinstitutional quality assurance programme for haematopoietic stem cell assessment. Provision of a reliable source of testing material will be a necessary next step.     

Instrument- and protocol-dependent variation in the enumeration of CD34+ cells by flow cytometry

BACKGROUND: The information regarding the minimum number of CD34+ cells that are necessary to reconstitute hematopoiesis in patients undergoing peripheral blood progenitor cell transplantation is quite controversial. Some of the differences in these figures might be due to the selection of antibodies, staining protocols, and acquisition strategies for the flow cytometric enumeration of these cells. STUDY DESIGN AND METHODS: Twenty-seven human umbilical cord blood samples and 33 leukapheresis products were consecutively collected for this study. Cells were stained following two different protocols, both using monoclonal antibodies to CD45 and CD34, and analyzed by the same operator in two different flow cytometers to enumerate the percentage of CD34+ mononuclear cells. RESULTS: Relevant differences in the proportion of cells were encountered, and the correlation between the results yielded by both instruments and protocols, although statistically valid, was suboptimal. CONCLUSIONS: Both interinstrument and interprotocol variation can provide additional explanation for the redundantly reported discrepancies concerning the numbers of CD34 cells that suffice to secure hemopoietic grafting. These results point to the need for new and different standardization approaches in this clinically relevant field.     

Semi-automated flow cytometric analysis of CD34-expressing hematopoietic cells in peripheral blood progenitor cell apheresis products

BACKGROUND: The measurement of CD34+ cells is the most important step in the quality control of peripheral blood progenitor cell apheresis products. For this purpose, flow cytometry is applied. Recently, a new test kit has been introduced for the enumeration of CD34-expressing cells, in combination with software support for semi-automation of data acquisition and analysis. STUDY DESIGN AND METHODS: This study evaluated the ProCOUNT kit. Ninety samples obtained from peripheral blood progenitor cell apheresis products from 39 patients with hemato-oncologic diseases were analyzed. For data acquisition and analysis, ProCOUNT software was used. Data comparison was performed with parallel measurements according to the International Society for Hematotherapy and Graft Engineering (ISHAGE) guidelines and the German reference protocol for analysis of CD34-expressing cells. RESULTS: Correlation of the German and ISHAGE techniques was excellent (r2 = 0.99). The initial correlation coefficient of ProCOUNT analysis with the German protocol was r2 = 0.89. In 21 (23.3%) of 90 ProCOUNT analyses, a warning message was encountered from the ProCOUNT software. Following manual reevaluation of these data with CellQUEST software, a correlation of r2 = 0.96 with the German protocol and r2 = 0.97 with the ISHAGE analyses was obtained. ANOVA testing revealed significant differences between ProCOUNT and ISHAGE techniques (p<0.05) and between ProCOUNT and the German protocol (p<0.05). No statistically significant difference between ISHAGE and German protocol was observed (p = 0.19). CONCLUSION: The ProCOUNT kit and software for semi-automated data acquisition and analysis represents a further step toward standardization of CD34 cell quantitation in peripheral blood progenitor cell apheresis products. However, the occurrence of software warnings is high, and analysis or data reevaluation by experienced staff is still mandatory. Therefore, currently there is no definite advantage of the kit and software over the existing guidelines for CD34+ analysis in peripheral blood progenitor cell grafts.     

Consistency of the initial cell acquisition procedure is critical to the standardization of CD34+ cell enumeration by flow cytometry: results of a pairwise analysis of umbilical cord blood units and cryopreserved aliquots

BACKGROUND: The CD34+ cell content is a predictive factor for engraftment and survival after umbilical cord blood (UCB) transplantation. The high variability in the CD34 assay results in different recommended cell doses for infusion across transplant centers and also limits the clinical utility of the CD34+ cell counts provided by cord blood banks (CBBs). This bi-institutional study was intended to understand the sources of this variability.
STUDY DESIGN AND METHODS: The level of CD34 agreement between the University of Minnesota (UM) and the Madrid CBB (MCBB) was evaluated on 50 UCB units before and after cryopreservation. Two cryopreserved vials per unit were thawed and processed at both laboratories. Dual-platform ISHAGE-based flow cytometry was used for CD34 enumeration.
RESULTS: Postthaw nucleated cell recoveries were similar. However, whereas CD34+ cell enumeration before freezing was 0.35 ± 0.22 percent, the results after thawing were 0.98 ± 0.65 and 0.57 ± 0.39 percent at UM and MCBB, respectively. Bland-Altman plots analysis ruled out the interchangeability of MCBB and UM CD34 values. Differences in the initial cell acquisition settings accounted for most of the CD34 discrepancy, which was no longer present after normalization of the forward scatter threshold for cell acquisition.
CONCLUSIONS: The standardization of CD34+ cell enumeration by flow cytometry is strongly reliant on a consistent initial cell acquisition procedure. The interlaboratory variation can be minimized by using frozen cell aliquots as reference samples. Both requisites should be considered for CD34 testing and UCB unit selection by regulatory institutions involved with cord blood banking and transplantation.     

Flow cytometry assessment of apoptotic CD34+ cells by annexin V labeling may improve prediction of cord blood potency for engraftment

BACKGROUND: Nonviable CD34+ cells are commonly assessed by standard flow cytometry using the nuclear stain 7‐aminoactinomycin D (7AAD). 7AAD, however, only detects necrotic and late apoptotic cells, not earlier apoptosis, which engraft poorly in animal models of cord blood (cord) transplantation. The standard method, therefore, may overestimate engraftment potency of cord units under certain conditions. STUDY DESIGN AND METHODS: To detect apoptotic events, costaining with 7AAD and annexin V (AnnV), in parallel with the quantitative, standard enumeration, was used. Cord units were assessed before and after cryopreservation using both staining methods and colony‐forming units (CFU) to determine if graft potency can be predicted using a “functional flow cytometry” approach. RESULTS: Significant numbers of CD34+ AnnV+ events were found within the 7AAD‐gated population. Nonapoptotic cell dose (CD34+ AnnV?) correlated well with CFUs in both a small‐scale (n = 10) and a large‐scale banking study (n = 107). Finally, following samples postthaw with time showed increasing numbers of apoptotic CD34+ cells and consequently the AnnV assessed dose was better at predicting the CFU compared with just the standard enumeration. CONCLUSION: Defining the apoptotic population of CD34+ cells improved the prediction of CFU, making this method a rapid test of potency for assessment of cord units for clinical use.     

Direct volumetric flow cytometric quantitation of CD34+ stem and progenitor cells

Objectives: In this study, we compared a classic single‐platform (SP) method applying beads for enumeration of CD45+ or CD34+ cells with a new device allowing direct volumetric measurements of stem and progenitor cells. Background: Following apheresis and cyropreservation, the precise enumeration of CD34+ cells as key parameter of graft quality is mandatory for the clinical course after transplantation. Currently, flow cytometry with SP technique represents the ‘gold standard’ for such determinations. Methods/Materials: Fresh samples, 14 from mobilised peripheral blood (PB), 9 from apheresis products (AP) and 13 samples from frozen‐thawed (FT) haematopoietic progenitor cell grafts, were analysed for CD34+ cells, CD45+ cells, and in frozen‐thawed samples for viability by a bead‐based flow cytometric method and in parallel by a direct, volumetric flow cytometric method. Results: Comparison of CD34+ analyses revealed a significant correlation (P < 0·01) for each material between both techniques with r = 0·95 (PB), r = 0·933 (AP) and r = 0·929 (FT). Also, for analysis of CD45+ cells µL^?1, the measured numbers evaluated with the different techniques did not significantly differ for all three materials analysed. In frozen‐thawed samples, the analysis of viability was comparable for both techniques. Conclusions: The results of this study demonstrate that a direct volumetric analysis of CD34+ cells µL^?1 or CD45+ cells µL^?1 is feasible. This technique represents a simple and economical approach for standardisation of progenitor and stem cell analyses.     

Integration of biological, procedural, apheresis principles of peripheral blood stem cell transplantation programs

Collection and use of peripheral blood stem cells have rapidly replaced harvesting of pelvic bone marrow for transplant protocols. The mobilization of progenitor populations into the peripheral blood by chemotherapy and/or cytokine stimulation of marrow hematopoietic production has made it possible, in general, to collect larger quantities of progenitor populations than obtained in a single harvest of marrow. Technological advances through flow cytometry and generation of monoclonal antibodies to identify CD34 antigen expression on cells has provided a rapid means of assessing leukapheresis products for the presence of progenitor populations and has largely replaced the laborious 14 day culture assays' to measure colony forming units. Unlike apheresis platelet collection, where the yields are predictable through integration of donor biological variability, total volume of blood processed, and machine efficiency, CD34+ cell yields are not predictable. This has led to great diversity in stem cell collection procedures. Analyses of the same variables used to predict platelet yields, if applied to CD34+ cell collection, might lead to useful algorithms for development of standardized guidelines for stem cell collection.     

脐血造血干/祖细胞部分归巢受体功能缺陷的研究

本研究旨在了解来源于脐血造血干细胞表面部分归巢受体(homing receptor)的功能缺陷并探讨体外干预的效果和可行性。用流式细胞术对来源于脐血的CD34+造血干/祖细胞表面的P、E选择蛋白配体活性基团表达、CD26的表达及活性进行检测,同时检测骨髓和外周血作为对照。采用岩藻糖基转移酶体外处理脐血CD34+造血干/祖细胞并检测其表面选择蛋白配体活性基团的表达水平。结果表明,CD26在脐血、骨髓及外周血的CD34+造血干/祖细胞表面的表达率分别为:(7.62±0.63)%,(6.35±0.89)%和(6.18±0.91)%(p0.05),其活性分别为:67.15U/1 000个细胞(1U=1pmol/min),26.85U/1 000个细胞和20.95U/1 000个细胞,脐血CD34+造血干/祖细胞表面CD26的活性明显高于其它两者(p0.001)。P选择蛋白配体活性基团在脐血、骨髓和外周血CD34+造血干/祖细胞表面的表达率分别为:(83.46±6.33)%,(15.65±0.89)%和(80.17±6.85)%;E选择蛋白配体活性基团的表达率为:(25.31±1.03)%,(26.34±0.89)%和(29.79±1.78)%(p0.05)。脐血CD34+细胞体外行岩藻糖基化工程后,E选择蛋白配体活性基团表达率由(25.31±1.03)%提高至(63.23±1.08)%。结论:3种不同来源CD34+造血干/祖细胞表面的CD26分子表达无明显差别,但其活性不一,脐血的CD34+造血干/祖细胞CD26活性最高,骨髓的次之,外周血的最低。P选择蛋白配体活性基团在脐血及外周血CD34+造血干/祖细胞表面的表达无明显差别,但在骨髓干细胞表面表达偏低。体外经岩藻糖基化工程处理的脐血CD34+造血干/祖细胞可明显上调其表面E选择蛋白配体活性基团的表达。     

Effect of storage conditions on the CD34+ counts in flow cytometric analysis after anticoagulation of samples with EDTA

The transplantation of peripheral blood precursor cells (PBPC) is becoming of interest for autografting patients with a wide variety of haematological and other malignancies. For rapid quality control of PBPC apheresis products, flow cytometry is applied to quantify the number of CD34+ events. We studied the effect of different storage conditions on the number of CD34+ counts in EDTA-anticoagulated aliquots of PBPC grafts. Within 24 h, CD34+ signals decreased when samples were stored at room temperature (RT, 20 +/- 2 degrees C) compared to the results obtained directly after cytapheresis. The signal rate equalled or exceeded the baseline values after 24 h when aliquots were deposited at room temperature and subjected to agitation. Storage at 4 degrees C revealed no significant changes. These data indicate that quality control of PBPC samples by flow cytometry significantly depends on storages time, temperature and other conditions like the agitation of the specimen.