脐血造血干/祖细胞免疫表型的流式细胞术双标法分析及其意义

目的比较脐血和骨髓中造血干/祖细胞(HSPC)的免疫表型差异.方法使用流式细胞术(FCM)双标法对38份脐血及10份骨髓HSPC进行免疫表型分析.结果①脐血有核细胞中CD34+细胞所占比例与骨髓中相近,约为0.5%;②脐血CD34+细胞中CD34+CD38-[(17.C4±5.37)%]、CD34+HLA-DR-[(32.65±10.71)%]及CD34+H-CAM+(CD44+)[(77.84±7.69)%]亚群含量均高于骨髓[含量分别为(8.26±3.19)%、(14.05±1.67)%和(70.02±6.40)%],CD34+CD13+、CD34+CD19+亚群比例低于骨髓.结论脐血与骨髓CD34+细胞比例相近,但前者较原始的干细胞含量更高,故脐血是极具潜力的HSPC来源;而脐血CD34+细胞中髓系及淋系祖细胞含量低于骨髓,可能是脐血移植后造血及免疫重建缓慢的原因之一.     

Ten-year quality control of a semiautomated procedure of cord blood unit volume reduction

BACKGROUND: Volume reduction of cord blood units decreases the cost of cryogenic storage. This study reports the analysis of a 10-year quality control program of a semiautomated cord blood volume reduction procedure.
STUDY DESIGN AND METHODS: Cord blood was collected in a plastic bag containing 29 mL citrate-phosphate-dextrose, centrifuged at 2124 ×  g for 12 minutes, and processed with a semiautomated device. The procedure was aimed at removing most red blood cells and plasma and concentrating hematopoietic progenitors in the buffy coat (BC), thus reducing the unit volume and saving cryogenic space. Finally, the BC was cryopreserved with an equal volume of 20 percent dimethyl sulfoxide. Total nucleated cells (TNCs) were counted before and after processing in the 4311 units banked from 1998 through 2007, whereas CD34+ cells and colony-forming units–granulocyte-macrophage (CFU-GM) were counted in 420 random units from 2001 through 2007.
RESULTS: Mean postvolume reduction annual recoveries of TNCs, CD34+ cells, and CFU-GM ranged from 82.8 ± 12.3 (standard deviation) to 91.4 ± 6.4 percent, from 87.8 ± 14.1 to 95.2 ± 23.8 percent, and from 101.5 ± 51.4 to 117.8 ± 59.5 percent, respectively. Very strong correlations were found (r > 0.87) between postprocessing versus preprocessing TNCs, CD34+ cells, and CFU-GM; a moderate correlation between initial TNC count and unit's volume (r = 0.51); and no correlation between TNC percentage of recovery in the BC and initial unit's volume. The latter data indicate that most TNCs concentrate in the BC.
CONCLUSIONS: The semiautomated procedure of cord blood unit volume reduction used in this study provides high and stable cellular recoveries during several years of routine cord blood banking.     

人脐血CD34+CD38-细胞免疫表型和染色体核型特征分析

目的 分离脐血干／祖细胞(CD34^ CD38)进行体外长期培养，观察分析其增殖、细胞表面分子标志和染色体核型的特征。方法 用流式细胞仪分选CD34-FITC和CD38-PE标记的CD34^ CD38脐血原始细胞，在含细胞生长因子IL-3、IL-6、GM-CSF、EPO、SCF和胰岛素样生长因子的干细胞培养基中培养6个月，用流式细胞术检测体外培养30d的干／祖细胞表面标记，并用G显带方法分析其染色体核型。结果 在一定培养条件下，经7～12d培养，脐血干／祖细胞(CD34^ CD38)开始增殖。培养6个月后，每孔接种1个细胞，细胞数增殖至250～350个；每孔接种10个细胞，细胞数可增殖至400～500个。每孔接种1个细胞其细胞增殖峰持续时间(8～9代)比接种10个细胞(6～7代)长：经体外长期培养增殖，细胞仍强烈显示十／祖细胞表面分子标记(CD34^ CD38^-)；细胞染色体数目、结构未见异常。结论 脐血干／祖细胞(CD34^ CD38^ )经体外特异性培养增殖，可为大量脐血干／祖细胞移植提供细胞来源。     

人脐血及骨髓淋巴细胞免疫表型的比较及其意义

为比较脐血和骨髓淋巴细胞及祖细胞分化抗原，通过流式细胞术（FCM）双标法对38份脐血及10份骨髓免疫细胞表型进行了分析研究。研究发现：（1）脐血及骨髓淋巴细胞中均测到稚淋巴细胞（CD3^-CD4^ )，且前中含量较多，但脐血细胞毒T细胞含量（CTL，CD3^ CD16^ 56^ )低于骨髓；（2）脐血中NK细胞（CD3^-CD16^ 56^ )比例高于骨髓；（3）脐血有核细胞中CD34^ 细胞的比值接近于骨髓，但脐血CD34^ 细胞中髓系祖细胞（CD34^ CD13^ ,CD34^ HLA-DR^ )及淋巴系祖细胞（CD34^ CD19^ )含量均低于骨髓，结论：（1）脐血免疫细胞具有不成熟性，这估计是脐血移植后GVHD程度轻的主要原因；（2）脐血淋巴细胞中NK细胞含量较高，推测脐血移植后移植物抗白血病效应（GVL）并不会降低；（3）脐血CD34^ 细胞中髓系祖细胞及淋巴系祖细胞比例均低于骨髓，可能是脐血移植后造血及免疫重建速度较慢的原因之一。     

Competitive repopulation assay of two gene-marked cord blood units in NOD/SCID/gammac(null) mice.

In multiunit cord blood transplantation, hematopoietic stem cells from each unrelated cord blood (UCB) unit competitively reconstitute the hematopoietic system in a recipient. To evaluate the fate of the progeny of each UCB unit and to determine the effects of graft-versus-graft reaction, we established a novel competitive repopulation assay using NOD/SCID/gammac(null) mice in which human T lymphocytes develop from CD34+ cells. CD34+ cells from each UCB unit were labeled with recombinant lentivirus vectors carrying genes encoding either enhanced green fluorescent protein (EGFP) or enhanced yellow fluorescent protein (EYFP). Hematopoietic chimerism composed of both EGFP+ and EYFP+ cells was stably maintained up to 6 months after transplantation with purified CD34+ cells; the ratio of EGFP+ to EYFP+ cells in peripheral blood and bone marrow posttransplantation was equivalent to the ratio of these cells at transplantation. However, when mononuclear cells from two UCB units were cotransplanted with CD34+ cells, engraftment was highly competitive, with cells from only one or the other of the two UCB units surviving. Further subfractionations of mononuclear cells indicate that the skewed chimerism that is often observed in clinical multiunit cord blood transplantation may be mediated by the cooperation of both CD4+ and CD8+ T cells. The assay established here will be a useful tool for analyzing hematopoietic reconstitution in clinical multiunit cord blood transplantation.     

Mobilization kinetics of CD133+ hematoprogenitor cells for hematopoietic grafting

BACKGROUND: Quantification of CD34+ mononuclear cells is the most important quality control measure for hematopoietic stem cell (HSC) transplantation. A fraction of CD34+ cells also express the CD133 antigen. These cells constitute a group of earlier, less-differentiated HSCs with a potentially higher capacity for engraftment. The correlation between total CD34+ peripheral HSCs and the fraction of these cells that coexpress CD133 was determined before and after automated collection by leukapheresis, as well as the effect of HSC CD133+ dose on hematopoiesis recovery.
STUDY DESIGN AND METHODS: Granulocyte–colony-stimulating factor mobilization of HSCs from the marrow to the peripheral blood (PB) of allogeneic and autologous donors was followed by automated collection through leukapheresis on the fifth day. Quantification of CD34+ and CD133+ cells was performed on PB before collection and in the hematopoietic graft (HG) by flow cytometry.
RESULTS: There was a significant correlation between CD133+ and CD34+ HSCs in the PB before collection and in the final product for grafting (r = 0.62 and 0.64; p < 0.01). CD34+ HSCs per µL in PB and the HG was the only variable that did not correlate (r = 0.18). CD34+/CD133+ correlation increased from 0.33 on PB to 0.94 on the leukapheresis product (p < 0.01). Time to recovery was not related to CD133+ HSCs infused.
CONCLUSION: There was a significant correlation of both number per µL and percentage of CD34+/CD133+ HSCs before and after collection for transplantation; number of CD133+ cells had no apparent clinical impact on time to hematopoiesis regeneration.     

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.     

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.     

Nonobese diabetic-severe combined immunodeficient mice transplantation of volume-reduced and thawed umbilical cord blood transplants following closed-system immunomagnetic cell selection

BACKGROUND: Protocols for the expansion of human umbilical cord blood (UCB) progenitors begin with the selection of CD34+ cells from stored frozen and thawed units. Use of an immunomagnetic selection procedure within a closed blood bag system for volume-reduced UCB transplants was evaluated, and the influence of CD34 cell selection on in vivo engraftment potential was studied. STUDY DESIGN AND METHODS: Eleven thawed buffy coat-processed UCB units were processed within a standard blood bag with a washing solution. In six independent experiments, the same dosage of 2 x 104 CD34+ cells from paired selected and nonselected samples was transplanted into NOD-SCID mice. In two experiments, cells from the negative fraction were also transplanted. RESULTS: The purity of CD34+ cells after selection was correlated with the removal of supernatant after the first washing step and therefore with adequate removal of damaged or dead cells (r=0.86, p < 0.01). Mice transplanted with unselected UCB cells had more human cells within their marrow than animals transplanted with selected cells (8.6 +/- 5.9% selected group vs. 19.8 +/- 14.2% unselected group; p=0.04), whereas no engraftment could be observed transplanting cells from the two negative fractions. A higher percentage of human CD45+ cells in the unselected group were found to be positive for CD38, CD14, CD33, and CD19, indicating a higher potential for these unselected progenitors to differentiate into myeloid cells and B cells. CONCLUSIONS: Processing of volume-reduced and thawed UCB transplants within a closed-bag system before immunomagnetic CD34+ cell selection allows for the preparation of CD34+ cells of significant purity at technically useful cell recoveries. However, these experiments indicate a potential impairment of engraftment capacity for the CD34+ cell-enriched fraction.     

间充质干细胞与人脐血CD34+细胞共移植对NOD/SCID小鼠造血重建的影响

目的 观察间充质干细胞(MSC)与不同比例脐血CD34+细胞共移植对NOD/SCID小鼠造血重建的影响,明确MSC与脐血CD34+细胞共移植的最适数量.方法 给60Coγ射线照射的雌性NOD/SCID小鼠共移植人MSC和不同比例的脐血CD34+细胞,观察共移植后42 d内小鼠外周血白细胞和血小板变化,并于移植后42 d处死小鼠,用流式细胞术检测外周血、骨髓和脾脏人源细胞含量.结果 与单纯脐血CD34+细胞移植相比较:①脐血CD34+细胞与1、5和10倍数量的MSC共移植时,可明显减轻外周血白细胞和皿小板的下降幅度(P<0.01),提前1周使白细胞和血小板恢复至正常水平(P<0.05),三组间差异无统计学意义(P>0.05);②MSC与不同比例的脐血CD34+细胞共移植均可明显提高外周血、骨髓和脾脏造血细胞植入率.比例为10:1时,外周血、骨髓和脾脏中的人源细胞(huCD45+细胞)含量分别增加了(2.8±0.6)倍、(3.5±0.9)倍和(5.2±0.6)倍,增加倍数差异均有统计学意义(P<0.01),达到了最佳的植入效果.结论 脐血CD34+细胞与10倍数量的MSC共移植可达到最佳的促进造血重建作用.     

Developmental variation in stem-cell markers from human fetal liver and umbilical cord blood leukocytes

Fetal tissues containing haematopoietic stem cells (HSC) are of potential value for allogeneic transplantation and gene therapy. Flow cytometry was used to investigate CD34+ cells from human fetal livers and umbilical cord (placental) blood (UCB). CD34+ cells, expressed as a proportion of CD45-positive leukocytes, were much more abundant in fetal livers (mean 38%) than in UCB (mean 0.3%), but fetal liver cells had lower proportions of CD34+HLA-DR+ and CD34+ CD38+ subsets. In fetal liver, there was a strong and highly significant inverse correlation between CD34+ cells (as a proportion of total leukocytes) and gestational age; no such relationship was found for subsets of CD34+ cells coexpressing CD38 or CDw90 (Thy-1), but CD34+HLA-DR+ cells were less abundant in first- compared to second-trimester livers. In UCB, a trend towards decreasing CD34+ cells (as a proportion of total leukocytes) with increasing gestational age in late pregnancy was also observed. The composition of fetal leukocytes changes during development, and therefore the timing of fetal HSC harvesting could be of relevance to transplantation outcome.     

Cesarean section due to fetal distress increases the number of stem cells in umbilical cord blood

BACKGROUND: Umbilical cord blood (UCB) can be used as hematopoietic stem cell source for transplantation. The success of a transplantation is highly correlated with the number of total nucleated cells (TNCs) and CD34+ cells in the UCB. Certain obstetric factors increase the yield of stem cells in the UCB. It is necessary to evaluate optimal conditions in labor to decrease the rate of sample rejection due to low cell count. No data exist regarding the difference between primary and secondary Cesarean sections in terms of efficacy of stem cell harvesting. STUDY DESIGN AND METHODS: Seventy-nine consecutive UCB units from women who had a Cesarean section between 1997 and 2003 were included. The number of TNCs, CD34+ cells, colony-forming units (CFUs), white blood cells (WBCs), nucleated red blood cells (NRBCs), and the total collection volume were compared between cases with primary and secondary Cesarean section. RESULTS: UCB obtained after a Cesarean section due to fetal distress has significantly higher numbers of TNCs, CD34+ cells, NRBCs, and WBCs compared to elective Cesarean section. Of the cases with secondary Cesarean section due to fetal distress, 67 percent resulted in UCB units with sufficient TNC numbers (> or =80 x 10(7) TNCs) compared to 42 percent of the cases with primary Cesarean section. CONCLUSION: Fetal distress increases the number of hematopoietic stem cells mobilized into UCB. Particular effort should be made to collect UCB from newborns who experienced fetal distress.     

Higher infused CD34+ hematopoietic stem cell dose correlates with earlier lymphocyte recovery and better clinical outcome after autologous stem cell transplantation in non-Hodgkin's lymphoma

BACKGROUND: Lymphocyte recovery after autologous stem cell transplantation (ASCT) has been shown to be associated with positive clinical outcome in non-Hodgkin's lymphoma (NHL). This study sought to identify variables that affect lymphocyte recovery and survival after ASCT.
STUDY DESIGN AND METHODS: A retrospective analysis of outcomes in 97 consecutive patients with NHL who underwent ASCT in a single center from August 1999 to January 2008 was conducted.
RESULTS: A significant relationship was not observed between infused lymphocyte count and days to recovery of absolute lymphocyte count 500 × 10⁶/L or greater after ASCT (ALC500; r = 0.139, p = 0.176), but there was a significant inverse correlation between infused CD34+ cell count and days to ALC500 (r = −0.333, p = 0.001). Univariately, infused CD34+ cell count and recovery of ALC500 by 20th day after ASCT were significant predictors of survival. The median overall survival (OS) and event-free survival (EFS) were significantly longer in patients who received 8.2 × 10⁶ CD34+ cells/kg or more than in those who received fewer than 8.2 × 10⁶ CD34+ cells/kg (OS, not reached vs. 11.6 months, p = 0.001; EFS, not reached vs. 4.8 months, p = 0.003). Multivariate analysis confirmed that infused CD34+ cell count was an independent prognostic factor for OS (p = 0.017) and EFS (p = 0.002).
CONCLUSION: These data suggest that infused CD34+ cell count is predictive of kinetics of lymphocyte recovery after ASCT and is an independent prognostic factor for OS and EFS after ASCT in patients with NHL.     

体外扩增脐血CD34+细胞的实验研究

目的 探讨体外扩增脐血干／祖细胞用于成人脐血移植的可能性。方法 从１０份新鲜的脐血标本中纯化的ＣＤ３４＾＋细胞接种于含体积分数为２０％的胎牛血清（ＦＢＳ）的ＩＭＤＭ培养基的悬 培养体系中,分别加入由ＳＣＦ、Ｆｌｔ－３Ｌｉｇａｎｄ（ＦＬ）与ＩＬ－１β、ＩＬ－３、ＩＬ－６、Ｇ－ＣＳＦ、Ｅｐｏ（合称１３６ＧＥ）组成的３组细胞因子（Ａ组：１３６ＧＥ＋ＦＬ;Ｂ组：ＳＣＦ＋１３６ＧＥ;Ｃ组：ＦＬ＋ＳＣＦ＋１３     

Multiple-laboratory comparison of in vitro assays utilized to characterize hematopoietic cells in cord blood

BACKGROUND: Understanding the variability in results obtained by multiple laboratories is important because cord blood units are distributed worldwide for transplantation. STUDY DESIGN AND METHODS: Four exercises were conducted by multiple laboratories to assess assay variability on nucleated cell (NC), mononuclear cell (MNC) by hematology analyzers [HAs], and CD34+ cell (flow cytometry) measurements. Exercise 1 was an intralaboratory exercise in which the reproducibility of cell measurements was determined. Exercises 2 and 3 involved the shipment of identical processed cord blood samples. In Exercise 2, laboratory-specific methods were utilized. In Exercise 3, two commercial CD34+ cell methods (Stem-Kit and TruCOUNT) were used. In Exercise 4, CD34+ cell levels were determined on repetitive regating of identical list-mode files. RESULTS: Intralaboratory reproducibility was highest for NC measurements and lowest for CD34+ cell measurements. In Exercise 2, all laboratories except one utilized HA with an impedance technology and determined comparable results for NC and MNC levels, whereas the other laboratory utilized a HA with an optical counting method. Substantial variation was observed on measuring CD34+ cells with ranges of 32 to 141, 32 to 66, and 25 to 116 CD34+ cells per microL for the three identical samples. In Exercise 3, on the use of one specific commercial assay, the ranges of CD34+ levels were 214 to 411 and 62 to 178 cells per microL for the two identical samples. Nearly all participating laboratories determined comparable CD34+ levels on the use of identical list-mode files. CONCLUSION: These studies indicate that substantial variability in CD34+ cell levels were determined with flow cytometry. The variability in NC and MNC levels was minimal with HA methodology.     

不同时相移植人骨髓间充质干细胞对人脐血CD34^＋细胞植入NOD／SCID小鼠的影响

为了观察不同时相移植人骨髓间充质干细胞（MSC）对脐血（UCB）CD34^＋细胞移植的NOD／SCID小鼠造血重建的影响，明确最佳的移植时机，将体外培养扩增的人骨髓MSC分别于UCBCD34^＋细胞移植同时、移植前48小时及移植后48小时输入经^60Coγ射线照射的NOD／SCID小鼠，观察共移植后42天内小鼠外周血白细胞和血小板变化，并于移植后42天处死小鼠，用FACS检测外周血、骨髓和脾脏人源细胞含量。结果表明：（1）MSC和UCBCD34^＋细胞同时输注可明显降低外周血白细胞和血小板下降幅度，缩短白细胞和血小板恢复时间；二者不同时输注均不降低白细胞和血小板下降幅度，且输注UCBCD34^＋细胞后48小时输注MSC时外周血血小板恢复时间明显晚于同时输注者。（2）与单纯UCBCD34^＋细胞移植相比较，不同时相输注MSC均可促进UCBCD34^＋细胞的植入，三个共输注组间促进骨髓各系造血植入效应无明显差异。结论：人骨髓MSC与UCBCD34^＋细胞共移植时，以同时移植效果最佳，此结果为MSC的临床应用提供了实验依据。     

Long-term cryopreservation of autologous stem cell grafts: a clinical and experimental study of hematopoietic and immunocompetent cells

BACKGROUND: Autologous stem cell transplantation (ASCT) is used in the treatment of several malignancies. Harvesting sufficient peripheral blood progenitor cells (PBPCs) for a potential second autotransplantation at the time of relapse several years after diagnosis is becoming an increasingly common practice.
STUDY DESIGN AND METHODS: Cryopreserved PBPCs were prepared with different concentrations of dimethyl sulfoxide (DMSO; 2, 4, 5, and 10%) and stored for at least 5 years before the recovery of CD34+ cells and various T- and natural killer (NK)-cell subsets were analyzed by flow cytometry. Furthermore, clinical variables for myeloma patients having a second autotransplantation with long-term-stored autografts were evaluated.
RESULTS: The number of viable CD34+ cells in long-term-stored grafts was higher when autografts were cryopreserved with 4 or 5% than with 2 and 10% DMSO. The number of viable CD34+ cells was reduced by 13.9% after 5 years of cryostorage in 5% DMSO. Lymphocyte viability was also higher with 4 or 5% DMSO. However, the frequencies of several T-cell subsets showed DMSO-dependent differences, whereas NK-cell subsets did not. Furthermore, after a second autotransplantation with long-term-stored PBPC grafts at the time of myeloma relapse (median storage time, 42 months) all 17 patients reached neutrophil counts exceeding 0.5 × 10⁹/L and platelet counts exceeding 20 × 10⁹/L within 15 days. There was no difference in engraftment between patients receiving autografts preserved with 5 and 10% DMSO.
CONCLUSION: PBPC autografts can safely be stored for at least 5 years in 5% DMSO and used for ASCT.     

Cell loss and recovery in umbilical cord blood processing: a comparison of postthaw and postwash samples

BACKGROUND: Engraftment after umbilical cord blood (UCB) transplantation is highly dependent on nucleated cell (NC) and CD34+ cell content. Current standard postthaw (PT) processing includes a wash step to remove dimethyl sulfoxide (DMSO), lysed red cells, and stroma. The contribution of the wash step to cell loss and ultimately the dose of cells available for transplant have yet to be systematically reported. This study examines the effect of the wash step as well as that of PT storage on various quality control variables of UCB units. STUDY DESIGN AND METHODS: Ten units were thawed and washed based on the New York Blood Center method. Samples were removed from each unit at six time points: prefreeze (PF), immediately PT, immediately postwash (PW), and 1, 2, and 5 hours PW. On each sample, total nucleated cell (TNC) count, CD34+ cell enumeration, colony-forming unit (CFU)-granulocyte-macrophage, and viability assays (fluorescence microscopy [acridine orange/propidium iodide, or AO/PI] and flow cytometry [7-aminoactinomycin]) were obtained. RESULTS: TNC counts decreased PT and at subsequent time points; the PT TNC recovery was 89 percent compared to 82 percent PW (p < 0.01). TNC recovery decreased to 90 percent of PW (82% of PT) values (p < 0.01) and 83 percent of PW (76% of PT) values (p < 0.001), at 2 and 5 hours PW, respectively. CD34+ cell loss PT was not significant. Viability by AO/PI decreased PT and plateaued over time. In contrast, viability by flow cytometry remained higher and increased slightly over time. CFUs were significantly lower PT, recovering PW. CONCLUSIONS: Our data indicate that the thawing and washing results in a substantial loss of cells, with TNC loss approaching 20 percent when compared with PF counts; the wash step was responsible for nearly half of the cell loss. The reduced PT viability was expected. Elapse of time PW resulted in further loss of NCs but no detectable significant changes in CD34+ cell content and viability and/or CFU.     

Assessment of cord blood hematopoietic cell parameters before and after cryopreservation

BACKGROUND: The testing of cord blood (CB) progenitor and stem cell units for transplantation suitability involves enumeration of total nucleated cells before freezing. CD34+ cell counts may also be a means of determining suitability. Studies have been conducted to evaluate how specific storage conditions influence cell counts. STUDY DESIGN AND METHODS: CB units were processed by hydroxyethyl starch volume reduction. Cryopreserved-thawed samples were diluted 1:3 without washing. CD34+ cells were measured with three commercially available assay methods. In specific studies, apoptosis-indicating reagents were included. CB units were analyzed for nucleated cells, aldehyde dehydrogenase-containing cells, and progenitor colonies. RESULTS: CD34+ cell levels and nucleated cells were retained during storage in test tubes at 1 to 6 degrees C for 3 days. Cryopreserved-thawed samples showed a reduction in CD34+ cells relative to prefreeze levels with the largest decrease with the Stem-Kit (Beckman Coulter) restricted gating procedure. Prefreeze samples contained minimal numbers of presumed apoptotic cells detected with 7-aminoactinomycin D or SYTO16, but after cryopreservation-thawing there was an increase. Nucleated cell levels determined with a hematology analyzer or flow cytometry were reduced after thawing. Cryopreservation-thawing reduced the percentage of CD34+ cells positive for the presence of aldehyde dehydrogenase and the number of progenitor colonies. These differences were significant. CONCLUSION: These studies indicate that CD34+ cell counts were maintained when CB samples were stored at 1 to 6 degrees C in test tubes for 3 days. Cryopreservation-thawing resulted in changes in a number of parameters including the percentage of CD34+ cells that were aldehyde dehydrogenase(+) and the number of 7-aminoactinomycin D(+) cells and SYTO16(low) cells.     

CD34+CD38- is a good predictive marker of cloning ability and expansion potential of CD34+ cord blood cells

BACKGROUND : Ex vivo expansion of HPCs is an attractive approach to overcoming the current limitations of human cord blood transplantation. It is important not only to define the optimal culture conditions but also to know the number of progenitor cells that can be obtained. CD34+ cells have a great variability in their cloning capacity and in their ability to expand HPCs. This study was carried out to assess whether this variability could be due to intrinsic or extrinsic factors.
STUDY DESIGN AND METHODS : CD34+ cells were analyzed for the expression of CD38, CD133, and CD117 and cultured in serum-free culture medium with four cytokine combinations: SCF plus thrombopoietin plus flt3 ligand (STF), STF plus IL-3, STF plus IL-6, and STF plus IL-6 plus IL-3. After a 1-week culture, the numbers of CD34+ cells and CFUs were determined.
RESULTS : The variability observed both in the cloning ability of CD34+ isolated cells and in their expansion capacity was inversely related to the frequency of the more immature CD34+CD38– cells. When more mature CD34+CD38+ cells were present within CD34+-isolated cells, a higher cloning ability, measured as CFUs, and a higher expansion capacity were observed.
CONCLUSION : Enumeration of CD34+CD38– cells is correlated with the number of committed progenitors and the capacity of generating CD34+ cells, an important parameter if expansion protocols must be used in clinical transplantation.