Significant Differences In CD34 Research Articles

The impact of the donors' COVID-19 status on the outcomes of allogeneic hematopoietic stem cell transplantation: a multi-center retrospective study.

To explore the impact of donors' COVID-19 status on allogeneic stem cell transplantation (allo-HSCT), we compared the transplant outcomes of 74 participants. This multi-center retrospective study included nine participants receiving grafts from COVID-19 positive donors (CPD), 45 from COVID-19 experienced donors (CED), and 20 from COVID-19 naive donors (CND). We evaluated engraftment, complications, and survival rates among the three groups. All apheresis procedures were successful with no significant differences in CD34+ cells or lymphocytes in grafts among the three groups. All patients achieved engraftment by day 30 post-HSCT. The incidence of grade II-IV acute graft-versus-host disease (aGVHD) was 55.6%, 20%, and 10% in the CPD, CED, and CND groups, respectively (p = 0.024). Multivariate analysis indicated that COVID-19 positivity in donors at the time of apheresis was an independent risk factor for II-IV aGVHD (p = 0.020, OR = 12.159, 95% CI 1.783 -135.760). No differences were observed among the groups in terms of chronic GVHD, viral infection, or sinusoidal obstruction syndrome. The 6-month overall survival and disease-free survival rates were also similar among the three groups. Our results suggest that the COVID-19-positive status of donors might not impact graft collection, engraftment, or short-term survival of allo-HSCT recipients but might increase the risk of aGVHD. Further research is needed to explore the influence of donors' COVID-19 status on long-term complications and survival in allo-HSCT recipients.

Comparative Efficacy of Autologous Hematopoietic Stem Cell Mobilization Regimens in Multiple Myeloma Patients

Aim. To compare the efficacy of mobilization regimens (MR) differing in their composition and intensity, with the purpose of defining the criteria to personalize the choice of MR based on clinical and hematological characteristics of multiple myeloma (MM) patients.
 Materials & Methods. A retrospective analysis of the autologous hematopoietic stem cell (HSC) mobilization and autograft harvesting results was performed in 177 patients with newly diagnosed MM. The patients were divided into 4 groups. Group 1 included 62 patients with the median age of 53 years who were treated with single injection of cyclophosphamide (CF) dose 3 g/m2 as MR. Group 2 consisted of 71 patients with the median age of 58 years who received vinorelbine 35 mg/m2. Granulocyte colony-stimulating factor (G-CSF) as a monoregimen was administered to group 3 consisting of 33 patients with the median age of 55 years. Group 4 included 11 patients with the median age of 57 years who received G-CSF enhanced by plerixafor administration. G-CSF 10 µg/kg was used as MR. In all chemomobilization cases, daily G-CSF 10 µg/kg started on Day 4 from the administration of the chemotherapy drug prescribed as MR.
 Results. In the analyzed groups, the median time from MR start to the first leukocyte apheresis session was 11, 8, 5, and 5 days, respectively. On the first leukocyte apheresis day, the median CD34+ cell collection in group 3 was significantly lower than in groups 1, 2, and 4: 2.2 × 106/kg vs. 3.79 × 106/kg, 7.22 × 106/kg, and 3.9 × 106/kg, respectively. The total CD34+ cell collection after two leukocyte apheresis sessions was also the lowest in group 3 compared with groups 1, 2, and 4: 3.22 × 106/kg vs. 5.2 × 106/kg, 4.95 × 106/kg, and 7.5 × 106/kg, respectively. In the analyzed groups, the rate of mobilization with CD34+ cell collection < 2.0 × 106/kg was 6.5 %, 5.6 %, 18.2 %, and 9.1 %. The evaluation of the results in all patients showed a direct correlation of CD34+ cell collection with lenalidomide administered before autologous HSC mobilization. A significant difference in CD34+ cell collection in lenalidomide recipients vs. non-recipients was reported when vinorelbine as MR and G-CSF as monoregimen (р = 0.001 and р = 0.022, respectively) were used. No significant differences were observed either with CF or G-CSF combined with plerixafor treatment.
 Conclusion. Based on the findings, age of a MM patient, comorbidities, and prior lenalidomide administration can be regarded as key criteria for choosing one of 4 MRs.

Safety of Cryopreserved Stem Cell Infusion through a Peripherally Inserted Central Venous Catheter.

The management of patients undergoing stem cell transplantation requires a multipurpose central venous catheter (CVC) to facilitate drug administration, parenteral nutrition, transfusion of blood products, and collection of blood samples. Peripherally inserted central venous catheters (PICCs) appear to meet these requirements but are rarely used for stem cell infusion. We aimed to retrospectively assess the safety and feasibility of stem cell infusion through PICC and to evaluate its impact on transplantation kinetics. We retrospectively analyzed the outcomes of peripheral blood stem cell (PBSC) transplantation in patients receiving cryopreserved autologous or allogeneic PBSC by PICCs and compared the results with patients receiving transplants through a conventionally inserted central venous catheter (CICC). Despite statistically significant differences in CD34+ dose, infusion rate, and total length of administration, the clinical outcomes of transplantation, exemplified by platelet and neutrophil engraftment, along with the length of hospitalization, were not affected by the prolonged infusion time and lower infusion velocity in the PICC group. Our study showed that the clinical outcomes of PBSC transplantation did not differ between the PICC and CICC groups, suggesting that both types of catheters can be implemented in a PBSC transplantation setting.

The Effectivity of Supplementation Artemisia vulgaris for Adenocarcinoma Mammae Chemotherapy to Reduce CD 34 and Tumor Massa Diameter (Study in C3H Mice Given Adriamycin - Cyclophosphamide Chemotherapy)

Introduction: Breast cancer is still a major health problem in the world. In the case of breast cancer, surgery is the main treatment option besides chemotherapy, radiation, and immunotherapy such as Artemisia vulgaris (AV). AV is cytotoxic selectively acts as a supplement to breast adenocarcinoma chemotherapy given the Adriamycin-Cyclophosphamide regimen, to improve chemotherapy response.The study was aimed to proving AV extract enhances the chemotherapy response in C3H mice with adenocarcinoma mammae given Adriamycin-Cyclophosphamide Chemotherapy.
 Method: This study used Post test only control group design on 24 females C3H mice that were randomly selected and divided into four groups: group K (control), P1 (chemotherapy), P2 (extract), and P3 (combination). Adenocarcinoma mammae comes from the inoculation of donor mice. Chemotherapy of Adriamycin 60 mg / m 2 and Cyclophosphamide 600 mg / m 2 were given in two cycles. AV 13 mg (0.2 ml) was given once daily orally. CD34 were evaluated by imunohistochemical staining and tumor mass diameter were counted by calipers.
 Result: The microvascular density CD34 and tumor mass diameter were obtained in groups of K, P1, P2, P3 respectively 60.76 ± 1.5; 39.70 ± 2.00; 57.10 ± 1.29; 35.26 ± 2.06 and 12.52 ± 1.49; 6.20 + 1.04; 9,94 + 1.21; 3.94 + 0.76. Statistical analysis showed significant differences in CD34 between groups K vs P1, P2, P3 (p = 0.001, p = 0.014, p = 0.001), P1 vs P2 and P3 (p = 0.001, p = 0.033) and P2 (P = 0.001). Tumor mass diameter between groups K vs P1, P2, P3 (p=0.001; p=0.014; p=0,001), P1 with P2 (p= 0.001) P1 with P3 (p = 0.033) and P2 with P3 (p = 0.001). Correlation analysis between CD34 with tumor mass diameter was found to have significant correlation (p = 0.001 and r = 0.932).
 Conclusion: Artemisia vulgaris is a potential to reduce angiogenesis in terms of decreasing the microvascular density CD34 and tumor mass diameter of adenocarcinoma mammae of C3H mice treated with Adriamycin-Cyclophosphamide chemotherapy and can improve the effectivity.

The Effectivity of Supplementation Artemisia vulgaris for Adenocarcinoma Mammae Chemotherapy to Reduce CD 34 and Tumor Massa Diameter (Study in C3H Mice Given Adriamycin - Cyclophosphamide Chemotherapy)

Introduction: Breast cancer is still a major health problem in the world. In the case of breast cancer, surgery is the main treatment option besides chemotherapy, radiation, and immunotherapy such as Artemisia vulgaris (AV). AV is cytotoxic selectively acts as a supplement to breast adenocarcinoma chemotherapy given the Adriamycin-Cyclophosphamide regimen, to improve chemotherapy response.The study was aimed to proving AV extract enhances the chemotherapy response in C3H mice with adenocarcinoma mammae given Adriamycin-Cyclophosphamide Chemotherapy.
 Method: This study used Post test only control group design on 24 females C3H mice that were randomly selected and divided into four groups: group K (control), P1 (chemotherapy), P2 (extract), and P3 (combination). Adenocarcinoma mammae comes from the inoculation of donor mice. Chemotherapy of Adriamycin 60 mg / m 2 and Cyclophosphamide 600 mg / m 2 were given in two cycles. AV 13 mg (0.2 ml) was given once daily orally. CD34 were evaluated by imunohistochemical staining and tumor mass diameter were counted by calipers.
 Result: The microvascular density CD34 and tumor mass diameter were obtained in groups of K, P1, P2, P3 respectively 60.76 ± 1.5; 39.70 ± 2.00; 57.10 ± 1.29; 35.26 ± 2.06 and 12.52 ± 1.49; 6.20 + 1.04; 9,94 + 1.21; 3.94 + 0.76. Statistical analysis showed significant differences in CD34 between groups K vs P1, P2, P3 (p = 0.001, p = 0.014, p = 0.001), P1 vs P2 and P3 (p = 0.001, p = 0.033) and P2 (P = 0.001). Tumor mass diameter between groups K vs P1, P2, P3 (p=0.001; p=0.014; p=0,001), P1 with P2 (p= 0.001) P1 with P3 (p = 0.033) and P2 with P3 (p = 0.001). Correlation analysis between CD34 with tumor mass diameter was found to have significant correlation (p = 0.001 and r = 0.932).
 Conclusion: Artemisia vulgaris is a potential to reduce angiogenesis in terms of decreasing the microvascular density CD34 and tumor mass diameter of adenocarcinoma mammae of C3H mice treated with Adriamycin-Cyclophosphamide chemotherapy and can improve the effectivity.

Improving the characterization of endothelial progenitor cell subsets by an optimized FACS protocol.

The characterization of circulating endothelial progenitor cells (EPCs) is fundamental to any study related to angiogenesis. Unfortunately, current literature lacks consistency in the definition of EPC subsets due to variations in isolation strategies and inconsistencies in the use of lineage markers. Here we address critical points in the identification of hematopoietic progenitor cells (HPCs), circulating endothelial cells (CECs), and culture-generated outgrowth endothelial cells (OECs) from blood samples of healthy adults (AB) and umbilical cord (UCB). Peripheral blood mononuclear cells (PBMCs) were enriched using a Ficoll-based gradient followed by an optimized staining and gating strategy to enrich for the target cells. Sorted EPC populations were subjected to RT-PCR for tracing the expression of markers beyond the limits of cell surface-based immunophenotyping. Using CD34, CD133 and c-kit staining, combined with FSC and SSC, we succeeded in the accurate and reproducible identification of four HPC subgroups and found significant differences in the respective populations in AB vs. UCB. Co-expression analysis of endothelial markers on HPCs revealed a complex pattern characterized by various subpopulations. CECs were identified by using CD34, KDR, CD45, and additional endothelial markers, and were subdivided according to their apoptotic state and expression of c-kit. Comparison of UCB-CECs vs. AB-CECs revealed significant differences in CD34 and KDR levels. OECs were grown from PBMC-fractions We found that viable c-kit+ CECs are a candidate circulating precursor for CECs. RT-PCR to angiogenic factors and receptors revealed that all EPC subsets expressed angiogenesis-related molecules. Taken together, the improvements in immunophenotyping and gating strategies resulted in accurate identification and comparison of better defined cell populations in a single procedure.

CD34+ cell count predicts long lasting life in the oldest old

Circulating progenitor cells (CPCs) represent a pool of cells capable of differentiating into mature cells of different organs and systems, promoting tissue maintenance and repair. Among CPCs, CD34+cells (CD34+CPCs) seem to predict outcome in CV disease, also in elderly people. A decline in CD34+CPCs was reported with advancing age. Moreover, aging is associated with a state of chronic inflammation, influencing life expectancy. Our purpose was to investigate a 10-year predictive ability of CD34+CPCs, inflammatory marker levels, classic CV risk factors (CVRFs), and Framingham Risk Score (FRS) in a population of healthy, self-sufficient octogenarians. We found that baseline CD34+CPCs was strongly associated with mortality, showing a significant difference in CD34+CPC numbers between deceased and living patients. Moreover, by dividing our patients into tertiles based on age reached, this difference was more remarkable the higher the age reached. Regressive analyses suggested that the chances of reaching an older age depend on higher CD34+CPCs at baseline and are not significantly affected by inflammatory markers levels, FRS, CVFRs, or HDL-C levels. We found that higher CD34+CPCs predict longer life also in the oldest old, providing additional insights on the predictive role of CD34+CPCs in subjects aged 80 years or more.

Comparison of the Effects of Different Cryoprotectants on Stem Cells from Umbilical Cord Blood.

Purpose. Cryoprotectants (CPA) for stem cells from umbilical cord blood (UCB) have been widely developed based on empirical evidence, but there is no consensus on a standard protocol of preservation of the UCB cells. Methods. In this study, UCB from 115 donors was collected. Each unit of UCB was divided into four equal parts and frozen in different kinds of cryoprotectant as follows: group A, 10% ethylene glycol and 2.0% dimethyl sulfoxide (DMSO) (v/v); group B, 10% DMSO and 2.0% dextran-40; group C, 2.5% DMSO (v/v) + 30 mmol/L trehalose; and group D, without CPA. Results. CD34+, cell viability, colony forming units (CFUs), and cell apoptosis of pre- and postcryopreservation using three cryoprotectants were analyzed. After thawing, significant differences in CD34+ count, CFUs, cell apoptosis, and cell viability were observed among the four groups (P < 0.05). Conclusion. The low concentration of DMSO with the addition of trehalose might improve the cryopreservation outcome.

Influence Of Race and Ethnicity On The Collection Of G-CSF Mobilized Peripheral Blood Stem Cells From Unrelated Donors, a Center For International Blood and Marrow Transplant Research Analysis

▪Peripheral blood stem cell (PBSC) collection is increasingly used in allogeneic stem cell transplantation. However, a small percentage of healthy donors have a poor mobilization response to G-CSF. Very little information exists on the effect of donor race or ethnicity on PBSC mobilization. We analyzed 10776 unrelated donors from the National Marrow Donor Program (NMDP) who underwent G-CSF mobilized PBSC collection from 2006–2012. We investigated the effect of self-reported donor race/ethnicity on collection efficiency, defined as number of CD34+ cells/L (of donor blood processed), number of mononuclear cells (MNC)/L and CD34+ cells/MNC collected on the first day of apheresis. Categorical variables were analyzed by the Chi-square test and the Kruskal-Wallis test was used for continuous variables. A linear regression model was used to compare the various race/ethnic groups while controlling for potential confounding factors (such as age, BMI, gender, and year of apheresis). The result of our analysis is shown in Table 1. Univariate analysis revealed statistically significant differences in CD34+ cells/L, MNC/L and CD34+/MNC in all races analyzed. In general, African Americans (AA) had the highest collection efficiency while Caucasians had the lowest. Other races/ethnicities had collection efficiencies between the two groups. On multivariate analysis, statistically significant differences in CD34+ cell/L were seen in Hispanics, AA and Asian/Pacific Islanders (API), primarily in the obese (Hispanic, AA, API) and overweight (AA, API) donors. In the API group the differences in collection efficiency were predominately seen in males. No differences were seen between Caucasians and Native Americans. This study reveals significant racial/ethnic differences in the efficiency of collection of CD34+ cells in unrelated donors. Although these differences do not appear to interfere with the ability to collect adequate numbers of PBSC, it is currently unknown why they exist. This is an area for continued research. Disclosures:No relevant conflicts of interest to declare.

Pre- and post-cryopreservation enumeration of viable CD34+ haemopoietic stem cells in autologous peripheral blood stem cell products: do viable CD34+ stem cell numbers reduce with cryopreservation time?

<h3>Introduction</h3> Autologous peripheral blood stem cell (PBSC) transplantation has an established role in the treatment of some haematological and non-haematological malignancies. The safety and efficacy of this procedure are dependent on the fact that haematopoietic stem cells can be mobilised, harvested, cryopre-served and accurately enumerated prior to transplantation. The effect of the cryopreservation procedure on the PBSC product is evaluated. <h3>Methods</h3> We analysed the number of CD34+ peripheral blood cell progenitors obtained by aphaeresis pre- and post-cryopreser-vation to determine if there is significant loss of CD34+ cells during freezing, processing and thawing. We then further correlated CD34+ cell degradation and its effect on neutrophil or platelet engraftment. <h3>Results</h3> Twenty-eight CD34 products from 28 individual patients, most undergoing treatment for haematological malignancy, were analysed for viable CD34 number using the standard ISHAGE single platform enumeration assay immediately post-harvest and pre-infusion. CD34 numbers were then calculated and expressed in the standard form of CD34+ cells/kg body weight. Mean pre-cryopreservation CD34+ cell product numbers were 4.04±2.17x106/kg (mean+SD). Mean post-cryopreservation CD34+ cell product numbers were 2.80±1.39x106/kg. Mean CD34+ ‘loss' with cryopreservation=1.24 ± 106/kg. Mean time to neutrophil and platelet engraftment was 10.6±0.04 and 13.52±0.13 days, respectively. There was no significant difference in CD34+ cell loss in products stored for >100 days compared with those stored for <100 days, with no change seen in engraftment potential. <h3>Conclusion</h3> Despite a documented decrease in viable CD 34 numbers arising form the cryopreservation and processing process, generally adequate CD34 numbers are preserved and no effect is seen on either neutrophil or platelet engraftment. The length of cryopreservation appears to have no effect on viable CD 34+ cell numbers inferring that cell loss, when it occurs, is during the initial processing of CD34+ products.

Plerixafor mobilization leads to a lower ratio of CD34+ cells to total nucleated cells which results in greater storage costs

Plerixafor (Mozobil, AMD3100) with granulocyte-colony stimulating factor (G-CSF) mobilizes more CD34+ cells/kg compared to G-CSF alone. Given that plerixafor enhances mobilization of multiple white blood cell lineages, we determined if more storage space is required for products collected from patients mobilized with plerixafor. A review of the medical records of 15 patients mobilized with chemotherapy and G-CSF (control) and 14 patients mobilized with plerixafor plus G-CSF (plerixafor) was performed. Data on demographics, baseline characteristics, CD34+ cells/kg, total nucleated cells, total mononuclear cells, total apheresis sessions, and total bags for storage were collected. Mean values were determined and compared using Student's t-test. We found that the proportion of CD34+ cells among total nucleated cells was less in the plerixafor group compared to the control group (P = 0.0427). More nucleated cells (10.7 x 10(10) vs. 7.1 x 10(10), P =0.0452) and mononuclear cells (9.7 x 10(10) vs. 5.9 x 10(10), P = 0.0059) were mobilized with plerixafor plus G-CSF. However, there was no significant difference in CD34+ cells/kg, total CD34+ cells or the proportion of mononuclear cells among total nucleated cells between the two groups. More storage bags were required for the plerixafor group compared to the control group (15 vs. 9, P = 0.0299). Mobilization with plerixafor plus G-CSF resulted in a smaller proportion of CD34+ cells collected and a greater number of storage bags. An increase in the number of bags required for stem cell storage may be logistically problematic and will also lead to increased costs for storage of stem cells.

Mobilization of Blood Stem Cells with G-CSF in Systemic Light-Chain Amyloidosis: Dominant Organ Involvement Significantly Affects Stem Cell Collection.

Systemic light-chain (AL) amyloidosis is both a protein deposition disorder and a monoclonal plasma cell dyscrasia. Effective treatment depends on reduction of the clonal plasma cells that produce the toxic light chains. Although high-dose melphalan with autologous stem cell transplant (MEL SCT) is effective for AL patients, the mobilization and collection of peripheral blood stem cells with G-CSF can be problematic despite their lack of prior chemotherapy and minimal marrow infiltration with plasma cells. In addition, unusual toxicities of G-CSF mobilization such as hypotension have been noted in AL patients. As new therapies show efficacy in AL, the role of stem cell mobilization and MEL SCT will likely be re-evaluated, increasing the importance of understanding the variability in stem cell mobilization. Recent work has shown that the mechanism of G-CSF mobilization likely involves the sympathetic nervous system (Cell 2006; 124:407). Interestingly, amyloid is well known to cause autonomic neuropathy. In addition, involvement of the heart in amyloidosis has also been shown to cause cardiac adrenergic denervation. If G-CSF mobilization depends upon the sympathetic nervous system, then the type of organ involvement could predict the yield of stem cell collection in AL patients mobilized with G-CSF. With these concerns in mind, we retrospectively assessed the results of stem cell mobilization and collection in all patients with AL mobilized with G-CSF (6ug/kg bid) for 4 days, with collections beginning on day 5 with a collection target of 10x106 CD34+ cells per kg in up to 4 leukaphereses. At initial evaluation, we assessed patients using standard criteria to identify the dominant organ of involvement (Am J Hematol 2005; 79:319) and to determine candidacy for risk-adapted MEL SCT (Blood 2002; 99:4276). Over a 7-year period 105 patients with AL were mobilized and collected as described, including 40 with kidney (K), 21 with liver/GI or soft-tissue (L/GI/ST), 31 with cardiac (H) and 13 with peripheral nervous system (PNS) involvement. We recorded the number of leukaphereses per patient and the dose of CD34+ cells/kg collected per leukapheresis. There were no significant differences in the number of leukaphereses per group. The K, L/GI/ST and H groups had a median of 2, and the PNS group of 3, collections. There were significant differences in CD34+ cells/kg collected based on dominant organ involvement: patients with H or PNS involvement had significantly fewer CD34+ cells/kg collected (see figure below; K vs H p=0.03, K vs PNS p=0.02, L/GI/ST vs H p=0.07, L/GI/ST vs PNS p=0.04; two tailed Mann-Whitney). Moreover, 15/44 H+PNS patients failed to collect at least 4x106 CD34+cells/kg compared to 7/61 K+L/GI/ST patients (p<0.01). These observations provide a basis for directed studies of the sympathetic nervous system in AL, particularly in association with stem cell mobilization with G-CSF. [Display omitted]

Bmi-1 Protein Expression in CD34 Positive Acute Myelogenous Leukemia Cells as a Prognostic Factor.

Background: Bmi-1 is a novel protein marker, one of the Polycomb group proteins which are involved in self-renewal, senescence, apoptosis and cell cycle regulation of both normal and leukemic haematopoietic stem cells (HSCs). Bmi-1 expression is restricted to primitive bone marrow progenitors and has a critical role in regulating the proliferative output of this cell population. The downstream target of bmi-1 gene is Ink4a locus encoding p16Ink4a and p19Arf repressed in a Bmi1 dependent manner. Enforced expression of p16Ink4a and p19Arf in HSCs leads to senescence and apoptosis, respectively. Recent data suggest bmi-1 to be responsible for immortalization of acute leukemia cells in murine model and one of the potential targets for therapeutic approaches.Aims: In this study we analyze expression of bmi-1 protein in acute myelogenous leukemia (AML) cells in humans. We tried to establish the prognostic significance and survival prediction due to bmi-1 expression in AML cells.Patients: 43 patients (F/M -18/25, median age 55) with AML (FAB subtypes M0,M1,M2,M4 and M5 in 3, 11, 19, 5 and 5 cases, respectively) diagnosed in our unit in two recent years and 8 healthy bone marrow volunteer donors were included into the study. All patients received anthracyclines and cytarabine based induction treatment according the guidelines of Polish Leukemia Study Group. Eight patients died during the follow up observation (early deaths related to treatment toxicity and infection complications were previously excluded).Methods: Bone marrow specimens were collected from patients during routine diagnostic procedure. Mononuclear cells were permeabilised and incubated with anti-bmi1 antibody (Upstate), followed by surface staining with anti-CD34 (Dako).Results: We estimated expression of bmi-1 in CD34+ AML cells and its correlation with overall survival (patients who did not complete induction therapy and deaths not related to AML were excluded from the study). On the other hand the differences in bmi expression in CD34+ population between AML subtypes were evaluated. We found significant negative correlation (Spearman test) between bmi-1 expression in CD34+ and overall survival (r= −0.39, p=0,04). The U Mann-Whitney test revealed significant difference in CD34+bmi-1+ cells expression between M0 with M1 patients on one side and M4 with M5 patients on the other side (p=0,02). The mentioned result is restricted to double positive cells, the difference in CD34+ cells alone was not significant. The difference in bmi1 expression in CD34+ cells between AML patients and healthy controls was statistically significant (p=0,003).Conclusions: Bmi-1 has been known for its role in repression Ink4a locus encoding p16Ink4a and p19Arf which are capable of inducing cell cycle arrest, cellular senescence and apoptosis. In this context bmi-1 is responsible for immortalization of HSCs. In this study we tried to establish the potential role of bmi-1 expression in CD34 positive AML cells, the differences according to FAB classification and its prognostic value. The follow up observation is still open. The further analysis is obviously required to determine if bmi-1 could be a potential target for future therapeutic approaches.

The Effect of Cryopreservation on Clonogenic Capacity and In Vitro Expansion Potential of Umbilical Cord Blood Progenitor Cells

Background Umbilical cord blood progenitor cells have been demonstrated to possess significant advantages over bone marrow in terms of proliferative capacity and immunologic reactivity. But the low number of hematopoeitic stem cells (HSC) is the most important limitation of its use. The ex vivo expansion of cord blood progenitor cells is the current strategy to overcome this problem. Futhermore, among the factors that enable successful cord blood transplantation is the ability to store and subsequently recover a sufficient number of viable cells. Since it would be costly to expand umbilical cord blood (UCB) progenitor cells, it is important to determine the feasibility and reproducibility of progenitor cell expansion after cryopreservation. We evaluated whether cryopreservation procedures might impair the clonogenic capacity and in vitro expansion of UCB. Materials and methods We evaluated the cell viability, clonogenic capacity, CD34 +38 − content and in vitro expansion potential of progenitor cells from UCB ( n = 10) separated mononuclear cells (MNC), before and after 1 month of cryopreservation by programmed rate freezing. Results Although cell viability decreased after cryopreservation ( P < .05), there was no significant difference in CD34 + or CD34 +38 − absolute count, colonogenic capacity and in vitro expansion potential of cord blood progenitor cells ( P > .05). Conclusions Since the survival of CD34 + cells was greater than other elements, CD34 + cells seem more tolerant to cryopreservation than the other nucleated populations. Moreover in vitro expansion of UCB progenitor cells may be obtained following cryopreservation. Our results suggest that cryopreservation procedures do not impair the clonogenic capacity and in vitro expansion potential of cord blood stem/progenitor cells.

Large‐volume leukapheresis yields more viable CD34+ cells and colony‐forming units than normal‐volume leukapheresis, especially in patients who mobilize low numbers of CD34+ cells

Large-volume leukapheresis (LVL) differs from normal-volume leukapheresis (NVL) by increased blood flow and altered anticoagulation regimen. LVL is now regarded as a safe procedure for collection of peripheral blood progenitor cells (PBPCs), but it is not known whether the procedure will alter CD34+ cell quality or will be useful for patients who mobilize few CD34+ cells into peripheral blood. The results from 82 LVL and 125 NVL (4.0-5.3 and 2.7-3.5 times the patients' blood volumes processed, respectively) were retrospectively analyzed in altogether 112 consecutive patients with malignant diseases. The LVL yielded significantly more CD34+ cells (4.2 x 10(6) vs. 3.1 x 10(6)/kg, p = 0.006, all patients; and 1.8 x 10(6) vs. 1.3 x 10(6)/kg, p = 0.004, bad mobilizers) and significantly higher colony-forming units (77 x 10(4) vs. 33 x 10(4)/kg; all patients and 33 x 10(4) vs. 20 x 10(4)/kg, p < 0.001, both groups). Significantly fewer leukapheresis procedures were required to obtain 2 x 10(6) CD34+ cells per kg (one vs. two, p = 0.001, all patients; and two vs. three, p = 0.009, bad mobilizers). No significant differences in CD34+ cell viability and time to hematologic recovery were observed between the patients who received PBPCs harvested by NVL and LVL. Although a median platelet loss of 36 percent can be expected, LVL can be recommended as the standard apheresis method for PBPC collections in patients with malignant diseases. LVL is particularly useful in patients who mobilize a low number of CD34+ cells into the peripheral blood.

Comparison of Different Lymphoma Cell Purging Modalities: An Experimental Study with K422 Lymphoma Cells

Blood-derived autografts from patients with non-Hodgkin’s lymphoma (NHL) are frequently contaminated with clonogenic lymphoma cells. In order to obtain a more efficient lymphoma cell depletion from the transplants we compared the efficiency of different purging techniques: B-cell-directed depletion, CD34+ selection by immunomagnetic beads (IMB selection) or by immunoadsorption with a biotin-avidin column (BAC selection). Furthermore, two combination approaches were investigated: IMB selection after B-cell depletion as well as BAC selection after B-cell depletion. To assess purging efficiency, fluorescence-tagged (PKH26) K422 follicular NHL cells were admixed to the respective samples of leukapheresis products. BAC selection following B-cell depletion compared to BAC selection alone showed no significant differences in CD34+ purity (77 ± 12.5% vs. 70.7 ± 5.4%) (mean ± SE) or CD34+ recovery (35.3 ± 8.5% vs. 32.8 ± 10.4%), but a significantly (p < 0.005) higher lymphoma cell purging efficiency (log 4.32 ± 0.15 vs. log 2.92 ± 0.19). IMB selection following B-cell depletion and IMB selection alone resulted in a CD34+ purity of 40.8 ± 8.7% and 64.7 ± 7.9%, a CD34+ recovery of 47.2 ± 8.3% and 26.5 ± 6.5% and a lymphoma cell purging efficiency of log 3.68 ± 0.16 and log 3.48 ± 0.21, respectively. Lymphoma cell purging efficiency after either CD34 selection method alone as well as after either double purging method was significantly higher (p < 0.0005 and p < 0.00005, respectively) compared to B-cell depletion alone (log 1.44 ± 0.23). Our results argue for the combination of different purging modalities to achieve a maximal lymphoma cell depletion.

Gene transfer of alpha interferon into hematopoietic stem cells

Gene transfer or gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. IFN-α has been used in the management of leukemia, and gene transfer of the IFN-α gene into hematopoietic progenitor cells may have great potential for the treatment of chronic myelogenous leukemia (CML). Therefore, we examined the ability of adenovirus (Ad)-IFN-α gene construct to transfect normal bone marrow hematopoietic CD34 + stem cells and the production of IFN-α protein by these cells. Ad-cytomegalovirus (CMV) promoter-driven IFN-α at multiple doses was assessed to transfect highly purified CD34 + cells in liquid culture. Optimal transduction of CD34 + cells with the AdCMV-IFN-α construct was achieved using 120 plaque forming units (pfu). Flow cytometric determinations revealed that there was no significant difference in CD34 + cell viability for the 8 or 12-h transfection periods. Immunoassay of IFN-α produced by CD34 + cells shows that IFN-α levels increased several fold in transfected cells and this was not seen in CD34 + cells transfected with the heme oxygenase gene (HO-1). These in vitro data suggest that adenovirus-mediated gene transfer of IFN-α into hematopoietic stem cells can be achieved and that the IFN-α protein is produced by viable CD34 progenitor cells.

Pediatric peripheral blood progenitor cell collection: haemonetics MCS 3P versus COBE Spectra versus Fresenius AS104.

An increasing number of apheresis machines are becoming available for peripheral blood progenitor cell (PBPC) collection in children. At the Children's Hospital of Florence (Italy), three apheresis machines were evaluated: MCS 3P (Haemonetics) (10 procedures in 4 patients, aged 10-12 years, weight 23.5-64 kg), Spectra, (COBE) (8 procedures in 3 patients, aged 4-17 years, weight 19-59 kg), and AS104 (Fresenius) (24 procedures in 9 patients, aged 2-16 years, weight 13.6-60 kg). For PBPC quantitative analysis, CD34 cytofluorimetry was employed. Relevant variables analyzed included efficiency of CD34+ cell extraction and enrichment, mononuclear cell purity and red cell contamination of the apheresis components, and platelet count decreases after leukapheresis. No significant differences in CD34+ cell-extraction abilities were found. However, the AS104 provided consistently purer leukapheresis components in terms of mononuclear cell and CD34+ cell enrichment (441 +/- 59%, vs. 240 +/- 35% and 290 +/- 42% for MCS 3P and Spectra, respectively). Postapheresis platelet counts dropped the least with the AS104. The smallest patient who underwent apheresis with MCS 3P (the only machine working on discontinuous flow and hence with greater volume shifts) weighed 23.5 kg and tolerated the procedure well, with no signs of hemodynamic instability. No significant complications were observed. All machines seem to have comparable PBPC extraction efficiency, but the AS104 seems to give the component with the greatest PBPC enrichment. This feature might be relevant for further ex vivo cell processing (CD34+ cell selection, expansion, and so on).

Analysis of the effect of prior therapy on progenitor cell yield: use of a chemotherapy scoring system.

A quantitative analysis of peripheral blood stem cell (PBSC) yield, measuring absolute numbers of CD34+ cells x 10(6)/kg and CFU-C x 10(4)/kg was performed in 74 consecutive patients. The interval or 'gap' from the end of previous chemotherapy to the date of priming was recorded in weeks. Geometric mean CD34 and CFU-C values were significantly higher in patients with a score of < or = 60 compared to those with score > 60 (P = 0.003 and 0.02, respectively) and a significant difference in CD34 values was also found when scores of < or = 38 were compared with scores > 38 (P = 0.003), with the difference in CFU-C values approaching significance (P = 0.08). Patients exposed to toxicity factor 4 drugs had significant lowering of both CD34 and CFU-C values (P < 0.001 and P = 0.038) and this emerged as the only independent factor when analysed using linear regression. No significant difference in the geometric mean CD34 or CFU-C values of patients was found in any of the gap categories analysed. Prior exposure to toxicity factor 4 drugs had a significant adverse effect on stem cell yield and should be avoided or minimized prior to stem cell harvesting.