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.     

Storage characteristics of cord blood progenitor cells: report of a multicenter study by the cellular therapies team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative

BACKGROUND: Most hematopoietic progenitor cell (HPC) products are infused or processed shortly after collection, but in some cases this may be delayed for up to 48 hours. A number of variables such as temperature and cell concentration are of critical importance for the integrity of HPCs during this time. STUDY DESIGN AND METHODS: We evaluated critical variables using cord blood HPC units that were divided equally and stored at 4°C versus room temperature (RT) for up to 96 hours. Total nucleated cell (TNC) and mononuclear cell (MNC) counts, viable CD34+ cell counts, and CD45+ cell viability as well as colony‐forming unit–granulocyte‐macrophage (CFU‐GM) present over time at each temperature were determined. RESULTS: Overall, the data indicate that with the exception of viable CD34+ cells, there was a significant decrease in each variable measured for 72 to 96 hours and, with the exception of viable CD34+ cells and CFU‐GM, the reductions were significantly greater in RT units than 4°C units. There was an increase in viable CD34+ count for units where TNC count was greater than 8.5 × 10⁹/L, compared with units where TNC count was less than 8.5 × 10⁹/L, that was different for each storage temperature. CONCLUSIONS: Cord blood HPC collections maintained at 4°C retained higher TNC counts, MNC counts, and CD45+ cell viability over a 72‐ to 96‐hour storage period.     

Characteristics of thawed autologous umbilical cord blood

BACKGROUND: Autologous umbilical cord blood (AutoUCB) has historically been cryopreserved for potential use in hematopoietic transplantation. Increasingly, private AutoUCB banking is performed for therapies unavailable today. A Phase I trial using AutoUCB treatment for early pediatric Type 1 diabetes afforded us an opportunity to analyze characteristics of AutoUCBs. STUDY DESIGN AND METHODS: Twenty AutoUCBs from AABB‐accredited private cord blood banks (CBBs) were evaluated for collection, processing, cryopreservation, and thaw characteristics. Using a standardized thaw‐wash method, AutoUCBs were assessed for viable total nucleated cells (vTNCs), viable CD34+ (vCD34+), and colony‐forming unit–granulocyte‐macrophage counts. Postthaw %vTNC recoveries were compared against processing characteristics and analyzed according to processing method, cryopreservation volume, concentration, container, and length of storage. RESULTS: AutoUCB collection volumes (19.9‐170 mL), cryopreserved TNC counts (7.6 × 10⁷‐3.34 × 10⁹), %TNC processing recoveries (39%‐100%), postthaw %vTNC recoveries (58%‐100%), and %vCD34+ recoveries (26%‐96%) varied widely. Regarding cell dose requirements, only 11% of evaluable AutoUCBs achieved the minimum TNC count of at least 9.0 × 10⁸ to meet the National Cord Blood Inventory banking threshold, and only 50% met the minimum of 5.0 × 10⁸ TNC count for Food and Drug Administration cord blood licensure eligibility. %vTNC recoveries correlated with %vCD34+ recoveries (R = 0.7; p = 0.03). Length of storage, cryopreservation volume, concentration, and container type did not affect postthaw %vTNC recoveries. CBB processing method, however, was associated with %vTNC postprocessing recoveries, with unmanipulated and plasma‐depleted AutoUCBs having the highest postthaw %vTNC recovery, followed by RBC‐depleted and density gradient–separated AutoUCBs. CONCLUSION: The high variability and low counts found in AutoUCB banking suggest that further standardization of characterization, collection, and processing procedures is needed.     

CD133+ cell content does not influence recovery time after hematopoietic stem cell transplantation

BACKGROUND: Infusion of an adequate dose of CD34+ mononuclear hematopoietic stem cells (HSCs) is the single most important variable to assure success in hematopoietic grafting. CD133+ HSCs constitute the CD34+ subgroup with higher differentiation potential. The number of granulocyte–colony‐stimulating factor (G‐CSF)‐mobilized CD133+ HSCs administered during hematopoietic grafting and its relationship with the number of days needed to regain hematopoiesis was determined. STUDY DESIGN AND METHODS: Thirty‐eight patients with malignant hematologic diseases who received an autologous (n = 15) or allogeneic (n = 23) HSC transplant were prospectively evaluated. G‐CSF was administered for 5 days at 10 µg/kg/day. Hematopoietic progenitors were recovered from peripheral blood on day 5 by leukopheresis. CD34+ and CD133+/CD34+ cell populations were quantified by flow cytometry; the number of days to hematologic recovery was documented. RESULTS: A median dose of 4.56 × 10⁶/kg CD34+ HSCs (range, 1.35 × 10⁶‐14.6 × 10⁶) was recovered and transplanted; of these grafted cells, a median 3.25 × 10⁶ were also CD133+ (range, 1.25 × 10⁶‐14.3 × 10⁶). In the autologous group, the median number of days to reach a platelet (PLT) count of 20 × 10⁹/L or greater was 12, and 15 days to obtain a neutrophil count of 0.5 × 10⁹/L or greater; in the allogeneic group 13 and 16 days, respectively, were required (p > 0.05). A median 76.5% of G‐CSF–mobilized CD34+ HSCs coexpressed the CD133+ antigen (range, 23.1‐97.9). CONCLUSIONS: A higher number of CD133+/CD34+ HSCs in the graft was not clearly associated with a shorter neutrophil or PLT recovery time in either allogeneic or autologous recipients.     

Effect of nucleated cell count and cryopreservation on engraftment post autologous stem cell transplant

Using 753 collections from 426 adult haematology patients, we conducted a retrospective, analysis into the effects of overnight storage and nucleated cell counts (NCC) on viable, CD34⁺ (vCD34⁺) recovery and engraftment kinetics post autologous stem cell, transplant (ASCT) with peripheral blood stem cells (PBSC). There were significant, differences in vCD34 + recovery ( P < 0.01) after cryopreservation associated with, the fresh NCC of ≥ 300 × 10 ⁶ /mL in products stored overnight, but no association, with time to platelet or neutrophil engraftment post-ASCT was observed for these, products. There was no association of vCD34⁺ numbers or engraftment kinetics with cryopreserved NCC with either below or greater than the local recommended concentration of 400 × 10⁶ /mL of product. However, there was significant difference in engraftment kinetics in relation to the viable CD34⁺ dose given at ASCT, in relation to the time to early engraftment and the amount of platelet support given during the engraftment period post-ASCT. We conclude the vCD34⁺ dose at ASCT is of great importance to early engraftment kinetics and that NCC is an important factor during overnight storage, but not for cryopreservation of PBSC. In light of our findings, we recommend that apheresis products collected in a closed system can safely be stored undiluted overnight.     

Thawing of cryopreserved hematopoietic progenitor cells from apheresis with a new dry‐warming device

BACKGROUND: Current thawing techniques of cryopreserved progenitor cells are based on the use of a water bath. The aim of this study has been to assess the progenitor cell viability and the time of hematopoietic engraftment after transplantation of cell products thawed with a new dry‐thawing device. STUDY DESIGN AND METHODS: In the preclinical phase, two cryobags from the same patient were thawed with the standard technique and with the dry system method in parallel (n = 5, Protocol A and Protocol B, respectively). In the clinical phase, cryobags were thawed with the dry system and the time to hematopoietic engraftment after autologous transplantation (n = 52) was compared with those of a control group of patients whose progenitor cell products were thawed with the standard technique (n = 52). RESULTS: There were no statistical differences in nuclear and CD34+ cell viability, total colony‐forming cells, and cloning efficiency after thawing with Protocols A and B. Days to neutrophil (>0.5 × 10⁹ and >1 × 10⁹/L) and platelet engraftment (>20 × 10⁹ and >50 × 10⁹/L) were not different between patients transplanted with products thawed with Protocols A and B. CONCLUSION: Progenitor cell viability and function are preserved with this dry‐thawing system. The time to hematopoietic engraftment of patients after transplantation is comparable to those infused with progenitor cells thawed with the water bath technique. Thawing cell products without the use of water and in a dry environment might favor the use of this dry method.     

Cryopreservation of hematopoietic progenitor cells from apheresis at high cell concentrations does not impair the hematopoietic recovery after transplantation

BACKGROUND: The high number of nuclear cells (NCs) from hematopoietic progenitor cells-apheresis (HPC-A) requires cryopreservation in large volumes or at high NC concentrations. The effect of NC concentration during cryopreservation has yet to be examined. STUDY DESIGN AND METHODS: In the experimental arm (n = 610, Protocol B), the first HPC-A sample from the patient was cryopreserved in two cryobags and subsequent collections in one cryobag, resulting in high NC concentrations (>100 x 10(6) NCs/mL) in most cases. The effect of NC concentrations at freezing in NC recovery after thawing and engraftment kinetics was analyzed and compared with a group of HPC-A cryopreserved at standard NC concentrations (n = 455, Protocol A). RESULTS: The mean (SD) NC concentration at freezing was 78 (28) x 10(6) per mL (median, 82 x 10(6)/mL; range, 12 x 10(6)-156 x 10(6)/mL) and 183 (108) x 10(6) per mL (median, 156 x 10(6)/mL; range, 16 x 10(6)-678 x 10(6)/mL), for HPC-A cryopreserved according to Protocols A and B, respectively. The NC viabilities of the test vials and HPC-A components after thawing were 88 percent versus 85 percent and 85 percent versus 82 percent, and the cloning efficiency was 49 percent versus 33 percent for Protocols A and B, respectively (p < 0.001). Significant differences were not observed in the recovery of NCs. Days to neutrophil and platelet engraftment were not different between patients transplanted in the standard- (n = 143) or high-cell-concentration group (n = 238). CONCLUSION: The cryopreservation of HPC-A at higher than standard NC concentrations has no adverse impact on hematopoietic reconstitution after transplantation.     

Successful hematopoietic stem cell collection in patients who fail initial plerixafor mobilization for autologous stem cell transplant

We report our experience of collecting stem cells in patients who failed to mobilize sufficient hematopoietic stem cell (HSC) using plerixafor (P) in the initial mobilization attempt. Twenty four patients were identified who failed a first mobilization attempt using P. Of these, 22 patients received granulocyte colony stimulating factor (G‐CSF) and two patients received cyclophosphamide (CY) + G‐CSF in combination with P for the initial attempt. The agents used for second collection attempt were granulocyte macrophage colony stimulating factor (GM‐CSF) + G‐CSF (19 patients), G‐CSF + P (three patients), CY + G‐CSF (one patient), and bone marrow harvest (one patient). A median of 0.6 × 10⁶ CD34⁺ cells/kg (range 0–1.97) were collected in the initial attempt. A second collection was attempted at a median of 22 days (range 15–127) after the first failed mobilization. The median CD34⁺ cell dose collected with the second attempt was 1.1 × 10⁶ CD34⁺ cells/kg (range 0–7.2). A third collection was attempted in six patients at median of 51 days (range 34–163) after the first failed mobilization. These patients collected a median of 1.1 × 10⁶ CD34⁺ cells/kg (range 0–6.5). Total of 16 patients (67%) collected sufficient cells to undergo autologous stem cell transplant and eight patients (33%) were able to collect ≥2 × 10⁶ CD34⁺ cells/kg in a single subsequent attempt. Our experience suggests that a majority of patients who fail primary mobilization despite use of P can collect sufficient HSC with a subsequent attempt using combination of G‐CSF with either P or GM‐CSF. J. Clin. Apheresis 29:293–298 2014. © 2014 Wiley Periodicals, Inc.     

Preparation of autologous bone marrow grafts for cryopreservation using the AS104 cell separator

We evaluated the AS104 cell separator (Fresenius AG, Bad Homburg, Germany) for ex vivo processing of bone marrow (BM) grafts of 43 patients suffering from germ cell cancer (GCC, n = 22), acute lymphocytic leukemia (ALL, n = 13) and malignant lymphoma (ML, n = 8). Recoveries of total nucleated cells (TNC), mononuclear cells (MNC) and colony-forming units granulocyte-macrophage (CFU-GM) were determined in the BM concentrates prepared for cryopreservation. Hematopoietic reconstitution was analyzed in patients who underwent autologous transplantation following high-dose radio-/chemotherapy (HDRCT). Processing of the BM suspension with a median volume of 1,013 ml (range: 422–1,574) resulted in 156 ml (80–186) within 50–120 min (median: 90). In the BM concentrates, medians of 28.6% TNC (10.6–69.6), 37.9% MNC (22.3–86.4), and 52.4% CFU-GM (20.8–96.4) were recovered. Twenty-six patients underwent HDRCT with reinfusion of autologous BM and were evaluable for engraftment. They received a median of 0.8 × 10⁸ MNC/kg (0.3–1.6 × 10⁸) and 2.2 × 10⁴ CFU-GM/kg (0.6–12.8 × 10⁴) for hematopoietic rescue. Engraftment with neutrophils >500/μl occurred in a median time of 12 days (8–33) in all patients. We conclude that ex vivo processing of autologous BM with median recovery rates of 37.9% for MNC, and 52.4% for CFU-GM, results in a cell population that can rescue patients from HDRCT. The described technique is convenient, time-efficient, and provides reliable results in preparing BM autografts for cryopreservation. J. Clin. Apheresis 12:179–182, 1997. © 1997 Wiley-Liss, Inc.     

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.     

A randomized clinical trial comparing granulocyte-colony-stimulating factor administration sites for mobilization of peripheral blood stem cells for patients with hematologic malignancies undergoing autologous stem cell transplantation

BACKGROUND: A study was undertaken to investigate whether granulocyte–colony‐stimulating factor (G‐CSF) injection in lower adipose tissue–containing sites (arms and legs) would result in a lower exposure and reduced stem cell collection efficiency compared with injection into abdominal skin. STUDY DESIGN AND METHODS: We completed a prospective randomized study to determine the efficacy and tolerability of different injection sites for patients with multiple myeloma or lymphoma undergoing stem cell mobilization and apheresis. Primary endpoints were the number of CD34+ cells collected and the number of days of apheresis. Forty patients were randomly assigned to receive cytokine injections in their abdomen (Group A) or extremities (Group B). Randomization was stratified based on diagnosis (myeloma, n = 29 vs. lymphoma, n = 11), age, and mobilization strategy and balanced across demographic factors and body mass index. RESULTS: Thirty‐five subjects were evaluable for the primary endpoint: 18 in Group A and 17 in Group B. One evaluable subject in each group failed to collect a minimum dose of at least 2.0 × 10⁶ CD34+ cells/kg. The mean numbers of CD34+ cells (±SD) collected were not different between Groups A and B (9.15 × 10⁶ ± 4.7 × 10⁶/kg vs. 9.85 × 10⁶ ± 5 × 10⁶/kg, respectively; p = NS) after a median of 2 days of apheresis. Adverse events were not different between the two groups. CONCLUSION: The site of G‐CSF administration does not affect the number of CD34+ cells collected by apheresis or the duration of apheresis needed to reach the target cell dose.     

Current strategies for the management of autologous peripheral blood stem cell mobilization failures in patients with multiple myeloma

Multiple myeloma (MM) is the leading indication of autologous hematopoietic stem cell transplantation (AHSCT) worldwide. The collection of PBSCs is the essential step for AHSCT. The limits for minimum and optimum CD34⁺ cells collected have been accepted as 2 × 10⁶/kg and ≥4 × 10⁶/kg for single AHSCT; 4 × 10⁶/kg and ≥8‐10 × 10⁶/kg for double AHSCT. Despite the success of conventional methods for PBSC mobilization in MM, mobilization failure is still a concern depending on patient age, duration of disease, and the type of induction therapy. By definition, “proven poor mobilizer” is the occurrence of mobilization failure (CD34+ cell peak <20/µL peripheral blood) after adequate preparation (after 6 days of G‐CSF 10 µg/kg body weight alone or after 20 days of G‐CSF >5 µg/kg body weight following chemotherapy) or a CD34+ cell yield of <2.0 × 10⁶/kg body weight after three consecutive apheresis. “Predicted poor mobilizer” involves (1) a failure of a previous collection attempt OR (2) a previous history of extensive radiotherapy or full courses of therapy affecting mobilization OR (3) the presence of at least two of the following features: advanced disease (>2 lines of chemotherapy), refractory disease, extensive bone marrow involvement or cellularity of 30% at the time of mobilization or age >65 years. This article aims at discussing the risk factors for mobilization failure in the era of novel antimyeloma drugs, defining the poor mobilizer concept and summarizing the current and future strategies for the prevention and management of mobilization failures in MM.     

Effects of cell concentration,time of fresh storage,and cryopreservation on peripheral blood stem cells: PBSC fresh storage and cryopreservation

IntroductionPeripheral blood stem cells are widely used in autologous or allogeneic transplantation. The quality of the product directly impacts clinical outcomes, and the cell quality and/or functionality may be influenced by the storage conditions as time, temperature, total nucleated cells (TNC) concentration and cryopreservation requirement.ObjectiveTo verify the effects of time, cell concentration, and cryopreservation/thawing in the viability and functionality of stem cells for transplantation.MethodsWe evaluated TNC, CD45⁺ viable cells, CD34⁺ viable cells, and cell viability and functionality of 11 samples. Measurements were performed immediately and 24 h, 48 h, 72 h, and 96 h after sample collection at high and low TNC concentrations. The same parameters were also evaluated after cryopreservation and thawing of the samples.ResultDuration of storage and TNC concentration exhibited a negative effect on cell quality (CD45⁺ viable cells, CD34⁺ viable cells and functionality). Moreover, the association of these parameters increased the negative effect on graft quality. Cryopreservation and thawing also negatively affected the collected sample regarding viable CD34⁺ cells (recovery 66.2 %), viable CD45⁺ cells (recovery 56.8 %), and 7-AAD viability. No significant losses in viable CD45⁺/CD34⁺ cells and functionality were observed in the first 24 h in both TNC conditions.ConclusionThese results emphasize the importance to consider carefully the storage conditions until transplantation, measuring TNC/μL until 24 h after collection (diluting the product when TNC > 300 × 10³/μL) and infusing fresh graft as soon as possible.     

Hematopoietic recovery in cancer patients after transplantation of autologous peripheral blood CD34+ cells or unmanipulated peripheral blood stem and progenitor cells

BACKGROUND: A study of CD34+ cell selection and transplantation was carried out with particular emphasis on characteristics of short- and long-term hematopoietic recovery. STUDY DESIGN AND METHODS: Peripheral blood stem and progenitor cells (PBPCs) were collected from 32 patients, and 17 CD34+ cell-selection procedures were carried out in 15 of the 32. One patient in whom two procedures failed to provide 1 × 10(6) CD34+ cells per kg was excluded from further analysis. After conditioning, patients received CD34+ cells (n = 10, CD34 group) or unmanipulated (n = 17, PBPC group) PBPCs containing equivalent amounts of CD34+ cells or progenitors. RESULTS: The yield of CD34+ cells was 53 percent (18–100) with a purity of 63 percent (49–82). The CD34+ fraction contained 66 percent of colony-forming units-granulocyte- macrophage (CFU-GM) and 58 percent of CFU of mixed lineages, but only 33 percent of burst-forming units-erythroid (BFU-E) (p < 0.05). Early recovery of neutrophils and reticulocytes was identical in the two groups, although a slight delay in platelet recovery may be seen with CD34+ cell selection. Late hematopoietic reconstitution, up to 1.5 years after transplant, was also similar. The two groups were thus combined for analyses of dose effects. A dose of 40 × 10(4) CFU-GM per kg ensured recovery of neutrophils to a level of 1 × 10(9) per L within 11 days, 15 × 10(4) CFU of mixed lineages per kg was associated with platelet independence within 11 days, and 100 × 10(4) BFU-E per kg predicted red cell independence within 13 days. However, a continuous effect of cell dose well beyond these thresholds was apparent, at least for neutrophil recovery. CONCLUSION: CD34+ cell selection, despite lower efficiency in collecting BFU-E, provides a suitable graft with hematopoietic capacity comparable to that of unmanipulated PBPCs. In both groups, all patients will eventually show hematopoietic recovery of all three lineages with 1 × 10(6) CD34+ cells per kg or 5 × 10(4) CFU-GM per kg, but a dose of 5 × 10(6) CD34+ cells or 40 × 10(4) CFU-GM per kg is critical to ensure rapid recovery.     

Obtaining of CD34+ cells from healthy blood donors: development of a rapid and efficient procedure using leukoreduction filters

BACKGROUND: Human CD34+ cells are mandatory to study many aspects of human hematopoiesis. Their low frequency in blood or marrow and ethical reasons limit their obtainment in large quantities. Leukoreduction filters (LRFs) are discarded after preparation of red blood cells. The CD34+ cell concentration in healthy donor blood is low (1 × 10³‐4 × 10³/mL), but their number trapped in one LRF after filtration of 400 to 450 mL of blood is high (0.4 × 10⁶‐1.6 × 10⁶). STUDY DESIGN AND METHODS: To develop a procedure allowing obtainment of purified CD34+ cells from LRFs with a good yield, white blood cell (WBC) recoveries after a 500‐mL continuous or after sequential elution (50‐ or 20‐mL fractions) were compared. Different WBC and mononuclear cell (MNC) centrifugation methods were tested to minimize their PLT contamination before the CD34+ cell immunomagnetic selection. Cell functionality was finally analyzed under various culture conditions. RESULTS: The 20‐mL back‐flushing of LRFs allowed the most efficient WBC recovery. The next steps (110 × g centrifugation, MNC separation on Ficoll, and washes) resulted in a cell suspension in which the lymphocyte recovery was approximately 76 ± 10% and the PLT contamination below 1.6%. After immunomagnetic selection, 4 × 10⁵ to 6 × 10⁵ cells containing approximately 85% of functional CD34+ cells were obtained. CONCLUSION: This procedure allows the easy, rapid (<5 hr), and efficient preparation of large quantities of CD34+ cells having functional activities similar to those of CD34+ cells from other sources. Therefore, easily available and virally safe, LRFs represent an important and regular WBC source to work with human CD34+ cells, but also with other WBC types.     

Mobilization effects of G‐CSF,GM‐CSF,and darbepoetin‐α for allogeneic peripheral blood stem cell transplantation

The effects of GM‐/G‐CSF and darbepoetin‐α on stem cell mobilization were investigated. From February 2005 to March 2007, 30 allogeneic sibling donors were randomly assigned to a G‐CSF group (5 μg/kg/day for 5–7 days) or triple group (GM‐CSF 10 μg/kg/day on 1st and 2nd day, G‐CSF 5 μg/kg/day for 5–7 days, and darbepoetin‐α 40 mg on 1st day). The MNCs and CD34⁺ cells were not different between the two groups, although the doses (×10⁸/kg of recipient body weight) of CD3⁺ cells (3.64 ± 1.75 vs. 2.63 ± 1.36, P = 0.089) and CD8⁺ cells (1.07 ± 0.53 vs. 0.60 ± 0.30, P = 0.006) were lower in the triple group. The engraftments, frequency of RBC transfusions, and hemoglobin recovery were not different between the two groups. The cumulative incidence of overall and Grades II–IV aGVHD was 64.3% vs. 61.1% and 25.9% vs. 27.1% in the G‐CSF and triple regimen group, respectively, whereas the cumulative incidence of cGVHD was 20.8 ± 1.3% and 24.4 ± 1.7%, respectively. In conclusion, the triple regimen did not seem to be superior to G‐CSF alone in terms of the CD34+ cell dose, hemoglobin recovery, and GVHD. However, the CD8+ cell count was significantly lower in the triple regimen group. The role of a lower CD8+ cell count in the graft may need to be elucidated in the future. J. Clin. Apheresis, 2009. © 2009 Wiley‐Liss, Inc.     

Delayed recovery after autologous peripheral hematopoietic cell transplantation: potential effect of a high number of total nucleated cells in the graft

BACKGROUND: Some patients demonstrate delayed recoveries after autologous hematopoietic stem cell transplantation despite infusion of an adequate number of CD34+ cells/kg and clinically stable status. Factors considered being possible predictors of this outcome in this context were explored. STUDY DESIGN AND METHODS: A total of 246 patients were evaluated in terms of engraftment. Delayed recovery was defined by white blood cell recovery time exceeding mean + 1 SEM. Clinical factors and graft characteristics were examined. Comparisons between patients with normal or delayed engraftment were made. Proinflammatory cytokines and proteolytic enzyme quantification and CXCR4+ and CD44+ cell enumeration were performed on peripheral hematopoietic stem cells (PHSC) product samples of patients with delayed engraftment and patients with usual engraftment time. RESULTS: Sixteen patients, who received at least 3 × 10⁶ CD34+ cells/kg without known clinical factors likely to affect engraftment, demonstrated a delayed recovery time of over 20 days. Some graft variables were found to be significantly increased in these patients by univariate analysis. One variable was the total number of nucleated cells cryopreserved and infused. Among the nucleated cells, the absolute number of granulocytes before and after cryopreservation also differed significantly between the two groups. A multivariate analysis showed that the main predictive factor for delayed recovery was the number of nucleated cells in the graft (p = 0.0044). The influence of contaminating cells might be related to the release of elastase, matrix metalloproteinase‐9, interleukin (IL)‐1β, and IL‐6 involved in stem cell homing. CONCLUSION: Therefore, the numeration of total nucleated cells and granulocytes should be considered as a possible quality control variable of PHSCs submitted for cryopreservation.     

Granulocyte colony‐stimulating factor (G‐CSF) depresses angiogenesis in vivo and in vitro: implications for sourcing cells for vascular regeneration therapy

Summary. Background: The most common source of hematopoietic progenitor cells (HPCs) for hematopoietic reconstitution comprises granulocyte colony‐stimulating factor (G‐CSF)‐mobilized peripheral blood stem cells (PBSCs). It has been proposed that endothelial progenitor cells (EPCs) share precursors with HPCs, and that EPC release may accompany HPC mobilization to the circulation following G‐CSF administration. Objective: To investigate EPC activity following HPC mobilization, and the direct effects of exogenous G‐CSF administration on human umbilical vein endothelial cells (HUVECs) and endothelial outgrowth cells (EOCs), using in vitro and in vivo correlates of angiogenesis. Patients/Methods: Heparinized venous blood samples were collected from healthy volunteers and from cord blood at parturition. G‐CSF‐mobilized samples were collected before administration, at apheresis harvest, and at follow‐up. PBSCs were phenotyped by flow cytometry, and cultured in standard colony‐forming unit (CFU)‐EPC and EOC assays. The effect of exogenous G‐CSF was investigated by addition of it to HUVECs and EOCs in standard tubule formation and aortic ring assays, and in an in vivo sponge implantation model. Results: Our data show that G‐CSF mobilization of PBSCs produces a profound, reversible depression of circulating CFU‐EPCs. Furthermore, G‐CSF administration did not mobilize CD34+CD133? cells, which include precursors of EOCs. No EOCs were cultured from any mobilized PBSCs studied. Exogenous G‐CSF inhibited CFU‐EPC generation, HUVEC and EOC tubule formation, microvessel outgrowth, and implanted sponge vascularization in mice. Conclusions: G‐CSF administration depresses both endothelial cell angiogenesis and monocyte proangiogenic activity, and we suggest that any angiogenic benefit observed following implantation of cells mobilized by G‐CSF may come only from a paracrine effect from HPCs.     

Leukapheresis in non–cytokine‐stimulated donors with a new apheresis system: first‐time collection results and evaluation of subsequent cryopreservation

BACKGROUND: Adoptive cell therapy based on mononuclear cells (MNCs) became an important modality of cancer immunotherapy. Data about collection results and donor response of leukapheresis with the Spectra Optia v.5.0 (Terumo BCT) in nonmobilized donors are required. STUDY DESIGN AND METHODS: Twelve MNC collections were performed using the Spectra Optia v.5.0 in non–cytokine‐stimulated donors. Leukapheresis products and peripheral blood samples from donors were assayed for CD45+, CD34+, CD3+, and CD14+ cells by flow cytometry. Prefreeze and postthaw cell counts, cell viability, and numbers of colony‐forming units were assessed in cryobags and compared to data from cryovials. RESULTS: Leukapheresis yielded a mean of 5.26 × 10⁹ ± 2.2 × 10⁹ CD45+ cells, 1.5 × 10⁹ ± 0.77 × 10⁹ CD14+ monocytes, and 2.28 × 10⁹ ± 1.2 × 10⁹ CD3+ T cells by processing 6690 ± 930 mL of whole blood. A significant positive correlation between yield of CD3+ T cells and residual platelets (PLTs) and red blood cells (RBCs) was observed. This did not apply for CD34+ and CD14+ white blood cell subsets. Mean collection efficiencies for CD14+ monocytes and CD3+ T cells were 61.8 ± 17 and 37.2 ± 18%, respectively. Recovery of CD14+ cells after cryopreservation was 75.2 ± 8.2%, which was significantly lower than recovery of CD45+ cells (81.4 ± 5.5%; p = 0.01). CONCLUSION: This study of a small cohort demonstrates that the Spectra Optia v.5.0 is capable of collecting low product volumes with satisfactory MNC yields and low residual RBCs and PLTs in non–cytokine‐mobilized apheresis. Our data suggest that cryovials can serve as a representative surrogate for the primary product cryobag.