Transplant-associated thrombotic microangiopathy is an endothelial complication associated with refractoriness of acute GvHD.

There is increasing evidence that endothelial dysfunction is involved in refractoriness of acute GvHD (aGvHD). Here we investigated the hypothesis that another endothelial complication, transplant-associated thrombotic microangiopathy (TMA), contributes to the pathogenesis of aGvHD refractoriness. TMA was retrospectively assessed in 771 patients after allogeneic stem cell transplantation (alloSCT). Incidences of TMA and refractory aGvHD were correlated with biomarkers of endothelial damage obtained before alloSCT for patients receiving or not receiving statin-based endothelial prophylaxis (SEP). Diagnostic criteria for TMA and refractory aGvHD were met by 41 (5.3%) and 76 (10%) patients, respectively. TMA was overrepresented in patients with refractory aGvHD (45.0 vs 2.3% in all other patients, P<0.001). TMA independently increased mortality. Elevated pretransplant suppressor of tumorigenicity-2 and nitrates along with high-risk variants of the thrombomodulin gene were associated with increased risk of TMA. In contrast, SEP abolished the unfavorable outcome predicted by pretransplant biomarkers on TMA risk. Patients on SEP had a significantly lower risk of TMA (P=0.001) and refractory aGvHD (P=0.055) in a multivariate multistate model. Our data provide evidence that TMA contributes to the pathogenesis of aGvHD refractoriness. Patients with an increased TMA risk can be identified pretransplant and may benefit from pharmacological endothelium protection.

GvL effects in T-prolymphocytic leukemia: evidence from MRD kinetics and TCR repertoire analyses.

Allogeneic stem cell transplantation (alloSCT) is used for treating patients with T-prolymphocytic leukemia (T-PLL). However, direct evidence of GvL activity in T-PLL is lacking. We correlated minimal residual disease (MRD) kinetics with immune interventions and T-cell receptor (TCR) repertoire diversity alterations in patients after alloSCT for T-PLL. Longitudinal quantitative MRD monitoring was performed by clone-specific real-time PCR of TCR rearrangements (n=7), and TCR repertoire diversity assessment by next-generation sequencing (NGS; n=3) Although post-transplant immunomodulation (immunosuppression tapering or donor lymphocyte infusions) resulted in significant reduction (>1 log) of MRD levels in 7 of 10 occasions, durable MRD clearance was observed in only two patients. In all three patients analyzed by TCR-NGS, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. Novel clonotypes that could explain a clonal GvL effect did not emerge. In conclusion, TCR-based MRD quantification appears to be a suitable tool for monitoring and guiding treatment interventions in T-PLL. The MRD responses to immune modulation observed here provide first molecular evidence for GvL activity in T-PLL which, however, may be often only transient and reliant on a poly-/oligoclonal rather than a monoclonal T-cell response.

Allogeneic hematopoietic stem cell transplantation for poor-risk CLL: dissecting immune-modulating strategies for disease eradication and treatment of relapse.

To elucidate factors contributing to the effectiveness of allogeneic hematopoietic stem cell transplantation (alloHCT) in high-risk CLL, immune interventions, GvHD and clinical outcome of 77 consecutive patients allografted for CLL were analyzed. Immune modulation (immunosuppression tapering, rituximab-augmented donor lymphocyte infusions) was guided by minimal residual disease (MRD) monitoring and commenced at a median of 91 (22-273) days after alloHCT, resulting in a probability of being event free and MRD-negative 1 year after transplant of 57% (84% in those encountering chronic GvHD). Patients who were event free and MRD-negative at the 12-month landmark had a 4-year PFS of 77% and largely remained durably MRD-negative if MRD clearance had occurred subsequent to immune modulation. Three-year overall survival, PFS, relapse incidence and non-relapse mortality of all 77 patients were 69, 57, 26 and 24%, respectively. Survival was not affected by EBMT risk category but by active disease at alloHCT, which could not be overcome by intensification of conditioning. Twenty-three patients who experienced relapse post alloHCT had a survival of 56% at 2 years after CLL recurrence. In conclusion, MRD-guided immune modulation after alloHCT for high-risk CLL can provide durable MRD clearance in more than half of the patients.

The impact of allogeneic stem cell transplantation on the natural course of poor-risk chronic lymphocytic leukemia as defined by the EBMT consensus criteria: a retrospective donor versus no donor comparison.

BACKGROUND: In a single-center retrospective donor versus no-donor comparison, we investigated if allogeneic stem cell transplantation (alloSCT) can improve the dismal course of poor-risk chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS: All patients with CLL who were referred for evaluation of alloSCT within a 7-year time frame and had a donor search indication according to the EBMT criteria or because of Richter's transformation were included. Patients for whom a matched donor could be found within 3 months (matches) were compared with patients without such a donor (controls). Primary end point was overall survival measured from the 3-month landmark after search initiation. RESULTS: Of 105 patients with donor search, 97 (matches 83; controls 14) were assessable at the 3-month landmark. Matches and controls were comparable for age, gender, time from diagnosis, number of previous regimens, and remission status. Disregarding if alloSCT was actually carried out or not, survival from the 3-month landmark was significantly better in matches versus controls [hazard ratio 0.38, 95% confidence interval (CI) 0.17-0.85; P = 0.014]. The survival benefit of matches remained significant on multivariate analysis. CONCLUSION: This study provides first comparative evidence that alloSCT may have the potential to improve the natural course of poor-risk CLL as defined by the EBMT criteria.

Risk factors and containment of respiratory syncytial virus outbreak in a hematology and transplant unit.

Respiratory syncytial virus (RSV) usually causes self-limiting upper respiratory tract infections, but can be associated with severe lower respiratory tract infection disease (LRTID) in infants and in patients with hematologic malignancies. We have analyzed the risk factors and the measures for containment within an outbreak of nosocomial RSV infections in a hematology and SCT unit. A total of 56 patients were affected (53 RSV-A and 3 RSV-B) including 32 transplant patients (16 allogeneic and 16 autologous). Forty (71%) of the 56 patients suffered from LRTID and 14 (35%) of the patients with LRTID subsequently died. However, because of concomitant infections with fungal and bacterial pathogens, the impact of RSV on the fatal outcome was difficult to assess. Multivariate analysis showed that low levels of IgG were significantly associated with fatal outcome (P=0.007), treatment with oral ribavirin represented a protective factor (P=0.02). An extremely protracted viral shedding was observed in this cohort of patients (median=30.5 days, range: 1-162 days), especially pronounced in patients after allogeneic transplantation (P=0.002). Implementation of rigorous isolation and barrier measures, although challenged by long-term viral carriers, was effective in containment of the outbreak.

Azacitidine and donor lymphocyte infusions as first salvage therapy for relapse of AML or MDS after allogeneic stem cell transplantation.

The combination of azacitidine and donor lymphocyte infusions (DLI) as first salvage therapy for relapse after allogeneic transplantation (allo-HSCT) was studied in 30 patients with acute myeloid leukemia (AML; n=28) or myelodysplastic syndromes (MDS; n=2) within a prospective single-arm multicenter phase-II trial. Treatment schedule contained up to eight cycles azacitidine (100 mg/m(2)/day, days 1-5, every 28 days) followed by DLI (from 1-5 × 10(6) to 1-5 × 10(8) CD3(+)cells/kg) after every second azacitidine cycle. A median of three courses azacitidine (range 1-8) were administered, and 22 patients (73%) received DLI. Overall response rate was 30%, including seven complete remissions (CRs, 23%) and two partial remissions (7%). Five patients remain in CR for a median of 777 days (range 461-888). Patients with MDS or AML with myelodysplasia-related changes were more likely to respond (P=0.011), and a lower blast count (P=0.039) as well as high-risk cytogenetics (P=0.035) correlated with the likelihood to achieve CR. Incidence of acute and chronic graft-versus-host disease was 37% and 17%, respectively. Neutropenia and thrombocytopenia grade III/IV occurred during 65% and 63% of treatment cycles, while infections were the most common grade III/IV non-hematological toxicity. Azacitidine and DLI as salvage therapy is safe, induces long-term remissions and may become an alternative for patients with AML or MDS relapsing after allo-HSCT.

Pentostatin for treatment of steroid-refractory acute GVHD: a retrospective single-center analysis.

Acute GVHD (aGVHD) remains a major cause of mortality in patients undergoing allo-SCT. In particular, the outcome of those patients who fail first-line therapy with glucocorticosteroids is poor. Preliminary reports suggested that the purine analogue pentostatin might be effective for treatment of steroid-refractory aGVHD. Here, we report on our single-center experience with pentostatin in this condition. From 2005 to 2008, a total of 24 consecutive patients, who had undergone first-line salvage treatment for steroid-refractory aGVHD of the gastrointestinal tract with pentostatin, were identified from 301 patients allografted during that period and retrospectively analyzed. Response to treatment, defined as CR or very good PR (VGPR), was observed in nine patients (38%), with a median time to response of 10 days. Although pentostatin was associated with only moderate myelosuppressive toxicity, if any, 2-year survival was only 17% with five of the nine responders dying from infection (four patients) or recurrent GVHD (one patient). We conclude that pentostatin is a moderately effective therapy for steroid-refractory aGVHD, showing response and outcome rates similar to other clinically used regimens.

Molecular mechanisms mediating gastrin-releasing peptide receptor modulation of memory consolidation in the hippocampus.

Although the gastrin-releasing peptide-preferring bombesin receptor (GRPR) has been implicated in memory formation, the underlying molecular events are poorly understood. In the present study, we examined interactions between the GRPR and cellular signaling pathways in influencing memory consolidation in the hippocampus. Male Wistar rats received bilateral infusions of bombesin (BB) into the dorsal hippocampus immediately after inhibitory avoidance (IA) training. Intermediate doses of BB enhanced, whereas a higher dose impaired, 24-h IA memory retention. The BB-induced memory enhancement was prevented by pretraining infusions of a GRPR antagonist or inhibitors of protein kinase C (PKC), mitogen-activated protein kinase (MAPK) kinase and protein kinase A (PKA), but not by a neuromedin B receptor (NMBR) antagonist. We next further investigated the interactions between the GRPR and the PKA pathway. BB-induced enhancement of consolidation was potentiated by coinfusion of activators of the dopamine D1/D5 receptor (D1R)/cAMP/PKA pathway and prevented by a PKA inhibitor. We conclude that memory modulation by hippocampal GRPRs is mediated by the PKC, MAPK, and PKA pathways. Furthermore, pretraining infusion of BB prevented beta-amyloid peptide (25-35)-induced memory impairment, supporting the view that the GRPR is a target for the development of cognitive enhancers for dementia.

Molecular monitoring of tumour load kinetics predicts disease progression after non-myeloablative allogeneic stem cell transplantation in multiple myeloma.

BACKGROUND: Non-myeloablative allogeneic stem cell transplantation followed by immunomodulatory therapies is considered a potentially curative approach in the treatment of multiple myeloma and most effective in a minimal residual disease setting. PATIENTS AND METHODS: The aim of this study was to find the most sensitive real-time PCR assay (TaqMan), based on the IGH rearrangement, to quantify the tumour load of 11 patients with multiple myeloma after non-myeloablative allogeneic transplantation. Patient-allele specific primers (ASO) and the TaqMan probe were derived from CDR2 and CDR3 hypervariable regions of IGH, while consensus primers were located within the FR3 and FR4/JH regions. Four different approaches of primer combinations were tested. RESULTS: ASO-forward and -reverse primers together with the clone-specific TaqMan probe were the most sensitive approach compared with the JH (P=0.071) or the FR3 consensus primer (P <0.001). The detection limit amounted to 1/10(4)-1/10(5) cells. Consecutively, 120 samples from 11 patients prior and post allogeneic transplantation were analysed. Three patients reached complete clinical remission accompanied by molecular remission. Disease progression or relapse was seen in six patients. In five, molecular progressive disease was detected prior to the clinical diagnosis of progression or relapse. CONCLUSION: Patient-specific real-time IGH-PCR provides the opportunity for earlier treatment intervention.

Thrombotic microangiopathy after combined autografting-allografting for multiple myeloma - report of three cases.

Thrombotic microangiopathy (TMA) has been described as a complication of bone marrow or stem cell transplantation. It is usually associated with high dose therapy for autologous transplantation or myeloablative conditioning in the allogeneic setting. Here we report three cases of TMA after reduced intensity conditioning and allogeneic peripheral blood stem cell transplantation. All three patients had high dose Melphalan therapy with autologous stem cell support preceding the allogeneic transplantation for several weeks, which may have contributed to endothelial damage and subsequent development of TMA.

Stable mixed chimerism after T cell-depleted allogeneic hematopoietic stem cell transplantation using conditioning with low-dose total body irradiation and fludarabine.

Although reduced intensity conditioning (RIC) before allografting is associated with low treatment-related morbidity and mortality, graft-versus-host disease (GVHD) remains a significant complication of hematopoietic stem cell transplantation (HSCT). T cell depletion (TCD) has been successfully used in conventional allotransplantation to reduce the incidence of GVHD, but was associated with an increased rate of engraftment failure. In a small cohort of six patients at high risk of developing GVHD we have determined whether sustained engraftment could be achieved using reduced intensity conditioning and T cell depletion in combination. All patients engrafted and 5/6 developed high levels (i.e. > or =95%) of donor chimerism, even though mismatched related or matched unrelated donors were used. Only one patient developed acute GVHD, as he received donor lymphocyte infusions (DLI) for relapse. In summary, TCD might be a useful prophylactic tool in RIC allogeneic HSCT. Although TCD after RIC might be associated with high relapse rate, as 5/6 patients are not in remission, this combined strategy might be appropriate for patients with less aggressive malignant or non-malignant diseases in which high transplant-related morbidity and mortality is not acceptable.

Exogenous peptides presented by transporter associated with antigen processing (TAP)-deficient and TAP-competent cells: intracellular loading and kinetics of presentation.

This study investigates the differential capacity of TAP-deficient T2 cells, TAP-competent EBV cells, and immature and mature dendritic cells to present peptides to preformed CTL lines. It demonstrates that presentation of exogenous peptides involves peptide uptake and loading onto newly synthesized MHC class I molecules. This mechanism was best demonstrated for low affinity peptides in the presence of irrelevant peptides competing for HLA binding sites. Under these circumstances, inhibition of protein synthesis with cycloheximide or vesicular trafficking with brefeldin A significantly reduced the presentation of low affinity peptides. This was not restored by adding exogenous beta(2)-microglobulin to stabilize the MHC complex on the cell surface. In contrast, presentation of high affinity peptides was not sensitive to cycloheximide or brefeldin A, which suggests that different mechanisms may operate for presentation of high and low affinity peptides by TAP-competent cells. High affinity peptides can apparently compete with peptides in preloaded MHC class I molecules at the cell surface, whereas low affinity peptides require empty MHC molecules within cells. Accordingly, very high concentrations of exogenous low affinity peptides in conjunction with active MHC class I metabolism were required to allow successful presentation against a background of competing intracellular high affinity peptides in TAP-competent cells. These findings have implications for the design of peptide and protein-based vaccines.

Interleukin (IL)-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human dendritic cells.

Interleukin (IL)-12 may be secreted as a bioactive T helper type 1 (Th1) cell-inducing heterodimer, as a monomer, or as an antagonistic homodimer. We analyzed the IL-12 produced by mouse splenic dendritic cells (DCs), human thymic DCs, and cultured human monocyte-derived DCs. IL-12 production required both a microbial or T cell-derived stimulus and an appropriate cytokine milieu. The different IL-12 forms were differentially regulated by the cytokines present rather than the stimulus used. IL-4 alone or together with granulocyte/macrophage colony-stimulating factor or interferon gamma effectively enhanced the production of the bioactive heterodimer and selectively reduced the antagonistic homodimer of IL-12. Therefore, IL-4, the major Th2-driving cytokine, provides a negative feedback causing DCs to produce the major Th1-inducing cytokine, bioactive IL-12.

Type I IFNs enhance the terminal differentiation of dendritic cells.

This study identifies type I IFNs as activating cytokines in a serum-free system in which human dendritic cells (DC) were generated from CD34+ progenitor cells. After 14 days of culture in GM-CSF, TNF-alpha, and IL-4, CD34+ progenitors gave rise to a population of large, immature DC expressing CD1a and CD11b but lacking CD14, CD80, CD83, CD86, and CMRF44. During the next 2 wk, this population spontaneously matured into nonadherent, CD1a(low/-), CD11b(low/-), CD14-, CD80+, CD83+, CD86+, CMRF44+ DC with high allostimulatory activity in the MLR. To examine which factors influenced this maturation, 25 different cytokines or factors were added to the immature DC culture. Only type I IFNs (alpha or beta) accelerated this maturation in a dose-dependent manner, so that after only 3 days the majority of large cells acquired the morphology, phenotype, and function characteristics of mature DC. Furthermore, supernatants from cultures containing spontaneously maturing DC revealed low levels of endogenous IFN production. Because of the similarity of the activation of DC in our culture system with the phenotypic and functional changes observed during Langerhans cells activation and migration in vivo, we investigated the effect of IFN-alpha on human Langerhans cell migration. IFN-alpha also activated the migration of human split skin-derived DC, demonstrating that this effect was not limited to DC derived in vitro from hemopoietic progenitor cells. DC activation by type I IFNs represents a novel mechanism of immunomodulation by these cytokines, which could be important during antiviral responses and autoimmune reactions.

A serum-free culture model for studying the differentiation of human dendritic cells from adult CD34+ progenitor cells.

The antigen-presenting capacity of dendritic cells (DCs) makes them attractive potential cellular adjuvants for vaccination strategies. Currently, most in vitro culture systems for the production of these DCs include serum. However, this is undesirable because serum contains growth factors that vary between individuals and could affect DC development. Unless the patient's own serum is used, foreign antigens and the risk of infection will detract from the usefulness of these cells in clinical strategies. In this study we investigated the production of DCs from CD34+ progenitor cells of cancer patients or normal donors under serum-free conditions. We have established a model system for the investigation of DC development and maturation. Dendritic cells that developed from myeloid precursors accumulated after 2 weeks in an intermediate CD1a , CD80-, CD83-, CD86- stage. Intermediate DCs adhered to plastic surfaces, expressed Birbeck granules, and were negative for CD2 and CD14. In the presence of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha, interleukin-4 promoted the development of these stages. Spontaneous maturation of intermediate DCs into fully activated DCs expressing CD83 and costimulatory molecules occurred asynchronously over the ensuing 2 to 3 weeks. This maturation involved increased expression of CD80, CD83, CD86, CMRF-44, HLA-A, -B, -C, and -DR as well as downregulation of CD1a and CD11b. Activated DCs are characterized by the lack of adherence to plastic surfaces and the absence of Birbeck granules. By day 28, these cells were nonphagocytic, potent antigen-presenting cells with an irreversible phenotype. This serum-free system offers advantages in that the process of differentiation and maturation of committed DCs is extended over a period of more than 28 days, allowing investigators to study the effects of individual cytokines or other supplements during distinct phases of DC development in a defined environment.

Mobilisation kinetics of primitive haemopoietic cells following G-CSF with or without chemotherapy for advanced breast cancer.

BACKGROUND: The objective of this study was to determine the optimal conditions for blood progenitor cell harvest for transplantation, with main emphasis on the mobilisation kinetics of primitive, marrow repopulating cells. PATIENTS AND METHODS: Sixteen patients with advanced breast cancer were treated with 4 cycles of dose escalating FAC chemotherapy (5-fluorouracil, adriamycin, cyclophosphamide) each followed by 10 micrograms/kg/d G-CSF for 13 days. We assessed the number of colony-forming cells (CFC), and estimated the long-term culture initiating cells (LTC-IC) and CD34+ cells during the recovery phase of cycle 1 and 4 of chemotherapy, and during additional periods of G-CSF administration either preceding or following the full course of chemotherapy. RESULTS: The highest peak numbers of CFC per ml of blood (median 10489, range 860-39282) were mobilised after the first cycle of chemotherapy. The lowest peak numbers of CFC were obtained during the recovery phase from cycle 4 (median 4739, range 40-26789). In contrast, the numbers of CD34+ cells per ml of blood were significantly higher in cycle 4 (median 650, range 30-2600 x 10(2)) compared to those of cycle 1 (median 240, range 20-770 x 10(2)). The peak numbers of CFC mobilised by G-CSF before commencement and after the cessation of chemotherapy were equivalent, with a median of 5470 (range 1056-25669) and 5948 (range 2710-38975) per ml of blood, respectively. However, while mononuclear cells (MNC) collected at the days of maximal CFC mobilization following G-CSF administration before or after cycle 1 were similar to normal bone marrow MNCs in their ability to generate haemopoiesis when seeded onto performed irradiated stroma, those collected after cycle 4 or during G-CSF administration after the cessation of chemotherapy were markedly compromised in this respect. CONCLUSIONS: Our results indicate that repeated cycles of FAC chemotherapy followed by G-CSF result in a far lower number of LTC-IC than of CFC mobilised into the circulation. Furthermore although the combination of chemotherapy and G-CSF mobilised the highest numbers if CFC, G-CSF alone pre-chemotherapy was more effective at mobilising LTC-IC. These data indicate that neither the numbers of CFC mobilised nor the numbers of CD34+ cells are necessarily a reliable indicator for the putative marrow repopulating capability of the blood cells mobilised with chemotherapy plus G-CSF.

Comparison of purity and enrichment of CD34+ cells from bone marrow, umbilical cord and peripheral blood (primed for apheresis) using five separation systems.

Interest in the isolation and characterization of primitive hemopoietic cells in both the clinical and research fields has rapidly increased. In parallel, different purification systems have been developed to isolate these cells. We have compared five different methods for separation of CD34+ cells from human umbilical cord blood, normal bone marrow and apheresis harvests and analyzed purity, recovery, yield and enrichment of colony forming cells (CFC) for each individual system. Our results indicate that the most reliable methods of purification for all samples were fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS) which consistently yielded high purities (> 70%) and enrichment of CFC. In this respect the enrichment of CFC from the MACS was superior to all the other systems including FACS. Similar results (> 70%) for purity were obtained using avidin affinity columns and a biotinylated antibody but neither yield nor CFC enrichment approached the values achieved with MACS. On average CFC enrichment using these affinity columns was greater than that observed for FACS while the purity was comparable. Both CELLector flasks and immunomagnetic beads coated with CD34 antibodies were less effective in our hands in separating purified populations of progenitor cells. Both purity and CFC enrichment of CD34+ cells using these methods were at least 50% lower than obtained with either FACS, MACS or affinity columns.

Effects of interleukin-6 on mobilization of primitive haemopoietic cells into the circulation.

Twenty-seven patients with advanced adenocarcinoma were studied. Groups of three patients received interleukin-6 (IL-6) in doses ranging from 0.5 to 20 micrograms/kg by daily subcutaneous injection on days 1-7 and 22-49. Four patients received IL-6 2.5 micrograms/kg/d with GM-CSF 5 micrograms/kg/d and three patients received IL-6 2.5 micrograms/kg/d with IL-3 5 micrograms/kg/d. Circulating platelet numbers increased 1.65-fold during IL-6 treatment, in a dose-dependent fashion (P = 0.01). This increase is inferior to that expected from laboratory studies. No significant change in total WBC was seen after IL-6 alone. After treatment with IL-6, significant increases in numbers of circulating mononuclear cells (2.2-fold, P = 0.006) and GM-CFC numbers (3.2-fold, P = 0.01) were seen, but there were no changes in circulating megakaryocyte-CFC numbers. In contrast, after treatment with IL-6 and GM-CSF, larger increases in both circulating GM-CFC (20-fold, P = 0.04) and megakaryocyte-CFC numbers (18-fold, P = 0.03) were seen. Increases in blood progenitors after treatment with IL-6 and IL-3 did not achieve statistical significance. The ability of peripheral blood mononuclear cells to generate and sustain long-term haemopoiesis in vitro was similar in IL-6-treated patients to that in untreated control subjects. No significant changes in the incidence of bone marrow progenitors or their cycling status (assessed by thymidine suicide) were seen. These data suggest that IL-6 alone will not be clinically useful to mobilize blood progenitor cells in cancer patients.

Direct comparison by limiting dilution analysis of long-term culture-initiating cells in human bone marrow, umbilical cord blood, and blood stem cells.

Limiting-dilution analysis of long-term culture-initiating cells (LTCIC) is a quantitative method of estimating hematopoietic stem cell activity in clinical samples. We compared the numbers of LTCIC in bone marrow (BM), umbilical cord blood, and blood progenitor cells (obtained from patients with solid tumors at leukapheresis after mobilization with induction chemotherapy and filgrastim administration), using a two-stage long-term culture system and a limiting-dilution technique, scoring cobblestone areas of greater than 15 hematopoietic cells weekly for up to 8 weeks. Samples were obtained from 30 normal BMs, 20 human umbilical cords, and 32 leukapheresis products. Direct comparison of LTCIC in the three sources showed that the median proportions of cells generating hematopoietic foci from unfractionated mononuclear cells at 5 and 8 weeks, respectively, were 1:13,314 and 1:33,949 for BM, 1:12,506 and 1:34,546 for umbilical cord blood, and 1:10,302 and 1:12,891 for leukapheresis product. The estimated proportions of LTCIC from unfractionated mononuclear cells and CD34+ cells were similar in experiments with leukapheresis product. Leukapheresis product was superior to umbilical cord blood and cord blood to BM at 5 and 8 weeks of culture (P = .01). In two-stage long-term cultures, more colonies per flask and CD34+ cells were found in assays of leukapheresis product than in BM or umbilical cord blood cultures (P = .0005). Results obtained by this simplified limiting-dilution analysis correlated well with standard long-term cultures and can be used as a measure of the stem cell population. These data suggest that the incidence of putative stem cells in leukapheresis product and umbilical cord blood are at least comparable with that of BM.