Peripheral Blood Stem Cell Products Research Articles

Autologous Peripheral Blood Stem Cell Products from Patients with Hematologic Malignancies Have Increased Frequency of Regulatory T Cells (CD4+FoxP3+ Treg).

Increased numbers of regulatory T cells (Treg) with immune suppressive function have been observed in patients with hematological malignancies. Many of these patients will undergo stem cell mobilization with high dose cyclophosphamide and G-CSF to prepare for autologous stem cell transplantation. Although infusion of low dose cyclophosphamide (Cy) seems to reduce the number of circulating Tregs, mobilization with high dose Cy and G-CSF was shown to increase the frequency of immunosuppresive Treg in stem cell autografts of myeloma patients. Treg populations have been defined by different phenotypic criteria, including CD4+FoxP3+ and CD4+CD25+ CD127lo. In this study, we compare the impact of donor types and stem cell mobilization regimens on CD4+FoxP3+ and CD4+CD25+ CD127lo Treg frequencies in 63 apheresis products.Methods: Apheresis products from 23 healthy donors (17 donors received G-CSF and 6 had no stem cell mobilization) and 40 patients with hematological malignancies (1 AML, 2 HD, 25 NHL, 10 MM, 2 neuroblastoma) were evaluated. Patients with malignancies were mobilized with chemotherapy (5 RICE, 25 Cy, 1 Ara-c+Etoposide) + G-CSF (N=31), AMD3100 + G-CSF (N=5), or G-CSF alone (N=2). Samples from each product were incubated with fluorochrome-conjugated antibodies against cell surface makers, CD45, CD4, CD14, CD127, and CD25, followed by intracellular staining with anti-FoxP3 antibody. Six-color flow cytometry and sequential gating was performed for each sample to determine the frequency of CD4+Foxp3+ and CD4+CD25+CD127lo Treg population.Results: A strong correlation was found between CD4+Foxp3+ and CD4+CD25+CD127lo Treg subsets (r=0.67, p<0.001).The percentage of both Treg populations was not different between mobilized or non-mobilized products from healthy donors (p=0.94) or between NHL and MM patients (p=0.81). Higher CD4+FoxP3+ Treg frequency was observed in autologous grafts from patients compared to healthy donors (p<0.001). The median CD4+FoxP3+ Treg frequency among mobilized products from patients and healthy donors were 8.5% (range, 1.8–38.9) and 4.3% (range, 1.8–15.0), respectively. Autografts from patients who received AMD3100+G-CSF compared to those who received chemotherapy + G-CSF also revealed higher CD4+FoxP3+ Treg content (median 11.3 vs. 7.5, p=0.04). Similar analysis using CD4+CD25+CD127lo Treg subset did not demonstrate any statistical significance.Conclusions: Although correlation was observed between CD4+FoxP3+ and CD4+CD25+CD127lo subsets, CD4+FoxP3+ is a more sensitive marker for the Treg population in stem cell products. Higher CD4+FoxP3+ Treg frequency was observed in grafts from patients with hematological malignancies compared to healthy donors. The addition of a novel mobilization agent, AMD3100, to Cy mobilization may further increase the Treg content of autologous stem cell products.

The Combination of AMD3100 Plus GROβ Rapidly Mobilizes Hematopoietic Stem Cells with Enhanced Homing, Adhesion and Survival Properties

Mobilized peripheral blood stem cells (PBSC) are the primary cell source for hematopoietic transplantation. In mice, combined single administration of AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4 with GROβ, a CXCR2 agonist, rapidly mobilizes hematopoietic stem and progenitor cells (HSPC) within 15 minutes of administration that is equal or greater in magnitude to a multiday regimen of G-CSF and demonstrates significant synergy compared to either compound used alone. In non-competitive transplant models, AMD3100 plus GROβ mobilized 6.2±0.3 CRU/105 PBSC compared to 1.8±0.2, 1.9±0.2 and 2.0±0.3 CRU, respectively, for G-CSF, AMD3100 or GROβ used alone. Furthermore, AMD3100 plus GROβ mobilized long-term repopulating cells that show superior hematopoietic engrafting capacity in lethally irradiated mice. Analysis of chimerism at 6 months post competitive transplant of mobilized PBSC compared to normal bone marrow at a ratio of 1:1, showed 15±2%, 10±2% and 28±2% donor chimerism for G-CSF, AMD3100 and GROβ mobilized PBSC, respectively, whereas the equivalent number of AMD3100 plus GROβ mobilized PBSC produced 55±6% donor chimerism. In order to define the mechanism(s) responsible for increased engraftment of AMD3100 plus GROβ mobilized PBSC, we performed extensive phenotypic and functional analysis of combination AMD3100 plus GROβ mobilized HSPC and compared them to HSPC mobilized by G-CSF, AMD3100 or GROβ used alone. Flow cytometry analysis of highly enriched HSC using SLAM family receptor markers demonstrated that the AMD3100 plus GROβ mobilized PBSC population contained 1.8±0.1, 3.2±0.15 and 1.6±0.18 fold more CD150+ CD48− SKL cells, respectively, than the PBSC products mobilized by G-CSF, AMD3100 or GROβ used alone. In addition, a higher proportion of AMD3100 plus GROβ mobilized CD150+ CD48− SKL cells expressed higher levels of the adhesion receptors CD11a, CD49e and CD49d. Cell cycle analysis demonstrated that AMD3100 plus GROβ mobilized PBSC contained more cells (92±4 %) in G0/G1 phase of the cell cycle than PBSC mobilized by G-CSF (82±6%), AMD3100 (76±4%) and GROβ (85±2%) alone. In homing studies using CFSE labeled mobilized mononuclear cells injected into lethally irradiated mice, ~2-fold more SKL cells mobilized by AMD3100 plus GROβ homed to bone-marrow compared to SKL cells mobilized by G-CSF, AMD3100 or GROβ used alone. In addition, analysis of apoptosis in homed CFSE+ SKL cells indicated that 2.92±0.4% of homed SKL cells mobilized by AMD3100 plus GROβ were Annexin-Vpositive vs. 6.4±1.5%, 10.4±2.6% and 4.5±1.2%, respectively, for homed SKL cells mobilized by G-CSF, AMD3100 or GROβ alone, suggesting that mobilization by AMD3100 and GROβ in combination results in enhanced survival of mobilized HSC. Our results suggest that the enhanced engraftment observed upon transplantation of PBSC mobilized by combination treatment with AMD3100 plus GROβ in results from mobilization of a greater number of more primitive quiescent HSC that have enhanced intrinsic homing, adhesion and survival properties.

A randomized phase III trial of myeloablative autologous peripheral blood stem cell (PBSC) transplant (ASCT) for high-risk neuroblastoma (HR-NB) employing immunomagnetic purged (P) versus unpurged (UP) PBSC: A Children’s Oncology Group study

10011 Background: ASCT improves outcome for HR-NB, but potential risk of reinfusion of viable tumor in PBSC could affect event-free survival (EFS). We assessed the impact of ASCT with immunomagnetic purging of PBSC, compared to UP product, on EFS, PBSC tumor content, engraftment, and ASCT toxicity. Methods: Between 2/2001 and 3/2006, 489 eligible newly diagnosed HR-NB pts were randomized at study entry to have P or UP PBSC obtained and cryopreserved after 2 cycles of induction chemotherapy. Purging was performed centrally using carbonyl iron depletion of phagocytes followed by 2 cycles of magnetic beads with 5 anti-NB monoclonal antibodies. After 6 chemotherapy cycles, pts received melphalan (180–210 mg/m2), carboplatin (AUC 4.1 to max 1700 mg/m2), and etoposide (800–1352 mg/m2) based on GFR followed by PBSC support. Post-ASCT pts had radiation to primary and MIBG+ sites, then 6 cycles of 13-cis-retinoic acid. EFS and overall survival (OS) from enrollment [primary endpoint] were analyzed as intent-to- treat. Results: Median age at study entry was 3.1 yrs (range 91 days-29 years), 44% of 392 tumors tested had MYCN amplification and 424 were INSS stage 4. There were 368 pts transplanted (178 P and 190 UP). Crossover from P to UP arm occurred in 30 pts and from UP to P arm in 5 pts. Only 5 pts (1 P and 4 UP) had NB detected in PBSC by immunocytology at collection, none after purging. RT- PCR analysis of tumor in PBSC products is ongoing. The median time to ANC>500 was 13 days for P and 11 days for UP (p= 0.0001). Toxic death following ASCT was 3.2%, (8 P, 4 UP) mainly due to infection. There was no difference in toxicities between arms. Conclusions: Centralized immunomagnetic purging of PBSC in the context of dose-intensive chemoradiotherapy and biologic therapy in first response did not impact EFS or OS at 2 years. RT-PCR analyses of PBSC products will determine if purging of rare tumor cells occurred. Cohorts N 2 yr EFS ± SE (%) 2-sided p-value 2 yr OS ±SE (%) 2-sided p-value Purged 244 47 ± 3 0.7881 69 ± 3 0.2920 Unpurged 245 49 ± 3 73 ± 3 No significant financial relationships to disclose.

Increased graft content of vascular progenitor cells is associated with reduced toxicity following autologous hematopoietic transplantation

Endothelial-like vascular progenitor cells (VPCs) can be collected in peripheral blood stem cell (PBSC) products that are used in hematopoietic stem cell transplantation (HSCT). The association between VPCs in PBSC products and transplant-related toxicity caused by high-dose chemo/radiotherapy was assessed to identify potential mediators of vascular repair. PBSC grafts in 29 patients (mean age: 48 years; range, 20-67 years) undergoing autologous HSCT were analyzed using a cell culture assay for VPC cluster formation in fibronectin-coated dishes in serum-rich angiogenic conditions. Transplant toxicity was estimated using total length of hospital stay (LOS) following HSCT and the Seattle criteria for transplant-related organ toxicity for 8 organ systems (grade 0-4). LOS following graft reinfusion was lower (14.7 vs 20.0 days, p = 0.002) and the mean number of organs with any toxicity (1.0 vs 2.4, p = 0.016) or with toxicity grade > or = 2 was reduced (0.2 vs 1.6 organs, p = 0.007) in patients with high graft VPC content (n = 10, >2.0 x 10(3) VPCs/kg) compared with reduced VPC content (n = 19, < or = 2.0 x 10(3) VPCs/kg). An association between graft CD34(+) levels and LOS or organ toxicity was not observed. In addition, graft VPC levels were independent of graft CD34 counts, peripheral blood monocytes and hemoglobin levels, age, and disease (p = NS). PBSC products enriched for VPCs are associated with reduced toxicity following HSCT. Identifying specific factors that contribute to high graft VPC levels is needed.

251: Impact of Storage Conditions on Marrow and Peripheral Blood Stem Cell Products Stored Over 72 Hours

251: Impact of Storage Conditions on Marrow and Peripheral Blood Stem Cell Products Stored Over 72 Hours

223: Alternative Donor Hematopoietic Stem Cell Transplantation for Pediatric Patients with Acute Lymphoblastic Leukemia Using Campath in a Myeloablative Conditioning Regimen

Allogeneic transplantation from a matched sibling donor (MSD) offers curative therapy for pediatric patients with acute lymphoblastic leukemia (ALL); however, less than 30% of patients will have a MSD. Alternative donor (AD) transplantation is a viable option for these patients, but the risk for GVHD remains much higher. From 2001 to present, we have used Campath in the conditioning regimen for AD transplants to achieve in vivo T-cell depletion, in an effort to reduce the risk of GVHD. Here we report the outcome of pediatric ALL patients receiving AD transplants using Campath as part of a myeloablative conditioning regimen. Risk assessment is assigned prior to transplant – standard risk (SR) includes patients with ALL in 1st or 2nd complete remission. Since 1997, 39 patients with SR ALL were transplanted with MSD grafts, and since instituting Campath in 2001, 39 patients have received AD grafts. AD patients received bone marrow or peripheral blood stem cell products from up to 1 antigen mismatched related or unrelated donor. Myeloablative conditioning included cyclophosphamide, cytarabine arabinoside and total body irradiation (1200 cGy for MSD and 1400 cGy for AD) in all patients with the addition of Campath in the AD recipients. Additional GVHD prophylaxis consisted of FK506 or cyclosporine with either methotrexate or prednisone. All but one evaluable patient achieved neutrophil engraftment. The 5-year disease-free survival for patients with MSD was 83% compared to 60% for AD recipients. The 100-day mortality was 8% in the AD group and 3% in the MSD group. GVHD was low in the AD cohort – 13% developed grade II-IV acute GVHD and none developed extensive, chronic GVHD. Consistent with other published reports using Campath, we did observe a high incidence of viral infections – 28% developed adenovirus reactivation; 28% developed CMV reactivation; and 3% developed EBV-LPD. We conclude that Campath used with a myeloablative conditioning regimen offers a curative option for pediatric patients with acute lymphoblastic leukemia requiring alternative donor transplantation with a low incidence of GVHD.

Difficulty of Peripheral Blood Stem Cell Collection after Multiple Consolidation Therapy in Acute Myeloid Leukemia

Autologous hematopoietic stem cell transplantation (autoSCT) is one therapeutic option after complete remission of acute myeloid leukemia (AML). Due to ease of collection and rapid engraftment, peripheral blood stem cells (PBSC) are now preferred over bone marrow for the auto-SCT procedure. To minimize both the total body leukemia burden and the contamination of the PBSC product, a high-dose-cytarabinebased consolidation regimen prior to the collection of granulocyte colony-stimulating factor (G-CSF) mobilized PBSCs could prove useful for in vivo purging. However, the optimal schedule for this consolidation therapy is unknown. We conducted a PBSC harvest in four patients with AML who were hospitalized in Toyohashi Municipal Hospital between April and November 2004. After complete remission was achieved in the patient by a single course of induction therapy, with intermediate-risk or good-risk cytogenesis excluding t(15,17), we conducted a G-CSF mobilized PBSC harvest both after the first consolidation therapy (cytarabine 4 g/m/day x 4 days and daunorubicin 45 mg/m/day x 2 days) and after the second consolidation therapy cytarabine 200 g/m/day x 5 days and mitoxantrone 7 mg/m/day x 3 days). The patients received 510 mg/kg of lenograstim before PBSC harvest. Cytogenetic classification of risk grouping was done according to the previously described method. If the CD34 dose in the PBSCs collected was > 2.0 x 10 CD34 cells/kg recipient body weight after each of the first and second consolidation therapy, we conducted autologous peripheral blood stem cell transplantation (auto-PBSCT). The PBSC product collected after the second consolidation therapy was infused into the patient, while the PBSC product collected after the first consolidation therapy was kept for “backup.” Informed consent was obtained for all medical procedures. The goal of this study was to evaluate the effect of the second consolidation on the dose of CD34 cells collected. The summary data for the four patients are shown in Table 1. In all cases in which two PBSC collections were performed (Patients 1, 2, and 4) the dose of collected CD34 cells after one cycle of consolidation therapy was higher than that after two cycles. Only in Patient 1 did the PBSCs collected after the second consolidation therapy contain > 2.0 x 10 CD34 cells/kg. This patient underwent auto-PBSCT. Prior to consolidation therapy, minimal residual disease (MRD) was found in this patient as evidenced by detection of AML1/ MTG8 by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Minimal residual disease could not be detected in the PBSC product that was collected after the first and second consolidation therapies, nor in the bone marrow samples collected after the first and second consolidation therapies. MRD was not evaluable in Patients 24, because they had AML with normal karyotype. Patients 24 did not undergo autologous transplantation. All four patients were alive in complete remission at median of 8.7 months (range, 6.0-13 months) after diagnosis. The concentration of leukemia-contaminated cells in the collected PBSC product after multiple courses of consolidation therapy is presumably smaller than that after a single course of consolidation therapy. However, the collection of an adequate dose of PBSC after the second consolidation therapy was difficult. We did not use high dose cytarabine in the second cycle consolidation regimen, because we were uncertain of its efficacy as a preparative regimen for PBSC harvest. This difference between the first and second consolidation regimens may have affected the dose of the collected CD34 cells.

Predictive parameters for granulocyte colony‐stimulating factor‐induced peripheral blood stem cell mobilization

To improve the selection of donors for allogeneic stem cell transplantation, it is important to identify reliable parameters that predict CD34+-cell yields after granulocyte-colony stimulating factor (G-CSF)-induced peripheral blood stem cell (PBSC) mobilization. We retrospectively investigated the peripheral blood (PB) kinetics of white blood cells (WBCs), CD34+ cells, matrix metalloproteinases (MMP)-9 and -2, and tissue inhibitors of metalloproteinases (TIMP)-1 and -2 in 15 healthy donors during their treatment with G-CSF. All donors received 10 microg/kg of recombinant human G-CSF once a day subcutaneously. Leukapheresis was initiated after 4 days of G-CSF treatment, and G-CSF treatment continued until the last day of leukapheresis. WBC and CD34+ cell numbers in the PB rose after 2 and 3 or 4 days of G-CSF treatment, respectively. The PB CD34+ cell numbers on day 4 correlated weakly with the increase in WBC counts from day 1 to day 2 (R(2) = 0.254, P = 0.056). There were also positive correlations between the CD34+ cell numbers in the PBSC products on day 4 and the CD34+ cells in the PB on days 1 and 4 (R(2) = 0.768, P < 0.0001 and R(2) = 0.816, P < 0.0005, respectively). The MMP-9 plasma levels on days 1 and 4 also correlated positively with the day 4 circulating CD34+ cell numbers (R(2) = 0.393, P < 0.05 and R(2) = 0.406, P = 0.01, respectively). In conclusion, the CD34+ cell numbers in the PB steady state may be a useful parameter selecting allogeneic PBSC donors.

Hematopoietic stem cell mobilization with G-CSF induces innate inflammation yet suppresses adaptive immune gene expression as revealed by microarray analysis

Granulocyte colony-stimulating factor (G-CSF) is used to boost granulocyte counts in immunocompromised patients, but its effects on the immune system may be counterproductive. We tested the hypothesis that G-CSF-mobilized peripheral blood stem cell (PBSC) products are immunologically downregulated based on gene microarray analysis. Ten peripheral blood samples from normal donors for allogeneic PBSC transplantation were obtained before and after administration of G-CSF and tested on Affymetrix Human U133 Plus 2.0 GeneChip microarrays and by flow cytometry. Significant changes in gene expression after G-CSF were reported by controlling the false discovery rate at 5%. The quantitative real-time polymerase chain reaction method was used to validate expression of representative genes. All immune cells measured, including neutrophils, monocytes, lymphocytes, and dendritic cells, were significantly increased after G-CSF. In terms of gene expression, inflammatory and neutrophil activation pathways were upregulated after G-CSF. However, adaptive immune-related gene expression, such as antigen presentation, co-stimulation, T-cell activation and cytolytic effector responses, were downregulated. Despite significant increases in lymphocytes and antigen-presenting cells, G-CSF-mobilized PBSC allografts exhibit a suppressive adaptive immune-related gene-expression profile. However, innate and inflammatory responses are elevated. Our data provides an explanation for the potentially immunosuppressive effects observed after G-CSF administration.

Contamination of Peripheral Hematopoeitic Stem Cell Products With aMycobacterium mucogenicum–Related Pathogen

A gram-positive rod with a restriction pattern closely related to the published nucleotide sequence of Mycobacterium mucogenicum was isolated from 6 of 45 units of peripheral blood stem cell products. The source of the contamination was traced to ice cubes used in processing the peripheral blood stem cell products. Substituting reusable ice trays for ice from an ice machine terminated the outbreak.

Immune reconstitution after autologous hematopoietic transplantation with Lin −, CD34 +, Thy-1 lo selected or intact stem cell products

In sequential studies, we compared immune reconstitution following high-dose chemotherapy (HDT) and stem cell transplantation (SCT) using intact mobilized peripheral blood stem cell (PSC) in intermediate grade non-Hodgkin's lymphoma (NHL) patients and CD34 +, lineage-negative (Lin −), Thy-1 lo (CD34 +Lin −Thy-1 lo) stem cells in low-grade NHL patients. Cytokine expression and cellular phenotype and function were used as the basis of comparison. Despite differences in cellular composition of the stem cell grafts, immune reconstitution in both groups was similar. Significantly higher levels of type 1- and 2-associated cytokine messenger ribonucleic acid (mRNA) were observed both prior to and following transplant in the peripheral blood (PB) of both cohorts as compared to normal individuals. Similar levels of interleukin (IL)-4, IL-10, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) messenger ribonucleic acid (mRNA) were seen in PB mononuclear cells following transplant with either product. In contrast, patients receiving isolated CD34 +Lin −Thy-1 lo cells expressed significantly higher IL-2 levels at all times examined post-transplant. Despite the high levels of cytokine gene expression and rapid restoration to pretransplant levels of CD3 cell number by day 30, T cell function and CD4:CD8 and CD4 +CD45RA:CD4 +CD45RO + ratios were significantly depressed in both cohorts compared to normal donors, and significantly lower in patients transplanted with CD34 +Lin −Thy-1 lo compared to patients receiving an intact PSC product. These data suggest that the peripheral tolerance in patients receiving HDT and an autologous SCT occurs independent of graft composition, although immune function and CD4 recovery are better facilitated by transplantation of an intact product.

CD34 + Hematopoietic Progenitor Cell Selection of Bone Marrow Grafts for Autologous Transplantation in Pediatric Patients

CD34 +-selection of hematopoietic grafts for patients undergoing autologous hematopoietic stem cell transplantation (HSCT) is frequently used to obtain a tumor-free graft. The majority of published experience is with peripheral blood stem cell (PBSC) products; only scant information has been published on bone marrow (BM) grafts. We reviewed our experience using CD34 + selection of BM grafts in children undergoing autologous BM transplantation. After obtaining institutional approval, we performed a retrospective review of the medical records of patients who underwent autologous stem cell collection at St. Jude. From January 1, 1999, to December 31, 2003, 373 patients underwent autologous HSCT; 131 received marrow grafts, 237 received PBSC grafts, and 5 received a combination. Seventeen patients underwent BM harvests for CD34 + selection of their stem cell grafts. Sixteen patients received 19 CD34 purified grafts processed on the Isolex 300i Magnetic Cell Selection System ® device. Four patients were not included in the engraftment analysis as 1 did not receive the collected product, 1 received a tandem product, and 2 received products that were composed of 2 or 3 combined purified products. Following selection, marrow grafts contained a median of 1.4 × 10 6 CD34 + cells/kg (range: 0.09-8.3 × 10 6/kg) and a median of 0.014 ×10 8 total nucleated cell cells/kg (range: 0.001-0.09 × 10 8/kg). The median CD34% recovery was 30.9% (range: 9.3%-57.1%), with the median CD34 purity being 95.5% (range: 62.2%-98.8%). All patients engrafted. The median time to absolute neutrophil count ≥500/mm 3 was 19 days (range: 12-35 days), and to platelet recovery was 28 days (range 18-37 days). No patient died from transplant-related complications. Our study demonstrates that CD34 +-selection of marrow grafts is feasible, and these grafts are able to successfully reconstitute hematopoiesis in patients undergoing autologous BMT.

Long‐term safety of filgrastim (rhG‐CSF) administration

This journal recently included an article by Bennett et al (2006) reporting five cases of haematological malignancy occurring in normal individuals following exposure to haematopoietic growth factors. Three cases described lymphoid malignancies (non-Hodgkin lymphoma and chronic lymphocytic leukaemia) occurring months to years after receipt of an investigational agent (pegylated, recombinant megakaryocyte growth and development factor). The two additional cases, however, were identified among 200 individuals who had received filgrastim (recombinant human granulocyte colony-stimulating factor; rhG-CSF, Neupogen®) as a mobilising agent for collection of peripheral blood stem cells (PBSC) for allogeneic transplantation. The transplant recipients for these latter two donors were their siblings, each with acute myelogenous leukaemia (AML). The donors themselves also developed AML 4–5 years following filgrastim exposure. It is known that siblings of persons with leukaemia have a 2–5-fold increased annual incidence of leukaemia (Pottern et al, 1991; Shpilberg et al, 1994; Rauscher et al, 2002). In some families, multi-generational occurrence of leukaemia, in the absence of any known syndrome, e.g. Fanconi anaemia, suggests direct inheritance of susceptibility genes (Segel & Lichtman, 2004). Given these patterns, the contribution of filgrastim exposure to the development of acute leukaemia within families remains speculative. Documenting the safety of filgrastim as a mobilising agent for PBSC donation has long been a matter of importance for the transplantation community, particularly in the context of donations made by volunteer, unrelated adult donors. Since 1997, the US National Marrow Donor Program (NMDP) has maintained an Investigational New Drug (IND) application accepted by the Food and Drug Administration for manufacture of PBSC products from unrelated donors. Filgrastim is administered for PBSC mobilisation at a total dose of c. 10 μg/kg donor weight per day for 5 d. Under the IND protocols, every donor provides informed consent for the research, which includes agreement for perpetual annual follow-up. Among 4015 donors who have passed the first anniversary of their PBSC donation, we have accumulated 9785 years of follow-up (range 1–9 years with 897 donors ≥4 years). Twenty cases of cancer have been reported, occurring in various organ systems, consistent with the age-adjusted US incidence of cancer in adults and in support of the applicability of data obtained from the NMDP follow-up system (Ries et al, 2006). There have been no reports of leukaemia or lymphoma in this donor cohort, which US statistics suggest should comprise 9% of all malignancies in this age group. National Marrow Donor Program donor consent forms approved by the Institutional Review Board contain the following information: Normal individuals are at risk for developing cancer, including leukaemia, lymphoma or other blood diseases throughout their lifetime. It is unknown whether filgrastim increases or decreases an individual's risk of developing cancer. The data being collected during follow-up will help establish if there are any positive or negative long-term effects from receiving filgrastim. Based on limited long-term data from healthy people who have received filgrastim, no long-term risks have been found so far. The low occurrence of leukaemia and lymphoma in our cohort of volunteer, unrelated PBSC donors should provide reassurance to individuals who receive filgrastim for PBSC mobilisation and should encourage their participation in carefully designed programmes for follow-up monitoring. As data from these and other studies mature, a more complete assessment of overall donor safety will become available to all interested parties.

Appropriate Limits for Microbial Contamination after PBSC Processing and the New European Pharmacopoeia Method for Microbiological Control of Cellular Products.

Aim: Patients undergoing haemopoietic stem cell transplantation are at risk of developing life-threatening infections. Peripheral Blood Stem Cell (PBSC) products represent a potential infection source and previously reported contamination rates of PBSC range from 0 – 4.5%. Our objective was to retrospectively investigate PBSC processing at the Centre for Blood Cell Therapies (CBCT) to determine its effectiveness in preventing contamination and to determine if the contamination rate limit is appropriate, as well as determining if the new European Pharmacopoeia (EP) method for microbiological control of cellular products (2.6.27) will have any bearing on these contamination rates.Method: CBCT employs multiple systems to prevent PBSC contamination including functional closed processing steps and appropriate aseptic process validation. A 4 sample (post collection, sterile additive, pre freezing, administration line) microbial testing system with inoculation of PF BacTAlert bottles (BioMerieux) allows identification of contamination sources. Any products with measurable contamination with initial (post apheresis) negative results was deemed to have processing-associated contamination. Our unit, by agreement with our manufacturing regulator (Therapeutic Goods Administration) has imposed a process-associated contamination rate limit of 1% of all procedures. A retrospective analysis from 2001 to 2005 collated all positive microbial events and classified contamination as process-associated or derived from other sources. The EP method was used to validate PF and pairs of adult BacTAlert FA (aerobic) and FN (anaerobic), and BA(aerobic) and BN(anaerobic) bottles with a final inoculum of 1% of the product volume containing 10 CFU and 100 CFU of E. coli, P. aeruginosa, S. aureus, C. albicans, C. pseudodiphtheriticum, B. fragilis, A. fumigatus and C. sporogenes. The final dilution was in representative patient cryopreserved PBSC consented for donation for this purpose to demonstrate the suitability of the method for detecting microbial contamination in PBSC in the presence of cryoprotectant. Bottles were incubated for at least 7 days for automated detection, all detected organisms were typed, and dilute inoculums were plated on TSA plates to confirm actual CFU inoculum.Results:1770 PBSC collections were processed over the 4-year period (May 2001–June 2005). Overall, 20/1770 (1.7%) PBSC products were contaminated, with 4/1770 (0.34%) deemed as process associated. The remainder resulted from other sources, including endogenous (11/1770, 0.94%), detected at infusion (1/1770, 0.09%) and false positives (4/1770, 0.34%). The EP method was positive for all inoculums into PF, FA and BA bottles save for B. fragilis and C. sporogenes, whilst FN was negative for all inoculums save for E. coli, S. aureus, B. fragilis and C. sporogenes and BN was similarly negative to FN for all inoculums with the addition of P. aeruginosa.Conclusion: We have demonstrated that whilst there is a process-associated contamination rate of 0.34%, this is within our regulator agreed limit of 1%. We have also demonstrated that a routine BacTAlert (FA and FN) detection method with 1% of PBSC will readily detect 10 CFU with no discernable inhibitory effects according to a pharmacopoeia method.

Prevalence and Reversibility of Cardiac Dysfunction in Systemic Lupus Erythematosus: Improved Ventricular and Valvular Function after Disease Remission Following Hematopoietic Stem Cell Transplantation.

Patients with cardiac abnormalities undergoing hematopoietic stem cell transplantation (HSCT) are considered at increased risk of cardiac toxicity which has traditionally precluded them from undergoing HSCT. However, cardiac involvement in patients with systemic lupus erythematosus (SLE) is not uncommon with a reported prevalence of up to 75%, often disease-related and may even be the indication for HSCT. Therefore, we undertook a retrospective analysis of all SLE patients who had undergone autologous HSCT (auto-HSCT) to determine the prevalence and nature of cardiac involvement, safety and effect of HSCT on cardiac function. Between April 1997 and January 2006, a total of 55 patients with SLE underwent auto-HSCT. Stem cell mobilization was with cyclophosphamide 2g/m^2 with subcutaneous filgrastim and peripheral blood stem cells (PBSC) were harvested via apheresis upon neutrophil recovery. The PBSC product was lymphocyte-depleted via CD34-positive selection with an Isolex cell separator in 51 patients and unmanipulated in the other 4 patients. HSCT conditioning regimens were cyclophosphamide 200mg/kg over 4 days and equine antithymocyte globulin (ATG) 90mg/kg over 3 days. The PBSC product was re-infused 36h after the completion of cyclophosphamide on day 0 and the patient received subcutaneous filgrastim from either day 0 or day+6 until neutrophil recovery. Fourteen patients were found to have baseline cardiac abnormalities, either clinically or on baseline radionuclide technetium multi-gated acquisition scans or two-dimensional echocardiogram. Of these, 13 were female and the median age at the time of HSCT was 39.8 years (range 15.7 to 55.5 years). The median baseline SLE Disease Activity Index of this cohort was 16 (range 10 to 35). The cardiac abnormalities were impaired left ventricular function (n=6), pulmonary hypertension (n=5), mitral valve dysfunction (n=3), significant pericardial effusion (n=1) and accelerated coronary artery disease (n=1). At a median follow up of 23.5 months (8 to 103 months), 2 of the patients are dead, one from disease progression/relapse at 11.5 months after HSCT and the other died from an accident at 8 months post-transplant. There were no cardiac or transplant-related deaths or significant cardiac toxicities. All patients with impaired ejection fraction (range 25% to 47%) stabilized or improved post-HSCT induced remission. The median pulmonary artery systolic pressure was 39mmHg (range 27 to 50 mmHg). Among the 5 patients with pulmonary hypertension, 3 improved paralleling remission from SLE. Again paralleling disease remission, 2 patients with severe disability from symptomatic mitral valve disease improved, obviating the need for valve replacement which had been planned pre-HSCT for one. In conclusion, we found that our patients with ventricular systolic and valvular dysfunction, and possibly pulmonary hypertension, improved following HSCT if they achieved remission from SLE. Our experience suggests auto-HSCT may be safely performed in selected patients with SLE with cardiac dysfunction and furthermore, improvement of cardiac function may occur following HSCT-induced disease remission. This warrants further study in a larger cohort of patients.

Immune Dysregulation after Hematopoietic Stem Cell Transplantation for Autoimmune Diseases Leading to Development of a Second Autoimmune Disorder: Role of Conditioning Regimen Used?.

Secondary autoimmune disorders following hematopoietic stem cell transplantation (HSCT) for malignancies occur at a reported frequency of 2 to 5%. Patients undergoing HSCT for autoimmune disorders may however have an added propensity to this complication, given the genetic predisposition to autoimmunity. Therefore, we undertook a retrospective analysis of all patients who underwent an autologous HSCT (auto−HSCT) for an autoimmune disease to determine the occurrence of a second autoimmune disorder, its nature, outcome and risk factors. In all, 154 patients underwent auto−HSCT for various autoimmune diseases in the period from August 1996 to March 2006. The peripheral blood stem cell product was either CD34−selected (n=87) or unmanipulated (n=67), depending on disease−specific protocols. The conditioning regimens consisted of various combinations of cyclophosphamide with either antithymocyte globulin (ATG) or alemtuzumab, or cyclophosphamide with intravenous busulfan or total body irradiation, again depending on disease−specific protocols. The autoimmune diseases represented were systemic lupus erythematosus (SLE) (n=60, 38.9%), systemic sclerosis (SSc) (n=13, 8.4%), multiple sclerosis (MS) (n=42, 27.3 %), rheumatoid arthritis (n=6, 3.9%), Crohn's disease (n=20, 13.0%) and others (n=13, 8.4%). There were 5 patients (2 with SLE, one each with MS, SLE and SSc) who developed an autoimmune disorder distinct from their underlying autoimmune disease at a median of 5 months (2 to 30 months) after HSCT. The frequency of a secondary autoimmune disease was 3 of 24 among patients receiving alemtuzumab, 2 of 102 among those receiving ATG and none (0 of 28) of those who received neither of these lympho−depleting antibodies (p=0.018). The difference between the frequency of this complication was also significantly higher among those receiving alemtuzumab compared to ATG (p=0.017). The frequency of a secondary autoimmune disease among those who received a CD34−selected graft was 2 of 87 versus 3 of 67 of those who received unselected stem cells (p=0.449). Two patients with SLE who received an ATG−containing conditioning regimen, developed acquired factor VIII inhibitors with severe bleeding. One patient each with MS, SSc and SLE, who received an alemtuzumab−containing conditioning regimen, developed severe autoimmune cytopenias. The 5 patients required multiple immunosuppressants to control the secondary autoimmune disorder. ATG and alemtuzumab induce a protracted post−HSCT lymphopenia, with particularly delayed reconstitution in T cell function. The resultant immune dysregulation in an autoimmune−prone patient may predispose to a second autoimmune disease. The increased likelihood of this complication seen with alemtuzumab recommends against its use in auto−HSCT for autoimmune disease.Conditioning Regimen and Occurrence of Secondary Autoimmune DiseaseRegimenPatients Treated (n)Primary Autoimmune DiseaseSecondary Autoimmune Disease/numberCy/rATG51SLE, SSc, CD, MS, othersNoneCy/eATG51SLEAcquired FVIII inhibitor (n=2)Cy/Al24MS, SLEAutoimmune cytopenias (n=3)Cy/TBI22MSNoneBu/Cy6RANoneCy:Cyclophosphamide; rATG:rabbit antithymocyte globulin; eATG:equine ATG; Al:Alemtuzumab; Bu:IV Busufan; SLE:systemic lupus erythematosus; SSc:systemic sclerosis; CD:Crohn's disease; MS:multiple sclerosis; RA:rheumatoid arthritis

Apheresis Safety and Product Yield among Elderly Donors for Allogeneic Sibling Hematopoietic Cell Transplantation (HCT).

With the advent of reduced-intensity conditioning regimens and the improved supportive care after high-intensity conditioning, elderly patients are increasingly offered allogeneic HCT using peripheral blood stem cells (PBSC) from older siblings. Little is known, however, about the potential impact of pre-collection medical fitness on the safety of the apheresis procedure and product yield with elderly donors. To address these issues, we reviewed the experiences of 140 sibling PBSC donors with a median age of 64 (range 60–83) who underwent collection between 1993 and 2006. About half of the donors were male. All donors were mobilized using granulocyte colony-stimulating factor, at 16 mg/kg/day, and underwent 12-liter apheresis procedures. Pre-apheresis medical comorbidities were scored by the HCT-specific comorbidity index (HCT-CI, Sorror et al. Blood. 2005; 106, 2912–2919) and post-apheresis toxicities were graded per the Common Toxicity Criteria of National Cancer Institute. Donors acquired HCT-CI scores of 0 (62%), 1 (20%), 2 (8%), 3 (9%), and ≥4 (1%). Eighty-four percent of donors underwent 2 PBSC collections (roughly half were mandatory) and 18% required 3 collections. A central venous catheter (CVC) was placed in 27 elderly donors (19%), and 28% of those requiring 3 collections had a CVC. There was no difference in the median age of donors requiring a CVC compared to those who did not. Longer apheresis procedure times were required during the 3rd collection (median of 149 minutes) compared to the 1st (136 minutes) and 2nd collections (131 minutes). Among the 15 donors with three procedures, significantly fewer CD3+ cells were collected across time (median of 1.7, 1.2, and 0.7 × 108 cells/kg recipient weight after the 1st, 2nd, and 3rd collections, respectively, p<0.001). There was a decreasing trend in both CD3+ and CD34+ cell yields across groups of donors with increasing numbers of collections (p<0.001 for both). Donors with HCT-CI scores of ≥3 had higher, but non-significant, median age (67 years) compared to those with scores of 0, 1, and 2 (64, 62, and 63, respectively). There were no correlations between increasing donor age above 60 years and procedure-related toxicities of grades ≥I (p=0.62) or ≥II (p=0.62). Donors with comorbidity scores of 2 or ≥3 had slightly higher incidence rates of grades ≥I toxicities (55% and 57%) compared to donors with scores of 0 and 1 (40% and 48%; p=0.16). There were no correlations between increasing donor age above 60 years and pre-collection white blood cell counts (p=0.31), apheresis procedure times (p=0.41), CD34+ cell yields (p=0.24), and CD3+ cell yields (p=0.26). We conclude that: 1) apheresis collection of PBSC is a safe procedure for donors with an age of ≥60 years, including those with medical comorbidities; 2) among elderly donors, increasing age did not significantly affect the need for CVC, risk of procedure-related toxicity or product yields; 3) sequential collections yield fewer CD3+ cells among donors of this age category. These latter findings need to be compared to those from multiple collections among donors with younger age. In addition, it will be important to compare the engraftment capacity of the PBSC products from elderly versus younger donors.

Allografting in patients with severe, refractory aplastic anemia using peripheral blood stem cells and a fludarabine-based conditioning regimen: The Mexican experience

We studied the effectiveness of a fludarabine/cyclophosphamide-based conditioning regimen without anti-thymocyte globulin in 23 aplastic anemia patients who had no response to previous conventional pharmacologic treatment. Patients received oral busulphan 4 mg/kg/day/2 days, IV cyclophosphamide 350 mg/m(2)/day/3 days, and fludarabine 30 mg/m(2)/day/3 days. For GVHD prophylaxis, patients received MTX 5 mg/m(2) days +1, +3, +6, and +11 and oral cyclosporin A (CyA) 5 mg/kg/day, starting on day -1. Peripheral blood stem cell products were used with a median dose of 5.5 x 10(6) CD34(+)/kg. The patients were followed for an average of 25 months. By a median of day +11, an ANC > 0.5 x 10(9)/L was reached; and by day +12, the platelet count had reached >20,000 x 10(9)/L. Acute grade I-II GVHD occurred in 4 patients, whereas limited chronic GVHD presented in 6 cases. Twenty-one patients (91.3%) achieved engraftment. Two patients failed to engraft, and 4 developed late rejection; 2 of these individuals died, 2 have survived with high transfusion requirements, whereas 2 received a second peripheral blood stem cell infusion and achieved sustained engraftment. Currently 21 (91%) of the 23 patients are alive, whereas 19 of 21 (90%) remain in complete remission. The average cost was about USD 15,000 for this kind of reduced-intensity allotransplant. Reduced-intensity stem cell transplantation represents an affordable alternative to traditional more cytotoxic conditioning for severe aplastic anemia (SAA) patients. Long-term effects however, remain to be evaluated.

Comparing Platelet Loss in 2 Common Apheresis Machines for Collecting Autologous Peripheral Blood Stem Cells (PBSC).

Chemotherapy together with G-CSF is commonly used to mobilize patients prior to leukapheresis (LP) of PBSC for autologous transplantation. For many patients, peripheral blood platelet counts (PLT) were below 75,000/μl when LP was initiated. Minimizing PLT loss is an important consideration, especially for these patients. The aim of this retrospective analysis is to compare PLT loss during LP for autologous PBSC in two common apheresis machines (Baxter Amicus and Cobe Spectra). Between August 2004 and May 2005, 57 autologous PBSC products were collected from 28 adult patients (weight > 40 kg) with results (mean ± SD) summarized in Table 1. LP were collected using automated procedures and 3 times blood volume was processed. Collection efficiencies (EFF) were calculated as total cells (CD34+ or PLT) in product divided by the circulating count prior to LP times processed volume. Statistical significance was determined by unpaired Student's t-test. There were no significant differences (p > 0.1) (NS) in patient age, weight, CD34+ cell count (data not shown) and PLT count prior to LP. There were also no differences in the proportion and the total CD34+ cells in the PBSC products collected by either machines. While both devices collected CD34+ cells with similar EFF (data not shown), the Amicus PBSC products have a trend (p = 0.08 and 0.07 when all LP or only those initiated with PLT count below 75,000/μl, respectively)towards a smaller volume (VOL) and contained significantly (p < 0.05) fewer PLT than those collected by Spectra. The PLT EFF was also significantly lower with Amicus, indicating a smaller PLT loss. The same findings were observed whether all LP or only those LP initiated with low PLT counts (< 75, 000/μl) were considered. Our results showed that lower PLT loss is achieved with Amicus during autologous PBSC collection. The machine would be more appropriate for collecting patients with low PLT counts.Table 1:PLT Loss during Autologous PBSC Collection in Amicus or SpectraNPre LP PLT (x103/μ l)LP VOL (ml)LP Total PLT (x1010)PLT EFF (%)ALL LPSpectra1749.5 ± 20.2178.0 ± 41.78.7 ± 3.517.6 ± 16.9Amicus4057.0 ± 32.9164.5 ± 16.56.8 ± 2.68.4 ± 4.1p valueNS≤ 0.08≤0.05≤0.05Low PLTSpectra1749.5 ± 20.2178.0 ± 41.78.7 ± 3.517.6 ± 16.9Amicus3546.8 ± 15.4162.9 ± 16.86.5 ± 2.49.0 ± 3.9p valueNS0.07≤ 0.05≤ 0.05

Merit of Initiating Peripheral Blood Stem Cell (PBSC) Collections at Low Level of Circulating CD34+ Cells.

Most patients or donors undergoing leukapheresis (LP) for autologous or allogeneic PBSC transplantation requires multiple collections to achieve a sufficient CD34+ cell dose. LP is usually initiated when peripheral blood (PB) CD34+ reached a certain level, such as 20/μl. The aim of this retrospective analysis is to summarize our institutional experience of initiating LP at a low PB CD34+ cell level of 5/μl and investigate the merit of the practice. All patients or donors underwent LP (using Cobe Spectra or Baxter Amicus) processing 3 times the blood volume. A total of 170 LP procedures (118 autologous and 52 allogeneic) was performed in 74 adult patients or donors (> 40 kg) between Jan 2004 and May 2005. Autologous patients were mobilized with chemotherapy and G-CSF while allogeneic donors with G-CSF alone. A “good” LP product is defined as one having ≥ 1 x 106 CD34+ cells/kg so that a minimum dose of 3 x 106/kg can be achieved in 3 collections. Our result showed that each PBSC product contained 6.07 x 108 WBC/kg (median, range: 0.13–17.5) and 1.59 x 106 CD34+ cells/kg (0.14–24.9). Total CD34+ cells in PBSC products were correlated to PB CD34+ cell counts (r = 0.79, data not shown). As shown in Table 1, initiating LP at higher levels of PB CD34+ cell increased the proportion of good LP. Nevertheless, 76% of collections initiated at > 5 CD34+ cells/μl achieved good LP criterion. It is possible that the level of PB CD34+ cells was still increasing in many patients or donors after initiation of LP at the low level. However, some patients /donors still achieved minimim CD34+ cell dose when second LP day (Day 2) PB CD34+ cell level was lower than that of first LP day (Day 1) (Table 2). These patients /donors would likely NOT have been collected if higher levels of PB CD34+ cells were used as guideline for start of LP. Eleven patients /donors whose Day 2 CD34+ cell count was below that of Day 1 achieved minimum CD34+ cell dose when LP was initiated at < 20/μl. When LP was initiated at < 10/μl, four individuals achieved minimum dose. All 4 were autologous patients mobilized with chemotherapy and G-CSF (3 AML and 1 NHL). In conclusion, our results showed that initiating LP at low PB CD34+ cells can be helpful to some individuals. The guideline may be especially useful in those patients that can only be mobilized marginally.Table 1:Initiating LP at Different Levels of PB CD34+ CellsPB CD34+ Cells# LP% Good LP> 5/μl15675.9> 10/μl11987.4> 20/μl6595.4Table 2:Patient /Donor Achieving Dose and Levels of PB CD34+ CellsPB CD34+ Cells# Patients & Donors# Patients & Donors Achieving Dose# Patients & Donors that achieved dose when Day 2 PB CD34+ cells were lower than that of Day 1< 20/μ l4635 (82.6%)11< 10/μ l1410 (71.4%)4