Peripheral Blood Progenitor Cell Donors Research Articles

Plerixafor As a Salvage Regimen to Mobilize Allogeneic Stem Cells in Healthy Volunteers (NCT01954914)

Introduction: Insufficient yield of Peripheral Blood Progenitor Cells (PBPC) from healthy donors can result in higher mortality after allogeneic transplantation. Plerixafor (Mozobil®) is approved for the mobilization of PBPC in combination with G-CSF for collection and subsequent autologous transplantation in patients with non-Hodgkin lymphoma and multiple myeloma. Clinical data on G-CSF and Plerixafor in healthy donors are still limited. In a prospective single arm phase 2 trial we investigated the safety and efficacy of a single dose of Plerixafor as a salvage regimen in poor mobilizing allogeneic PBPC donors (NCT01954914).Methods: We enrolled healthy unrelated PBPC donors who had received G-CSF (Lenograstim) at a targed dose of 10 µg/kg bodyweight (bw) for five days but donated less than 2x 106 CD34+ cells/kg recipient bw during the 1st apheresis on day 5 of the mobilization regimen. Plerixafor was injected subcutaneously at a dose of 240µg/kg once on day 5 in the evening before the second leukapheresis. The target for the processed blood volume during each apheresis procedure was 3x donor blood volume +/- 25%. Mobilization success was defined by a donation of more than 4.5x 106CD34+ cells/kg recipient bw. Primary endpoint was the number of mobilization successes. The null hypothesis (success rate of less than 50%) was tested with an exact single sample binomial test at a one-sided significance level of 5%.Results: Between 1/2014 and 12/2015 37 allogeneic unrelated PBPC donors (54% women, 46% men, median age 34 years) were enrolled into the study. The median donor bw was 69 kg (IQR, 61 kg to 76 kg) and the median recipient bw was 85 kg (IQR, 74 kg to 102 kg). Application of Plerixafor was well tolerated in most donors with only moderate side effects such as nausea, diarrhea and occasional vomiting. The median CD34+ count in peripheral blood was 15.4/µl (IQR, 12.0-18.3) on day 5 after G-CSF alone and 44.0/µl (IQR, 38.0-60.5) on day 6 after G-CSF plus Plerixafor. The collected total nucleated cell count was 187 x 106 (IQR, 159.9-223.4) cells per ml volume of the apheresis product on day 5 and 289 x 106 (IQR, 248.0-348.0) cells per ml volume of the apheresis product on day 6. The yield of CD34+ cells in the apheresis products was 1.05 x 108 (range, 0.21-1.80) on day 5 and 2.8 x108(range, 0.93-5.26) on day 6. The percentage of CD3+ cells per TNC was 31% (IQR 27%-40%) and 31% (IQR, 23% - 35%) on days 5 and 6, respectively.In total, a median number of 5.16 x 106 CD34+ cells (IQR 3.06-6.10) per kg recipient weight were collected during both aphereses. Twenty-one out of 37 donors reached the target cell count (56.8%, 95% confidence interval (CI) 39.5% - 72.9%) by donating a CD34+ cell dose of >4.5x 106 CD34+ cells per kg recipient bw. Nine donors had donated ≤0.8 106 CD34+ cells per kg recipient bw during their first leukaphersis after stimulation with G-CSF alone. Notably, only one out of these nine very poor mobilizing donors failed to donate at least 2.0 x 106CD 34+ cells per kg recipient bw after salvage mobilization with Plerixafor. Nevertheless, the null hypothesis (H0: rate of mobilization success <50%) could not be rejected at the 5% level (p= 0.2557).Conclusions: In this trial in poor mobilizing healthy unrelated donors the rate of mobilization success (donation of >4.5x 106 CD34+ cells per kg recipient bw) was 56.8% (95%-CI 39.5% - 72.9%) after a single dose of Plerixafor on day 5 of G-CSF administration. After injection of Plerixafor the CD34+count in the peripheral blood and the yield of CD34+ cells in the leukapheresis product increased by more than factor 2. The application of G-CSF and Plerixafor was well tolerated by most of the donors. Like for autologous donors, the administration of Plerixafor appears promising to optimize PBPC mobilization in allogeneic poor mobilizing donors. DisclosuresHoelig:Janssen Cilag GmbH: Honoraria; Therakos: Honoraria, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Schetelig:Sanofi: Honoraria.

Severe Pulmonary Adverse Events in Healthy Unrelated Donors of Peripheral Blood Stem Cells Following G-CSF Administration,

Abstract 4057Short-term treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF) followed by leukapheresis (LPH) is the common procedure to obtain CD34+ peripheral blood progenitor cells (PBPC) from allogeneic donors. While bone pain, headache and flu-like symptoms are well-known G-CSF associated side effects, gastrointestinal, cardiovascular and pulmonary symptoms are rare.We are reporting about two cases of healthy unrelated PBPC donor with severe lung injury/ interstitial pneumonia. Case 1:A 31years old female PBPC donor was mobilized with G-CSF (Lenograstim 8.5 μg/kg, 5 days) and donated PBPC without unexpected adverse events. Three days after leukapheresis she experienced symptoms of common cold. Two days later she was admitted to hospital due to fever, thoracic pain and dyspnoea. At this time WBC count was 41.0 × 10^9/L. Chest-CT scan demonstrated bipulmonary disseminated cloudy infiltrates. In addition, papular skin lesions, muscle pain and impairment of renal function occurred. Due to worsened respiratory distress and pleural-effusion, she had to be monitored on the ICU temporarily.Because the symptoms seemed to be associated with disseminated overwhelming inflammatory reaction after G-CSF administration, high-dose steroid treatment was initiated. Bronchoscopy demonstrated normal respiratory mucosa. Biopsies from lung tissue and skin lesions showed infiltration by neutrophiles. Microbiologic and virologic analyses, as well as auto-antibody testing remained negative. While exanthema and pain disappeared with prednisone, lung injury continued (Horowitz index 245). In follow-up chest CT scan the pattern of infiltrates was changed and now compatible with lobular pneumonia. Antibiotic treatment was re-initiated with Piperacillin/Tazobactam and Clarithromycin, and steroids were tapered. During the following days the donor recovered and could be discharged from hospital in a good condition. Case 2:A 20 year old male presented to pre-donation health check-up with symptoms of acute respiratory tract infection. At this time, influenza A (H1N1) was detected in nasal swab. He received oseltamivir 75 mg bid for 5 days and recovered completely.Three weeks later mobilization with Lenograstim (8.5 μg/kg/day) was started. On day 3 of G-CSF treatment the donor developed dyspnoea and haemoptysis without any symptoms of infection. Because of stable respiratory and circulatory parameters G-CSF-application was continued and leukapheresis performed without complication on day 5 (WBC count 42.0 × 10^9/L). Subsequent diagnostic revealed disseminated, bipulmonary, interstitial infiltrates in CT corresponding to severe bronchitis. While relevant microbes could not be found in bronchoalveolar lavage, the PCR from nasal swabs was positive for influenza B. Therefore oseltamivir was restarted again. Because of possible bacterial superinfection levofloxacin was added. Some days later he could be discharged from hospital free from symptoms. Discussion:It is well known that G-CSF not only regulates the numbers of circulating neutrophiles but also modulates their function, i.e. adherence, movement, migration, and cytokine release. Increased cytokine release might induce pulmonary edema and lung damage.We describe two cases of severe respiratory disturbances following G-CSF application for PBPC. In both cases the severe pulmonary injury based on a coexisting infection. Therefore we hypothesize that an inflammatory pulmonary reaction induced by a bacterial/viral infection is aggravated by persisting neutrophilia after G-CSF treatment. Conclusion:A different pathophysiology of infectious lung diseases in PBPC donors, caused by increased numbers of circulating neutrophiles, could lead to severe pulmonary failure. Therefore antiinfective treatment in PBPC donors with respiratory infections should be initiated early and consistently. Under careful monitoring and appropriate treatment stem cell collection should be possible to be finished successfully in most cases. Disclosures:No relevant conflicts of interest to declare.

African American adult apheresis donors respond to granulocyte–colony‐stimulating factor with neutrophil and progenitor cell yields comparable to those of Caucasian and Hispanic donors

Granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPCs) are the most common source of cells used for hematopoietic transplantation. Benign ethnic neutropenia has been found in persons of African descent, affecting circulating white blood cells (WBCs), but not WBC production within marrow. Persons of African descent have reduced neutrophil mobilization after steroid administration, and newborns have fewer nucleated and progenitor cells in their cord blood. Twenty-two African American (AA) and 12 Hispanic PBPC donors were age, sex, and weight matched with 34 Caucasian donors. Groups were compared based on WBC and neutrophil counts after mobilization and numbers of CD34+ cells collected on Day 5 of G-CSF mobilization. AA donors had significantly lower baseline WBC (6.1±1.1 vs. 7.1±1.7, p=0.04) and neutrophil (3.4±1.1 vs. 4.5±1.3, p=0.01) counts compared to matched Caucasian donors. G-CSF-stimulated AAs had a significantly greater increase in WBC and neutrophil counts compared to matched Caucasians (889±293% vs. 665±230% neutrophils, p=0.02). There was no significant difference in product cell counts when comparing total nucleated, CD3+, CD34+, and mononuclear cells or colony-forming units (CFUs) between Caucasians and Hispanics or AAs and trends to greater numbers of neutrophils in products from AA donors. When stimulated by G-CSF, AAs are able to increase WBC and neutrophil counts to a higher degree than Caucasians, achieving similar numbers of neutrophil and progenitor cells in apheresis products despite starting from lower baseline blood counts.

Stem cell harvesting and transplantation

Allogeneic haematopoietic stem cell transplantation is an accepted treatment for a variety of malignant and non‐malignant diseases. Today, peripheral blood progenitor cells (PBPC) are increasingly used instead of bone marrow (BM) as stem cell source. Additionally, umbilical cord blood (UCB) has been established as an alternative source of donor cells. Many differences between the distinct stem cell sources do exist. PBPC contain a higher amount of CD34 + cells compared with BM and UCB, leading to more rapid engraftment with the disadvantage of a higher incidence of chronic graft‐versus‐host disease. In contrast to adults, children seem to have a better outcome after BM than PBPC transplantation. Therefore, especially patients with non‐malignant diseases and children should be transplated with BM. The disadvantage of the significantly lower CD34 + cells count in UCB products can be overcome by transplantation of two different units. The risk for allogeneic BM and PBPC donors is considered negligible. Lethal and serious side‐effects because of the harvest procedure are rarely reported with an incidence of 1/10 000 and 7·25/10 000, respectively. Granulocyte colony‐stimulating factor (G‐CSF) is widely administered to PBPC donors, but questions have been raised about its safety in respect of development of haematologic malignancies. To evaluate this concern, more long‐term safety data from larger prospective studies are needed. In the therapy with adult stem cells, a genotypic human leucocyte antigen (HLA) class I and class II identity between donor and recipient for the loci HLA‐A, HLA‐B, HLA‐C, HLA‐DR and HLA‐DQ is optimal; for UCB, HLA matching seems to be less important because of the relative low number of mature T cells in cord blood. The influence of other HLA‐linked or not HLA‐linked loci in stem cell transplantation (HLA‐DP, killer cell immunoglobulin receptor, …) is under investigation.

Exploratory analysis describing ultrasonic (US) evaluation of spleen size during peripheral blood progenitor cell (PBPC) mobilization by filgrastim in normal donors

7102 Background: Filgrastim is used to mobilize CD34+ cells into the peripheral blood that are collected by apheresis for allogeneic transplantation. We prospectively evaluated spleen size during PBPC mobilization Methods: Donors ≥18 yrs eligible to be PBPC donors per institutional guidelines enrolled. Splenic assessments were done before, during, and after PBPC mobilization. Filgrastim dose and schedule and leukapheresis (LK) procedures were per local practice. Spleen size by US was measured in 3 dimensions: longitudinal (craniocaudal), transverse, and diagonal (perpendicular to transverse). Splenic volume was estimated by taking the cross-product of 3 dimensions and multiplying by 0.52, approximating an ellipse volume. Stiff (ASH 2007) reported the primary endpoint, fold change from baseline in splenic volume during mobilization. Exploratory analyses, including a linear regression evaluating the effect of age and baseline spleen size on fold-change, were performed. Results: 309 enrolled, median age 44yrs, 56% male. Median fold volume change from baseline to first LK was 1.47, resolving to near baseline 1 week after last LK. No significant clinical sequelae, including splenic rupture, were reported. Older donors appeared to have the smallest baseline spleen volume and the largest fold change (table). Linear regression analyses indicated age was a significant predictor for both baseline spleen volume (p=0.0031) and spleen volume fold change from baseline at first LK (p=0.0499). Conclusions: During mobilization, spleen volume transiently increased from baseline to day of 1st LK and returned to near baseline 1 week after last LK. Older donors tended to have smaller baseline splenic volume and greater fold changes in spleen size. Preclinical models suggest decreasing hematopoietic stem cell homing after mobilization with aging (Morrison 1996; Wagers 2002), which could result in splenic accumulation of progenitor and stem cells. [Table: see text] [Table: see text]

Platelet count is a sensitive predictor of autologous peripheral blood progenitor cell collection yield in previously treated plasma cell disease patients

It is often a clinical dilemma to determine when to collect autologous peripheral blood progenitor cells (PBPCs) in patients who received prior chemotherapy. It is also challenging to predict if the collected cells will be enough for one or two transplants. A total of 103 PBPC donors were followed to evaluate factors that predict poor autologous PBPC collection. The donors were categorized into three groups: plasma cell disorders (PCDs), lymphomas, and normal allogeneic donors. Our evaluation showed that platelet (PLT) count before growth factor administration significantly correlated with total CD34+ cell yield (Spearman r = 0.38, p < 0.001). Further analysis showed this correlation was only significant in plasma cell disease patients who received prior chemotherapy (Spearman r = 0.5, p = 0.008). Baseline PLT counts did not correlate with PBPC collection yield in untreated PCD, lymphoma, and normal allogeneic donors. In addition, daily PLT count during PBPC harvest correlated with CD34+ cell yield for that day (Spearman r = 0.41, p < 0.001). With a multiple linear regression model (adjusted R(2) = 0.31, AIC = 63.1), it has been determined that the baseline PLT count significantly correlates with total CD34+ cell yield in treated PCD patients. Baseline PLT count is a sensitive indicator of autologous PBPC mobilization in PCD patients who received prior chemotherapy. This finding may be considered before growth factor administration to determine the optimal period to mobilize treated PCD patients and to predict if enough cells can be collected for one or two transplants.

Biologic and molecular effects of granulocyte colony-stimulating factor in healthy individuals: recent findings and current challenges

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used in healthy donors for collection of peripheral blood progenitor cells (PBPCs) for allogeneic transplantation and granulocytes for transfusion. The spectrum of its biologic and molecular activities in healthy individuals is coming into sharper focus, creating a unique set of challenges and clarifying the need to monitor and safeguard donor safety. Accumulating evidence indicates that rhG-CSF effects are not limited to the myeloid cell lineage. This may reflect the presence of functional G-CSF receptors on other cell types and tissues, as well as rhG-CSF-induced modulation of cytokine networks. While most rhG-CSF-induced effects are transient and self-limiting, preliminary, provocative data have suggested the possibility of a more durable effect on the chromosomal integrity of lymphocytes. While these reports have not been validated and have been subject to criticism, they are prompting prospective studies and monitoring efforts to determine whether there is a significant risk of long-term adverse events (eg, hematologic malignancies) in healthy PBPC and granulocyte donors. Based on the totality of information that is currently available, the administration of rhG-CSF to healthy donors for the purpose of PBPC donation continues to have a favorable risk-benefit profile.

Clinical and Radiographic Evaluation of Spleen Size during Peripheral Blood Progenitor Cell (PBPC) Mobilization by Filgrastim: Results of an Open-Label Trial in Normal Donors.

Introduction: Filgrastim is widely used for mobilizing CD34+ cells into the peripheral blood that are easily collected by apheresis for allogeneic transplantation. With case reports documenting splenomegaly with life-threatening complications in normal donors, we prospectively evaluated spleen size using ultrasonography and clinical examination during PBPC mobilization and collection in a single-arm trial.Methods: Subjects ≥18 yrs eligible to be PBPC donors per institutional guidelines enrolled. Splenic assessments were done before, during, and after PBPC mobilization. Filgrastim dose and schedule and leukapheresis (LK) procedures were per institutional practice. The primary endpoint was fold change from baseline in splenic volume in post-baseline measurements during mobilization (measured by ultrasound [US]). Spleen size by US was measured in 3 dimensions similarly by all centers: longitudinal (craniocaudal), transverse, and diagonal (perpendicular to transverse in transverse image) diameters. Splenic volume was estimated by taking the cross-product of 3 dimensions and multiplying by 0.52, approximating the volume of an ellipse. Physical examination was performed on US days, assessing spleen palpability. US and palpation results were blinded from each other at assessment times. Timepoints included baseline (before first filgrastim dose), first LK (done before LK, typically day 4 or 5 of filgrastim), 2 and 4 days after first LK, and 7 days after last LK. Timepoints in the post-amendment cohort (n=219) were reduced to facilitate enrollment and were baseline and day of first LK (before LK).Results: 309 donors enrolled, median age 44yrs (range 18 to 74), 56% male. Mean daily filgrastim dose was 11.4mcg/kg (SD=3.0). Median number of LK was 1.5 (range 1 to 4). In all donors, the median increase in each measured dimension on first LK day was 1.4cm, 1.4cm, and 0.6cm (12.8%, 12.6%, and 15.0%), and the median fold volume increase from baseline to first LK was 1.47, resolving to near baseline 1 week after last LK. There was no apparent relationship between volume fold change and filgrastim dose, ANC, or CD34+ yield. Of 861 splenic palpation assessments reported in all donors, 98% were reported as nonpalpable (842 assessments), and 2% were palpable (19 assessments, 2 at baseline). Reporting of palpable spleens did not correlate with increased spleen size. Tenderness or guarding upon splenic palpation was reported in 2 donors with a spleen considered palpable and in 6 donors with nonpalpable spleens. No donor experienced a splenic rupture. Adverse events related to filgrastim were generally mild to moderate.Conclusion: During PBPC mobilization with filgrastim in normal donors, the spleen increased a median of approximately 50% from baseline to day of first LK and returned to near baseline 1 week after last LK. Size change was not associated with significant clinical sequelae.TimepointMedian fold change from baseline in splenic volume (Q1, Q3)First LK (n=304)1.47* (1.27, 1.68)Day 2 after first LK (n=88)1.41* (1.21, 1.61)Day 4 after first LK (n=86)1.19* (1.10, 1.38)Day 7 after last LK (n=89)1.08* (0.96, 1.21)*statistically significant (p<0.05); Q1=1st quartile, Q3=3rd quartile

Splenic rupture in a patient with plasma cell myeloma following G-CSF/GM-CSF administration for stem cell transplantation and review of the literature

Recipients of granulocyte-colony stimulating factor/granulocyte macrophage-colony stimulating factor are not only individuals with underlying disorders, but also healthy donors undergoing peripheral blood progenitor cell (PBPC) mobilization. In addition to the known adverse effects associated with G-CSF, complications such as splenic rupture have also been reported. A review of the English literature, with addition of a patient with plasma cell myeloma, reveals that splenic rupture occurs not only in patients with underlying disease, but also in healthy PBPC donors. Although the cause of splenic rupture does not appear to be associated with any specific condition, physicians should be alerted to the possibility of this potentially fatal complication in individuals receiving G-CSF therapy.

Granulocyte–colony‐stimulating factor–mobilized prophylactic granulocyte transfusions given after allogeneic peripheral blood progenitor cell transplantation result in a modest reduction of febrile days and intravenous antibiotic usage

It was hypothesized that transfusion of two granulocyte-colony-stimulating factor (G-CSF)-mobilized prophylactic granulocyte components into allogeneic peripheral blood progenitor cell (PBPC) transplant patients during the regimen-related neutropenic interval would result in clinical benefit. HLA-matched sibling PBPC donors (n=151) were biologically randomized based on ABO mismatch to donate granulocyte components (Cohort G) or not donate granulocytes (control group, Cohort C). ABO-matched donors who did not meet other study-specific criteria were reassigned to Cohort C. Feasibility, defined as the proportion of ABO-matched donors who underwent granulocyte collections, was 42 percent (53 of 125). The percentage of patients who developed fever during the initial hospitalization was greater in Cohort C versus Cohort G (82.7% vs. 64.2%; p=0.03). In the interval from when granulocyte transfusions were initially given in Cohort G (Day +3 or Day +5) until neutrophil engraftment, the number of febrile days was less in Cohort G versus Cohort C (median, 0 vs. 1; Mann-Whitney p=0.003). The median number of days of intravenous antibiotics given during the initial hospitalization was less in Cohort G versus Cohort C (9 vs. 11; Mann-Whitney p=0.03), a difference accounted for in the interval from Day +3 or Day +5 to neutrophil recovery. There was no significant difference in length of the initial hospital stay, acute graft-versus-host disease rates, or 100-day survival between the two cohorts. This prospective study demonstrates a modest, but significant, benefit of G-CSF-mobilized HLA-matched prophylactic granulocyte transfusions in neutropenic allogeneic PBPC recipients.

Transient respiratory disturbance by granulocyte–colony‐stimulating factor administration in healthy donors of allogeneic peripheral blood progenitor cell transplantation

Allogeneic peripheral blood progenitor cell (PBPC) transplantation requires granulocyte-colony-stimulating factor (G-CSF) administration to mobilize PBPCs in healthy donors. The effects of G-CSF on pulmonary functions, however, have not been clearly elucidated in PBPC donors. Respiratory status by measurements of arterial blood gas was prospectively evaluated serially in 25 healthy donors (9 men, 16 women; age, 18-61 years) administered a dose of 10 microg per kg for 5 days. White blood cell (WBC) counts increased in all the subjects after G-CSF administration; means on Days 0, 3, and 5 were 6 x 10(9), 33.4 x 10(9), and 33.6 x 10(9) per L, respectively. The mean PaO(2) values on the respective days were 93.1, 85.8, and 81.8 mmHg, and these changes were significant (p < 0.0001), remaining significant after adjustment for the WBC count. Levels of both PaCO(2) and AaDO(2) were significantly higher after G-CSF administration than those before G-CSF administration (p < 0.0001 and p = 0.0004, respectively). SaO(2) was significantly decreased after G-CSF administration (p = 0.0002). Age was identified as a significant predictive factor for the increase of AaDO(2) and PaO(2) decline. These observations clearly indicate that the gas exchange was significantly affected during G-CSF administration in healthy PBPC donors. Considering an increasing use of PBPC mobilization by G-CSF, careful monitoring of the respiratory status is important to ensure safety of PBPC donors, especially elderly donors.

Changes in Bone Metabolism Induced by Short-Term Treatment with G-CSF for the Mobilization of Allogeneic Hematopoietic Progenitor Cells.

Short-term treatment with granulocyte colony-stimulating factor (G-CSF) has become a standard procedure for the mobilization of allogeneic peripheral blood progenitor cells (PBPC) in healthy donors. While osteopenia was observed after G-CSF long-term treatment in patients with chronic neutropenia and in G-CSF overexpressing animal models, the information about the effects of G-SCF priming in PBPC donors is limited. Bone pain as the most frequent acute toxicity and the transient increase of serum alkaline phosphatase suggest an effect on bone metabolism also during G-CSF treatment for mobilization. A total of 93 unrelated stem cell donors (21 female, 72 male; median age 35 years) were included in a prospective study to investigate the changes in bone metabolism induced by short-term treatment with G-CSF. Mobilization treatment consisted of 7.5 μg/kg body weight lenograstim (Granocyte™, Chugai Pharma Inc., Tokyo, Japan) for five days. Leukaphereses were performed on day 5 and 6. Samples of blood and urine were taken at pre-donation health-check (before rhG-CSF stimulation, baseline), at the first day of apheresis (leukapheresis) and four weeks after donation (follow-up) and tested for a panel of biochemical markers for bone formation and bone resorption. Stem cell collections were not performed in all donors who were included at baseline, therefore data from leukapheresis are available from 73 donors, and 63 donors were examined at follow-up. Serum bone alkaline phosphatase (BAP) and serum ostase which are both markers for bone formation significantly increased from baseline to leukapheresis (p<0.0001). Both returned to baseline values within 4 weeks after donation. In contrast tartrat-resistent alkaline phosphatase (TRAP) 5b, which is produced by osteoclasts, was found to be decreased at leukapheresis (p=0.001) and at follow-up (p=0.005). The collagen type I crosslinks in urine increased significantly from baseline to follow-up (telopeptides p=0.022; deoxypyridinoline p=0,012). The ligand of receptor activator of NF-kB (RANKL) and osteoprotergerin (OPG), which are members of the TNF receptor family and play a role in the regulation of osteoclast recruitment were also affected by G-CSF treatment, which was reflected by a significant increase of OPG/RANKL-ratio (p=0.01). Serum parathormon (PTH) showed a highly significant increase from baseline to leukapheresis (p<0.0001), but was found at normal ranges in all donors at follow-up. Pearson's correlation of bone markers with circulating CD34+ hematopoietic progenitors before first leukapheresis did not reveal an impact of these parameters on mobilization efficacy.In conclusion the results confirm the influence of G-CSF short-term treatment on bone metabolism in allogeneic stem cell donors. An initial osteoblastic stimulation is followed by a period of increased bone resorption which seems to be not finished 4 weeks after G-CSF treatment. However, the changes in urine crosslink concentrations occurred within the normal ranges of the used assays in the vast majority of donors and therefore we do not assume an impact on the safety of the mobilization procedure. The complex regulation of bone metabolism by humoral and cellular effectors and the cause of transient hyperparathyroidism need further investigations.

Placebo‐controlled study of intravenous magnesium supplementation during large‐volume leukapheresis in healthy allogeneic donors

Marked decreases in ionized magnesium (iMg) levels occur during large-volume leukapheresis (LVL); however, the effect of intravenous (IV) magnesium supplementation in this setting has not been carefully studied. Thirty healthy allogeneic peripheral blood progenitor cell donors receiving citrate anticoagulant with IV calcium prophylaxis were randomized to receive either IV magnesium (0.2 mg Mg per mL acid citrate dextrose-A) or placebo during LVL, with a double-blind design. Thirty subjects underwent 75 LVL pro- cedures, 37 with magnesium and 38 with placebo. Group characteristics were similar for sex, weight, citrate infusion rate (1.36 mg/kg/min vs. 1.37 mg/kg/min), and volume processed (16 L vs. 17 L). Serum iMg levels remained within the reference range with magnesium supplementation, but decreased 39+/-11 percent below baseline (p<10(-10)) after placebo, with greater decreases after consecutive procedures. Subjects receiving magnesium had more vigorous parathyroid hormone responses and higher glucose levels and also tended to have higher serum potassium and ionized calcium levels. Mild paresthesias, coldness, and nausea occurred in 28, 20, and 7 percent of donors, respectively, with no significant differences between groups. Severe symptoms (chest tightness) occurred in only one subject receiving placebo. IV magnesium supplementation exerts a significant impact on serum magnesium levels, but does not reduce the frequency or severity of the relatively mild citrate-related effects observed in LVL performed with continuous IV calcium prophylaxis.

Innovative strategies for PBPC mobilization

Stem cell transplantation, whether autologous or allogeneic, improves the outcome of patients with a number of hematologic malignancies or solid tumors. A relevant proportion of these patients are excluded from this treatment because sufficient numbers of hematopoietic stem cells cannot be obtained by standard cytokine-assisted mobilization. In this article we review the physiology of peripheral blood progenitor cell (PBPC) mobilization and discuss the role of adhesion molecules, such as integrins and selectins, chemokines and their ligands, such as SDF-1alpha and CXCR4, and proteolytic enzymes. Based on this knowledge, several innovative pharmacologic approaches have been proposed to boost the stem cell harvest. Some of them (CTCE, C3a receptor agonist and GrobetaT) are still subject of pre-clinical development, others, such as chemokine receptor ligand AMD3100, have recently been introduced in clinical trials and already deliver promising results. It appears possible to harvest PBPC successfully in poor mobilizers and to cut down the number of collections required in the remaining PBPC donors.

Detection of free hepatitis C virus core antigen by enzyme‐linked immunosorbent assay is not suitable for screening of granulocyte colony‐stimulating factor‐mobilized hematopoietic progenitor donors

Recent studies have shown that hepatitis C virus (HCV) can be detected in peripheral blood mononuclear cells of patients who are negative for the presence of anti-HCV and serum HCV RNA. The aim of the study was to evaluate the prevalence of HCV viremia in granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood progenitor cell (PBPC) donors by the use of a free HCV core antigen enzyme-linked immunosorbent assay (ELISA). A total of 28 samples from consecutive PBPC donors that were mobilized with G-CSF, and 13 samples from patients presenting with leukocytosis of greater than 20 x 10(9) per L from other causes, were tested by a free HCV core antigen ELISA. Positive samples were confirmed by use of neutralization assays. The specificity of the assay was studied in 48,911 healthy blood donors negative for the presence of anti-HCV. The free HCV core antigen assay showed a 46.4 percent positivity in PBPC donors mobilized with G-CSF and 61.5 percent in patients exhibiting leukocytosis in the absence of G-CSF treatment. All the samples were found to be false-positive samples, and those related with growth factor treatment did not react when G-CSF was discontinued. Overall specificity by the test in freshly collected blood donor specimens was 99.62 percent. Data indicate that the free HCV core antigen ELISA is not a valid test in diagnosing HCV infection in G-CSF-treated PBPC donors. Moreover, false-positive results of this test on blood donors might be indicative of elevated white blood cell numbers. The low specificity of this assay in the PBPC mobilization setting suggests that molecular assays should be the test of choice in the screening of G-CSF-treated donors.

Insertion of Retroviral Vectors in NOD/SCID Repopulating Human Peripheral Blood Progenitor Cells Occurs Preferentially in the Vicinity of Transcription Start Regions and in Introns

Reports on insertional “genotoxicity” in patients have created intense interest in characterizing retroviral vector integrations on the genomic level. The retroviral vector SF91m3 was used for transduction of human peripheral blood progenitor cells (PBPC). These PBPC were transplanted into nonobese diabetic/severe combined immunodeficient mice. A total of 186 retroviral vector integration sites were isolated by ligation-mediated PCR from chimeric mouse bone marrow of five PBPC donors, sequenced, and blasted against the human genome. Preferred integration near the transcription start regions, within CpG islands, and within Alu regions was observed. Detailed analysis of targeted RefSeq genes showed a favored integration within the first intron. Integrations were most common in genes coding for signaling proteins, transcription factors, and kinases. In all genes targeted independently multiple times the respective orientation of the provirus within the gene was identical, indicating integration hot spot regions and similar steric determinants for integration sites. Possible explanations for these findings could be nonrandom vector integration, clonal selection due to gene expression interference, or engraftment issues related to gene insertion in signaling and cell cycle genes. The low frequency of integrations in exons may be reassuring as to the safety of retroviral gene therapy with normal human PBPC.

Transient spleen enlargement in peripheral blood progenitor cell donors given G-CSF

The administration of granulocyte colony-stimulating factor (G-CSF) to peripheral blood progenitor cell (PBPC) donors causes spleen length to increase, but the duration of enlargement is not known. Eighteen healthy subjects were given 10 μg/kg of G-CSF for 5 days and a PBSC concentrate was collected by apheresis. Ultrasound scans were used to assess craniocaudal spleen length before and after G-CSF administration. Mean spleen length increased from a baseline length of 10.7 ± 1.3 cm to 12.1 ± 1.2 cm on the apheresis day (p < 0.001). Ten days after apheresis, spleen length fell to 10.5 ± 1.2 cm and did not differ from baseline levels (p = 0.57), but in 3 subjects remained 0.5 cm greater than baseline length. Increases in spleen length in PBPC donors are transient and reversible.

G-CSF-induced spleen size changes in peripheral blood progenitor cell donors.

PBPC donors given G-CSF experience splenic enlargement and, rarely, spontaneous rupture of the spleen. This study evaluated the incidence and time course of splenic enlargement in PBPC concentrate donors and assessed factors affecting size changes. Twenty healthy adults were given G-CSF (10 microg/kg/day) for 5 days and a PBPC concentrate was collected by apheresis. Ultrasound was used to assess craniocaudal spleen length before giving G-CSF, on the day of apheresis and 3 or 4 days after apheresis. The effects of donor age, gender, race, and changes in blood chemistries, blood counts, and CD34+ cell counts on spleen length change were assessed. Spleen length increased in 19 of 20 donors. Mean length changed from 10.9 +/- 2.0 cm before G-CSF to 12.3 +/- 2.1 cm on the day of apheresis (p < 0.001). The mean increase in length was 1.5 +/- 0.9 cm or 13.8 +/- 9.1 percent. Spleen length increased 20 percent or more in six subjects. The spleen length fell to 11.3 +/- 1.8 cm (p < 0.001) 3 or 4 days after apheresis, but it remained greater than baseline levels (p = 0.03). Spleen length change was not affected by donor gender, race, or age. There was no relationship between changes in spleen length and 1) baseline and apheresis-day blood counts and chemistries, or 2) changes in blood counts and chemistries. Spleen size increases in almost all PBPC donors. Enlargement is transient but may be marked in some donors and may place them at risk for splenic rupture.

Long-term follow-up of normal peripheral blood progenitor cell donors treated with filgrastim: no evidence of increased risk of leukemia development.

Data on the long-term safety of filgrastim administration in peripheral blood progenitor cell (PBPC) donors are scarce. The main theoretical risk is believed to be the possible development of leukemia. We conducted a survey of filgrastim-treated related donors to determine the incidence of leukemia after PBPC donation. Of the 343 PBPC donors eligible for inclusion in the survey, 281 (82%) were interviewed by telephone between December 1998 and February 2000. The mean age at donation was 44 years. The median time elapsed after PBPC donation was 39 months, and in 278 (99%) of the interviewed donors it was at least 1 year. At the time of the interview none of the donors had been diagnosed with acute or chronic leukemia. Although the sample size is small and the follow-up duration is limited, these data suggest that exposure to filgrastim is not associated with any notable risk of leukemia development in PBPC donors.

T-cell apoptosis induced by granulocyte colony-stimulating factor is associated with retinoblastoma protein phosphorylation and reduced expression of cyclin-dependent kinase inhibitors.

Peripheral blood progenitor cells (PBPC) mobilized by granulocyte colony-stimulating factor (G-CSF) promptly engraft allogeneic recipients after myeloablative chemotherapy for hematologic malignancies. Surprisingly, no exacerbation of acute graft-vs-host disease has been observed despite a 10-fold higher T-cell content in PBPC compared with bone marrow allografts. Because G-CSF can suppress T-cell proliferation in response to mitogens and enhance their activation-induced apoptosis, we examined the molecular mechanisms underlying G-CSF-induced immune dysfunction. Normal allogeneic lymphocytes were challenged with phytohemagglutinin in the presence of serum collected after G-CSF administration (postG) to healthy PBPC donors, and the expression of key components of the cell cycle and apoptotic machineries was investigated by flow cytometry and Western blotting. Lymphocyte stimulation was associated with collapse of mitochondrial transmembrane potential, hypergeneration of reactive oxygen intermediates, and activation of caspase-3 and DNA fragmentation. Lymphocytes were arrested in a G(1)-like phase of the cell cycle, as measured by G(1)-phase cyclin expression and bromodeoxyuridine (BrdUrd) incorporation. Cell tracking experiments confirmed the occurrence of a lower number of population doublings in postG compared with preG cultures. Unexpectedly, the phosphorylation state of the protein encoded by the retinoblastoma susceptibility gene (pRB) was unaltered in postG cultures, and the inhibition of cell cycle progression occurred without the recruitment of the cyclin-dependent kinase inhibitors p15(INK4B), p16(INK4A), and p27(Kip1). We eventually evaluated the ability of antioxidant/cytoprotectant agents to prevent the G-CSF-induced mitochondrial dysfunction and inhibition of cell cycle progression. Of interest, both N-acetylcysteine and amifostine reduced apoptotic cell death by 45% on average, inhibited the activation/processing of caspase-3, and increased BrdUrd incorporation in postG cultures. Based on these experimental findings, a model is proposed in which T-cell activation in the presence of serum immunoregulatory factor(s) induced by G-CSF is associated with a molecular phenotype mimicking the G(1)-S transition and consisting of pRB phosphorylation, lack of CDKI recruitment, and reduced cyclin-E expression. The putative relationship between lymphocyte mitogenic unresponsiveness and apoptosis induction would occur at the level of key molecules shared by the cell cycle and apoptotic machineries. Whether the G-CSF-mediated modulation of lymphocyte functions in vitro is beneficial in transplantation medicine remains to be determined.