Acute and endothelial-specific Robo4 deletion affect hematopoietic stem cell trafficking independent of VCAM1.

Hematopoietic stem cell (HSC) trafficking is regulated by a number of complex mechanisms. Among them are the transmembrane protein Robo4 and the vascular cell adhesion molecule, VCAM1. Endothelial VCAM1 is a well-known regulator of hematopoietic cell trafficking, and our previous studies revealed that germline deletion of Robo4 led to impaired HSC trafficking, with an increase in vascular endothelial cell (VEC) numbers and downregulation of VCAM1 protein on sinusoidal VECs. Here, we utilized two Robo4 conditional deletion models in parallel with Robo4 germline knockout mice (R4KO) to evaluate the effects of acute and endothelial cell-specific Robo4 deletion on HSC trafficking. Strikingly similar to the R4KO, the acute deletion of Robo4 resulted in altered HSC distribution between the bone marrow and blood compartments, despite normal numbers of VECs and wild-type levels of VCAM1 cell surface protein on sinusoidal VECs. Additionally, consistent with the R4KO mice, acute loss of Robo4 in the host perturbed long-term engraftment of donor wild-type HSCs and improved HSC mobilization to the peripheral blood. These data demonstrate the significant role that endothelial Robo4 plays in directional HSC trafficking, independent of alterations in VEC numbers and VCAM1 expression.

Changes in Hematopoietic Cell Transplantation Practices in Response to COVID-19: A Survey from the Worldwide Network for Blood & Marrow Transplantation.

SARS-CoV-2 has spread rapidly worldwide, but the full impact of the COVID-19 pandemic on the field of hematopoietic cell transplantation (HCT) remains unknown. To understand this better, an 18-item online survey was disseminated by the Worldwide Network for Blood & Marrow Transplantation with questions exploring SARS-CoV-2 testing algorithms, mobilization, and cryopreservation strategies and COVID-19 infections in allogeneic related and autologous hematopoietic progenitor cell (HPC) donors. The aim of this survey was to assess the impact of the outbreak on policies relating to HPC mobilization, collection, and processing with respect to changes in daily routine. A total of 91 individual responses from distinct centers in 6 continents were available for analysis. In these centers, the majority (72%) of allogeneic related and autologous donors are routinely tested for SARS-CoV-2 before HPC collection, and 80% of centers implement cryopreservation of allogeneic HPC grafts before commencing conditioning regimens in patients. Five related and 14 autologous donors who tested positive for COVID-19 did not experience any unexpected adverse events or reactions during growth factor administration (eg, hyperinflammatory syndrome). These data are limited by the small number of survey respondents but nonetheless suggest that centers are following the recommendations of appropriate scientific organizations and provide some preliminary data to suggest areas of further study.

Safety profile of autologous hematopoietic stem cell mobilization and transplantation in patients with systemic sclerosis.

Autologous hematopoietic stem cell transplantation (AHSCT) is thought to be effective therapeutic approach in patients with poor prognosis systemic sclerosis; however, the toxicity remains a challenge. Between years 2003 and 2016, we enrolled 18 patients with systemic sclerosis at median age at transplant of 52 years (range 24-68). The median duration of disease before AHSCT was 14 months (range 2-85). Peripheral blood stem cells were mobilized with cyclophosphamide (CY) and granulocyte colony-stimulating factor. Conditioning regimen included CY (200 mg/kg) and alemtuzumab (median dose, 60 mg) [n = 11], melphalan (MEL; 140 mg/m2) and alemtuzumab [n = 2], CY and rabbit anti-thymocyte globulin (rATG; 7.5 mg/kg) [n = 4], and CY alone (n = 1). Four deaths occurred early after transplant. There were three males and one female at median age at death of 51 years (range 24-68). The AHSCT-related deaths have been observed on days + 1, + 4, + 9, and + 15 after procedure. The causes of death included bilateral pneumonia followed by multi-organ failure in three patients and myocardial infarction in one. Three patients expired late during post-transplant follow-up, after 5, 21, and 42 months. The causes of death were disease progression in two patients and sudden heart attack in one. Eleven patients are alive after median follow-up after AHSCT of 42.0 months (range 0-95). Before proceeding to AHSCT in systemic sclerosis, there is a strong need to optimize patient selection to reduce toxicity. The administration of alemtuzumab should be avoided due to high risk of life-threatening infectious complications.

Improved Prediction of CD34+ Cell Yield before Peripheral Blood Hematopoietic Progenitor Cell Collection Using a Modified Target Value-Tailored Approach.

The most commonly used stem cell source for both autologous and allogeneic transplantation is mobilized peripheral blood hematopoietic progenitor cells collected by apheresis. In the 1990s, an Italian group used the correlation between the preapheresis peripheral blood CD34+ cell count and the final number of CD34+ cells collected to devise a formula for "target value-tailored" (TVT) apheresis. Using local patient data, the Canadian Blood Services Stem Cell Laboratory created a similar model to determine the blood volume to process during apheresis collection. The objectives of this study were to determine the correlation between the number of CD34+ cells predicted by the TVT formula and the actual number of CD34+ cells collected and to determine whether the TVT formula remains predictive when applied to an external data set. All apheresis collections performed at the Ottawa Hospital between January 1, 2003 and October 2, 2013 were reviewed. The primary outcome was the correlation between the number of CD34+ cells predicted by the TVT formula and the actual number of CD34+ cells collected on day 1 of apheresis. For the external data set, all autologous collections performed at the London Health Sciences Centre between December 1, 2008 and December 1, 2013 were reviewed. The external data set was divided into test and validation sets to determine whether a model could be created to predict the final number of CD34+ cells collected on day 1 based on the preapheresis CD34+ count. A total of 1252 collections were included in the analysis. The Ottawa data set included 1012 collections, 836 of which were autologous and 176 of which were from donors. Of the autologous collections in Ottawa, 764 (92.5%) were first collections. In 759 (91%) collections, chemotherapy plus granulocyte colony-stimulating factor (G-CSF) was used as the mobilization regimen. In 747 collections (89%), only 1 collection day was required to achieve the desired number of CD34+ cells. The TVT estimate was highly predictive of the number of CD34+ cells × 10(6)/kg actually collected on apheresis day 1 (r = .90, P < .0001). The London data set included 240 autologous collections. All mobilizations were with G-CSF alone. For the test set, the precollection CD34+ count was highly predictive of the number of CD34+ cells × 10(6)/kg collected on day 1 of apheresis. Applying this model to the validation set, the correlation between the predicted and final and day 1 CD34+ cells × 10(6)/kg count was .9186 (P < .0001). Using a modified TVT approach, the preapheresis CD34+ count can be used to accurately predict the number of CD34+ cells × 10(6)/kg collected on day 1. This approach can be applied at other centers and for different diseases and mobilization regimens. This method can be used to individualize the blood volume processed and, thus, optimize resource utilization.

Increased red cell distribution width is associated with poor stem cell mobilization in patients with advanced chronic heart failure.

Parameters associated with poor CD34(+) stem cell mobilization in advanced chronic heart failure (CHF) patients were investigated. Forty-four CHF patients underwent bone marrow stimulation with granulocyte colony stimulating factor. Poor cell mobilization presents in 32% of patients. Poor and good mobilizers did not differ significantly regarding age, gender, left ventricular ejection fraction, kidney or liver function and exercise capacity. Significant differences were found regarding NT-proBNP levels and red cell distribution width (RDW). Increased RDW was the only independent predictor of poor CD34(+) stem cell mobilization on multivariable analysis and may serve as a biomarker of poor stem cell mobilization in CHF patients.

Incidence and risk factors of poor mobilization in adult autologous peripheral blood stem cell transplantation: a single-centre experience.

BACKGROUND AND OBJECTIVES: Collection of sufficient CD34+ cells for autologous peripheral blood stem cell (PBSC) transplantation is frequently failed in patients with lymphoma or multiple myeloma (MM). We investigated the incidence and the predictive factors for poor mobilization. MATERIALS AND METHODS: A total of 205 adult patients (101 lymphoma and 104 MM) were retrospectively included for identifying the incidence of mobilization failure and the predictive factors for poor mobilization in conventional G-CSF-based mobilization regimen. Another 17 patients who used plerixafor for mobilization were included. RESULTS: Overall, 14·1% of patients (21·8% of patients with lymphoma, 6·7% of patients with MM) were poor mobilizers. Univariate analysis and multivariate analysis revealed an interval from G-CSF administration to PBSC collection exceeding 10 days and peripheral blood mononuclear cells count on the first day of collection were predictive factors for poor mobilization in lymphoma, but not in MM. Among plerixafor-treated patient group, 9 of 11 poor mobilizers who received second-cycle plerixafor mobilization were able to collect higher number of CD34+ cells than that of CD34+ cells during the G-CSF-based first mobilization. All patients who had received initial plerixafor mobilization reached 2·0 × 10(6) CD34+ cells/kg during the four leukaphereses. CONCLUSION: In conventional G-CSF-based mobilization, early PBSC collection after G-CSF administration might enhance CD34+ cell yield. A combination of a new mobilizing agent, plerixafor, would be helpful to harvest sufficient number of CD34+ cells for successful transplantation outcome while reducing the effort of collection procedures in poor mobilizers.

Mobilization and collection of peripheral blood stem cells in healthy donors: risks, adverse events and follow-up.

Allogeneic haematopoietic stem cell transplantation is the choice treatment for many haematological malignancies. Granulocyte-colony-stimulating factor (G-CSF) has been widely used to mobilize stem cells into the peripheral blood from healthy siblings or volunteer unrelated donors. To a large extent, the use of mobilized peripheral blood haematopoietic stem cells has replaced marrow-derived stem cells as the preferred source of donor haematopoietic stem cells. Clinicians have been aware since the first clinical use, that administration of G-CSF, even in a single short course, could possibly be a risk for healthy donors either in short-term or as a delayed effect. The immediate side effects of G-CSF have been established for a long time, most of them are frequent but transient, self-limited and without long-term consequences. Questions have been raised about potential long-term adverse effects such as an elevated risk of haematological malignancies after G-CSF administration. More long-term safety data from registries are needed to adequately evaluate such a relationship. Our objective in this article is to provide an in-depth review of reported adverse events associated with the use of G-CSF in healthy donors and to focus attention on unanswered questions related to their long-term follow-up.

Clinical impact and resource utilization after stem cell mobilization failure in patients with multiple myeloma and lymphoma.

High-dose chemotherapy in conjunction with auto-SCT is the preferred treatment of relapsed Hodgkin disease and non-Hodgkin lymphoma and newly diagnosed multiple myeloma. Failure to achieve optimal stem cell mobilization results in multiple subsequent attempts, which consumes large amounts of growth factors and potentially requires antibiotics and transfusions. We retrospectively reviewed the natural history of stem cell mobilization attempts at our institution from 2001 to 2007 to determine the frequency of suboptimal mobilization in patients with hematologic malignancy undergoing autologous transplant and analyzed the subsequent resource utilization in patients with initially failed attempts. Of 1775 patients undergoing mobilization during the study period, stem cell collection (defined by the number of CD34+ cells/kg) was 'optimal' (> or = 5 x 10(6)) in 53%, 'low' (> or = 2-5 x 10(6)) in 25%, 'poor' (<2 x 10(6)) in 10%, and 'failed' (<10 CD34+ cells/microl) in 12%. In the 47% of collections that were less than optimal, increased resource consumption included increased use of growth factors and antibiotics, subsequent chemotherapy mobilization, increased transfusional support, more apheresis procedures, and more frequent hospitalization. This usually unappreciated resource utilization associated with stem cell mobilization failure highlights the need for more effective mobilization strategies.

Older age but not donor health impairs allogeneic granulocyte colony-stimulating factor (G-CSF) peripheral blood stem cell mobilization.

We evaluated stem cell mobilization in 195 consecutive sibling donors who underwent a uniform mobilization regimen of granulocyte colony-stimulating factor (G-CSF) at 10 microg/kg/day divided into twice daily dosing. On day 5, peripheral blood (PB) CD34 cells/microL were measured immediately prior to peripheral blood stem cell (PBSC) apheresis. Failed mobilization was defined as <20 CD34 cells/microL on day 5. The median age was 52 years and 73 (37%) were 55 years or greater. Comorbid conditions by the Charlson Comorbidity Index (CCI) occurred in 13%, but only 3% had Karnofsky performance status (PS) <100%. Eight (4%) failed mobilization, defined as <20 CD34 cells/microL on day 5. Older age was associated with fewer CD34 cells/microL (P=.002). In addition, 6/73 (8.2%) older donors failed mobilization compared to 2/122 (1.6%) younger donors (P=.054). Comorbidity, sex, race, and donor weight did not influence mobilization. Although low PS was very uncommon, it was associated with reduced mobilization (P=.021), but not mobilization failure. A small fraction of older donors mobilize poorly, and this is not explained by standard measures of comorbidity or PS.

Adverse events among 2408 unrelated donors of peripheral blood stem cells: results of a prospective trial from the National Marrow Donor Program.

Limited data are available describing donor adverse events (AEs) associated with filgrastim mobilized peripheral blood stem cell (PBSC) collections in unrelated volunteers. We report results in 2408 unrelated PBSC donors prospectively evaluated by the National Marrow Donor Program (NMDP) between 1999 and 2004. Female donors had higher rates of AEs, requiring central line placement more often (17% vs 4%, P< .001), experiencing more apheresis-related AEs (20% vs 7%, P< .001), more bone pain (odds ratio [OR]=1.49), and higher rates of grades II-IV and III-IV CALGB AEs (OR=2.22 and 2.32). Obese donors experienced more bone pain (obese vs normal, OR=1.73) and heavy donors had higher rates of CALGB toxicities (>95 kg vs <70 kg, OR=1.49). Six percent of donors experienced grade III-IV CALGB toxicities and 0.6% experienced toxicities that were considered serious and unexpected. Complete recovery is universal, however, and no late AEs attributable to donation have been identified. In conclusion, PBSC collection in unrelated donors is generally safe, but nearly all donors will experience bone pain, 1 in 4 will have significant headache, nausea, or citrate toxicity, and a small percentage will experience serious short-term adverse events. In addition, women and larger donors are at higher risk for donation-related AEs.

The expansion of megakaryocyte progenitors from CD34+-enriched mobilized peripheral blood stem cells is inhibited by Flt3-L.

This study aimed to determine the optimal growth factor combination for expansion of megakaryocyte (Mk) progenitors with clonogenic potential from CD34+-enriched mobilized peripheral blood stem cells (PBSC). Mobilized PBSC were monocyte depleted and CD34+ enriched, then cultured with various combinations of interleukin-3 (IL-3), IL-6, IL-11, Flt3 ligand (Flt3-L), stem cell factor (SCF), granulocyte-macrophage colonystimulating factor (GM-CSF), and erythropoietin (EPO), using a 2(7-3) IV fractional factorial design. Expansion of Mk committed progenitors (CD41+) and primitive precursors (CD61+ CD34+) was determined using FACS and colony-forming assays. Amplification of Mk progenitor production was attributed to IL-3 (p < 0.002), SCF (p < 0.001), and GM-CSF (p < 0.05). Flt3-L inhibited the production of total CD61+ cells (p < 0.05), CD61+CD34+ cells (p < 0.03), and total CD41a+ cells (p < 0.01). Addition of Flt3-L to the optimum growth factor combination of megakaryocyte growth and development factor (MGDF), SCF, IL-3, and GM-CSF caused the greatest increase in total nucleated cells but reduced Mk progenitor expansion. There was also a 20% reduction in Mk+ colonies from cells expanded in the presence of Flt3-L. Factorial analysis identified the optimal combination of growth factors required to expand Mk precursors with clonogenic potential. The addition of Flt3-L to the optimal combination of MGDF, SCF, IL-3, and GM-CSF reduced both the fold expansion of Mk progenitors and Mk colony numbers.

Primed marrow for autologous and allogeneic transplantation: a review comparing primed marrow to mobilized blood and steady-state marrow.

Mobilized peripheral blood collections, obtained following either chemotherapy (with or without granulocyte colony-stimulating factor (G-CSF)) or G-CSF administration alone, are rapidly replacing traditional bone marrow harvests as the source of cells for hematopoietic stem cell transplantation. According to the Autologous Blood and Marrow Transplant and the International Bone Marrow Transplant Registries, for the years 1998 through 2000, blood stem cell (BSC) transplants accounted for about 80% of autologous transplants in the pediatric age group and more than 90% of the autologous transplants among adults. In allogeneic transplantation, where the donor is a healthy family member or normal volunteer, G-CSF-mobilized BSC transplants are being used more and more frequently, accounting for about 20% of allogeneic transplants in the pediatric age range and more than 40% of allogeneic transplants among adults during the same time period. It is not, therefore, too great a stretch to imagine that BSC transplants will soon be, if not already, in the majority for allogeneic transplantation among adults. The principal reason why this is happening is the prevailing view that BSC engraft more rapidly than marrow stem cells (MSC). However, this view is based on comparisons between primed circulating blood cells (BSC) and unprimed resident marrow cells in the steady state (SS-MSC). If the reason why BSC engraft faster than SS-MSC were a consequence of G-CSF used for mobilization, then would priming of MSC by G-CSF (Prim-MSC) accelerate engraftment of marrow as well? We reviewed the literature of the last 10 years to see if there were enough data to answer this question.

Immunophenotypic analysis of peripheral blood stem cell harvests from patients with multiple myeloma.

BACKGROUND AND OBJECTIVES: Four-color multiparameter immunophenotyping has recently proven to be an attractive technique for evaluating the plasma cell (PC) compartment since it allows discrimination between myelomatous and normal PC. This study was designed to investigate: i) whether peripheral blood is less contaminated than bone marrow as a source for an autologous transplant; ii) the effect of growth factors on mobilizing myelomatous PC into peripheral blood; iii) the degree of contamination by myelomatous PC in apheresis samples; and iv) whether the number of PC increases during the last days of apheresis. DESIGN AND METHODS: Using 4-color antigen staining we investigated the composition of the PC compartment in 90 apheresis products from 40 patients with MM; in 17 cases bone marrow and peripheral blood samples were also simultaneously evaluated. RESULTS: (i) All pre-mobilization bone marrow samples analyzed were always contaminated with myelomatous PC whereas only 41% of the post-mobilization peripheral blood samples were contaminated. Moreover, the use of peripheral blood would lead to a reduction of >5x10(5) infused myelomatous PC; (ii) mobilization with cytokines increased the number of circulating PC, generally because of an expansion of the normal PC population; (iii) forty-eight percent of all peripheral blood stem cell harvests were contaminated with myelomatous PC, although normal PC usually represented the predominant population; (iv) no significant changes were observed in the amount of contaminating myelomatous PC during the first three days of apheresis. INTERPRETATION AND CONCLUSIONS: Multiparameter immunophenotyping is a useful approach for investigating the PC compartment in apheresis products.

Factors predicting peripheral blood progenitor cell collection from pediatric donors for allogeneic transplantation.

BACKGROUND AND OBJECTIVES: Although several studies have reported on the use of children as donors for peripheral blood progenitor cells (PBPC), no specific characteristics have been identified as predictors of PBPC collection in this population. In this study we analyzed predictive factors for PBPC collection in pediatric donors. DESIGN AND METHODS: We retrospectively analyzed factors predicting the yield for a target CD34+ cell dose of > or =4x10(6)/Kg donor or recipient body weight, in 105 aphereses from 76 healthy pediatric donors (36 boys and 40 girls) included in the Spanish National Donor Registry. Mobilization consisted of granulocyte colony-stimulating factor (G-CSF) in single doses of 10 microg/kg per day subcutaneously for 4 or 5 days. Apheresis started after the fourth dose of G-CSF. RESULTS: Median age and body weight were 10 years (range 1-18) and 42 kg (range 9-89), respectively. The median number of CD34+ cells/kg recipient body weight was 4.22 (range 0.1-32). On multivariate analysis variables that had a significant negative impact on the CD34+ cell yield, considering the recipient's body weight were the total blood volume processed (regression coefficient (RC): 0.41, 95% CI: 0.21-0.81; p=0.01) and day of apheresis other than first (RC: 0.16, 95% CI: 0.07-0.34; p<0.0001). When considering donor's body weight the variables that positively influenced collection were younger age (RC: 6.79, 95% CI: 1.57-29.25; p<0.01) and large volume leukapheresis (RC: 3.33, 95% CI: 1.13-9.77; p<0.02). INTERPRETATION AND CONCLUSIONS: Our data suggest that pediatric donors mobilized by G-CSF may donate sufficient numbers of CD34+ cells for allogeneic transplantation. The variables that influenced the yield were the donor's age, blood volume processed and the first day of the apheresis.

Impact of stem cell donation modality on normal donor quality of life: a prospective randomized study.

As part of a previously reported trial comparing granulocyte-colony stimulating factor (G-CSF) stimulated bone marrow and peripheral blood stem cells (PBSCs) in allogeneic stem cell transplantation, we included a questionnaire to compare donor morbidity and long-term complications between the two donation procedures. Bone marrow donation was associated with significantly more donors experiencing localized pain at the donation site compared to PBSC collection. However, this was not associated with any increased delay in returning to normal activity. Although a minority of bone marrow donors suffered chronic pain at the donation site, no serious long-term side effects relating to G-CSF stimulated stem cell donation were identified.