Effect of voxelotor on murine bone marrow and peripheral blood with hematopoietic progenitor cell mobilization for gene therapy of sickle cell disease.

In preparation for hematopoietic stem cell mobilization and collection, current ex vivo gene therapy protocols for sickle cell disease require patients to undergo several months of chronic red cell transfusion. For health care equity, alternatives to red cell transfusion should be available. We examined whether treatment with GBT1118, the murine analog of voxelotor, could be a safe and feasible alternative to red cell transfusion. We found that 3 weeks of treatment with GBT1118 increased the percentage of bone marrow hematopoietic stem cells and upon plerixafor mobilization, the percentage of peripheral blood hematopoietic stem cells. Our data suggest that voxelotor should be further explored for its potential safety and utility as preparation for hematopoietic stem cell mobilization and collection.

Type I interferon is induced by hemolysis and drives antibody-mediated erythrophagocytosis in sickle cell disease.

Patients with sickle cell disease (SCD) suffer from intravascular hemolysis-associated vascular injury and tissue damage. Classical monocytes (CMo), which are the most abundant of circulating monocytes, are activated in SCD, but the cause and consequences of activation remain incompletely understood. We found a positive correlation between total plasma heme levels and circulating interferon-α (IFN-α) in patients with SCD along with upregulation of the type I IFN (IFN-I) inducible genes in sort-purified SCD patients' CMo by transcriptome analysis. We demonstrated that hemolysis led to IFN-I expression, predominantly by mouse liver monocyte and macrophages (Mⲫ), primarily through Tank kinase binding 1 (TBK1)/IκB kinase-ε (IKKε) but not TLR4. In response to hemolysis-induced IFN-I, mouse CMo migrated to the liver and differentiated into monocyte-derived Mⲫ, increasing their numbers by sixfold with acute hemin treatment. Hemolysis-driven IFN-I activity also led to the induction of Fc receptor CD64 expression on monocyte and Mⲫ populations, enhancing alloantibody-mediated erythrophagocytosis in SCD both in vivo in mice and in in vitro human cultures. Altogether, these data demonstrate IFN-I response to hemolysis as a novel activation pathway in monocytes and Mⲫ in SCD, opening the possibility for development of IFN-I-based diagnostics and therapeutics against alloantibody-mediated erythrophagocytosis.

Hemolysis inhibits humoral B-cell responses and modulates alloimmunization risk in patients with sickle cell disease.

Red blood cell alloimmunization remains a barrier for safe and effective transfusions in sickle cell disease (SCD), but the associated risk factors remain largely unknown. Intravascular hemolysis, a hallmark of SCD, results in the release of heme with potent immunomodulatory activity, although its effect on SCD humoral response, specifically alloimmunization, remains unclear. Here, we found that cell-free heme suppresses human B-cell plasmablast and plasma cell differentiation by inhibiting the DOCK8/STAT3 signaling pathway, which is critical for B-cell activation, as well as by upregulating heme oxygenase 1 (HO-1) through its enzymatic byproducts, carbon monoxide and biliverdin. Whereas nonalloimmunized SCD B cells were inhibited by exogenous heme, B cells from the alloimmunized group were nonresponsive to heme inhibition and readily differentiated into plasma cells. Consistent with a differential B-cell response to hemolysis, we found elevated B-cell basal levels of DOCK8 and higher HO-1-mediated inhibition of activated B cells in nonalloimmunized compared with alloimmunized SCD patients. To overcome the alloimmunized B-cell heme insensitivity, we screened several heme-binding molecules and identified quinine as a potent inhibitor of B-cell activity, reversing the resistance to heme suppression in alloimmunized patients. B-cell inhibition by quinine occurred only in the presence of heme and through HO-1 induction. Altogether, these data suggest that hemolysis can dampen the humoral B-cell response and that B-cell heme responsiveness maybe a determinant of alloimmunization risk in SCD. By restoring B-cell heme sensitivity, quinine may have therapeutic potential to prevent and inhibit alloimmunization in SCD patients.

How do I initiate and maintain a mobile apheresis service in the era of cellular therapy.

BACKGROUND: Mobile delivery of apheresis services is an increasingly important component of health care equity, as patients should not have to transfer care providers or travel far distances to receive critical therapeutic apheresis procedures or cell therapy-based treatments. Therefore, the availability of such services should be expanded. STUDY DESIGN AND METHODS: In this "How Do I" article, we provide a detailed overview of the elements necessary to initiate and maintain a successful mobile apheresis service, including challenges and potential solutions. RESULTS: Safe and efficient operation of a mobile apheresis service must consider acquisition of physical assets, such as apheresis sites, personnel, equipment and supplies, communication devices, and transportation vehicles, and optimize organizational aspects, such as staff responsibilities, service partnerships, logistics management, case scheduling and triage, and billing. In the era of cellular therapy, additional critical considerations include regulatory compliance and facility accreditation. DISCUSSION: To our knowledge, no previous publication provides the extensive details described herein to set up and maintain a successful mobile apheresis service, and thus will be very helpful to those facilities wishing to initiate or expand mobile apheresis services.

Umbilical cord blood: an undervalued and underutilized resource in allogeneic hematopoietic stem cell transplant and novel cell therapy applications.

PURPOSE OF REVIEW: The purpose of this review is to primarily discuss the unwarranted decline in the use of umbilical cord blood (UCB) as a source of donor hematopoietic stem cells (HSC) for hematopoietic cell transplantation (HCT) and the resulting important implications in addressing healthcare inequities, and secondly to highlight the incredible potential of UCB and related birthing tissues for the development of a broad range of therapies to treat human disease including but not limited to oncology, neurologic, cardiac, orthopedic and immunologic conditions. RECENT FINDINGS: When current best practices are followed, unrelated donor umbilical cord blood transplant (CBT) can provide superior quality of life-related survival compared to other allogeneic HSC donor sources (sibling, matched or mismatched unrelated, and haploidentical) through decreased risks of relapse and chronic graft vs. host disease. Current best practices include improved UCB donor selection criteria with consideration of higher resolution human leukocyte antigen (HLA) typing and CD34+ cell dose, availability of newer myeloablative but reduced toxicity conditioning regimens, and rigorous supportive care in the early posttransplant period with monitoring for known complications, especially related to viral and other infections that may require intervention. Emerging best practice may include the use of ex vivo expanded single-unit CBT rather than double-unit CBT (dCBT) or 'haplo-cord' transplant, and the incorporation of posttransplant cyclophosphamide as with haploidentical transplant and/or incorporation of novel posttransplant therapies to reduce the risk of relapse, such as NK cell adoptive transfer. Novel, non-HCT uses of UCB and birthing tissue include the production of UCB-derived immune effector cell therapies such as unmodified NK cells, chimeric antigen receptor-natural killer cells and immune T-cell populations, the isolation of mesenchymal stem cells for immune modulatory treatments and derivation of induced pluripotent stem cells haplobanks for regenerative medicine development and population studies to facilitate exploration of drug development through functional genomics. SUMMARY: The potential of allogeneic UCB for HCT and novel cell-based therapies is undervalued and underutilized. The inventory of high-quality UCB units available from public cord blood banks (CBB) should be expanding rather than contracting in order to address ongoing healthcare inequities and to maintain a valuable source of cellular starting material for cell and gene therapies and regenerative medicine approaches. The expertise in Good Manufacturing Practice-grade manufacturing provided by CBB should be supported to effectively partner with groups developing UCB for novel cell-based therapies.

Evidence for continued dose escalation of plerixafor for hematopoietic progenitor cell collections in sickle cell disease.

We present data from our study of plerixafor mobilization (NCT02193191) relevant to the question of whether further dose escalation of plerixafor can address inconsistent adequacy of CD34+ mobilization for gene therapy of sickle cell disease (SCD). We found that, in the same patient, higher plerixafor dosing was associated with higher fold increases in PB CD34+ count, but not necessarily higher absolute CD34+ counts. Variation in pre-apheresis absolute CD34+ counts was related to intra-individual variation in baseline PB CD34+ counts and inter-individual variation in responsiveness to plerixafor. Overall, our results support further studies of continued dose escalation of plerixafor for autologous HPC collection in SCD.

Prognostic factors associated with COVID-19 related severity in sickle cell disease.

BACKGROUND: Equipoise exists regarding sickle cell disease (SCD) as a risk factor for COVID-19 disease severity and variables that increase risk of COVID-19 severity in SCD. Given our health system's large SCD patient catchment, we analyzed our own experience in this regard. STUDY METHODS: Retrospective analysis of the clinical course and factors associated with need for hospitalization and ICU admission of SCD patients diagnosed with COVID-19 through the Northwell Health system from March 1 to Dec 31, 2020. RESULTS: Of 1098 patients with SCD, 3.3% were diagnosed with COVID-19. Overall rates of hospitalization, ICU admission, cohort mortality, and in-hospital mortality were 80%, 19%, 2.5%,and 3.1%, respectively. By multivariable analysis, hospitalization risk was decreased by 60% for every 1 g/dL increase in admission Hb. ICU admission risk was increased by 84% as a health care worker; increased by 45% for every 1000/uL increase in admission immature granulocyte count; and decreased by 17% with hydroxyurea use. DISCUSSION: High hospitalization rates are compatible with worsened severity upon COVID-19 infection in SCD compared to the general population. Patients should be placed on hydroxyurea to increase their Hb and perhaps lower their neutrophil counts. Health care workers with SCD may warrant special safety precautions.

Process and procedural adjustments to improve CD34+ collection efficiency of hematopoietic progenitor cell collections in sickle cell disease.

BACKGROUND: Adequate CD34+ collection efficiency (CE) is critical to achieve target CD34+ cell doses in hematopoietic progenitor cell (HPC) collections. Autologous HPC collection in sickle cell disease (SCD) is associated with unstable collection interfaces and low CD34+ CEs. We hypothesized that variables specific to SCD, activation of blood cells and elevated viscosity, might contribute to these issues and made adjustments to the collection process and procedure to address our hypothesis. STUDY DESIGN AND METHODS: In two patients with SCD undergoing autologous HPC collection on our clinical trial (NCT02193191), we therefore implemented adjustments to the process and procedure in the following areas: proximity of RBC exchange to HPC collection, the type of anticoagulation, and the packing factor setting. RESULTS: There was no collection interface instability. Our CD34+ CE1s were high at 70% and 51%, and granulocyte CE, platelet CE, and product granulocyte % were remarkably low. Product hematocrits were not as high as previously reported to be required to obtain adequate CEs. Interestingly, one HPC product showed a hemoglobin S (HbS) of 91% at the same time that the peripheral blood (PB) showed a HbS of 22%. DISCUSSION: Adjustments to the HPC collection process and procedure were associated with adequate CD34+ CEs and low granulocyte and platelet contamination in HPC products from SCD patients. Given the discrepancy in the percentage of sickle RBCs in the product versus the PB, we hypothesize that CD34+ cells and RBCs may aggregate. Our interventions and hypothesis should be further investigated in larger studies.

Potentially modifiable predictors of cell collection efficiencies and product characteristics of allogeneic hematopoietic progenitor cell collections.

BACKGROUND: Hematopoietic progenitor cell (HPC) and immune effector cell (IEC) therapies often require high doses of mononuclear cells (MNCs), whether CD34+ cells, lymphocytes, or monocytes. Cells for IEC can be sourced from HPC products. We thus examined potentially modifiable variables affecting collection efficiencies (CEs) of MNC subsets in HPC collection and also of the typically undesired cell types of platelets, granulocytes, and red cells, which hinder downstream processing. Finally, we sought to confirm previously indeterminate studies of the effect of an adjusted collect flow rate (CFR) on CD34+ CE. STUDY DESIGN AND METHODS: We performed univariate and multivariate regression analyses of all 135 National Marrow Donor Program (NMDP) HPC collections in 2019 and compared these fixed CFR procedures to previous NMDP collections using adjusted CFRs. RESULTS: Target cell CEs decreased with increasing peripheral blood (PB) concentration and were associated with different cell type locations within the MNC layer. CEs of undesired cell types varied with standard procedural parameters (inlet flow rate, whole blood processed, etc.). Interestingly, some CEs increased with preapheresis hematocrit. Finally, adjusting the CFR by PB MNC count improved MNC CE but not CD34+ CE. CONCLUSION: Correlation of target cell CEs with their PB concentration and different cell type locations by depth within the MNC layer indicates the importance of investigating the compensatory fine-tuning of procedure variables to improve CE. Correlation of CEs with PB hematocrit, and CFR adjustment by a modified PB MNC and/or PB CD34 algorithm should be further explored. Adjusting standard procedural parameters may reduce product contamination.

Safety and benefits of automated red cell depletion-exchange compared to standard exchange in patients with sickle cell disease undergoing chronic transfusion.

BACKGROUND: The Spectra Optia allows automated performance of red blood cell reduction and isovolemic hemodilution (IHD) prior to standard red cell exchange (RCE), and is primarily intended for patients with sickle cell disease (SCD) undergoing chronic RCE. Data on the safety of inducing transient further anemia and the benefits of IHD-RCE is limited and occasionally contradictory. STUDY DESIGN AND METHODS: In this retrospective crossover analysis of six patients with SCD who underwent chronic exchange with standard RCE (Cobe Spectra) followed by IHD-RCE (Spectra Optia), we compared safety and benefit outcomes with IHD-RCE vs standard RCE. RESULTS: There were statistically but not clinically significant drops in blood pressure in the post-IHD phase. With IHD-RCE, there were significant reductions in red blood cell (RBC) usage and/or lower fraction of cells and significant increases in postprocedure hematocrit (Hct) associated with increased preprocedure Hct. There were no differences achieved in the time interval between procedures or in the net RBC gain with IHD-RCE. Overall, there were also no significant differences in pre- and postprocedure percentage of hemoglobin S, reticulocyte count, interval daily hemoglobin A decrement, or postprocedure white blood cell, neutrophil, or platelet counts. CONCLUSIONS: Our study supports that IHD-RCE can be safely used in patients with stroke risk and compared to standard RCE, results in benefits of lower RBC usage and/or fraction of cells remaining and higher postprocedure Hct associated with higher preprocedure Hct. These findings support wider use of IHD-RCE, especially in the current environment with reduced availability of minority units.

Characteristics of coronavirus disease 19 convalescent plasma donors and donations in the New York metropolitan area.

BACKGROUND: Convalescent plasma (CP) is an important initial treatment in pandemics and the New York (NY) metropolitan area is likely to remain a hotspot for collection and distribution of such units. This study reports characteristics of coronavirus disease 19 CP (CCP) donors and their donations to the New York Blood Center (NYBC). STUDY DESIGN AND METHODS: All CCP data from our first day of collection on March 26th through July 7th, 2020 are included in this retrospective analysis. Donor and donation data were extracted from NYBC electronic databases. SARS-CoV-2 antibody testing was initially performed by the NY State Department of Health, and later by NYBC using Ortho and Abbott platforms. RESULTS: CCP donor age and ABO distributions were consistent with reported lower COVID-19 susceptibility in O blood types. CCP versus whole blood donors had similar on-site deferrals, but higher post-donation deferral rates. CCP versus routine plasmapheresis donations had higher vasovagal reactions but similar product rejection rates. Changes in antibody (Ab) test platforms resulted in significant changes in the percent of donors regarded as antibody positive. Donor correlates with higher anti-spike total Ig S/CO ratios were Hispanic ethnicity, overweight body mass index, and longer symptom duration; and with higher anti-nucleocapsid IgG S/CO ratios were male gender, older age, Hispanic ethnicity, and fewer days between symptom onset and first donation. DISCUSSION: We identify donor characteristics not previously reported to correlate with Ab titer. Our analysis should assist with donor outreach strategies, blood center operating logistics, and recruitment of high titer donors.

Further evidence for the benefit of therapeutic plasma exchange for acute multi-organ failure syndrome refractory to red cell exchange in sickle cell disease.

Acute multiorgan failure syndrome (MOFS) remains a significant cause of mortality in sickle cell disease (SCD) patients despite red cell exchange (RCE). In small case series and reports, therapeutic plasma exchange (TPE) has shown benefit in MOFS. As further support for consideration of this modality, we present two patients with SCD and MOFS refractory to RCE who were subsequently treated with TPE. Fresh frozen plasma was used as the replacement fluid. Despite estimated hospital mortality of 40% at the time of intensive care unit admission, both patients showed marked clinical improvement with TPE treatment. Our cases add to the evidence supporting the potential inclusion of MOFS secondary to acute SCD as an indication for TPE in the next edition of the American Society of Apheresis Guidelines on the Use of Therapeutic Apheresis in Clinical Practice.

Optimizing leukapheresis product yield and purity for blood cell-based gene and immune effector cell therapy.

PURPOSE OF REVIEW: A critical common step for blood-based ex-vivo gene and immune effector cell (IEC) therapies is the collection of target cells for further processing and manufacturing, often accomplished through a leukapheresis procedure to collect mononuclear cells (MNCs). The purpose of this review is to describe strategies to optimize the apheresis product cell yield and purity for gene and IEC therapies. Relevant data from the conventional bone marrow transplant literature is described where applicable. RECENT FINDINGS: Product yield is affected by three main factors: the peripheral blood concentration of the target cell, optimized by mobilizing agents, donor interventions or donor selection; the volume of peripheral blood processed, tailored to the desired product yield using prediction algorithms; and target cell collection efficiency, optimized by a variety of device and donor-specific considerations. Factors affecting product purity include characteristics of the donor, mobilizing agent, device, and device settings. SUMMARY: Strategies to optimize product yield and purity for gene and IEC therapies are important to consider because of loss of target cell numbers or function with downstream steps and detrimental effects of nontarget cells on further manufacturing and patient outcome.

Validation of simple prediction algorithms to consistently achieve CD3+ and postselection CD34+ targets with leukapheresis.

BACKGROUND: Cellular therapies using engineered T cells, haploidentical transplants, and autologous gene therapy are increasing. Specified CD3+ or high CD34+ doses are typically required for subsequent manufacturing, manipulation, or CD34+ selection. Simple, practical, and reliable lymphocyte and hematopoietic progenitor cell (HPC) collection algorithms accounting for subsequent CD34+ selection have not been published. STUDY DESIGN AND METHODS: In this analysis of 15 haploidentical donors undergoing tandem lymphocyte and HPC collections, we validated one-step, practical prediction algorithms (Appendix S1, available as supporting information in the online version of this paper) that use conservative facility-specific collection efficiencies, CD34+ selection efficiency, and donor-specific peripheral counts to reliably achieve the target CD3+ and CD34+ product doses. These algorithms expand on our previously published work regarding predictive HPC collection algorithms. RESULTS: Ninety-three percent of lymphocyte and 93% of CD34+ collections achieved the final target CD3+ and CD34+ product dose when our algorithm-calculated process volumes were used. Linear regression analysis of our algorithms for CD3+, preselection CD34+, and postselection CD34+ showed statistically significant models with R2 of 0.80 (root mean square error [RMSE], 31.3), 0.72 (RMSE, 385.7), and 0.56 (RMSE, 326.0), respectively, all with p values less than 0.001. CONCLUSION: Because achievement of CD3+ or CD34+ dose targets may be critical for safety and efficacy of cell therapies, these simple, practical, and reliable prediction algorithms for lymphocyte and HPC collections should be very useful for collection facilities.

Red cell exchange for patients with sickle cell disease: an international survey of current practices.

INTRODUCTION: Red cell exchange (RCE) therapy is increasingly used to treat patients with acute or chronic manifestations of sickle cell disease (SCD). However, little is known regarding the most safe and effective practice parameters associated with this particular therapy. METHODS: A SCD subcommittee of members of the American Society for Apheresis (ASFA) developed a 122-question survey and administered it via email to other ASFA members. The survey inquired about clinical indications for treatment, practice patterns, and transfusion policies for RCE when used for patients with SCD. RESULTS: Ninety-nine distinct institutions completed the survey. Twenty-one (21%) were from outside of the US. Twenty-two (22%) provided chronic transfusion therapy to >10 patients, and both adult (25%) and pediatric-focused services (20%) were represented. Common acute indications for RCE included acute chest syndrome, acute ischemic stroke, and pre-surgical prophylaxis. Common chronic indications included primary stroke prophylaxis, secondary stroke prophylaxis, and recurrent acute chest syndrome. Respondents most commonly set a post-RCE treatment target of 30% for the hematocrit and hemoglobin S levels, regardless of the therapeutic indication. Units for RCE were phenotypically matched in 95% of cases. About 40% of respondents reported using isovolemic hemodilution. CONCLUSIONS: This survey solicited the current practice variations in RCE from a diverse range of practice sites. Many sites reported similar practice patterns and challenges but some variations emerged. To our knowledge, this survey represents the largest and most in-depth investigation of the use of RCE for patients with SCD, and could inform future studies in the field.

Emerging roles of adjunct therapies in acquired thrombotic thrombocytopenia purpura.

Acquired thrombotic thrombocytopenia purpura (aTTP) is caused by autoantibody-mediated severe deficiency of the von Willebrand factor (vWF) cleaving protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13), with subsequent accumulation of ultra-large vWF-multimers that spontaneously form platelet-VWF complexes and microthrombi within the microcirculation. Therapeutic plasma exchange (TPE), by removing autoantibodies and excess ultra-large vWF multimers and replenishing ADAMTS13 activity, remains the urgent primary initial treatment. Although heterogeneity in treatment exists, most centers add upfront immunosuppression with steroids, and many also add upfront rituximab. Refractoriness, exacerbation and relapse are commonly treated with adjunct rituximab. Despite adjunct steroids and rituximab, TTP refractoriness, exacerbation, relapse, morbidity, and mortality remain problematic. Newer adjunct therapies include suppression of ADAMTS13 autoantibody production via plasma cell depletion, inhibition of vWF-platelet interaction, and replenishment of ADAMTS13 function with recombinant ADAMTS13 protein.

A dual strategy to optimize hematopoietic progenitor cell collections: validation of a simple prediction algorithm and use of collect flow rates guided by mononuclear cell count.

BACKGROUND: Previous prediction algorithms to achieve target CD34+ goals have not been widely adopted, with many centers still using a set volume to process for hematopoietic progenitor cell collections. This may be because previous algorithms are challenging to implement. Additionally, no study has yet examined the utility of adjusting the collect flow rate (CFR) based on the donor's preprocedure total mononuclear cell (MNC) count, which correlates with CD34+ yield. STUDY DESIGN AND METHODS: In this retrospective analysis of mobilized allogeneic donors collected using MNC (COBE Spectra, Terumo BCT) or continuous mononuclear cell collection (CMNC) (Spectra Optia, Terumo BCT) procedures, we validated a one-step prediction algorithm to achieve the target CD34+ product dose (Appendix S1, available as supporting information in the online version of this paper). The COBE Spectra MNC Collect Flow Tool (Appendix S2, available as supporting information in the online version of this paper) was used to select the collect flow rate for both MNC and CMNC procedures. Procedural collection efficiency (CE) was compared to that of historical procedures utilizing fixed CFRs (1.0-1.5 mL/min). RESULTS: Ninety-three percent of collections achieved the target CD34+ goal using our algorithm-calculated process volumes. The remaining 7% of cases had CEs lower than the algorithm CE (0.40), and thus were below goal. Second, an MNC-based CFR improved MNC and CD34+ CEs in patients with higher MNC counts compared to our historical controls. CONCLUSION: We validated that this simple, single-step prediction algorithm achieves the target CD34+ goal in most HPC collections. Secondly, we showed that an MNC-based CFR for hematopoietic progenitor cell collections improves CE at higher MNCs; this may be preferable to a WBC-based CFR because of variability of MNC counts at a given WBC count.

Combined heparin/acid citrate dextrose solution A anticoagulation in the Optia continuous mononuclear cell protocol for pediatric lymphocyte apheresis.

Collecting a sufficient number of T-cells is a critical first step in the production of chimeric antigen receptor (CAR)-T cells. Herein, we report a successful implementation of anticoagulation with combined heparin and acid citrate dextrose solution A (ACD-A) for the continuous mononuclear cell (CMNC) protocol on the Spectra Optia in a 20-month-old, 7.5 kg patient with refractory acute lymphoblastic leukemia for manufacture of tisagenlecleucel, a CAR-T cell therapy. Combined heparin/ACD-A was used following clotting issues when ACD-A was used alone during initial CMNC collections. To our knowledge, this is the first reported case in pediatrics of combined heparin/ACD-A anticoagulation with the Spectra Optia CMNC protocol.

Safety and efficacy of plerixafor dose escalation for the mobilization of CD34+ hematopoietic progenitor cells in patients with sickle cell disease: interim results.

Gene therapy for sickle cell disease is limited by the yield of hematopoietic progenitor cells that can be harvested for transduction or gene editing. We therefore performed a phase I dose-escalation study of the hematopoietic progenitor cell mobilizing agent plerixafor to evaluate the efficacy and safety of standard dosing on peripheral blood CD34+ cell mobilization. Of 15 patients enrolled to date, only one was chronically transfused and ten were on hydroxyurea. Of eight patients who achieved a CD34+ cell concentration >30 cells/µL, six were on hydroxyurea. There was no clear dose response to increasing plerixafor dosage. There was a low rate of serious adverse events; two patients developed vaso-occlusive crises, at the doses of 80 µg/kg and 240 µg/kg. Hydroxyurea may have contributed to the limited CD34+ mobilization by affecting baseline peripheral blood CD34 counts, which correlated strongly with peak peripheral blood CD34 counts. Plerixafor administration did not induce significant increases in the fraction of activated neutrophils, monocytes, or platelets. However, increased neutrophils positive for activated ß2 integrin and Mac-1 were associated with serious adverse events. In summary, plerixafor was well tolerated but did not achieve consistent CD34+ cell mobilization in this cohort of patients, most of whom were being actively treated with hydroxyurea and only one was chronically transfused. The study will continue with escalation of the dose of plerixafor and modification of hydroxyurea administration. Clinicaltrials.gov identifier: NCT02193191.