Apheresis Collection Research Articles

Human-derived raw materials: Controlled, consistent collections enable successful manufacturing of cell-based regenerative medicine products (TECH1P.847)

Abstract Human cells are critical raw materials for manufacturing cell therapy products, but introduce significant variability. HemaCare controls apheresis procedures and collection sites under a formal quality system, with GMP-compliant, validated procedures and equipment, and GTP-compliant donor screening and tracking. HemaCare performed 86,799 cellular apheresis collections in the last 7 years for research, clinical trials, and commercial products. HemaCare’s unmobilized apheresis products showed consistently high MNC purity, with 93.8% of products containing ≥75% MNC, and an average of 85.66% MNC±7.1% (mean±1 SD). RBC contamination was low, with hematocrit averaging 1.78%±0.7%. Approximately 85% of HemaCare donors have donated apheresis products 5 or more times. HemaCare’s laboratory is equipped with Miltenyi Biotec technology for isolation of raw material into purified cellular subpopulations. The consistency and viability of the purified end products are measured with flow cytometry. Using BioLife Solutions’ serum-free and protein-free cGMP CryoStor™ cryopreservation media with purified cells, post-thaw recovery rates of cell fractions have been above 95%; dendritic cell and macrophage have demonstrated recovery rates of ≥90%. CryoStor™, in combination with freezing in the BioCision CoolCell™ freezing container, has enabled HemaCare to standardize the cryopreservation process, reducing variability while optimizing post-thaw viable cell recovery of its research products.

Isolation of Highly Suppressive CD25+FoxP3+ T Regulatory Cells from G-CSF-Mobilized Donors with Retention of Cytotoxic Anti-Viral CTLs: Application for Multi-Functional Immunotherapy Post Stem Cell Transplantation

Previous studies have demonstrated the effective control of cytomegalovirus (CMV) infections post haematopoietic stem cell transplant through the adoptive transfer of donor derived CMV-specific T cells (CMV-T). Strategies for manufacturing CMV immunotherapies has involved a second leukapheresis or blood draw from the donor, which in the unrelated donor setting is not always possible. We have investigated the feasibility of using an aliquot of the original G-CSF-mobilized graft as a starting material for manufacture of CMV-T and examined the activation marker CD25 as a targeted approach for identification and isolation following CMVpp65 peptide stimulation. CD25+ cells isolated from G-CSF-mobilized apheresis revealed a significant increase in the proportion of FoxP3 expression when compared with conventional non-mobilized CD25+ cells and showed a superior suppressive capacity in a T cell proliferation assay, demonstrating the emergence of a population of Tregs not present in non-mobilized apheresis collections. The expansion of CD25+ CMV-T in short-term culture resulted in a mixed population of CD4+ and CD8+ T cells with CMV-specificity that secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. Furthermore CD25 expanded cells retained their suppressive capacity but did not maintain FoxP3 expression or secrete IL-10. In summary our data indicates that CD25 enrichment post CMV stimulation in G-CSF-mobilized PBMCs results in the simultaneous generation of both a functional population of anti-viral T cells and Tregs thus illustrating a potential single therapeutic strategy for the treatment of both GvHD and CMV reactivation following allogeneic haematopoietic stem cell transplantation. The use of G-CSF-mobilized cells as a starting material for cell therapy manufacture represents a feasible approach to alleviating the many problems incurred with successive donations and procurement of cells from unrelated donors. This approach may therefore simplify the clinical application of adoptive immunotherapy and broaden the approach for manufacturing multi-functional T cells.

Analysis of predictive factors of complete blood count for a high-yield hematopoietic stem cell apheresis collection

Introduction: In medical practice, quantification of peripheral blood CD34+ cells is a method of choice to calculate apheresis yield. Nevertheless, other predictive factors have been evaluated. The objective of this study was to investigate the association between CD34+ cells and number of apheresis collections from patients undergoing autologous bone marrow transplantation based on complete blood count parameters. Methods: Retrospective analysis of 113 patients of the Department of Pediatric Oncology of Hospital de Clinicas de Porto Alegre (HCPA) who had autologous bone marrow transplantation between 2004 and 2011 and underwent mobilization with granulocyte-colony stimulating factor in combination or not with chemotherapy. The following parameters were assessed: Total leukocyte count, platelets, hemoglobin, absolute neutrophil count, lymphocytes, monocytes, and immature granulocytes (IG). Statistical tests were used for asymmetric variables. Results: The correlation between CD34+ × 106/kg and leukocyte count (rs = 0.082; P = 0.394), platelets (rs = 0.078; P = 0.418), hemoglobin level (rs = −0.05; P = 0.564), neutrophils (rs = 0.042; P = 0.665), lymphocytes (rs = 0.048; P = 0.619), and IG (rs = 0.165; P = 0.083) revealed no significant result. In relation to monocytes, there was a weak but significant correlation (rs = 0.255; P = 0.007). In addition, patients with leukocyte count higher than 30 × 109/L and monocyte count higher than 1.8 × 109/L had good collection yield. Conclusion: Although there was no significant association between CD34 × 106/kg and blood parameters, we found that leukocyte count higher than 30 × 109/L and monocyte count higher than 1.8 × 109/L may be predictive factors of efficient collection. However, these values cannot be considered absolute factors because patients with lower counts also had satisfactory collections.

Serum Alkaline Phosphatase Level On Day 4 Of G-CSF Administration Could Be a Simple Yet Practical Surrogate Marker For CD34 Mobilization and Collection

Introduction Granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSCs) have become the primary source of stem cells in the majority of allogeneic stem cell transplantations, mainly because of ease of collection and rapid hematopoietic reconstitution. G-CSF for 4 days is the preferred stem cell mobilization method for normal donors. Peripheral blood CD34+ cell count on day 4 of G-CSF is used as a guide to initiate apheresis and PBSC collection. The actual cutoff for PBSC collection varies by institutional preferences. At our center, we collect PBSCs when the CD34+ cell count is greater than 15/ml. Since the turnaround time for CD34 count is about 3 hours; there is a delay in the start of apheresis which extends the PBSC collection to a second day. A reliable and faster method which estimates CD34+ cell count will enable us to avoid delays in collection to the next day. We report the data from our center on the utility of serum alkaline phosphatase (ALK.ph) level as a surrogate marker for the adequacy of peripheral blood CD 34+ cell count suitable for stem cell collection. Methods A retrospective chart review on normal PBSC donors for allogeneic stem cell transplant between January 1, 2009 and March 31, 2013, was performed after obtaining IRB approval. Data was collected at baseline to include sex, age, BMI, WBC, ALK.ph and day 4 ALK.ph, WBC and CD34 count. Results 51 PBSC donors were evaluated, 2 patients were excluded for baseline ALK.ph> 200 which was due to undetermined causes. None of these donors had a history of hematologic malignancies or underwent previous apheresis procedures. Of the 49 evaluable patients (23 were males); the median age was 53 years (range 11-72), and the median body mass index (BMI) was 29.1 (range 14-78). Median baseline ALK.ph was 72 (range 42-150), the median day 4 ALk.ph was 164 (54-279). The median CD34 count on first day of collection was 47 (5-218) and there were no mobilization failures. A positive correlation was observed between CD34 count and increase in ALK ph levels 0.4171 (p=0.002). For every one unit/ liter increase in alkaline phosphatase there is 0.48 times (95% CI 0.17-0.78) increase in CD34 count. On univariate regression analysis, the factors that were correlated with CD34+ cell counts were day 4 alkaline phosphatase (p=0.015), day 4 WBC count (p<0.001) and change in alkaline phosphatase as compared to baseline (p=0.002). However, on multivariate robust regression analysis, factors that were correlated with CD34 counts were gender (p=0.04), day 4 WBC count (p<0.001) and baseline alkaline phosphatase (p=0.027). Conclusion Our analysis suggests a potential utility of the change in serum alkaline phosphatase level at day 4 after GCSF in predicting peripheral blood CD34+ cell count. Further studies are underway to utilize this information as part of an algorithm to improve efficiency and prevent delays in normal donors undergoing PBSC mobilization and harvesting. Disclosures: No relevant conflicts of interest to declare.

Frequency of adverse events in plateletpheresis donors in regional transfusion centre in North India

BackgroundAlthough automated cell separators have undergone a lot of technical refinements, attention has been focused on the quality of platelet concentrates than on donor safety. We planned this prospective study to look into donor safety aspect by studying adverse events in normal healthy plateletpheresis donors. Study design and methodsThe study included 500 healthy, first-time (n=301) and repeat (n=199) plateletpheresis donors after informed consent. The plateletpheresis procedures were performed on Trima Accel (5.1 version, GAMBRO BCT) and Amicus (3.2 version FENWAL) cell separators. The adverse events during procedure were recorded and classified according to their nature. The pre and post procedure hematological and biochemical profiles of these donors were also assessed with the help of automated cell counter and analyser respectively. ResultsA total of 18% (n=90) adverse events were recorded in 500 plateletpheresis donors, of which 9% of were hypocalcaemia in nature followed by hematoma (7.4%), vasovagal reaction (0.8%) and kit related adverse events in (0.8%). There was significant post procedure drop in Hb, Hct, platelet count of the donors (p<0.0001) whereas WBC count showed a statistically significant rise (p<0.0001). Divalent cations (iCa+, TCa+, TMg+) also showed a statistically significant decline after donation (p<0.0001). However there were no statistically significance difference between adverse events in Trima Accel (5.1 version, GAMBRO BCT) and Amicus (3.2 version FENWAL) cell separators. ConclusionDonor reactions can adversely affect the voluntary donor recruitment strategies to increase the public awareness regarding constant need for blood and blood products. Commonly observed adverse events in plateletpheresis donors were hypocalcemia, hematoma formation and vasovagal reactions which can be prevented by pre-donation education of the donors and change of machine configuration. Nevertheless, more prospective studies on this aspect are required in order to establish guidelines for donor safety in apheresis and also to help in assessing donor suitability, especially given the present trend of double product apheresis collections.

Discordant CD34+ cell results in peripheral blood and hematopoietic progenitor cell–apheresis product: implications for clinical decisions and impact on patient treatment

A 50-year-old male with T-cell lymphoma presented for autologous peripheral blood stem cell transplantation. After granulocyte-colony-stimulating factor (G-CSF) mobilization, his peripheral blood CD34+ cell count was 166 × 10(6) /L on the day before collection, which predicted a high yield of CD34+ cells in the apheresis product. The first two collections had yields much lower than expected, triggering an investigation and changes to the apheresis collection methods since mobilization appeared adequate from the peripheral CD34+ values. Changes to the apheresis collection variables and instrumentation did not improve the yields in the next three collections. The laboratory investigation demonstrated that there was an interfering substance in the patient's plasma that was causing falsely high peripheral blood CD34+ cell values and that the low CD34+ cell yields in the collections were consistent with the actual peripheral blood CD34+ cell value. Based on this finding and after discussion with the clinical service, the patient then received plerixafor to increase the number of circulating CD34+ cells before Collections 6 and 7. The patient went on to achieve the target CD34+ cell dose and subsequently underwent a successful autologous transplant with full hematopoietic engraftment. This case demonstrates the importance of timely and critical review of laboratory results in the context of the specific patient. This case exemplifies how diligent review of laboratory results and open communication among the various teams can positively affect patient outcomes.

Study of serum ferritin in donors of two red blood cells units collected by apheresis

ObjectiveTo analyze the recovery of iron stores without supplementation, when keeping an interval of six months between donations. Material and methodsFrom April 2007 to May 2011, 308 regular and voluntary donors were selected. The apheresis collections were performed using ALYX® Component Collection System-Fenwal™. The hematological parameters were analyzed using the Cell DIN Sapphire – Abbot Diagnostics, and the serum ferritin by sandwich immunoassay method with fluorescence detection in final phase (ELFA) – Vidas® Ferritin–Biomérieux SA. A descriptive statistical analysis was performed for each hematological parameters and serum ferritin. ResultsThe median hemoglobin concentration was 15.6g/dL (14, 18.4) in the first procedure and remains constant at subsequent donations. The ferritin median concentration was 64.6μg/L (7.2, 886). A decrease of 15.6% was observed when compared the first to the second procedure with a median 54.6μg/L (8.3, 213.7). Paradoxically, this decrease is not evident in the subsequent procedures, where an increase of 14.6% and 3.4% for the third and fourth procedure respectively was observed. Changes in ferritin values show statistically significant differences between the first and second collection, but this difference disappeared in subsequent donations. The analysis of MCH in each collection indicates that the significant difference between first and second donation (p1–2<0.05) and between first and third (p1–3=0.015), agree with the greatest decline of the ferritin found between procedures and the beginning of the stabilization of ferritin levels. CommentaryThe determination of ferritin appears not to be the most important parameter to consider at the time of donor selection and suggests that other factors unrelated to the donation may play a significant role. A decrease in serum ferritin was observed at the beginning, but it seems to attend a recovery and stabilization in the successive procedures.

Human blood-derived raw material: enabling controlled, consistent collection (P4415)

Abstract Human cells are critical raw materials for manufacturing cell therapy products, but often introduce significant variability. Rigorous operational controls and quality systems, however, enable optimal collection of high-quality, consistent cellular material. HemaCare, a long-standing supplier of human-derived blood components, controls apheresis procedures and collection sites under a formal quality system, with GMP-compliant, validated procedures and equipment, and GTP-compliant donor screening and tracking. HemaCare performed 69,658 cellular apheresis collections in the last five years, including patient and normal-donor MNCs, G-CSF-mobilized PBPCs and plateletpheresis products, for use in research, clinical trials, and commercial products. Expanded capabilities include bone marrow, umbilical cord blood, and cord tissue collection, immunomagnetic selection, cryopreservation, and analysis by flow cytometry. HemaCare unmobilized apheresis products showed consistently high MNC purity, with 93.8% of products containing ≥75% MNC, and an average of 85.2% MNC ± 6.6% (mean ± 1 SD). Red blood cell contamination was low, with hematocrit averaging 1.8% ± 0.8%. Approximately 85% of HemaCare donors have donated apheresis products 5 or more times, and this repeat-donor pool also contributes to product consistency, as MNC content of individual donor apheresis products had an average coefficient of variation of 3.5%, compared to a CV of 7.7% for all apheresis products.

Donor lymphocyte collections using the spectra Optia MNC version 5

Methods and Materials: 10 non-mobilized allogeneic donors have had DLI apheresis collections on Optia MNC version 5. Collection preference was set to deliver a low haematocrit final product. ACD-A anticoagulation was used and apheresis completed at an AC ratio of 13.5 and infusion rate of 0.8mL/min/L total blood volume (TBV). Run data were recorded collected from the Spectra Optia apheresis reporting system at the end of each procedure. Full blood count data and CD3+/CD19+ target cell numbers were completed for the donor before and after apheresis and from the final product. Performance was measured by target cell yield and collection efficiency together with final product volume and haematocrit. Platelet loss to the donor was also measured after apheresis.

Expanded Natural Killer (NK) Cells for Immunotherapy: Fresh and Made to Order

AbstractAbstract 1912 IntroductionRemarkable increases in the dose and activity of NK cells can be achieved by co-culture with the HLA class I deficient cell line K562 that has been genetically modified to express membrane-bound IL15 and the co-stimulatory molecule 41BB-ligand (K562-mb15-41BBL; Fujisaki et al. Cancer Res. 2009;69:4010–4017). We are conducting a clinical trial utilizing these ex-vivo expanded NK cells (ENK) which are produced at the Center for Cell and Gene Therapy (CAGT) at Baylor and then shipped to the University of Arkansas for Medical Sciences (UAMS) for infusion to high-risk relapsed multiple myeloma (MM) patients using the NHLBI-PACT mechanism. Here we report on the characteristics of the ENK cell products sent fresh versus frozen. MethodsApheresis products were collected from MM patients or healthy donors (HD), cryopreserved, and then shipped to CAGT for GMP grade production, as described (Lapteva et al. Cytotherapy 2012; in press). Briefly, mononuclear cells from thawed and ficolled apheresis products were cultured in Stem Cell Growth Medium (CellGenix) supplemented with 10% fetal bovine serum and 10 U/mL IL2 with stimulator cells at a ratio of 1 NK cell to 10 irradiated K562-mb15-41BBL cells (developed at St. Jude Children’s Research Hospital, Memphis, TN). Cells were harvested on day 8–9; products from HD were CD3-depleted. Clinical-grade products were shipped to UAMS overnight either cryopreserved in a dry shipper (n=7) or fresh in 5% human serum albumin on cold packs at 1–11°C (n=4). Cell purity, expression of activating molecules, and viability by 7AAD exclusion was assessed by flow cytometry. Standard 4h chromium-release assays were used to assess potency against K562 cells at a 20:1 ENK: K562 ratio. Student’s t-Test was used to determine significance. ResultsFrom 0.9–1.5×107 starting NK cells, the total number of ENK cells produced was 5.4×109 (range 1.8–24×109). The fold NK-cell expansion was significantly lower for MM patients (n=5, median 22, 12–70 fold) than for HD (n=6, median 95, 31–160 fold; p<0.05). At harvest, median CD3+/CD56+ NK cell purity was 70% (52–88); CD3 depletion of HD products increased CD3+/CD56+ purity to 93% (86–95) resulting in a median CD3+/CD56- T cell content of 0.02% (0.04–1.02). Overall, median viability was 93% (67–98) and potency (defined as lysis of K562 cells at a 20:1 E:T ratio) was 74% (26–92). One product derived from a patient with 21% CD138+ MM cells in the apheresis collection had low expansion (12-fold), viability (66.7%) and potency (26%). For cryopreserved products, viability immediately after thawing was acceptable (median 94%, 75–99) but recovery of viable cells varied from 61% to 100% and thawed ENK failed to lyse K562 cells unless rested overnight. Further, recovery was extremely poor after overnight incubation (median 16%, 10–21). We therefore validated shipment of fresh ENK products. In contrast with frozen NK cells, the median recovery for fresh clinical products post-shipping was 101% (87–151). We confirmed that NK purity, viability, potency and expression of the key activating molecules NKG2D, NKp30, NKp44 and CD226 were retained up to 48h after transfer. ENK further increased by 34% after 72h in vitro incubation in the presence of IL2. Significant in vivo expansion of ENK was observed after infusion of fresh ENK cell products (n=3) but not after infusion of thawed products (n=3, see separate abstract). An additional advantage was that the fresh cells arrived ready to infuse and changes in release criteria relying on rapid and in process testing significantly reduced the time from apheresis collection to ENK infusion, an important consideration when treating high-risk MM patients who can experience rapid disease progression. ConclusionWe conclude that large numbers of clinical grade ENK cells can be generated from both MM patient and HD derived apheresis products by co-culture with IL2 and K562-mb15-41BBL although less vigorous expansion was observed with patient-derived cells. Upon thawing, cryopreserved ENK cells exhibited inferior recovery and potency, and survived poorly during further in vitro culture. In contrast, freshly formulated and shipped ENK cells have excellent recovery and retain cytolytic ability. Robust in vivo expansion was only seen after infusion of fresh ENK cells. Production assistance by CAGT allowed for the rapid implementation of a novel therapy utilizing fresh ENK cells for poor prognosis MM patients. Disclosures:No relevant conflicts of interest to declare.

High Dose Sargramostim (GM-CSF) Combined with IV Plerixafor for the Mobilization of Peripheral Blood Stem Cells (PBSC) From Normal HLA-Matched Allogeneic Sibling Donors Results in Hypercoagulability

AbstractAbstract ▪4095▪This icon denotes a clinically relevant abstractWe report an interim safety analysis of a phase II trial evaluating GM-CSF and plerixafor for the mobilization of peripheral blood stem cells in normal HLA-matched allogeneic sibling donors, performed due to unexpected toxicity observed with this combination.We have previously reported a lower than expected incidence of acute GVHD in recipients of sibling donor allogeneic HSC transplants mobilized with GM-CSF alone (Devine et al. Blood), as well as similar results using a mouse model of GVHD, in which increased Tregs as well as putative myeloid derived suppressor cells (MDSCs) were observed. However, the use of GM-CSF as a single agent for stem cell mobilization has been limited by an unacceptably high (∼30%) incidence of failure to mobilize and collect adequate stem cell numbers in a single 20 L apheresis procedure. Based upon observed synergy between GM-CSF and plerixafor for HSC mobilization in mice, we hypothesized that this combination in humans might overcome the low stem cell yields observed with either agent alone and result in a PBSC product enriched in Tregs and MDSCs that could reduce GvHD after transplant.A phase II trial was designed, which is ongoing, to evaluate the efficacy of GM-CSF, initially at a dose of 10 mcg/kg SC daily × 5 – 6 days, and plerixafor 320 mcg/kg IV daily starting on day 5, with the primary objective of reduction in the day 1 mobilization failure rate from 33% to <10%, and a planned enrollment of 17 patients. To date 14 donors have been mobilized and 13 recipients transplanted. Initial mobilization with GM-CSF 10mcg/kg was complicated by a high incidence (4/4 donors) of unexpected catheter and IV tubing-associated thrombosis at the time of apheresis, requiring line revision, and in some cases placement of central venous access. The trial was halted for an interim safety analysis, resulting in reduction in the dose of GM-CSF to 5 mcg/kg SC daily × 5–6 days. Since this dose reduction, no significant problems with thrombosis precluding a full 20 L apheresis have been observed among 13 subsequent donors. No evidence of systemic thrombosis has been observed in any donors with this combination. The most common adverse effects have included first dose reaction of severe acute onset bone pain (only in 10mcg/kg dose of GM-CSF), diarrhea, nausea, fatigue, bone pain, injection site reaction, and cardiovascular disorders including bradycardia and transient hypotension. There have been no grade 3–4 adverse events related to mobilization.A preliminary analysis of mobilization in 10 donors with a 20 L minimum apheresis volume demonstrated a failure to collect ≥2×106 CD34/kg recipient weight in 1/10 donors. The mean CD34/Kg recipient weight collected from these donors was 5.25 × 106 CD34/Kg. Among donors who failed to undergo a minimum 20 L apheresis collection, only 2/4 required a second day of aphereis to achieve a collection goal of 2 × 106 CD34/kg, and no donor failed to reach this goal after two days of apheresis. CD34 cell mobilization peaked at 4–9 hours after plerixafor infusion with mean CD34/uL = 38 at 6 hours (SD = 10.35).No recipients had primary graft failure. The median time to neutrophil engraftment was 13 days (range 11 – 15), and median time to platelet engraftment was 24 days (range 19 – 28). All evaluable recipients had 100% peripheral blood donor chimerism at day 30 post transplant. One recipient developed secondary graft failure at day +51, and required reinfusion of G-CSF-mobilized stem cells from his initial donor.The incidence of grades II–IV and III-IV acute GvHD, respectively, among recipients of a GM+P mobilized product was 45% and 18%. The incidence of cGvHD was not evaluable due to the short duration of follow-up. CMV viremia incidence was 38%.Peripheral blood from donors was collected for correlative studies including phenotyping and functional studies, but has not yet been analysed. Further studies evaluating the hypercoagulability associated with higher doses of GM-CSF are ongoing.GM-CSF combined with IV plerixafor is a feasible combination. Higher doses of GM-CSF (10mcg/kg) are associated with first dose reactions, hypercoaguability and catheter thrombosis. Disclosures:Schroeder:Sanofi Oncology/Genzyme: Research Funding. DiPersio:genzyme: Honoraria.

Pediatric apheresis with a novel apheresis device with electronic interface control

Cancer in children, and specifically cancer requiring autologous stem cell transplantation, is rare. As a consequence, though, experience with pediatric stem cell apheresis collections is limited. Challenges of apheresis in small children (<20 kg) include small total blood volume, issues with venous access, concerns about tolerable anticoagulant doses, and limitations in product volumes that can safely be collected. This article presents a small series of autologous "stem cell" apheresis procedures in infants and toddlers weighing between 5.5 and 20 kg, the first ones performed with a novel leukapheresis device (Spectra Optia MNC v.3.0, Terumo BCT) to be reported. Some features of the system are described that can be used to achieve favorable apheresis outcomes in small children. Apheresis procedures were uneventful and successful with similar extraction efficiencies (median preapheresis collection efficiency [CE2], 36%) as in adult patients. At 58%, platelet attrition was considerable. Our data indicate that stem cell apheresis with the Spectra Optia MNC v.3.0 in very small donors is feasible, safe, and associated with very small product volumes.

Successful collection and engraftment of autologous peripheral blood progenitor cells in poorly mobilized patients receiving high‐dose granulocyte colony‐stimulating factor

Granulocyte Colony-Stimulating Factor (G-CSF) alone or in conjunction with chemotherapy is commonly used to mobilize hematopoietic progenitor cells (HPC) into peripheral blood for progenitor cell harvest for autologous HPC transplantation. However, in up to 30% of patients, HPC are not effectively mobilized. In this study, we report the efficacy and safety profiles of a mobilization strategy using high-dose (up to 36 μg/kg) G-CSF in poorly mobilized patients. Retrospective medical record reviews were performed for 392 patients who underwent autologous peripheral blood progenitor cell collection. A total of 56 patients were given high-dose G-CSF due to very ineffective mobilization and 35 of these patients underwent autologous HPC transplantation. The efficacy of mobilization, apheresis collection, and infusion were reviewed and analyzed. More than 2.5 × 10(6) CD34/Kg were collected in 88% of patients (49 of 56) who were placed on high-dose G-CSF due to very ineffective mobilization. Of the 35 patients who underwent HPC transplantation using the progenitor cells that were mobilized with high-dose G-CSF due to very ineffective mobilization, all had rapid and complete neutrophil and platelet engraftment comparable with good mobilizers. We conclude that collection of HPC using hyperstimulation with G-CSF is an effective alternative approach for HPC harvest for poorly mobilized patients.

Prediction of Peripheral Blood Progenitor Cell Numbers In Leukocyte Apheresis Collection

Many allogeneic donors are donating hemopoietic progenitor cells(HPC) from peripheral blood. If the HPCs collected in a standard 12 L blood processing is less than target, donor can choose to do a 2‐day collection or 1‐day collection with extended time with a maximum of 24 L blood processed. We presented a formula for predicting the number of CD34 cells(est‐CD34) in a 12 L collection based on pre‐harvest CD34(/uL) and body weight(Wt):Est‐CD34(×106)=pre‐CD34xkx(Donor Wt/Recipient Wt), where k is a coefficient related to the efficiency of the collection and we use k=0.085 in our facility. We tested 19 donors aged 22 to 54 y/o including 12 men and 7 women. 11 donors had an est‐CD34 above target. Only 1 of them didn't meet the goal, which was 1.9% less. Among the 8 donors with an est‐CD34 below target, we met the goal in 1 donor after 15 L collection. None of the 4 donors who were predicted to have a low est‐CD34 even with 24 L collection met the goal, although the actual numbers were close to our prediction. We didn't extend collection in 3 donors for various reasons and their final HPCs were below the targets. Our prediction formula may help collection facilities to predict who needs extended collection.

CD133+CD34+ and CD133+CD38+ blood progenitor cells as predictors of platelet engraftment in patients undergoing autologous peripheral blood stem cell transplantation

BackgroundHematopoietic stem cells (HSC) have been characterized by CD34+ expression and an adequate dose of CD34+ cells is associated with a complete engraftment. CD133 is a more specific marker of HSC. Materials and methodsWe studied the relationship between graft content of CD34+, CD133+, and CD38+ cells and trilineage engraftment after autologous stem cell transplantation in patients with different hematological disorders. Blood samples were obtained before and after mobilization with recombinant granulocyte-colony stimulating factor (G-CSF, 16μg/kg), from apheresis collections, and after transplantation. ResultsCell subsets were quantified by flow cytometry, and the dose of each population infused was correlated with success of engraftment. G-CSF induced mobilization of CD133+CD38+ cells (12.6-fold) and CD133+CD34+ cells (14.7-fold). A correlation was observed between the infused dose of CD133+CD34+ and CD133+CD38+ cells and platelet engraftment. ConclusionCD133+CD34+ and CD133+CD38+ cells were mobilized with G-CSF and these cell subsets were correlated with platelet engraftment.

The use of plerixafor in hematopoietic progenitor cell collection in pediatric patients: a single center experience

Plerixafor was recently approved for use in combination with granulocyte-colony-stimulating factor (G-CSF) for hematopoietic progenitor cell (HPC) collection by apheresis in adults with multiple myeloma (MM) or non-Hodgkin lymphoma (NHL). However, its efficacy in pediatric patients is not well-studied; thus, we report on our institutional experience with this population. A retrospective observational analysis was performed using both stem cell-processing laboratory information as well as apheresis charts and medical records on all pediatric patients who received plerixafor as part of the mobilization regimen between December 2006 and December 2010. The primary outcome was collection yield. Secondary outcomes included the ability to undergo autologous hematopoietic stem cell transplantation (auto-HSCT) and engraftment status. Eighteen HPC collections by apheresis representing seven mobilization courses were performed on five pediatric patients with poor mobilization status (three males, two females; median age 14 years). Median pre-harvest peripheral blood CD34(+) cell (PB CD34(+)) count was 6.88/μL. A strong correlation between pre-harvest PB CD34(+) count and collection yield was observed. Median total collection yield was 2.26 × 10(6) CD34(+) cells/kg. Four patients achieved a minimum collection of 2 × 10(6) CD34(+) cells/kg. Three patients underwent auto-HSCT with a median neutrophil and platelet engraftment of 12 and 34 days, respectively. No major adverse events with plerixafor administration or apheresis collections were reported. Plerixafor in combination with G-CSF is a safe and potentially helpful mobilization agent in poor mobilizers. Further studies should be done to evaluate the true efficacy of plerixafor in the pediatric population.

Peripheral Blood CD34+ Cell Enumeration as a Predictor of Apheresis Yield: An Analysis of More Than 1,000 Collections

The role of the peripheral blood (PB) CD34(+) cell count in predicting the CD34(+) cell yield in hematopoietic progenitor cell apheresis collections is well established. However, sometimes unexpectedly poor CD34(+) cell yields are obtained. To determine the effect, if any, of a range of factors on the ability of the PB CD34(+) count to predict collection CD34(+) cell count, we performed a retrospective analysis on consecutive hematopoietic progenitor cell apheresis collections between 2004 and 2008. Factors investigated included mobilization regimen, PB white blood cell count, body weight, and disease. After exclusion of collections involving apheresis complications, a total of 1,225 PB CD34(+) cell results with corresponding collection CD34(+) cell results from 458 patients were analyzed. Although differences in the median PB CD34(+) cell counts and collection CD34(+) cell counts were seen between mobilized collections with chemotherapy plus granulocyte colony-stimulating factor and those with granulocyte colony-stimulating factor alone, the predictive capability of the PB CD34(+) cell count for the collection CD34(+) cell yield remained similar. Although poorer collection efficiencies were observed in the myelodysplastic syndrome/myeloproliferative disorder diagnostic subgroup, our findings confirm that PB CD34(+) cell analysis remains a powerful and irreplaceable tool for predicting hematopoietic progenitor cell apheresis CD34(+) cell yield.

High Correlation of Pre-Apheresis Peripheral Blood CD34+ Cell Counts with Final Apheresis Product CD34+ Cell Count by Using a More Accurate Determination of Patient Blood Volume

Abstract 2996The final apheresis product CD34+ cell count per Kg of recipient weight is used to determine if additional apheresis procedures are necessary to collect the targeted amount of hematopoietic stem cells for autologous peripheral blood stem cell transplantation. Flow cytometric analysis of CD34+ cells can take several hours. A more timely technique to predict apheresis product CD34+ cell counts during apheresis may help determine if further administration of cytokines is necessary or if apheresis catheters can be removed, and overall improve the efficiency of patient (pt) care. We performed a retrospective review of all pts undergoing autologous peripheral blood stem cell mobilization with granulocyte-colony stimulating factor (G-CSF) alone, G-CSF and plerixafor, or with chemotherapy followed by G-CSF, from July 2010 through May 2011 who underwent peripheral blood stem cell collection with apheresis on the COBE Spectra cell separator. Linear regression models were used to formulate the calculation of pt blood volume based on the pre-apheresis CD34+ cell count per micro liter of blood, the final apheresis product CD34+ cell counts, and the amount of blood processed during the apheresis procedure. This calculated blood volume is expressed by the formula BV = 82.5(patients weight in Kg) + 793. We then prospectively evaluated the next consecutive pts who underwent stem cell mobilization and apheresis in June and July 2011. Twenty-seven apheresis collections were done on 26 pts. Fourteen pts were female, and 12 pts were male. Seventeen pts were diagnosed with myeloma, 6 pts with NHL, and 3 pts with other diseases. Twenty pts were mobilized with G-CSF (10 ug/kg daily) with apheresis to begin on day 5. Fifteen of the 20 pts required plerixafor on day 4 because of low peripheral blood CD34+ cell counts (< 10/ul). Six pts were mobilized with chemotherapy followed by G-CSF 10 ug/kg daily until peripheral CD34 cell counts recovered greater than 10/ul and then apheresis was started. Each pt had their blood volume calculated according to the formula above and the peripheral blood CD 34+ cell count was measured on the first and second day of apheresis. The peripheral blood CD 34+ cell count/ul was multiplied by 1000 and this product was multiplied by the calculated blood volume and then divided by the pts weight [(PBCD34+ cell count × 1000) × BV]/Kg to determine the predicted apheresis product CD34+ cell count, which was then compared to the actual apheresis product final CD34+ cell count. On the first day of collection the mean for the predicted product CD34+ cell count was 4.98 × 106 +/− 3.1 × 106, and the actual apheresis product CD34+ cell count was 4.61 × 106 +/− 2.90 × 106 (Pearson correlation r value of 0.913 and a p value <0.001)(see figure). Nineteen collections were evaluable on the second day of collection with the mean for the predicted product CD34 + cell count of 2.08 × 106 +/− 1.64 × 106, and the mean for the actual apheresis product CD34+ cell count of 2.29 × 106 +/− 0.768 × 106 (Pearson correlation r value of 0.620 with a p value of 0.005). There was no significant difference in the correlation between patients mobilized with G-CSF alone, G-CSF and plerixafor or after chemotherapy and G-CSF. In conclusion, a more accurate determination of patient blood volume allowed for a high degree of correlation on the first day of peripheral blood stem cell collection on the COBE Spectra machine between the predicted product CD34+ cell count and the actual apheresis product CD34+ cell count. An accurate prediction of the final apheresis product CD34+ cell count may allow for less cytokine administration, quicker removal of apheresis catheters, and more efficient disposition of patients undergoing peripheral blood stem cell collection. [Display omitted] Disclosures:Shaughnessy:Otsuka: Honoraria, Speakers Bureau; Millenium: Honoraria, Speakers Bureau; Genzyme: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Outcomes of Patients Receiving Allogeneic Peripheral Blood Stem Cell Products Mobilized with Intravenous Plerixafor

Abstract 314We have previously reported mobilization data of hematopoietic stem and progenitor cells (HSPCs) in donors treated with IV plerixafor in a phase I/II open label trial (Rettig et al. Blood 2010 116: a2261). Here we report the final outcomes of donors and recipients of the phase II portion of the trial evaluating the use of 0.32 mg/kg plerixafor IV as a single agent for stem cell mobilization. We hypothesized, based on preclinical studies in the mouse, that IV plerixafor would have more rapid and prolonged mobilization and increase stem cell yields so that failure to collect ≥2×106 CD34+ /Kg recipient weight in one apheresis would be reduced from 33% (seen with 240 ug SC plerixafor) to 11%. We also hypothesized based on our prior observations of increased plasmacytoid dendritic cells (pDCs) in plerixafor mobilized products (Rettig et al. Blood 2009 114: a32), that peripheral blood stem cell products would be enriched in pDCs which may lead to improved immunity in the recipient after transplant and reduced incidence of CMV. Methods.In this phase II open label trial donors were mobilized with 0.32 mg/Kg IV plerixafor (over 30 minutes), followed by a 20 L apheresis starting 4 hours after injection. The primary outcome was failure to collect ≥2×106 CD34+/Kg actual recipient weight on day 1. Secondary outcomes included donor toxicity, successful collection in 2 aphereses, pharmacodynamics of mobilization, engraftment, graft-versus-host disease (GvHD), treatment related mortality (TRM), incidence of CMV, relapse free survival, and overall survival. The study was powered to detect a difference in failure rate from 33% to 11%. Sibling donors were 18 – 70 years of age and 6/6 HLA matched with recipients. Recipients were 18 – 67 years of age with a hematologic malignancy (AML n=14, NHL n=9, CLL n=3, MDS n=3, ALL n=2, CML n=1, and HD n=1). 33 recipients were transplanted (5 reduced-intensity and 28 myeloablative conditioning). A total of 27 donor recipient pairs were initially enrolled and 7 additional pairs of subjects were enrolled to replace 6 donors who failed to collect goal apheresis volume (>17.5L) due to technical problems related to initial apheresis collections. Median follow up of recipients is 248 days (range 37–695 days). Results.The primary outcome of failure to reach ≥2×10e6 CD34+/Kg recipient weight was analyzed on a per protocol basis. CD34 HSPC mobilization kinetics were previously reported for the phase I cohorts (ASH 2010). 29 donors were evaluable for the primary outcome. 34% (10/29) failed to reach the day 1 collection goal of ≥2×106 CD34/Kg recipient weight. 10% (3/29) failed to reach goal after 2 apheresis procedures. Toxicity in donors was mild with the most common reported side effects being grade 1 GI toxicity of abdominal bloating (30%) and grade 1 bradycardia (30%). All recipients engrafted and there was no graft failure. The median time to neutrophil engraftment (ANC>500K/uL × 2 days) was 14 days, with 31/33 engrafting by day 21. The median time to platelet engraftment (Platelets>50K/uL × 2 days) was 25 days, with 23/33 engrafting by day 30. The incidence of grade II-IV acute GvHD was 7/33 (21%). The incidence of grade III-IV acute GvHD was 4/33 (12%). The cumulative incidence of chronic GvHD in those living beyond 100 days was 7/25 (28%) and 4/25 (16%) had severe disease. The incidence of CMV viermia >10,000 copies in at risk recipients (defined as serology positive in donor or recipient) was 3/20 (15%). The incidence of CMV disease was 1/20 (5%). Median overall survival was 258 days, with 43% one-year survival. Median relapse free survival was 238 days. The day 100 TRM was 1/33 (3%). Summary.IV administration of plerixafor was well tolerated by donors. However, based on our prior experience with subcutaneous dosing with 0.24mg/kg (Devine et al Blood, 2008) there was no improvement in the percent of normal donors who successfully collected ≥2×106 CD34+/Kg after a single IV infusion of 0.32 mg/Kg plerixafor (67% vs. 66% respectively). Failure to reach collection goal in one apheresis procedure remains high with single agent plerixafor regardless of route of administration. Incidence of CMV viremia was low and could possibly be related to increased plasmacytoid dendritic cells in the product. The incidence of aGvHD was lower than historical controls for G-CSF mobilized peripheral blood transplantation and deserves further evaluation in future prospective trials involving CXCR4 antagonists such as plerixafor. [Display omitted] [Display omitted] Disclosures:Schroeder:Genzyme: Research Funding. Rettig:Genzyme: Honoraria. Uy:Genzyme: Consultancy, Speakers Bureau. DiPersio:Genzyme: Honoraria.

Autologous Stem Cell Transplant in Patients with Multiple Myeloma Previously Treated with Lenalidomide: A Single-Institution Experience Using An Intermediate-Dose Cyclophosphamide-Based Regimen

Abstract 4391 Background:High dose chemotherapy followed by autologous transplantation of peripheral blood stem cells plays an important role in the management of intermediate- and advanced-stage multiple myeloma. In order for successful engraftment to occur, adequate numbers of high quality peripheral stem cells must be harvested prior to transplantation. Mobilization of PBSC in patients treated with lenalidomide has been associated with poor apheresis collections after G-CSF mobilization. The purpose of this study is to present our institution's experience utilizing an intermediate-dose cyclophosphamide-based mobilization regimen. Methods:We retrospectively analyzed data for patients with multiple myeloma who underwent autologous stem cell transplant at UCLA between 2006 and 2010. Data were obtained from the database of the UCLA Heme Malignancy/Stem Cell Transplant Unit, the electronic health record system and stem cell processing laboratory. All patients underwent mobilization with a regimen of cyclophosphamide 2.5g/m2 IVPB, G-CSF 10 mcg/kg/day for 4 days SQ, and prednisone 2mg/kg/day for 4 days po. The number of CD 34+ cells was used as a marker for the number of peripheral stem cells collected. Minimum dose collected to ensure adequate engraftment was 2×106/kg CD34+ cells. Patients were conditioned with melphalan 100mg/m2/d x2 (unless there was evidence of renal failure, in which case the dose was reduced to 100mg/m2) with subsequent infusion of stem cells. Neutrophil engraftment was defined as the first day of absolute neutrophil count greater than 500×106/L ≥ 7 days after transplant. Results:Autologous stem cell transplant was performed in 103 patients with multiple myeloma at UCLA between 2006 and 2010. Median number of apheresis procedures was 1 (1–12) with a median of 4.4×106/kg (1.4–33.5) CD34+ cells collected. Median time to engraftment was 10 (8–18) days. Thirty-five patients received lenalidomide at some point in their pretransplant treatment. Median number of apheresis procedures was 1 in both lenalidomide and non lenalidomide treated groups. In the lenalidomide treated group 54% required only one collection versus 75% in the non lenalidomide treated group (p=0.033). In the lenalidomide treated group 31% required 3 more or more collections versus 10% in non lenalidomide treated group (p=0.0075). Three patients in the lenalidomide group had subsequent mobilization with plerixafor with one of these requiring bone marrow harvesting. Median CD34+ cells collected was 3.5×106/kg and 4.9×106/kg (p=0.246) in the lenalidomide and non-lenalidomide groups respectively. Both groups had a median time to neutrophil engraftment of 10 days with a similar range. Conclusion:Pre-transplant use of lenalidomide adversely affected the number of stem cell apheresis procedures required to procure adequate stem cell dose as evidenced by a greater percentage requiring 3 or more collections. However, despite prior lenalidomide exposure, the use of our mobilization regimen permitted adequate collection, with the majority of patients requiring only one apheresis procedure, and led to an equivalent time to neutrophil recovery. Disclosures:Off Label Use: Cyclophosphamide and G-CSF for mobilization of stem cells.