Hematopoietic Progenitor Cell Collection Research Articles

Handling the different requirements for commercial and investigational MNC collections by apheresis

BackgroundWith the success of chimeric antigen receptor T-cell (CAR-T) and similar cellular-based therapies, the demand for collection of autologous mononuclear cells by apheresis (MNC(A)) from blood by apheresis has increased. From an apheresis technical standpoint, the collection of MNC(A) is relatively straightforward, especially when compared with collection of hematopoietic progenitor cells (HPC(A)). Most of the collection for MNC(A) are performed for the commercial entities, who use the product for manufacturing cellular therapeutics. We have noticed discrepancies in the handling and apheresis processes required by different companies in obtaining essentially the same product (all companies in the study manufacture CAR-T-based products). We have analyzed the MNC collection requirements from all FDA-approved CAR-T cellular products and some investigational products collected at University of Nebraska Medical Center. We identified discrepancies in the process and suggested mitigation strategies. MethodsStep-by-step analysis of the collection requirements. Review of the current guidelines and recommendations on this issue. ResultsMultiple discrepancies in the collection process have been identified, even in the products collected for the same company. Practical approach of satisfying all the requirements based on University of Nebraska Medical Center experience has been suggested. ConclusionThe current recommendations from multiple sources were reviewed in discussion.

Resource Utilization Analysis of Two Algorithms for Hematopoietic Progenitor Cell Mobilization in Multiple Myeloma

Background: Autologous hematopoietic cell transplantation (HCT) is a standard of care after induction therapy for multiple myeloma (MM). A key step in this procedure is the mobilization and collection of hematopoietic progenitor cells (HPC). HPC mobilization typically uses G-CSF with or without the CXCR4 antagonist Plerixafor (P). Although P increases the effectiveness of mobilization and may reduce the number of apheresis sessions, it is associated with significantly increased cost per dose. There is significant heterogeneity to HPC collection algorithms across institutions and there is no clear standard. At the Cleveland Clinic, our HPC collection algorithm was updated in an effort to reduce the use and associated cost of P. The criteria for initiating P became more stringent and far fewer patients were initiated on P prior to their first session of HPC collection apheresis. Prior to October 2017, patients were initiated on P if any 1 of the following criteria were met: age>60 years, prior treatment with melphalan or other alkylating therapy, prior treatment with lenalidomide >2 cycles, extensive bone marrow disease, or platelet count <100K. Starting October 1, 2017, patients were preemptively initiated on P prior to first collection only if all 3 of the following criteria were met: age > 65 years, lenalidomide > 6 cycles, and platelet count < 100K. However, P was added after the first apheresis session if peripheral blood CD34+ cell count was <20/uL or HPC yield was unsatisfactory (<2 x 10^6 CD34+ cells). Furthermore, target CD34 yield was decreased from 5 to 4 x 10^6 CD34+ cells. Herein, we perform a comparative resource utilization analysis of HPC collection at our center. Methods: We retrospectively collected HPC mobilization data on patients undergoing HCT for MM from 1/12014 to 12/31/2020. We determined the number of P doses, number of apheresis sessions, and HPC collection totals per patient. Patient characteristics were summarized in median, interquartile range (IQR), or frequencies and percentages as appropriate. Fisher's exact test and Wilcoxon rank sum test were used to compare variables in patients undergoing mobilization prior to versus after 10/1/2017. All tests were two-sided and p-values of 0.05 or less were considered statistically significant. Results: 219 patients underwent HPC mobilization from 1/1/2014 to 9/30/2017 and 251 patients underwent mobilization after algorithm change from 10/1/2017 to 12/31/2020. As anticipated, the protocol change decreased the number of patients receiving P prior to their first apheresis session. 91% received P prior to the first apheresis session before the change while 14% received this initial dose of P after the change. Prior to the change 51% of patients completed collection in 1 day, 30% completed in 2 days, 12 % completed in 3 days, and 7% completed in 4 days or more. These figures changed to 20%, 41%, 23%, and 17% respectively following the change (p<0.0001) Median day 1 peripheral blood CD34 count decreased from 63 to 19 cells/uL while median day 1 CD34 collection yield fell from 4.8 to 1.46 x 10^6 after the change (p<0.0001) (Table 1). Total CD34 collection yield also decreased from a median of 6.64 to 4.6 x 10^6 CD34+ cells (p<0.0001). However, the target yield was decreased from 5 to 4 x 10^6 CD34+ cells as described above. The number of doses of P per patient decreased after the change (pre-change median dose / patient = 1, IQR = 1-2) (post-change median dose / patient = 1, IQR = 0-2) (p=0.004). The average number of apheresis sessions required per patient increased following the change (pre-change median apheresis sessions = 1, IQR = 1-2) (median post change apheresis sessions = 2, IQR = 2-3) (p<0.0001) (Figure 1). Conclusion: Changes to our institutional HPC mobilization algorithm led to a substantial decrease in the use of preemptive P prior to day 1 of collection by design. The resulting decrease in total doses of P per patient was smaller than expected, although still statistically significant. However, this was offset by a concomitant significant increase in the number of apheresis sessions required per patient despite a lower collection target. Based on these results, a cost analysis is ongoing in order to re-evaluate our institutional algorithm.

Collection efficiency in apheresis

Significant advances in procedural information displayed by current apheresis machines have been made, but analyses of cell collection efficiency (CE) still rely on calculations done by apheresis professionals. Accordingly, understanding CE equations can support the optimization of apheresis techniques and identification of incidents that could impact the procedure’s effectiveness. This report summarizes classical and novel CE analyses applied to apheresis exemplified by an actual case of hematopoietic progenitor cell collection. In addition to the apheresis yield and most common CE1 and CE2 formulas, we present the instantaneous and corrected CE, fold enrichment, collection throughput, collection rate and its variants, average inlet rate, classical and adjusted captured cells, recruitment pool, recruitment factor, recruitment coefficient, blood component loss, predictive apheresis yield, and performance ratio calculations. Moreover, the mathematical relationship between these CE equations is also shown, which can be helpful in many apheresis procedures.

Cryopreservation and storage patterns of hematopoietic progenitor stem cells for multiple myeloma

Autologous hematopoietic stem cell transplantation (HCT) has been a standard of care treatment for eligible patients with newly diagnosed multiple myeloma (MM). Guidelines generally recommend hematopoietic progenitor cell (HPC) harvest for two potential HCT. There is a paucity of data reporting use of such collections in the era of novel approved therapies. In this single-center retrospective study, our goal was to determine the HPC utilization rate and costs associated with leukocytapheresis, collection, storage, and disposal to guide future HPC collection planning.We included 613 patients with MM who underwent HPC collection over a nine-year period. The patients were separated into four groups based on HPC utilization: 1) patients who never proceeded to HCT, or Harvest and Hold (14.8 %), 2) patients who proceeded to one HCT with banked HPC remaining (76.8 %), 3) patients who proceeded to one HCT without HPC remaining (5.1 %), and 4) patients who proceeded to two HCTs (3.3 %). After collection, 73.9 % of patients underwent HCT within 30 days. Of patients with banked HPC, defined as not undergoing HCT within 30 days of leukocytapheresis, the overall utilization rate was 14.9 %. At 2- and 5-years post HPC collection, utilization rate was 10.4 % and 11.5 %, respectively.In conclusion, our results suggest very low utilization of stored HPC, raising into question the current HPC collection targets. Given advances in MM therapy, as well as significant costs associated with harvest and storage, collection for unplanned future use warrants reconsideration. As a result of our analysis, our institution has reduced our HPC collection targets.

Adverse events related to central venous catheters (CVC) and the influence of CVC characteristics on peripheral blood hematopoietic progenitor cell collection in children.

The use of peripheral blood progenitor cells (PBPCs) as a source for hematopoietic stem cell transplantation (HSCT) in pediatric healthy donors is still under debate. The risk of a central venous catheter (CVC) placement and catheter-related complications continue to be the main arguments to discourage its use. we present a retrospective analysis of 140 PBPC collections in pediatric patients and donors, describing adverse events (AE) related to CVCs as well as the influence of catheterrelated variables on the efficiency of the leukapheresis. 14 CVC-related AEs were recorded (10%). The most common was fever in 5 patients, 4 of which had a catheter-related bacteriemia. Thrombotic events were only observed in 3 patients with active malignancy. A healthy donor presented a moderate bleeding after catheter withdrawal that resolved with local measures, and none of the rest presented any AE. Regarding variables related to the development of AEs, the subject group (patient or donor) was the only one significantly associated (p < 0.0001). Of interest, efficiency was also related to catheter location, being worse in those located in the femoral vein than in into the jugular or the subclavian veins (p < 0.05). In a multivariate analysis, the only variable significantly associated was catheter size (beta 0.238, p < 0.01). Placing a CVC for PBPC collection in pediatric subjects is overall safe; CVC-related complications in pediatric healthy donors are very rare. Furthermore, we should try to place catheters of the largest caliber possible, since the efficiency of the collection is related to this variable.

Collection and processing of hematopoietic progenitor cell products at risk of presenting with cold agglutination

Background aimsCold agglutinins are commonly identified in transfusion laboratories and are defined by their ability to agglutinate erythrocytes at 3–4°C, with most demonstrating a titer >64. Similarly, cryoglobulins can precipitate from plasma when temperatures drop below central body temperature, resulting in erythrocyte agglutination. Thankfully, disease associated from these autoantibodies is rare, but unfortunately, such temperature ranges are routinely encountered outside of the body's circulation, as in an extracorporeal circuit during hematopoietic progenitor cell (HPC) collection or human cell therapy laboratory processing. When agglutination occurs ex vivo, complications with the collection and product may be encountered, resulting in adverse events or product loss. Here, we endeavor to share our experience in preventing and responding to known cases at risk of or spontaneous HPC agglutination in our human cell therapy laboratory. Case reportsFour cases of HPC products at risk for, or spontaneously, agglutinating were seen at our institution from 2018 to 2020. Planned modifications occurred, including ambient room temperature increases, tandem draw and return blood warmers, warm product transport and extended post-thaw warming occurred. In addition, unplanned modifications were undertaken, including warm HPC product processing and plasma replacement of the product when spontaneous agglutination of the product was identified. All recipients successfully engrafted after infusion. ConclusionsWhile uncommon, cold agglutination of HPC products can disrupt standard processes of collection and processing. Protocol modifications can circumvent adverse events for the donor and minimize product loss. Such process modifications should be considered in individuals with known risks for agglutination going to HPC donation/collection.

Ethylene oxide-type hypersensitivity reactions in G-CSF mobilized, peripheral blood hematopoietic progenitor cell donors and review.

Ethylene oxide (EtO) is a volatile, ringed toxic ether used to sterilize heat-labile plastics including apheresis sets. In the 1980s, EtO-associated severe hypersensitivity reactions during hemodialysis led to widespread adoption of alternative sterilization for dialysis kits but not apheresis tubing sets. We now report several cases of EtO-type hypersensitivity reactions in autologous donors undergoing hematopoietic progenitor cell collection (HPCC). A 10-year retrospective review of allergic EtO-type reactions in adults undergoing HPCC on the COBE Spectra and SPECTRA Optia was performed. Donor medical history and absolute eosinophil counts were compared between cases and 34 HPCC controls. Published EtO reactions during extracorporeal procedures were reviewed with statistical analysis. Graphics and statistics were performed using commercial software. Three autologous HPCC donors experienced EtO-type reactions within 15 min of initiating HPCC, for a 10-year incident rate of 0.08% per procedure and 0.18% per donor. All three reactions occurred using the Spectra Optia and IDL tubing set, for an Optia/IDL specific rate of 0.2% per procedure and 0.5% per donor. There was no correlation between EtO reactions, eosinophil counts, or saline prime dwell times. No patient had classic predisposing risk factors for EtO hypersensitivity. Two patients required medical intervention whereas the third responded by pausing the procedure and slowing the inlet rate. EtO-type hypersensitivity reactions can be observed during HPCC, especially with the Optia IDL tubing set. EtO reactions may be missed due to their rarity and staff unfamiliarity with this clinical entity.

233 - Impact of the COVID-19 Pandemic on Unrelated Allogeneic Hematopoietic Peripheral Blood Progenitor Cell Collections in Canada

233 - Impact of the COVID-19 Pandemic on Unrelated Allogeneic Hematopoietic Peripheral Blood Progenitor Cell Collections in Canada

Hypercoagulability in pediatric autologous hematopoietic progenitor cell collection – Is it the time to reconsider screening? – A case report

Hypercoagulability in pediatric autologous hematopoietic progenitor cell collection – Is it the time to reconsider screening? – A case report

Handling of Allogeneic HPC Grafts in European Transplant Centers during the COVID-19 Pandemic - a Survey from the Infectious Diseases and Cellular Therapy & Immunobiology Working Parties of the EBMT

Background: For more than two years, the COVID-19 pandemic has had detrimental impact on medical practices. Because of its intrinsic complexity, the delivery of allogeneic hematopoietic cell transplantation (HCT) in this context represents an enormous challenge. Transplant centers have adapted promptly and developed strategies to enable safe HCT, by introducing significant changes in their procedures, e.g. through changes in donor types and an unprecedented use of cryopreservation for allogeneic hematopoietic progenitor cell (HPC) grafts. Methods: To explore how centers modified their strategies in terms of donor selection and implementation of new procedures (e.g. SARS-CoV-2 test strategy, cryopreservation), a 14-item survey was developed by the Infectious Disease (IDWP) & Cellular Therapy & Immunobiology (CTIWP) working parties of the European Society for Blood and Marrow Transplantation (EBMT). The survey was sent to 509 EBMT-affiliated allogeneic HCT in Q2, 2011 and re-opened between January 31st and March 18th, 2022 to also cover changes during to the infection period with the Omicron variant. Results: One hundred thirty-five of 509 (27%) centers from 31countries responded. Seventy-four percent of centers introduced COVID-19 mitigation measures for their donors (e.g. physical and social distancing) to minimize the risk of infection. Thirty-four percent of centers changed their donor search strategy, and favored recruitment of related haploidentical donors over unrelated donors (URD). In addition, 33% and 50% of centers searched for a backup donor in the related and URD settings, respectively. Only 10 (7.4 %) of centers resorted to cord blood (CB) as an alternative source to URD. Of these, four centers where pediatric centers and three, pediatric and adult transplant centers. Before COVID-19, only ten of 135 centers (7.4%) were routinely cryopreserving HPC products, whereas this percentage increased to 90% (122/135) during the pandemic. In details, 84/122 (69%) of centers cryopreserved both related and URD grafts, 34 (28%) only URD and 4 (3%) related grafts only. One hundred twenty centers responded during the re-opening of the survey to cover the situation during the Omicron wave. Of those, 78% were still considering graft cryopreservation, whereas 18/26 (70%) of centers not using cryopreservation during the Omicron wave used it before that period. In the majority of centers, changes in policies were introduced based on EBMT recommendations (88/117; 75%) and/or national guidelines (73/117; 62%). Bone marrow (BM) grafts were avoided by 52 (39%) centers (possibly because of the notorious low recovery percentage of cryopreserved BM progenitor cells). In the study period, a total of 4,443 related and 4,586 URD transplants have been reported. Of these transplants, 62% (n=2,748) related and 71% (n=3,254) of URD grafts have been cryopreserved pre-emptively and infused in the vast majority of cases. Sixty-four (2.3 %) related and hundred-eight (3.3 %) URD cryopreserved products have not been infused during the observation period. Release criteria for cryopreserved products in terms of COVID-19 were based on a negative SARS-CoV-2 test (not further specified) in 58/121 (48%), interview of the donor (medical history) in 4/121 (3%), and both in 38/121 (31%) of centers. Eighty-eight centers reported on their test strategy for related donors (multiple answers possible). A SARS-CoV-2 test was performed during check-up in 43% (38/88), before HPC mobilization in 63% (55/88) and before HPC collection in 41% (36/88) centers. Test strategies for URD were not covered by the survey. Conclusions: These data are limited by the low number of respondents, but show a historical increase in cryopreservation of allogeneic HPC grafts during the first three waves of the SARS-CoV-2 pandemic, more in URD than in related donor grafts. The use of BM decreased, and CB grafts were not used as an alternative at most centers. A significant proportion of cryopreserved products (2.8%) were not infused. The reason for this is under investigation. The clinical impact of allogeneic cryopreserved HPCs has to be further analyzed in a larger cohort of patients.

Long-Term Patient-Reported Outcomes Following Treatment with betibeglogene autotemcel in Patients with Transfusion-Dependent β-Thalassemia

Introduction: Patients (pts) with transfusion-dependent β-thalassemia (TDT) have increased morbidity and mortality and reduced quality of life (QoL) compared with the general population owing to the mental, physical, and time demands of chronic transfusions and iron chelation. Autologous gene therapy with betibeglogene autotemcel (beti-cel) therapy uses a modified β-globin gene that establishes stable production of functional adult hemoglobin (Hb), thus allowing for transfusion independence (TI). Pts with TDT were treated with beti-cel in 2 completed phase 1/2 studies (HGB-204, HGB-205) and 2 ongoing phase 3 studies as of Aug 2021 (HGB-207, HGB-212). After 2 years of follow-up, pts could enroll in a long-term follow-up study (LTF-303 [NCT02633943]) for up to an additional 13 years. QoL improvement in the 1-2 years post beti-cel treatment was previously described. Here, we evaluate long term (3 years post-treatment) patient reported outcomes after beti-cel treatment in pts with TDT enrolled in LTF-303. Methods: Pts with TDT underwent hematopoietic stem progenitor cell collection followed by myeloablative busulfan conditioning and beti-cel infusion. TI is defined as weighted average Hb ≥9 g/dL without packed red blood cell transfusions for ≥12 months. Generic health related QoL (HRQoL) over time was assessed using the Pediatric Quality of Life Inventory (PedsQL; range, 0-100) for pediatric and adolescent pts (<18 years old [YO]). Short Form-36 Health Survey Physical Component Summary and Mental Component Summary (SF-36 PCS and MCS, respectively; range 0-100), and the EuroQol (EQ-5D-3L) visual analog scale (VAS; range 0-100) composite index score (range 0-1) for adult (≥18 YO) pts. HRQoL was assessed at baseline (BL), Months (M) 6, 12, 18, 24 (in the parent HGB studies), and M 36, 48, and 60 (in LTF-303) after beti-cel infusion. Pts were excluded from HRQoL analyses if BL or M36 HRQoL assessment was missing as of data cut (Aug 2021). Higher scores indicated improvement in HRQoL. A bespoke questionnaire assessing β-thalassemia and pt-centric aspects of daily living (i.e., ability to work or attend school, physical activity, and perceived benefit from treatment) was completed at M 36. Results: As of August 18, 2021, 57 pts were enrolled in LTF-303 (phase 1/2: 22 pts; phase 3: 35 pts), of which 26 (45.6%) were pediatric / adolescent pts and 31 (54.4%) were adult pts. Among pts <18 YO with PedsQL data who achieved TI, the mean (standard error (SE)) PedsQL total score (population norm = 80.9) increased from 77.4 (3.4) at BL (n=19) to 92.1 (4.2) at M 36 (n=4). Among adult pts with SF-36 data who achieved TI, the mean (SE) SF-36 PCS and MCS scores (US general population norm = 50) increased from 53.8 (1.4) and 50.9 (1.7), respectively, at BL (n=12) to 55.7 (1.7) and 56.3 (1.4) at M 36 (n=9), respectively. The mean (SE) EQ-5D-3L Composite Index and VAS scores increased from 0.92 (0.04) and 85.2 (3.0), respectively, at BL (n=12), to 0.95 (0.03) and 91.4 (2.2) at M 36 (n=9) (Table 1). Data from the M 36 testimonial questionnaire (Figure 1) showed the ability to seek employment or be employed, increased from 68.8% (11/16) of pts at BL to 93.8% (15/16) at M 36. There was a reduction in school absences at M 36 (44.4% [4/9] missing school) compared with BL (83.3% [15/18] missing school). 50% (10/20) of pts still required management of disease symptoms at M 36. 80% (16/20) of pts reported improvement in physical activity at M 36. 100% (19/19) of pts reported an overall benefit from undergoing treatment with beti-cel. Conclusions: Published data shows beti-cel is potentially curative in pts with TDT, defined as having TI with normal or near-normal Hb. It was previously shown that with high-range QoL at BL, QoL improved at 1-2 years post-beti-cel. The updated data indicates durability of QoL improvement through year 3. A trend towards a positive impact on employment, school attendance, physical activity, and other aspects of life is also observed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Sysmex XN series hematopoietic progenitor cell (XN-HPC) as a predictive marker of stem cell enumeration and products: a systemic review and meta-analysis

ABSTRACT Objectives: Sysmex® XN series hematopoietic progenitor cell (XN-HPC) is a sensitive, fast, and economic analytical method for predicting the yields of peripheral blood stem cell enumeration and products, and does not require a sophisticated or expensive workflow. However, various studies have shown that the characteristics of its diagnostic performance were non-uniform. Methods: We performed a systematic inquiry using PubMed, Embase, and Cochrane Library, to comprehensively search for studies published before November 21, 2021. The pooled specificity (SPE), sensitivity (SEN), negative likelihood ratio (NLR), positive likelihood ratio (PLR), diagnostic odds ratio (DOR) and receiver operating characteristic (ROC) curves were summarized to appraise the diagnostic merit of XN-HPC. A forest plot was used to research the sensitivities and specificities of XN-HPC performance. Subgroup analysis was performed to investigate heterogeneity where of importance. Results: Our research included four studies that assessed the diagnostic performance of XN-HPC in hematopoietic progenitor cell collection. The pooled accuracy was 95.4% (95% CI, 94.3–96.3), SPE was 0.81 (95% CI, 0.71–0.88), SEN was 0.95 (95% CI, 0.75-0.99), NLR was 0.06 (95% CI, 0.01–0.37), PLR was 5.0 (95% CI, 3.0–8.5), DOR was 78 (95% CI, 9–707) and the summary of the area under the ROC was 0.90 (95% CI, 0.87–0.92). Forest plot of sensitivities and specificities from XN-HPC test accuracy studies indicated the existence of high heterogeneity. We deduced that the patients were the source of heterogeneity via subgroup analysis. Conclusions: XN-HPC is an excellent diagnostic marker for quantitative detection of peripheral blood hematopoietic progenitor cells.

PB2182: CHARACTERISTICS THAT INFLUENCE THE STEM CELL COLLECTION BY APHERESIS IN PATIENTS WHO ARE CANDIDATES FOR AUTOLOGOUS STEM CELL TRANSPLANTATION

Background: High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a consolidation treatment for several hematologic malignancies such as multiple myeloma (MM), Hodgkin lymphoma (LH) and non-Hodgkin lymphomas (diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), NK/T- cell lymphoma (NKTL), etc.). However, between 5-40% of patients fail to obtain an adequate collection of hematopoietic progenitor cells (HPCs), this is known as mobilization failure. This failure can be due to multiple factors that may be associated with the previous treatment of the disease (number of treatment lines, type of chemotherapy or radiotherapy), the patient (age, bone marrow cellularity) or the collection procedure and other biochemical parameters like pre-count CD34+ cell/ul, pre-harvest hemoglobin and platelet count. Aims: -To determine the clinical characteristics of patients and the haracteristics of apheresis procedures in patients who had fail to obtain an adequate collection of HPCs by apheresis of peripheral blood and who are candidates for ASCT. Methods: A retrospective study was carried out, in patients aged between 18 and 65 years old, with hematological onco diseases who went to the Blood Bank of the National Cancer Institute for the collection of HPCs from peripheral blood by apheresis for ASCT, between January 2017 and December 2020. A review of the electronic medical record of each patient was carried out, as well as the records of the procedure for collecting HPCs. The nominal variables were compared by chi-square test and the quantitative variables with Student´s t-test, a value less than 0.05 was considered statistically significant. Results: The study included 127 patients, who underwent HPCs collection for ASCT; 15% (19 patients) had failure to mobilize (63% men; mean age 48 years). The most frequent diagnosis was MM 47.4%, followed by non-Hodgkin lymphoma 42.1% (37.5% DLBCL, 25% MCL, 37.5% NKTL, follicular lymphoma and peripheral T-cell lymphoma) and Hodgkin lymphoma with 10.5%. The most common used method of mobilization was chemotherapy with filgrastim (57.8%), followed by filgrastim. In the pre-harvest studies, 26% had anemia and 42% thrombocytopenia; pre-count CD34+ less than 20/ul was presented in 94%. More than 50% required two or more days for collection. During the process to obtain HPCs, the median duration of this procedure was 273 minutes (range 193 to 311 minutes), with an average processing speed of 81.1 ml/h. While the mean volume extracted was 16 879 ml (range 4 087 to 20 463ml) with an average number of complete cycles of 14.6. It was determined that age >50 years, two lines of treatment, mobilization with chemotherapy and filgrastim, pre-count CD34+ less than 20/ul and more days of harvesting were associated with HPCs harvest failure. While other factors such as sex, bone marrow status, radiotherapy site, pre-harvest hemoglobin, platelet count did not have a statistically significant association. Image:Summary/Conclusion: In our study, harvest failure (15%) is within the range reported in the literature and the factors associated with this failure have also been documented in other studies. With these data obtained, it could be established that patients at high risk of failure to harvest, such as those with multiple myeloma, are considered to be harvested receiving a smaller number of lines of treatment and consider stimulation with other stimulants such as plerixafor.

Comparison of hematopoietic progenitor cell collection using different inlet flow rates with the Fenwal Amicus.

We have used a hematopoietic progenitor cell (HPC) algorithm (standard [STD]) that restricted the inlet flow rate to 65 mL/min for peripheral white blood cell count (PWBC) >35 × 109 /L (STD). In this study, we evaluated a technique that allows 85 mL/min, regardless of the PWBC count (high). For patients with PWBC >35 × 109 /L, a prospective, randomized comparison of the high flow rate vs the STD PWBC-based flow rate (65 mL/min) was performed, comparing CD34+ and lymphocyte yields, collection efficiencies (CE1), mononuclear cells (MNC), and granulocytes, red blood cell (RBC), and platelet content. The Fenwal Amicus version 4.5 with a heparinized ACD-A anticoagulant (AC) delivered at a 26:1AC ratio was used. Paired comparisons between high and STD techniques were assessed with Wilcoxon signed rank tests, with P < .05 considered significant. Data are summarized as medians. Forty patient pairs (autologous) were compared. Diagnoses included primarily multiple myeloma (60%) and lymphoma (37.5%). High had significantly higher median average inlet rates (69 vs 55 mL/min), whole blood processed (20 vs 16 L), and cycles (15 vs 14) than STD. There were no significant differences in pre-procedure counts. Collection contents were (high/STD): 306/328 × 106 CD34+ cells, 48/59% CD34+ CE1 (significant), 0.2/0.2 × 109 /kg lymphocytes, 45/57% lymphocyte CE1, 63/59 × 109 WBC, 15/16 × 109 granulocytes, and 1.9/1.7 × 1011 platelets. The simpler, standardized high flow technique did not significantly increase or decrease CD34+ cells or lymphocyte yields, but did significantly decrease CD34+ CE1. The effects on cross-cellular content were minimal and not clinically significant.

Optimization of Autologous Hematopoietic Progenitor Stem Cell Apheresis Collection from Plerixafor-Mobilized Patients with Sickle Cell Disease

IntroductionAutologous hematopoietic progenitor cell (HPC)-based gene therapy and genome editing are emerging therapeutic strategies for patients with sickle cell disease (SCD). Given the high cell loss incurred during production of genetically modified HPCs, a large number of HPCs (10-15 x10 6 cells/kg) are required. Obtaining sufficient autologous HPCs from patients with SCD is a critical rate limiting step for the success of these novel genetic therapies. Plerixafor, a CXCR4 antagonist, has been shown to be safe and efficient for HPC mobilization in patients with SCD. However, impaired red blood cell (RBC) rheology and chronic inflammation associated with SCD make HPC apheresis collection challenging. For example, HPCs can potentially form aggregates with sickled RBCs traveling deep in the buffy coat upon density-based separation. In addition, elevated levels of activated platelets can increase cell clumping during apheresis. Thus, autologous HPC collection using standard apheresis setting is generally less efficient in patients with SCD and often associated with clumping and unstable cell interfaces during apheresis. It has been reported that standard HPC apheresis collections yield <0.7 x 10 6 CD34+ cells/kg per procedure in SCD patients despite adequate HPC mobilization (peak CD34 count 36-65/µL; Esrick, EB et al., 2018). To address these challenges, we modified our apheresis collection settings to improve the HPC collection efficiency and yield from plerixafor-mobilized patients with SCD.MethodsAdult patients with SCD who were enrolled on two clinical trials (NCT03226691 and NCT04443907) at St. Jude Children's Research Hospital and the National Heart, Lung and Blood Institute at the National Institutes of Health were included. Hydroxyurea was discontinued at least 2 weeks and an RBC exchange was performed 3-7 days prior to HPC mobilization. The participants received 240 µg/kg of Plerixafor subcutaneously, and underwent HPC collection 2-4 hours later using the Spectra Optia continuous mononuclear cell collection. Acid-citrate-dextrose-solution A was used for anticoagulation and calcium gluconate was infused during apheresis to prevent citrate-induced hypocalcemia symptoms. Up to 4 total blood volumes were processed for each collection with an adjusted collection preference (CP) of 10-40 and AC/Inlet ratio of 6-12. Collection efficiency (CE) 1 was calculated as CD34+ cell yield / [(pre-CD34 count + post-CD34 count)/2 x whole blood volume processed]. CE 2 was calculated as CD34+ cell yield / (pre-CD34 count x whole blood volume processed).ResultsWe performed 46 autologous HPC collections in 26 participants (age of 20 to 50 years). Mean (±1SD) pre-collection blood indices were: hematocrit (Hct) 27.4 ±3.8%, platelet count 226 ± 111 x10 9/L, and hemoglobin S 25.7 ± 6.0%, WBC count 13.82 ± 6.0 x10 9/L, and CD34+ cell count 62 ± 49 /µL. To address the altered HPC sedimentation and clumping associated with SCD, we targeted a deep buffy coat collection by lowing CP from standard 50 to 10-40, and used more anticoagulant by lowering Inlet/AC ratio from standard 12 to 6-12 respectively. The adjustments increased HPC yield, and 5.60 ± 5.48 x10 6/kg CD34+ cells were collected per procedure. The CE1 was 49.21 ± 28.34 % and CE2 42.74 ± 26.24%, which are comparable to autologous HPC CEs for non-SCD individuals. A deep collection led to highly efficient collections, although this tended to increase RBC contamination of the products (product Hct 6.0 ±2.2% versus < 3% for standard collection). Nevertheless, the participants experienced minimal loss of RBCs with a post-collection Hct of 26.6 ± 2.9%. Platelet loss was also relatively low, as demonstrated by a post-collection platelet count of 152 ± 69 x 10 9/L. No participants required platelet or blood transfusions, had bleeding or severe hypocalcemia-associated symptoms during and after the collections.ConclusionThe adjusted apheresis settings with a low buffy coat collection and increased anticoagulant dosage allowed safe and efficient autologous HPC collection in the patients with SCD. Further optimization of apheresis settings is required to increase HPC collection efficiency. DisclosuresSharma: Novartis: Other: Salary support paid to institution; Spotlight Therapeutics: Consultancy; Medexus Inc: Consultancy; CRISPR Therapeutics: Other, Research Funding; Vindico Medical Education: Honoraria; Vertex Pharmaceuticals/CRISPR Therapeutics: Other: Salary support paid to institution. Hankins: Bluebird Bio: Consultancy; UpToDate: Consultancy; Vindico Medical Education: Consultancy; Global Blood Therapeutics: Consultancy. Weiss: Cellarity Inc.: Consultancy; Novartis: Consultancy; Forma Therapeutics: Consultancy; Beam Therapeutics: Current holder of stock options in a privately-held company.

Long-Acting, Oxidation-Resistant IL-33 Mobilizes High-Quality Hematopoietic Stem and Progenitor Cells More Efficiently Than Wild-Type IL-33, G-CSF or AMD3100

▪Mobilization and collection of peripheral blood hematopoietic stem and progenitor cells (HSPC) has become increasingly important for hematopoietic cell transplantation. Granulocyte-colony stimulating factor (G-CSF) alone or in combination with the CXCR4-inhibitor AMD3100 are the most common mobilization approaches. These are however limited since they (a) fail to mobilize a sufficient number of cells in a significant fraction of patients, and (b) are biased towards myeloid immune reconstitution. A novel, single drug mobilization agent that results in a more balanced - myeloid and lymphoid - reconstitution would therefore be highly favorable to improve transplantation outcome.Interleukin (IL)-33 has recently been described as a potent mobilizer of HSPCs with long-term reconstitution potential. IL-33 is an intracellular protein that is believed to be enzymatically activated, released upon cell death, and inactivated upon oxidation. Whereas mobilization of HSPCs by G-CSF has been linked, at least in-part, to modulating the CXCR4-CXCL12 axis, the mobilizing function of IL-33 has been attributed to a different chemokine, CCL7.Here we asked whether IL-33 mobilizes HSPCs better than current clinical mobilizing agents, G-CSF and/or AMD3100, and how the quality of the mobilized cells compares. By flow cytometry and colony-forming assays, we observed for the first time that IL-33 treatment in mice mobilized Lin- Sca-1+ c-kit+ (LSK) cells and common myeloid progenitors (CMP/CFU-GEMMs) more efficiently than G-CSF or AMD3100 alone, and that IL-33 acts synergistically with both drugs. Next we engineered IL-33 variants lacking native cysteine residues, thereby rendering these variants resistant to oxidative inactivation. These variants had equal or better in vitro activity (a) in cell-based reporter assays and (b) in inducing CCL7 protein production by primary human umbilical vein endothelial cells (HUVECs). In vivo, these variants mobilized LSK cells and, interestingly also common lymphoid progenitor (CLP) and endothelial progenitor cells, more efficiently than wild-type IL-33 or G-CSF. We then engineered a one-armed IL-33-Fc fusion molecule comprising a single oxidation-resistant IL-33 fused to an Fc domain. Interestingly, a single dose of this oxidation-resistant IL-33-Fc variant was sufficient to significantly increase the mobilization of LSK, CLP, and endothelial progenitor cells, whereas oxidation-resistant IL-33 or G-CSF were not.In conclusion, our findings suggest that a single dose of long-acting, oxidation-resistant IL-33 may allow efficient mobilization of high-quality HSPCs including CLP cells, and may thus represent a novel mechanism to improve bone marrow transplantation.All authors were employed by Bayer at the time the studies were conducted. DisclosuresAlt:Bayer: Equity Ownership; Global Blood Therapeutics: Employment, Equity Ownership. Schopies:Bayer: Employment. Liu:Global Blood Therapeutics: Employment. Kauser:Global Blood Therapeutics: Employment, Equity Ownership. Bringmann:Cerus Corporation: Employment. Thompson:Bayer: Employment. Yuan:Bayer: Employment.

O-Fucose and Fringe-Modified Recombinant Notch Peptides As Decoy Molecules to Promote Hematopoietic Progenitor Mobilization

Nearly all autologous hematopoietic cell therapy (HCT) procedures are performed using mobilized hematopoietic progenitor cells (HPCs). Successful outcome is dependent on infusing an adequate number of functionally active HPCs. Mobilization strategy of AMD3100, in combination with G-CSF, increases total CD34+ cells mobilized, and often is used for HPC mobilization in myeloma and non-Hodgkin lymphoma patients. Unfortunately, this approach does not always lead to adequate HPC collection in patients who previously have been exposed to extensive cytotoxic therapy. Furthermore, suboptimal number of HPCs mobilized in some patients may result in slower hematopoietic reconstitution and increased risk of complications during the transplant. Hence developing new mobilization strategy is highly desired for patients who are considered poor-mobilizers. Notch is a signaling molecule important for stem cell self-renewal and fate determination in many tissues including the hematopoietic system. An important feature of Notch is its adhesive nature which was first described by cell aggregation assays in Drosophila studies. We previously reported that hematopoietic stem cell and progenitors (HSPCs) with faulty Notch-ligand interaction display increased cell cycling, decreased adhesion to marrow osteoblastic lineage cells, and enhanced egress from the marrow.Since Notch2 is the major Notch isotype expressed on HSPCs, we showed that prior treatment in mice with Notch2 blocking antibody but not Notch1 blocking antibody sensitizes HSPC to the mobilizing stimuli of G-CSF and AMD3100 with a 3~4 - fold increase in mobilization. We showed that transient Notch2 blockade leads to decreased CXCR4 expression by HSPCs with CXCR4 transcription being directly regulated by the Notch transcriptional protein RBPJ. Notch2 blockade also increases cell cycling of HPCs during mobilization temporarily without affecting hematopoietic stem cell (HSC) homeostasis and self-renewal. Moreover, transient Notch2 blockade leads to greater HSPC homing to the marrow that is accompanied with a faster hematopoietic recovery and a competitive repopulating advantage of HSPCs.Herein, to search for alternative agents to Notch blocking antibody, we evaluate the ability of recombinant Notch peptides as decoy molecules to promote disengagement of Notch2-ligand interaction between HSPCs and niche supporting cells. By using OP9 cell-based binding assay and surface plasmon resonance (SPR) analyses, we showed that recombinant Notch111-13, Notch19-13, Notch18-13, Notch211-13, Notch29-13, and Notch28-13 peptide comprising EGF-like repeats 11-13, 8-13, and 9-13, of Notch1 or Notch2, respectively, all bind to Notch ligand Dll4, Dll1 and Jag2. Because EGF-like repeat 12 (EGF 12) has been shown located within the essential ligand binding domain, and the O -fucose attached to the threonine residue of Notch1 EGF12 acts as a surrogate amino acid to make specific and functional contact to residues of Notch ligand, we generated peptides in CHO cells with O -fucose attached to the threonine residue of EGF12 and further extended by Fringe. We found that binding affinities of the glycosylated peptides further increased compared to those of non-glycosylated peptides. Compared to Notch2 peptides, O -fucose and Fringe-modified Notch111-13 peptide shows the highest biding affinity to Notch ligand Dll4 with dissociation constant (Kd) reaching 34 nM. Similar to Notch2 blocking antibody, the O -fucose and Fringe-modified Notch111-13 peptide effectively outcompetes Notch2-ligand binding and thus blocks the adhesion of marrow HSPCs to the primary osteoblasts or OP9 cells expressing Notch ligands. Further, O -fucose and Fringe-modified Notch111-13 peptide can effectively induce HSPCs emigration from a 3D osteoblastic “spheroid niche”, an in vitro model of HSC niche. Compared to Notch2 blocking antibody, O -fucose and Fringe-modified Notch111-13 peptide caused a further decrease of HSPC cells remained in the “spheroid niche” and more HSPC emigration out of the “niche”.In summary, our findings suggest that recombinant Notch decoy peptides may be considered as alternatives to Notch2 blockade to be combined with the mobilizing regimen of AMD3100 and G-CSF for more effective HSPC mobilization. DisclosuresNo relevant conflicts of interest to declare.

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.

Improved collection of hematopoietic stem cells and progenitors from Fanconi anemia patients for gene therapy purposes

Difficulties in the collection of hematopoietic stem and progenitor cells (HSPCs) from Fanconi anemia (FA) patients have limited the gene therapy in this disease. We have investigated (ClinicalTrials.gov, NCT02931071) the safety and efficacy of filgrastim and plerixafor for mobilization of HSPCs and collection by leukapheresis in FA patients. Nine of eleven enrolled patients mobilized beyond the threshold level of 5 CD34+ cells/μL required to initiate apheresis. A median of 21.8 CD34+ cells/μL was reached at the peak of mobilization. Significantly, the oldest patients (15 and 16 years old) were the only ones who did not reach that threshold. A median of 4.27 million CD34+ cells/kg was collected in 2 or 3 aphereses. These numbers were markedly decreased to 1.1 million CD34+ cells/kg after immunoselection, probably because of weak expression of the CD34 antigen. However, these numbers were sufficient to facilitate the engraftment of corrected HSPCs in non-conditioned patients. No procedure-associated serious adverse events were observed. Mobilization of CD34+ cells correlated with younger age, higher leukocyte counts and hemoglobin values, lower mean corpuscular volume, and higher proportion of CD34+ cells in bone marrow (BM). All these values offer crucial information for the enrollment of FA patients for gene therapy protocols.

Infectious Disease Testing to Optimize Safety of Hematopoietic Stem Cell Transplant Products

Over 1 million hematopoietic stem cell transplants (HSCTs) have been performed during the last 50 years, with the number of transplants and clinical indications expected to continue to increase. Although advances in the field have improved outcomes of HSCT, it still carries a high risk due to the immunocompromised state of HSCT recipients. Multiple steps are involved from the initial collection of donor hematopoietic progenitor cells to the final infusion of the cellular product in a recipient. This review focuses on the infectious risk associated with each of these steps, as well as infectious disease testing strategies that can be used to mitigate these risks and to ensure a safe HSCT procedure.