Hematopoietic Progenitor Cell Apheresis Research Articles

CD34+ cell yield among healthy donors: Large-scale model development and validation.

Successful engraftment in hematopoietic stem cell transplantation necessitates the collection of an adequate dose of CD34+ cells. Thus, the precise estimation of CD34+ cells harvested via apheresis is critical. Current CD34+ cell yield prediction models have limited reproducibility. This study aims to develop a more reliable and universally applicable model by utilizing a large dataset, enhancing yield predictions, optimizing the collection process, and improving clinical outcomes. A secondary analysis was conducted using the Center for International Blood and Marrow Transplant Research database, involving data from over 17 000 healthy donors who underwent filgrastim-mobilized hematopoietic progenitor cell apheresis. Linear regression, gradient boosting regressor, and logistic regression classification models were employed to predict CD34+ cell yield. Key predictors identified include pre-apheresis CD34+ cell count, weight, age, sex, and blood volume processed. The linear regression model achieved a coefficient of determination (R2) value of 0.66 and a correlation coefficient (r) of 0.81. The gradient boosting regressor model demonstrated marginally improved results with an R2 value of 0.67 and an r value of 0.82. The logistic regression classification model achieved a predictive accuracy of 96% at the 200 × 106 CD34+ cell count threshold. At thresholds of 400, 600, 800, and 1000 × 106 CD34+ cell count, the accuracies were 88%, 83%, 83%, and 88%, respectively. The model demonstrated a high area under the receiver operator curve scores ranging from 0.90 to 0.93. This study introduces advanced predictive models for estimating CD34+ cell yield, with the logistic regression classification model demonstrating remarkable accuracy and practical utility.

Cryopreservation of Allogeneic Hematopoietic Cell Products During COVID-19 Pandemic: Graft Characterization and Engraftment Outcomes

The COVID-19 pandemic triggered the deployment of unfamiliar measures to safeguard successful allogeneic hematopoietic cell transplantation (allo-HCT). Among these measures, cryopreservation offered logistical benefits that could outlast the pandemic, including graft availability and timely clinical service. The purpose of this study was to evaluate graft quality and hematopoietic reconstitution in patients transplanted with cryopreserved allogeneic stem cell products during the COVID-19 pandemic. We evaluated 44 patients who underwent allo-HCT using cryopreserved grafts consisting of hematopoietic progenitor cells (HPC) apheresis (A) and bone marrow (BM) products at Mount Sinai Hospital. Comparative analyses of 37 grafts infused fresh during the one-year period preceding the pandemic were performed. Assessment of cellular therapy products included total nucleated cell and CD34+ cell enumeration, viability, and post-thaw recovery. The primary clinical endpoint was the evaluation of engraftment (absolute neutrophil count [ANC] and platelet count) and donor chimerism (presence of CD33+ and CD3+ donor cells) at day +30 and +100 post-transplant. Adverse events related to cell infusion were also analyzed. Patient characteristics were comparable between the fresh and cryopreserved groups with 2 exceptions in the HPC-A cohort: the number of patients in the cryopreserved group that received haploidentical grafts was 6 times that in the fresh group, and the number of patients in the fresh group with a Karnofsky performance score >90 was double that in the cryopreserved group. The quality of HPC-A and HPC-BM products was not affected by cryopreservation, and all grafts met the release criteria for infusion. The pandemic did not affect the time between collection and cryopreservation (median, 24 hours) and time in storage (median, 15 days). Median time to ANC recovery was significantly delayed in recipients of cryopreserved HPC-A (15 vs 11 days, P=.0121), and there was a trend toward delayed platelet engraftment (24 vs 19 days, P=.0712). The delay in ANC and platelet recovery was not observed when only matched graft recipients were compared. Cryopreservation did not affect the ability of HPC-BM grafts to engraft and reconstitute hematopoiesis, and there was no difference in the rates of ANC and platelet recovery. Achievement of donor CD3/CD33 chimerism was not affected by cryopreservation of either HPC-A or HPC-BM products. Graft failure was observed in only 1 case, a recipient of cryopreserved HPC-BM. Three recipients of cryopreserved HPC-A grafts died before ANC engraftment from infectious complications. Remarkably, 22% of our studied population had myelofibrosis, and almost half received cryopreserved HPC-A grafts with no graft failure observed. Finally, patients receiving cryopreserved grafts were at a higher risk of infusion-related adverse events than those receiving fresh grafts. Cryopreservation of allogeneic grafts results in adequate product quality with minimal impact on short-term clinical outcomes, except for an increased risk of infusion-related adverse events. Cryopreservation is a safe option in terms of graft quality and hematopoietic reconstitution with logistical benefits, but additional data are needed to determine long-term outcomes and assess whether this is a suitable strategy for at-risk patients.

Comparison between peripheral blood progenitor cell collection on the 4th or 5th day of granulocyte colony-stimulating factor treatment in allogeneic stem cell donors: implications for hematopoietic progenitor cell apheresis guidelines.

Comparison between peripheral blood progenitor cell collection on the 4th or 5th day of granulocyte colony-stimulating factor treatment in allogeneic stem cell donors: implications for hematopoietic progenitor cell apheresis guidelines.

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.

Peripheral blood stem cell collection in children with extremely low body weight (≤8kg). What have we learned over the past 25 years and where are the limits?

Hematopoietic progenitor cells-apheresis (HPC-A) collection is now a routine procedure for autologous hematopoietic stem cell transplantation. Here we present our 25 years' experience of HPC-A collection in children weighing 8kg or less, with a focus on the evolution of our standard operating procedures, and the safety limits for these young patients, in the Pediatric Apheresis Unit of Clermont-Ferrand University Hospital (France). Fifteen children weighing 8kg or less underwent 26 HPC-A collections over 25 years. Median CD34+ cell yield by leukapheresis was 4.4 106 /kg. No procedure-related complications were encountered during or after the collection. No patient had profound thrombocytopenia or anemia that needed post-collection transfusions. Our experience in pediatric oncology patients who underwent HPC-A collections shows that this procedure can be performed even in the smallest of children with no increase in toxicity provided all precautions are taken to ensure that the procedure is carried out under the ideal conditions.

Low CD45 mean fluorescence intensity predicts poor Post-Thaw CD34 cell viability

Background & Aim Background Reduced CD34 cell viability following cryopreservation is associated with suboptimal engraftment in stem cell transplantation. Collection of extra hematopoietic progenitor cells (HPC) can compensate for the potential loss, but subjects patients to unnecessary procedures, and drives up healthcare costs. Aim The goal of this study was to identify variables available on the day of HPC collection that help predict the likelihood of a significant post-thaw drop in viable CD34 cells (vCD34). A prediction model was generated to recommend appropriate extra-day collections. Methods, Results & Conclusion Methods We collected data on 235 consecutive autologous HPC apheresis collections in a single pediatric hospital and analyzed variables that are patient-related, product-related, and technical in nature (Table 1). Both continuous and discrete variables were analyzed and used to construct a prediction model with a false negative rate (missing a call to predict a significant drop in vCD34%) of zero. Results In 5.5% of apheresis products, post-thaw vCD34% decreased by >50%, 15% had 25-50% drop, 29% showed 50%) post-thaw vCD34% decrease included a diagnosis of Hodgkin lymphoma and a low mononuclear cell (MNC) percentage. A simple algorithm combining these three factors correctly identified 9 out of 11 (81%) unnecessary repeat collections, and did not miss any patients for whom a recollection was necessary. Retrospective analysis suggests that using this algorithm, 33% of repeat collections could be avoided. Conclusion Low CD45 nMFI of CD34+ cells is a novel marker of poor post-thaw CD34 cell recovery. A predictive model that combines CD45 nMFI, Hodgkin lymphoma disease status, and MNC percentage has shown good power for predicting unnecessary repeat collections in our patient population. Our predictive model of post-thaw CD34 cell recovery, implemented as a clinical support tool, could help spare unnecessary procedures in a significant percentage of patients. Prospective validation of the algorithm is ongoing, and data will be presented at the meeting.

Optimized peripheral blood progenitor cell mobilization for autologous hematopoietic cell transplantation in children with high-risk and refractory malignancies.

Autologous hematopoietic stem cell transplantation (aHSCT) using hematopoietic progenitor cells (HPCs) has become an important therapeutic modality for patients with high-risk malignancies. Current literature on standardized method for HPC apheresis in children is sparse and failure rate reported as high as 30%. A retrospective study of 125 pediatric patients with high-risk malignancies undergoing aHSCT in Western Australia between 1997 and 2016 was conducted. Mobilization was achieved by means of chemotherapy and granulocyte colony-stimulating factor (G-CSF). Patients underwent apheresis the day after CD34+ counts reached ≥20/µL and an additional dose of G-CSF. Peripheral arterial and intravenous lines were inserted in pediatric intensive care unit under local anesthetic and/or sedation, omitting the need for general anesthesia as well as facilitating an uninterrupted apheresis flow. Larger apheresis total blood volumes were processed in patients weighing ≤20kg. The minimal dose of ≥2×106 CD34+ cells/kg was successfully collected in 98.4% of all patients. The optimal dose of 3-5×106 CD34+ cells/kg was collected in 96% of patients scheduled for a single aHSCT, 87.5% for tandem, and 100% for triple aHSCT. All HPC collections were completed in one apheresis session. Mobilization after ≤3 chemotherapy cycles and cycles including cyclophosphamide resulted in a significantly higher yield of CD34+ cells. Our approach to HPC mobilization by means of chemotherapy and single myeloid growth factor combined with optimal collection timing facilitated by continuous apheresis flow resulted in highly effective HPC harvest in children and adolescents with high-risk cancers.

Limited Utility of Flow Cytometry Immunophenotyping for Assessment of Plasma Cells in Hematopoietic Progenitor Cell Apheresis Product From Patients With Multiple Myeloma

Abstract Aim Flow cytometric immunophenotyping (FCI) is routinely performed in our laboratory to assess the clonality of plasma cells in hematopoietic progenitor cell apheresis (HPC-A) products as part of the quality control for multiple myeloma patients undergoing autologous stem cell transplantation. The purpose of this project is to investigate whether FCI is indicated for HPC-A when FCI is also performed on a bone marrow biopsy sample obtained before or after HPC-A collection. Materials and Methods The FCI results of HPC-A samples were retrieved from the laboratory database and analyzed. Relevant FCI results of the corresponding bone marrow biopsy samples were also retrieved and analyzed. All FCI was performed using BD FACSCalibur/FACSCanto with a four-color antibody panel, and the listmode data were analyzed by BD FACSDiva. Results FCI was performed on a total of 1,621 HPC-A samples in our laboratory from 02/01/2012 to 02/02/2018. A total of 58 HPC-A samples were positive for a monoclonal plasma cell population (3.8% of the total sample). Among those positive samples, 55 had bone marrow biopsy done before or after HPC-A collection: 38 within a month, 8 between 1 and 2 months, and 9 between 3 and 6 months. Fifty-four of 55 bone marrow samples were positive for a monoclonal plasma cell population by FCI. Conclusion The utility of FCI in the quality assessment of HPC-A products from patients with multiple myeloma is a very limited when FCI is performed on a bone marrow biopsy obtained within 6 months of HPC-A collection.

Cellular collection by apheresis.

Cellular collection is an important and increasingly used apheresis procedure. These collections are performed by leukocytapheresis, a procedure involving the removal of a patient's or donor's white blood cells, and are used to collect hematopoietic progenitor cells, specific cell populations (such as T-lymphocytes), and granulocytes. Hematopoietic progenitor cell apheresis and T-lymphocyte collection are performed by procedures that enrich for mononuclear cells. Hematopoietic progenitor cells are used for autologous and allogeneic hematopoietic stem cell transplantation, whereas T-cell collection is being used increasingly in novel cellular therapy approaches and for donor lymphocyte infusions to induce graft-versus-leukemia effect. Granulocytes are collected from healthy donors to treat severe sepsis in patients who are refractory to antimicrobial therapies. Less frequently, cellular depletion of leukocytes may be indicated in leukemic patients who have severe hyperleukocytosis resulting in leukoaggregation and decreased tissue perfusion. In these procedures, establishing and maintaining adequate vascular access are critical prerequisites to ensuring a successful procedure. For most types of leukocytapheresis procedures, efforts should be made to ensure that they are performed using peripheral veins, and the use of an intravascular access device should be considered only after it is determined that peripheral access is not feasible or desirable. However, in some settings (such as in patients undergoing autologous hematopoietic stem cell transplantation), intravascular access devices are often used to facilitate both the leukocytapheresis procedure and the subsequent transplant. Here, different types of vascular access approaches used in cellular collections are discussed, and this information is supplemented by the author's experience and practice in areas where published information is limited.

LB22 - Process validation of cryopreservation procedures of hematopoietic progenitor cells, apheresis (HPC-A) at a new processing facility for clinical bone marrow transplant program

LB22 - Process validation of cryopreservation procedures of hematopoietic progenitor cells, apheresis (HPC-A) at a new processing facility for clinical bone marrow transplant program

Use of allogeneic apheresis stem cell products as an interlaboratory proficiency challenge.

AABB Standards requires that laboratories participate in a proficiency test (PT) program for critical analytes. Institutions can purchase commercial PT materials; however, PT can also be performed through interlaboratory exchange. We investigated the utility of allogeneic hematopoietic progenitor cell apheresis (HPC-A) products as an interlaboratory PT challenge for total nucleated cell count (TNC) and CD34 assessment. Three-year retrospective and comparative review of unrelated allogeneic HPC-A products received by the University of Michigan between January 2011 and December 2013. Internal TNC and CD34 count were compared to the external collecting facility by paired t test and linear regression. The absolute and percent difference between external and internal counts and 95% limits of agreeability (95% LA) were determined. Results were analyzed relative to donor center location (international, domestic), time zone (domestic), and calendar year. There was a strong correlation between internal and external TNC, regardless of donor center location or year. For CD34, there was a good correlation between centers (R = 0.88-0.91; slope = 0.95-0.98x) with a median difference of -1% (95% LA, -50%, +47%). This was considerably better than commercial PT challenges, which showed a persistent negative bias for absolute CD34 and CD3 counts. Allogeneic HPC-A products represent an interlaboratory PT exchange for all critical analytes, including TNC and CD34 count, cell viability, and sterility. Allogeneic HPC-A products, which are fresh and transported under validated conditions, are less subject to preanalytical variables that may impact commercial PT samples such as aliquoting and sample homogeneity, commercial additives, and sample stability during manufacturing and transport.

Total leukocyte count-based predictor tool for calculating hematopoietic progenitor cell dose in bone-marrow harvest

Background: Hematopoietic progenitor cell transplantation is increasingly being used as the curative therapy in India for various clinical conditions. There are three main sources of hematopoietic progenitor cells; hematopoietic progenitor cell-apheresis (HPC-A), hematopoietic progenitor cell-bone marrow (HPC-M) and hematopoietic progenitor cell-Umbilical Cord (HPC-C). The number of CD34+ cells in HPC-C is fixed. In HPC-A, the number of CD34+ cells collected is based on the baseline peripheral blood counts and a second harvest can be easily performed. The trickiest calculation of CD34+ cells adequacy is in case of HPC-M harvest, where the end point of harvest cannot be predetermined. Aim: The aim was to study the accuracy of a total leukocyte count (TLC)-based predictor tool in predicting CD34+ dose in comparison to the actual CD34+ cell counts in the harvest. Materials and Methods: This was a prospective study to validate the tool. The data captured included patient and donor demographic data, disease condition, mobilization regimen of the donor, progenitor cell harvest data, dose collected, cryopreservation if any, progenitor cell infusion data, engraftment, and follow-up of the patient including day thirty and day hundred chimerism in a tertiary care hospital. Results: Five patients were included in the study. For each patient, the target dose and volume were collected in a single HPC-M harvest attempt, and no repeat harvests were required. The average volume of HPC-M harvest collected was 195 ml. The average CD-34+ cell dose in HPC-M harvest achieved was 4.3 × 106/kg (Range = 3.39–6.42). This was 85.7% of the targeted dose calculated on the basis of TLC-based predictor tool. Conclusion: This study reiterates the importance of a simple TLC-based predictor tool (formula) for estimation of HPC-M volume to be harvested.

Phase I Study Combining Ibrutinib with Rituximab, Ifosfamide, Carboplatin, and Etoposide (R-ICE) in Patients with Relapsed or Primary Refractory Diffuse Large B-Cell Lymphoma (DLBCL): NCI-Cancer Therapeutics Evaluation Program (CTEP) #9588

Background: In the post-rituximab era, half the patients with relapsed or refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL) fail to achieve a chemosensitive response with standard salvage therapy, and are thus ineligible to proceed to consolidative autologous stem cell transplantation (ASCT) with curative intent. The Bruton's tyrosine kinase inhibitor ibrutinib demonstrates single-agent activity in rel/ref DLBCL, predominately of the non-germinal center B-cell (non-GCB) phenotype, with minimal toxicity. This single center, NCI-CTEP sponsored phase I study is the first to evaluate the combination of ibrutinib with standard salvage therapy of rituximab, ifosfamide, carboplatin and etoposide (R-ICE) in transplant-eligible rel/ref DLBCL patients.Methods: Patients with rel/ref DLBCL, including transformed B-cell non-Hodgkin lymphoma, are eligible for study. The phase I study design is a standard 3 x 3 dose escalation of ibrutinib at 420 mg (dose level [DL] #1), 560 mg (DL #2) and 840 mg (DL #3) on days 1-21 with standard dosing of R-ICE for 3 cycles, every 21 days. The primary objective is to determine safety and the maximum tolerated dose (MTD) of ibrutinib in combination with R-ICE. Secondary objectives include response rate according to computed tomography (CT) and functional imaging (FDG-PET) per Deauville criteria.Results: To date, 16 patients are evaluable for toxicity, and 15 are evaluable for response. The median age of the 16 evaluable patients is 51 years (range 19-75 years). Histologies of the patients evaluable for response are: GCB DLBCL n=3, non-GCB DLBCL n=4, primary mediastinal large B-cell lymphoma n=4, and transformed chronic lymphocytic leukemia/small lymphocytic lymphoma (tCLL/SLL) n=4. There were no dose-limiting toxicities (DLTs) seen at DL #1 (n=3), 2 (n=3), or 3 (n=10, currently in expansion). Of the 16 patients evaluable for toxicity, 15 experienced expected and transient grade 3 or 4 hematologic toxicities with hematopoietic recovery prior to each cycle. The median number of cumulative platelet transfusions per patient for 3 cycles was 2 (range 0-11). Eleven of the 15 patients evaluable for response underwent chemotherapy-primed CD34+ hematopoietic progenitor cells (HPCs) apheresis procedures on study; 10 of the 11 patients successfully collected HPCs with a median of 5.6 x 106CD34+/kg (range 1.7-8.6). The only patient that failed to collect HPCs was an HIV-positive patient at DL #3 in the setting of febrile illness. One patient experienced grade 3 atrial fibrillation/flutter and was subsequently removed from study per treating physician's decision. Per CT criteria, five patients achieved complete remission (CR), eight patients achieved partial remission (PR), and two patients had stable disease for an overall response rate of 87%. All eight patients with non-GCB DLBCL and tCLL/SLL that were evaluable for response achieved chemosensitive remission per CT criteria (CR=4, PR=4). Seven of the 15 total patients (47%) evaluable for response achieved a complete metabolic remission (Deauville 1-3) per FDG-PET, including all 4 patients of non-GCB phenotype.Conclusions: Currently, no DLTs have been observed with ibrutinib at dosing up to 840 mg daily in combination with R-ICE. We are currently expanding DL #3. Manageable and expected hematologic toxicities have been observed. Importantly, hematologic toxicity has not resulted in failure to complete protocol therapy on-schedule or mobilize HPCs. Encouragingly, 87% of patients achieved a CT response (CR/PR) and 47% of patients achieved a complete metabolic remission per FDG-PET, including 100% of patients with non-GCB phenotype. These results compare favorably to historic cohorts. Given the safety and efficacy observed in this phase I, later phase studies for this treatment program are warranted. DisclosuresNo relevant conflicts of interest to declare.

Low-molecular-weight carbohydrate Pentaisomaltose may replace dimethyl sulfoxide as a safer cryoprotectant for cryopreservation of peripheral blood stem cells.

Cryopreserved hematopoietic stem cell products are widely used for certain hematologic malignancies. Dimethyl sulfoxide (DMSO) is the most widely used cryoprotective agent (CPA) today, but due to indications of cellular toxicity, changes of the cellular epigenetic state, and patient-related side effects, there is an increasing demand for DMSO-free alternatives. We therefore investigated whether Pentaisomaltose (PIM), a low-molecular-weight carbohydrate (1 kDa), can be used for cryopreservation of peripheral blood stem cells, more specifically hematopoietic progenitor cell apheresis (HPC(A)) product. We cryopreserved patient or donor HPC(A) products using 10% DMSO or 16% PIM and quantified the recovery of CD34+ cells and CD34+ subpopulations by multicolor flow cytometry. In addition, we compared the frequency of HPCs after DMSO and PIM cryopreservation using the colony-forming cells (CFCs) assay. The mean CD34+ cell recovery was 56.3 ± 23.7% (11.4%-97.3%) and 58.2 ± 10.0% (45.7%-76.9%) for 10% DMSO and 16% PIM, respectively. The distribution of CD34+ cell subpopulations was similar when comparing DMSO or PIM as CPA. CFC assay showed mean colony numbers of 70.7 ± 25.4 (range, 37.8-115.5) and 67.7 ± 15.7 (range, 48-86) for 10% DMSO and 16% PIM, respectively. Our findings demonstrate that PIM cryopreservation of HPC(A) products provides recovery of CD34+ cells, CD34+ subpopulations, and CFCs similar to that of DMSO cryopreservation and therefore may have the potential to be used for cryopreservation of peripheral blood stem cells.

Comparison of manual hematocrit determinations versus automated methods for hematopoietic progenitor cell apheresis products

Allogeneic hematopoietic stem cell donor selection is based primarily on human leukocyte antigen degree of match and it often occurs without regard to the red blood cell (RBC) compatibility between donor and recipient. When major ABO-mismatched grafts are infused, it is imperative that an accurate determination of the incompatible RBC content is made to ensure that the product is safe for infusion. RBC content determination requires the hematocrit (Hct) parameter which can be obtained via manual (directly measured) or automated (calculated) methods. Ninety-seven apheresis hematopoietic progenitor grafts were assessed for Hct by manual testing and by four commercially available automated hematology analyzer instruments. A clinical model was developed to assess the frequency of unnecessary RBC reductions or alteration in standard infusion practice. Significant (p < 0.001) differences were observed where the manual Hct value was markedly lower than automated Hct values. At stringent incompatible RBC threshold of 10 mL, the number of preventable RBC reduction procedures ranged from 18% to 69%. Accurate determination of RBC content of hematopoietic progenitor grafts is essential for patient safety. Despite the rapidity and convenience offered by automated Hct methods, they significantly overestimate the incompatible RBC content of grafts, which may trigger unnecessary RBC reduction procedures or split infusions. In products where automated Hct methods indicate excessive amounts of incompatible RBCs are present, we advise the performance of confirmatory testing with a manual Hct method to ensure that the automated Hct value is not a false positive.

Utility Of An Algorithm For Use Of Plerixafor In Filgrastim-based Hematopoietic Progenitor Cell Mobilization In Patients With Plasma Cell Myeloma Treated With Carfilzomib-Lenalidomide-Dexamethasone.

▪Mobilization of hematopoietic progenitor cells (HPC) for subsequent autologous transplantation is difficult in patients with plasma cell myeloma (PCM) due to poor marrow reserve. Targeted HPC yields are generally not achieved in a single apheresis procedure without use of plerixafor as a supplement to standard filgrastim. Strategies to limit use of plerixafor, due to its expense, to cases of poor CD34 mobilization have been developed, but their applicability in patients receiving the novel induction regimen carfilzomib-lenalidomide-dexamethasone (CRD) (Blood 2012;120:1801) has not been described. We prospectively studied the CD34 cell mobilization responses of PCM patients following CRD induction, using a CD34 cell predictive algorithm to determine when plerixafor should be added to the mobilization regimen. Thirty patients, including 23 with PCM and 7 with smoldering PCM, mean age 55 (range 40-72), 47% male, received 4 to 7 cycles of CRD (median, 5 cycles), with the last dose of lenalidomide given at least one week prior to mobilization. Filgrastim 10-16 mcg/kg/day was given as a single evening dose for 5 days, with circulating CD34 count assessed 12 hours after the 4th dose. The pre-apheresis CD34 count after the 5th dose of filgrastim was predicted to be 10% greater than that after the 4th dose; this prediction was validated with an actual pre-apheresis CD34 count obtained the following day. Prior mobilization data derived from healthy HPC apheresis donors was used to generate a regression formula, y=0.45x+0.86, where x=the pre-apheresis circulating CD34 count after the 5th dose of filgrastim, and y=the expected yield of the apheresis procedure, expressed as millions of CD34 cells harvested per liter processed. Targeted yield was ≥ 4 x 106 CD34 cells/kg, with minimum acceptable yield ≥ 2 x 106 CD34 cells/kg. Plerixafor 240 mcg/kg was given with the 5th dose of filgrastim, 8-10 hours prior to apheresis, if the regression equation predicted a CD34 cell yield of < 4 x 106 CD34 cells/kg in a single procedure with a maximum of 30 liters processed. The actual volume processed was based on the stat blood CD34 count drawn immediately prior to apheresis. Procedures were performed on the Cobe Spectra device; continuous intravenous calcium was used to mitigate citrate toxicity. Central lines were required in 67% of subjects.Mean CD34 cell count in the entire group after the 4th dose of filgrastim was 29/uL (range 2-88/uL). Using the regression formula as a guide, 17/30 (57%) of patients received plerixafor. CD34 counts increased 4.2-fold in patients receiving plerixafor, from 15 ± 9/uL (m ± SD) on the day prior to apheresis to 53 ± 30/uL immediately pre-apheresis; CD34 counts did not change in patients who received filgrastim alone (from 48 ± 17/uL to 45 ± 19/uL). Guided by the stat pre-apheresis CD34 count, the volume processed in the first apheresis procedure was the same, 23 ± 7 (range 12-30) liters, with or without plerixafor. CD34 cells were collected with 72 ± 14% efficiency. First-procedure CD34 cell yields were 6.4 ± 2.5 x 106/kg (range 2.5-10.1) with supplemental plerixafor vs 5.8 ± 2.5 x 106/kg (range 1.1-9.3) with filgrastim alone. Only 2/30 patients underwent a second procedure; neither received plerixafor prior to the first procedure, and both received it prior to the second. In one patient, criteria for plerixafor administration were met, but the drug was inadvertently not given prior to the first procedure; in the second patient, an unexpectedly low pre-apheresis CD34 count was traced to inadequate self-administration of the 5th dose of filgrastim. All 30 patients achieved the minimum CD34 collection goal of ≥ 2 x 106 cells/kg and 29/30 did this in one procedure. The higher targeted collection goal of ≥ 4 x 106 CD34 cells/kg was achieved in a single procedure by 76% of patients in both the plerixafor group and the filgrastim-alone group. There was a trend for higher cumulative lenalidomide and carfilzomib doses to be associated with lower CD34 mobilization responses to filgrastim.Induction treatment with CRD does not appear to impair HPC mobilization response to filgrastim in patients with PCM, compared to older regimens. An algorithm that uses the CD34 cell count after 4 doses of filgrastim to predict the following day's pre-apheresis CD34 count and thus determine whether plerixafor supplementation is needed, was useful in identifying the 40% of CRD-treated myeloma patients who do not need plerixafor. Disclosures:No relevant conflicts of interest to declare.

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.

Hematopoietic progenitor cell mobilization using low‐dose cyclophosphamide and granulocyte colony‐stimulating factor for multiple myeloma

High-dose chemotherapy (HDT) supported by autologous stem cell transplantation (ASCT) has long been one of the standards of care for younger patients with multiple myeloma (MM). Cyclophosphamide (CY) plus granulocyte colony-stimulating factor (G-CSF) has been the conventional preparation for hematopoietic progenitor cell (HPC) mobilization, although the optimal dosage of CY in this setting has not yet been clearly defined. This study investigated the efficacy and safety of low-dose (LD-)CY (1.5 g/m(2)) plus G-CSF for conditioning for HPC apheresis harvest (HPC-A) in 18 MM patients, and compared it with a regimen consisting of intermediate-dose (ID)-CY (4 g/m(2)) plus G-CSF for 13 MM patients. Eleven patients in the former and six in the latter were treated with bortezomib (BTZ) during the induction therapy. Both regimens were comparably effective in terms of CD34(+) cell yields, while adverse events, such as leukopenia, thrombocytopenia, and febrile neutropenia, occurred significantly less frequently in the LD-CY cohort. All patients in LD-CY cohort started and completed their apheresis on day 7 or 8, whereas for the ID-CY cohort the day of first apheresis varied widely from day 8 to 15. These findings indicate that the LD-CY regimen is as effective as ID-CY for HPC mobilization, while the former is clearly more practicable and convenient than the ID-CY regimen for patients with MM.

A new system for quality control in hematopoietic progenitor cell units before reinfusion in autologous transplant

In our Center, the cell viability, the integrity of the bag, and the clonogenic assay were evaluated before the reinfusion of hematopoietic progenitor cells-apheresis (HPC-A). This quality control (QC) should be made 14 days before the reinfusion to the patient to have the result of the functional test on the proliferative capacity of hematopoietic progenitors. This study was designed to assess the potential of an automatic cell counting system (NucleoCounter NC-3000, ChemoMetec) in our clinical routine as a support of the clonogenic assay and the cytofluorimetric analysis for the QC of the cryopreserved HPC-A. The cell viability was evaluated by flow cytometry using the modified International Society of Hematotherapy and Graft Engineering protocol. The proliferative potential was assessed by specific clonogenic tests using a commercial medium. Furthermore, we evaluated the cellular functionality with NucleoCounter NC-3000, by using two protocols: "vitality assay" and "mitochondrial potential assay." The evaluation of the total nucleated cells in preapoptosis measured by 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazol-carbocyanine iodide (JC-1) assay showed a negative correlation (r=-0.43) with the total number of colonies (colony-forming unit [CFU]-granulocyte-macrophage progenitors plus burst-forming unit-erythroid progenitors plus CFU-granulocyte, erythroid, macrophage, megakaryocyte progenitors) obtained after seeding of 50 × 10(6) /L viable total nucleated cells. We observed a significant difference (p<0.0001) comparing the median number of colonies (166.70; SD, ± 136.36) obtained with a value of JC-1 less than 30% to the number of colonies (61.75; SD, ± 59.76) obtained with a value of JC-1 more than 30%. The evaluation of cell functionality by the use of the NucleoCounter NC-3000 is in agreement with results from clonogenic assay and can be considered an effective alternative in the routine laboratory.

A Paradigm Shift in Hematopoietic Progenitor Cell Apheresis: From Apheresis Technicians to Trained Oncology Nurses - Improving Patient Outcomes

A Paradigm Shift in Hematopoietic Progenitor Cell Apheresis: From Apheresis Technicians to Trained Oncology Nurses - Improving Patient Outcomes