Peripheral Blood Stem Cell Apheresis Research Articles

Autologous peripheral blood stem cell mobilization and apheresis in pediatric patients with cancer: A single-center report of 64 procedures.

The published experience concerning autologous peripheral blood stem cell collection in children is very limited. The data of pediatric patients who underwent autologous stem cell mobilization and apheresis between January 2011 and April 2020 were analyzed retrospectively. We studied retrospectively 64 mobilization and apheresis procedures in 48 pediatric patients (34 males, 14 females), mean age of 7.31 ± 5.38 (range, 1.5-19.7) years, the underlying disease was mostly neuroblastoma (NBL). The body weight of 21 patients (43.75%) was 15 kg or less. The targeted autologous peripheral stem cell apheresis (APSCA) was successfully achieved in 98% of patients. Neuroblastoma patients were younger than the rest of the patients and underwent apheresis after receiving fewer chemotherapy cycles than others and all of them mobilized within the first session successfully. Plerixafor was added to mobilization in nine heavily pretreated patients (18.7%), median two doses (range, 1-4 doses). 11 patients (22.9%) underwent radiotherapy (RT) before mobilization with doses of median 24 Gy (range, 10.8-54.0 Gy). Patients with RT were older at the time of apheresis and had received more chemotherapy courses than patients without RT. As a result, patients with a history of RT had significantly lower peripheral CD34+ cells and CD34+ yields than those without RT. In 17 patients (35.4%), 22 different complications were noted. The most common complications were catheter-related infections (n:10, 20.8%), followed by catheter-related thrombosis in eight patients (16.7%). Patients who had far less therapy before apheresis were more likely to mobilize successfully. Our study provides a detailed practice approach including complications during APSCA aiming to increase the success rates of apheresis in transplantation centers.

Efficacy of prophylactic antibiotics for the prevention of neutropenic fever in patients with multiple myeloma receiving high-dose cyclophosphamide for stem cell mobilization

High-dose cyclophosphamide (HD-Cy) (3 g/m2) plus granulocyte colony-stimulating factor (G-CSF) is a very effective regimen for peripheral blood stem cell (PBSC) mobilization. Unfortunately, it is associated with an increased risk of neutropenic fever (NF). We analyzed the effect of NF on PBSC apheresis results and the efficacy of prophylactic antibiotics for the prevention of NF associated with HD-Cy plus G-CSF for PBSC mobilization in patients with newly diagnosed multiple myeloma (MM). First, patients were divided into NF ( +) and NF ( −) groups according to whether they suffered from NF during mobilization. Second, we divided patients into an antibiotic prophylaxis group and a nonantibiotic prophylaxis group according to whether antibiotic prophylaxis was used during the mobilization period. Our study showed that NF( +) patients (n = 44) had lower CD34 + cell dose collection (median 2.60 versus 5.34 × 106/kg, P < 0.001) and slower neutrophil engraftment and platelet engraftment (median 11 versus 10 days, P = 0.002, and median 13 versus 11 days, P = 0.043, respectively) than NF( −) patients (n = 234). Of note, the nonantibiotic prophylaxis group patients (n = 30) had a 26.7% incidence of NF. In the patients receiving antibiotic prophylaxis (n = 227), the incidence was reduced to 9.3% (P = 0.01). The antibiotic prophylaxis patients had higher CD34 + cell collection (median 5.41 versus 2.27 × 106/kg, P < 0.001) and lower hospitalization cost of mobilization ($ median 3108.02 versus 3702.39, p = 0.012). Thus, our results demonstrate that NF is associated with lower CD34 + cell collection and that antibiotic prophylaxis can reduce the incidence of NF and improve stem cell mobilization and collection outcomes, which reduces the hospitalization cost of mobilization.

Assessment and comparison of viability assays for cellular products

Background aimsAccurate assessment of cell viability is crucial in cellular product manufacturing, yet selecting the appropriate viability assay presents challenges due to various factors. This study compares and evaluates different viability assays on fresh and cryopreserved cellular products, including peripheral blood stem cell (PBSC) and peripheral blood mononuclear cell (PBMC) apheresis products, purified PBMCs and cultured chimeric antigen receptor and T-cell receptor-engineered T-cell products. MethodsViability assays, including manual Trypan Blue exclusion, flow cytometry-based assays using 7-aminoactinomycin D (7-AAD) or propidium iodide (PI) direct staining or cell surface marker staining in conjunction with 7-AAD, Cellometer (Nexcelom Bioscience LLC, Lawrence, MA, USA) Acridine Orange/PI staining and Vi-CELL BLU Cell Viability Analyzer (Beckman Coulter, Inc, Brea, CA, USA), were evaluated. A viability standard was established using live and dead cell mixtures to assess the accuracy of these assays. Furthermore, precision assessment was conducted to determine the reproducibility of the viability assays. Additionally, the viability of individual cell populations from cryopreserved PBSC and PBMC apheresis products was examined. ResultsAll methods provided accurate viability measurements and generated consistent and reproducible viability data. The assessed viability assays were demonstrated to be reliable alternatives when evaluating the viability of fresh cellular products. However, cryopreserved products exhibited variability among the tested assays. Additionally, analyzing the viability of each subset of the cryopreserved PBSC and PBMC apheresis products revealed that T cells and granulocytes were more susceptible to the freeze–thaw process, showing decreased viability. ConclusionsThe study demonstrates the importance of careful assay selection, validation and standardization, particularly for assessing the viability of cryopreserved products. Given the complexity of cellular products, choosing a fit-for-purpose viability assay is essential.

Comparison of the efficiency of peripheral blood stem cell apheresis on the blood cell separators.

Stem cells apheresis is a key step in the process of the autologous stem cell transplantation. Available blood cell separators (BCS) have different efficiency due to the technical characteristics and influence of the operator. Retrospectively, data were collected of the peripheral blood stem cells apheresis performed using available BCS manufactured by Fresenius (ComTec and Amicus) in the National Cancer Institute Ukraine from 2017 to 2020. The collection efficiency coefficient (CEC) was calculated, the formula for predicting the total volume of processed blood (TVPB) was adapted for each separator. The analysis included data from 60 patients (total of 92 apheresis procedures). The mean CEC was established at the level of (53.8 ± 36.6) % for the Amicus device and (44.2 ± 37.3) % for the ComTec device; P = 0.22. The lower product volume was obtained using the Amicus device compared to the ComTec device; P = 2×10-7. The amount of collected stem cells was comparable in both groups (5.8 ± 5.7) ×106/kg and (4.1 ± 3.1) ×106/kg, respectively; P = 0.064. The adaptation of the formula for predicting the TVPB to achieve the optimum amount of stem cells was performed. The CEC for each device was within the generally accepted limits of 30-50%, and did not differ significantly. Nevertheless, using of the Amicus BCS allowed to collect lower volumes of the product, maintaining other characteristics of the product competitive.

Peripheral blood stem cells collection by apheresis in very low-weight children with malignant diseases-A single center experience.

Hematopoietic stem cell transplantation is used in the treatment of children with malignant and non-malignant diseases. However, apheresis of peripheral blood stem cells (PBSC) represents a challenge in children below 10kg. A retrospective trial was conducted in the Cellular Therapy Department of Portuguese Oncology Institute of Porto. children with body weight inferior to 10kg who underwent autologous PBSC apheresis until 2021. Demographic and clinical data were collected and our institutional protocol was described. COBE Spectra apheresis system (TerumoBCT, Lakewood, Colorado) until 2012 and then Spectra Optia (TerumoBCT) were used. Sixteen leukocytaphereses were performed in 13 patients-nine females (69%). Mean age and weight were 13.31 months (±5.26) and 8.31 kg (±1.17), respectively. The initial CD34+ cells/μL in peripheral blood was 70.8 (±61.9). A central venous catheter (CVC) was exclusively used in all but one patient, in whom a peripheral vein was also required. In 10 procedures, both heparin and anticoagulant citrate dextrose solution-formula A (ACD-A) were used; in the remaining ones, only ACD-A was employed. The median duration of each procedure was 168 minutes (±45) and 2.96 blood volumes (±0.31) were processed. Median CD34+ cells yield per leukocytapheresis was 6.52 × 106 /kg (±9.87 × 106 ). CD34+ cell extraction rates were not significantly different between the two apheresis systems. Platelet and magnesium levels were significantly lower after collection (P < .001 and P=.009, respectively). Although recommendations are lacking, we have successfully and safely performed leukocytaphereses in children with body weight below 10kg. The authors believe that a permanent and dynamic comprehensive evaluation of each child is paramount for attaining good results.

Hematopoietic progenitor cell counting can optimize peripheral blood stem cell apheresis process.

Monitoring of stem cell concentration in transplantation settings is crucial to determine the optimal time of apheresis and is currently based on enumeration of CD34+ cells by flow cytometry. No surrogate marker has replaced CD34+ cell enumeration to date. The aim of this study was the evaluation of the hematopoietic progenitor cell (HPC) parameter of the Sysmex XN-1000 analyzer in terms of optimizing the peripheral blood stem cell apheresis process. Results of flow cytometric CD34+ cell and Sysmex HPC count were compared in 208 preharvest samples and 139 apheresis products. HPC and CD34+ cell counts showed significant differences in the multiple myeloma (MM) group. The correlation between preharvest HPC and CD34+ cell counts was good in the MM group (rho=.613) and strong in the lymphoma group (rho=.802), allogeneic donors (rho=.923), and other group of samples (rho=.816). The HPC positive cutoff demonstrating 100% specificity and positive predictive value for MM patients was high for ≥20/μL and ≥10/μL CD34+ cell counts, and therefore of limited value. The HPC negative cutoff demonstrating 100% sensitivity and negative predictive value was approximately <4/μL, irrespective of diagnosis. Based on proposed HPC positive cutoffs (≥31/μL in the lymphoma group and ≥11/μL in the other group of samples), routine HPC enumeration could improve the workflow by replacing CD34+ cell counting in allogeneic donors as well as non-MM patients. Furthermore, based on proposed HPC negative cutoff (<4/μL), CD34+ cell counting could be fully omitted in donors and patients that are not adequately mobilized.

Innovator Filgrastim versus Generic Filgrastim in Hematopoietic Stem Cell Transplantation Mobilization.

Methods This is a retrospective study. G-CSF was administered in the dose of 10 μg/kg subcutaneous as a single dose for 4 days. On day 5, peripheral blood stem cell (PBSC) apheresis was performed using Haemonetics MCS plus or COBE Spectra apheresis machine through a double-lumen central venous catheter. Primary outcome parameters were the total number of CD34+ HSCs/kg of recipient weight mobilized in peripheral blood and the number of days required for neutrophil and platelets engraftment, respectively. Objective We compared the effectiveness and safety of innovator filgrastim versus generic filgrastim in patients who underwent hematopoietic stem cell transplantation (HSCT). Results A total of 91 stem cell mobilizations was analyzed. There were 58 normal healthy donors for allogeneic HSCT and 33 patients for autologous HSCT. There was no statistically significant difference among groups in terms of total collected CD34+ cells value ( p = 0.609). The mean time to neutrophil engraftment was 13.7 days in the innovator group and 13.2 days in the Grafeel group ( p = 0.518). The mean time to platelet engraftment was 16.2 days in the innovator group and 14.8 days in the generic group ( p = 0.435). The patient who received generic filgrastim had more febrile episodes during the course of transplantation ( p = 0.020). Conclusion Generic filgrastim was found to be comparable to original filgrastim for peripheral blood stem cell mobilization in normal healthy donors for allogeneic HSCT and patients for autologous HSCT.

Platelet decrease and efficacy of platelet-rich plasma return following peripheral blood stem cell apheresis.

Peripheral blood stem cell (PBSC) transplantation is a key treatment option for hematological diseases and is widely performed in clinical practice. Platelet loss is one of the major complications of PBSC apheresis, and platelet-rich plasma (PRP) return is considered in case of platelet decrease following apheresis; however, little is known about the frequency and severity of platelet loss and the efficacy of PRP return postapheresis. We assessed changes in platelet counts following PBSC-related apheresis in 270 allogeneic (allo)- and 105 autologous (auto)-PBSC settings. We also evaluated the efficacy of PRP transfusion on platelet recovery postapheresis. In both allo- and auto-PBSC settings, the preapheresis platelet count (range, 84-385 and 33-558 × 109 /L, respectively) decreased postapheresis (range, 57-292 and 20-429 × 109 /L, respectively), whereas severe platelet decrease (<50 × 109 /L) was only observed in auto-PBSC patients (n=9). We confirmed that platelet count before apheresis was a risk factor for severe platelet decrease (<50 × 109 /L) following auto-PBSC apheresis (odds ratio 0.749, P < .049). PRP return postapheresis facilitated platelet recovery in more than 80% of cases in both allo and auto settings. Lower platelet count preapheresis is a useful predictor of severe platelet decrease following auto-PBSC apheresis and PRP return is an effective process to facilitate platelet recovery postapheresis.

Simple Predictors of Peripheral Blood Stem Cell Yield in Healthy Donors

BACKGROUND: Peripheral blood stem cells (PBSCs) are commonly used for hematopoietic stem cell transplant (HSCT) over other stem cell sources. The hematopoietic stem cells (HSCs) are mobilized from marrow by granulocyte colony-stimulating factor (G-CSF) and then harvested by apheresis technique. The HSC yield differs in donors that may be due to inadequate mobilization or difficulty in harvesting the mobilized stem cells. MATERIALS AND METHODS: We retrospectively analyzed donor demographic and pre-apheresis hematological variables with circulating CD34+ cell (cir CD34) count and HSC yield in product in 100 normal donors. G-CSF was given for 5 consecutive days, and the stem cells were harvested on day 5. The cir CD34 count and pre-apheresis variables were recorded a day before harvest. RESULTS: Of 100 donors, 77% were males and 23% were females. Male sex, younger age, and donor weight were significantly associated with better CD34 yield in the product. Among the pre-apheresis hematological variables, absolute neutrophil count, hematocrit, and absolute nucleated red blood cell count significantly correlated with post-GCF circulating CD34 and CD34 yield in the product. Donors with mean corpuscular volume &lt;80 fL showed relatively poor CD34 cell harvest as compared to normal donors, though with adequate mobilization. CONCLUSION: Selection of donors for PBSC apheresis is crucial for a good transplant outcome and recovery. Alternate strategies that improve the final CD34 yield should be employed for donors with high risk for poor CD34+ cell yield.

Analysis of laboratory parameters for optimal autologous peripheral blood stem cell collection from lymphoma and myeloma patients.

Peripheral blood stem cell (PBSC) collection is important for successful hematopoietic stem cell transplantation. This study aimed to investigate the laboratory parameters associated with the optimal timing of autologous PBSC collection from lymphoma and multiple myeloma patients. We retrospectively evaluated data from 1105 PBSC apheresis procedures performed on 379 adult patients at the National Cancer Center between June 2005 and December 2019. Laboratory parameters, including cutoff values for the number of hematopoietic progenitor cells (HPCs) and circulating CD34+ cells, were analyzed to determine their association with CD34+ cell yield. The pre-apheresis HPC and CD34+ cell count were statistically significant variables associated with harvested CD34+ cell in lymphoma and MM patients. The optimal cutoff values were 18 × 106 /L for pre-HPC count (66.8% sensitivity, 66.4% specificity) and 11/μL for pre-CD34+ cell count (85.8% sensitivity, 87.2% specificity), to achieve CD34+ cell yields ≥ 1.0 × 106 /kg for each apheresis procedure. Moreover, the optimal cutoff values were 23 × 106 /L for pre-HPC count (71.0% sensitivity, 69.0% specificity) and 18/μL for pre-CD34+ cell count (87.5% sensitivity, 86.3% specificity) to achieve CD34+ cell yields ≥ 2.0 × 106 /kg for each apheresis procedure. HPC count is a potential surrogate marker for monitoring the starting time for PBSC collection. Applying cutoff values for the number of HPC and CD34+ cells may be clinically useful for optimizing the timing of PBSC collection.

Platelet Decrease and Efficacy of Platelet-Rich Plasma Return for Peripheral Blood Stem Cell Apheresis

Peripheral blood stem cell (PBSC) transplantation is a key treatment option for hematological diseases and widely performed in clinical practice. Platelet loss is the major complication of PBSC apheresis, and platelet-rich plasma (PRP) return is recommended in case of severe platelet decrease following apheresis; however, little is known about the frequency and severity of platelet loss nor the efficacy of PRP return post-apheresis. To address these questions, we assessed changes in platelet counts following PBSC-related apheresis in 270 allogeneic (allo)- and 105 autologous (auto)-PBSC settings. We also evaluated efficacy of PRP transfusion on platelet recovery post-apheresis. Platelet counts reduced up to 70% post-apheresis in both allo- and auto-PBSC settings, while severe platelet count decrease (&amp;lt; 50 x 109/L) was only observed in auto-PBSC patients (Figure 1). We next analyzed the relationship between severe platelet (&amp;lt; 50 x 109/L) after apheresis and several clinical factors by using univariate and multivariate analysis for auto-PBSC patients. As shown in Table 1, in univariate analysis, severe platelet counts following auto-PBSC apheresis was found more frequently in patients with lower platelet count, lower percentage of CD34+ cells in PB at pre-apheresis, repeated round of apheresis, and smaller number of collected CD34+ cells. On the other hand, in multivariate analysis, the white blood cell (WBC) counts pre-apheresis was the only significant risk factor of severe platelet count following apheresis (p = 0.038). We finally analyzed the transitions of platelet counts in the setting of apheresis. The median platelet counts at pre-apheresis, post-apheresis, and post-PRP return were 187.0 x 109/L, 132.0 x 109/L, and 154.0 x 109/L for allo-PBSC apheresis, and 147.0 x 109/L, 111.0 x 109/L, and 127.0 x 109/L for auto-PBSC apheresis (p &amp;lt; 0.0001 for all, allo-PBSC donors and auto-PBSC patients, respectively) (Figure 2), indicating that PRP return post-apheresis facilitated a rapid platelet recovery in both allo- and auto-settings. Collectively, our data suggest that WBC counts pre-apheresis is a useful predictor for severe platelet decrease following auto-PBSC apheresis and that PRP return is an effective mean to facilitate platelet recovery post-apheresis. Disclosures No relevant conflicts of interest to declare.

Timing of Daratumumab Administered Pre-Mobilization in Multiple Myeloma Impacts Pre-Harvest Peripheral Blood CD34+ Cell Counts and Plerixafor Use

Background: Daratumumab (DARA) is a monoclonal antibody which targets CD38 on plasma cells and B cell progenitors. DARA has been effectively combined with other therapies in newly diagnosed and relapsed/refractory multiple myeloma (RRMM), while DARA-based induction regimens in transplant-eligible patients (pts) are increasingly being used in clinical practice. Given that hematopoietic stem cells also express CD38, DARA may potentially affect stem cell mobilization and hematopoietic reconstitution following autologous stem cell transplant (ASCT). Although no clinically significant impact of DARA on stem cell mobilization or hematopoietic recovery was described in large phase 3 trials of triplet induction regimens +/- DARA in newly diagnosed MM, stem cell yields were lower and plerixafor more commonly used in the DARA-containing arms [Moreau et al, Lancet 2019; Voorhees et al, Blood 2020]. Significantly longer time to neutrophil (PMN) engraftment was also reported in pts receiving DARA-based induction who underwent upfront ASCT [Al Saleh et al, Am J Hematol 2020]. In this study, we examine the impact of timing of DARA administration pre-mobilization on day 4 pre-harvest peripheral blood CD34 cell count, stem cell apheresis yield, and post-ASCT engraftment. Methods: Between 1/1/2016 and 12/31/2019, newly diagnosed and RRMM pts receiving DARA-based induction regimens with ≥1 dose of DARA administered within 1 month prior to stem cell mobilization were identified retrospectively and compared to matched controls receiving similar induction regimens without DARA. Granulocyte colony-stimulating factor (G-CSF) was administered per institutional standards and plerixafor added based on day 4 pre-harvest peripheral blood CD34 counts. PMN and platelet engraftment post-ASCT was defined as the first of 3 consecutive days with sustained PMN count &amp;gt;500 x 106/L and independence from platelet transfusion in the preceding 7 days with a count &amp;gt;20 x 109/L, respectively. Pre-harvest peripheral blood CD34 counts and stem cell apheresis yields were obtained from the Cellular Therapy Laboratory at NewYork-Presbyterian Hospital. The study was approved by the Weill Cornell Medicine IRB. Results: We identified 16 pts who received DARA-based induction with ≥1 dose of DARA administered within 1 month of apheresis (DARA group) and 16 non-DARA-containing regimen-matched controls (non-DARA group). Demographics of the DARA and non-DARA groups were well matched (Table 1). DARA pts received their last dose of DARA a mean of 17.3 days prior to the first day of apheresis, with 8 pts receiving their last dose within 2 weeks and the remaining 8 pts between 2 weeks and 1 month prior. Overall, mobilization outcomes were inferior in the DARA group (Table 2). DARA pts had significantly lower day 4 pre-harvest peripheral blood CD34 counts compared to non-DARA pts (17.2 vs 35.4 cells/µL; P=0.0146). Institutional algorithm required plerixafor to be given for day 4 CD34 count ≤40 cells/µL. Fifteen of the 16 DARA pts received plerixafor vs. 11 non-DARA pts (P=0.07). Additionally, DARA pts required significantly more apheresis days (2.4 vs 1.6 days; P=0.0279). Differences in stem cell yield were not significant (8 vs 10 x106cells/kg; P=0.1391). Hematopoietic recovery post-ASCT was not affected by DARA administered in the month preceding mobilization. Conclusions: In summary, we report lower day 4 pre-harvest peripheral blood CD34 count, increased requirement for plerixafor, and longer apheresis duration in newly diagnosed and RRMM pts receiving DARA within 1 month ofstem cell mobilization. These limitations are largely overcome by plerixafor usage which, combined with G-CSF, resulted in successful stem cell collection in all patients. Limitations in our study include small sample sizes, retrospective control selection, and fewer pts in the DARA group achieving ≥VGPR prior to mobilization. Nevertheless, our findings are consistent with inferior mobilization outcomes reported in the DARA-containing arms of phase 3 trials of triplet induction +/- DARA and highlight the nearly universal requirement for plerixafor usage when DARA is administered within a month prior to apheresis. Prospective study of day 4 pre-harvest peripheral blood CD34 counts and other predictors of stem cell yield should be incorporated into future clinical trials of CD38 monoclonal antibody-based induction regimens for transplant-eligible MM pts. Disclosures Rossi: Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Niesvizky:GSK: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Rosenbaum:Amgen: Research Funding; GlaxoSmithKline: Research Funding; Akcea: Honoraria; Celgene: Honoraria; Janssen: Research Funding.

Dose-Adjusted Granulocyte Colony-Stimulating Factor Plus Dexamethasone Achieves More CD34+ Cell Mobilization and Earlier Engraftment in Haploidentical Hematopoietic Stem Cell Transplantation

Introduction: Previous studies have showed that higher doses of CD34+ cell were associated with more rapid neutrophil and platelet engraftment, lower probabilities of graft rejection, as well as reduced transplant-related mortality. The aim of this study was to investigate the effects of G-CSF (Filgrastim) plus dexamethasone in CD34+ cell mobilization and engraftment in T-cell replete haploidentical hematopoietic stem cell transplantation(HHSCT) which was based on G-CSF-primed bone marrow and peripheral blood graft. Methods: A total of 79 healthy donors, who underwent bone marrow (BM) harvest and peripheral blood Stem Cells (PBSCs) collection between January 2015 and June 2019, were investigated. In G-CSF group, G-CSF was administered subcutaneously at a dose of 5μg/kg once a day from 1st to 5th day, while BM and PBSC were harvested on the 4th day and 5th day, respectively. In Dose-Adjusted G-CSF+Dex group, G-CSF was administered subcutaneously at a dose of 5μg/kg once a day on the 1st and 2nd day, then twice a day from the 3rd to 5th day; 5mg dexamethasone was injected intravenously before BM collection on the 4th day and before PBSC apheresis on the 5th day, respectively. All 79 recipients with hematological malignancies underwent HHSCT based on modification of BU/CY (busulfan/cyclophosphamide) and Anti-human T Lymphocyte Rabbit Immunoglobulin (ATG-F). All recipients received cyclosporine A, mycophenolate mofetil, and short-term cyclophosphamide as GVHD prophylaxis. Results: There were no significantly statistical differences between these two groups on characteristics of both recipients and donors. In Dose-Adjusted-G-CSF+Dex group, more mono nuclear cells (MNCs) were collected from BM and PB in comparison to the cells collected in the G-CSF group (p&lt;0.001). There was a significant difference between the two groups on CD34+ cell counts from PB (p=0.002), which led to a significant difference on CD34+ cells in mixture allografts (p=0.04). In DA-G-CSF + Dex group, more CD34+ cells achieved earlier neutrophil (p=0.001) and platelet (p&lt;0.001) engraftment compared with G-CSF group. Conclusion: Compared to G-CSF alone, dose-adjusted G-CSF plus dexamethasone on healthy donors can lead to more collection of MNCs and CD34+ cells in mixture allografts, which achieves earlier neutrophil and platelet engraftment. Disclosures Zheng: Pfizer: Research Funding.

Immunomagnetic selective donor-derived CD4+CCR7+ T cell depletion procedure for peripheral blood stem cells graft

Purpose of the studyWhile acute graft-versus-host-disease (GVHD) is a T cell-mediated disease caused by alloreactive donor T cells, we and others have highlighted that patients who received higher proportion of donor CD4+ naïve and central memory T cells expressing the chemokine receptor 7 (CCR7) more often developed acute GVHD than those who did not. Consequently, we then investigated in vitro the impact of selective CD4+ CCR7+ T cell depletion on immune reactions and showed that such a depletion reduced alloreactivity without altering acquired anti-infectious reactions.In order to translate these findings to clinic, we now developed a compliant procedure for a selective reduction of the CD4+ naïve and central memory T cell subset relevant to peripheral blood stem cell (PBSC) allografts. Patients and methodsWe performed a two-step immunomagnetic depletion of CD4+ CCR7+ T cells from ten G-CSF-mobilized PBSC apheresis samples. ResultsA median of 89% (82–94%) of CD4+ CCR7+ T cells could be depleted. This allowed a marked reduction of the alloreactive immune response against allogenic dendritic cells compared with unmanipulated cells. The preservation of CD34+ cell number and the hematopoietic progenitor function were controlled. Functional tests showed that the selection procedure did not interfere with the capacity of pathogen-specific T cells to produce interferon-gamma in response to certain viral pathogens. ConclusionOur results pave the way to a feasible procedure that can be used in patients undergoing allo-hematopoietic cell transplantation and particularly for improving haploidentical transplant results by controlling GVHD, the main immune complication.

Mobilized peripheral blood stem cell apheresis via Hickman catheter in pediatric patients.

Autologous stem cell transplantation remains an integral treatment tool for certain childhood malignancies. In children, a central venous catheter is typically necessary to provide adequate flow rates for preparative apheresis. In this study, the feasibility and efficiency of collecting CD34+ cells via an indwelling Hickman catheter, preimplanted for chemotherapy, instead of placing an additional temporary central venous catheter was evaluated. Forty-eight pediatric leukaphereses for autologous hematopoietic stem cell transplantation using Spectra Optia MNC, Version 3.0 were reviewed. We compared preimplanted Hickman catheters with a temporary Shaldon catheter, inserted for apheresis. Apheresis was considered successful if a dose of 2 × 106 CD34+ peripheral blood stem cells/kg BW was achieved. In 43 (89.6%) of the 48 patients, a Hickman catheter was used for leukapheresis. Only 5 patients (10.4%) received a temporary Shaldon catheter. In both groups, apheresis was performed without apparent adverse reactions. The dose of collected CD34+ peripheral blood stem cells was 12.7 × 106 (range, 2.3-70.7 × 106 ) cells/kg BW in the Hickman group and 16.2 × 106 (range, 3.8-48.4 × 106 ) cells/kg BW in the Shaldon group, showing no statistically significant difference (p = 0.58). In both groups, the primary endpoint of a minimal CD34+ cell concentration of 2 × 106 cells/kg BW was achieved at a maximum of two leukapheresis sessions. Apheresis efficacy was further confirmed by the collection efficiency of 40.2% in the Hickman group and 27.8% in the Shaldon group (p = 0.32). These data indicate the reliable feasibility and efficacy of mobilized apheresis via an indwelling Hickman catheter. In light of this, the routine insertion of a dialysis catheter for the purpose of leukapheresis should be critically reconsidered.

Change of apheresis device decreased the incidence of severe acute graft-versus-host disease among patients after allogeneic stem cell transplantation with sibling donors.

The composition of the graft used for allogeneic hematopoietic stem cell transplantation (HSCT) is important for the treatment outcome. Different apheresis devices may yield significant differences in peripheral blood stem cell graft cellular composition. We compared stem cell grafts produced by Cobe Spectra (Cobe) and Spectra Optia (Optia) with use of the mononuclear cell (MNC) protocol, and evaluated clinical outcome parameters such as graft-versus-host disease (GvHD), transplant-related mortality (TRM), relapse, and overall survival. During 5 years, 31 Cobe Spectra and 40 Spectra Optia grafts were analyzed for CD34, CD3, CD4, CD8, CD19, and CD56 cell content. Clinical outcome parameters were correlated and compared between the two patient groups using different apheresis devices. Optia grafts contained fewer lymphocytes compared to Cobe (p < 0.001). Optia grafts had a significantly lower incidence of acute GvHD Grades II through IV (Cobe 45% vs. Optia 23%; p = 0.039) and TRM (16% vs. 2.5%; p < 0.05) but higher chronic GvHD (32% vs. 67%; p = 0.005) compared to Cobe grafts. Finally, the multivariate analysis showed a significant correlation among the different apheresis devices and both acute GvHD II through IV and severe chronic GvHD. The multivariate analysis also showed a significant correlation between the CD3+ cell dose and the incidence of severe acute GvHD. Optia-obtained grafts yielded a lower acute GvHD Grades II-IV and TRM risk, but had no impact on relapse or overall survival in this study. Understanding and further improvement of peripheral blood stem cell (PBSC) apheresis techniques may be used in the future to personalize HSCT by, for example, fine-tuning the GvHD incidence.

Comprehensive technical and patient-care optimization in the management of pediatric apheresis for peripheral blood stem cell harvesting

BackgroundPediatric apheresis for peripheral blood stem cell transplantation should be carried out with due concern for low corporeal blood volume and vulnerability to hypocalcemia-related complications, hypovolemic shock, and hypervolemic cardiac overload. Study Design And MethodsWe retrospectively investigated a total of 267 apheresis procedures from 1990 to 2013 on 93 children between 0 and 10 years old, including 89 patients and 4 healthy donors, with body weights of 6.3 to 44.0 kg. ResultsThe median CD34+ cell yield per apheresis procedure was 2.3 × 106 CD34+ cells/kg (0.2–77.9 × 106 CD34+ cells/kg). Adverse events occurred in 11.6% of procedures (n = 31), including mild perivascular pain (n = 12), emesis (n = 9), hypotension (n = 3), urticaria (n = 2), numbness (n = 2), chest pain (n = 1), facial flush (n = 1), and abdominal pain (n = 1). Among hypotensive events, shock in a 9.6 kg one-year-old boy required emergency treatment in 1996. Thereafter, we adopted continuous injection of calcium gluconate, ionized calcium monitoring, central venous catheter access and circuit priming with albumin in addition to concentrated red cells. Since then we have had fewer complications: 16.4% per apheresis during 1990–1997 versus 5.8% during 1998–2013. No healthy pediatric donors suffered from any late-onset complications related to apheresis or G-CSF administration. ConclusionBy employing appropriate measures, peripheral blood stem cell apheresis for small children can have an improved safety profile, even for children weighing <10 kg.

Abstract 1626: Evaluation of the expression of neuroblastoma-associated genes for bone marrow (BM) involvement and minimal residual disease (MRD) detection

Abstract Methods. Panel of 4 tumor-associated genes (TAGs): PHOX2B, TH, ELAVL4 and GD2 was created. Normalized expression (CtABL-CtTAG) of these genes was analyzed in neuroblastoma (NB) cell lines, in 26 BM samples from patients without malignancies, in the discovery (331 BM samples from 57 patients) and validation cohorts of NB patients (311 BM samples from 55 patients) and in 23 peripheral blood stem cells (PBSC) preparations. For performing of ROC analysis BM samples were defined as positive in case of detectable PHOX2B expression or tumor cells presence in BM smears. Threshold levels (TL) of each gene expression were established and subsequently applied for overall correct prediction (OCP) computation. Prognostic significance of the BM positivity for the evaluated markers was determined using event-free (EFS) and overall survival (OS) rates with median of follow-up time 2.45 years. Results. Neither PHOX2B nor TH expression was detected in the intact BM, while expression of ELAVL4 was revealed in 20 and GD2 - in 15 of 26 BM samples of patients without malignancies. In the discovery cohort 105 analyzed BM samples were positive for PHOX2B expression, while 101 for TH. TH expression was revealed in 5 out of 224 negative BM samples. ELAVL4 and GD2 expression was detected in all 107 positive BM samples as well as in the majority of negative BM samples (209 and 197 of 224, respectively). TL of TH, ELAVL4 and GD2 expression established by ROC analysis were -16.535, -7.130 and -8.459. OCP values calculated using TL achieved 0.952, 0.828 and 0.767 correspondingly. OCP for PHOX2B was 0.994. Due to high OCP values for PHOX2B and TH expression of these genes was analyzed in the validation cohort, where TL for TH was -13.995, while OCP values achieved 0.997 for PHOX2B and 0.939 for TH. Presence of PHOX2B/TH expression in BM at the time of primary NB diagnostics led to decreased both EFS (.31SE.12 vs .81SE.06, p&amp;lt;.001) and OS (.31SE.13 vs .87SE.05, p&amp;lt;.001). Persistence of TAGs expression during the treatment demonstrated trend to reduced EFS (.27SE.12 vs .43SE.19, p = .08). No expression of TAGs was find in the PBSC preparations while positivity for PHOX2B/TH expression before PBSC apheresis had strongly negative prognostic impact (EFS .00 vs .35SE.14, p = .04; OS .00 vs .36SE.15, p = .03) despite of CD34+ selection. Predominance of PHOX2B expression over TH in BM of primary NB patients with the difference in normalized expression greater then 1.68 had negative prognostic significance: EFS .00 vs .56SE.12, p = .017, OS .00 vs .72SE.11, p = .006. Conclusion. PHOX2B and TH are the most appropriate markers for BM involvement and MRD detection in NB patients. Presence of these TAGs expression in the BM at the time of primary diagnostics and before PBSC apheresis had strongly negative prognostic impact. Predominance of PHOX2B expression over TH in the BM can define high risk patients. Citation Format: Alexander E. Druy, Egor V. Shorikov, Grigory A. Tsaur, Alexander M. Popov, Leonid I. Saveliev, Larisa G. Fechina. Evaluation of the expression of neuroblastoma-associated genes for bone marrow (BM) involvement and minimal residual disease (MRD) detection. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1626. doi:10.1158/1538-7445.AM2015-1626

Automated CD34+ cell isolation of peripheral blood stem cell apheresis product

Background aimsImmunomagnetic enrichment of CD34+ hematopoietic “stem” cells (HSCs) using paramagnetic nanobead coupled CD34 antibody and immunomagnetic extraction with the CliniMACS plus system is the standard approach to generating T-cell-depleted stem cell grafts. Their clinical beneficence in selected indications is established. Even though CD34+ selected grafts are typically given in the context of a severely immunosuppressive conditioning with anti-thymocyte globulin or similar, the degree of T-cell depletion appears to affect clinical outcomes and thus in addition to CD34 cell recovery, the degree of T-cell depletion critically describes process quality. An automatic immunomagnetic cell processing system, CliniMACS Prodigy, including a protocol for fully automatic CD34+ cell selection from apheresis products, was recently developed. We performed a formal process validation to support submission of the protocol for CE release, a prerequisite for clinical use of Prodigy CD34+ products. MethodsGranulocyte-colony stimulating factor–mobilized healthy-donor apheresis products were subjected to CD34+ cell selection using Prodigy with clinical reagents and consumables and advanced beta versions of the CD34 selection software. Target and non-target cells were enumerated using sensitive flow cytometry platforms. ResultsNine successful clinical-scale CD34+ cell selections were performed. Beyond setup, no operator intervention was required. Prodigy recovered 74 ± 13% of target cells with a viability of 99.9 ± 0.05%. Per 5 × 10E6 CD34+ cells, which we consider a per-kilogram dose of HSCs, products contained 17 ± 3 × 10E3 T cells and 78 ± 22 × 10E3 B cells. ConclusionsThe process for CD34 selection with Prodigy is robust and labor-saving but not time-saving. Compared with clinical CD34+ selected products concurrently generated with the predecessor technology, product properties, importantly including CD34+ cell recovery and T-cell contents, were not significantly different. The automatic system is suitable for routine clinical application.

Endocan in Hypertension and Cardiovascular Disease

In their pilot study, Balta et al reported that circulating endocan (endothelial cell specific molecule 1 [ESM-1]) levels may represent a new marker of essential hypertension. They found that in 18 patients with newly diagnosed hypertension, serum endocan levels correlated significantly with carotid intimamedia thickness (cIMT) and high-sensitivity C-reactive protein (hsCRP) compared with 23 normotensive controls. Therefore, Balta et al suggested that circulating endocan levels may represent a new marker for essential hypertension. Endocan is a protein encoded by the ESM-1 gene in humans. Due to possible confusion, it must be clarified that endocan is not related to endocannabinoids that are endogenous mediators that elicit multiple biological effects similar to those of marijuana. The focus of this editorial is specifically on endocan and its CV relationships. Endocan is a recombinant proteoglycan that can be produced from engineered HEK293 cells with efficient longterm production. This availability opens new opportunities to study the structure and functions of endocan. Abid et al tested the hypothesis that endocan, as a novel soluble dermatan sulfate proteoglycan, is differentially expressed in the intact endothelium and that site-specific expression is mediated by signals in the local microenvironment. Using a combination of analytical techniques, they reported that endocan is preferentially expressed in the endothelial lining of tumor xenografts, including human nonsmall cell lung cancer, rat glioma, and human renal cell carcinoma. They found that in vitro expression of endocan in human umbilical vein endothelial cells (HUVECs) was upregulated by a tumor cell-conditioned medium and that this was inhibited by the addition of neutralizing antibody to vascular endothelial growth factor (VEGF). The authors also found that treatment of HUVEC with VEGF resulted in a doseand time-dependent increase in messenger RNA (mRNA) in endocan. It therefore appeared to them that endocan is preferentially expressed in tumor endothelium in vivo with its expression regulated by tumor-derived factors. Akkok et al reported that peripheral blood stem cell (PBSC) collection in patients with multiple myeloma using PBSC apheresis decreased the angioregulatory cytokines, angiopoietin 22 and VEGF, which had been found to be increased in the patients with myeloma, while at the same time, PBSC apheresis resulted in an increase in endocan levels as a microvascular endothelial marker. Rennel et al appeared to show that among angiogenic growth factors, VEGF-A is a specific inducer of endocan transcription. This translates into increased protein levels in VEGF-A-treated endothelial cells. Rennel et al suggested that increased endocan protein expression in human renal cancer supports its role in tumor growth. Similar supportive data are available for colorectal cancer. When colorectal cancer is present, the expression of endocan is downregulated. However, endocan is positively correlated with tissue differentiation in colorectal cancer, which suggests that endocan is associated with the development and differentiation of colorectal cancer. Zhang et al studied endocan as a novel human endothelial cell-specific molecule whose expression is regulated by cytokines and VEGF. They found that endocan was expressed in actively proliferative or neogenic tissues and cells such as the germinal centers of lymph nodes, bronchial epithelium, neovasculature, endothelium, and glandular tissues. Endocan was not present in silent or resting tissues of cells such as the endothelium of the spleen and large arteries. Zhang et al considered that endocan may function as a marker for angiogenesis or oncogenesis and that the gene for endocan might be a candidate gene for the development of inflammatory tissue, metastases, neoplasia, and the development of tumors such that the expression level of endocan may be of value in early diagnosis and prognosis of some tumors. In line with this, Roudnicky et al found that both endocan and VEGF-A levels were more elevated in the plasma of patients with invasive bladder cancer than in healthy individuals. They therefore postulated that endocan might function as a useful biomarker for monitoring disease progression and the efficacy of therapies to target VEGF-A in patients with bladder cancer. Endocan has also been found to be overexpressed in renal clear cell carcinomas and as a result it may be a target marker for follow-up of these cancers and a potential way to evaluate their response to antiangiogenic therapies. Serum endocan has