Collection, cryopreservation and thawing of stem cells for children weighing less than 25 Kg with high-risk neuroblastoma: A single center results in Morocco

ABSTRACT Introduction: An important component of the advances made in neuroblastoma treatment has been the use of peripheral blood stem cells to support high-dose chemotherapy. In this study, we report our experience on a series of small children who have undergone standard and large volume leukaphersis (LVL) procedures, provide an update on a single institution's experience with cryopreservation of autologous peripheral blood stem cells (PBSCs), using 10% dimethyl sulfoxide (DMSO) and applying post-thaw DMSO depletion and analyze a number of variables that may affect viability. Methods: A total of 36 aphereses were performed on 29 children weighing less than 25 kg between July 2016 and October 2019 at the Ibn Sina university hospital. Results: Seven females and twenty-two males, median bodyweight 14 kg (9 - 22). A single apheresis was sufficient to obtain at least 3 × 106/kg body weight (BW) of CD34+ cells in 82.8% of the cases. The LVL was performed in 22 aphereses. A median number of 5.9 × 106/ kg CD34 cells were collected per apheresis. A total of 60 PBSC samples were cryopreserved and 46 samples were infused. The mean cell viability percentage decreased from 94.75 ± 1.14% before freezing to 70.84 ± 8.6% after thawing (p < 0.001). No correlation was found between post-thaw viability and storage time (r = -0.233; p = 0.234) or number of total nucleated cells (r = 0.344; p = 0.073). Conclusion: Leukapheresis is safe and feasible in small pediatric patients if the appropriate measures are used. Cryopreservation poses numerous challenges, especially a decrease in cell viability after thawing.

Prospective identification of potential factors influencing stem cell mobilization and the necessity for plerixafor use in newly diagnosed multiple myeloma patients undergoing autologous stem cell transplantation

ABSTRACT Introduction: To study the efficacy and safety of single large volume leukapheresis by using generic G-CSF or G-CSF plus Plerixafor in achieving adequate stem cell yield and various factors influencing thereof in newly diagnosed multiple myeloma patients undergoing autologous stem cell transplant . Method: This prospective study was undertaken among 55 newly diagnosed multiple myeloma patients undergoing autologous stem cell transplant and aged between 18 and 75 years. Mobilization and harvesting of stem cells were performed by using GCSF or GCSF plus Plerixafor and large volume leukapheresis, respectively. A stem cell yield of ≥2 × 106 kg-1 and the number of apheresis procedures were primary efficacy endpoints, while the ideal stem cells yield >5 × 106 kg-1, the engraftment day and D100 response/graft sustainability were secondary endpoints. Result: The primary endpoint was achieved in all cases in both the groups by using a single LVL leukapheresis procedure. Fulfillment of all the secondary endpoints was satisfactory and comparable in both the groups. Age, pre-apheresis CD34+ count and number of interruptions during the LVL were significant factors influencing the stem cell yield (p < 0.05). Adverse drug reactions during the apheresis and post-ASCT period were manageable. Conclusion: The LVL is safe and cost-effective in attaining a minimum of CD34+ cells in a single procedure with manageable adverse reactions. Judicious intervention during the procedure may be helpful in ensuring the adequate yield.

Usefulness of Calcium Injection in Large-volume Leukapheresis / 대한수혈학회지

BACKGROUND: Hypocalcemia is the most common side effect in large-volume leukapheresis (LVL) for collection of peripheral blood stem cells (PBSCs). We evaluated the effect of intravenous calcium infusion on the hypocalcemic symptoms during LVL. METHODS: Seventy-six LVLs with hypocalcemic symptoms were participated in this study. LVLs were performed using Gambro Spectra (blood rate, 80 mL/min; whole blood to ACD-A with heparin, 24:1) until 20 L of blood was processed. Blood flow rate was slowed to 60-70% of initial in 35 LVLs (group A) and 20 mL of 3% CaCl2 was infused intravenously in 41 LVLs (group B). Serum levels of ionized calcium, total magnesium, and electrolytes were determined before and after each LVL and analyzed with students?-test. RESULTS: The observed hypocalcemic symptoms were perioral paresthesias (71%), digital numbness (17%), chest tightness (4%), chills (4%), headache (3%), and vomiting (1%). Serum ionized calcium decreased by 15.9% in group A (P<0.001) and increased by 1.3% in group B. Total magnesium in group A (11.4%, P=0.002) and potassium in both groups (9.5%, P<0.001 and 8.6%, P=0.02, respectively) were significantly reduced. In group A, hypocalcemic symptoms were not completely relieved but weakened in 63%, and blood flow rate had to be kept below 55 mL/min in 37%. In group B, the symptoms were relieved by 20 mL of 3% CaCl2 in 27% and by 40 mL in 73%. CONCLUSION: Reduction of blood flow for relief of hypocalcemia symptoms was only helpful in lightening the symptoms. Intravenous infusion of calcium was more effective and convenient to alleviate hypocalcemic symptoms and did not affect LVL procedures nor serum levels of ionized calcium.

Allogeneic Peripheral Blood Stem Cell Transplantation from An HLA-identical Sibling Donor in Children / 대한소아혈액종양학회지

PURPOSE: G-CSF-mobilized peripheral blood is one of the sources of allogeneic hematopoietic stem cells. We report our experiences on allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from an HLA-identical sibling donor in children. METHODS: From August 1998 to January 2003, 8 patients underwent allo-PBSCT. Median age of recipients and donors were 4 yr 10 mo (range 3 yr 2 mo 15 yr) and 5 yr 1 mo (range 1 yr 11 mo 19 yr 8 mo), respectively. Seven patients (3 ALL, 2 neuroblastomas, 1 AML, 1 Gaucher disease) had failed from previous allogeneic or autologous transplant and 1 patient had refractory acute biphenotypic leukemia. Only 2 patients were in complete remission at allo-PBSCT. G-CSF 10mug/kg/day was injected subcutaneously to the donor for 5 days and large volume leukapheresis was performed on 4th and 5th days. RESULTS: Median number of CD34 and CD3 cells infused was 18.55 106 (range 9.47 84.76) /kg and 8.26 108 (range 0.88 24.58) /kg, respectively. All patients achieved ANC > 0.5 109/L after a median of 9 days and 6 patients eventually achieved platelet engraftment. There was no grade II-IV acute GVHD but limited chronic GVHD developed in 6 evaluable patients. There was no CMV antigenemia. Three patients died from either transplant-related mortality (n=2) or relapse (n=1). The remaining 5 patients are alive disease-free for 7, 8, 15, 16, and 19 months from allo-PBSCT, respectively. CONCLUSION: Our results suggest that mega-dose allo-PBSCT from an HLA-identical sibling donor is expected to improve transplant outcome especially in very high risk pediatric patients.

The Efficacy and Safety of Large Volume Leukapheresis in Allogeneic Peripheral Blood Stem Cells Collection / 대한수혈학회지

BACKGROUND: Transplantation of allogeneic peripheral blood stem cells (PBSCs) may have advantage over bone marrow transplantation with regards to the speed of hematopoietic and immunologic recovery. Recently to overcome the need for multiple leukaphereses to collect enough PBSCs for autologous transplantation, large volume leukaphereses (LVL) are used to process multiple blood volumes per session. Experience with this technique in healthy individuals after mobilization with colony stimulating factor (CSF) is limited. We have investigated the efficacy and safety of LVL for the collection of G-CSF and GM-CSF mobilized PBSCs from healthy donors. METHODS: This study was done on 40 healthy donors who were mobilized with G-CSF and GM-CSF for allogeneic peripheral blood stem cells transplantation (allo-PBSCT). After 5 days of mobilization treatment, PBSCs were collected by LVL with Fenwal CS-3000 Plus (Baxter Co, USA). In LVL, heparin was administered in addition to ACD-A. Patients underwent of LVL procedures daily to obtain a target cell dose of >or= 4x10(8)/kg MNCs and >or= 6x10(6)/kg CD34+ cells. RESULTS: 66 LVL procedures were done on 40 donors. Of these donors, 31 (77.5%) reached the collection target with one leukapheresis. The product per LVL contained a mean 5.79+/-2.47 10(8)MNCs/kg and 11.6+/-10.62x10(6) CD34+ cells/kg respectively. Mean percentages of MNC were 79.88+/-22.15% and collection efficiencies of MNCs were inversely related to the starting MNC count (r=-0.536, P<0.001). After LVL, although none of the donors exhibited bleeding complications, platelets decreased from 187.4+/-52.68x10(3)/microL to 74.88+/-13.7x10(3)/microL and activated partial thromboplastin time (APTT) prolonged from 29.13+/-3.77 seconds to 67.51+/-54.26 seconds. CONCLUSION: We conclude that LVL after mobilization treatment with G-CSF and GM-CSF in normal healthy donors was tolerable and efficient methods for PBSCs collection, but long-term risk of adverse effects in normal donors needs to be carefully addressed by individual institutions as well as national and international registries.

Peripheral Blood Stem Cell Collections in Childhood Tumor Patients Weighing Less than 25 kg Using Large Volume Leukapheresis / 대한소아혈액종양학회지

BACKGROUND: Peripheral blood stem cell (PBSC) transplantation has been widely used to support the hematopoietic recovery after high dose chemotherapy in patients with advanced malignancies. The procedure of PBSC collection in pediatric patients is similar to that in adults, but needs the 'fine tuning' of the volume shift of each procedure and the technical factors to achieve specific target goals. This article provides our experience with fourteen collections of PBSC from five patients less than 25 kg in weight. METHODS: Patient's diagnoses were 2 stage IV neuroblastoma, 1 non-Hodgkin's lymphoma, 1 stage IV Ewing sarcoma, and 1 stage IV rhabdoid tumor of kidney. Their age ranged from 2 to 7 years old. Collections were performed using COBE Spectra or CS3000 plus that had been primed with leukoreduced, irradiated red blood cells. Patients underwent large volume leukapheresis. Radial artery was used as draw line and subclavian vein was used as return line. The blood to ACD ratio was 24:1 with 3000 units of heparin added to each 500 mL of ACD, in addition, heparin (1000 units) was added to collection bag when performed with COBE Spectra. Simultaneously, calcium chloride solution was dripped into an another venous line. During one course of large volume leukapheresis, about 5,000 mL of blood (>three total blood volume) were processed at a flow rate of 25~35 mL/min. RESULTS: The mean of total WBCs in collected components per procedure was 5.9+/-2.9x109 (3.0-10.5x109) with yield of 3.6+/-2.0x108 per kg of body weight. The mean of total CD34+ cells was 5.2+/-4.5x106 per kg (1.6-14.6x106/kg) for each collection. The patients tolerated well during the procedure without any apparent symptoms related to anemia or volume deficit or overload. CONCLUSION: In children weighing less than 25 kg, peripheral blood progenitor cell collection can be safely and efficiently performed with continuous flow blood cell separators, primed with red cells and additional heparin anticoagulation.

Large-Volume Leukapheresis for Collection of Peripheral Blood Stem Cells: A Comparison of Two Continous Flow Cell Separators / 대한혈액학회지

BACKGROUND: Mobilized peripheral blood stem cells (PBSCs) are now used increasingly in patients with hematologic and solid tumors to reconstitute hematopoiesis after dose-intensive chemotherapy. We evaluated the efficacy of large-volume leukapheresis (LVL) and compared the ability of Fenwal CS3000 Plus and Cobe Spectra to collect mononuclear cells (MNCs) for PBSCT. METHODS: Twenty liters of whole blood per LVL were processed in 22 patients with acute leukemia and lymphoma. LVL were performed in rapid recovery phase (white cells >3x109/L, or CD34+ cells > 1% of white cells) after chemotherapy followed by granulocyte-colony stimulating factor. The end point of LVL was mononuclear cells (MNCs) >8x108/kg, or CD34+ cells > 6x106/kg. A-35 collection chamber was used in Fenwal CS3000 Plus and whole blood flow was set at 85mL/min, whole blood to anticoagulant ratio 11~13:1, interface offset 150. MNC procedure was used in Cobe Spectra, whole blood flow was 90~100mL/min, whole blood to anticoagulant ratio 24:1 with heparin to anticoagulant and product bags, collection rate 1mL/min, and hematocrit 2~3%. RESULTS: Total 53 LVL (35 with Fenwal CS3000 Plus and 18 with Cobe Spectra) were performed on 22 patients. An average of 2.4 LVL per patient (range 1~4) were performed. With Fenwal CS3000 Plus, post-LVL values of hematocrit, platelets and MNCs were reduced by 12.4%, 53.1%, and 33.0% and with Cobe Spectra, 9.2%, 36.1%, and 39.6%, respectively. Mean collection volume of Fenwal CS3000 Plus and Cobe Spectra were 135.7mL and 175.2mL per LVL, respectively. There was no statistical significant difference in the yields of LVL between Fenwal CS3000 Plus (3.4+/-1.9x108/kg MNCs, 7.2+/-11.2x106/kg CD34+ cells) and Spectra (4.7+/-2.1x108/kg MNCs, 7.4+/-9.6x106/kg CD34+ cells). The yields of LVL were correlated well with patients' pre-MNC counts in both cell separators. Mean percentages of MNC were 95.4% with Fenwal CS3000 Plus and 74.0% with Cobe Spectra (P 0.05). LVL product with Cobe Spectra contained less red cells (10.5+/-2.7mL) than Fenwal CS3000 Plus (34.1+/-10.8mL) (P<0.001). Platelet contamination was not different for Fenwal CS3000 Plus (2.3+/-2.1x1011) and Cobe Spectra (3.1+/-1.0x1011). CONCLUSION: LVL could be conveniently used for PBSC collection with good collection efficiency and safety without serious citrate toxicities. LVL products with Fenwal CS3000 Plus showed less collection volume and granulocyte contamination. The products with Cobe Spectra showed less red cell contamination and less decrease in patients' platelet counts.