An innovation in stem cell harvesting: Heparin use.

BACKGROUND AND OBJECTIVES: Stem cell transplantation is a growing treatment strategy for most malignant and non- malignant hematological diseases. Plerixafor and granulocyte colony stimulating factor (G-CSF) are usually used in mobilization regimens to increase the CD34+ cell count in the harvest. Heparin is a sulphated glycosaminoglycated polymer with 12-15 kDa mass. Heparin inhibits the CXCR4/SDF1 axis, as does plerixafor. In this study, our aim was to investigate the effect of using heparin on stem cell mobilization and harvesting. MATERIALS AND METHODS: We administered 5000 units of unfractioned heparin intravenously in 150 mL (mL) of isotonic sodium chloride solution, 15 min before the stem cell harvesting procedure to 141 patients who underwent bone marrow transplantation between the years of 2018 and 2019 at our Stem Cell Transplantation Unit. Thirty patients were included as a control group, and they were not given heparin. The study population included patients with multiple myeloma and lymphoma equally in each group. RESULTS: In all patients hematopoeitic stem cells were successfully harvested in a single cycle of apheresis. In multiple myeloma patients who received heparin, the mean collected CD34+ cell number was 8 × 106/kg, and the mean CD34+ cell number yield was 12,555/µl. In the control group, the mean collected CD34+ cell number was 4,2 × 106/kg, and mean CD34+ cell number in yield was 492/µl. In lymphoma patients who received heparin, the mean collected CD34+ cell number was 6,8 × 106/kg, and the mean CD34+ cell number was 1421/µl. In the control group the mean collected CD34+ cell number was 4,3 × 106/kg, and the mean CD34+ cell number was 358/µl. The effect of heparin on the collected stem cell number in both myeloma and lymphoma patients was statistically significant (p < 0.01). CONCLUSIONS: Our results have shown that heparin increases harvested stem cell numbers significantly. Heparin may be a promising agent for stem cell harvesting.

Plerixafor added to G-CSF allows mobilization of a sufficient number of hematopoietic progenitors without impacting the efficacy of TCR-alpha/beta depletion in pediatric haploidentical and genoidentical donors failing to mobilize with G-CSF alone.

BACKGROUND: Collection of a large number of early hematopoietic progenitors is essential for allogeneic apheresis products intended for TCR-alpha/beta depletion. MATERIALS AND METHODS: We added plerixafor 0.24 mg/kg body weight (bw) on day 4 of high-dose filgrastim mobilization 10 hours prior to apheresis in 16 (30.5%) pediatric allogeneic donors who failed to recover a sufficient number of CD34+ cells. RESULTS: On day 4 of G-CSF, the median CD34+ cell count in peripheral blood was 6 per µL (range 4-9 per µL) in 6 poor mobilizers and 16 per µL (range 12-19 per µL) in insufficient mobilizers. In all donors, the threshold of 50 CD34+ cells/µL was achieved, and the median increase was 14.8-fold in poor mobilizers and 6.5-fold in insufficient mobilizers, whereas it was 3.45-fold increase in those mobilized with G-CSF alone. DISCUSSION: In all donors, a predefined number of >10 × 106 CD34+ cells/kg of recipient bw before depletion was reached in the apheresis product. The use of plerixafor did not affect the purity of further TCR-alpha/beta depletion. Side effects were mild to moderate and consisted of nausea and vomiting. CONCLUSION: Thus, the safety and high efficacy of plerixafor was proven in healthy pediatric allogeneic hematopoietic cell donors.

Comparison of spectra optia and amicus cell separators for autologous peripheral blood stem cell collection.

INTRODUCTION: Autologous peripheral blood stem cell (PBSC) transplantation has become a standard treatment option for many oncology patients. The aim of this study was to evaluate the performance of two cell separators, Spectra Optia (Terumo BCT, Japan) and Amicus (Fresenius-Kabi) for autologous PBSC collection. METHODS: We retrospectively evaluated 56 apheresis by Spectra Optia with Continuous Mononuclear Cell Collection (cMNC) from 20 patients, and 50 apheresis by Amicus from 27 patients between December 2018 and December 2019. CD34+ collection efficiency (CE2) and platelet (PLT) loss were evaluated. RESULTS: There was no significant difference in CD34+ CE2 between Spectra Optia with cMNC (median, 28.8%) and Amicus (median, 33.1%; P = 0.537). PLT loss was significantly lower in Amicus (median, 28.6%) than in Spectra Optia with cMNC (median, 37.8%; P = 0.009). CONCLUSION: CD34+ CE2 was comparable between Spectra Optia and Amicus, and PLT loss was significantly lower in Amicus. To the best of our knowledge, this is the first report comparing autologous PBSC collection of the Spectra Optia and Amicus. These results may provide general guidance with regard to device selection to apheresis clinics that use both separators for optimal outcomes depending on each patient's characteristics.

Evaluation of the predictive value of the hematopoietic progenitor cell count using an automated hematology analyzer for CD34+ stem cell mobilization and apheresis product yield.

INTRODUCTION: We evaluated the value of hematopoietic progenitor cells (HPCs) counted in Sysmex XN analyzers to predict the mobilization and collection of CD34+ cells in apheresis for stem cell transplantation. METHODS: Eighty patients who underwent stem cell transplantation were enrolled (50 autologous and 30 allogeneic). In the autologous group, patients were considered poor mobilizers when the CD34+ count was <10 × 106 /L or <20 × 106 /L in patients with multiple myeloma who were going to undergo two transplants. ROC curves were generated, and HPC cutoffs were calculated. RESULTS: The correlation between the HPC and CD34+ cell counts was good. Two algorithms were proposed. In the first algorithm, samples collected the day before apheresis, negative and positive HPC cutoffs were selected to detect poor and good mobilization and, therefore, the need or not to administer plerixafor. In the second algorithm, samples collected pre-apheresis, the negative HPC cutoff was an indication to delay apheresis; an HPC higher than the optimal cutoff was an indication to start apheresis. When the HPC values were between these cutoffs, there was an indication to count CD34+ cells for a better decision-making. Finally, in samples collected pre-apheresis, HPC counts could be used to predict patients who would have poor CD34+ cell collections. In the allogeneic group, all the donors mobilized well, and very few needed two apheresis procedures. CONCLUSIONS: The HPC count is useful for decision-making in the management of patients subjected to apheresis procedures to collect peripheral blood stem cells.

Collection of hematopoietic CD34 stem cells in rhesus macaques using Spectra Optia.

BACKGROUND: Nonhuman primates, particularly rhesus macaques, are ideal preclinical large animal models to investigate organ tolerance induction protocols using donor hematopoietic stem cells (HSCs) to induce chimerism. Their relatively small size poses some challenges for the safe and effective collection of peripheral blood HSCs through apheresis procedures. We describe our experiences using the Spectra Optia apheresis unit to successfully obtain HSCs from mobilized peripheral blood of rhesus macaques. METHOD: Mobilization of peripheral blood HSCs was induced using granulocyte stimulating factor (G-CSF) and Mozobil. The Spectra Optia unit was used in 18 apheresis procedures in 13 animals (4.9-10 kg). Animal health was carefully monitored during and after the procedure. Changes in peripheral blood cells before, during and after procedure were determined by complete blood count and flow cytometry. RESULTS: The automatic settings of the Spectra Optia unit were applied successfully to the procedures on the rhesus macaque. All animals tolerated the procedure well with no mortality. Mobilization of HSCs were most consistently achieved using 50 µg/kg of G-CSF for 5 days and a single dose of Mozobil on the 5th day, followed by collection of cells 3 h after Mozobil injection. The final apheresis product contained an average of 23 billion total nucleated cells with 47% granulocytes, 3,871 million total CD3 cells and 77 million CD34 cells which resulted in an average of 10 million CD34+ cells/kg of donor weight. CONCLUSION: Apheresis of peripheral blood mobilized HSCs in rhesus macaques using Spectra Optia is a safe and effective procedure.

Monitoring blood for CD34+ cells to determine timing of Hematopoietic Progenitor Cells Apheresis.

Hematopoietic Progenitor Cell (HPC) Apheresis generally results in a mononuclear cell product that is highly enriched for hematopoietic stem and progenitor cells when performed on autologous patients in whom autologous stem cell transplant is planned who have been mobilized with cytotoxic chemotherapy and exogenous hematopoietic growth factors (cytokines) and possibly CXCR4 antagonists. Alternatively, patients scheduled for autologous transplants may be mobilized with cytokines only or a combination of cytokines and CXCR4 antagonists. Allogeneic Donors, either matched related donors (MRD) or matched unrelated donors (MUD), are typically mobilized with cytokines only. The HPC Apheresis product, enriched for hematopoietic progenitor cells collected from the patient/donor's peripheral blood via an apheresis system, is used for restoring hematopoiesis in the patient/recipient who has received myeloablative therapy. Timing of the collection of an HPC Apheresis product from allogeneic donors is based on the schedule of the recipient's myeloablative regime. However, the optimal timing of collection on HPC Apheresis product from a patient scheduled for an autologous stem cell transplant can be complex.

Use of the haematopoietic progenitor cell parameter in optimizing timing of peripheral blood stem cell harvest.

BACKGROUND AND OBJECTIVES: Timing of peripheral blood stem cell (PBSC) harvest is typically based on quantification of peripheral blood (PB) CD34+ cells. CD34 enumeration is expensive, requires expertise and takes a minimum of 1-2 h to perform. The Sysmex XE2100 is an automated haematology analyser that can rapidly and inexpensively identify haematopoietic progenitor cell (HPC) populations in PB. The aim of this study was to examine if HPC can be used to optimize timing of PBSC harvest. MATERIALS AND METHODS: White blood cell (WBC), HPC and CD34 counts were determined in a total of 60 mobilized donors. Data were analysed to examine the utility of WBC and HPC counts in predicting preharvest CD34+ counts. RESULTS: In adults presenting for autologous collection, a PB HPC threshold of > 30/microl predicts a preharvest CD34+ count of > 20/microl with sensitivity of 86% and positive predictive value (PPV) of 100%. Among paediatric patients with a diagnosis of neuroblastoma, an HPC threshold of > 16/microl yielded sensitivity and PPV of 100%, while in children with other diagnoses, an HPC cut-off of > 44/microl yielded sensitivity and PPV of 67% and 100%, respectively. Eighty per cent of adequately mobilized allogeneic donors were identified using an HPC threshold > 15/microl, with a PPV of 100%. PB WBC can also aid in predicting CD34 counts in most patient groups, albeit with lower sensitivity than HPC. CONCLUSION: By virtue of being a sensitive and accurate predictor of preharvest CD34+ counts, our data support the use of the HPC parameter in optimizing the timing of PBSC harvest.

Phase II study of a single pegfilgrastim injection as an adjunct to chemotherapy to mobilize stem cells into the peripheral blood of pretreated lymphoma patients.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate the efficacy of pegfilgrastim, in combination with salvage chemotherapy, in mobilizing CD34(+) stem cells into the peripheral blood of pretreated lymphoma patients. DESIGN AND METHODS: This was an open-label phase II study including 25 pretreated patients (Hodgkin's disease=4; aggressive non-Hodgkin's lymphoma=21). The primary end-point of the study was the successful mobilization of a target cell dose of 2x10(6) CD34(+) cells/kg in lymphoma patients receiving ifosfamide, epirubicin and etoposide (IEV) chemotherapy and a fixed dose (6 mg) of pegfilgrastim given as single subcutaneous injection. RESULTS: Following chemotherapy, all patients had grade 4 neutropenia that lasted a median of 1.5 days (1-3). Pegfilgrastim treatment was well tolerated and only 2/25 patients required pain-control medication. CD34+ cells were mobilized in all patients. The median (range) peak value of peripheral blood CD34+ cells after IEV chemotherapy and pegfilgrastim was 141x10(6)/L (12.8-386) and occurred almost invariably on day +14 (13-16). Twenty-three of the 25 patients underwent a single standard volume leukapheresis to collect a median of 8.7x10(6) CD34(+) cells/kg (1.78-17.3). Twenty four/25 patients (96%) reached the target cell dose of 2x10(6) CD34(+) cells/kg. High concentrations of circulating CD34+ cells (> 50x10(6)/L) were observed for several days after the achievement of the peak value. All the study patients were transplanted with their pegfilgrastim-mobilized CD34(+) cells and showed a rapid and sustained engraftment after high-dose chemotherapy. INTERPRETATION AND CONCLUSIONS: Our results show that pegfilgrastim as an adjunct to chemotherapy is a predictable and highly effective mobilization regimen in pretreated lymphoma patients.

Comparison of two different methods for CD34+ selection and T cell depletion in peripheral blood stem cell grafts--our experiences with CellPro, E rosetting and CliniMACS technique.

The aim of this study was to establish a suitable method for in vitro T cell depletion in peripheral blood stem cell grafts for mismatched/haploidentical transplantation in children and adults with severe hematological disorders and for autologous transplantation in patients with autoimmune diseases refractory to conventional immunosuppressive treatment. Two different selection techniques have been used: CD34+ selection using immunoaffinity columns (CellPro Ceprate) followed by T cell depletion by E-rosetting or CD34+ selection using submicroscopic paramagnetic beads (CliniMACS device) with T cell depletion in a one step procedure. The mean purity and recovery of CD34+ cells and efficiency of T cell removal in the final product were compared. From March 1995 to December 1998 we prepared twelve allografts using Cell Pro system for eight children with high-risk hematological malignancies and six autografts for six patients with severe autoimmune diseases. From January 1999 to October 2000 we prepared fifteen allografts using CliniMACS system for ten children with high-risk hematological diseases and inborn metabolic disorders or primary immunodeficiences, five allografts for three adult patients with high-risk hematological malignancies and two autografts for two patients with autoimmune diseases. In allogeneic transplantation the median purity of CD34+ cells in the final products after CellPro and E-rosetting was 85.6% (55.3%-95.7%); median recovery was 24.8% (17%-35%), median transplanted doses of T cells per kilogram of body weight were 0.66x10(4) (0-2.8); in autologous transplantation the median purity of CD34+ was 92.6% (55.6%-96%), median recovery was 28% (22%-46.2%), median transplanted doses of T cells per kilogram of body weight were 0.39x10(4) (0.0-3.6). After CliniMACS technique the median purity of CD34+ cells was 94.87% (69.15%-99%),medianrecoverywas 58% (30%-79.6%), median transplanted doses of T cells per kg of body weight were 0.254x10(4) (0-14.15); in autologous transplantation the median purity of CD34+ was 94% (94%-94%, median recovery was 97.4% (95%-99.8%), median transplanted doses of T cells per kilogram of body weight were 0.87x10(4) (0.49-1.24). We consider both methods of CD34+ selection and T cell depletion suitable for peripheral blood stem cell processing before mismatched hemopoietic stem cell transplantation in patients without identical donor or before autologous transplantation for severe autoimmune diseases. However, magnetic separation using CliniMACS system results in higher levels of purity and recovery with efficient T cell depletion.

Femoral catheters: safety and efficacy in peripheral blood stem cell collection.

Central venous access is necessary in patients candidate for peripheral blood stem cell (PBSC) collection. We report our experience with a dual lumen femoral catheter (Gamcath, 11 french), initially designed for hemodialysis. We studied 147 patients and performed 488 collections after mobilization with either G-CSF alone or chemotherapy + G-CSF, when the white blood cell count exceeded 1 x 10(9)/L, or when a measurable population of CD34+ cells (20/microL) was detected in peripheral blood. All patients received systemic anticoagulation with a low weight heparin and ultrasound examination was performed after the removal of the catheter. Seven patients developed thrombosis (4.7%), ten experienced hematomas at the site of catheter placement (6.8%) despite prophylactic platelet transfusions, while only one patient (0.6%) had a catheter-related infection. In conclusion, the short-term use of large bore femoral catheters in setting up PBSC collection seems to be associated with minimal risk of infection and low thrombotic incidence.

Isolation of highly purified autologous and allogeneic peripheral CD34+ cells using the CliniMACS device.

The CliniMACS CD34+ selection device was used for positive selection of apheresis products for autologous transplantation from 10 patients with malignant diseases and for allogeneic transplantation from 26 healthy donors. A total of 71 separations were performed. In 1 allogeneic donor, CD34+ progenitors were also isolated from bone marrow. Between 0.27 and 8.9 x 10(10) nucleated cells (median 4.9 x 10(10)) containing 0.09%-10.8% (median 0.67%) CD34+ progenitor cells were separated. After separation, a median number of 227 x 10(6) mononuclear cells (MNC) (51-524) were recovered, with a median viability of 99% (22%-100%) and a median purity of 97.0% (68.3%-99.7%) CD34+ cells. Depletion of T cells was extensive, with a median of 0.04% residual CD3+ cells (range <0.01%-0.92%). Residual CD19+ cells were between <0.01% and 17%, including CD34+CD19+ cells. Recovery of CD34+ cells was calculated according to the ISHAGE guidelines and ranged from 24% to 105% (median 71%). We conclude that with the CliniMACS device CD34+ cells with high purity and recovery can be isolated with concomitant effective T cell depletion in the allogeneic setting and with a high purging efficacy in the autologous setting.

Large scale purification of human blood CD34(+) cells using a nylon-fiber syringe system and immunomagnetic microspheres.

BACKGROUND: To establish an available, economical technique for the large-scale purification of CD34(+) cells we used a nylon-fiber syringe (NF-S) for depletion of adherent cells and then selected CD 34(+) cells from peripheral blood mobilized by G-CSF, using MAb and magnetic heads. METHODS: PBSC were mobilized and collected from adult, healthy volunteers. With the effect of concentration of anti-CD34 MAb (9C5) on the purification of CD34(+) cells from 1 2 10(8) NF-S treated cells (NF cells), the recovery and the purity of CD34(+) cells was identical for 5, 10, 20, 40 microg of 9C5. In this study, therefore, the concentration of 9C5 was maintained at >5 microg/10(8) NF cells, with a cellhead ratio of 1:10. RESULTS: When half to one-third of leukapheresis product containing 121.5 + or - 26.0 2 10(8) mononuclear cells (mean + or - SD; n = 6) was processed for CD34(+) cell purification, 5.26 + or - 3.01 2 10(7) total purified cells were obtained with a purity of CD34(+) cells at 94.9 - 8.5%. The numbers of CD34(+) cells and total progenitor cells recovered in this process were 4.71 + or - 2.33 2 10(7) cells and 145.9 + or - 121.8 2 10(5) cells, respectively. The total recovery of CD34(+) cells was 37.0 + or - 21.0%. The depletion of monocyte by NF-S reduced the 9C5 anti-human CD34 MAb needed for purification of CD34(+) cells to one-third. Two patients were grafted with peripheral blood CD34(+) cells selected by this procedure and achieved a rapid and consistent hematopoiesis. DISCUSSION: Our clinical data show that these cells are capable of rapid reconstitution of hematopoiesis after high-dose chemotherapy. The procedure contains an additional step; however, consistent high purity of CD34(+) cells in the purified population and cost reduction were achieved, both critical issues in the better purging of malignant cells and for a wide clinical application.

CD34+ cell selection from frozen cord blood products using the Isolex 300i and CliniMACS CD34 selection devices.

Ex vivo expansion of cord blood (CB) cells requires CD34+ cell selection before expansion to obtain optimal numbers of progenitor cells. As a preliminary step to preclinical development of CB expansion, we have evaluated two clinical scale selection devices, the Isolex 300i (Baxter Healthcare, Immunotherapy Division) and the CliniMACS (Miltenyi Biotech Inc.), for CD34+ cell selection from frozen CB products. As expansion of CB results in differentiation of cells, there may be a depletion of stem cells. Therefore, only a fraction of the CB should be expanded while a portion of the CB is maintained unmanipulated for infusion. After thawing of 40% fractions of each CB product, we observed >95% viable cells, with a median total WBC count of 1.8 x 10(8) cells. Use of the Isolex 300i resulted in a median purity of 51% CD34+ cells (n=8) and a median recovery of 34% CD34+ cells. Use of the CliniMACS resulted in a median purity of 54% CD34+ cells (n=10) and a median recovery of 80% CD34+ cells. The absolute number of CD34+ cells recovered after selection varied with samples from 6.7 x 10(4) to 3.2 x 10(6) CD34+ cells. Expansion of CD34+ cells from both systems resulted in >20-fold expansion of CFU-GM, with a median of 44-fold expansion. These data demonstrate the feasibility of selecting small fractions of frozen CB products using clinical scale CD34+ cell selection devices.

Comparison of performance of six different cell separators in collecting peripheral blood mononuclear cells.

We compared the efficacy of six different cell separators in collecting peripheral mononuclear cells to be used for autologous or homologous peripheral stem cell transplantation. The product obtained with the Dideco Vivacell cell separator showed a low percentage of mononuclear cells (38%) in the final product and a high platelet efficiency (38%). The Baxter CS3000 Plus cell separator required the longest time to load and prime the kit (18 min), it showed a high MNC efficiency (68%), with the highest percentage of MNC in the final product, the highest platelet efficiency (45%), a low red blood cell contamination in the final product (2.7 mL), the highest extracorporeal volume (450 mL) and a high percentage of technical failures (15%). The product obtained with the Fresenius AS104 cell separator with P1Y kit showed the highest final volume (297 mL), the lowest platelet efficiency (12%) and the lowest extracorporeal volume (230 mL). The same cell separator with C4Y kit showed a lower MNC efficiency (52 vs 60%) and a higher percentage of MNC in final product (63 vs 41%). The platelet contamination in final product was the lowest (18 x 10(9)/100 mL). The Haemonetics MCS3p cell separator required the lowest time to load and prime the kit (5 min), it showed the highest MNC efficiency (71%). The blood volume processed per hour (1328 mL) and the percentage of MNC in final product was lowest (32%), the extracorporeal volume (450 mL) was the highest. The Cobe Spectra cell separator allowed to process the highest blood volume per hour (3383 mL) and the final product had the lowest red blood cell contamination (2.3 mL/100 mL). The Dideco Excel cell separator required the longest time to load and prime the kit (18 min), the lowest MNC efficiency (38%), the highest platelet contamination in final product. Furthermore this machine showed the highest percentage of technical failure (20%). None of the six instruments have all the required preconditions and the ideal cell separator for peripheral stem cell apheresis at present is not available on the market.

Collection of platelets and peripheral progenitor cells with the fresenius AS.TEC 204 blood cell separator.

The aim of the present study was to clinically evaluate the blood cell separator AS.TEC 204. One hundred fifteen platelet collections were carried out with the dual or single needle procedure. Platelet yield was 3.21 +/- 0.80 x 10(11) (mean +/- standard deviation) and 59.1% of the collections showed platelet counts > or = 3.0 x 10(11). Leukocyte contamination was 1.77 +/- 2.81 x 10(6) and 89.0% of the platelet concentrates had a white blood cell content < 5 x 10(6). Using a dual needle technique with an alternating interface adjustment, all of the products were contaminated with less than 1 x 10(6) leukocytes. Furthermore, 23 peripheral progenitor cell collections were performed in 12 patients and three allogeneic donors. Median numbers of harvested CD 34 antigen expressing cells/kg body weight were 0.78 (range 0-4.7) and 3.67 x 10(6) (range 2.2-5.23), respectively. We conclude that platelet and progenitor cell collections can be carried out with efficient results. The collections were well tolerated by the donors.