Cryopreservation Of Peripheral Blood Stem Cells Research Articles

The impact of cryoprotectant exposure time on post-thaw viability of autologous and allogeneic hematopoietic stem cells and leukocyte subpopulations.

Although the use of cryoprotectant dimethyl sulfoxide (DMSO) is the gold standard in cryopreservation of hematopoietic stem cells, it is well known that it has a negative effect on cell viability. The aim of this prospective study was to examine how the length of post-thaw exposure to DMSO affects the cell viability and stability of peripheral blood stem cell (PBSC) samples. Additionally, the effects of donor type and pre-cryopreservation storage time on post-thaw viability during the stability study were evaluated. In 30 autologous and 30 allogeneic PBSC samples viable CD34+, CD14+, CD19+, CD16+/56+, and CD3+ cells were determined immediately after thawing, and one-and three-hours post-thaw. Analysis of the absolute count of viable cells in thawed samples showed a significant difference between all measurement points for CD34+ (p < 0.001), CD14+ (p < 0.001), and CD19+ cells (p < 0.001). No significant differences were observed for post-thaw stability of allogeneic samples analysed between products stored before cryopreservation ≥ 24 hours (N = 20), and those stored < 24 hours (N = 10), except for viable CD3+/CD4+ cells after three hours post-thaw (p = 0.028). In conclusion, DMSO had different effects on leukocyte subpopulations in cryopre-served PBSC samples. The type of donors and the length of storage before cryopreservation did not affect the post-thaw stability of cryopreserved PBSC samples.

The Peripheral Blood Stem Cell Immunome Demonstrates Abnormal Immune Effector Cells Early in the Disease Course of Multiple Myeloma and Contributes to Inferior Outcomes and Secondary Myeloid Neoplasms

Background: Upfront autologous stem cell transplant (ASCT) is the standard-of-care for newly diagnosed multiple myeloma (MM) patients. However, relapse and the development of therapy-related myeloid neoplasms (t-MN) post ASCT results in considerable morbidity and mortality. We hypothesized that enrichment of abnormal myeloid progenitors and immune effector cells (IEC) in peripheral blood stem cells (PBSCs) of patients with MM is associated with a higher risk of relapse and development of t-MN. Methods: After institutional review board approval, we screened patients with active MM that were evaluated at Mayo Clinic, Rochester, MN between 01/01/2003 and 12/31/2020. Patients that underwent an ASCT and had cryopreserved PBSC product available for research were included. Using mass cytometry, we performed a comprehensive immunophenotyping of mobilized cryopreserved PBSCs from 54 MM patients undergoing 1 st ASCT, using validated antibody panel of 37 lymphoid- and myeloid-based markers each. Flow cytometry standard files were normalized and concatenated using the Fluidigm acquisition software. Flow cytometry standard files were uploaded to the OMIQ software from Dotmatics (www.omiq.ai, www.dotmatics.com), where transformation and cleaning (doublets, debris) were performed. To correct for between-sample technical variations (“batch effects”), we used fdaNorm within the omiq.ai platform. Clustering and visualization of normalized and concatenaded flowcytometry files were performed within the omiq.ai platform using PhenoGraph and UMAP, respectively. We performed principal component analysis (PCA) to identify major immunomic drivers of variability across patients. We explored the associations between immune subsets and the dichotomized clinical outcomes of interest: progression-free (PFS), myeloid neoplasm-free (MNFS) and overall survival (OS) from ASCT. A 2-sided false discovery rate adjusted P-value of &amp;lt;0.05 was considered significant when multiple comparisons were performed; otherwise, a P-value of &amp;lt;0.05 was considered significant. Results: The most abundant cell populations were T-cells (49% of the CD45 +) and myeloid cells (43% of the CD45 + cells). Clustering on the CD45 + cells revealed 30 unique lymphoid clusters and 27 unique myeloid clusters. Subclustering on NK-cells identified an additional 16 NK-cell clusters. On PCA, principal components-1 (PC-1) and 2 (PC-2) explained 15.2% and 12.5% of the variability in the data, respectively. A high PC-2 score was associated with inferior median PFS (20 vs. 52 months; P=0.005, Figure 1A), MNFS (52 vs. 85 months, P=0.03, Figure 1B) and OS (64 vs. 106 months, P=0.01) from ASCT. Immune subsets enriched in the high PC-2 cohort included the ‘NKT-like’ (CD56 +) T-cell subset which expressed the inhibitory CD159a receptor and TIGIT; a terminally differentiated (CD27/CD28 -, CD57/KLRG1 +) and exhausted (TIGIT/PD-1 +) T cell subset, 2 exhausted (TIGIT/PD-1 +) T cell subsets; an immunosenescent (CD27/CD28 -, CD57/KLRG1 +) T cell subset as well as a terminally exhausted (C27/C28 -, KLRG1/PD-1/TIGIT +) TCRgamma-delta subset. Immune subsets with high loadings within PC-1 (associated with improved clinical outcomes) consisted exclusively of CD4 + cells with early memory phenotypes (CD27/CD28/CD127 +). Abnormal expression of CD7 and HLA-DR expression on myeloid progenitor cell subsets was associated with an inferior PFS, MNFS, and OS. A NK-cell subcluster, characterized by an immature (CD56 high), inhibitory (CD159a +, NKG2A) phenotype, was enriched in patients developing t-MN (0.33% vs. 0.17%, P=0.006). Conclusion: We identify abnormal myeloid and immune effector cells in the PBSCs of patients with MM, even prior to ASCT. Patients with a PBSC product that is enriched in immunosuppressive T and myeloid cell subsets, and with a lower proportion of antigen presenting cells, progenitor populations, and anti-tumor macrophages have a shorter PFS and MNFS. This suggests that the predisposition to t-MN and inferior outcomes for MM are possibly determined early in the disease course. These findings need external validation and have the potential to guide sequencing of future treatment modalities.

Long-Term Cryopreservation of Peripheral Blood Stem Cell Harvest Using Low Concentration (4.35%) Dimethyl Sulfoxide with Methyl Cellulose and Uncontrolled Rate Freezing at -80 °C: An Effective Option in Resource-Limited Settings

Long-term cryopreservation of peripheral blood stem cells (PBSCs) is highly useful in the setting of tandem/multiple transplantations or treatment of relapse in the autologous hematopoietic stem cell transplantation (HSCT) setting. Even in allogeneic HSCT, donor lymphocyte infusions may be stored for months to years if excess stem cells are collected from donors. Cryopreservation is a delicate, complex, and costly procedure, and higher concentrations of dimethyl sulfoxide (DMSO), a commonly used cryoprotectant, can be toxic to cells and cause adverse effects in the recipient during infusions. In this study, we examined the effect of long-term cryopreservation using 4.35% DMSO (as final concentration) with methyl cellulose and uncontrolled rate freezing in a mechanical freezer (-80 °C) on the viability and colony-forming ability of CD34+ human PBSCs. For patients undergoing autologous HSCT, PBSCs were cryopreserved using DMSO (final concentration of 4.35%) with methyl cellulose. The post-thaw viability of PBSCs was determined using Trypan blue exclusion and flow cytometry-based 7-amino-actinomycin-D (FC-7AAD) methods. Concentrations of CD34+ stem cells and immune cell subsets in post-thaw PBSC harvest samples were assessed using multicolor flow cytometry, and the clonogenic potential of post-thaw stem cells was studied using a colony-forming unit (CFU) assay. CD34+ stem cell levels were correlated with the prestorage CD34 levels using the Pearson correlation test. The viability results in the Trypan blue dye exclusion method and the flow cytometry-based method were compared using Bland-Altman plots. We studied 26 PBSC harvest samples with a median cryopreservation duration of 6.6 years (range, 3.8 to 11.5 years). The median viability of post-thaw PBSCs was >80% using both methods, with a weak agreement between them (r=.03; P=.5). The median CD34+ stem cell count in the post-thaw samples was 9.13×106/kg (range, .44 to 26.27×106/kg). The CFU assay yielded a good proliferation and differentiation potential in post-thaw PBSCs, with a weak correlation between granulocyte macrophage CFU and CD34+ stem cell levels (r=.4; P=.05). Two samples that had been cryopreserved for >8 years showed low viability. Cryopreservation of PBSCs using 4.35% DMSO with methyl cellulose and uncontrolled freezing in a mechanical freezer at -80 °C allows the maintenance of long-term viability of PBSC for up to 8 years.

P-205: The use of non cryopreserved peripheral blood stem cells in multiple myeloma: ten years experience from a single center in Algeria

P-205: The use of non cryopreserved peripheral blood stem cells in multiple myeloma: ten years experience from a single center in Algeria

Cryopreservation of unrelated donor hematopoietic stem cells: the right answer for transplantations during the COVID-19 pandemic?

Cryopreservation was recommended to ensure continuity of unrelated donor (UD) hematopoietic stem cell transplantation (HSCT) during COVID-19 pandemic. However, its impact on clinical outcomes and feasibility was not well known. We compared 32 patients who underwent UD HSCT using cryopreserved peripheral blood stem cells (PBSC) during the COVID-19 pandemic with 32 patients who underwent UD HSCT using fresh PBSC in the previous period. Median neutrophil engraftment was 17.5 and 17.0 days with cryopreserved and fresh grafts, respectively. Non-significant delays were found in platelet recovery days (25.5 versus 19.0; P = 0.192) and full donor chimerism days (35.0 and 31.5; P = 0.872) using cryopreserved PBSC. The rate of acute graft-versus-host disease at 100 days was 41% (95% CI [21–55%]) in cryopreserved group versus 31% (95% CI [13–46%]) in fresh group (P = 0.380). One-hundred days progression-relapse free survival and overall survival did not differ significantly. During COVID-19 pandemic, six frozen UD donations were not transfused and logistical and clinical issues regarding cryopreservation procedure, packaging, and transporting appeared. In summary, UD HSCT with cryopreserved PBSC was safe during this challenging time. More efforts are needed to ensure that all frozen grafts are transplanted and cryopreservation requirements are harmonized.

Non-cryopreserved hematopoietic stem cells in autograft patients with lymphoma: a matched-pair analysis comparing a single center experience with the use of cryopreserved stem cells reported to the European Society for Blood and Marrow Transplantation registry

Background aimsAround 50 000 autologous stem cell transplantations are done each year worldwide using cryopreserved peripheral blood stem cells (PBSCs). Cryopreservation is time-consuming and expensive. Since 2007, several retrospective studies have shown that PBSCs can be stored at 4°C for 2–3 days, allowing autologous stem cell transplantation in patients with multiple myeloma receiving high-dose melphalan. Data with non-cryopreserved PBSCs in patients autografted for lymphoma following longer pre-conditioning regimens are limited. In addition, no controlled comparison has been able to detect unforeseen differences. MethodsThe authors compared outcomes of 94 consecutive adult patients with lymphoma (66 with Hodgkin lymphoma) autografted in our department in Oran (Algeria) using PBSCs stored at 4°C, from 2009 to 2018, with patients receiving cryopreserved stem cells reported to the European Society for Blood and Marrow Transplantation registry. Patients autografted in Oran were matched with patients receiving cryopreserved PBSCs in the registry (four controls per patient in Oran). ResultsNeutrophil engraftment was significantly faster with cryopreserved PBSCs (P = 0.003). By day 10, only 17% of patients receiving non-cryopreserved PBSCs engrafted versus 48% for cryopreserved PBSCs. Likewise, platelet recovery to 20 000/mm3 was significantly faster in patients receiving cryopreserved PBSCs (P = 0.01). However, all patients in both groups had recovered by day 20. There were no significant differences in non-relapse mortality (9% versus 7%, P = 0.4), relapse incidence (22% versus 32%, P = 0.13), progression-free survival (70% versus 61%, P = 0.4) or overall survival (85% versus 75%, P = 0.3). ConclusionsThis analysis suggests that, in patients with lymphoma receiving pre-transplant regimens such as carmustine, etoposide, cytarabine and melphalan, PBSCs stored at 4°C for up to 6 days can be used safely in centers with no cryopreservation facility. However, the kinetics of hematopoietic recovery showed a significant, albeit small, delay in engraftment for both neutrophils and platelets, which favors the use of cryopreservation if available.

Outcomes of Non-Cryopreserved Versus Cryopreserved Peripheral Blood Stem Cells for Autologous Stem Cell Transplantation in Multiple Myeloma.

BackgroundAutologous stem cell transplantation (ASCT) has become a standard procedure in multiple myeloma (MM) patients. Cryopreservation (CRYO) of stem cells may be associated with adverse reactions of dimethyl sulfoxide. Previous studies showed that stem cell storage at 4°C (non-cryopreserved [NC] method) may have some advantages. This analysis focused on comparing the transplant-related outcomes of the 2 preservation methods.Material/MethodsThis was a cohort study of consecutive MM patients who underwent ASCT at Chiang Mai University from 2014 to 2019. Primary outcomes were time to neutrophil and platelet engraftment. Key secondary outcomes were the incidence of infusion reactions, duration of hospitalization, cost, and survival.ResultsA total of 42 MM patients underwent ASCT. Of these, 26 patients and 16 patients underwent NC and CRYO stem cell collections, respectively. There was no difference in time to neutrophil engraftment (median 12 vs. 10.5 days, P=0.203) or platelet engraftment (median 14 vs. 12 days, P=0.809) between groups. The incidence of infusion reactions and duration of hospitalization were similar in both groups. The average cost of ASCT was 10% lower in the NC group. There was no difference in progression-free survival (median 16 vs. 22 months, P=0.701) or overall survival between NC and CRYO groups.ConclusionsASCT in MM using the NC preservation method is effective and safe compared to the CRYO method in both short-term and survival outcomes.

NON Cryopreserved Hematopoietic STEM CELLS to Autograft Patients with Lymphomas: A PAIR Matched Analysis Comparing a Single Center Experience to the Use of Cryopreserved STEM CELLS Reported to the EBMT Registry

Introduction: Around fifty thousand autologous stem cell transplantations are done each year worldwide, using cryopreserved peripheral blood stem cells (PBSC). Cryopreservation is time consuming and expensive. Since 2007, several retrospective studies have shown that PBSC can be stored at 4Dg C for two to three days, allowing autologous stem cell transplantation (ASCT) in patients with multiple myeloma receiving high dose melphalan. Data with non-cryopreserved PBSC in patients autografted for lymphoma following longer preconditioning regimens are limited. In addition, there has been no controlled comparison able to possibly detect unforeseen differences. Patients and methods: We compared outcomes of 94 consecutive adult patients with lymphomas (66 with Hodgkin Lymphoma) autografted in our department in Oran (Algeria), using PBSC stored at 4 dg C, from 2009 to 2018 with patients receiving cryopreserved stem cells reported to the EBMT registry. Patient's autografted in Oran were matched with patients receiving cryopreserved PBSC in the registry (4 controls per Oran Patient). Results: Neutrophil engraftment was significantly faster with cryopreserved PBSC (p= 0.003): by day 10, only 17% of patients receiving non-cryopreserved PBSC engrafted versus 48% for cryopreserved PBSC. Likewise, platelet recovery to 20 000/mm3 was significantly faster in patients receiving cryopreserved PBSC (p=0.01). However, all patients in both groups had recovered by day 20. There were no significant differences in Non Relapse Mortality (9% versus 7%; p=0.4), Relapse Incidence (22% versus 32%; p=0.13), Progression Free Survival (70% versus 61%; 0.4) or Overall Survival (85% versus 75%; p=0.3). Conclusion: This analysis suggests that, in patients with lymphomas receiving pretransplant regimens such as the BEAM, PBSC stored at 4dg C for up to six days can be used safely in centers with no cryopreservation facility. The kinetics of recovery of hematopoiesis however did show a significant, albeit small, delay of engraftment for both neutrophils and platelets, which favors the use of cryopreservation if available. Disclosures Blaise: Jazz Pharmaceuticals: Honoraria. Peggs:Autolus: Consultancy. Salles:Karyopharm: Consultancy; F. Hoffman-La Roche Ltd: Consultancy, Honoraria, Other; Bristol Myers Squibb: Consultancy, Other; Celgene: Consultancy, Honoraria, Other: Participation in educational events; Autolus: Consultancy; Abbvie: Consultancy, Honoraria, Other: Participation in educational events; Genmab: Consultancy; Novartis: Consultancy, Honoraria, Other; Kite: Consultancy, Honoraria, Other; Takeda: Consultancy, Honoraria, Other; Debiopharm: Consultancy; Amgen: Honoraria, Other: Participation in educational events; MorphoSys: Consultancy, Honoraria, Other; Gilead: Consultancy, Honoraria, Other: Participation in educational events; Janssen: Consultancy, Honoraria, Other: Participation in educational events; Epizyme: Consultancy. Milpied:Roche: Honoraria, Other: Travel support; Astellas: Honoraria; Sandoz: Honoraria, Other: consultancy or advisory role; Janssen: Honoraria; Gilead Sciences: Other: consultancy or advisory role; Celgene: Other: Travel support.

Fresh or Cryopreserved CD34+-Selected Mobilized Peripheral Blood Stem and Progenitor Cells for the Treatment of Poor Graft Function after Allogeneic Hematopoietic Cell Transplantation

CD34+-selected stem cell boost (SCB) without conditioning has recently been utilized for poor graft function (PGF) after allogeneic hematopoietic stem cell transplantation with promising results. Unfortunately, many patients have been unable to receive the boost infusion as their donors were unwilling or unable to undergo an additional stem cell collection. Therefore, we conducted this study utilizing either fresh or cryopreserved peripheral blood stem cell products to create CD34+-selected boost infusions for the treatment of PGF. Additionally, to explore relationship of CD34+ dose and response, we included a cohort of donors mobilized with plerixafor in addition to the standard granulocyte colony–stimulating factor (G-CSF). Twenty-six patients with PGF were included in this study. Seventeen donor-recipient pairs were enrolled onto the prospective study; an additional 9 patients treated off protocol were reviewed retrospectively. Three different donor products were used for CD34+ selection: (1) fresh mobilized product using G-CSF only, (2) fresh mobilized products using G-CSF and plerixafor, and (3) cryopreserved cells mobilized with G-CSF. CD34+ cell selection was performed using a CliniMACS. The infusion was not preceded by administration of any chemotherapy or conditioning regimen. The primary objective was hematologic response rate and secondary objectives included CD34+ yields, incidence and severity of acute and chronic graft-versus-host disease (GVHD), overall survival (OS), and relapse-free survival (RFS). The median post-selection CD34+ counts per kilogram of recipient weight were 3.1 × 106, 10.9 × 106, and 1 × 106 for G-CSF only, G-CSF plus plerixafor, and cryopreserved products, respectively. The median CD34+ yields (defined as the number of CD34+ cells after selection/CD34+ cells before CD34+ selection) were 69%, 66%, and 28% for G-CSF only, G-CSF plus plerixafor, and cryopreserved products, respectively. After SCB, 16 of the 26 recipients (62%) had a complete response, including 5 of 8 (63%) who received cryopreserved products. Five had a partial response (19%), resulting in an overall response rate of 81%. One-year RFS and OS were 50% and 65%, respectively. There was no treatment-related toxicity reported other than GVHD: 6 (23%) developed acute GVHD (2 grade I and 4 grade II) and 8 (31%) developed chronic GVHD (2 limited and 6 extensive). Cryopreserved products are viable alternatives to create SCB for the treatment of PGF. When collecting fresh products is an option, the addition of plerixafor increases CD34+ yield over G-CSF alone; however, it is currently unclear if the CD34+ cell dose impacts the efficacy of the SCB.

Prior harvesting and cryopreservation of peripheral blood stem cells from related donors: current situations in Japan

Although peripheral blood stem cell (PBSC) transplantations in an unrelated transplant setting have been performed since 2010, prior harvesting and cryopreservation of PBSCs from unrelated donors has not been approved in Japan. There are no restrictions with regard to related donors. Therefore, in April 2015, we conducted a nationwide survey and obtained written answers from 123 transplant hospitals throughout Japan. Our survey revealed that as much as 81.3% of transplant hospitals routinely perform prior harvesting and cryopreservation of PBSCs from related donors and that both cell processing and quality management of cryopreserved products have been appropriately conducted in line with domestic guidelines, although post-thaw quality control and storage period setting require further improvements. Moreover, informed consent obtained from both patients and donors with regard to cryopreservation of PBSCs was not always sufficient in some hospitals. We found that the average number of unused or discarded cryopreserved PBSCs is 1.09 per hospital, and the overall nonuse or discard rates of cryopreserved PBSCs were estimated to be as low as 2.67%.

CD34+-Selected Infusions of Fresh or Cryopreserved Peripheral Blood Stem Cells for the Treatment of Poor Graft Function Following Allogeneic Hematopoietic Stem Cell Transplant

▪Introduction: CD34+-selected peripheral blood stem cell infusions without conditioning have recently been utilized for poor graft function (PGF) with promising results. Unfortunately, many patients have been unable to receive the boost infusion as their donors were unwilling or unable to undergo an additional stem cell collection. Therefore, we conducted this pilot trial utilizing either fresh or cryopreserved peripheral blood stem cell products to create CD34+-selected peripheral blood stem cell infusions for the treatment of PGF. Additionally, to explore relationship of CD34+ dose and response we included a cohort of donors mobilized with plerixafor in addition to the standard G-CSF.Methods: Poor graft function (PGF) was defined as ANC < 0.5k/μL, platelets < 30k/μL, or platelet or RBC transfusion dependence despite full donor chimerism and in the absence of GVHD more than 60 days following allogeneic stem cell transplant (allo-SCT). Three different donor products were used for CD34+ selection: (1) fresh mobilized product using G-CSF only (SC at a dose of 10 μg/kg daily for 5 days), (2) fresh mobilized products using G-CSF (SC at a dose of 10 μg/kg daily for 5 days, days-4 through day 0) and plerixafor (IV at a dose of 320 μg/kg on day 0), and (3) cryopreserved cells mobilized with G-CSF (SC at a dose of 10 μg/kg daily for 5 days). Seventeen donor-recipient pairs were enrolled onto this prospective study. The original allo-SCT donor either underwent an additional peripheral blood stem cell (PBSC) mobilization and collection with G-CSF only (n=5) or G-CSF plus plerixafor (n=9) or a cryopreserved product (n=3) from a previous collection (using G-CSF only) was used to create the CD34+ cells for infusion. CD34+ cell selection was performed using a CliniMACS. The infusion was not preceded by administration of any chemotherapy or conditioning regimen. Neutrophil improvement was defined as neutrophil count > 500/μl without growth factor support for >7 days; platelet improvement was defined as platelet count ≥ 50,000/µl without platelet transfusion support for > 7 days; and RBC improvement was defined as hemoglobin > 9 g/dL and transfusion independence. Complete response was defined as improvement of all involved cells lines; partial response was defined as improvement of platelets and/or neutrophils with continuing RBC transfusion dependence.Results: The mean post-selection CD34+ count per kg of recipient weight was 3.7x106, 12x106, and 1.7x106 for G-CSF only, G-CSF plus plerixafor, and cryopreserved products, respectively. Mean CD34+ yields (defined as the number of CD34+ cells after selection/CD34+ cells prior to CD34 selection) was 58%, 67%, and 31% for G-CSF only, G-CSF plus plerixafor, and cryopreserved products, respectively (Table 1 and Figure 1). The mean number of CD3+ T cells/kg infused in each product was 0.004 x 106, 0.032 x 106, and 0.015 x 106 for G-CSF only, G-CSF plus plerixafore and cryopreserved groups respectively (Table 1). Following the CD34+-selected stem cell infusion, 12 of the 17 recipients (71%) had a complete response including all 3 who received cryopreserved products; 3 had a partial response (17%) and 2 patients (12%) did not respond. One year relapse-free and overall survival was 71% and 65%. There was no treatment related toxicity reported other than graft-versus-host-disease (GVHD); three (18%) developed acute GVHD (1 grade I localized to the skin, 2 grade II with gut involvement), 6 chronic GVHD (2 limited and 4 extensive) (Table 1).Conclusion: Cryopreserved products seem to be a viable alternative to create CD34+ -selected peripheral blood stem cell infusions for the treatment of PGF. When collecting fresh products is an option, the addition of plerixafor increases CD34+ yield over G-CSF alone. [Display omitted] [Display omitted] DisclosuresAbboud:Teva Phamaceutical: Research Funding. Uy:Novartis: Research Funding.

Cryopreserved Allogeneic Peripheral Blood Stem Cells Result in Outcome Equivalent to Those of Fresh Infusions Enabling Rational Scheduling of Donations,

Abstract 4052 Introduction:Donor stem cells are traditionally infused fresh into recipients in the setting of allogeneic hematopoietic stem cell transplantation (allo-SCT). In this study, we investigated outcomes of 133 sibling allografts using cryopreserved peripheral blood stem cells. We demonstrate that cryopreservation did not have a negative impact on engraftment when compared to data from the IBMTR/EBMT, Seattle and the Canadian registry studies. Non-relapse mortality (NRM) and overall survival (OS) were within acceptable limits. Patients and Methods:We identified all recipients of HLA-matched sibling peripheral stem cells cryopreserved for a minimum of 7 days, who underwent allo-SCT at Hammersmith Hospital from January 1998 until May 2011 (n = 133). The median age at allo-SCT of 78 (59 %) males and 55 (41 %) females was 48 (17 – 65) yrs. Thirty-five (26 %) were transplanted for CML (including accelerated and blast phase), 42 (31 %) for AML, 11 (8 %) for ALL, 14 (11 %) for myeloma and 13 (10%) for other causes. Fifty-six (42 %) had myeloablative and 77 (58 %) had reduced intensity conditioning, with 23 (17 %) having in vivo T-cell depletion with monoclonal anti-CD52 antibody (alemtuzumab). Using validated institutional protocols hematopoietic progenitor cell collections were cryopreserved on the day of collection or on following morning. Cells are were mixed with 10 % v/v dimethyl sulfoxide, frozen to -−00°C at a controlled rate and then transferred to vapor phase liquid nitrogen ≤ −150°C. Thawing and infusion of cells were performed in accordance with a standard protocol defining thawing temperature and the maximum time between thawing and infusion. Median CD-34+ cell dose infused was 9.83 × 106/kg (range 2.4 – 33 × 106/kg). All cryopreserved peripheral blood stem cell collections were infused into the recipients. Engraftment was defined as a peripheral absolute neutrophil count (ANC) of 0.5 × 109/L for 2 successive days and platelet count of > 50 × 109/L for 2 consecutive days, both without support. G-CSF was used only in delayed neutrophil engraftment (> 30 days). Results:Overall 125 (93 %) achieved neutrophil engraftment and median time to engraftment was 19 (range 10 – 42) days. Delayed neutrophil engraftment (>30 days) was present in 4 patients. Results are comparable to the registry data which showed neutrophil engraftment in a median of 14 – 19 days (Table 1). Cumulative probability of achieving ANC > of 0.5 × 109/L for the whole cohort was 94 % (88 – 95). Eight of the 133 patients who died early (< day 30) failed to achieve neutrophil engraftment prior to death. The causes of death were sepsis (n = 6), myocardial ischemia (n = 1) and renal failure (n = 1).Three recovered counts with G-CSF and one patient required stem cell rescue. One hundred and thirteen patients (84 %) recovered platelets to >50 × 109/L within a median time of 21 (range 0 – 240) days, which again is similar to registry data as shown in table 1. The cumulative probability of achieving platelets of 50 × 109/L was 84 % (77 – 88). Twenty patients (16 %) failed to achieve this threshold and causes were multiple. There was no association between CD34+ cell doses infused and delayed or non engraftment of platelets. The incidence of acute and chronic GvHD were 44 % (grade II-IV GvHD 31 %) and 30 % (50 % extensive) respectively. Day 100 NRM was 23 % and OS at 3 years was 50 %. Conclusion:This study provides evidence that cryopreservation and subsequent infusion of peripheral blood stem cell harvests is safe, ensures durable engraftment and is comparable to fresh stem cell infusions (Table 1). We do not routinely use growth factors to aid count recovery and as such the time to engraftment data is consistent. Cryopreservation importantly allows for flexibility in arranging admissions and scheduling regimens for both the donors and the transplant units. Although not observed in our cohort, there is a theoretical risk of not utilizing the cryopreserved cells, thus unnecessarily harvesting donors. One way to circumvent this possibility is by timing the collection close to the transplant.Table 1Comparison between cryopreserved (Hammersmith) and fresh PBSC infusionsStudyNumber of PatientsANC to 0.5 × 109/lPlatelets to 50 × 109/lReferenceHammersmith13319 (10–42)21 (0–180)IBMTR/EBMT28814 (10–40)a19 (11–70)Champlin et al, Blood 2000Seattle8116 (11–29)NABensinger et al, NEJM 2001Canadian10919 (12–35)NACouban et al, Blood 2000aregular use of G-CSF Disclosures:No relevant conflicts of interest to declare.

Cryopreservation of peripheral blood stem cell: the influence of cell concentration on cellular and hematopoietic recovery

The optimal cryopreservation cell concentration during the peripheral blood stem cell (PBSC) collection is a controversial topic. We evaluated the influence of cryopreservation concentration on the recovery of hematopoietic progenitor cells and the kinetics of hematopoietic recovery of autologous stem cell transplant patients. In this retrospective study, we compared two different cryopreservation protocols: 1×10(8) cells/mL (Protocol A) and 2×10(8) cells/mL (Protocol B). A total of 419 PBSCs were analyzed with regard to the number of viable cells and colony-forming units-granulocytes-monocytes (CFU-GM) progenitors. The hematopoietic recovery of 275 patients who received PBSCs cryopreserved at a dose of 1×10(8) cells/mL (Group A) and 2×10(8) cells/mL (Group B) were compared. There were no significant differences in recovery of viable cells between Protocol A and Protocol B. The median of recovery of CFU-GM progenitors was significantly higher in Protocol B (41.2 vs. 57.3, p<0.01). The median times to neutrophil recovery (≥500×10(6) /L) and platelet (PLT) recovery (≥20×10(9) /L) in Groups A and B were 11 days versus 11 days and 12 days versus 12 days, respectively. However, by Kaplan and Meier analyses, Group B recovered neutrophils with a little delay (p=0.01). No difference was observed with regard to time to PLT recovery. On multivariate analysis, we found that the number of CD34+ cells and CFU-GM progenitors had a significant influence on hematopoietic recovery. Cryopreservation of PBSCs at a dose of 2×10(8) cells/mL did not affect the recovery rate of viable cells or the hematopoietic recovery of autologous stem cell transplant patients.

Transient loss of consciousness in pediatric recipients of dimethylsulfoxide (DMSO)-cryopre-served peripheral blood stem cells independent of morphine co-medication

Toxicity related to the infusion of dimethylsulfoxide-cryopreserved peripheral blood stem cells (DMSO-PBSC) manifests mostly as cardiovascular side effects. Neurotoxicity1 including transient global amnesia,2,3 seizures,4,5 and stroke6,7 has been reported as a rare complication primarily in adults. In children, data8 are sparse. In light of a recent report implicating morphine co-medication as a major contributing factor,8 we evaluated retrospectively our own data base, including all infusions of DMSO-PBSC applied in our pediatric center between January 1st 2002 and December 31st 2008. We report on 2 incidences of transient loss of consciousness following 131 infusions of DMSO-PBSC.

Similar Outcomes of Cryopreserved Allogeneic Peripheral Stem Cell Transplants (PBSCT) Compared to Fresh Allografts

The BMT program at Princess Margaret Hospital performed 105 transplants using cryopreserved peripheral blood stem cells (PBSC) from related allogeneic donors. The outcomes were compared with those of a historic control of 106 patients transplanted with freshly procured PBSC. The infusions were tolerated with limited toxicity related to nausea/vomiting or bradycardia, correlated with the total amount of DMSO infused. The average viability of the total nucleated cell (TNC) population after thawing was 71%. The survival of clonogenic progenitors amounted to 75% for colony-forming unit-granulocyte-macrophage (CFU-GM), 69% for burst-forming units erythroid (BFU-E), and 78% for colony-forming units granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM). In contrast, colony-forming units megakaryocyte (CFU-MEG) was significantly more cryosensitive with recovery rates of 39%. The number of viable CD34 + cells transplanted was correlated with the number of transplanted viable CFU-GM ( P < .001), BFU-E ( P < .001), CFU-MEG ( P < .001), and CFU-GEMM ( P = .049), but not with the TNC dose. The number of transplanted CD34 + cells was correlated with engraftment of neutrophils ( P = .012) and platelets ( P = .013). The outcomes of cryopreseved or fresh PBSC transplants (PBSCT) with respect to engraftment of neutrophils ( P = .178) and platelets ( P = .785), lymphocyte recovery ( P = .926), acute ( P = .113), and chronic graft-versus-host disease ( P = .673), recurrence ( P = .295), nonrelapse mortality ( P = .340), and overall survival ( P = .668) were not significantly different. It is therefore reasonable to consider the option of cryopreserved allografts.

Sequential detection of alphafetoprotein-bearing cells in blood stem cell fraction of germ cell tumour patients.

High-dose chemotherapy with peripheral blood stem cell (PBSC) transplantation in advanced germ cell tumour (GCT) patients is widely applied. The aims of this study were: (1) To examine the presence of alphafetoprotein (AFP) bearing tumour cells in PBSC harvests from advanced GCT patients obtained after multiple cycles of induction chemotherapy. (2) To determine whether induction chemotherapy contributed to in vivo purging of the tumour. We evaluated cryopreserved PBSC samples from 5 patients with advanced stage II/III AFP producing GCT. PBSC were separated after the first, second and third cycles of induction chemotherapy. Those samples were analysed using the nested reverse transcription polymerase chain reaction (RT-PCR) method to detect AFP mRNA. Although, in all patients, AFP mRNA was detected in PBSC samples after the first or second cycle of induction chemotherapy, but was not detected in 3 of 4 samples after the third cycle of chemotherapy. Although it is not clear whether tumour cells contaminating PBSC fraction contribute to disease relapse, PBSC harvested after at least 3 cycles of induction chemotherapy might be recommended to avoid such a possibility. © 2001 Cancer Research Campaignhttp://www.bjcancer.com

Peripheral blood stem cell autografts for the treatment of childhood cancer: a review of the Japanese experience. Japanese Cooperative Study Group of PBSCT (JCSG/PBSCT).

Harvesting peripheral blood stem cells (PBSC) for autografts (PBSCT) in children with active cancers is a safe and reliable procedure with a low incidence of serious morbidity. A significant correlation between the number of infused CFU-GM and the time to both granulocyte and platelet engraftment was found. Cells induced by G-CSF could speed the recovery of granulocyte or platelet counts after PBSCT. In terms of preserving engraftment potential, cryopreservation of PBSC by a simplified uncontrolled-rate method is at least as effective as the traditional controlled-rate freezing procedure with a programmed freezer. As the serum G-CSF level increases immediately following infusion of PBSC graft, therapy with G-CSF may have only a limited ability to further enhance hematopoietic recovery after PBSCT. Preliminary results of high-dose chemotherapy without TBI and PBSCT for the treatment of children with relapsed ALL are encouraging.

Endotoxin-induced tumor necrosis factor formation in man and its reversal by pentoxifylline: [formula omitted] Forschungsinstitut Borstel, D-2061 Borstel, FRG and Hoechst Werk Albert, D-6000 Wiesbaden, FRG

The BMT program at Princess Margaret Hospital performed 105 transplants using cryopreserved peripheral blood stem cells (PBSC) from related allogeneic donors. The outcomes were compared with those of a historic control of 106 patients transplanted with freshly procured PBSC. The infusions were tolerated with limited toxicity related to nausea/vomiting or bradycardia, correlated with the total amount of DMSO infused. The average viability of the total nucleated cell (TNC) population after thawing was 71%. The survival of clonogenic progenitors amounted to 75% for colony-forming unit-granulocyte-macrophage (CFU-GM), 69% for burst-forming units erythroid (BFU-E), and 78% for colony-forming units granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM). In contrast, colony-forming units megakaryocyte (CFU-MEG) was significantly more cryosensitive with recovery rates of 39%. The number of viable CD34+ cells transplanted was correlated with the number of transplanted viable CFU-GM (P < .001), BFU-E (P < .001), CFU-MEG (P < .001), and CFU-GEMM (P = .049), but not with the TNC dose. The number of transplanted CD34+ cells was correlated with engraftment of neutrophils (P = .012) and platelets (P = .013). The outcomes of cryopreseved or fresh PBSC transplants (PBSCT) with respect to engraftment of neutrophils (P = .178) and platelets (P = .785), lymphocyte recovery (P = .926), acute (P = .113), and chronic graft-versus-host disease (P = .673), recurrence (P = .295), nonrelapse mortality (P = .340), and overall survival (P = .668) were not significantly different. It is therefore reasonable to consider the option of cryopreserved allografts.