Our previous in vitro studies proved a higher clonogenic potential of peripheral blood progenitor cells cryopreserved in 7.5% dimethyl sulfoxide (Me2SO) than in 10% Me2SO containing medium. Based on ...this findings 7.5% Me2SO cryopreservation medium was introduced to our protocol and both the hematopoietic recovery and infusion-related toxicity were compared with that obtained with standard 10% Me2SO containing solution. Two cohorts of consecutive patients treated with autologous hematopoietic stem cell transplantation were included in the analysis: 56 patients with PBPCs cryopreserved in 7.5% Me2SO solution and 52 patients who obtained cells cryopreserved in 10% Me2SO. Both study groups did not differ significantly with regard to age, diagnosis, and the number of transplanted CD34+ cells. The time to leukocyte recovery was shorter for patients in the 7.5% Me2SO treated group than in the 10% one. Reconstitution of platelets and the frequency of adverse events did not differ in both groups. Reduction of Me2SO concentration from 10% to 7.5% in cryoprotective mixture has a beneficial impact on leukocyte recovery. These findings require verification in a prospective, randomized trial.
Peripheral blood is a preferable source of hematopoietic stem and progenitor cells (HSPCs) used for autologous transplantation. HSPCs are mobilized to peripheral blood and collected by leukapheresis. ...Prior to cryopreservation the cells need to be processed including the addition of cryoprotective mixture as dimethyl sulfoxide (DMSO) prediluted in human serum albumin solution (HSAS). In Europe there is no commercially available albumin manufactured in packs with tubing which would enable the use of sterile tubing welder. Alternatively cryoprotective solution can be prepared using autologous plasma (AP) obtained during the same leukapheresis, allowing for the preparation of HSPCs in a completely closed system and hence to reduce the risk of contamination. The goal of our study was to test if the HSAS may be replaced by autologous plasma without negative impact on cell recovery and clonogenicity.
Samples were prospectively collected from 18 patients with multiple myeloma (n=13) and lymphomas (n=5) mobilized with chemotherapy combined with G-CSF. Small volumes (1.5 ml) of cell suspensions obtained from the leukapheresis products were divided into 2 parts (0,5ml) placed in separate small vials, each containing different cryoprotective mixture - 5% HSAS or AP with a final 7.5% DMSO concentration. The final volume of cell suspensions equaling 1 ml, the cell concentration (0.7–1.5 × 108 /ml). The cells were frozen in IceCube, using a computer controlled cooling program and stored in liquid nitrogen for 2 - 4 months. Concentration of total protein and individual electrophoretic fractions of plasma proteins were measured. The quality of cryoprotective mixtures was evaluated by cell recovery and clonogenic potential. The recovery was determined by comparing number of living cells before and after cryopreservation, using trypan blue staining. Clonogenic potential was carried out by colony forming unit (CFU) assays. Depending on CD34+ percentage, 5-40 × 103 living cells were plated (in triplicates) in MethoCult medium and cultured for 14 days.
The median recovery of nucleated cells for AP was 68.3% (range 40.6-96.1) and was similar to HSAS 68.5%, (41.7-100); (p=0.3; Wilcoxon matched pairs test). The number of CFUs calculated per 100 000 cryopreserved cells did not differ significantly between tested cryoprotective mixture: 187.3 (11.3-806.3) for albumin, 130.5 (15-924.2) for autologous plasma (p=0.5; Wilcoxon matched pairs test). No significant differences were observed when the number of specific types of CFUs were compared. Neither total protein nor albumin concentration of plasma correlated with the clonogenic potential of the leukapheresis product cryopreserved in AP when samples from patients with higher concentration than median were compared with the other (Mann-Whitney U test). Between January and July 2013 more than 50 successful transplants of autologous HSPCs cryopreserved with 7.5% DMSO prediluted in autologous plasma were performed in our Department.
Commercially available human serum albumin can be replaced by autologous plasma in procedure of HSPCs cryopreservation. The use of autologous plasma for cryoprotective mixture preparation does not appear to negatively affect cell recovery and clonogenic potential of leukapheresis product. The advantage of such solution is possibility of HSPCs preparation in closed system to reduce risk of auto-HSCT product contamination to the minimum.
No relevant conflicts of interest to declare.
Abstract 3018
Cryopreservation of autologous peripheral blood progenitor cells (PBPCs) in 10% DMSO is a routine in most transplantation centers. During ASH 2011 Meeting, we presented the results of ...in vitro research, concerning the optimization of DMSO concentrations for recovery and clonogeneic potential of PBPCs after thawing. We concluded, that reduction of DMSO concentration from 10% to 7.5% may have favorable impact for cell clonogeneicity. Accordingly, we implemented new cryoprotective mixture (7.5% instead of 10% DMSO) into clinical practice. The purpose of this study was to clinically evaluate the changed protocol.
In our department, between Jan 2012-Aug 2012, 56 patients were transplanted with autologous PBPCs cryopreserved in 7.5% DMSO solution (median of age: 57 years, range: 21–66). We compared the data concerning hematopoietic engraftment and the frequency of side effects with historical control – 52 subsequent patients treated with transplantation of PBPSc cells cryopreserved in 10% DMSO (median of age: 57 years, range: 21–66) in a preceding period. Both study groups did not differ significantly with regard to the diagnosis (mostly lymphoproliferative disorders) or disease status at transplantation. As well, the number of transplanted CD34+ cells was comparable: median 6.5′106/kg (range 1.5–24.7) for 7.5% DMSO and 7.5′106/kg (2.1–24.6) for 10% DMSO group, p=0.68. All received G-CSF (filgrastim) starting on day +7 after transplantation.
The volume of infused DMSO was significantly lower in patients who obtained PBPBc cryopreserved in 7.5% DMSO (median 22.5 ml, range 7.5–45) than 10% DMSO (median: 30 ml, range, 10–160); p=0.02. The time to leukocyte recovery >1′109/L was faster for 7.5% DMSO (median: 11 days, range: 9–12) than in 10% DMSO (median 11 days, range: 10–13), p=0.03. Similarly, reconstitution of neutrophils >0.5′109/L was faster for 7.5% DMSO group: median 11 days (range 9–13 days) vs. 11 days (range 10–13 days), respectively; p = 0.04. We didn’t observe significant difference with regard to platelet recovery >50′109/L (median 12 days, range: 0–21 days for 7.5% DMSO vs. median 12.5, range: 0–19 days for 10% DMSO). Hospital stay since HSCT was shorter in case of 7.5% group (median: 14 days, range: 11–21) than 10% group (median: 15 days, range: 13–25); p=0.04. Number of RBC and platelets transfusions as well as transfusion-related complications did not differ between the groups. Adverse events after transplantation were mild and transient, usually grade 1 nausea, and occurred in 20 (38%) patients in 10% DMSO group compared to 24 (43%) in 7.5% DMSO group (p=0.7).
The analysis of newly implemented cryopreservation protocol suggest that reduction of the DMSO concentration from 10% to 7.5% is associated with faster leukocyte and neutrophil recovery as well as shorter hospital stay. These findings require verification in a prospective, randomized trial. Display omitted
No relevant conflicts of interest to declare.
Abstract 1930
Hematopoietic stem cells mobilized to peripheral blood and collected by leukapheresis are predominantly used for autologous transplantation. Prior to cryopreservation the cells need to ...be processed including the addition of DMSO (dimethyl sulfoxide), which enhance cell survival, but is potentially toxic to stem cell recipient. The most commonly used concentration of DMSO is 10%. The goal of our study was to test if the concentration may be reduced without negative impact on cell recovery and clonogenicity.
Samples were prospectively collected from 12 patients with lymphomas mobilized with chemotherapy combined with G-CSF. Small volumes (2–3 ml) of cell suspensions obtained from the leukapheresis product were divided into 4 parts placed in separate small vials, each containing different cryoprotective mixture - with 10%, 7.5%, 5% and 2.5% DMSO. The final volume of cell suspensions equaling 1 ml, the cell concentration (0.8–1 × 108 /ml) and the proportions of human albumin and plasma were the same in all vials. The cells were frozen in IceCube, using a computer controlled cooling program and stored in liquid nitrogen for 2 weeks – 4 months. The quality of cryoprotective mixtures was evaluated by cell recovery and clonogenic potential. The recovery was determined by comparing number of living cells before and after cryopreservation, using trypan blue staining. Clonogenic potential was carried out by colony forming unit (CFU) assays. Depending on CD34+ percentage, 5 or 10 × 103 living cells were plated (in triplicates) in medium MethoCult and cultured for 14 days.
The median recovery of nucleated cells for 10% DMSO was 62.4% (range 41.2–86.8) and was significantly higher compared to 7.5% DMSO (54.9%, 41.2–89.1; p=0.04), 5% DMSO (49.2%, 28.1–69.8; p=0.002) and 2.5% DMSO (37.2%, 19.3–54.3; p=0.002). The number of CFUs calculated per 100 000 cryopreserved cells did not differ significantly according to DMSO concentration: 217 (14–1795) for 10% DMSO, 225 (27–2718) for 7.5% DMSO, 196 (26–2761) for 5% DMSO, and 178 (14–2208) for 2.5% DMSO. Neither cell recover nor clonogenic potential correlated with the percentage of CD34+ cells in the leukapheresis product.
Reduction of DMSO concentration to equal or below 7.5% is associated with impaired recovery of nucleated cells after cryopreservation. However, it does not appear to negatively affect clonogenic potential of leukapheresis product, suggesting relative resistance of hematopoietic progenitor cells. Therefore, considering potential toxicity of DMSO to stem cell recipient, its lower concentrations may be clinically beneficial. This hypothesis requires prospective verification in a setting of autologous transplantation.
No relevant conflicts of interest to declare.
