Summary:The number of CD34 þ cells infused into patients at the time of autologous or allogeneic transplantation is a clinically important variable, but the viability of these cells has not been extensively documented. In this study, we analyzed the recovery of viable CD34 þ cells before and after cryopreservation on 79 autologous stem cell products, using a novel flow cytometry assay without red cell lysis. For 70 PBSC harvest samples, the mean viable CD34 þ cell count was 5.98 Â 10 6 /kg (range 0.3-23 Â 10 6 / kg) before freezing and 5.4 Â 10 6 /kg (range 0.2-23 Â 10 6 / kg) after thawing. The median recovery was 93% (range 48-107%), with 90% recovery for NHL (range 48-100%, n ¼ 34), 83% for multiple myeloma (range 56-106%, n ¼ 11), 92.3% for acute leukemia (range 71-100% n ¼ 7) and 94.5% for nonhematological malignancies (range 50-107% n ¼ 18). Similarly, for autologous bone marrows (n ¼ 9) the median recovery of viable CD34 þ cells was 90% (range 68-100%). The recovery of viable CD34 þ cells for adult (n ¼ 51) and pediatric (n ¼ 28) stem cell collections was 91 and 94%, respectively. Further examination of the correlation between the kinetics of hematological recovery and the number of viable progenitor cells infused, particularly at the lower end of the accepted dose range, may be warranted.
The recovery of viable CD34+ cells reinfused into patients at the time of autologous or allogeneic transplantation is clinically an important variable, which can determine graft success or failure. In this study we analyse the recovery of viable CD34+ cells /kg pre and post cryopreservation on a total of 86 autologous stem cell products from adult and paediatric patients as well as 4 cryopreserved stem cell products from allogeneic donors. CD34 enumeration was performed on all samples pre and post cryopreservation using a novel in-house no-lyse CD34 assay (previously described ASH 2003 abstract no.1685). Cells were labelled with CD45, CD34 and 7AAD in TRUCOUNT tubes using a modified single platform ISHAGE protocol. The absolute number of viable CD34 + cells per Kg was determined. For the 77 PBSC harvest samples the mean viable CD34+ cell count was 6.0 x10^6/Kg (range 0.3 – 25.2 x 10^6/Kg) before freezing. For post thaw samples the mean viable CD34+ cell count was 5.5 x 10^6/Kg (range 0.2 – 24.6 x 10^6/Kg). The median recovery was 95% (range 48–124%). This represents a median loss post freeze/thaw of 5%. Further analysis showed a median recovery of 90% for NHL (range 48–119%, n=34), 87% for MM (range 56–115%, n=12), 92.5% for acute leukaemia (range 71–124% n=8) and 97% for non-hematological malignancies (range 50–120% n=21). There was no significant difference in the recovery of viable CD34+ cells within the four groups of malignancies (p>0.17 for all groups tested). Similarly, autologous bone marrow collections (n=9) also showed a good recovery of viable CD34+ cells post thaw. The median viable CD34+ cell count was 8.1x10^6 /Kg (range 0.6–30.3x10^6/kg) pre-cryopreservation, compared to a median viable cell count of 6.5 x10^6/Kg CD34+ cells (range 0.6–26x10^6/Kg) post thaw, this represents a median recovery of 90% viable CD34+cells from autologous bone marrow collections. There was no significant difference in the recovery of viable CD34+ cells from autologous PBSC harvests and autologous bone marrow collections (p=0.169). We also compared the recovery of viable CD34+ cells post thaw between adult and pediatric stem cells collections. The median recovery of viable CD34+ cells from 56 adult stem cell products post thaw was 91% (range 48–120%), compared to a median recovery of 96.5% (range 50–124%) viable CD34+ cells from 30 pediatric stem cell products (p=0.06). Interestingly the greatest loss occurred in allogeneic donors, where viable CD34+ counts on fresh samples averaged 5.7 x 10^6/Kg (range 3.1–11.8 x 10^6/Kg, n=4), whereas post freeze/thaw averaged 2.2 x 10^6/Kg (range 1.2–3.3 x 10^6/Kg). Representing a mean loss of 58% of CD34+ cells. Twenty-nine patients were transplanted with a median number of 3.8x10^6 viable CD34+ cells per Kg (range 1.8–18.4x10^6/Kg), The median time to neutrophil and platelet engraftment was 12 days (range 10–18) and 14 days (range 8–65) respectively. Assaying the viability of CD34+ cells post cryopreservation may identify patients at risk of poor haematological recovery that could benefit from further stem cell collections.
Recovery of neutrophil numbers after peripheral blood stem cell transplantation (PBSCT) is closely associated with graft CD34+ cell dose. Predicting the speed of platelet recovery is more difficult but would be of value given that a significant minority of patients experience delayed platelet recovery and bleeding complications after transplantation. In this study we retrospectively analysed the graft composition of 29 patients who underwent autologous transplantation, using blood stem cells mobilized with cyclophoshamide and G-CSF, to assess the utility of c-mpl expression on CD34+ cells as a predictor of platelet engraftment (ie, time to platelet count greater than 20 x 109/L for three consecutive days without the need for platelet transfusion). Absolute CD34+ cells and CD34 subsets expressing c-mpl were enumerated using a published single platform viable CD34 flow cytometry assay (BMT, 36: 199–204,2005). Of the 29 patients, 7 required at least 21 days for platelet engraftment. These patients received a median graft dose of 5.7 x 104 CD34+CD110+ cells/kg compared with a median dose of 13.4 x 104 cells/kg received by patients who experienced platelet engraftment within 21 days of transplant (p=0.013). In contrast, there was no difference in the number of CD34+ cells/kg infused (4.0 v 4.9 x 106/kg for > or < 21 days for platelet engraftment respectively, p=0.23). There was a poor correlation between the absolute number of CD34+ cells and the number of CD34+CD110+ cells in the graft (r2 = 0.48). Similarly there was no correlation between the percentage of CD34+ cells expressing c-mpl and the speed of platelet engraftment (8.1 v 5.8%) for > or < 21 days for platelet engraftment respectively, p=0.39). Patients with >21 days for platelet engraftment received platelet transfusions more often than those with <21 days for platelet engraftment (median 9 v 2 transfusions, p <0.001). The absolute number of CD34+/CD110+ cells/kg infused at time of transplantation appears to be an important factor identifying patients at risk of delayed (>21 days) platelet engraftment. Those with <6 x 104 CD34+/CD110+ cells/kg are at particularly high risk of delayed platelet engraftment, requiring multiple transfusions after transplantation.
Background and Aim: The optimum storage and transport of freshly harvested hemopoetic progenitor cells (HPC) in the liquid state is not specified in the JACIE and FACT guidelines. Depending upon transplant centre, there is a range of reported ideal temperatures (1°C to 24°C) for HPC storage and transport but little data exists to justify the recommendations. Due to the limitations of Trypan Blue viability assays and CFU-GM colony assays, we used a no-lyse, CD34 assay (Sartor et al, Bone Marrow Transplantation 2005) to determine the optimum storage and transport temperature for maintaining viability of CD34+ stem cells in freshly harvested HPC. Method: Samples were aseptically removed from 46 fresh HPC harvests (34 PBSC & 12 BM) and stored at refrigerated temperature (2°– 8°C), room temperature (18°– 24°C) and 37°C, for up to 72 hours. Samples were analysed for viable CD34+ cells/ml at 0, 24, 48 and 72 hours. Results: The mean viable CD34+ yield prior to storage was 7.7x106/kg (range: 0.7 – 30.3). No viable CD34+ cells remained after storage at 37°C for 24 hours. The mean % loss of viable CD34+ cells at refrigerated and room temperatures can be summarized as follows: Conclusion: These results demonstrate that the optimum temperature to maintain the viability of CD34+ stem cells for up to 72 hours during storage and transport of freshly harvested HPC is 2°– 8°C. Mean % loss or gain of CD34+ cells on storage Time Refrigerated Temperature Room Temperature N Mean Range N Mean Range 24 hrs 39 −9.4 +15 to −42 23 −21.9 +17 to −59 48 hrs 31 −19.4 +7 to −69 20 −30.7 +3 to −68 72 hrs 29 −28.0 +4 to −53 18 −43.3 +11 to −70
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