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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.

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In this study, we retrospectively analysed the utility of CD110 expression on CD34 þ cells as a predictor of delayed platelet transfusion independence in 39 patients who underwent autologous peripheral blood stem cell transplantation. Absolute CD34 þ cells and CD34 þ subsets expressing CD110 were enumerated using flow cytometry. Of the 39 patients, 7 required 21 days or more to achieve platelet transfusion independence. Six of the seven patients received a dose of CD34 þ CD110 þ cells below 6.0 Â 10 4 /kg while 30 of 32 patients who achieved platelet transfusion independence in o21 days received a dose of CD34 þ CD110 þ cells 46.0 Â 10 4 /kg (Po0.001). Patients with delayed platelet engraftment received a median dose of 5.2 Â 10 4 CD34 þ CD110 þ cells/kg compared with a median dose of 16.4 Â 10 4 cells/kg for those engrafting within 21 days (P ¼ 0.003). Further analysis showed that 46.0 Â 10 4 CD34 þ CD110 þ cells/ kg was highly sensitive (93.8%) and highly specific (85.7%) for achieving platelet transfusion independence within 21 days. Delay in platelet transfusion independence translated into an increased requirement for platelet transfusion (median 6 vs 2 transfusions, Po0.0001). The dose of CD34 þ /CD110 þ cells/kg infused at time of transplantation appears to be an important factor identifying patients at risk of delayed platelet engraftment.

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.

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