Frances Garvin

Fresh PBSC harvests, but not BM, show temperature-related loss of CD34 viability during storage and transport

BACKGROUND The optimum conditions for storage and transport of freshly harvested HPC in the liquid state are uncertain. It is not specified in commonly applied standards for stem cell transplantation. We used a viable CD34 assay to determine the optimum temperature for maintaining progenitor cell viability in freshly harvested BM and PBSC. Our aim was to identify standardized conditions for storage and transport of marrow or peripheral blood products that would optimize CD34 recovery, leading to better transplant outcomes. METHODS Samples were aseptically removed from 46 fresh HPC harvests (34 PBSC and 12 BM) and stored at refrigerated temperature (2-8 degrees C), room temperature (18-24 degrees C) and 37 degrees C for up to 72 h. Samples were analyzed for viable CD34+ cells/microL at 0, 24, 48 and 72 h. RESULTS The mean viable CD34+ yield prior to storage was 7.7 x 10(6)/kg (range 0.7-30.3). The mean loss of viable CD34+ cells in HPC products at refrigerated temperature was 9.4%, 19.4% and 28% at 24, 48 and 72 h, respectively. In contrast, the mean loss of viable CD34+ cells at room temperature was 21.9%, 30.7% and 43.3% at 24, 48 and 72 h, respectively. No viable CD34+ cells remained after storage at 37 degrees C for 24 h. Only PBSC products and not BM showed temperature-related loss of CD34 viability. Greater loss of viable CD34+ cells was observed for allogeneic PBSC compared with autologous PBSC. DISCUSSION These results demonstrate that the optimum temperature for maintaining the viability of CD34+ cells, during overnight storage and transport of freshly harvested HPC, is 2-8 degrees C. These findings will allow the development of standard guidelines for HPC storage and transport.

Recovery of viable CD34 + cells from cryopreserved hemopoietic progenitor cell products

Summary:The number of CD34+ 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+ 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+ cell count was 5.98 X 106/kg (range 0.3–23 X 106/kg) before freezing and 5.4 X 106/kg (range 0.2–23 X 106/kg) after thawing. The median recovery was 93% (range 48–107%), with 90% recovery for NHL (range 48–100%, n=34), 83% for multiple myeloma (range 56–106%, n=11), 92.3% for acute leukemia (range 71–100% n=7) and 94.5% for nonhematological malignancies (range 50–107% n=18). Similarly, for autologous bone marrows (n=9) the median recovery of viable CD34+cells was 90% (range 68–100%). The recovery of viable CD34+ cells for adult (n=51) and pediatric (n=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.

Failure to achieve a threshold dose of CD34(+)CD110(+) progenitor cells in the graft predicts delayed platelet engraftment after autologous stem cell transplantation

In this study, we retrospectively analysed the utility of CD110 expression on CD34+ cells as a predictor of delayed platelet transfusion independence in 39 patients who underwent autologous peripheral blood stem cell transplantation. Absolute CD34+ cells and CD34+ 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+CD110+ cells below 6.0 × 104/kg while 30 of 32 patients who achieved platelet transfusion independence in <21 days received a dose of CD34+CD110+ cells >6.0 × 104/kg (P<0.001). Patients with delayed platelet engraftment received a median dose of 5.2 × 104 CD34+CD110+ cells/kg compared with a median dose of 16.4 × 104 cells/kg for those engrafting within 21 days (P=0.003). Further analysis showed that >6.0 × 104 CD34+CD110+ 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, P<0.0001). The dose of CD34+/CD110+ cells/kg infused at time of transplantation appears to be an important factor identifying patients at risk of delayed platelet engraftment.

Clinical-scale elutriation as a means of enriching antigen-presenting cells and manipulating alloreactivity

BACKGROUND AIMS Clinical-scale elutriation using the Elutra(c) has been shown to enrich monocytes reliably for immunotherapy protocols. Until now, a detailed assessment of the four (F1-F4) non-monocyte fractions derived from this process has not been performed. METHODS Using fluorescence-activated cell sorting (FACS), we performed phenotypic analyses to investigate the possible enrichment of T, B, natural killer (NK) and dendritic cells (DC) or their subsets in one or more Elutra fractions. RESULTS Blood DC were enriched up to 10-fold in some fractions (F3 and F4) compared with the pre-elutriation apheresis product. This increased the number of DC that could be isolated from a given cell number by immunomagnetic separation. It was also found that CD62L(-) effector memory CD4(+) T cells were enriched in later fractions. In four of five cases tested, cells from F3 demonstrated decreased alloreactive proliferation in a mixed lymphocyte reaction compared with cells from the apheresis product. B cells were enriched in F1 compared with the apheresis product. CONCLUSIONS In addition to providing enrichment of monocytes for the generation of DC, the Elutra enriches cell subsets that may be incorporated into and enhance existing immunotherapy and stem cell transplantation protocols.