The effects of the composition of PBPC grafts from matched related donors (MRDs) and matched unrelated donors (MUDs) have not been compared. In a single-center study, the compositions of 55 MRD PBPC grafts and 33 MUD grafts were studied for their effect on the rate of engraftment in patients who had evidence of donor cell engraftment on day þ 28. The MUD grafts came more frequently from young male donors and contained more CD34 þ cells but similar numbers of colony-forming units granulocyte-macrophage (CFU-GM) and burst forming units-erythroid. The recovery of neutrophils to 4500/mm 3 was equally fast in both groups, but recovery of platelets to 420 000/mm 3 was significantly delayed in the MUD group (Po0.001). The MUD group also required more transfusions of platelets and red cells. Patients receiving grafts containing low numbers of CFU-GM had markedly delayed platelet recovery. The patients with the slowest engraftment tended to have prolonged transportation times. Storage experiments suggested a major loss of viable CD34 þ cells and CFU-GM when undiluted PBPC products are stored at room temperature. The data suggest that a fraction of the MUD grafts suffer during transportation. In vitro proliferation assays should be part of the validation and auditing of transportation of MUD grafts.
Phosphatidic acid and its hydrolysis product, diacylglycerol, play potentially vital roles as extracellular messengers in numerous cellular systems and may play a key role in regulating hematopoiesis. In this study, we describe an ecto‐phosphatidic acid phosphohydrolase that potentially regulates cellular responses to phosphatidic acid on bone marrow derived human hematopoietic progenitors. We partially purified hematopoietic progenitor ecto‐PAPase using a novel in‐gel phosphatase assay and then characterized the enzyme on phenotypically defined subpopulations of hematopoietic CD34+ progenitors isolated by flow cytometry. The most pronounced PAPase activity was confined to uncommitted CD34+/CD38+ hematopoietic progenitors, which lacked the expression of other lineage‐associated antigens. We conclude that hematopoietic progenitor cells at various stages of maturation possess a potent ecto‐PAPase, an enzyme well positioned to regulate progenitor cell growth and differentiation induced by phosphatidic acid and related lipids.
The viability of transported PBPC products has not been studied extensively. Commonly, PBPC products are transported at a concentration of >200 x109/l in containers with −20oC ice packs. Continuous temperature monitoring has shown that the temperatures of these products stays at <10oC for less than 24 hours and reaches room temperature by 48 hours. Samples of freshly collected PBPC from 12 allogeneic donors were studied for various viability parameters during storage for up to 96 hours. The effects of storage time, concentration of cells, temperature, and storage in gas-permeable bags were studied. Trypan-blue exclusion and double fluorescence for 7-AAD and CD34 were used for viability assessment. Over a wide range of temperatures and storage times, the viable CD34+ assay was more sensitive to damage than trypan-blue exclusion (mean Δ 10.7%, p<0.0001 in paired t-test). The viable CD34+ assay was routinely used in parallel with CFU-GM cultures. No difference in survival of viable CD34+ cells or CFU-GM was found whether cells were incubated for 48hr in test-tubes or in gas-permeable bags. When cells at 200 x 109/l were incubated for 48hr at room temperature, the mean viability decreased to 19% and 6% of starting values of viable CD34+ cells and CFU-GM, respectively. Serial dilution to 25 x 109/l improved the survival to 81% and 51% respectively. Similarly, incubation at lower temperatures led to better survival of CD34+ cells and CFU-GM: 67% and 18% at 17oC, 80% and 50% at 13oC, and 95% and 86% at 4oC. At 200 x109/l and 22oC the survivals of CD34+ cells and CFU-GM were 74% and 21% at 24hr, 19% and 7% at 48hr, 7% and 6% at 72hr, and 3% and 13% at 96hr. The effects of concentration, temperature and duration of storage were all significant (p<0.05). Transportation at 4oC leads to the best survival of CD34+ cells and CFU-GM, in particular at a low concentration. If transportation at a slightly higher temperature is necessary, dilution of the PBPC product will enhance the survival of CD34+ cells and CFU-GM. Proliferative assays such as CFU-GM appear the most sensitive parameters of PBPC survival, and should be included in the validation process of PBPC transportation.
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