James M. Thompson

Transplantation of hematopoietic stem cells from the peripheral blood

Hematopoietic stem cells can be collected from the peripheral blood. These hematopoietic stem cells (HSC), or better progenitor cells, are mostly expressed as the percentage of cells than react with CD34 antibodies or that form colonies in semi‐solid medium (CFU‐GM). Under steady‐state conditions the number of HSC is much lower in peripheral blood than in bone marrow. Mobilization with chemotherapy and/or growth factors may lead to a concentration of HSC in the peripheral blood that equals or exceeds the concentration in bone marrow. Transplantation of HSC from the peripheral blood results in faster hematologic recovery than HSC from bone marrow. This decreases the risk of infection and the need for blood‐product support. For autologous stem‐cell transplantation (SCT), the use of peripheral blood cells has completely replaced the use of bone marrow. For allogeneic SCT, on the other hand, the situation is more complex. Since peripheral blood contains more T‐lymphocytes than bone marow, the use of HSC from the peripheral blood increases the risk of graft‐versus‐host disease after allogeneic SCT. For patients with goodrisk leukemia, bone marrow is still preferred, but for patients with high‐risk disease, peripheral blood SCT has become the therapy of choice.

Prediction of engraftment after autologous peripheral blood progenitor cell transplantation: CD34, colony‐forming unit–granulocyte‐macrophage, or both?

BACKGROUND: The rate of hematologic recovery after peripheral blood progenitor cell (PBPC) transplantation is influenced by the dose of progenitor cells. Enumeration of cells that express CD34+ on their surface is the most frequently used method to determine progenitor cell dose. In vitro growth of myeloid progenitor cells (colony‐forming unit–granulocyte‐macrophage [CFU‐GM]) requires more time and resources, but may add predictive information.

Transportation of peripheral blood progenitor cell products: effects of time, temperature and cell concentration

BACKGROUND AIMS Peripheral blood progenitor cell (PBPC) products are often transported at high cell concentrations (>200x10(9)/L) over long distances, requiring >36 h transport time. METHODS Fresh PBPC samples from 12 healthy donors were studied with various viability assays regarding the effects of temperature, cell concentration and duration of storage. RESULTS Trypan blue exclusion was far less sensitive to cell damage than two-color fluorescence for CD34 and 7-AAD, and colony-forming unit-granulocyte-macrophage (CFU-GM) assays; the latter assay proved the most sensitive. All products stored at 4 degrees C maintained their viability for up to 4 days. Thus, at 96 h, recovery of viable CD34(+) cells was still 82%, and of CFU-GM 57%, even at concentrations of 200x10(9)/L. Higher storage temperatures rapidly decreased the viability, with extensive variation between donors. At room temperature 80% of viable CD34(+) cells and >90% of CFU-GM were lost after 48 h of storage at 200x10(9)/L. Lower cell concentrations allowed storage at higher temperatures: at 17 degrees C a concentration of 50x10(9)/L resulted in only 5% loss of viable CD34(+) cells after 48 h, while the loss was >30% at 200x10(9)/L. CONCLUSIONS PBPC products should be transported at 4 degrees C. Dilution of the product may partly compensate for slightly higher temperatures. Trypan blue exclusion should be abandoned as a method for assessing viability after prolonged transportation. Proliferative assays should be used to validate transportation conditions.

Matched-pair analysis of hematopoietic progenitor cell mobilization using G-CSF vs. cyclophosphamide, etoposide, and G-CSF: Enhanced CD34+ cell collections are not necessarily cost-effective

Using matched-pair analysis, we compared two popular methods of stem cell mobilization in 24 advanced-stage breast cancer patients who underwent two consecutive mobilizing procedures as part of a tandem transplant protocol. For the first cycle, 10 microg/kg/day granulocyte colony-stimulating factor (G-CSF) was given and apheresis commenced on day 4 and continued for < or =5 days (median 3 days). One week after the first cycle of apheresis, 4000 mg/m2 cyclophosphamide, 400 mg/m2 etoposide, and 10 microg/kg G-CSF were administered for < or =16 days (cycle 2). Apheresis was initiated when the white blood cell (WBC) count exceeded 5000 cells/microL and continued for < or =5 days (median 3 days). Mean values of peripheral blood WBC (31,700+/-3200 vs. 30,700+/-3300/microL) were not significantly different between cycles 1 and 2. Mean number of mononuclear cells (MNC) collected per day was slightly greater with G-CSF mobilization than with the combination of chemotherapy and G-CSF (2.5+/-0.21x10(8) vs. 1.8+/-0.19x10(8) cells/kg). Mean daily CD34+ cell yield, however, was nearly six times higher (12.9+/-4.4 vs. 2.2+/-0.5x10(6)/kg; p = 0.01) with chemotherapy plus G-CSF. With G-CSF alone, 13% of aphereses reached the target dose of 5x10(6) CD34+ cells/kg in one collection vs. 57% with chemotherapy plus G-CSF. Transfusions of red blood cells or platelets were necessary in 18 of 24 patients in cycle 2. Three patients were hospitalized with fever for a median of 3 days after cycle 2. No patients received transfusions or required hospitalization during mobilization with G-CSF alone. Resource utilization (cost of drugs, aphereses, cryopreservation, transfusions, hospitalization) was calculated comparing the median number of collections to obtain a target CD34+ cell dose of 5x10(6) cells/kg: four using G-CSF vs. one using the combination in this data set. Resources for G-CSF mobilization cost

Hematopoietic growth factor after autologous peripheral blood transplantation: comparison of G-CSF and GM-CSF

7326 vs.

Impaired PBPC collection in patients with myeloma after high-dose melphalan

8693 for the combination, even though more apheresis procedures were performed using G-CSF mobilization. The cost of chemotherapy administration, more doses of G-CSF, transfusions, and hospitalizations caused cyclophosphamide, etoposide, and G-CSF to be more expensive than G-CSF alone. A less toxic and less expensive treatment than cyclophosphamide, etoposide, and G-CSF is needed to be more cost-effective than G-CSF alone for peripheral blood progenitor cell mobilization.

GROWTH FACTOR INDUCTION OF CYTOSOLIC PROTEIN TYROSINE KINASE ACTIVITY IN HUMAN HAEMOPOIETIC PROGENITOR CELLS ISOLATED BY FLOW CYTOMETRY

Autologous peripheral blood stem cell (PBSC) transplantation results in rapid hematologic recovery when sufficient numbers of CD34+ cells/kg are infused. Recent studies suggest that filgrastim (G-CSF) administration following transplantation leads to more rapid neutrophil recovery and lower total transplant costs. This study compares the use of G-CSF (5 μg/kg/day) with sargramostim (GM-CSF) 500 μg/day from day 0 until neutrophil recovery (ANC >1500/mm3) in patients with breast cancer or myeloma who had PBSC mobilized with the combination of cyclophosphamide, etoposide, and G-CSF. Twenty patients (13 breast cancer and seven myeloma) received GM-CSF and 26 patients (14 breast cancer and 12 myeloma) received G-CSF. The patients were comparable for age and stage of disease, and received stem cell grafts that were not significantly different (CD34+×106/kg was 12.5 ± 11.1 (mean ± s.d.) for GM-CSF and 19.8 ± 18.5 for G-CSF; P = 0.10). The use of red cells (2.8 vs 2.3 units), and platelet transfusions (2.5 vs 3.1) was similar for the two groups, as was the use of intravenous antibiotics (4.3 vs 4.6 days) and the number of days with temperature >38.3°C (2.3 vs 1.8). Platelet recovery was also similar in both groups (platelets >50 000/mm3 reached after 11.8 vs 14.9 days). The recovery of neutrophils, however, was faster using G-CSF. ANC >500/mm3 and >1000/mm3 were reached in the GM-CSF group at 10.5 ± 1.5 and 11.0 ± 1.7 days, respectively, whereas with G-CSF only 8.8 ± 1.2 and 8.9 ± 2.2 days were required (P < 0.001). as a result, patients given g-csf received fewer injections than the gm-csf patients (10.9 vs 12.3). Resource utilization immediately attributable to the use of growth factors and the duration of pancytopenia, excluding hospitalization, were similar for the two groups. This study suggests that neutrophil recovery occurs more quickly following autologous PBSC transplant using G-CSF in comparison to GM-CSF, but the difference is not extensive enough to result in lower total cost.

Slow platelet recovery after PBPC transplantation from unrelated donors

BACKGROUND Tandem stem cell transplantation is an important treatment option for patients with myeloma and some additional tumors. In an attempt to reduce the contamination of the stem cell graft with tumor cells, patients with myeloma who entered complete remission after the first transplant underwent a second episode of mobilization to obtain progenitor cells for the second transplant. METHODS Twenty-two patients with myeloma participated in the study. The first mobilization utilized CY, etoposide and filgrastim. The second mobilization used the same regimen, but seven patients received only filgrastim. The interval between the two collection periods was 6 months (median; range 4-9 months). The preparative regimen for the first transplant consisted of melphalan 200 mg/m(2). RESULTS The number of total white cells collected during the two collection episodes was similar: 10.8+/-1.6 x 10(8)/kg white cells vs. 11.8+/-1.7 x 10(8)/kg white cells (P=0.63). The collected CD34(+) cell dose was much larger during the first collection: 45.2+/-8.4 x 10(6)/kg vs. 6.9+/-2.7 x 10(6)/kg (P<0.001). Similarly, the collected colony-forming unit (CFU)-GM dose was much larger during the first collection: 295.4+/-59.3 x 10(4)/kg vs. 67.3+/-21.6x10(4)/kg (P<0.001). While the CD34(+) cells collected during the two collection episodes correlated significantly (r=0.55, P<0.01); the first dose was a median of 14.9-fold larger. DISCUSSION No laboratory parameter was able reliably to predict the results of the second collection. A second mobilization/collection episode as part of a tandem transplant approach carries a considerable risk of failing to obtain sufficient progenitor cells.

Delayed ABLC prophylaxis after allogeneic stem-cell transplantation

We employed a highly sensitive method to assay protein tyrosine kinase activity in extracts of subpopulations of CD34+ bone marrow progenitor cells isolated by fluorescence activated cell sorting in an attempt to better define how growth‐factor induction of enzymatic activity relates to progenitor cell maturation. FACS analysis confirmed that, under the conditions employed, essentially all of the CD34+ cells in adult human marrow that lacked the CD38 antigen were devoid of the myeloid maturation marker CD33 as well as the lineage antigens: CD10, 13, 14, 15, 16, 19, 71 and glycophorin A. A variable portion (50–90%) of these CD34+, CD38− progenitor cells expressed HLA‐DR. CD34+, CD38− cells that did not express HLA‐DR were found to lack detectable levels of either membrane or cytosolic tyrosine kinase activity. HLA‐DR+ progenitor cells that lacked CD38 possessed elevated levels of cytosolic tyrosine kinase activity but only low levels of plasma membrane activity. In contrast, CD34+ cells that expressed CD38 (and HLA‐DR) possessed high levels of membrane‐associated tyrosine kinase activity. A cocktail of haemopoietic growth factors that included IL‐3, IL‐6 and stem cell factor effectively induced tyrosine kinase activity in CD34+, CD38−, HLA‐DR− progenitor cells. Growth factor induction of tyrosine kinase activity in these cells was not inhibited by actinomycin D or cyclohexamide. Most of the tyrosine kinase activity induced by these growth factors was recovered from the cytosolic fraction of disrupted cells. Thus, induction of cytosolic tyrosine kinase activity is an early event in the response of uncommitted haemopoietic cells to haemopoietic growth factors. Subsequent activation of membrane tyrosine kinases may initiate key transduction processes as these cells begin to differentiate.

Transportation of peripheral blood progenitor cell products: effect of ambient temperature

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 +28. The MUD grafts came more frequently from young male donors and contained more CD34+ cells but similar numbers of colony-forming units granulocyte-macrophage (CFU-GM) and burst forming units-erythroid. The recovery of neutrophils to >500/mm3 was equally fast in both groups, but recovery of platelets to >20 000/mm3 was significantly delayed in the MUD group (P<0.001). 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+ 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.