Citrate ratio in collection of peripheral blood progenitors.
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Anticoagulants↗
Biomedical subjects
S D Rowley
Publications and source records attributed to S D Rowley.
High-dose therapy followed by autologous hematopoietic stem-cell infusion for patients with multiple myeloma.
PURPOSE: To evaluate the outcome of patients with multiple myeloma (MM) who received high-dose therapy followed by autologous bone marrow (BM) or peripheral-blood stem-cell (PBSC) infusion. PATIENTS AND METHODS: Sixty-three consecutive patients with MM received autologous BM (n = 13) or PBSC with or without BM (n = 50) following regimens that contained busulfan (Bu) and cyclophosphamide (Cy) (n = 18), modified total-body irradiation (TBI) followed by Bu and Cy (n = 36), or Bu, melphalan, and thiotepa (n = 9). Two thirds of the patients had resistant disease and 69% had received more than 6 months of previous chemotherapy. RESULTS AND CONCLUSION: Recovery of peripheral-blood cell counts was more rapid in patients who received PBSC with or without BM than in patients who received BM alone. Sixteen of 63 patients (25%) died of complications of treatment within 100 days. Nineteen (40%) of 48 assessable patients achieved a complete response (CR), 23 (48%) had a partial response (PR), and six (12%) had no response. The probabilities of survival and survival without relapse or progression for all 63 patients at 3.0 years were .43 and .21, respectively. The probability of relapse or progression at 3 years was .69, and 17 patients (27%) have died of progressive MM. The probabilities of survival and relapse-free survival at 3 years for the 19 patients who achieved a CR were .42 and .17, respectively. In the multivariate analysis, beta2-microglobulin levels more than 2.5 micrograms/mL, more than two regimens of prior therapy and eight cycles of treatment, time to transplant longer than 3 years from diagnosis, and prior radiation were associated with adverse outcomes. Additional strategies, such as intervention earlier in the disease course, improved treatment regimens, sequential high-dose treatments, and posttransplant therapies may improve outcome of selected patients with MM.
Adult↗
Long-term cryopreservation of bone marrow for autologous transplantation.
Little is known about the effect of long-term cryopreservation on the viability of hematopoietic stem cells (HSC) or on the success of autologous bone marrow transplantation. Although progenitor cell assays such as culture of CFU-GM after thawing can be predictive of engraftment, the most rigorous assay for the cryosurvival of HSC is engraftment after reinfusion of stem cells. We retrospectively evaluated the engraftment data for 36 patients with hematologic malignancies or solid tumors treated at the Fred Hutchinson Cancer Research Center between 1981 and 1993 who received bone marrows stored for 2 years or more. The median duration of cryopreservation for this study group was 2.7 years (range 2.0-7.8). Ninety-seven percent of patients in the study group achieved a granulocyte count of > or = 0.5 x 1.0(9)/1 at a median of 19 days (range 10-115) vs 86% of control group (selected by diagnosis and date of storage) at a median of 20 days (P = 0.14). Seventy percent of patients in the study group achieved a platelet count > or = 20 x 10(9)/1 at a median of 27 days (range 9-69) vs 74% of control group at a median of 23 days (P = 0.47). Also, samples of 28 marrows cryopreserved for a median of 4.4 years (range 2.0-7.8) were cultured to determine if a loss of hematopoietic progenitors relative to duration of storage could be detected. The storage length was not predictive for the quantity of colonies formed (P = 0.57 for BFU-E-derived colonies; P = 0.65 for CFU-GM-derived colonies). We found no consistent detrimental effect of long-term cryopreservation on the success rate of autologous bone marrow transplantation. This report confirms previous reports that marrow cells cryopreserved for several years are capable of engrafting. Therefore, bone marrow cells may be stored at an early appropriate time before the side-effects of multiple cycles of chemotherapy and radiotherapy on hematopoietic tissues are incurred.
Adolescent↗
Microbial contamination of peripheral blood stem cell collections.
We prospectively evaluated microbiologic cultures for a series of 1263 peripheral blood stem cell harvests collected from 376 sequential patients. The incidence of microbial contamination was 0.23% of all samples, or 47% of samples from patients not receiving systemic antibiotics. This incidence of positive microbial cultures is less than that for cultures of bone marrow (3.8%) after harvesting and before further processing. Of the three positive cultures, two grew coagulase-negative Staphylococcus and the third, Streptococcus viridans. Two patients were reinfused with cultured-positive PBSC components, and neither exhibited the same organism in subsequent blood cultures. Although microbial contamination may occur during peripheral blood stem cell collection and cryopreservation, this report indicates that PBSC contamination does not play a conspicuous role in the infectious complications of PBSC transplantation.
Bacteriological Techniques↗
High-dose fractionated total-body irradiation, etoposide and cyclophosphamide for treatment of malignant lymphoma: comparison of autologous bone marrow and peripheral blood stem cells.
Consecutive patients with non-Hodgkin's lymphoma (NHL, n = 133) or Hodgkin's disease (HD, n = 20) were treated with 12.0 Gy of fractionated total body irradiation, etoposide 60 mg/kg, and CY 100 mg/kg followed by infusion of autologous hematopoietic stem cells. Seventy-nine patients received purged (n = 62) or unpurged BM (n = 17), and 74 received unpurged PBSCs alone (n = 56) or with BM (n = 18). The median day for achieving a sustained granulocyte count of 0.5 x 10(9)/I was 14 range (7-66) for BM recipients and 10 (7-30) for PBSC +/- BM recipients (P = 0.03). A platelet count of 20 x 10(9)/I was achieved at a median of day 24 (6-145) in BM recipients and day 11 (range, 7-56) in PBSC +/- BM recipients (P = 0.007). The median number of platelet units transfused was 86 (0-1432) for BM recipients and 30 (6-786) for PBSC +/- BM recipients (P = 0.001). The median number of hospital days was 36 (10-88) for BM recipients and 27 (14-76) for PBSC +/- BM recipients (P = 0.0001). The unadjusted Kaplan-Meier (KM) estimates of survival, event-free survival (EFS) and relapse at 2 years were 0.57, 0.45 and 0.43 for patients receiving BM and 0.55, 0.36 and 0.59 for patients receiving PBSC +/- BM. After adjusting for confounding variables, the estimated relative risk (RR) of death from any cause was 0.92 (P = 0.75), of relapse was 1.25 (P = 0.39), of non-relapse mortality was 0.71 (P = 0.42) and of mortality and/or relapse was 1.17 (P = 0.48) for patients receiving PBSC +/- BM as compared to BM. For 46 patients with NHL receiving unpurged PBSC alone, the unadjusted KM estimate of relapse was 0.61 compared with 0.48 for 52 comparable patients receiving purged BM, while the RR for relapse for patients receiving unpurged PBSCs was 1.37 (P = 0.33) after adjusting for other significant covariates. These data confirm previous observations that patients who receive PBSC +/- BM have faster engraftment, fewer transfusions and shorter hospital stays than patients who receive only BM. There were no statistically significant differences between the two groups in survival, relapse, death from causes other than relapse and event-free survival.
Adolescent↗
Rapid engraftment by peripheral blood progenitor cells mobilized by recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in nonhuman primates.
We have previously shown that administration of low-dose recombinant human stem cell factor (rhSCF) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) to baboons mobilizes greater numbers of progenitor cells in the blood than does administration of rhG-CSF alone. The purpose of the present study was to determine whether marrow repopulating cells are present in the blood of nonhuman primates administered low-dose rhSCF plus rhG-CSF, and if present, whether these cells engraft lethally irradiated recipients as rapidly as blood cells mobilized by treatment with rhG-CSF alone. One group of baboons was administered low-dose rhSCF (25 micrograms/kg/d) plus rhG-CSF (100 micrograms/kg/d) while a second group received rhG-CSF alone (100 micrograms/kg/d). Each animal underwent a single 2-hour leukapheresis occurring the day when the number of progenitor cells per volume of blood was maximal. For baboons administered low-dose rhSCF plus rhG-CSF, the leukapheresis products contained 1.8-fold more mononuclear cells and 14.0-fold more progenitor cells compared to the leukapheresis products from animals treated with rhG-CSF alone. All animals successfully engrafted after transplantation of cryopreserved autologous blood cells. In animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells, we observed a time to a platelet count of > 20,000 was 8 days +/- 0, to a white blood cell count (WBC) of > 1,000 was 11 +/- 1 days, and to an absolute neutrophil count (ANC) of > 500 was 12 +/- 1 days. These results compared with 42 +/- 12, 16 +/- 1, and 24 +/- 4 days to achieve platelets > 20,000, WBC > 1,000, and ANC > 500, respectively, for baboons transplanted with rhG-CSF mobilized blood cells. Animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells had blood counts equivalent to pretransplant values within 3 weeks after transplant. The results suggest that the combination of low-dose rhSCF plus rhG-CSF mobilizes greater numbers of progenitor cells that can be collected by leukapheresis than does rhG-CSF alone, that blood cells mobilized by low-dose rhSCF plus rhG-CSF contain marrow repopulating cells, and finally that using a single 2-hour leukapheresis to collect cells, the blood cells mobilized by low-dose rhSCF plus rhG-CSF engraft lethally irradiated recipients more rapidly than do blood cells mobilized by rhG-CSF alone.
Animals↗
Recombinant human deoxyribonuclease for hematopoietic stem cell processing.
Numerous reagents are used in the collection processing and storage of hematopoietic progenitor cells for transplantation. To decrease potential variations in the final component for transplantation, these reagents should be uniform in safety, potency, and efficacy. Pharmaceutical-grade reagents are ideal but often are not available. Recently, recombinant human deoxyribonuclease (DNase) was approved for the treatment of patients suffering from the pulmonary complications of cystic fibrosis. We tested this pharmaceutical for toxicity to hematopoietic progenitor cells. These cells were exposed to a range of incubation concentrations for both the recombinant enzyme and bovine DNase previously used in this laboratory. No loss of nucleated cells or hematopoietic progenitors was observed after short-term incubation (1 h) or with direct addition to the culture medium. No incremental toxicity was observed in using recombinant enzyme with murine anti-B cell antibodies and rabbit complement in an immunologic purge technique. A variable effect on cell recovery after thawing of cryopreserved bone marrow cells was observed for both enzyme sources. These data suggest that the pharmaceutical-grade, recombinant human DNase may substitute for previously used reagent-grade protein from animal sources.
Animals↗
Effect of cell concentration on bone marrow and peripheral blood stem cell cryopreservation.
The effects of cell concentration during cryopreservation on bone marrow (BM) or peripheral blood (PB)-derived hematopoietic progenitor cells have not been described. The much greater numbers of cells harvested for autologous PB stem cell (PBSC) transplantation requires that the cells be frozen at higher cell concentrations, or in much greater volumes, compared with BM. We cryopreserved 108 PBSC collections from 30 patients at an average (+/- SD) cell concentration of 3.7 +/- 1.9 x 10(8) nucleated cells per mL in 127 +/- 45 mL. The proportion of mononuclear cells was 52.9% +/- 27.2%. The products also contained 2.9 +/- 2.1 x 10(9) platelets/mL and an average red cell proportion of 12.9% +/- 7.2%. The nucleated cell recovery after thawing was 75.4% +/- 13.0%. The nucleated cell concentration during freezing was not predictive for the postthaw recoveries of nucleated cells (P = .38), granulocyte-macrophage colony-forming unit (P = .06) or CD34+ cells (P = .54), or for the viability of mononuclear cells (P = .81). The platelet and red cell concentrations similarly were not predictive for these endpoints. Samples (3 BM, 7 PBSC) from 10 patients were simultaneously cryopreserved at two-fold, and from 5 additional patients (PBSC) at 6- to 24-fold differing cell concentrations. A lower recovery of erythroid burst forming unit was found for samples frozen at higher cell concentrations (P = .04), but no significant differences were found in the other endpoints listed above. The average cell concentration during freezing for each patient's PBSC collections (n = 34 patients) did not predict time to achieve a PB count of > 500 granulocytes/microL (P = .51) or platelet transfusion independence (P = .39). Patients achieved these endpoints of engraftment at medians of 12 and 13 days, respectively. The infusion of these products was generally well tolerated. Similarly, the cell concentration at which BM cells were frozen did not predict for the duration of granulocyte (P = .63) or platelet (P = .36) aplasias for 54 patients undergoing autologous BM transplantation. These data suggest that PBSC or BM cells collected for transplantation may be cryopreserved at very high cell concentrations without loss of engraftment potential or undue infusion-related toxicity.
Antigens, CD↗
Hematopoietic precursors resistant to treatment with 4-hydroperoxycyclophosphamide: requirement for an interaction with marrow stroma in addition to hematopoietic growth factors for maximal generation of colony-forming activity.
We tested the ability of CD34+lin- precursor cells isolated from marrow after treatment with 4-hydroperoxycyclophosphamide (4HC) to generate colony-forming cells (CFC). In liquid cultures, recombinant human stem cell factor (SCF), in combination with interleukin-1 (IL-1), IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, or granulocyte colony-stimulating factor caused untreated, but not 4HC-treated, CD34+lin- cells to form CFC. However, generation of CFC from CD34+lin- cells treated with 60 micrograms/mL of 4HC was possible in the presence of an irradiated allogeneic stromal cell layer. This generation was increased when combinations of hematopoietic growth factors including SCF and IL-3 were added. Maximal generation of CFC was seen after 11 to 21 days of culture. At that time, generation of CFC from CD34+lin- 4HC-treated cells equalled that from untreated cells. The phenotype of these 4HC-resistant CD34+lin- precursors was also further defined as CD38-. These studies show that the generation of CFC from the 4HC-resistant, highly immature population of CD34+lin- cells requires an as yet undefined interaction with marrow stroma in addition to known hematopoietic growth factors.
ADP-ribosyl Cyclase↗
Bone marrow purging, 4-hydroperoxycyclophosphamide, and the FDA.
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Acute Disease↗
Effect of DMSO exposure without cryopreservation on hematopoietic progenitor cells.
Colligative cryoprotectants must be non-toxic at the high concentrations required for protection of cells from freeze-injury. Human hematopoietic stem cells are usually cryopreserved in a solution containing 1.6 molal (10% v/v) DMSO. We studied the chemical toxicity of this agent to myeloid and erythroid progenitor cells from healthy donors. No DMSO toxicity was found at concentrations of 5% or 10% at either 4 degrees or 37 degrees C for incubation durations up to 1 h. DMSO at 20% did not decrease the number of progenitor cell-derived colonies per 5 x 10(4) cells cultured, but did result in cell clumping during DMSO washout, resulting in a net loss of progenitor cells. At a concentration of 40% DMSO, a direct toxicity to hematopoietic progenitors was found. Delay in removal of DMSO after thawing of cryopreserved cells for periods up to 1 h was also non-toxic to hematopoietic progenitor cells. Direct addition of DMSO at 1% or 10% final concentration (v/v) to the culture dishes suppressed colony formation. These data suggest that DMSO is not toxic to haematopoietic progenitor cells after short-term exposure at the concentrations used for cryopreservation of marrow and peripheral blood stem cells.
Cell Aggregation↗
Hematopoietic stem cell processing and cryopreservation.
Either bone marrow or peripheral blood may be harvested to provide hematopoietic stem cells (HSC) for autologous transplantation. Both, however, comprise heterogeneous cell populations. The HSC necessary for successful engraftment constitute a very small fraction of the cells harvested. After collection, the harvested cells usually undergo several processing steps to reduce the product volume, remove cells (such as mature blood cells or tumor cells), or to cryopreserve the cells for later reinfusion. Granulocytes and red blood cells, for example, survive cryopreservation poorly using freezing techniques designed for HSC. Therefore, bone marrows being cryopreserved must be depleted of mature blood cells to avoid toxicity from infusion of damaged mature blood cells. Mature blood cells may also impede the variety of tumor cell purging techniques currently being studied. These processings are designed to minimize the loss of HSC while achieving an appropriate HSC product for the individual patient. A number of apheresis devices and cell washers simplify the enrichment of HSC in the harvested cell products. In contrast, tumor cell purging techniques are not standardized between the various transplant centers.
Blood Component Removal↗
Low-temperature storage of bone marrow in nitrogen vapor-phase refrigerators: decreased temperature gradients with an aluminum racking system.
Large temperature gradients may exist in nitrogen vapor-phase refrigerators. Cryopreserved cells stored at higher levels may be considerably warmer than those stored closer to the liquid nitrogen reservoir. To decrease this temperature gradient, racking systems constructed of aluminum were placed in marrow storage refrigerators. The higher heat conductivity of aluminum resulted in a vapor-phase temperature gradient of only 5.9 degrees C at 22.5 inches above the liquid, as compared to the gradient of 86 degrees C seen with steel frames in a similar refrigerator. Temperature fluxes were minimal with lid opening or nitrogen filling. The thicker frame size and loss of the lowest storage level resulted in a storage capacity 63 percent of that achievable with steel frames and liquid immersion. Consumption of nitrogen was estimated to be 174 to 220 percent of the static usage in this model of refrigerator with 6 inches of nitrogen, but comparable to the consumption expected with full immersion of the racking system, regardless of frame construction. These data demonstrate the feasibility of achieving very low, stable, cryogenic temperatures in a vapor-phase refrigerator.
Aluminum↗
Hematopoietic stem cell cryopreservation: a review of current techniques.
Hematopoietic stem cells (HSC) can be stored for prolonged periods at cryogenic temperatures. The techniques currently used were derived from the initial report in 1949 of cryopreservation of bovine sperm in glycerol. The addition of this penetrating cryoprotectant protected the cells from the injury associated with ice formation. Current cryopreservation techniques (with minor variations) suspend cells in an aqueous solution of salts, protein, and one or more cryoprotectants. Cells are frozen at slow rates and stored generally below -120 degrees C in mechanical freezers or nitrogen refrigerators. That these techniques are successful in maintaining HSC viability is evident from the engraftment of these cells in patients treated with marrow-lethal conditioning regimens. However, issues such as the composition of the cryoprotectant solution, cell concentration during freezing, cryoprotectant toxicity, and storage temperatures have not been adequately studied, primarily because of a lack of appropriate assays for HSC cryosurvival. HSC cryobiology will become an increasingly important subject as new HSC collection and processing techniques are developed. Improved cryosurvival of HSC using modified cryoprotectant solutions may improve engraftment kinetics and decrease the cost and morbidity of autologous transplantation.
Bone Marrow Cells↗
Purging with 4-hydroperoxycyclophosphamide combinations.
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Antineoplastic Combined Chemotherapy Protocols↗
Incidence of bacterial contamination of bone marrow grafts.
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Bacteria↗
Correlation of occult clonogenic leukemia drug sensitivity with relapse after autologous bone marrow transplantation.
Despite initial complete remission rates exceeding 70%, the majority of patients with acute myeloid leukemia (AML) and adults with acute lymphocytic leukemia (ALL) eventually relapse. Improving the therapeutic results in acute leukemia requires detecting, and understanding the biology of, the minimal residual leukemia remaining after therapy and responsible for relapse. To investigate the biologic relevance of an in vitro assay for clonogenic leukemia (leukemia colony-forming units [CFU-L]) as a measure of minimal residual leukemia, we studied 58 consecutive patients with acute leukemia in complete remission undergoing autologous bone marrow transplantation (BMT) with cyclophosphamide-based therapy. CFU-L were cultured from the pretransplant remission marrows in 45 of 58 patients: 35 of 43 patients with AML and 10 of 15 with ALL. Clonal rearrangements, identical to the patients' overt leukemia when available, were detected in the occult CFU-L from four of the eight patients with ALL in whom adequate DNA for analysis could be obtained from the CFU-L. None of the uncultured pretransplant remission marrows from the 15 ALL patients showed clonal gene rearrangements. We also determined the in vitro sensitivity of the occult CFU-L to 4-hydroperoxycyclophosphamide (4HC), and correlated these results with the outcome of the patients. The sensitivity of the occult CFU-L to 4HC was the only factor that predicted relapse following BMT. The actuarial probability of relapse was 18% in the 23 patients whose CFU-L were sensitive to 4HC compared with 77% in the 22 patients whose CFU-L were resistant (P less than .001). The only factor that influenced the CFU-L sensitivity to 4HC was the type of leukemia. The CFU-L from the AML patients were more sensitive to 4HC than the CFU-L from the ALL patients (P = .001). Occult CFU-L genetically and functionally represent occult leukemia. Therefore, the CFU-L assay should provide a means for studying the biology of minimal residual leukemia and improving the therapeutic results in patients with acute leukemia.
Adolescent↗
High-dose chemotherapy with reinfusion of purged autologous bone marrow following dose-intense induction as initial therapy for metastatic breast cancer.
We assessed the toxicity and efficacy of high-dose chemotherapy consolidation with reinfusion of purged autologous bone marrow in women with metastatic breast cancer responding to a dose-intense outpatient regimen. Thirty women with hormone-unresponsive metastatic breast cancer, previously untreated with adjuvant doxorubicin or with any chemotherapy for metastatic disease, were treated with cyclophosphamide, methotrexate, doxorubicin, fluorouracil, vincristine, and leucovorin for 16 weeks. Twenty-four patients responded to therapy; 8 showed a complete response, and 16 showed a partial response. These patients proceeded to the next phase of the protocol, ie, marrow harvest and treatment with 6000 mg/m2 cyclophosphamide and 800 mg/m2 thiotepa given over 4 days. Harvested marrow was purged with 100 micrograms/mL 4-hydroperoxycyclophosphamide, and all patients engrafted satisfactorily. The predominant side effects were myelosuppressive and gastrointestinal, and there were no deaths from toxic effects. Three of the 16 patients who showed a partial response after the outpatient phase of treatment achieved a complete response after high-dose therapy. The partial response seen in two more patients converted to a complete response at all sites except bone. The median time to disease progression for all patients in this study was 13 months, and the median survival was 22 months. Four of the original 30 patients remained without disease progression a median of 27 months from entry into the study. This study indicates that this dose-intense regimen can be safely administered, even with the use of purged marrow, with an acceptable toxicity profile. This approach results in a high response rate in women with metastatic breast cancer and could form the basis for a regimen to be tested in the high-risk adjuvant setting.
Adult↗