CFU-mix are no better than CFU-GM in predicting hemopoietic reconstitutive capacity of peripheral blood stem cells collected in the very early remission phase of acute nonlymphoblastic leukemia.

High levels of circulating myeloid progenitor cells (CFU-GM) occur during the very early remission phase of acute nonlymphoblastic leukemia (ANLL). Autologous stem cell rescue using blood cells collected during this phase has shown that successful hemopoietic reconstitution can be achieved, but a higher CFU-GM dose appears to be required than when bone marrow cells are used. This suggests that during very early remission, the level of marrow repopulating pluripotent stem cells (PSC) in blood does not undergo the same amount of increase as does the CFU-GM. This study set out to determine whether the levels of the multilineage progenitor cell (CFU-Mix) would be better indicators of the PSC in these cells than the CFU-GM. Serial peripheral blood CFU-Mix and CFU-GM measurements were carried out in six ANLL patients during very early remission. The levels of peripheral blood CFU-Mix showed a mean 12-fold increase, as compared to a mean 20-fold increase in the CFU-GM. The timing of the increase in the CFU-Mix paralleled that of the CFU-GM. These findings suggest that the CFU-Mix is no better than the CFU-GM in predicting PSC levels during very early remission of ANLL, and is closer to the CFU-GM than to the PSC in ontogeny.

Single high doses of cyclophosphamide enable the collection of high numbers of hemopoietic stem cells from the peripheral blood.

We used single high doses of cyclophosphamide (4 g/m2) to produce rebound increases in peripheral blood (PB) stem cells (PBSC) during recovery from myelosuppression, enabling their collection by apheresis for later autotransplantation. Thirty-three courses of cyclophosphamide were given to 30 patients with malignant lymphoma, multiple myeloma, or solid tumors. The neutrophil count was less than 0.5 x 10(9)/liter for a mean of 6.9 days (median 7 days), and fever occurred in 17 of 33 courses. Positive blood cultures occurred in two patients, one of whom died. The mean peak level of PB granulocyte-macrophage colony-forming units (CFU-GM) was 1517 x 10(3)/liter (median 2447 x 10(3)/liter), a 14-fold increase above the mean in normal subjects. The peak occurred at a mean of 16.6 days (median 16 days) after cyclophosphamide, generally coinciding with the time to reach 1.0 x 10(9) neutrophils per liter. Normal or minimally involved bone marrow and a rapid rise in leukocyte count during recovery were independent variables correlated to the peak of the rebound increase in PB CFU-GM levels. Previous chemotherapy and the duration of neutropenia were additional independent variables in the group with peak PB CFU-GM levels of greater than 1000 x 10(3)/liter. The mean total CFU-GM collected after a mean of five aphereses was 43.8 x 10(4)/kg body weight (BW) (median 35.5 x 10(4)/kg BW), significantly correlated with the mononuclear cell yield. We conclude that single 4 g/m2 doses of cyclophosphamide effectively produce high levels of PBSC, particularly but not exclusively in patients with normal or minimally involved bone marrow and who have not had intensive recent chemotherapy.

Peripheral blood stem cells mobilized from patients with acute myeloid leukaemia have different platelet repopulating abilities compared with those mobilized from patients with other diseases.

Peripheral blood stem cell (PBSC) transplantation gives rapid recovery of neutrophils and platelets and sustained haemopoiesis. However in patients with acute myeloid leukaemia (AML) platelet recovery has a distinctive rapid rise and then secondary fall between 3 to 8 weeks post-transplant. This study compares platelet and neutrophil recovery after PBSC transplantation in 15 patients with AML and 29 patients with other diseases consecutively transplanted in a single unit. PBSC were collected during recovery from consolidation chemotherapy in AML patients and after cyclophosphamide or cytokine administration in the other patient groups. Mononuclear cell numbers collected were similar but CFU-GM numbers were greater from the AML patients. A significant secondary fall occurred only in the platelet count and only in AML patients. Long-term recovery of the platelet count was the same in AML as in the other patients. In AML patients, the fall was the same in the long term remitters as in those who eventually relapsed. Previous studies have not, demonstrated a difference in type of precursors mobilized by differing methods, but have not included AML patients. Megakaryocyte precursors were assayed in this study and showed no consistent differences in number between patient groups however pre-progenitor assays are not yet established especially in the megakaryocytic lineage. The possible explanation for this secondary fall in AML patients is discussed.

Increased levels of megakaryocyte progenitors in peripheral blood mobilised by chemotherapy and/or haemopoietic growth factor protocols.

We have quantitated colony-forming unit megakaryocyte, (CFU-Mk), burst-forming unit megakaryocyte, (BFU-Mk), colony-forming unit granulocyte-macrophage (CFU-GM), and CD34+ cells in 98 mobilised PB samples from 53 patients mobilised by one of six protocols, including myelosuppressive chemotherapy alone (n = 22), or in combination with recombinant haemopoietic growth factors (n = 32), and growth factors alone (n = 17) or in combination (n = 27). The frequency of megakaryocyte progenitors (total Mk = CFU-Mk + BFU-Mk) in mobilised PB (mean 356, range 0-3240/10(6)) was similar to that in steady-state BM (mean 429, range 0-3315/10(6) n = 45). The levels of total Mk in mobilised PB (mean 1509, range 0-36 099/ml) showed a mean 75-fold increase compared with steady state PB (mean 20, range 0-86/ml, n = 15). In mobilised PB the levels of CFU-Mk were significantly correlated with levels of BFU-Mk (rs = 0.71, P < 0.0001) and the levels of megakaryocyte progenitors correlated significantly with those of myeloid progenitors (rs = 0.59, P < 0.0001) and CD34+ cells (rs = 0.69, P < 0.0001). The mobilisation of megakaryocyte progenitors into the circulation in response to high-dose chemotherapy and/or haemopoietic growth factors contributes to an understanding of the rapid platelet recovery following PBSC transplantation and suggests that the measurement of megakaryocyte progenitors may be a useful indicator for platelet reconstitutive capacity.

Peripheral blood stem cells collected in very early remission produce rapid and sustained autologous haemopoietic reconstitution in acute non-lymphoblastic leukaemia.

Haemopoietic reconstitution was achieved in a patient with acute non-lymphoblastic leukaemia (ANLL) in relapse who was autografted with blood-derived stem cells collected during very early remission. The patient received a myeloid progenitor cell dose of 230 x 10(4) CFU-GM/kg body weight. Engraftment was evident in the bone marrow 7 days post-graft. Normal neutrophil and platelet counts were attained by day 14 and blood counts remained normal thereafter. An overshoot in peripheral blood haemopoietic progenitor levels occurred at the end of the second week, presumably the progeny of a family of early progenitor cells. The completeness of haemopoietic reconstitution is further illustrated by the satisfactory nucleated cell and myeloid progenitor cell yield when a bone marrow harvest was performed 4 1/2 months post-graft. Seven months post-graft, the patient remained in complete remission with normal blood counts and bone marrow cellularity, although haemopoietic progenitor levels were slightly reduced. The rapid recovery minimises aplasia-related risks and suggests that such autografting can be carried out safely in first remission. We propose that autografting using very early remission blood cells is a new therapeutic option for patients with acute ANLL.

CD34+ selection of autologous peripheral blood stem cells for transplantation following sequential cycles of high-dose therapy and mobilization in multiple myeloma.

A potential problem of autologous transplantation in the treatment of multiple myeloma (MM) is the infusion of tumor cells. CD34+ selection has been used to purge autografts in MM and it is also possible to reduce tumour cell contamination of autografts by cytotoxic drug therapy prior to peripheral blood stem cell (PBSC) collection. To evaluate the effectiveness of a protocol combining multiple cycles of high-dose therapy and CD34+ selection to reduce tumour contamination of PBSC autografts, 34 MM patients were entered on a treatment schedule comprising two sequential cycles of mobilisation, CD34+ selection, and transplantation following high-dose therapy. In the second cycle of mobilisation there was a five-fold reduction in tumour contamination of the stem cell harvest (0.5 x 106/kg) compared with the first cycle (2.5 x 106/kg). In the 97 CD34+ selection procedures performed a median of 185 x 108 mononuclear cells (MNC) were processed yielding a median of 0.98 x 108 CD34+-enriched cells. CD34+ cells were enriched 68-fold from 1. 3% to 88.6%. The median yield of CD34+ cells was 42.2%. Following CD34+ selection the tumour cell contamination of the leukapheresis product was reduced by a median of 2.7 logs. This study demonstrates that in multiple myeloma a significant reduction in the malignant contamination of stem cell autografts can be achieved by combining the in vivo purging effect of cytotoxic therapy with in vitro purging by CD34+ selection.

An unusual pattern of hemopoietic reconstitution in patients with acute myeloid leukemia transplanted with autologous recovery phase peripheral blood.

Fourteen patients with acute myeloid leukemia (AML) were autotransplanted with peripheral blood cells collected during early remission. Seven were autotransplanted in first relapse and seven in first remission. They received a median of 3.3 X 10(8) nucleated cells/kg body weight (BW) and 92 X 10(4) myeloid progenitor cell (CFU-GM) per kg BW. Rapid hemopoietic reconstitution (HR) occurred in all patients with median time to reach normal neutrophil and platelet counts 13 and 18 days post re-infusion respectively. However, in three patients neutrophil counts fell to less than 1.0 x 10(9)/l and in seven patients platelet counts fell to less than 25 x 10(9)/l between 26 and 40 days post-transplant (trough count). In all but two patients who received the lowest CFU-GM dose the counts returned to normal or near normal levels (steady count). There were significant correlations between the CFU-GM dose and the trough and the steady platelet counts (p = 0.04 and 0.01 respectively). Patients receiving more than 50 x 10(4) CFU-GM/kg BW had higher steady neutrophil and platelet counts (p = 0.011 and 0.033 respectively) although some patients receiving greater than 50 x 10(4) CFU-GM/kg still experienced thrombocytopenia during the second month post graft. There was no significant correlation between the nucleated cell dose and HR. The cause of the fall in platelet and neutrophil counts in the second month post graft is not clear but is probably a reflection of a proliferative defect in the recovery phase stem cells in AML.

The optimization of collection of peripheral blood stem cells for autotransplantation in acute myeloid leukaemia.

Between November 1982 and November 1986 31 patients with acute myeloid leukaemia underwent peripheral blood stem cell apheresis during haemopoietic regeneration following induction chemotherapy. A retrospective analysis of the factors affecting the efficacy of stem cell harvest and of the clinical outcome of these patients was performed. The mean number of myeloid progenitor cells (CFU-GM) collected was significantly higher in the complete remission group (n = 22) than in the partial remission group (n = 9). Fifty x 10(4) CFU-GM/kg body weight or more, which produced rapid, complete and sustained haemopoietic reconstitution after autografting in our patients, were collected from six of nine patients who underwent three or four 7-litre aphereses over 5-7 days using a lymphocyte collection procedure on the Fenwal CS3000 [Protocol B] but only from two of 12 patients who underwent three or four 5-litre aphereses over 3-5 days using the Aminco Celltrifuge [Protocol A] (p less than 0.05). No adverse effects on the rates of neutrophil, platelet and lymphocyte recovery after induction chemotherapy or on long-term disease-free survival for patients who achieved a complete remission could be attributed to apheresis when compared with a historical control group of 39 patients who achieved complete remission following the same induction chemotherapy but did not undergo apheresis. We conclude that sufficient numbers of peripheral blood stem cells to produce safe and rapid haemopoietic reconstitution can be collected from most patients who achieve complete remission using apheresis Protocol B without impairment of haemopoietic recovery or adversely affecting the length of complete remission.

Granulocyte colony-stimulating factor (G-CSF) dose-dependent efficacy in peripheral blood stem cell mobilization in patients who had failed initial mobilization with chemotherapy and G-CSF.

For 10 consecutive patients in our unit who did not show a significant rise in blood progenitor cells within 14 days following chemotherapy and G-CSF, we increased the G-CSF dose from 5 to 10 microg/kg/day (n = 9) or from 10 to 15 microg/kg/day (n = 1). As a result, there were significant increases in total yield as well as yield per apheresis of mononuclear cells, CD34+ cells and CFU-GM (P < 0.025, <0.01 and <0.005, respectively). After G-CSF dose escalation, six of the 10 patients had sufficient CD34+ cells for performing transplantation. These results demonstrate a dose-dependent response of progenitor cell mobilization by G-CSF when used in combination with chemotherapy. Moreover, increasing the dose of G-CSF as late as the third week of mobilization may still provide sufficient cell yield even with patients who did not show a significant mobilization with conventional doses of G-CSF.

Comparison of haematological recovery times and supportive care requirements of autologous recovery phase peripheral blood stem cell transplants, autologous bone marrow transplants and allogeneic bone marrow transplants.

The haematological recovery time, infection rate and supportive care requirements of patients receiving recovery phase autologous peripheral blood stem cell transplants (APBSCT) (n = 38), autologous bone marrow transplants (autoBMT) (n = 13) and allogeneic bone marrow transplants (alloBMT) (n = 14) were compared with respect to the time post-transplant to reach 0.1, 0.5 and 2.0 x 10(9) neutrophils/l and 50 and 150 x 10(9) platelets/l, the length of hospitalization, fever and antibiotic use, the incidence of documented infection and the number of red cell and platelet transfusions. The APBSCT group had a significantly more rapid recovery of neutrophils and platelets and their supportive care requirements were significantly less than the autoBMT and the alloBMT groups. There was no difference between the latter two groups. The most significant variables contributing to the differences in haematological recovery times were the granulocyte-macrophage progenitor (CFU-GM) dose infused and, to a lesser extent, patient age. The APBSCT group received a higher CFU-GM dose of 87 +/- 12 x 10(4)/kg BW compared with 12 +/- 5 and 17 +/- 3 x 10(4)/kg BW in the autoBMT and the alloBMT groups, respectively (p = 0.0001). Patient age showed a negative correlation with the rate of recovery because the APBSCT group, which recovered faster was also older (48 +/- 2 years, compared with 33 +/- 3 and 31 +/- 2, respectively, p = 0.0001). On multivariate analysis, CFU-GM dose was the only variable to show a significant correlation with all the haematological recovery endpoints studied in these 65 patients.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of the APAAP technique as a rapid indicator of peripheral blood progenitor cell levels.

Rapid and sustained engraftment following autotransplantation with peripheral blood stem cells (PBSC) depends on adequate numbers of stem cells and progenitor cells. In this study we have compared the number of myeloid progenitor cells quantitated using the colony forming units-granulocyte macrophage (CFU-GM) clonogenic assay with the number of CD34+ cells estimated both by flow cytometry and by the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. We have analysed 15 peripheral blood mononuclear cells (PBMNC) samples from 13 normal subjects and 179 PBMNC from 32 patients undergoing PBSC harvests during the recovery phase of high dose cyclophosphamide chemotheraphy. The number of CD34+ cells measured by the APAAP technique correlated well with the number of CD34+ cells measured by flow cytometry (r = 0.727, p = 0.0001), and also with the number of CFU-GM measured in the clonogenic assay (r = 0.721, p = 0.0001). The APAAP method provides a rapid, reliable measure of progenitor cell levels that can be used to monitor the optimal time to harvest peripheral blood stem cells (PBSC), and to estimate the marrow repopulating ability (MRA) of stem cell preparations used for transplantation.

Standardization of the CFU-GM assay using hematopoietic growth factors.

The colony-forming unit-granulocyte-macrophage (CFU-GM) assay is used commonly to assess adequacy of progenitor number in bone marrow transplantation. The assay is poorly standardized, resulting in variability of results between and within laboratories. We assessed three variables that contribute to the lack of standardization. The colony-stimulating activity of human placental-conditioned medium (HPCM) was compared with combinations of recombinant hematopoietic growth factors (HGF) in 5 normal bone marrow donors. A protocol for batch testing of fetal calf serum (FCS) is described. In addition, a rigid training program has been introduced to minimize interstaff and intrastaff variability in the counting of colonies. We show that a five-factor combination of interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and stem cell factor (SCF) produces a mean increase of 85% in colony number. Some combinations of three HGF produce similar growth to HPCM, and all four HGF combinations are equivalent or superior to HPCM. Batch testing of FCS shows variability between batches. We show significant interstaff and intrastaff variability between a new and experienced staff member that improves following a period of training. In summary, the use of recombinant HGF in association with a rigorous program of batch testing of FCS and staff training results in a CFU-GM assay that can be standardized between laboratories.

A discrepancy between the instantaneous and the overall collection efficiency of the Fenwal CS3000 for peripheral blood stem cell apheresis.

The collection efficiency (CE) of the Fenwall CS3000 continuous flow blood cell separator in the apheresis of peripheral blood stem cells during haemopoietic recovery following myelosuppressive chemotherapy was analysed. Ninety-three apheresis were performed in 19 patients using procedure 3 on the Fenwal CS3000. The overall CE was calculated from the pre-apheresis cell counts and the stated blood volume processed. Instantaneous CE was calculated from cell counts in the inlet and return lines. The overall mononuclear cell and granulocyte-macrophage colony forming unit CE were 64.0% and 55.8%, respectively, significantly lower than the instantaneous CEs of 94.5% and 95.4%, respectively (P = 0.0001, t test, for both comparisons). Three factors unrelated to machine performance contributed to the lower overall CE despite a high instantaneous CE: (1) A fall in the patient's mononuclear cell counts during apheresis leading to an overestimation of the cells available for collection, (2) dilution of blood by anti-coagulant, and (3) the operational dead space of the Fenwal CS3000. The overall CE corrected for these 3 factors approximated the instantaneous CE closely. Thus there is little room for further enhancement of machine performance because the Fenwal CS3000 is already operating with a very high instantaneous CE. To achieve major improvement in the yield of peripheral blood stem cell harvests, more effective mobilization protocols and better timing of apheresis are required.

Stimulation of proliferation, differentiation, and function of human cells by primate interleukin 3.

Cloned gibbon interleukin 3 (gIL-3) was found to stimulate the proliferation and differentiation of human bone marrow cells to produce day-14 granulocyte, macrophage, granulocyte-macrophage, and eosinophil colonies in semisolid agar. In the presence of normal human plasma, gIL-3 stimulated megakaryocytes. In methylcellulose cultures, it stimulated erythroid colonies in the presence, but not in the absence, of erythropoietin. When mature human leukocytes were used, gIL-3 stimulated the function of purified mature eosinophils as measured by the capacity to kill antibody-coated target cells, to produce superoxide anions, and to phagocytize opsonized yeast particles in a manner similar to recombinant human granulocyte-macrophage colony-stimulating factor. In contrast, gIL-3 did not significantly stimulate any of the neutrophil functions tested, whereas human recombinant granulocyte-macrophage colony-stimulating factor was active in these assays. Among cytokines that are active on human hematopoietic cells, gIL-3 thus has a distinct set of functions and may predict the range of actions of the human molecule.

Recombinant human interleukin-3 stimulation of hematopoiesis in humans: loss of responsiveness with differentiation in the neutrophilic myeloid series.

Recombinant human (rh) interleukin-3 (IL-3) stimulated the proliferation and differentiation of erythroid, granulocyte, macrophage, eosinophil (Eo), and mixed colonies as well as megakaryocytes from human bone marrow cells. rh IL-3 was a weaker stimulus than rh granulocyte-macrophage colony-stimulating factor (GM-CSF) for day 14 myeloid cell colonies. At day 7 of incubation, rh IL-3 stimulated a few G, M, and Eo clusters but no colonies. This loss of responsiveness of myeloid cells to rh IL-3 was accentuated with further differentiation of the cells. rh IL-3 stimulated very few or no clones after five-day incubation with enriched promyelocytes and myelocytes, whereas rh GM-CSF was an efficient stimulus. Responsiveness to rh IL-3 was completely lost in postmitotic mature neutrophils. Incubation of these cells with rh IL-3 did not result in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells or superoxide anion production after stimulation with formyl-methyl-leucyl-phenylalanine (FMLP), although they could be stimulated by rh GM-CSF. In addition, preincubation of neutrophils with different concentrations of rh IL-3 failed to increase or decrease their response to rh GM-CSF. In contrast to neutrophils, mature Eos could be stimulated by rh IL-3 to kill antibody-coated tumor cells. These results show that cells of the neutrophilic myeloid series lose their responsiveness to h IL-3 as they differentiate and suggest that although h IL-3 may be an important therapeutic agent to use for hematopoietic regeneration in vivo, the lack of stimulation of mature neutrophil function makes it an unlikely sole candidate as adjunct therapy for treatment of infectious diseases.