Marcelo Villa

Autologous peripheral blood CD133 + cell implantation for limb salvage in patients with critical limb ischemia

We report the safety and feasibility of autologous CD133+ cell implantation into the lower extremity muscles of patients with critical limb ischemia, whose only other option was limb amputation. Nine patients participated in the study: seven patients suffering from arteriosclerosis obliterans, one with thromboangiitis obliterans (Buergers disease) and one with thromboembolic disorder. Autologous PBSC were collected after the administration of G-CSF (10 mcg/kg/day). CD133+ cells were selected using the CLINIMACS cell separation device and were injected i.m. without earlier cryopreservation using a 22-gauge needle into multiple sites 3 cm apart in the gastrocnemius/soleus muscle, or depending on clinical circumstances, in the foot or quadriceps muscle, or both, of the involved leg. There were no complications from either leukapheresis or injection. Stem cell injection prevented leg amputation in seven of the nine patients. In this small cohort of patients with end-stage critical limb ischemia, quality of life (Short Form-36) physical component score improved significantly at 3 (P=0.02) and 6 (P=0.01) months, but not at 1 year (P=0.08). There was a trend towards the improvement in pain-free treadmill walking time (P=0.13) and exercise capacity (P=0.16) at 1 year. Lower extremity limb salvage was achieved for seven of the nine treated patients.

Phase I/II study of combined granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor administration for the mobilization of hematopoietic progenitor cells.

PURPOSE To study the toxicity and efficacy of combined granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) administration for mobilization of hematopoietic progenitor cells (HPCs). MATERIALS AND METHODS Cohorts of a minimum of five patients each were treated subcutaneously as follows: G-CSF 5 micrograms/kg on days 1 to 12 and GM-CSF at .5, 1, or 5 micrograms/kg on days 7 to 12 (cohorts 1, 2, and 3); GM-CSF 5 micrograms/kg on days 1 to 12 and G-CSF 5 micrograms/kg on days 7 to 12 (cohort 4); and G-CSF and GM-CSF 5 micrograms/kg each on days 1 to 12 (cohort 5). Ten-liter aphereses were performed on days 1 (baseline, pre-CSF), 5, 7, 11, and 13. Colony assays for granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) were performed on each harvest. RESULTS The principal toxicities were myalgias, bone pain, fever, nausea, and mild thrombocytopenia, but none was dose-limiting. Four days of treatment with either G-CSF or GM-CSF resulted in dramatic and sustained increases in the numbers of CFU-GM per kilogram collected per harvest that represented 35.6 +/- 8.9- and 33.7 +/- 13.0-fold increases over baseline, respectively. This increment was attributable both to increased numbers of mononuclear cells collected per 10-L apheresis and to increased concentrations of progenitors within each collection. The administration of G-CSF to patients already receiving GM-CSF (cohort 4) caused the HPC content to surge to nearly 80-fold the baseline (P = .024); the reverse sequence, ie, the addition of GM-CSF to G-CSF, was less effective. The CFU-GM content of the baseline aphereses correlated with the maximal mobilization achieved (r = .74, P = .001). CONCLUSION Combined G-CSF and GM-CSF administration effectively and predictably mobilizes HPCs and facilitates apheresis.

Mobilization, harvesting and selection of peripheral blood stem cells in patients with autoimmune diseases undergoing autologous hematopoietic stem cell transplantation

Peripheral blood stem cells (PBSC) were mobilized in 130 patients with autoimmune diseases undergoing autologous hematopoietic stem cell transplantation using cyclophosphamide 2 g/m2 and either granulocyte colony-stimulating factor (G-CSF) 5 mcg/kg/day (for systemic lupus erythematosus (SLE) and secondary progressive multiple sclerosis, SPMS) or G-CSF 10 mcg/kg/day (for relapsing remitting multiple sclerosis (RRMS), Crohns disease (CD), systemic sclerosis (SSc), and other immune-mediated disorders). Mobilization-related mortality was 0.8% (one of 130) secondary to infection. Circulating peripheral blood (PB) CD34+ cells/μl differed significantly by disease. Collected CD34+ cells/kg/apheresis and overall collection efficiency was significantly better using Spectra apheresis device compared to the Fenwall CS3000 instrument. Patients with SLE and RRMS achieved the lowest and the highest CD34+ cell yields, respectively. Ex vivo CD34+ cell selection employing Isolex 300iv2.5 apparatus was significantly more efficient compared to CEPRATE CS device. Circulating PB CD34+ cells/μl correlated positively with initial CD34+ cells/kg/apheresis and enriched product CD34+ cells/kg. Mean WBC and platelet engraftment (ANC>0.5 × 109/l and platelet count >20 × 109/l) occurred on days 9 and 11, respectively. Infused CD34+ cell/kg dose showed significant direct correlation with faster white blood cell (WBC) and platelet engraftment. When adjusted for CD34+ cell/kg dose, patients treated with a myeloablative regimen had significantly slower WBC and platelet recovery compared to non-myeloablative regimens.

Cobe Spectra is superior to Fenwal CS 3000 Plus for collection of hematopoietic stem cells

One hundred and seventy-seven stem cell apheresis procedures performed on 91 patients using the Fenwal CS 3000 Plus cell separator and 61 procedures performed on 37 patients using the Cobe Spectra cell separator were studied to compare the CD34+ cell collection efficiencies (CE; the proportion of the total CD34+ cell content in the blood volumes processed that is harvested) of the two machines. The absolute peripheral blood CD34+ cell count was comparable for the two groups (P = 0.27). A strong correlation was seen between the blood CD34+ cell count and the total number of CD34+ cells collected for the Spectra (r2 = 0.59; P < 10−6) and for the CS 3000 Plus (r2 = 0.60; P < 10−6). No significant correlation emerged between the peripheral blood CD34+ cell count and the CE of either machine. The total number of CD34+ cells collected per procedure was comparable (P = 0.51): median 113 × 106 for CS 3000 Plus and median 218 × 106for Spectra. CE was significantly higher with the Spectra (median 45.7%, range 9.8–98.6%) than the CS 3000 Plus (median 30.3%, range 1.7–89.3%; P < 0.00001). We conclude that the CD34+ cell CE of the Spectra is superior to that of the CS 3000 Plus. Therefore, under the usual clinical conditions, Cobe Spectra should be used preferentially for peripheral blood progentor cell collection to maximize the number of hematopoietic stem cells collected.

CD34(+) cell collection efficiency does not correlate with the pre-leukapheresis hematocrit.

One hundred and seventy-seven large-volume leukapheresis procedures performed on 91 patients over a 15 month period were reviewed to see if the pre-apheresis hematocrit (Hct) affected the CD34+ cell collection efficiency (CE) of the Fenwal CS 3000 Plus cell separator. The Hct was 0.174–0.461 (median 0.317), and the peripheral blood CD34+ cell count 2–2487 per μl (median 21). The total CD34+ cell quantity collected was 3.0–2677.2 × 106 (median 113.0). Based on the number of CD34+cells contained in the blood volume processed (23.3–37303.2 × 106; median 318.0), the CE was 1.7–87.5% (median 30.3). No correlation was found between the Hct and CE (r2 = 0.0034; P = 0.44) or the total CD34+ cell quantity collected (r2 = 0.0040; P = 0.40). CEs for Hct <0.25 (median CE 36%), Hct 0.25–0.299 (median CE 30%) and Hct 0.30 (median CE 30%) were comparable. As expected, highly significant correlations were seen between the CD34+ cell quantities collected and quantities processed (r2 = 0.59; P < 10−6) as well as the peripheral blood CD34+ cell counts (r2 = 0.60; P < 10−6). We conclude that the minimum acceptable Hct or hemoglobin level for leukapheresis should be dictated by clinical circumstances because it does not affect stem cell collection. Bone Marrow Transplantation (2001) 28, 597–601.

Stem cell component therapy: supplementation of unmanipulated marrow with CD34 enriched peripheral blood stem cells.

Eleven patients with hematologic malignancies and two with aplastic anemia were treated using unmanipulated marrow and immunoselected CD34+ blood cells. Donors began G-CSF (10 μg/kg) injections 1 day after undergoing bone marrow harvest. Blood stem cells were collected on day 5 of G-CSF. Peripheral blood lymphocytes were depleted via CD34-positive selection. If, after marrow and blood harvest, less than 2.0 × 106 CD34 cells/kg were mobilized, leukapheresis was repeated on day 6. Median time to an absolute neutrophil count greater than 500 μl was day 10; transfusion-independent platelet count greater than 20000/μl was day 13; average hospital discharge was day 14; and average inpatient hospital charges were 101870 US dollars. Acute GVHD grade II occurred in five of 13 patients. No patient developed grade III or IV acute GVHD. At a median follow-up of 10 months, no patient has developed extensive chronic GVHD. Allografts of unmanipulated bone marrow supplemented with G-CSF-mobilized and CD34 immunoselected blood cells may prevent an increased risk of GVHD while preserving the rapid engraftment kinetics of peripheral blood. Supplementation of marrow with CD34 enriched blood cells appears to result in rapid engraftment, early hospital discharge, lower inpatient charges, decreased regimen-related toxicity, and no apparent increase in GVHD.

Actual or ideal body weight to calculate CD34+ cell dose in patients undergoing autologous hematopoietic SCT for myeloma?

CD34+ cell dose calculations are usually based on actual body weight (ABW). We have shown that ideal body weight (IBW) may provide a better basis for this in a small population of patients with hematologic malignancies. This was studied further in 514 myeloma autografts. The CD34+ cell doses (106/kg) by IBW and ABW were 1.37–39.36 (median 6.03) and 1.15–29.67 (median 4.84), respectively. IBW-based cell doses correlated slightly better with engraftment than ABW-based doses (higher r2): 0.5 × 109/l neutrophils 0.83 versus 0.82, 1.0 × 109/l neutrophils 0.78 versus 0.77, 20 × 109/l platelets 0.54 versus 0.53 and 50 × 109/l platelets 0.57 versus 0.55. When outliers (hematologic recovery in <8 or >16 days) were excluded, the findings were similar: 0.5 × 109/l neutrophils 0.85 versus 0.84, 1.0 × 109/l neutrophils 0.85 versus 0.84, 20 × 109/l platelets 0.86 versus 0.85 and 50 × 109/l platelets 0.85 versus 0.84. CD34+ cell doses based on IBW as well as ABW significantly affected engraftment when analyzed separately as continuous variables. However, when analyzed together, only the dose based on IBW retained significance. We conclude that calculation of CD34+ cell numbers for autotransplantation should be based on IBW.

Immunohistochemical detection of breast cancer cells in paired peripheral blood progenitor cell specimens collected after cytokine or cytokine and myelosuppressive chemotherapy

Mobilized peripheral blood progenitor cells (PBPC) from 30 patients with advanced breast cancer were studied for the presence of tumor cell contamination using a highly sensitive immunohistochemical technique with the capacity to detect one tumor cell in one million mononuclear cells. Aliquots of PBPC were obtained after 4 days of G-CSF and/or GM-CSF and again during G-CSF-stimulated recovery from myelosuppressive doses of cyclophosphamide. The overall incidence of tumor cell contamination was 23%, occurring in PBPC specimens from seven of 30 patients. All four cases in which tumor cells were detected after mobilization with cytokine alone also had tumor cells detected in PBPCs collected following chemotherapy and G-CSF. There were three cases in which malignant contamination was detected only in the specimens collected after cyclophosphamide. There was a greater frequency of tumor cell contamination in aphereses performed during G-CSF-stimulated recovery from cyclophosphamide than in collections primed by cytokine alone (13% vs 23%; P = 0.08), although this did not reach statistical significance. This trend suggests that collection of PBPC during cytokine-stimulated recovery from myelosuppressive chemotherapy may be associated with a greater risk of contamination with malignant cells than apheresis during mobilization with cytokines in the steady state.