Allogeneic GM-CSF-secreting tumor vaccines are safe in patients with pancreatic adenocarcinoma. This vaccine approach seems to induce dose-dependent systemic antitumor immunity as measured by increased postvaccination DTH responses against autologous tumors. Further clinical evaluation of this approach in patients with pancreatic cancer is warranted.

Reinfusion of PBPC collected in a single leukapheresis accelerates engraftment in the majority of patients. Pretreatment bone marrow CD34+ cell content determines PBPC mobilization capacity and may help select hematopoietic rescue strategies.

The use of peripheral blood progenitor cells (PBPC) for hematopoietic rescue after high-dose chemotherapy is limited by the number of leukaphereses required to collect an adequate number of hematopoietic progenitors. To optimize the collection of PBPC, we evaluated a single large-volume leukapheresis protocol with citrate anticoagulation. A group of 23 patients received cyclophosphamide (4 g/m2) and GM-CSF (5 micrograms/kg/day for 15 days) as PBPC mobilization, with a single outpatient 6 h leukapheresis performed on the COBE Spectra 15 days later. Citrate (0.190 mmol/ml) was infused at 1.2 ml/L of blood/minute with a whole blood to citrate ratio between 17:1 and 25:1. Calcium chloride (50 mM) was administered at a citrate to calcium molar ratio between 10:1 and 5:1 to prevent hypocalcemia. A median 36.6 L (range 24.4-46.4) blood was processed using 338 mM citrate (269-473) and 50 mM calcium (25-75). A median 5 x 10(6) CD34+ cells/kg (< 0.3-24) and 6.2 x 10(5) CFU-GM/kg (< 0.001-29) were collected, representing 5.6 and 5.9 more PBPC, respectively, than were in circulation at the initiation of leukapheresis. We conclude that a 6 h large-volume leukapheresis following cyclophosphamide and GM-CSF mobilization is safe, can recruit hematopoietic progenitors into the circulatory compartment, and allows the collection of high numbers of PBPC in a single procedure.

A procedure is described for the measurement of the %CD34+ progenitor cells in bone marrow using directly conjugated antibodies. Staining cells with anti-CD45.FITC in conjunction with anti-CD34.PE allows the CD45- nucleated red blood cells and the CD45++ lymphocytes and monocytes to be separated from the CD45+ progenitor cells. Granulocytes are separated from the CD34+ cells based on differences in side scatter properties. A gated acquisition of CD34+ cells is used to define the boundaries of the CD34+ population in a plot of forward scatter vs side scatter and in a plot of anti-CD45.FITC vs anti-CD34.PE. Use of these regions during analysis reduces background staining and allows for a consistent identification of a CD34+ population. Acquisition of 50,000 cells provides adequate precision of the %CD34+ measurement. Acquisition and analysis procedures are presented for use of both a Becton Dickinson FACScan flow cytometer and a Coulter EPICS Profile II flow cytometer.

Bone marrow harvests were processed using two techniques; 20 mononuclear cell concentrations (MNC) were prepared on the COBE Spectra and compared to 10 light-density cell fractions isolated using the COBE 2991. Both procedures recovered essentially the same percentage of nucleated cells (22 versus 21%) and gave progenitor cell recoveries of 132 and 100% of the CFU-GM and 101 and 104% of the CD34+ cells in the MNC and light-density products, respectively. The advantage of the Spectra MNC concentrate is that it was prepared with reagents approved for injection by the Food and Drug Administration. However, the average hematocrit on the MNC concentrate was 4%, while it was unmeasurable in the light-density cell fractions. This difference is significant only when bone marrows require purging with 4-hydroperoxycyclophosphamide or erythrocytes otherwise interfere with processing. The Spectra procedure recovered a larger percentage of MNC cells and had less contamination with mature granulocytes than did the density-gradient technique. When erythrocytes do not affect the bone marrow processing protocol, a Spectra MNC concentrate is a safe substitute for a light-density cell preparation.