Ex vivo evaluation of PBMNCs collected with a new cell separator.

BACKGROUND: This study reports on an evaluation of the ability of a cell separator (Amicus, Baxter Healthcare) and the integral MNC computer software program to collect a variety of MNC subsets. The collection efficiency (CE) of the Amicus for these MNC subsets was compared to that of another cell separator (CS-3000 Plus, Baxter). The collected MNCs were also assayed ex vivo to determine if these cells remained functional. STUDY DESIGN AND METHODS: Healthy volunteer blood donors were recruited to provide PBMNCs for the isolation of CD3+, CD4+, CD8+, CD19+, NK, and gammadelta+ cells and monocytes. Cells were collected with an Amicus (test arm; n = 16) or a CS-3000 Plus (control arm; n = 11) cell separator. Cells were counted on a flow cytometer and CEs were calculated. For functional studies, the Amicus-collected MNC data were compared to CS-3000 Plus historical data. Functional studies performed included surface antigen expression assays (CD8+), proliferation assays (CD4+ and CD8+ cells), NK cytotoxicity assays for K562 and HUVE cells, and E-selectin induction on endothelial cells through NK+ contact dependency. Dendritic cells (DCs) were generated from CD34+ cells collected on the Amicus, positively selected by the use of antibody-bound, magnetic bead technology, and then cultured ex vivo with a combination of growth factors to generate the DCs. RESULTS: CEs were higher on the Amicus than on the CS-3000 Plus for CD3+ (68 vs. 54%), CD4+ (70 vs. 56%), CD8+ (68 vs. 52%), and CD19+ (60 vs. 48%) cells (p<0.05). For the two separators, CEs were equivalent for monocytes, NK+, and gammadelta+ cells. The Amicus separator collected significantly fewer platelets than did the CS-3000 Plus (p<0.00001). CD4+, CD8+, and NK cells proliferated normally. NK cells appropriately stimulated E-selectin expression on endothelial cells. Culture-generated DCs obtained by using Amicus-collected CD34+ cells expressed appropriate cell surface markers. CONCLUSION: The Amicus separator is acceptable for the collection of PBMNC subsets. The device collects CD3+, CD4+, CD8+, and CD19+ T- and B-cell subsets with greater efficiency and collects MNCs with significantly fewer contaminating platelets than does the CS-3000 Plus. Cells collected on the Amicus are suitable for use in a variety of research and clinical immunobiologic studies.

Four crossmatch methods to select platelet donors.

We employed four crossmatch techniques to select platelet donors for refractory patients. Forty-four donor-recipient pairs were studied in 32 patients. Analysis of effectiveness of platelet transfusions revealed that only 18 percent of transfusions gave a borderline response; the remainder were either effective or not effective at all. The corrected predictive values of three crossmatch tests were as follows: enzyme-linked immuno-specific assay, 81 percent; platelet immunofluorescence test, 73 percent; and lymphocytotoxicity, 70 percent (p greater than 0.05). The predictive value of these tests did not differ in HLA-matched versus unmatched platelet transfusions. Donor selection by lymphocytotoxicity compatibility did not appear to be useful if donors were selected by either of the other two methods. The fourth test, antiglobulin-modified lymphocytotoxicity, offered no advantage over lymphocytotoxicity. Our data suggest that platelet crossmatching assays are a useful adjunct to the selection process for the platelet donor in addition to ABO, Rh, and HLA matching.