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.


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.


CONCLUSIONS
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.
Snyder
E L
EL
Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut 06504, USA. edward.snyder@yale.edu
O'Donnell
L
L
Dengler
T J
TJ
Pomper
G J
GJ
Velleca
M A
MA
Dincecco
D M
DM
Baril
L L
LL
Min
K
K
Gudino
M D
MD
Bender
J R
JR
D001782:Blood Donors; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology; D002455:Cell Division / Q000502:physiology; D002469:Cell Separation / Q000295:instrumentation*; D003601:Cytotoxicity Tests, Immunologic; D019040:E-Selectin / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000166:cytology; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D007231:Infant, Newborn; D007694:Killer Cells, Natural / Q000502:physiology; D007937:Leukapheresis; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000502:physiology; D013997:Time Factors; D014471:Umbilical Veins
D019040:E-Selectin