Positive selection of human blood cells using improved high gradient magnetic separation filters.
High gradient magnetic separators (HGMS) create magnetic field gradients that can be used to attract much smaller and less magnetic particles than those required for conventional magnetic separation techniques. As a result cells can be labeled with submicron magnetic particles and still be separated using an HGMS filter. Typically, HGMS filters consist of random arrays of wire such as stainless steel wool. Wire elements arranged regularly in a filter should allow more efficient separation of cells. Filters were constructed containing ordered wire arrays composed of 430 series stainless steel wire mesh with wire diameters of 50, 100, or 150 microns. The ability of these filters to separate T cells from peripheral blood mononuclear cell suspensions was tested and found superior to random arrays of 302 series stainless steel wire (Thomas et al, 1992). Target cells recognized by OKT5 monoclonal antibody were cross-linked to dextran-iron particles of approximately 20 nm in diameter. Separation conditions were optimized and after one passage through the filter 88% of the OKT5+ cells were recovered in the enriched fraction with 85% purity (%OKT5+). Multiple passages (3 times) could achieve 99% purity with 68% recovery. Variations in separation flow rate had a large effect on the balance between purity and recovery. Optimum separation efficiencies were achieved only when > 10(8) cells were processed. The primarily cause of nonspecific entrapment of CD8- cells was not nonspecific magnetic labeling of cells but the physical (nonmagnetic) characteristics of the filter/filter chamber.
Thomas
T E
TE
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada.
Richards
A J
AJ
Roath
O S
OS
Watson
J H
JH
Smith
R J
RJ
Lansdorp
P M
PM
D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes; D003102:Colloids; D003911:Dextrans; D003973:Diatrizoate; D005069:Evaluation Studies as Topic; D005362:Ficoll; D005374:Filtration / Q000295:instrumentation; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G; D018189:Immunomagnetic Separation / Q000295:instrumentation*; D007501:Iron; D007958:Leukocyte Count; D051379:Mice; D010316:Particle Size; D013193:Stainless Steel
D000911:Antibodies, Monoclonal; D003102:Colloids; D003911:Dextrans; D007074:Immunoglobulin G; D003973:Diatrizoate; D013193:Stainless Steel; D005362:Ficoll; D007501:Iron