P-selectin-coated microtube for the purification of CD45+ hematopoietic cells directly from human peripheral blood.
Purified samples of CD45+ hematopoietic cells are a prerequisite for chimerism analysis in transplantation therapies, and are useful in various research and clinical settings such as functional and molecular analysis or disease diagnosis. Recently, we have established a flow-based adhesion molecule-dependent process for the purification of these cells from human bone marrow. However, for practical purposes, it is desirable to apply this approach to process small volumes of human blood. CD45+ cell purities were >94% when PBMNCs and plasma depleted blood were perfused through P-selectin coated microtubes. However, P-selectin surface failed to capture CD45+ cells when fresh blood prior to washing was perfused. The process requires a pre-step of plasma removal which otherwise inhibits interactions of cell surface PSGL-1 with immobilized P-selectin due to the presence of soluble PSGL-1 in plasma. We conclude that P-selectin can be used in a compact flow device to isolate and purify CD45+ cells directly from human peripheral blood. The process is simple, rapid, cost effective and represents a physiologic approach to the capture and purification of CD45+ MNCs from peripheral blood.
Narasipura
Srinivas D
SD
Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA.
King
Michael R
MR
D002448:Cell Adhesion; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods; D002470:Cell Survival; D002499:Centrifugation, Density Gradient; D005362:Ficoll; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism*; D017493:Leukocyte Common Antigens / Q000032:analysis*; D007962:Leukocytes / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D019007:P-Selectin / Q000378:metabolism*; D010477:Perfusion; D010949:Plasma; D011994:Recombinant Proteins / Q000378:metabolism
D055767:Immobilized Proteins; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D011994:Recombinant Proteins; D005362:Ficoll; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human