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Separation of the immune competent cells 8th seminar Slide 2 Characteristics of the separation: purity recovery, yield, lost viability of the cells Physical isolation of the cells of interest from a heterogeneous population Differences in the physical, biological or immunological properties of the cells are utilized to separate the cells (differences in cell surface receptor expression is often available there is a possibility to further investigate the separated living cells ) physical density, size cell biological adherence, phagocytosis, sensitivity to the medium immunological antigen differences (surface!) Cell separation Slide 3 Base strategies: positive separation labeling and separation of the cells of interest eg. Labeling of a cell surface molecule (receptor!) by a fluorescent antibody. The cells become affected both by the separation environment and the antibodies bound to the receptors. The purity of the separation is generally high. negative separation get rid of the labeled unwanted cells (depletion) The cells become affected only by the separation environment This is the preferred strategy in the functional examinations. Separation Slide 4 Separation of the plasma from the cellular components: Separation by filtration (simple membrane or holofiber membrane) Pore diameter for plasma separation: 0.2 to 0.6m Peripheral blood is an easily accessible source for the separation of human immune cells Slide 5 The different density parts of the anticoagulated blood has been separating to three parts in undisturbed tube: bottom: sedimented red blood cells top: cell free plasma the intermediate layer is called buffy coat contains the leukocytes, platelets The process can be accelerated by centrifugation Slide 6 Apheresis (ancient greek ) -to take away Separating out one particular component of the blood and returning the remainder to the circulation Donor apheresis Therapeutic apheresis Donor apheresis: Plasmapheresis processed (eg.IVIG) or fresh frozen plasma for immuondefficient patients or in acute infections as passive immunization Plateletpheresis (trombocytapheresis) concentrated platelets for inherited or induced thrombocytopenia (infections, chemotherapy, irradiation) or in the case of thrombocyte disfunction Red blood cells (erythrocytapheresis) - for patients with anemia (inherited or internal/external blood loss by surgery or trauma) Leukapheresis buffy coat, mainly for autotransplantation isolating and protecting the leukocytes before chemotherapy monocyte separation for dendritic cell therapy leukapheresis after the mobilization of bone marrow stem cells for stem cell therapy or bone marrow transplantation (autologous, allogeneic) Slide 7 Therapeutic apheresis: Removing abnormal/disfunctional components of the blood (humoral or cellular) Replacing a component with a healthy donor apheresis product Blood component alteration (ex vivo therapy) Leukapheresis Extreme high leukocyte number can lead to hemostatic disorders in leukemia (accompanying shortness of breath, vision changes). Inflammatory cell number can be decreased in autoimmune chronic inflammatory diseases (ulcerative colitis, rheumatoid arthritis) LDL apheresis Removal of low density lipoprotein from the plasma in patients with familial hypercholesterolemia (adsorption with ApoB affinity column or precipitation with acetate) Thrombocytapheresis In essential trombocythemia/e.trombocytosis (rare disease) the disfunctional very high thrombocyte number (with thrombosis and bleedings) can be lowered rapidly in the rare cases of life threatening emergency situations Erythrocytapheresis - Removing abnormal red blood cells in patients experiencing sickle cell crisis (in sickle-cell anemia) Plasmaexchange Removing/replacement the plasma with autoimmune antibodies in various autoimmune diseases (combined with immunosuppression) (eg. Myasthenia gravis, Guillain-Barr syndrome, lupus, Goodpasture syndrome, Antiphospholipid antibody syndrome, Behcet syndrome, etc.) Immunoadsorption with protein A/G removal of allo- and autoantibodies (in autoimmune diseases, transplant rejection, hemophilia) by directing plasma through protein A or G-agarose columns Slide 8 Continuous Flow Centrifugation (CFC) Cross section representation of an old fashioned washable apheresis centrifuge bowl Continuous Flow Apheresis Systems Thrombocytes and thrombocyte free plasma can be separated by appropriate CFC methods Slide 9 plasma production by filtration & WBC plasma, red blood cells, and buffy coat production by continuous flow centrifugation concentrated platelet production from pooled buffy coats Slide 10 Buffy coat contains too much erythrocytes to investigate the white blood cell part. Further separation is needed. Ficoll-Paque (1.077g/ml) Slide 11 (Nature Protocols http://www.nature.com/nprot/journal/v3/n6/images/nprot.2008.69-F1.jpg) (from Google pictures) Slide 12 Ficoll-Paque: density based cell separation peripheral blood pipetting cells on ficoll, or pipetting ficoll under the cells centrifugation separated cells plasma ficoll Red blood cells mononuclear cells (PBMC) neutrophyl granulocytes pipettig the ring containing the mononuclear cells to a new tube to get rid of ficoll Slide 13 Percoll for selecting neutrophyl granulocytes stepwise or continuous pecoll gradient for the selection of neutrophyls Romanian J. Biophys., Vol. 14, Nos. 14, P. 5358, Bucharest, 2004 Physical chemical characterization of Percoll. I-III. Laurent, T.C. et al. Colloid Interface Sci. 76, 124145 (1980). Slide 14 Ficoll separation combined with rosette formation can be used for the depletion of unwanted cells negative separation Slide 15 Isolating or depleting adherent cells (negative and positive separation) Cheap, simple, but only for adharent cells. Low purity and recovery. Slide 16 Antibody panning coated antibodies (negative and positive separation) Slide 17 Complement mediated lysis antibodies complement LYSIS (Red blood cells could be lysated in mild hypotonic ammonium-chloride buffer without any pretreatment) (negative separation) Slide 18 Magnetic immunoseparation (MACS) paramagnetic bead antigene specific antibody Simple magnetic cell separation Phagocyte cells can uptake small iron particles. These cells could be separated with a strong magnet. Slide 19 MACS Slide 20 Magnetic cell separation (MACS) MAGNET column depleting or selecting unlabeled cells (negative separation) separation of labeled cells (positive separation) Slide 21 Magnetic column Slide 22 CliniMACS closed system magnetic cell separator CliniMACS Prodigy coming soon CliniMACS Plus Slide 23 MicroBeads are very small, usually dont interfere with cellular functions. CD8+ T cells DETACHaBEAD Policlonal antibodies against the Fab fragment of the antigen specific antibodies can be produced (including anti-idiotype antibodies). These could compete with the cell surface antigens for binding to the magnetic antibodies. So the magnetic antibodies could detach from the cells. Slide 24 FACS (Fluorescence Activated Cell Sorting) any distinctive cell population can be gated, and the gated cells could be separated in one step PMT 1 PMT 2 PMT 4 Laser Flow cell PMT 3 Sample Different gating strategies can be combined: by light scattering by specific immunofluorescence Slide 25 CD antigencell typefunctionligand CD3T cells TCR signalling- CD4helper T sejtek, (monocytes, pDC) T cell coreceptor, (HIV receptor) MHC- II, HIV CD5T cells, (B cell subset: B1) adhesion, activation signals CD72 CD8cytotoxic T cells, (NK, T cells) T cell coreceptor MHC I CD14monocytes, macrophages, some granulocytes LPS binding LPS, LBP CD19B cells part of CR2, B cell coreceptor C3d, C3b CD28T cells costimulatory signals to T cells (B7-1, B7-2) CD80, CD86 CD34hematopoietic progenitor cell adhesion CD62L (L-selektin) CD56NK cell, (T and B cell subset) homoadhesion (N-CAM isoform) CD80, CD86 (B7-1, -2) APC: DC, B, monocyte, macrophage costimulatory signals CD28, CD152 Slide 26 Example: CD5+ B1 cell separation (CD19/CD5) NKT cell separation (CD3/CD56) NK cells NKT cells lymphocytes Try to figure out how to achieve this by magnetic separation Slide 27 The fluid stream break up into dropplets by the vibration of the flow cell. breakoff point Slide 28 Laser + - - - - + + + ++ ++ + - - - - - vibration (nozzle orifice of the flow cell) If the wanted cell reach the breakoff point, the stream become charged for the short time of drop formation, and the formed drop become charged charged deflection plate + + + + + + + + + collection tube waste Slide 29 Slide 30 Vge Slide 31 mozdulatlan