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Separation of the immune competent cells 8th
seminar
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Cell separation

• 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!)

• Characteristics of the separation
• purity
• recovery, yield, lost
• viability of the cells

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Separation
Base strategies positive separation labeling
and separation of the cells of interest e.g.
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.
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Peripheral blood is an easily accessible source
for the separation of human immune cells
Separation of the plasma from the cellular
components Separation by filtration (simple
membrane or holofiber membrane) Pore diameter
for plasma separation 0.2 to 0.6µm
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The different density parts of the anticoagulated
blood is separated 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
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• Apheresis (ancient greek ?fa??es??) -to take
  away
• Separating one particular component of the blood
  and returning the remainder to the circulation
• Donor apheresis
• Therapeutic apheresis

• Donor apheresis
• Plasmapheresis processed (e.g. IVIG) or fresh
  frozen plasma for immunodefficient 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)

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• 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-Barré 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

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Continuous Flow Apheresis Systems
Continuous Flow Centrifugation (CFC)
Cross section representation of an old fashioned
washable apheresis centrifuge bowl
Thrombocytes and thrombocyte free plasma can be
separated by appropriate CFC methods
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plasma, red blood cells, and buffy coat
production by continuous flow centrifugation
plasma production by filtration
WBC
concentrated platelet production from pooled
buffy coats
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Buffy coat contains too much erythrocytes to
investigate the white blood cell part. Further
separation is needed.
Ficoll-Paque (1.077g/ml)
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(Nature Protocols http//www.nature.com/nprot/jour
nal/v3/n6/images/nprot.2008.69-F1.jpg)
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Ficoll-Paque density based cell separation
pipettig the ring containing the mononuclear
cells to a new tube to get rid of ficoll
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Ficoll separation combined with rosette formation
can be used for the depletion of unwanted cells
negative separation
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Isolating or depleting adherent cells (negative
and positive separation)
Cheap, simple, but only for adharent cells. Low
purity and recovery.
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Antibody panning
(negative and positive separation)
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Complement mediated lysis
antibodies
complement
LYSIS
(negative separation)
(Red blood cells could be lysated in mild
hypotonic ammonium-chloride buffer without any
pretreatment)
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Simple magnetic cell separation
Phagocyte cells can uptake small iron
particles. These cells could be separated with a
strong magnet.
Magnetic immunoseparation (MACS)
antigene specific antibody
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MACS
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Magnetic cell separation (MACS)
column
depleting or selecting unlabeled cells (negative
separation)
separation of labeled cells (positive separation)
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CliniMACS closed system magnetic cell separator
automats
CliniMACS Plus
CliniMACS Prodigy
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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.
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CD antigen cell type function ligand
CD3 T cells TCR signalling -
CD4 helper T sejtek, (monocytes, pDC) T cell coreceptor, (HIV receptor) MHC- II, HIV
CD5 T cells, (B cell subset B1) adhesion, activation signals CD72
CD8 cytotoxic T cells, (NK, ?? T cells) T cell coreceptor MHC I
CD14 monocytes, macrophages, some granulocytes LPS binding LPS, LBP
CD19 B cells part of CR2, B cell coreceptor C3d, C3b
CD28 T cells costimulatory signals to T cells (B7-1, B7-2) CD80, CD86
CD34 hematopoietic progenitor cell adhesion CD62L (L-selektin)
CD56 NK cell, (T and B cell subset) homoadhesion (N-CAM isoform)
CD80, CD86 (B7-1, -2) APC DC, B, monocyte, macrophage costimulatory signals CD28, CD152
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FACS
Example NKT cell separation (CD3/CD56)
Try to figure out how to achieve this by magnetic
separation
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The fluid stream break up into dropplets by the
vibration of the flow cell.
breakoff point
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vibration (nozzle orifice of the flow cell)
Laser
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
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charged deflection plate

charged deflection plate


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collection tube
collection tube
waste
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