Transcript – a brief overview Flow sorting Ian Titley Haematology Oncology

Flow sorting – a brief overview
Ian Titley
Haematology Oncology
Leukaemia Research Fund
Institute of Cancer Research
London
Flow sorting - a form of fluorescence microscopy
whereby single cells in liquid suspension can be
identified and physically separated from each
other according to unique characteristics
History of cell sorter
1st prototype at Los Alamos National Laboratory (LANL) in 1965 developed by Mack
J Fulwyler by “joining” Coulter volume sensing (invented by Wallace and Joseph
Coulter) with the newly invented ink-jet printer technology.
Biologist Leonard Herzenberg at Stanford University was the first to recognise the
utility for this technology to biological applications. With colleagues from the Genetics
Dept Instrument Research group and using Fulwyler’s plans developed a machine to
sort fluorescently labelled cells. The 1969 instrument had a mercury arc lamp as light
source and in 1972 an argon ion laser.
Funding from the National Institutes of Health (NIH) allowed Herzenberg and the
Stanford engineers to interest the medical products company Becton Dickinson (BD)
to convert their prototypes into the first commercial instruments, the FACS
(Fluorescence Activated Cell Sorter) in 1975.
Smithsonian Institute Archives – Video History Collection
http://www.si.edu/archives/ihd/videocatalog/9554.htm
Electrostatic
Mechanical
“Lab-on-chip”
Electrostatic Sorting : Stream-in-air
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Hydrodynamic focusing in a nozzle
vibrated by a transducer produces a
stream breaking into droplets.
Laser interrogation and signal processing
followed by sort decision : white sort right,
blue sort left, green or black no sort.
Electronic delay until cell reaches break
off point. Then the stream is charged :
+ if white - if blue.
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+
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Charged droplets deflect by electrostatic field
from plates held at high voltage (+/- 3000 volts).
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left waste
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right
Various collection devices can be attached :
tubes, slides, multi-well plates.
[email protected]
http://www.cardiff.ac.uk/medicine/haematology/cytonetuk/introduction_to_fcm/cell_sorting.htm
BD VantageSE DiVa option
DakoCytomation MoFlo
Beckman Coulter Epics Altra Hypersort
BD FACSAria
Sorting into a 96 well plate
(Taken from BD FACSAria brochure)
Electrostatic
Pros
Fast (?x104cellssec-1)
Multiparameter
High purity
4-way
Cons
Expensive to buy and run
Needs trained operator
Aerosol – biohazard
Electrostatic
Mechanical
“Lab-on-chip”
Mechanical Sorting:
Takes place within a Flow cell
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or by applying
an acoustic
pulse to the
stream to
divert the cell
into the tube.
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Hydrodynamic focusing
takes place within a flow cell.
Laser beam interrogates cells
When a sort decision “Green cell” has been
made it is diverted into a ‘catcher’ tube
either by moving the tube into the stream:
o
o
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[email protected]
http://www.cardiff.ac.uk/medicine/haematology/cytonetuk/introduction_to_fcm/cell_sorting.htm
Partec PPCS
Union Biometrica COPAS
BD FACSCalibur
Mechanical
Pros
“Cheaper”
No aerosol
Easier to use
Multiparameter
Cons
Slow (?x102cellssec-1)
1(2) way sorting
“Dilute” sorted sample
Electrostatic
Mechanical
“Lab-on-chip”
Gawad, S., Heuschkel, M., Leung-Ki, Y., Iuzzolino, R., Schild, L., Lerch, Ph. & Renaud,
Ph.Fabrication of a microfluidic cell ananlyzer in a microchannel using impedance
spectroscopy in IEEE-EMBS Conference on Microtechnologies in Medicine & Biology 297-301
(Lyon, France, 2000).
Useful texts
Flow cytometry: a practical approach MG Ormerod IRL Press
Practical flow cytometry HM Shapiro WileyLiss
Current protocols in flow cytometry Ed JP Robinson et al Wiley
Some cell sorting web resources
Terry Hoy et al Cardiff University
http://www.cardiff.ac.uk/medicine/haematology/cytonetuk/introduction_to_fcm/cell_sorting.htm
Royal Microscopical Society
http://www.rms.org.uk/cyto.shtml
Derek Davies et al London Research Institute
http://science.cancerresearchuk.org/sci/facs/fac_labinfo/flow_sorting/?version=1
Cytometry E-mail archive Purdue USA
http://www.cyto.purdue.edu/hmarchiv/index.htm