Cell Sorting

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Cell Sorting
Svensk Förening FörFlödescytometri2005
Lasse GisselsonBD Biosciences
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Sorting - FACSCalibur
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Sorting - FACSVantage
Charging Electrode
Sample Injection Tube
Nozzle
Sheath Tube
Vent Tube
Deflection Plates
Collection Tubes
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FACSAria Cuvette Flow Cell

• Fixed optical alignment

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Sorting - FACSAria
Deflection Plates
Collection Tubes
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Sorting - FACSAria
Cuvette
Point of analysis
Nozzle
Drop Delay
Sort pulse (Drop Charge)
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Sorting - FACSAria
Nozzle
Sort pulse (Drop Charge)
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Sorting - FACSAria
Nozzle
Sort pulse (Drop Charge)
- - -
Charged droplet
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Sorting - FACSAria
Deflection Plates
-

0 - - -
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Droplet sorting

• What we want to do
• Sort FAST
• Sort PURE
• Sort with a high YIELD

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Sort speed
Sorting speed is dependent on fluid pressure and
nozzle size Example At 70 psi preessure, using
a 70 µm nozzle, the frequency of drop formation
can be up to 90 kHz. I.e. 90000 drops per
second can be formed.
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Sort speed
Drop formation frequency Jet velocity Jet
Velocity 250 KHz would require 500 psi
(!) Sequential cell sorting will not become
faster (?)
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Purity
Purity of the sort is mainly dependent on the
sample The major source of contamination during
a sort are DOUBLETS!
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Purity
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Purity
AND
!
AND
!
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Purity / doublet discrimination
Gate on singlets!
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Sort decisions

• A sort decision must be made for each particle
• The sort decision is based on the sort gate AND
  on sort masks.
• Sort masks can be defined based on the purpouse
  of the sort. There is a tradeoff between purity
  and yield.
• The effect of this tradeoff is dependent on sort
  speed and the frequency of the cells of interest.

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Yield

•  Yield mask , to minimize loss of cells due to
  position uncertainty
• Two drop sort

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Yield

• One drop sort

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Purity

•  Purity Mask , to avoid contamination
• No sort,contaminating cell in purity mask

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Purity

• Sort,no contaminating cell in purity mask

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Yield Purity conflict resolution

• Two drop sort

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Yield and purity conflict resolution

• Second drop sort decision is reversed

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Cloning count accuracy

•  Phase Mask , to ensure count accuracy
• No sort

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Cloning count accuracy

•  Phase Mask 
• Sort

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Summary

• Yield mask to recover cells that would be lost
  due to position uncertainty
• Purity mask to avoid contamination
• Phase mask for 100 count accuracy

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Speed Purity Yield tradeoff

• The purity/yield tradeoff increases in importance
  with increasing sort speed

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Speed Purity Yield tradeoff
80.000 Hz 80.000 ev/s
80.000 Hz 20.000 ev/s
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Speed Purity Yield tradeoff
80.000 Hz 80.000 ev/s
80.000 Hz 20.000 ev/s
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Speed Purity Yield tradeoff

• Sort twice (1. Yield, 2. Purity)
• Enrich (or deplete) cells magnetically